Articles in PresS. Am J Physiol Lung Cell Mol Physiol (January 27, 2012). doi:10.1152/ajplung.00293.2011
Acidosis and Vascular Function in Pulmonary Hypertension
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Improved Pulmonary Vascular Reactivity and Decreased Hypertrophic Remodeling
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during Non-Hypercapnic Acidosis in Experimental Pulmonary Hypertension
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Helen Christou1,4, Ossama M. Reslan2, Virak Mam2, Alain F. Tanbe2, Sally H. Vitali3,
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Marlin Touma1,4, Elena Arons1, S. Alex Mitsialis4, Stella Kourembanas1,4,
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Raouf A. Khalil2
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Division of Newborn Medicine, and 2Division of Vascular and Endovascular Surgery,
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Brigham and Women’s Hospital, and 3Department of Anesthesia and 4Division of Newborn
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Medicine, Children’s Hospital, Harvard Medical School, Boston, MA 02115
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Running Title: Acidosis and Vascular Function in Pulmonary Hypertension
Correspondence and Reprints:
Raouf A Khalil, MD, PhD
Harvard Medical School
Brigham and Women's Hospital
Division of Vascular Surgery
75 Francis Street
Boston, MA 02115
Tel: (617) 525-8530
Fax: (617) 264-5124
E-mail: [email protected]
List of Abbreviations: Ach, acetylcholine; ET-1, endothelin; FI, Fulton’s index; L-NAME, Nωnitro-L-arginine methyl ester; LVSP, left ventricular systolic pressure; NO, nitric oxide; NOS,
nitric oxide synthase; ODQ, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one; PDE-5,
phosphodiesterase-5; PGI2, prostacyclin; PH, pulmonary hypertension; Phe, phenylephrine;
RVH, right ventricular hypertrophy; RVSP, right ventricular systolic pressure; SNP, sodium
nitroprusside; VSMC, vascular smooth muscle cell
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Copyright © 2012 by the American Physiological Society.
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ABSTRACT
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Pulmonary hypertension (PH) is characterized by pulmonary arteriolar remodeling with
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excessive pulmonary vascular smooth muscle cell (VSMC) proliferation. This results in
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decreased responsiveness of pulmonary circulation to vasodilator therapies. We have shown
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that extracellular acidosis inhibits VSMC proliferation and migration in vitro. Here we tested
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whether induction of non-hypercapnic acidosis in vivo ameliorates PH and the underlying
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pulmonary vascular remodeling and dysfunction. Adult male Sprague-Dawley rats were
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exposed to hypoxia (8.5% O2) for two weeks, or injected s.c. with monocrotaline (MCT, 60
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mg/kg) to develop PH. Acidosis was induced with NH4Cl (1.5%) in the drinking water 5 days
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prior to and during the two weeks of hypoxic exposure (prevention protocol), or after MCT
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injection from day 21 to 28 (reversal protocol). Right ventricular systolic pressure (RVSP) and
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Fulton’s index were measured, and pulmonary arteriolar remodeling was analyzed. Pulmonary
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and mesenteric artery contraction to phenylephrine (Phe) and high KCl, and relaxation to
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acetylcholine (Ach) and sodium nitroprusside (SNP) were examined ex vivo. Hypoxic and
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MCT-treated rats demonstrated increased RVSP, Fulton’s index and pulmonary arteriolar
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thickening. In pulmonary arteries of hypoxic and MCT rats there was reduced contraction to
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Phe and KCl, and reduced vasodilation to Ach and SNP. Acidosis prevented hypoxia-induced
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PH, reversed MCT-induced PH, and resulted in reduction in all indices of PH including RVSP,
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Fulton’s index and pulmonary arteriolar remodeling. Pulmonary artery contraction to Phe and
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KCl was preserved or improved, and relaxation to Ach and SNP was enhanced in NH4Cl-
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treated PH animals. Acidosis alone did not affect the hemodynamics or pulmonary vascular
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function. Phe and KCl contraction, and Ach and SNP relaxation were not different in
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mesenteric arteries of all groups. Thus non-hypercapnic acidosis ameliorates experimental
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PH,
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responsiveness to vasoconstrictor and vasodilator stimuli.
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acidosis decreases VSMC proliferation, the results are consistent with the possibility that non-
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hypercapnic acidosis promotes differentiation of pulmonary VSMCs to a more contractile
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phenotype, which may enhance the effectiveness of vasodilator therapies in PH.
attenuates
pulmonary
arteriolar
thickening
2
and
enhances
pulmonary
vascular
Together with our finding that
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Key words: pulmonary artery, pulmonary circulation, nitric oxide, vascular smooth muscle,
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hypertension
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INTRODUCTION
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Pulmonary Hypertension (PH) is a serious disease and a major and expanding public
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health problem with approximately 1000 new patients diagnosed every year in the United
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States (6, 20). PH is characterized by increased pulmonary arterial pressure and right
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ventricular hypertrophy (RVH). Increased pulmonary vascular resistance and pulmonary
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vascular remodeling lead to progressive right ventricular failure and significantly compromise
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the quality of life and life expectancy in affected individuals (20, 21, 51).
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Although the etiology of PH is diverse and multi-factorial, the underlying pathology and
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pathophysiology are common among its various forms. Endothelial dysfunction, excessive
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pulmonary vascular smooth muscle cell (VSMC) proliferation, hypertrophy, and migration, as
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well as various degrees of pulmonary vasoconstriction and inflammation are major
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components of the pathobiology of PH, and therefore represent important targets for current
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and emerging therapies (9, 13). Vasodilator therapies such as prostacyclin (PGI2), endothelin
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(ET-1) receptor antagonists and phosphodiesterase-5 (PDE-5) inhibitors, either separate or in
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combination, are variably successful in slowing PH progression and prolonging survival (10,
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47, 51, 71, 80). Also, it is increasingly appreciated that alternative approaches such as anti-
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proliferative, pro-apoptotic, or immuno-modulatory therapies hold promise in further improving
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the outcome of PH (5, 18, 36, 52, 54, 64, 77). Importantly, accumulating evidence supports
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that a phenotypic switch of pulmonary VSMCs from a contractile to a proliferative phenotype
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may contribute to the pathogenesis of PH (4, 24, 45, 48, 53), and reversal of this pathology
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could enhance the effectiveness of vasodilator therapies.
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Rodent models of experimental PH recapitulate to a certain extent the histologic features
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of human PH, and provide a useful tool to study the efficacy of novel therapeutic approaches
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(17, 31, 46, 65). The hypoxic and monocrotaline (MCT) rat models are particularly useful in the
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evaluation of anti-proliferative and pro-apoptotic therapies since medial hypertrophy of the
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pulmonary arterioles is a key feature in these PH models (36, 52, 54, 64). We have previously
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shown that chronic hypoxia and MCT treatment in rats are associated with decreased
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pulmonary artery reactivity and significant pulmonary arteriolar remodeling (50). We have also
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reported that extracellular acidosis inhibits proliferation and migration of cultured rat and
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mouse VSMCs (8, 11, 30). Although some studies have raised the possibility that hypercapnic
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acidosis may be protective in the setting of PH (42, 58), the role of acidosis per se has not
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been examined, and the pulmonary vascular mechanisms involved have not been clearly
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identified.
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acidosis is protective in experimental PH by improving pulmonary artery reactivity and by
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decreasing hypertrophic remodeling. We measured the hemodynamics and examined the
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pulmonary arterial function in hypoxia and MCT-induced rat models of PH chronically-treated
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or non-treated with NH4Cl in order to determine whether: 1) non-hypercapnic acidosis
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improves hemodynamic parameters and ameliorates RVH in experimental PH, 2) mild acidosis
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improves pulmonary arterial relaxation via the endothelium-dependent NO-cGMP pathway in
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PH, 3) the pulmonary VSMC responsiveness to vasodilators is enhanced during non-
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hypercapnic acidosis in experimental PH, and 4) the beneficial effects of acidosis in
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experimental PH could be related to decreased pulmonary arterial remodeling.
The present study was designed to test the hypothesis that non-hypercapnic
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METHODS
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Animals: Adult (12 week) male Sprague-Dawley rats (250-300g) (Charles River Laboratories,
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Wilmington, MA) were housed in the animal facility in 12 hr/12 hr light/dark cycle, at 22±1°C
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ambient temperature and maintained on ad libitum normal Purina Rodent Chow (Purina, St.
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Louis, MO) and tap water. All experiments were approved by the Children’s Hospital Animal
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Care and Use Committee and the Harvard Medical Area Standard Committee on Animals.
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Hypoxic Exposure: Rats were exposed to chronic hypoxia at 8.5% O2 inside a chamber,
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where O2 is controlled to within a 0.2% range by an OxyCycler controller (BioSpherix, Redfield,
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NY) (76). Electronic controllers injected nitrogen into the hypoxic chamber to maintain the
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appropriate FiO2, and ventilation was adjusted to remove CO2 so that it did not exceed 5,000
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ppm (0.5%). Ammonia was removed by ventilation and activated charcoal filtration using an
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electric air purifier. The hypoxic chamber was opened twice a week to replenish food and
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water and to change the bedding. The duration of hypoxic exposure was two weeks. Normoxic
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control rats were kept in the same animal room outside the hypoxic chamber.
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Monocrotaline (MCT) Injection: For the MCT model of PH, age-matched rats were given a
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single subcutaneous injection of 60 mg/kg MCT (Sigma, St. Louis, MO). Control rats were
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injected with the same volume of vehicle (normal saline). The rats were assessed for
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development of PH 28 days after injection.
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Induction of Non-Hypercapnic Acidosis: Non-hypercapnic acidosis was induced in order to
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examine: 1) Whether non-hypercapnic acidosis can prevent experimental PH before the onset
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of the disease, and this was tested in the hypoxic model.
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acidosis can reverse experimental PH after the disease is already established, and this was
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tested in the MCT model. To induce non-hypercapnic acidosis in the hypoxic animals,
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ammonium chloride (NH4Cl, 1.5%) was added to the drinking water for 5 days prior to and
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continued during the two weeks of hypoxic exposure (prevention protocol). Sucrose (5%) was
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added to increase palatability of the drinking water for both hypoxic and control normoxic rats.
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Water consumption was monitored and was estimated to be ~20 ml per rat per day. For
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induction of non-hypercapnic acidosis in the MCT rats, NH4Cl and sucrose were added to the
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drinking water starting on day 21 after MCT injection and continued until day 28 (late reversal
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protocol).
2) Whether non-hypercapnic
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NH4Cl treatment for 3 to 5 days has been used to induce metabolic acidosis in
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experimental animals (22, 49). Our initial experiments showed that animals pretreated with
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NH4Cl for 5 days then subjected to hypoxia for one week with continuous NH4Cl treatment had
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significantly lower Fulton’s index (less RVH) than animals subjected to one week hypoxia
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without NH4Cl treatment. As prolonged hypoxia could cause a more severe form of PH, we
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tested whether induction of acidosis would ameliorate hypoxic PH after prolonged hypoxic
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exposure. Prolonged two weeks hypoxia caused further increase in Fulton’s index, and animal
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pretreatment with NH4Cl for 5 days and continuous NH4Cl treatment for the two weeks of
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hypoxia caused significant reduction in Fulton’s index (see Results section).
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Because acidosis was effective as a preventive strategy, we also tested whether it would
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be effective as a reversal strategy. While there are no established reversal protocols for the
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hypoxic rat model of PH, the MCT model has been utilized in established reversal protocols:
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treatments aimed at ‘early reversal’ last from day 14-28, and treatments aimed at ‘late reversal’
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last from day 21-28. We chose the ‘late reversal’ protocol because we reasoned that if our
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intervention was successful, this would be the most clinically relevant model. Also, given that
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MCT requires conversion to the toxic metabolite in order to cause the disease, we wanted to
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space the acidosis intervention at a sufficiently remote time from MCT metabolism so that the
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results would not be attributed to possible effects of NH4Cl on MCT metabolism.
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Right and Left Ventricular Systolic Pressure (RVSP and LVSP) Measurements: At the
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end of the experimental exposure, rats were anesthetized with 2% isoflurane inhalation and
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remained spontaneously breathing. A small, transverse incision was made in the abdominal
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wall, and the transparent diaphragm exposed.
