Cysteinyl Leukotriene 1 Receptors as Novel Mechanosensors
Mediating Myogenic Tone Together With Angiotensin II
Type 1 Receptors—Brief Report
Ursula Storch,* Stephanie Blodow,* Thomas Gudermann, Michael Mederos y Schnitzler
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Objective—Myogenic vasoconstriction is mediated by vascular smooth muscle cells of resistance arteries sensing
mechanical stretch. Angiotensin II AT1 receptors and in particular AT1BRs in murine vascular smooth muscle cells have
been characterized as mechanosensors that cannot fully account for myogenic vasoconstriction observed. Therefore, we
aimed at uncovering novel vascular mechanosensors by expression profiling and functional characterization of candidate
proteins.
Approach and Results—Analyzing myogenic tone of isolated murine mesenteric arteries of AT1A and AT1B receptor double
gene-deficient (AT1A/1B−/−) mice ex vivo, we observed a decreased myogenic tone at high intraluminal pressures and an
unexpected hyper-reactivity at low intraluminal pressures because of upregulation of cysteinyl leukotriene 1 receptors
(CysLT1Rs). Pharmacological blockade of CysLT1Rs with pranlukast significantly reduced myogenic tone not only in
AT1A/1B−/− but also in wild-type arteries. In wild-type arteries, additional blockade of angiotensin II AT1 receptors with
candesartan resulted in an additive reduction of myogenic tone. Furthermore, calcium imaging experiments were performed
with fura-2–loaded human embryonic kidney 293 cells overexpressing CysLT1Rs and with isolated mesenteric vascular
smooth muscle cells. Hypo-osmotically induced membrane stretch provoked calcium transients that were significantly
reduced by pranlukast. Incubations of isolated mesenteric vascular smooth muscle cells with the 5-lipoxygenase inhibitor
zileuton had no effect. Furthermore, the Gq/11-protein inhibitor YM 254890 profoundly reduced myogenic tone to the
same extent as induced by the application of pranlukast plus candesartan.
Conclusions—Here, we identify a novel, hitherto unappreciated role of CysLT1Rs in vascular regulation. We identified
CysLT1Rs as novel mechanosensors in the vasculature involved in myogenic vasoconstriction. Moreover, our findings
suggest that myogenic tone is determined by AT1 and CysLT1 receptors acting together as mechanosensors via
Gq/11-protein activation. (Arterioscler Thromb Vasc Biol. 2015;35:121-126. DOI: 10.1161/ATVBAHA.114.304844.)
Key Word: G-protein–coupled receptors
C
other G-protein–coupled receptors, such as the bradykinin
B2, the endothelin ETA, the parathyroid hormone type 1, the
histamine H1, the muscarinic M5, and the formyl peptide 1,
are discussed as mechanosensors.10 For some of these receptors, physiological roles emerged, for example, as sensors of
fluid shear stress in endothelial cells, for retraction of pseudopods in neutrophils or for bone growth. Altogether, there
is a growing evidence supporting inherent mechanosensitivity
of G-protein–coupled receptors. CysLT1Rs are well known as
constrictor receptors in bronchial smooth muscle. Any contribution of these receptors to myogenic vasoconstriction, however, is not known to date.
In contrast to humans, rodents express 2 isoforms of the
AT1 receptor: AT1A and AT1B. Investigating both murine AT1R
ysteinyl leukotriene 1 receptors (CysLT1Rs) are known
to mediate the contraction of bronchial smooth muscle.
However, a potential role of these receptors in the cardiovascular system has still remained elusive. Myogenic vasoconstriction or Bayliss effect1,2 is essential for the maintenance
of vascular tone, peripheral vascular resistance, and organ
perfusion.3 Vascular smooth muscle cells (VSMCs) but not
endothelial cells4 are considered sensors for mechanical stress
inducing myogenic vasoconstriction. Several proteins, such
as integrins, ion channels, or membrane-associated enzymes,
are discussed as mechanosensors. Recently, angiotensin II AT1
receptors (AT1Rs) emerged as mechanosensors in VSMCs
mediating myogenic tone and in cardiac myocytes inducing
cardiac hypertrophy independent of agonist.5–9 Numerous
Received on: April 30, 2014; final version accepted on: November 4, 2014.