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attached to a pressure transducer, was inserted through the diaphragm first into the right
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ventricle and then into the left ventricle, and pressure measurements were recorded in
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spontaneously breathing animals with heart rates over 300/min using PowerLab monitoring
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hardware and software (ADInstruments, Colorado Springs, CO). Mean RVSP and LVSP over
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the first 10 stable heart beats was recorded.
A 23-gauge butterfly needle, with tubing
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Arterial Blood Gas and Hematocrit Analyses: After hemodynamic measurements, a 0.2 ml
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blood sample was collected from the cardiac chambers for determination of hematocrit, pH
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and pCO2 using a blood gas analyzer (Roche Diagnostics Indianapolis, IN). Over-anesthetized
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animals with respiratory depression (PCO2 >55mmHg) were excluded from the analysis.
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Right Ventricular Weight and Fulton’s Index: Hearts and pulmonary vasculature were
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perfused in situ with cold 1X phosphate buffered Saline (PBS) injection into the right ventricle.
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The heart was excised, and both ventricles were weighed. The right ventricular free wall was
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then dissected and the remaining left ventricular wall and ventricular septum were weighed.
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RVH was assessed as Fulton’s Index (ratio of right ventricular weight to the left
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ventricular+septum weight) or as the ratio of right ventricular weight to total body weight.
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Tissue Preparation for ex vivo Vascular Function Studies: In euthanized rats, the thoracic
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cavity was opened, and the lung and pulmonary arteries were rapidly excised. The abdominal
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cavity was then opened and the mesentery and mesenteric arterial arcade were excised and
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placed in oxygenated Krebs solution. The right and left pulmonary artery, and 2nd order
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mesenteric arteries were carefully dissected and cleaned of connective tissue under
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microscopic visualization, and cut into 3 mm-wide rings.
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Isometric Contraction. Vascular segments were suspended between two tungsten wire hooks,
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with one hook fixed at the bottom of a tissue bath and the other hook connected to a Grass force
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transducer (FT03, Astro-Med Inc., West Warwick, RI). Pulmonary artery and mesenteric artery
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segments from the same rat were stretched under 1 g or 0.5 g of resting tension, respectively (as
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determined by preliminary tension-contraction curves to KCl), and allowed to equilibrate for 45
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min in a temperature controlled, water-jacketed tissue bath, filled with 50 ml Krebs solution
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continuously bubbled with 95% O2 5% CO2 at 37ºC. The changes in isometric contraction were
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recorded on a Grass polygraph (Model 7D, Astro-Med).
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After tissue equilibration, a control contraction in response to 96 mM KCl was elicited.
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Once maximum KCl contraction was reached the tissue was rinsed with Krebs 3 times, 10 min
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each. The control KCl-induced contraction followed by rinsing in Krebs was repeated twice.
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Vascular segments were stimulated with increasing concentrations of phenylephrine (Phe, 10-9
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to 10-5 M), concentration-contraction curves were constructed, and the maximal Phe
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contraction and the pED50 (-log M) were calculated. In other experiments, the tissues were
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precontracted with Phe (10-5 M), increasing concentrations (10-9 to 10-5 M) of acetylcholine
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(Ach) were added and the % relaxation of Phe contraction was measured. Parallel contraction
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and relaxation experiments were performed in endothelium-intact vascular rings pretreated
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with the NO synthase (NOS) inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 3x10-4 M) or
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the guanylate cyclase inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10-5 M) for
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10 min. In other experiments the relaxation to increasing concentrations (10-9 to 10-5 M) of the
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exogenous NO donor sodium nitroprusside (SNP) was measured in vascular rings
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precontracted with Phe.
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Lung Histology and Morphometric Analysis: In a subset of experimental animals, the lungs
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were perfused with PBS through the right ventricle to remove the blood from the pulmonary
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vessels, fixed with cold 4% paraformaldehyde through the trachea, then excised and fixed in
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4% paraformaldehyde overnight at 4° C followed by paraffin embedding. Lung sections (6 μm)
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were stained with hematoxylin and eosin and examined with light microscopy by two
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independent investigators (E.A. and M.T.) in a blinded fashion. Images of the arterioles were
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captured with a microscope digital camera system (Nikon) and analyzed using an image
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analysis program (NIH Image). At least 15 arterioles of comparable size (50-100 μm diameter)
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per rat, from the lungs of 5 different rats from each experimental group were evaluated. The
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percent wall thickness was determined by dividing the area occupied by the vessel wall by the
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total cross sectional area of the arteriole as previously reported (12). This method accounts for
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uneven vessel wall thickness and areas that have obliquely sectioned pulmonary arterioles.
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Solutions and Drugs. Krebs solution contained (in mM): NaCl 120, KCl 5.9, NaHCO3 25,
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NaH2PO4 1.2, dextrose 11.5, CaCl2 2.5, MgCl2 1.2, at pH 7.4, and bubbled with 95% O2 and 5%
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CO2. KCl (96mM) was prepared as Krebs solution with equimolar substitution of NaCl with KCl.
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Stock solutions of Phe, Ach and L-NAME (10-1 M, Sigma) were prepared in distilled water. Stock
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solution of ODQ (10-1 M, EMD Biosciences) was prepared in DMSO. Final concentration of
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DMSO in experimental solution was <0.1%. All other chemicals were of reagent grade or better.
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Statistical Analysis: Cumulative data from 6 to 12 rats per experimental group were analyzed
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and presented as means±SEM, with the “n” value representing the number of rats. For the
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hemodynamic and histology data, group comparisons were done with a one way ANOVA and
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Tukey-Kramer post test for multiple comparisons. Correlation was done with a non-parametric
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test (Spearman correlation, Graphpad Prism). For the ex vivo studies in vascular rings,
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contraction and relaxation experiments were performed on 2 to 4 rings of pulmonary artery and 2
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rings of mesenteric artery from each rat, and the data from different vascular rings from each
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vascular bed were averaged for each rat. Cumulative data from 6 to 8 different rats per
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experimental group were presented as means±SEM with the “n” value representing the number
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of rats.
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F=(variance between groups/variance within groups). When a statistical difference was
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observed, the data were further analyzed using Student-Newman-Keuls post-hoc test for
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multiple comparisons. Concentration-contraction curves and Phe ED50 were determined using
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non-linear regression best-fit sigmoidal curve (Sigmaplot).
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statistically significant if p<0.05.
Data were first analyzed using one way ANOVA with Scheffe’s F test, where
Differences were considered
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RESULTS
Effect of NH4Cl treatment on body weight, acid base status, and hematocrit
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Initial body weight in all rats was 250-300g. Treatment of rats under normoxia with NH4Cl
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for 5 days did not significantly affect body weight (Table 1). Hypoxic animals did not gain
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weight over a two week period. Also, following two weeks of hypoxic exposure, NH4Cl-treated
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rats had significantly lower body weight (243±11g) compared to non-treated rats (289±10g,
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p=0.004). In comparison, MCT-treated rats gained weight, and NH4Cl treatment did not
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significantly change their body weight (Table 1).
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In order to address the effectiveness of NH4Cl treatment in inducing non-hypercapnic
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acidosis we performed arterial blood gas analysis. As shown in Table 1, in rats under hypoxic
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exposure for two weeks treatment with NH4Cl resulted in significantly lower pH values
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compared to hypoxic rats without NH4Cl treatment. The mean pH of hypoxic rats after a two
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week hypoxic exposure (7.29±0.01) was significantly lower than that of normoxic animals
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(7.37±0.01, p=0.0001), and treatment with NH4Cl led to a significantly lower pH (7.08±0.02,
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p<0.0001) compared to non-treated hypoxic rats in the same hypoxic chamber. These
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changes were not due to hypercapnia because mean pCO2 in the NH4Cl-treated hypoxic rats
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(40±1.8 mmHg) was not significantly different from that in hypoxic rats in the same chamber
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and without NH4Cl treatment (45±2 mmHg, p=0.08). Of note, hypoxic rats that received NH4Cl
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had significantly lower HCO3- levels compared to hypoxic rats without NH4Cl treatment. In
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MCT-induced PH, treatment with NH4Cl for one week was associated with significantly lower
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systemic pH (7.26±0.04) and HCO3- (19.5±1.7) compared to MCT-treated rats without NH4Cl
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treatment (Mean pH 7.36±0.01 and mean HCO3- 27.5±0.7, p<0.05) (Table 1).
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Hematocrit, a sensitive indicator of hypoxia, was significantly greater in hypoxic animals
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(63.3±2.3% after two week hypoxic exposure) than normoxic animals (38.5±1.2%, p<0.001).
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Among hypoxic rats, there were no significant differences in hematocrits between NH4Cl-
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treated (66.1±2.4%) and non-treated rats (63.3±2.3% at two weeks). This indicates that
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treatment with NH4Cl did not interfere with the polycythemic response to hypoxia in our
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experimental animals. Hematocrits were not significantly different between MCT-treated and
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normoxic rats with or without treatment with NH4Cl (Table 1).
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Treatment with NH4Cl decreases RVSP in experimental PH
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Hemodynamic
measurements
were
performed
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under
isoflurane
anesthesia
in
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spontaneously breathing animals using direct right ventricular puncture. Measurements of
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hemodynamics revealed signs of PH in hypoxic rats.
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pressure in the pulmonary circulation, was significantly increased after hypoxic exposure
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(57.1±2.8 mmHg) compared to normoxia (26.3±4.9 mmHg, p<0.05) (Fig. 1A and 1B). In
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hypoxic rats, treatment with NH4Cl was associated with a small but significant reduction of
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RVSP (48.2±2.2 mmHg), although these rats still had significantly higher RVSP than normoxic
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controls (Fig. 1). Left ventricular systolic pressure (LVSP), an indicator of blood pressure in the
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systemic circulation, was not significantly different in hypoxic compared to normoxic rats, and
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treatment with NH4Cl did not significantly alter LVSP (Fig. 1C).
The RVSP, an indicator of blood
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We also examined whether NH4Cl treatment in established PH (21 days after MCT
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injection) would be effective in reversing MCT-induced PH. Hemodynamic measurements
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revealed significantly greater RVSP in MCT-treated compared to vehicle-treated rats (Fig. 1B).
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RVSP was not significantly different in the MCT- vs. hypoxia-induced model of PH. Treatment
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with NH4Cl resulted in a significant reduction in RVSP in MCT-treated rats, although the RVSP
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in these animals remained elevated compared to vehicle-treated controls (Fig. 1B). MCT-
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treated rats had slightly but not significantly lower LVSP compared to vehicle-treated controls,
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and treatment with NH4Cl was associated with significant decrease in LVSP (Fig. 1C).
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Induction of mild acidosis in experimental PH leads to amelioration of RVH
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In order to define the effect of NH4Cl treatment on the hypoxic response of the right
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ventricle, we next assessed RVH using Fulton’s index (FI) and the ratio of right ventricular
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weight to total body weight. FI was 0.28±0.007 in normoxic rats, and increased significantly
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after two weeks of hypoxic exposure (0.54±0.02, p<0.001) (Fig. 2A). Treatment with NH4Cl for
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5 days prior to and during the two week hypoxic exposure resulted in significant amelioration
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of RVH and reduction of FI (0.44±0.01, p<0.0001). Similar findings were observed when RVH
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was assessed as the ratio of right ventricular weight to total body weight (Fig. 2B). Importantly,
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among the rats exposed to hypoxia for two weeks with or without treatment with NH4Cl, there
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was a linear relationship between plasma pH and FI (Fig. 2C). It thus appears that the degree
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of protection by NH4Cl treatment against RVH is proportional to the degree of metabolic
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acidosis induced. Similarly, FI was significantly increased in MCT-treated rats compared to
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vehicle-injected controls, and treatment with NH4Cl significantly decreased FI in MCT-treated
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animals (Fig. 2A). Also, the right ventricular weight to total body weight was significantly
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increased in MCT-treated rats compared to vehicle-injected controls, and was significantly
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reduced during treatment with NH4Cl (Fig. 2B).
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Treatment with NH4Cl preserves pulmonary artery contraction in experimental PH
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In pulmonary artery rings of normoxic rats, the α-adrenergic agonist phenylephrine (Phe)
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caused concentration-dependent contraction that reached a maximum at 10-5 M (Fig. 3A).
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The Phe-induced contraction was significantly reduced in pulmonary artery rings from hypoxic
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and MCT-treated rats compared to normoxic rats (Fig. 3A and Table 2) suggesting reduced
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pulmonary vascular reactivity in PH. In hypoxic and MCT-treated rats that received NH4Cl,
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Phe-induced contraction was significantly improved to levels approaching, yet still less than,
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those observed in control normoxic rats (Fig. 3A and Table 2). When the Phe contraction was
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presented as % of max, and the ED50 was calculated, Phe appeared to be equally potent in
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inducing contraction in the pulmonary arteries of the various groups of rats (Fig. 3C and Table
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2). In comparison, parallel experiments on mesenteric arteries from the same animals
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demonstrated that Phe-induced maximum contraction and the Phe ED50 did not differ among
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the various experimental groups (Fig. 3B, 3D and Table 2).
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To examine the effect of acidosis on another vasoconstrictor stimulus, the contractile
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response to membrane depolarization by KCl was examined. Membrane depolarization by 96
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mM KCl caused significant contraction in pulmonary and mesenteric artery of normoxic rats
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(Fig. 4). KCl contraction was significantly reduced in pulmonary arteries of hypoxic and MCT-
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treated rats compared with normoxic rats, and treatment with NH4Cl significantly improved KCl
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contraction in the hypoxic and MCT-treated groups (Fig. 4A). KCl-induced contraction
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remained significantly lower in pulmonary arteries of hypoxia+acidosis rats compared with
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normoxia+acidosis rats, but was enhanced in MCT rats treated with NH4Cl to levels not
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significantly different from those in normoxia+acidosis rats. In contrast, KCl contraction was not
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significantly different in mesenteric arteries of normoxic, hypoxic and MCT-treated animals
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irrespective of NH4Cl treatment (Fig. 4B).
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Pretreatment of pulmonary artery with the NOS inhibitor L-NAME (3x10-4 M) or the
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guanylate cyclase inhibitor ODQ (10-5 M) for 10 min caused an increase in basal tension and
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slightly enhanced the magnitude of Phe-induced contraction in normoxic rats (Fig. 5A and
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Table 2). In pulmonary artery of normoxic rats treated with NH4Cl, L-Name did not cause any
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change in Phe contraction, and ODQ caused an apparent yet insignificant enhancement of
345
Phe contraction (Fig. 5A and Table 2). Treatment of pulmonary artery rings with L-NAME or
346
ODQ caused a small increase in basal tension in hypoxic and MCT-treated rats and minimally
347
enhanced the magnitude of Phe contraction (Fig. 5C, 5E and Table 2), and the Phe responses
348
were still less than those of control normoxic rats. Also, in pulmonary artery rings of hypoxic
349
and MCT-treated rats that received NH4Cl, L-NAME and ODQ caused little change in basal
350
tension and Phe contraction (Fig. 5D, 5F and Table 2). The Phe contractile response as % of
351
maximal Phe-induced contraction and the Phe ED50 were similar in L-NAME- and ODQ-
352
treated pulmonary artery from normoxic rats with or without NH4Cl treatment (Fig. 6A, 6B and
353
Table 2). Although the Phe ED50 was not significantly different in the pulmonary artery of the
354
different experimental groups (Table 2), the Phe contractile response as % of max was
355
significantly enhanced with ODQ in pulmonary artery segments of hypoxic and MCT-treated
356
rats with (Fig. 6D, 6F) or without treatment with NH4Cl (Fig. 6C, 6E). Also, presenting the Phe
357
contraction as % of Ca2+-dependent KCl contraction demonstrated that the Phe contraction
358
was slightly, yet significantly enhanced by ODQ in pulmonary arteries from normoxic rats
359
irrespective of NH4Cl treatment (Fig. 7A, 7B). In comparison, in the presence of L-NAME or
360
ODQ, the Phe contraction as % of KCl was dramatically enhanced in hypoxic and MCT-treated
14
Acidosis and Vascular Function in Pulmonary Hypertension
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361
rats with (Fig. 7D, 7F) or without NH4Cl treatment (Fig. 7C, 7E and Table 2). Combined,
362
these findings suggest that a potential compensatory mechanism involving the NO-cGMP
363
signaling pathway may be activated in experimental PH, and NH4Cl treatment did not
364
significantly alter or interfere with this potential rescue mechanism.
365
366
Treatment with NH4Cl improves pulmonary artery relaxation in experimental PH
367
Ach caused concentration-dependent relaxation in Phe-precontracted pulmonary artery
368
rings of normoxic rats that reached a maximum of 48.56±2.88% at 10-5 M (Fig. 8A). Ach
369
relaxation was not significantly different in pulmonary arteries of normoxic rats with or without
370
NH4Cl treatment (Fig. 8A). Ach-induced pulmonary artery relaxation was significantly reduced
371
in hypoxic and MCT-treated rats compared to normoxic rats (Fig. 8A and Table 2), suggesting
372
either reduced production of, or decreased responsiveness to, endothelium-derived
373
vasodilators such as NO in experimental PH.
374
hypoxic and MCT-treated rats that received NH4Cl to levels approaching, but still less than,
375
those observed in normoxic rats (Fig. 8A and Table 2). In contrast, Ach relaxation was not
376
significantly different in Phe-precontracted mesenteric artery rings of normoxic, hypoxic and
377
MCT-treated rats with or without NH4Cl treatment (Fig. 8B and Table 2).
Ach-induced relaxation was enhanced in
378
In pulmonary artery rings of normoxic rats with or without treatment with NH4Cl, the NOS
379
inhibitor L-NAME and the guanylate cyclase inhibitor ODQ abolished Ach relaxation,
380
suggesting the involvement of the NO-cGMP pathway (Fig. 9A, 9B). Pretreatment with L-
381
NAME or ODQ also abolished the remaining small Ach-induced relaxation in pulmonary artery
382
of hypoxic and MCT-treated rats (Fig. 9C, 9E), suggesting that the residual vasorelaxation
383
response to Ach in experimental PH is mediated by the NO-cGMP pathway. L-NAME and
384
ODQ also inhibited the improved Ach relaxation in hypoxic and MCT-treated rats that received
385
NH4Cl (Fig. 9D, 9F), suggesting that the enhanced Ach relaxation induced by NH4Cl involves
386
enhanced production of, or responsiveness to, the NO-cGMP signaling pathway.
15
Acidosis and Vascular Function in Pulmonary Hypertension
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387
In pulmonary artery segments precontracted with Phe (10-5 M), the exogenous NO donor
388
sodium nitroprusside (SNP) caused concentration-dependent relaxation that was significantly
389
reduced in hypoxic and MCT-treated rats compared to normoxic rats (Fig. 10A and Table 2),
390
suggesting decreased responsiveness of VSMCs to vasodilators. SNP-induced relaxation was
391
significantly improved in hypoxic and MCT-treated rats that received NH4Cl to levels
392
approaching those observed in normoxic rats (Fig. 10A and Table 2). In contrast, SNP-
393
induced relaxation was not significantly different in mesenteric arteries of normoxic, hypoxic
394
and MCT-treated rats with or without NH4Cl treatment (Fig. 10B and Table 2).
395
396
Treatment with NH4Cl ameliorates pulmonary vascular remodeling in experimental PH
397
To test for possible relation between the changes in vascular function in the pulmonary
398
vessels and structural remodeling of the pulmonary arterioles, lung histology and
399
morphometric analysis were performed on lung tissue sections from all experimental groups.
400
In lung tissue sections stained with hematoxylin and eosin, the % wall thickness of the
401
pulmonary arterioles was significantly greater in hypoxic rats as compared with control
402
normoxic rats (Fig. 11). In contrast, in hypoxic rats treated with NH4Cl the pulmonary vascular
403
remodeling was markedly reduced, and the thickness of the pulmonary arterioles was
404
comparable to that of the normoxic controls (Fig. 11A and 11B). The medial and adventitial
405
thickness was also increased in pulmonary arterioles from MCT-treated rats (day 28)
406
compared to vehicle-treated controls. In MCT rats treated with NH4Cl from day 21 to day 28
407
after MCT injection (late reversal) a significant reduction in pulmonary arteriolar wall thickness
408
was observed when compared to MCT-injected animals without NH4Cl treatment (Fig. 11B).
409
410
DISCUSSION
411
Extracellular pH has an important role in the regulation of systemic and pulmonary
412
vascular tone. Although hypercapnia and acidosis are clinically associated with pulmonary
413
vasoconstriction, studies in adult and newborn rats showed that hypercapnia was protective in
16
Acidosis and Vascular Function in Pulmonary Hypertension
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414
hypoxia-induced PH (42, 58). Ooi and colleagues reported that chronic hypercapnia inhibited
415
hypoxic pulmonary vascular remodeling in adult rats, but did not report the arterial pH of the
416
hypercapnic rats (58). The authors found improved Ach-induced relaxation in pulmonary
417
arteries of hypercapnic rats in the setting of hypoxia, which is in agreement with our vascular
418
function findings. However, they found that hypercapnia interfered with the polycythemic
419
response in hypoxic rats, whereas in our study non-hypercapnic acidosis did not interfere with
420
the hypoxic sensing and signaling leading to polycythemia. Kantores and coworkers reported
421
that hypercapnia attenuated oxidant stress and ameliorated PH in a hypoxic neonatal rat
422
model, possibly through prevention of hypoxic upregulation of ET-1 in distal airway epithelium
423
and pulmonary arteriolar wall (42). Although this study reported mean arterial pH of 7.10 in the
424
hypercapnic/hypoxic rats compared to 7.33 in hypoxic controls, the study did not address
425
whether the protection seen in the hypercapnic rats was mediated by acidosis. Thus although
426
acidosis was likely present in these studies (42, 58), neither study assessed the role of
427
acidosis in mediating the protective effects of hypercapnia. Interestingly, Hales and colleagues
428
suggested that pulmonary artery VSMC Na+/H+ exchanger and intracellular alkalinization may
429
play a pathogenetic role in experimental PH (60-62, 67). Together, these studies provide
430
indirect evidence that acidosis may be protective in experimental PH.
431
To test for a more direct evidence that acidosis is protective in experimental PH, the
432
present study demonstrates that induction of mild non-hypercapnic acidosis in hypoxic and
433
MCT-treated rats is associated with: 1) improved pulmonary hemodynamics and reduced
434
RVSP and RVH, 2) improved pulmonary vascular function and relaxation via the endothelium-
435
dependent NO-cGMP pathway, 3) enhanced pulmonary VSMC responsiveness to
436
vasodilators, and 4) decreased pulmonary arteriolar hypertrophic remodeling.
437
Chronic treatment with NH4Cl has been used to induce metabolic acidosis in experimental
438
animals (22, 28, 41, 49, 55). In the present study, treatment with 1.5% NH4Cl for 19 days in
439
the hypoxic group and 7 days in the MCT group was associated with a decrease in arterial pH,
440
without any significant changes in arterial pCO2 levels, and was well-tolerated. Induction of
17
Acidosis and Vascular Function in Pulmonary Hypertension
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441
non-hypercapnic acidosis in hypoxic rats was associated with improved indices of PH including
442
RVSP, RVH and FI, suggesting efficiency of acidosis in preventing the progression of PH.
443
Also, induction of acidosis in MCT-treated rats was associated with reduced RVSP, RVH, and
444
FI, supporting the efficiency of acidosis intervention in reversing the pathology of PH.
445
In order to identify the vascular mechanisms involved in the improved pulmonary
446
hemodynamics during non-hypercapnic acidosis in experimental PH, we examined vascular
447
function in pulmonary arteries from hypoxic and MCT rats with and without NH4Cl treatment.
448
Consistent with our previous report (50), pulmonary artery contraction to the α-adrenergic
449
receptor agonist Phe was reduced in hypoxic and MCT-treated rats. This is in agreement with
450
a report that ET-1-induced constriction of pulmonary artery is reduced in the hypoxic rat model
451
of PH (37). Other studies have shown that chronic hypoxia is associated with increased
452
vasomotor tone and enhanced production/activity of ET-1 and angiotensin II (AngII) in the lung
453
(56, 66). The difference in the results could be due to the vasoconstrictive agonist (Phe vs. ET-
454
1 or AngII) or the vascular preparation used (pulmonary artery vs. isolated perfused lung).