From the Walther Straub Institute of Pharmacology and Toxicology (U.S., S.B., T.G., M.MyS.), Ludwig Maximilians University of Munich, Munich,
Germany; and DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance (T.G., M.MyS.), Munich, Germany.
Current address for S.B.: Walter Brendel Centre of Experimental Medicine, Ludwig Maximilians University of Munich, 81377 Munich, Germany.
*These authors contributed equally to this article.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.304844/-/DC1.
Correspondence to Michael Mederos y Schnitzler, PhD, or Thomas Gudermann, MD, Walther Straub Institute for Pharmacology and Toxicology,
Ludwig Maximilians University of Munich, Goethestr 33, 80336 Munich, Germany. E-mail [email protected] or thomas.gudermann@lrz.
uni-muenchen.de
© 2014 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
121
DOI: 10.1161/ATVBAHA.114.304844
122 Arterioscler Thromb Vasc Biol January 2015
Nonstandard Abbreviations and Acronyms
AT1R
CysLT1R
IMMAs
VSMCs
WT
angiotensin II AT1 receptor
cysteinyl leukotriene 1 receptor
isolated murine mesenteric arteries
vascular smooth muscle cells
wild-type
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isoforms, we recently found that mechanosensitive AT1B but
not AT1A receptors are key elements for mechanosensation in
VSMC mediating myogenic tone.5 However, myogenic tone
was not completely lost in AT1B gene-deficient arteries or after
blockade of AT1Rs with candesartan or losartan, suggesting
that other signaling pathways might participate in myogenic
tone. The aim of this study was to analyze the total effect of
AT1A and AT1B receptors on myogenic tone by investigating
AT1A/1B double gene-deficient (AT1A/1B−/−) mice and to discover
novel candidate mechanosensors in the vasculature.
Materials and Methods
Materials and Methods are available in the online-only Data
Supplement.
Results
CysLT1Rs Together With AT1Rs Mediate
Myogenic Vasoconstriction via Gq/11-Proteins
To analyze myogenic tone of AT1A/1B−/− mice ex vivo, we performed pressure myography of isolated murine mesenteric
arteries (IMMAs) from AT1A/1B−/− and wild-type (WT) mice.
Unexpectedly, AT1A/1B−/− arteries were hyper-reactive with
significantly increased myogenic vasoconstriction at a premyogenic pressure range from 20 to 70 mm Hg (Figure 1A).
Only at higher pressures >100 mm Hg, myogenic tone was
significantly reduced as expected. Figure 1B shows exemplary vessel diameter time courses at incremental pressure
levels. Because vascular hyper-reactivity might be caused by
compensatory upregulation of other proteins, gene expression
profiling was performed showing that among all G-protein–
coupled receptors, only CysLT1Rs were significantly enriched
in AT1A/1B−/− IMMAs as confirmed by quantitative reverse-transcriptase-polymerase chain reaction (Figure 1C). Therefore,
we pursued a pharmacological approach and applied pranlukast, a selective CysLT1R antagonist, at maximally inhibitory concentrations. Administration of 50 nmol/L pranlukast
significantly reduced myogenic tone in the pressure range
≤90 mm Hg. At higher pressure levels, myogenic tone was
reduced similar to untreated AT1A/1B−/− IMMAs (Figure 1A and
1D), suggesting that the hyper-reactivity at low pressure levels
is mediated by CysLT1Rs, whereas the decrease of myogenic
responsiveness at higher pressures reflects the loss of AT1Rs.
Figure 1D shows the summary of changes of myogenic tone
at different pressure ranges. Of note, candesartan did not
reduce myogenic tone of AT1A/1B−/− IMMAs (data not shown),
ruling out side effects of candesartan on vascular tone. Next,
we applied pranlukast to WT arteries to determine the physiological effect of CysLT1Rs on myogenic vasoconstriction.