455
Importantly, Phe-induced contraction was improved in hypoxic and MCT rats treated with
456
NH4Cl. This is unlikely due to changes in the sensitivity of α-adrenergic receptors because the
457
Phe ED50 was not significantly different between normoxic, hypoxic and MCT-treated rats with
458
or without NH4Cl treatment. This is also unlikely due to increased expression of α-
459
adrenoreceptors because contraction to high KCl, a receptor-independent response, was
460
improved in hypoxic and MCT rats treated with NH4Cl, suggesting that non-hypercapnic
461
acidosis may improve a common post-receptor signaling pathway in pulmonary vessels.
462
To investigate whether the reduced pulmonary artery contraction in hypoxic and MCT rats,
463
and its improvement during non-hypercapnic acidosis involve changes in endothelium-
464
dependent NO-cGMP pathway (23, 35), we tested the effects of blockade of NO production by
465
L-NAME or inhibition of guanylate cyclase and cGMP production by ODQ. Even with NOS or
466
guanylate cyclase inhibition, Phe contraction remained significantly less in hypoxic and MCT
467
rats than normoxic rats, suggesting that the α-adrenergic post-receptor signaling mechanisms
18
Acidosis and Vascular Function in Pulmonary Hypertension
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468
or the pulmonary artery contractile machinery are less responsive to Phe. Although the Phe
469
ED50 was not significantly different in the pulmonary artery of different experimental groups,
470
the Phe contractile response as % of max was markedly enhanced by ODQ in hypoxic and
471
MCT-treated rats with and without acidosis. Also, Phe contraction as % of KCl contraction was
472
enhanced by L-NAME or ODQ in pulmonary arteries from hypoxic and MCT rats with or
473
without treatment with NH4Cl. These findings can be explained by possible activation of the
474
NO-cGMP pathway as a compensatory rescue mechanism in experimental PH, and suggest
475
that NH4Cl treatment does not interfere with this compensatory mechanism. Also, assuming
476
that KCl contraction is mainly due to Ca2+ influx (43), then the enhancing effects of blockers of
477
NO-cGMP on Phe contraction in pulmonary arteries of hypoxic and MCT rats with NH4Cl
478
treatment could be due to increased Ca2+-sensitization pathways of VSMC contraction such as
479
protein kinase C and Rho kinase. These Ca2+-sensitization pathways are likely obscured by
480
compensatory activation of NO-cGMP in experimental PH, but uncovered during treatment of
481
pulmonary arteries with blockers of NO-cGMP. This is supported by reports that the RhoA/Rho
482
kinase system plays a key role in PH (15), and that treatment with Rho-kinase inhibitors
483
reduces RVH and reverses pulmonary arterial remodeling in the hypoxic rat model of PH (79).
484
In concordance with previous reports (2, 50), Ach-induced relaxation was reduced in
485
pulmonary artery of hypoxic and MCT rats. Ach relaxation was improved during induction of
486
non-hypercapnic acidosis in rat models of PH. The enhanced Ach relaxation induced by NH4Cl
487
treatment is less likely due to changes in endothelial cholinergic receptors because the
488
relaxation to the exogenous NO donor SNP was also reduced in PH rats and improved during
489
induction of non-hypercapnic acidosis. Because Ach-induced relaxation was blocked by L-
490
NAME or ODQ, the enhanced Ach relaxation during NH4Cl treatment may be explained by
491
enhanced NO synthesis. This is unlikely the only mechanism, as blockers of NO-cGMP
492
enhanced Phe contraction in pulmonary arteries of hypoxic and MCT rats with or without
493
acidosis, suggesting possible compensatory activation of the NO-cGMP pathway in
494
experimental PH. The reduced Ach relaxation in the PH rats and its improvement with acidosis
19
Acidosis and Vascular Function in Pulmonary Hypertension
L-00293-2011-R2
495
is also unlikely due to decreased NO bioavailability due to increased oxidative stress in the
496
setting of hypoxia (16), because Ach-induced relaxation was also reduced in the MCT model,
497
and improved during induction of acidosis in MCT rats. A plausible explanation for the reduced
498
Ach relaxation in hypoxic and MCT-treated rats is possible structural changes in the pulmonary
499
vascular wall and decreased responsiveness of pulmonary VSMCs to vasodilators. This is
500
supported by the reduced pulmonary artery relaxation to the NO donor SNP in hypoxic and
501
MCT-treated rats, and consistent with the report that both endothelium-dependent and -
502
independent relaxation are reduced in rat model of MCT-induced progressive lung injury (25).
503
Consequently, the enhanced SNP-induced relaxation in pulmonary arteries of hypoxic and
504
MCT rats treated with NH4Cl can be explained by prevention or reversal of structural changes
505
in the pulmonary vasculature and improved responsiveness of pulmonary VSMCs to
506
vasodilators. However, other factors contributing to the vascular responsiveness to SNP may
507
include PDE-5 and protein kinase G activity and should be examined in future studies.
508
Increased thickness of pulmonary arterioles is a key structural feature of hypoxic PH, as
509
evidenced by remodeling of the small pulmonary arteries, vascular cell proliferation and
510
obliteration of the pulmonary microvasculature (9, 21, 53, 59). We and others have shown that
511
PH in hypoxic and MCT-treated rats is associated with reduced pulmonary responsiveness to
512
vasoconstrictors and endogenous and exogenous nitrovasodilators (2, 25, 27, 50, 67) and
513
extensive pulmonary arteriolar thickening and remodeling (9, 12, 44, 50, 53, 59). Multiple
514
mechanisms may contribute to pulmonary vascular remodeling in PH including resident medial
515
pulmonary VSMC hypertrophy and hyperplasia and a phenotypic switch from a contractile to a
516
synthetic phenotype, trans-differentiation of circulating and resident progenitor, adventitial or
517
endothelial cells to a VSMC-like phenotype, and intimal and adventitial changes.
518
Although evidence supports a role of pulmonary VSMC phenotypic switch in the
519
pathogenesis PH, an imbalance between pulmonary vasoconstrictors such as ET-1 and
520
vasodilators such as NO and PGI2 has also been implicated in PH (19, 26, 33, 34, 38, 64), and
521
vasodilators are a major component of the current therapy for PH (10, 47, 71, 80). However, a
20
Acidosis and Vascular Function in Pulmonary Hypertension
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522
large number of patients do not respond to vasodilators, possibly due to excessive pulmonary
523
vascular remodeling. Interventions to improve the responsiveness of the remodeled pulmonary
524
arteries to vasodilators could be a useful approach in PH. We found that acidosis prevented
525
and reversed established remodeling and improved pulmonary vascular responsiveness to
526
vasoactive mediators. Also, we previously reported that extracellular acidosis inhibited
527
proliferation and migration of cultured rat and mouse VSMCs (8, 11, 30). These observations
528
support that the reduced wall thickness in pulmonary arterioles and improved responsiveness
529
to vasoconstrictor and vasodilator stimuli in pulmonary arteries of PH rats treated with NH4Cl
530
may be related to restoration of VSMC phenotype from proliferative to contractile with
531
enhanced contraction mechanisms and increased plasticity and responsiveness to vasodilator
532
signaling.
533
A potential confounding factor is the body weight during hypoxia and NH4Cl treatment.
534
Hypoxic animals did not gain weight, and hypoxic animals treated with NH4Cl actually lost
535
weight. The protective effects of NH4Cl on hypoxia-induced PH are unlikely due to changes in
536
body weight because NH4Cl treatment was also protective in MCT-induced PH despite no
537
effect on animal weight. It is also unlikely that weight changes could have altered the
538
hemodynamic and structural indices of PH because the hemodynamic measurements did not
539
show a correlation with body weight, and the analysis of RVH took animal weight into
540
consideration. A potential limitation of the vascular function studies is that they were performed
541
on extra-lobar pulmonary arteries instead of intra-lobar resistance vessels, which are thought
542
to play a more important role in the regulation of pulmonary vascular resistance, and future
543
studies should compare the effects of non-hypercapnic acidosis in extra-lobar and intra-lobar
544
vessels. Another potential limitation is whether the present study directly addressed the
545
contribution of endothelial cells vs. VSMCs to the vascular responses to acidosis. Several pieces
546
of ex vivo and in vitro evidence support that acidosis elicits effects on VSMCs that may be, at least
547
in part, responsible for the protective pulmonary vascular effects observed. Although we did not
548
physically remove the endothelium, we tested the effects of chemical blockade of endothelium-
21
Acidosis and Vascular Function in Pulmonary Hypertension
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549
derived NO-cGMP and found that in pulmonary arteries treated with L-NAME or ODQ Phe
550
contraction was still reduced in hypoxia and MCT rat models of PH compared to normoxia rats, and
551
improved in hypoxia+acidosis and MCT+acidosis rats (Table 2). Also, the improved response to
552
SNP, an exogenous NO donor and endothelium-independent vasodilator, in pulmonary arteries
553
from hypoxia+acidosis and MCT+acidosis animals provides indirect evidence that NH4Cl treatment
554
improves VSMC sensitivity to exogenous NO independent of the endothelium. Additionally, the
555
histology data demonstrated thickening of the pulmonary arteriolar wall in hypoxia and MCT rats,
556
and amelioration of the vascular wall hypertrophy in hypoxia+acidosis and MCT+acidosis rats.
557
Furthermore, our previously published in vitro data demonstrated that acidosis decreased VSMCs
558
proliferation and migration and enhanced their susceptibility to apoptosis which may underlie the
559
ameliorating effects of acidosis on pulmonary vascular remodeling in experimental PH (8). While
560
these data point to an effect of acidosis on VSMCs, possible contribution of the endothelium to the
561
effects of acidosis on the hemodynamics and vascular responses cannot be ruled out and should
562
be examined in future studies. It is also important to define the effects of non-hypercapnic
563
acidosis on other tissues and organs such as the heart and kidney. In our models of PH, a 2-
564
week hypoxic exposure or 4-weeks after MCT treatment were not associated with significant
565
changes in LVSP or the responsiveness of mesenteric vessels to vasoconstrictor or
566
vasodilator stimuli, indicating specific changes in the pulmonary, but not the systemic
567
circulation in experimental PH. Similarly, NH4Cl treatment in the hypoxic model of PH was not
568
associated with significant changes in LVSP or the responsiveness of mesenteric vessels to
569
vasoconstrictor or vasodilator stimuli, supporting specific changes in the pulmonary but not
570
systemic vasculature. In MCT-induced PH, treatment with NH4Cl did not alter the
571
responsiveness of mesenteric vessels to vasoactive mediators, but caused a significant
572
reduction in LVSP, possibly due to direct cardiac effects of the combination of MCT (3) and
573
NH4Cl.
574
The rodent models of hypoxia- and MCT-induced PH share some hemodynamic and
575
histologic features with human PH, including increased pulmonary arterial pressure, RVH and
22
Acidosis and Vascular Function in Pulmonary Hypertension
L-00293-2011-R2
576
pulmonary vascular remodeling. Although these ‘classic’ animal models may not fully
577
recapitulate the pathologic changes in human PH (7, 73) and may lack some of the hallmarks
578
of the disease such as the plexogenic lesions, they have been useful especially in intervention
579
studies. All current therapies (including PGI2, ET-1 receptor antagonists and PDE-5 inhibitors)
580
for all categories of human PH have been tested and proven successful in these animal
581
models. Future work should test the effectiveness of the acidosis intervention in new models of
582
PH in which neointimal arteriopathy and plexogenic lesions more closely resemble the human
583
condition. These models include the S100A4/Mts1 protein-overexpressing mouse infected with
584
M1-γ-herpesvirus 68, left pneumonectomized MCT-injected rat, chronic hypoxic Sugen 5416
585
(VEGF receptor blocker)-injected rat or mouse, Sugen 5416-injected athymic nude rat, chronic
586
hypoxic athymic nude rat, MCT-injected endothelin-B receptor-deficient rat, and IL-6
587
overexpressing hypoxic mouse (1, 14, 29, 38-40, 57, 70, 72, 74, 75, 78).
588
589
Perspectives
590
Human PH is characterized by progressive fibro-proliferative obliteration of pulmonary
591
arterioles and various degrees of pulmonary vasoconstriction, inflammation and thrombosis,
592
leading to progressive increase in pulmonary vascular resistance and RVH and failure (9, 21,
593
44, 59). Current vasodilator therapies for PH include PDE-5 inhibitors, PGI2 analogs, ET-1 type
594
A receptor (ETAR) antagonists, and Ca2+ channel blockers (63, 68). To enhance their
595
effectiveness, these therapeutic approaches are often used in combination (32, 51). While
596
many vasodilators also have anti-proliferative effects on VSMCs, there is no definitive
597
evidence that pulmonary vascular remodeling in human PH is reversible. Also, current
598
vasodilator therapies are not universally successful in altering PH progression and increasing
599
survival. Therefore, novel approaches that directly target pulmonary vessel wall pathology are
600
needed in order to reverse the established pulmonary vascular pathology in PH patients.
601
Careful evaluation of the findings in animal models could spearhead studies in humans to
602
determine the effects of acidosis on the course of PH. Ample experimental evidence supports
23
Acidosis and Vascular Function in Pulmonary Hypertension
L-00293-2011-R2
603
that specific anti-proliferative, pro-apoptotic, immuno-modulatory and cell based therapies
604
could be effective in PH (5, 18, 36, 52, 54, 64, 69, 77); however, translation to clinical
605
application is lagging behind these new discoveries. Because vasodilators such as PGI2,
606
nitrates and PDE-5 inhibitors are commonly used as a first line of treatment in severe PH (10,
607
47, 71, 80), it is important to find interventions that could enhance the responsiveness of the
608
pulmonary circulation to these vasodilators. The present data suggest that induction of non-
609
hypercapnic acidosis could improve the pulmonary hemodynamics, pulmonary vascular
610
function, and reduce pulmonary artery remodeling, and therefore may provide a complimentary
611
approach to enhance the effectiveness of vasodilator therapy in PH. Further studies using
612
suitable experimental models of PH and additional methods of inducing acidosis in vivo are
613
needed to define the underlying mechanisms and translational potential of this approach.
614
615
ACKNOWLEDGEMENTS
616
This work was supported by grants from The National Heart, Lung, and Blood Institute
617
(HL-65998, HL-98724, HL-55454, HL-85446, and K08HL-77344) and The Eunice Kennedy
618
Shriver National Institute of Child Health and Human Development (HD-60702, and T32HD-
619
007466). Dr. Christou was supported by the Peabody Foundation.
620
621
24
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622
REFERENCES
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
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661
662
663
664
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666
1.
Abe K, Toba M, Alzoubi A, Ito M, Fagan KA, Cool CD, Voelkel NF, McMurtry IF,
and Oka M. Formation of plexiform lesions in experimental severe pulmonary arterial
hypertension. Circulation 121: 2747-2754, 2011.
2.
Adnot S, Raffestin B, Eddahibi S, Braquet P, and Chabrier PE. Loss of endotheliumdependent relaxant activity in the pulmonary circulation of rats exposed to chronic hypoxia. J Clin
Invest 87: 155-162, 1991.
3.
Akhavein F, St-Michel EJ, Seifert E, and Rohlicek CV. Decreased left ventricular
function, myocarditis, and coronary arteriolar medial thickening following monocrotaline
administration in adult rats. J Appl Physiol 103: 287-295, 2007.
4.
Aoshima D, Murata T, Hori M, and Ozaki H. Time-dependent phenotypic and contractile
changes of pulmonary artery in chronic hypoxia-induced pulmonary hypertension. J Pharmacol Sci
110: 182-190, 2009.
5.
Baber SR, Deng W, Master RG, Bunnell BA, Taylor BK, Murthy SN, Hyman AL, and
Kadowitz PJ. Intratracheal mesenchymal stem cell administration attenuates monocrotalineinduced pulmonary hypertension and endothelial dysfunction. Am J Physiol Heart Circ Physiol 292:
H1120-1128, 2007.
6.
Badesch DB, Raskob GE, Elliott CG, Krichman AM, Farber HW, Frost AE, Barst RJ,
Benza RL, Liou TG, Turner M, Giles S, Feldkircher K, Miller DP, and McGoon MD.
Pulmonary arterial hypertension: baseline characteristics from the REVEAL Registry. Chest 137:
376-387, 2010.
7.
Bauer NR, Moore TM, and McMurtry IF. Rodent models of PAH: are we there yet? Am J
Physiol Lung Cell Mol Physiol 293: L580-582, 2007.
8.
Brenninkmeijer L, Kuehl C, Miele Geldart A, Arons E, and Christou H. Heme
Oxygenase-1 does not mediate the effects of extracellular acidosis on vascular smooth muscle cell
proliferation, migration, and susceptibility to apoptosis. Journal of vascular research 48: 285-296,
2011.
9.
Budhiraja R, Tuder RM, and Hassoun PM. Endothelial dysfunction in pulmonary
hypertension. Circulation 109: 159-165, 2004.
10.
Burger CD. Pulmonary hypertension in COPD: a review and consideration of the role of
arterial vasodilators. COPD 6: 137-144, 2009.
11.
Christou H, Bailey N, Kluger MS, Mitsialis SA, and Kourembanas S. Extracellular
acidosis induces heme oxygenase-1 expression in vascular smooth muscle cells. Am J Physiol Heart
Circ Physiol 288: H2647-2652, 2005.
12.
Christou H, Morita T, Hsieh CM, Koike H, Arkonac B, Perrella MA, and
Kourembanas S. Prevention of hypoxia-induced pulmonary hypertension by enhancement of
endogenous heme oxygenase-1 in the rat. Circ Res 86: 1224-1229, 2000.
13.
Christou HA, and Khalil RA. Sex hormones and vascular protection in pulmonary arterial
hypertension. J Cardiovasc Pharmacol 56: 471-474, 2010.
14.
Ciuclan L, Bonneau O, Hussey M, Duggan N, Holmes AM, Good R, Stringer R, Jones
P, Morrell NW, Jarai G, Walker C, Westwick J, and Thomas M. A Novel Murine Model of
Severe Pulmonary Arterial Hypertension. Am J Respir Crit Care Med 2011.
15.
Connolly MJ, and Aaronson PI. Key role of the RhoA/Rho kinase system in pulmonary
hypertension. Pulm Pharmacol Ther 24: 1-14, 2011.
25
Acidosis and Vascular Function in Pulmonary Hypertension
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
L-00293-2011-R2
16.
Cowan DB, Jones M, Garcia LM, Noria S, del Nido PJ, and McGowan FX, Jr. Hypoxia
and stretch regulate intercellular communication in vascular smooth muscle cells through reactive
oxygen species formation. Arterioscler Thromb Vasc Biol 23: 1754-1760, 2003.
17.
Crossno JT, Jr., Garat CV, Reusch JE, Morris KG, Dempsey EC, McMurtry IF,
Stenmark KR, and Klemm DJ. Rosiglitazone attenuates hypoxia-induced pulmonary arterial
remodeling. Am J Physiol Lung Cell Mol Physiol 292: L885-897, 2007.
18.
Daley E, Emson C, Guignabert C, de Waal Malefyt R, Louten J, Kurup VP,
Hogaboam C, Taraseviciene-Stewart L, Voelkel NF, Rabinovitch M, Grunig E, and Grunig G.
Pulmonary arterial remodeling induced by a Th2 immune response. J Exp Med 205: 361-372, 2008.
19.
Fagan KA, Oka M, Bauer NR, Gebb SA, Ivy DD, Morris KG, and McMurtry IF.
Attenuation of acute hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension in
mice by inhibition of Rho-kinase. Am J Physiol Lung Cell Mol Physiol 287: L656-664, 2004.
20.
Farber HW. The status of pulmonary arterial hypertension in 2008. Circulation 117: 29662968, 2008.
21.
Farber HW, and Loscalzo J. Pulmonary arterial hypertension. N Engl J Med 351: 16551665, 2004.
22.
Fawcett J, Hsu FW, Tsao T, and Rabkin R. Effect of metabolic acidosis on the insulinlike growth factor-I system and cathepsins B and L gene expression in the kidney. J Lab Clin Med
136: 468-475, 2000.
23.
Fleming I, and Busse R. NO: the primary EDRF. J Mol Cell Cardiol 31: 5-14, 1999.
24.
Frid MG, Li M, Gnanasekharan M, Burke DL, Fragoso M, Strassheim D, Sylman JL,
and Stenmark KR. Sustained hypoxia leads to the emergence of cells with enhanced growth,
migratory, and promitogenic potentials within the distal pulmonary artery wall. Am J Physiol Lung
Cell Mol Physiol 297: L1059-1072, 2009.
25.
Fullerton DA, Hahn AR, and McIntyre RC, Jr. Mechanistic imbalance of pulmonary
vasomotor control in progressive lung injury. Surgery 119: 98-103, 1996.
26.
Geraci MW, Gao B, Shepherd DC, Moore MD, Westcott JY, Fagan KA, Alger LA,
Tuder RM, and Voelkel NF. Pulmonary prostacyclin synthase overexpression in transgenic mice
protects against development of hypoxic pulmonary hypertension. J Clin Invest 103: 1509-1515,
1999.
27.
Gillespie MN, Olson JW, Reinsel CN, O'Connor WN, and Altiere RJ. Vascular
hyperresponsiveness in perfused lungs from monocrotaline-treated rats. Am J Physiol 251: H109114, 1986.
28.
Green ML, Hatch M, and Freel RW. Ethylene glycol induces hyperoxaluria without
metabolic acidosis in rats. Am J Physiol Renal Physiol 289: F536-543, 2005.
29.
Greenway S, van Suylen RJ, Du Marchie Sarvaas G, Kwan E, Ambartsumian N,
Lukanidin E, and Rabinovitch M. S100A4/Mts1 produces murine pulmonary artery changes
resembling plexogenic arteriopathy and is increased in human plexogenic arteriopathy. The
American journal of pathology 164: 253-262, 2004.
30.
Guan J, Wu X, Arons E, and Christou H. The p38 mitogen-activated protein kinase
pathway is involved in the regulation of heme oxygenase-1 by acidic extracellular pH in aortic
smooth muscle cells. J Cell Biochem 105: 1298-1306, 2008.
31.
Guilluy C, Sauzeau V, Rolli-Derkinderen M, Guerin P, Sagan C, Pacaud P, and
Loirand G. Inhibition of RhoA/Rho kinase pathway is involved in the beneficial effect of sildenafil
on pulmonary hypertension. British journal of pharmacology 146: 1010-1018, 2005.
32.
Hoeper MM, Markevych I, Spiekerkoetter E, Welte T, and Niedermeyer J. Goaloriented treatment and combination therapy for pulmonary arterial hypertension. Eur Respir J 26:
858-863, 2005.
26
Acidosis and Vascular Function in Pulmonary Hypertension
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
L-00293-2011-R2
33.
Homma N, Nagaoka T, Morio Y, Ota H, Gebb SA, Karoor V, McMurtry IF, and Oka
M. Endothelin-1 and serotonin are involved in activation of RhoA/Rho kinase signaling in the
chronically hypoxic hypertensive rat pulmonary circulation. J Cardiovasc Pharmacol 50: 697-702,
2007.
34.
Hoshikawa Y, Voelkel NF, Gesell TL, Moore MD, Morris KG, Alger LA, Narumiya S,
and Geraci MW. Prostacyclin receptor-dependent modulation of pulmonary vascular remodeling.
Am J Respir Crit Care Med 164: 314-318, 2001.
35.
Ignarro LJ. Nitric oxide. A novel signal transduction mechanism for transcellular
communication. Hypertension 16: 477-483, 1990.
36.
Ito T, Okada T, Miyashita H, Nomoto T, Nonaka-Sarukawa M, Uchibori R, Maeda Y,
Urabe M, Mizukami H, Kume A, Takahashi M, Ikeda U, Shimada K, and Ozawa K.
Interleukin-10 expression mediated by an adeno-associated virus vector prevents monocrotalineinduced pulmonary arterial hypertension in rats. Circ Res 101: 734-741, 2007.
37.
Itoh H, Yokochi A, Yamauchi-Kohno R, and Maruyama K. Effects of the endothelin
ET(A) receptor antagonist, TA-0201, on pulmonary arteries isolated from hypoxic rats. Eur J
Pharmacol 376: 233-238, 1999.
38.
Ivy D, McMurtry IF, Yanagisawa M, Gariepy CE, Le Cras TD, Gebb SA, Morris KG,
Wiseman RC, and Abman SH. Endothelin B receptor deficiency potentiates ET-1 and hypoxic
pulmonary vasoconstriction. Am J Physiol Lung Cell Mol Physiol 280: L1040-1048, 2001.