Interestingly, pranlukast also significantly reduced myogenic
tone of WT IMMAs over a wide pressure range (Figure 1E and
1F), suggesting that CysLT1Rs are generally involved in myogenic vasoconstriction. Moreover, application of montelukast
caused a significant reduction of myogenic tone, which was
concentration dependent (Figure 1G and 1H). Interestingly,
although 50 nmol/L montelukast was ineffective, 100 nmol/L
montelukast caused the strongest reduction of myogenic
tone in the middle pressure range of 70 to 90 mm Hg in contrast to 200 nmol/L montelukast additionally suppressing
myogenic tone at high intraluminal pressures >100 mm Hg.
These findings suggest that mechanically activated CysLT1Rs
are particularly sensitive to selective antagonists at moderate intraluminal pressures. Furthermore, pranlukast-induced
reductions of myogenic tone in WT arteries were similar to
candesartan-induced reductions (Figure 1I and 1J). Moreover,
simultaneous blockade of CysLT1Rs and AT1Rs resulted in an
even more pronounced reduction of myogenic tone than that
induced by either drug alone (Figure 1I and 1J). The additive
effect of the blockers suggests an important role of both receptors for myogenic vasoconstriction and supports the notion
that both receptors act together as mechanosensors.
To analyze whether mechanical receptor activation during
myogenic vasoconstriction was subject to the same signaling pathway as initiated by agonist stimulation, we applied
YM 254890, a potent and selective Gq/11-protein inhibitor.11
Interestingly, YM 254890 suppressed myogenic tone to a similar extent as observed after simultaneous blockade of AT1Rs
and CysLT1Rs (Figure 1K and 1L) amounting to a relative
reduction of myogenic tone of >63%. These findings indicate
that myogenic vasoconstriction is mainly mediated by AT1Rs
and CysLT1Rs signaling through the Gq/11-protein pathway. To
analyze whether mechanically induced CysLT1Rs activation
during myogenic vasoconstriction is agonist independent, the
5-lipoxygenase inhibitor zileuton was incubated for ≥30 minutes to prevent leukotriene production. Interestingly, zileuton
slightly but significantly reduced myogenic tone only at high
pressures (Figure 1M and 1N), suggesting an involvement
of endogenously produced leukotrienes in myogenic vasoconstriction. Because endothelial cells can produce leukotrienes,12,13 we next analyzed whether the endothelium might
be responsible for zileuton-induced vasorelaxation. For this,
arteries were de-endothelialized by intraluminal administration of an air bubble. Interestingly, de-endothelialized, zileuton-treated vessels showed normal myogenic vasoconstriction
(Figure 1M and 1N), indicating that leukotriene release from
the endothelium might fine-tune myogenic tone. Because
5-lipoxygenase inhibition does not prevent release of eventually preformed leukotrienes, we next applied a neutralizing
monoclonal antibody against leukotriene C4 and D4,14 which
are the most potent CysLT1Rs agonists with EC50 values of
11 and 1 nmol/L, respectively. The administration of neutralizing monoclonal antibody had no effect on myogenic tone.
However, leukotriene E4, which is only a partial and weak
agonist with markedly higher EC50 value of 56 nmol/L, might
still be effective. Therefore, total leukotriene C4, D4, and E4
concentration in the bath solution superfusing the vessels
during myogenic vasoconstriction was determined. Because
leukotriene concentrations were below the detection limit of
Storch et al AT1 and CysLT1 Receptors Mediate Myogenic Tone 123
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Figure 1. Cysteinyl leukotriene 1 (CysLT1) together with angiotensin II type 1 (AT1) receptors mediate myogenic vasoconstriction via
Gq/11-proteins. A, Myogenic tone of isolated murine mesenteric arteries from wild-type (control) and AT1A/1B−/− mice normalized to the
maximal passive vasodilatation by Ca2+-free solution at increasing intraluminal pressures from 5 to 120 mm Hg in the presence (blue) or
absence (black and gray line) of pranlukast. Numbers in parentheses indicate the number of measurements and the number of mice.