39.
Ivy DD, McMurtry IF, Colvin K, Imamura M, Oka M, Lee DS, Gebb S, and Jones PL.
Development of occlusive neointimal lesions in distal pulmonary arteries of endothelin B receptordeficient rats: a new model of severe pulmonary arterial hypertension. Circulation 111: 2988-2996,
2005.
40.
Ivy DD, Yanagisawa M, Gariepy CE, Gebb SA, Colvin KL, and McMurtry IF.
Exaggerated hypoxic pulmonary hypertension in endothelin B receptor-deficient rats. Am J Physiol
Lung Cell Mol Physiol 282: L703-712, 2002.
41.
Jara A, Felsenfeld AJ, Bover J, and Kleeman CR. Chronic metabolic acidosis in azotemic
rats on a high-phosphate diet halts the progression of renal disease. Kidney Int 58: 1023-1032, 2000.
42.
Kantores C, McNamara PJ, Teixeira L, Engelberts D, Murthy P, Kavanagh BP, and
Jankov RP. Therapeutic hypercapnia prevents chronic hypoxia-induced pulmonary hypertension in
the newborn rat. Am J Physiol Lung Cell Mol Physiol 291: L912-922, 2006.
43.
Khalil RA, and van Breemen C. Sustained contraction of vascular smooth muscle: calcium
influx or C-kinase activation? J Pharmacol Exp Ther 244: 537-542, 1988.
44.
Klings ES, and Farber HW. Pulmonary hypertension as a risk factor for death in patients
with sickle cell disease. N Engl J Med 350: 2521-2522; author reply 2521-2522, 2004.
45.
Lagna G, Ku MM, Nguyen PH, Neuman NA, Davis BN, and Hata A. Control of
phenotypic plasticity of smooth muscle cells by bone morphogenetic protein signaling through the
myocardin-related transcription factors. J Biol Chem 282: 37244-37255, 2007.
46.
Laudi S, Trump S, Schmitz V, West J, McMurtry IF, Mutlak H, Christians U,
Weimann J, Kaisers U, and Steudel W. Serotonin transporter protein in pulmonary hypertensive
rats treated with atorvastatin. Am J Physiol Lung Cell Mol Physiol 293: L630-638, 2007.
47.
Lee JE, Hillier SC, and Knoderer CA. Use of sildenafil to facilitate weaning from inhaled
nitric oxide in children with pulmonary hypertension following surgery for congenital heart disease.
J Intensive Care Med 23: 329-334, 2008.
48.
Li X, Zhang X, Leathers R, Makino A, Huang C, Parsa P, Macias J, Yuan JX,
Jamieson SW, and Thistlethwaite PA. Notch3 signaling promotes the development of pulmonary
arterial hypertension. Nat Med 15: 1289-1297, 2009.
27
Acidosis and Vascular Function in Pulmonary Hypertension
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
L-00293-2011-R2
49.
Lucioni A, Womack C, Musch MW, Rocha FL, Bookstein C, and Chang EB. Metabolic
acidosis in rats increases intestinal NHE2 and NHE3 expression and function. Am J Physiol
Gastrointest Liver Physiol 283: G51-56, 2002.
50.
Mam V, Tanbe AF, Vitali SH, Arons E, Christou HA, and Khalil RA. Impaired
vasoconstriction and nitric oxide-mediated relaxation in pulmonary arteries of hypoxia- and
monocrotaline-induced pulmonary hypertensive rats. J Pharmacol Exp Ther 332: 455-462, 2010.
51.
McLaughlin VV, Benza RL, Rubin LJ, Channick RN, Voswinckel R, Tapson VF,
Robbins IM, Olschewski H, Rubenfire M, and Seeger W. Addition of inhaled treprostinil to oral
therapy for pulmonary arterial hypertension: a randomized controlled clinical trial. J Am Coll
Cardiol 55: 1915-1922, 2010.
52.
McMurtry MS, Archer SL, Altieri DC, Bonnet S, Haromy A, Harry G, Puttagunta L,
and Michelakis ED. Gene therapy targeting survivin selectively induces pulmonary vascular
apoptosis and reverses pulmonary arterial hypertension. J Clin Invest 115: 1479-1491, 2005.
53.
Mitani Y, Ueda M, Komatsu R, Maruyama K, Nagai R, Matsumura M, and Sakurai
M. Vascular smooth muscle cell phenotypes in primary pulmonary hypertension. Eur Respir J 17:
316-320, 2001.
54.
Nishimura T, Vaszar LT, Faul JL, Zhao G, Berry GJ, Shi L, Qiu D, Benson G, Pearl
RG, and Kao PN. Simvastatin rescues rats from fatal pulmonary hypertension by inducing
apoptosis of neointimal smooth muscle cells. Circulation 108: 1640-1645, 2003.
55.
Nowik M, Kampik NB, Mihailova M, Eladari D, and Wagner CA. Induction of
metabolic acidosis with ammonium chloride (NH4Cl) in mice and rats--species differences and
technical considerations. Cell Physiol Biochem 26: 1059-1072, 2010.
56.
Oka M, Homma N, Taraseviciene-Stewart L, Morris KG, Kraskauskas D, Burns N,
Voelkel NF, and McMurtry IF. Rho kinase-mediated vasoconstriction is important in severe
occlusive pulmonary arterial hypertension in rats. Circ Res 100: 923-929, 2007.
57.
Okada K, Tanaka Y, Bernstein M, Zhang W, Patterson GA, and Botney MD.
Pulmonary hemodynamics modify the rat pulmonary artery response to injury. A neointimal model
of pulmonary hypertension. The American journal of pathology 151: 1019-1025, 1997.
58.
Ooi H, Cadogan E, Sweeney M, Howell K, O'Regan RG, and McLoughlin P. Chronic
hypercapnia inhibits hypoxic pulmonary vascular remodeling. Am J Physiol Heart Circ Physiol
278: H331-338, 2000.
59.
Pietra GG, Edwards WD, Kay JM, Rich S, Kernis J, Schloo B, Ayres SM, Bergofsky
EH, Brundage BH, Detre KM, and et al. Histopathology of primary pulmonary hypertension. A
qualitative and quantitative study of pulmonary blood vessels from 58 patients in the National
Heart, Lung, and Blood Institute, Primary Pulmonary Hypertension Registry. Circulation 80: 11981206, 1989.
60.
Quinn DA, Dahlberg CG, Bonventre JP, Scheid CR, Honeyman T, Joseph PM,
Thompson BT, and Hales CA. The role of Na+/H+ exchange and growth factors in pulmonary
artery smooth muscle cell proliferation. Am J Respir Cell Mol Biol 14: 139-145, 1996.
61.
Quinn DA, Du HK, Thompson BT, and Hales CA. Amiloride analogs inhibit chronic
hypoxic pulmonary hypertension. Am J Respir Crit Care Med 157: 1263-1268, 1998.
62.
Quinn DA, Honeyman TW, Joseph PM, Thompson BT, Hales CA, and Scheid CR.
Contribution of Na+/H+ exchange to pH regulation in pulmonary artery smooth muscle cells. Am J
Respir Cell Mol Biol 5: 586-591, 1991.
63.
Rich S, Kaufmann E, and Levy PS. The effect of high doses of calcium-channel blockers
on survival in primary pulmonary hypertension. N Engl J Med 327: 76-81, 1992.
28
Acidosis and Vascular Function in Pulmonary Hypertension
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
L-00293-2011-R2
64.
Schermuly RT, Dony E, Ghofrani HA, Pullamsetti S, Savai R, Roth M, Sydykov A, Lai
YJ, Weissmann N, Seeger W, and Grimminger F. Reversal of experimental pulmonary
hypertension by PDGF inhibition. J Clin Invest 115: 2811-2821, 2005.
65.
Schermuly RT, Kreisselmeier KP, Ghofrani HA, Yilmaz H, Butrous G, Ermert L,
Ermert M, Weissmann N, Rose F, Guenther A, Walmrath D, Seeger W, and Grimminger F.
Chronic sildenafil treatment inhibits monocrotaline-induced pulmonary hypertension in rats.
American journal of respiratory and critical care medicine 169: 39-45, 2004.
66.
Shimoda LA, Sham JS, and Sylvester JT. Altered pulmonary vasoreactivity in the
chronically hypoxic lung. Physiol Res 49: 549-560, 2000.
67.
Silverman ES, Thompson BT, Quinn DA, Kinane TB, Bonventre JV, and Hales CA.
Na+/H+ exchange in pulmonary artery smooth muscle from spontaneously hypertensive and
Wistar-Kyoto rats. Am J Physiol 269: L673-680, 1995.
68.
Sitbon O, Humbert M, Jais X, Ioos V, Hamid AM, Provencher S, Garcia G, Parent F,
Herve P, and Simonneau G. Long-term response to calcium channel blockers in idiopathic
pulmonary arterial hypertension. Circulation 111: 3105-3111, 2005.
69.
Smith AM, Jones RD, and Channer KS. The influence of sex hormones on pulmonary
vascular reactivity: possible vasodilator therapies for the treatment of pulmonary hypertension. Curr
Vasc Pharmacol 4: 9-15, 2006.
70.
Spiekerkoetter E, Alvira CM, Kim YM, Bruneau A, Pricola KL, Wang L,
Ambartsumian N, and Rabinovitch M. Reactivation of gammaHV68 induces neointimal lesions
in pulmonary arteries of S100A4/Mts1-overexpressing mice in association with degradation of
elastin. Am J Physiol Lung Cell Mol Physiol 294: L276-289, 2008.
71.
Stehlik J, and Movsesian MA. Combined use of PDE5 inhibitors and nitrates in the
treatment of pulmonary arterial hypertension in patients with heart failure. J Card Fail 15: 31-34,
2009.
72.
Steiner MK, Syrkina OL, Kolliputi N, Mark EJ, Hales CA, and Waxman AB.
Interleukin-6 Overexpression Induces Pulmonary Hypertension. Circ Res 104: 236-244, 2009.
73.
Stenmark KR, Meyrick B, Galie N, Mooi WJ, and McMurtry IF. Animal models of
pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure. Am J
Physiol Lung Cell Mol Physiol 297: L1013-1032, 2009.
74.
Taraseviciene-Stewart L, Kasahara Y, Alger L, Hirth P, Mc Mahon G, Waltenberger
J, Voelkel NF, and Tuder RM. Inhibition of the VEGF receptor 2 combined with chronic hypoxia
causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary
hypertension. Faseb J 15: 427-438, 2001.
75.
Taraseviciene-Stewart L, Nicolls MR, Kraskauskas D, Scerbavicius R, Burns N, Cool
C, Wood K, Parr JE, Boackle SA, and Voelkel NF. Absence of T cells confers increased
pulmonary arterial hypertension and vascular remodeling. American journal of respiratory and
critical care medicine 175: 1280-1289, 2007.
76.
Vitali SH, Mitsialis SA, Liang OD, Liu X, Fernandez-Gonzalez A, Christou H, Wu X,
McGowan FX, and Kourembanas S. Divergent cardiopulmonary actions of heme oxygenase
enzymatic products in chronic hypoxia. PLoS One 4: e5978, 2009.
77.
Wang XX, Zhang FR, Shang YP, Zhu JH, Xie XD, Tao QM, and Chen JZ.
Transplantation of autologous endothelial progenitor cells may be beneficial in patients with
idiopathic pulmonary arterial hypertension: a pilot randomized controlled trial. J Am Coll Cardiol
49: 1566-1571, 2007.
78.
White RJ, Meoli DF, Swarthout RF, Kallop DY, Galaria, II, Harvey JL, Miller CM,
Blaxall BC, Hall CM, Pierce RA, Cool CD, and Taubman MB. Plexiform-like lesions and
29
Acidosis and Vascular Function in Pulmonary Hypertension
855
856
857
858
859
860
861
862
863
864
865
866
L-00293-2011-R2
increased tissue factor expression in a rat model of severe pulmonary arterial hypertension. Am J
Physiol Lung Cell Mol Physiol 293: L583-590, 2007.
79.
Xu EZ, Kantores C, Ivanovska J, Engelberts D, Kavanagh BP, McNamara PJ, and
Jankov RP. Rescue treatment with a Rho-kinase inhibitor normalizes right ventricular function and
reverses remodeling in juvenile rats with chronic pulmonary hypertension. Am J Physiol Heart Circ
Physiol 299: H1854-1864, 2010.
80.
Yin N, Kaestle S, Yin J, Hentschel T, Pries AR, Kuppe H, and Kuebler WM. Inhaled
nitric oxide versus aerosolized iloprost for the treatment of pulmonary hypertension with left heart
disease. Crit Care Med 37: 980-986, 2009.
30
Acidosis and Vascular Function in Pulmonary Hypertension
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867
868
Table 1. Body weight, arterial pH, blood gas, and hematocrit in control normoxic, hypoxic and
MCT-treated rats with or without treatment with NH4Cl (Acidosis).
Parameter
Normoxia
Normoxia
Hypoxia
Hypoxia
MCT
MCT
+Acidosis
+Acidosis
+Acidosis
Body weight (g)
373±12
363±5
293±12.5
291±16
289±10
243±11#
Arterial pH
pCO2 (mmHg)
-
7.37±0.01
7.32±0.03
7.29±0.01*
7.08±0.02#
7.36±0.01
7.26±0.04#
52±0.9
48±1.8
45±2
40±1.8
49.4±1.3
44.8±2.1
Plasma HCO3
28.8±0.6
24.6±1.9
21.2±0.6*
11.8±0.6
27.5±0.7
19.5±1.7#
Hematocrit (%)
38.5±1.2
36.5±0.8
63.3±2.3*
66.1±2.4*
42±1
43.2±1.3
869
870
871
872
873
874
875
876
#
Data represent means±SEM of cumulative data from 6 to 12 rats.
* Measurements in hypoxia or MCT-treated rats are significantly different (p<0.05) from
corresponding measurements in normoxic rats.
# Measurements in NH4Cl-treated (Acidosis) rats are significantly different (p<0.05) from
corresponding measurements in normoxic, hypoxia or MCT rats without NH4Cl treatment.
31
Acidosis and Vascular Function in Pulmonary Hypertension
L-00293-2011-R2
877
878
Table 2. Phe contraction, and Ach and SNP relaxation in pulmonary and mesenteric arteries
control normoxic, hypoxic and MCT-treated rats with or without treatment with NH4Cl (Acidosis).
Parameter
Normoxia
Normoxia
Hypoxia
Hypoxia
MCT
+Acidosis
+Acidosis
Pulmonary Artery
Phe Max (10-5 M)
Contraction (g/mg)
0.88±0.08
0.30±0.07*
0.89±0.12
0.24±0.03*
0.43±0.07*#
-4
+L-NAME (3x10 M)
0.31±0.05*
1.02±0.15
0.33±0.11*
0.88±0.17
0.37±0.09*
+ODQ (10-5 M)
0.35±0.06*
1.11±0.17
0.45±0.11*
1.33±0.30
0.34±0.06*
Phe pED50 (-log M)
+L-NAME
+ODQ
of
MCT
+Acidosis
0.63±0.16#
0.66±0.22#
0.73±0.23
7.71±0.08
7.56±0.13
7.81±0.10
7.68±0.09
7.82±0.13
7.66±0.11
7.67±0.09
7.47±0.17
7.67±0.14
7.46±0.09
7.32±0.14
7.59±0.15
7.58±0.16
7.20±0.18
7.76±0.10
7.53±0.08
7.42±0.11
7.61±0.15
94.97±2.23
106.17±4.67†
116.72±4.53†
96.46±4.09
113.93±11.72
114.25±7.18†
94.04±5.90
185.65±66.34
133.74±13.06†
93.64±5.10
113.51±11.15
135.85 ±15.50†
113.51±10.23
158.80±19.57*†
194.34±24.66*†
104.96±5.53
142.65±11.66†
155.33±10.28*†
Ach (10-5 M) %
Relaxation
48.56±2.88
45.33±4.81
14.35±5.14*
31.92±5.53*#
7.98±3.00*
18.54±2.42*#
SNP (10-5 M) %
Relaxation
91.86±2.78
95.30±1.56
48.18±10.72*
78.28±6.21#
58.45±6.07*
82.22±4.70#
1.26±0.14
6.28±0.12
0.93±0.07
6.57±0.15
0.97±0.12
6.15±0.12
1.06±0.16
6.26±0.09
1.08±0.16
6.63±0.25
1.00±0.14
6.55±0.16
93.26±3.19
77.43±12.21
94.18±4.10 14
88.68±7.35 12
87.45±6.68
95.53±2.74
96.20±2.02
98.85±1.15
92.86±7.14
100.0±0.00
90.48±7.14
97.74±1.45
Phe Contraction
% 96 mM KCl
+L-NAME
+ODQ
Mesenteric Artery
Phe Max (10-5 M)
Contraction (g)
pED50 (-log M)
Ach (10-5 M) %
Relaxation
SNP (10-5 M) %
Relaxation
879
880
881
882
883
884
885
886
Data represent means±SEM of cumulative data from 6 to 8 rats.
* Measurements in hypoxia or MCT-treated rats are significantly different (p<0.05) from
corresponding measurements in normoxic rats.
# Measurements in NH4Cl-treated (Acidosis) rats are significantly different (p<0.05) from
corresponding measurements in normoxic, hypoxia or MCT rats without NH4Cl treatment.
†
Measurements in L-NAME or ODQ-treated arteries are significantly different from corresponding
measurement in non-treated arteries.
887
888
32
Acidosis and Vascular Function in Pulmonary Hypertension
L-00293-2011-R2
889
FIGURE LEGENDS
890
Fig. 1. Effect of treatment with NH4Cl on right ventricular systolic pressure (RVSP) and left
891
ventricular systolic pressure (LVSP) in hypoxic and MCT-treated rat models of PH. A.
892
Representative RVSP tracings from individual rats. B. RVSP cumulative data. C. LVSP
893
cumulative data. Data represent means±SEM from 6 to 12 rats per experimental group.
894
* Measurements in hypoxic or MCT-treated rats are significantly different (p<0.05) from the
895
corresponding measurements in control normoxic or vehicle-treated rats.
896
# Measurements in NH4Cl-treated hypoxic or MCT rats are significantly different (p<0.05) from
897
the corresponding measurements in hypoxic or MCT rats without NH4Cl treatment.
898
899
Fig. 2. Effect of treatment with NH4Cl on right ventricular hypertrophy (RVH) in hypoxic and
900
MCT-treated rat models of PH. RVH was assessed by Fulton’s index (ratio of right ventricular
901
weight to left ventricular+septal weight) (A) and as the ratio of right ventricular weight to total
902
body weight (B). Data represent means±SEM from 6 to 12 rats per experimental group.
903
* Measurements in hypoxic or MCT-treated rats are significantly different (p<0.05) from the
904
corresponding measurements in control normoxic or vehicle-treated rats. # Measurements in
905
NH4Cl-treated hypoxic or MCT-treated rats are significantly different (p<0.05) from the
906
corresponding measurements in hypoxic or MCT rats without NH4Cl treatment. C. Linear
907
regression and 95% confidence intervals of the relationship between arterial pH and Fulton’s
908
index among rats exposed to hypoxia for two weeks with or without treatment with NH4Cl
909
(Acidosis). Each individual rat is represented by an individual data point.
910
911
Fig. 3. Phe-induced contraction in pulmonary artery (A and C) and mesenteric artery (B and
912
D) of normoxic, hypoxic and MCT-treated rats with or without treatment with NH4Cl (Acidosis).
913
Pulmonary artery and mesenteric artery rings were stimulated with increasing concentrations
914
of Phe. The contractile response was measured and presented in g/mg tissue weight (A) or in
915
g (B) or as % of maximum Phe contraction (C and D). Data represent means±SEM (n=6 to 8)
33
Acidosis and Vascular Function in Pulmonary Hypertension
L-00293-2011-R2
916
* Measurements in hypoxia or MCT-treated rats are significantly different (p<0.05) from
917
corresponding measurements in control normoxic rats.
918
#
919
corresponding measurements in hypoxia or MCT-treated rats without NH4Cl treatment.
Measurements in NH4Cl-treated (Acidosis) rats are significantly different (p<0.05) from
920
921
Fig. 4. KCl-induced contraction in pulmonary artery (A) and mesenteric artery (B) of normoxic,
922
hypoxic and MCT-treated rats with or without treatment with NH4Cl (Acidosis). Pulmonary and
923
mesenteric artery rings were stimulated with 96 mM KCl and the contractile response was
924
measured and presented in g/mg tissue (A) or in g (B). Data represent means±SEM (n=6 to
925
8).
926
* Measurements in hypoxia or MCT-treated rats are significantly different (p<0.05) from
927
corresponding measurements in control normoxic rats.
928
#
929
corresponding measurements in hypoxia or MCT-treated rats without NH4Cl treatment.
930
† Measurements in hypoxia+acidosis rats are significantly different (p<0.05) from
931
corresponding measurements in normoxia+acidosis rats.
Measurements in NH4Cl-treated (Acidosis) rats are significantly different (p<0.05) from
932
933
Fig. 5. Effect of blockade of the NO-cGMP pathway on Phe-induced contraction in pulmonary
934
artery of normoxic, hypoxic and MCT-treated rats with or without treatment with NH4Cl
935
(Acidosis). Pulmonary artery rings of control normoxic (A), normoxic+acidosis (B), hypoxic
936
(C), hypoxic+acidosis (D), MCT-treated (E) and MCT+acidosis rats (F) were either nontreated
937
(open circle) or pretreated with the NOS inhibitor L-NAME (3x10-4 M) (closed circles) or the
938
guanylate cyclase inhibitor ODQ 10-5 M (open triangles) for 10 min. The tissues were
939
stimulated with increasing concentrations of Phe, and the contractile response was measured
940
and presented as g/mg tissue weight. Data represent means±SEM (n=6 to 8).
941
* Measurements in L-NAME-treated pulmonary artery segments are significantly different
942
(p<0.05) from corresponding measurements in non-treated segments.
34
Acidosis and Vascular Function in Pulmonary Hypertension
L-00293-2011-R2
943
# Measurements in ODQ-treated pulmonary artery segments are significantly different
944
(p<0.05) from corresponding measurements in non-treated segments.
945
946
Fig. 6. Sensitivity to Phe-induced contraction during blockade of the NO-cGMP pathway in
947
pulmonary artery of normoxic, hypoxic and MCT-treated rats with or without treatment with
948
NH4Cl (Acidosis).
949
hypoxic (C), hypoxic+acidosis (D), MCT-treated (E) and MCT+acidosis rats (F) were either
950
nontreated (open circle) or pretreated with the NOS inhibitor L-NAME (3x10-4 M) (closed
951
circles) or the guanylate cyclase inhibitor ODQ 10-5 M (open triangles) for 10 min. The tissues
952
were stimulated with increasing concentrations of Phe, and the contractile response was
953
measured and presented as % of maximum Phe contraction. Data represent means±SEM
954
(n=6 to 8).
955
* Measurements in L-NAME-treated pulmonary artery segments are significantly different
956
(p<0.05) from corresponding measurements in non-treated segments.
957
# Measurements in ODQ-treated pulmonary artery segments are significantly different
958
(p<0.05) from corresponding measurements in non-treated segments.
Pulmonary artery rings of control normoxic (A), normoxic+acidosis (B),
959
960
Fig. 7. Phe-induced contraction as % of Ca2+-dependent 96 mM KCl-induced contraction
961
during blockade of the NO-cGMP pathway in pulmonary artery of normoxic, hypoxic and MCT-
962
treated rats with or without treatment with NH4Cl (Acidosis). Pulmonary artery segments of
963
control normoxic (A), normoxic+acidosis (B), hypoxic (C), hypoxic+acidosis (D), MCT-treated
964
(E) and MCT+acidosis rats (F) were either nontreated (open circle) or pretreated with the NOS
965
inhibitor L-NAME (3x10-4 M) (closed circles) or the guanylate cyclase inhibitor ODQ 10-5 M
966
(open triangles) for 10 min. The tissues were stimulated with increasing concentrations of Phe,
967
and the contractile response was measured and presented as % of the control Ca2+-dependent
968
96 mM KCl-induced contraction. Data represent means±SEM (n=6 to 8).
35
Acidosis and Vascular Function in Pulmonary Hypertension
L-00293-2011-R2
969
* Measurements in L-NAME-treated pulmonary artery segments are significantly different
970
(p<0.05) from corresponding measurements in non-treated segments.
971
# Measurements in ODQ-treated pulmonary artery segments are significantly different
972
(p<0.05) from corresponding measurements in non-treated segments.
973
974
Fig. 8. Ach-induced relaxation in pulmonary and mesenteric artery rings of control normoxic,
975
hypoxic and MCT-treated rats with or without treatment with NH4Cl (Acidosis). Pulmonary
976
artery (A) and mesenteric artery segments (B) were precontracted with Phe (10-5 M),
977
increasing concentrations of Ach were added and the % relaxation of Phe contraction was
978
measured. Data represent means±SEM (n=6 to 8).
979
* Measurements in hypoxic and MCT-treated rats are significantly different (p<0.05) from
980
corresponding measurements in control normoxic rats.
981
# Measurements in NH4Cl-treated (Acidosis) rats are significantly different (p<0.05) from
982
corresponding measurements in hypoxia or MCT-treated rats without NH4Cl treatment.
983
984
Fig. 9. Effect of blockade of the NO-cGMP pathway on Ach-induced relaxation in pulmonary
985
artery of normoxic, hypoxic and MCT-treated rats with or without treatment with NH4Cl
986
(Acidosis).
987
hypoxic (C), hypoxic+acidosis (D), MCT-treated (E) and MCT+acidosis rats (F) were either
988
nontreated (open circle) or pretreated with the NOS inhibitor L-NAME (3x10-4 M) (closed
989
circles), or the guanylate cyclase inhibitor ODQ (10-5 M) (open triangles) for 10 min. The
990
tissues were precontracted with Phe (10-5 M), increasing concentrations of Ach were added
991
and the % relaxation of Phe contraction was measured. Data represent means±SEM (n=6 to
992
8). * Measurements in L-NAME-treated pulmonary artery segments are significantly different
993
(p<0.05) from corresponding measurements in non-treated segments.
994
# Measurements in ODQ-treated pulmonary artery segments are significantly different
995
(p<0.05) from corresponding measurements in non-treated segments.
Pulmonary artery segments of control normoxic (A), normoxic+acidosis (B),
36
Acidosis and Vascular Function in Pulmonary Hypertension
L-00293-2011-R2
996
997
Fig. 10. SNP-induced relaxation in pulmonary and mesenteric artery of normoxic, hypoxic and
998
MCT-treated rats with or without treatment with NH4Cl (Acidosis). Pulmonary artery (A) and
999
mesenteric artery segments (B) were precontracted with Phe (10-5 M), increasing
1000
concentrations of SNP were added and the % relaxation of Phe contraction was measured.
1001
Data represent means±SEM (n=6 to 8).
1002
* Measurements in hypoxic and MCT-treated rats are significantly different (p<0.05) from
1003
corresponding measurements in normoxic rats.
1004
# Measurements in NH4Cl-treated (Acidosis) rats are significantly different (p<0.05) from
1005
1006
corresponding measurements in hypoxia or MCT-treated rats without NH4Cl treatment.
1007
Fig. 11. Effect of treatment with NH4Cl on pulmonary arteriolar remodeling in hypoxic and MCT
1008
models of PH. A. Representative Hematoxylin & Eosin-stained lung sections from control
1009
normoxic, hypoxic (2 weeks) and MCT-treated rats with or without treatment with NH4Cl
1010
(Acidosis). Pulmonary arterioles 50-100 μM diameter are indicated with arrows. Total
1011
magnification X20. Scale bar = 50 μm. B. Quantitative morphometric analysis of % wall
1012
thickness of pulmonary arterioles defined as the area occupied by the vessel wall divided by
1013
the total cross sectional area of the arteriole. Fifteen pulmonary arterioles (50-100 µM
1014
diameter) from 5 rats per experimental group were analyzed by two independent investigators.
1015
Percent wall thickness was measured and presented as means±SEM. * Measurements in
1016
hypoxic or MCT-treated rats are significantly different (p<0.05) from corresponding
1017
measurements in control normoxic or vehicle-treated rats. # Measurements in NH4Cl-treated
1018
hypoxic or MCT-treated rats are significantly different (p<0.05) from corresponding
1019
measurements in hypoxic or MCT-treated rats without NH4Cl treatment.
1020
1021
37
100
RVSP (mmH
Hg)
RVSP (mmH
Hg)
100
50
0
100
RVSP (mmH
Hg)
A
50
0
0
1 sec
Hypoxia
+ NH4Cl (Acidosis)
(A id i )
*
*#
*#
40
20
LVSP (mmHg)
*
w/o NH4Cl
150
80
RVSP (mmH
Hg)
Hypoxia+Acidosis
C
w/o NH4Cl
60
1 sec
1 sec
Normoxia
B
Hypoxia
Vehicle
MCT
+ NH4Cl (Acidosis)
(A id i )
100
#
50
0
0
Normoxia
50
Normoxia Hypoxia
Vehicle
MCT
A
B
w/o NH4Cl
w/o NH4Cl
+ NH4Cl (Acidosis)
(A id i )
+ NH4Cl (Acidosis)
1.5
*
*#
0.4
*#
0.2
0.0
Normoxia
Hypoxia
Vehicle
RV W
Weight / Body Weight
*
*
*
*#
1.0
0.0
MCT
Normoxia
Hypoxia
Vehicle
95% Confidence Interval
0.8
r = 0.5
P = 0.009
0.6
0.4
Hypoxia
0.2
0.0
Hypoxia+Acidosis
7.0
7.1
7.2
Arterial pH
*#
0.5
Linear Regression
C
Fulton's Inde
ex
Fulton's ind
dex
0.6
7.3
7.4
MCT
Pulmonary
B
1.2
0.8
#
#
0.4
*
*
Conttraction (g)
Contractio
on (g/mg Tissu
ue)
A
0.0
Mesenteric
1.6
1.2
0.8
0.4
0.0
-9
-8
-7
-6
-9
-5
-8
log [Phe] (M)
D
100
80
Contraction (% Max)
Contraction (% Max)
C
60
40
20
0
-7
-6
log [Phe] (M)
-5
100
80
60
Normoxia
Normoxia+Acidosis
Hypoxia
40
Hypoxia+Acidosis
MCT
20
MCT+Acidosis
0
-9
-8
-7
log [Phe] (M)
-6
-5
-9
-8
-7
log [Phe] (M)
-6
-5
Pulmonary
A
KCl Contracttion (g/mg Tissue)
1.2
0.8
#
# †
0.4
*
*
0
Normoxia
Hypoxia
MCT
w/o NH4Cl
+ NH4Cl (Acidosis)
Mesenteric
B
KCl Contraction (g)
1.2
0.8
0.4
0
Normoxia
Hypoxia
MCT
1.2
0.8
*
*
Control
+L-NAME
+ODQ
-8
Contractio
on (g/mg Tissue)
C
16
1.6
0.8
#
#
# #
*
-8
-7
-6
log [Phe] (M)
0.8
##
#
#
0.4
#
-8
-7
-6
log [Phe] (M)
-5
Hypoxia+Acidosis
16
1.6
1.2
0.8
0.4
-9
F
1.2
04
0.4
0.8
0.0
-5
MCT
1.6
1.2
D
1.2
0.4
Acidosis
1.6
0.0
-9
-5
Hypoxia
0.0
-9
Conttraction (g/mg Tiissue)
-7
-6
log [Phe] (M)
Contractio
on (g/mg Tissue)
0.4
Contraction (g/m
mg Tissue)
1.6
0.0
-9
E
B
Normoxia
Conttraction (g/mg Tiissue)
Contraction (g/m
mg Tissue)
A
-8
-7
-6
log [Phe] (M)
-5
MCT+Acidosis
1.6
1.2
0.8
04 # #
0.4
0.0
0.0
-9
-8
-7
-6
log [Phe] (M)
-5
-9
-8
-7
-6
log [Phe] (M)
-5
B
Normoxia
100
80
60
#
40
20
#
*
*
Control
+L-NAME
+ODQ
Contraction (% Max)
Contraction (% Max)
A
0
-9
-8
-7
-6
80
60
40
20
0
-5
Acidosis
100
-9
-8
100
Contra
action (% Max)
Contra
action (% Max)
D
Hypoxia
80
#
60
#
#
40
20
0
Hypoxia+Acidosis
100
80
#
60
40
#
#
#
-8
-7
-6
#
*
20
-5
-9
-8
F
MCT
100
80
#
60
#
#
#
#
-7
-6
-5
log [Phe] (M)
#
Co
ontraction (% Ma
ax)
Co
ontraction (% Ma
ax)
#
# #
*
log [Phe] (M)
40
-5
0
-9
E
-6
log [Phe] (M)
log [Phe] (M)
C
-7
#
20
MCT+Acidosis
100
80
#
60
40 #
20
#
#
#
#
#
*
0
0
-9
-8
-7
log [Phe] (M)
-6
-5
-9
-8
-7
log [Phe] (M)
-6
-5
Contraction (%
% 96 KCl)
B
Normoxia
200
150
#
# # #
##
100
50
#
*
#
#
# #*
#
Control
+L-NAME
+ODQ
*
0
-9
**
-8
-7
-6
Contraction (%
% 96 KCl)
A
150
50
-9
-8
#
100
*
*
Contractiion (% 96 KCl)
Contractiion (% 96 KCl)
D
150
50
0
-6
-5
Hypoxia+Acidosis
200
150
#
100
50
*
*
*
*
0
-9
-8
-7
-6
-5
-9
9
-8
8
log [Phe] (M)
F
MCT
200
*
* **
150
100
50
0
-9
-8
-7
log [Phe] (M)
-7
-6
6
-5
5
log [Phe] (M)
-6
-5
#
Con
ntraction (% 96 K
KCl)
E
Con
ntraction (% 96 K
KCl)
-7
log [Phe] (M)
Hypoxia
200
##
*
log [Phe] (M)
C
##
#
100
0
-5
Acidosis
200
MCT+Acidosis
200
#
150
*
100
50
0
-9
-8
-7
log [Phe] (M)
-6
-5
Pulmonary
A
0
0
*
20
20
#
#
#
Normoxia
Normoxia+Acidosis
Hypoxia
yp
Hypoxia+Acidosis
MCT
MCT+Acidosis
40
% Relaxatiion
*
% Relaxation
Mesenteric
B
40
60
80
100
60
-10
10
-9
9
-8
8
-7
7
log [ACh] (M)
-6
6
-5
5
10
-10
-9
9
-8
8
-7
7
log [ACh] (M)
-6
6
-5
5
Normoxia
A
*
20
40
60
-10
Control
+L-NAME
+ODQ
-9
-8
-7
log [ACh] (M)
-6
#
20
40
60
-10
-9
-8
-7
log [ACh] (M)
-6
-6
#
20
40
-9
-8
-7
log [ACh] (M)
-6
MCT+Acidosis
0
20
40
-9
-8
-7
log [ACh] (M)
-6
-5
-5
*
F
% Relaxation
% Relaxation
-8
-7
log [ACh] (M)
Hypoxia+Acidosis
60
-10
0
60
-10
-9
0
-5
MCT
E
40
D
%R
Relaxation
%R
Relaxation
0
#
20
60
-10
-5
Hypoxia
C
*
0
#
% Relaxa
ation
% Relaxa
ation
0
Acidosis
B
-5
*
#
20
40
60
-10
-9
-8
-7
log [ACh] (M)
-6
-5
Pulmonary
A
0
0
20
Normoxia
Normoxia+Acidosis
Hypoxia
Hypoxia+Acidosis
yp
MCT
MCT+Acidosis
20
40
*
60
*
80
#
#
100
% Relaxation
n
% Relaxation
n
Mesenteric
B
40
60
80
100
-10
-9
-8
-7
log [SNP] (M)
-6
-5
-10
-9
-8
-7
log [SNP] (M)
-6
-5
Normoxia
Normoxia+Acidosis
Hypoxia
MCT
Hypoxia+Acidosis
MCT+Acidosis
w/o NH4Cl
B
%W
Wall Thickness
A
60
40
+ NH4Cl ((Acidosis))
*
*#
*
#
20
0
Normoxia Hypoxia
Vehicle
MCT