Significance is tested in comparison with control (gray asterisks) and to AT1A/1B−/− arteries (blue asterisks). B, Exemplary time courses of
vessel diameter measurements during the second and third pressure ladder with indicated pressure steps (bottom, red) of AT1A/1B−/−
(top, gray) and wild-type (middle, black) arteries. Incubations with calcium-free solution (gray bar) are indicated. C, Summary of relative
mRNA expression levels determined by quantitative reverse-transcriptase-polymerase chain reaction. Numbers over bars indicate the
number of independent experiments. D, Summary of the absolute reduction and increase of myogenic tone at indicated pressure ranges
of AT1A/1B−/− arteries in the presence (blue) or absence (gray) of pranlukast. Numbers in parentheses indicate the number of measurements
and the number of mice. Significance is tested in comparison with control (black asterisks) and to AT1A/1B−/− arteries (gray asterisks).
E, Myogenic tone of control arteries in the presence (blue) or absence (black) of pranlukast. Significance tested compared with control
arteries. F, Summary of absolute reductions of myogenic tone by pranlukast at indicated pressure ranges. Significance tested compared
with control. G, Myogenic tone of control arteries in the presence (green and blue) or absence (black) of montelukast. Significance tested
compared with control arteries (green and blue) and to 100 nmol/L montelukast (black). H, Summary of absolute reductions of myogenic
tone by 100 and 200 nmol/L montelukast. Significance tested to control (black asterisks) and to 100 nmol/L montelukast (green asterisks).
I, Myogenic tone of control arteries in the absence (black) or presence of candesartan (green), pranlukast (blue), and candesartan plus
pranlukast (dark green). Significance tested compared with control arteries. J, Summary of absolute reductions of myogenic tone by indicated receptor blockers. Significance tested to control (black) and to pranlukast (blue) or candesartan (green). K, Myogenic tone of control arteries in the absence (black) or presence of YM 254890 (light blue) or candesartan plus pranlukast (dark green). Significance tested
compared with control. L, Summary of absolute reduction of myogenic tone. Significance tested compared with control. M, Myogenic
tone of control arteries in the absence (black) or presence of zileuton (orange), neutralizing LT antibody (red) or de-endothelialized arteries
in the presence of zileuton (dark yellow). Significance tested compared with control (orange asterisks). N, Summary of absolute reductions of myogenic tone. Significance tested compared with control (black asterisks). neAB indicates neutralizing monoclonal antibody.
124 Arterioscler Thromb Vasc Biol January 2015
≈100 pmol/L, these findings indicate that endogenously produced leukotrienes are not essential for myogenic vasoconstriction. In summary, our findings suggest a newly discovered
role of CysLT1Rs as mechanosensors in the vasculature, where
AT1 and CysLT1 receptors cooperate as mechanosensors mediating myogenic vasoconstriction via Gq/11-protein activation.
CysLT1Rs Are Mechanosensitive
Independent of Endogenous Agonist
To confirm the ex vivo results on a cellular level, fluorimetric
calcium measurements with fura-2–loaded mesenteric VSMCs
of WT and AT1A/1B−/− mice were performed. Interestingly, basal
intracellular calcium concentrations [Ca2+]i were significantly
increased in AT1A/1B−/− VSMCs (Figure 2A–2C) most likely
reflecting the hyper-reactivity of AT1A/1B−/− arteries at low pressure levels, which was verified measuring basal [Ca2+]i after
incubation of 50 nmol/L pranlukast for 10 minutes. Indeed,
basal [Ca2+]i of AT1A/1B−/− VSMCs were significantly reduced
by pranlukast (Figure 2C). Hypo-osmotically induced [Ca2+]i
increases were similar in both cell types, and 50 nmol/L pranlukast significantly suppressed membrane stretch-induced
[Ca2+]i transients to a similar extent (Figure 2A–2C). The
combined application of pranlukast and candesartan suppressed [Ca2+]i increases in WT VSMCs (Figure 2A and 2C)
even more prominently.
To analyze whether mechanical CysLT1R activation is
independent of endogenously produced leukotrienes, we
incubated cells with the 5-lipoxygenase inhibitor zileuton for
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Figure 2. Cysteinyl leukotriene 1 receptors (CysLT1Rs) are mechanosensitive independent from the endogenous agonist. A–E, Fluorimetric calcium measurements with fura-2–loaded freshly isolated mesenteric vascular smooth muscle cells (VSMCs) from wild-type (WT) and
AT1A/1B−/− mice. A, B, and D, Exemplary time courses of the intracellular calcium concentration [Ca2+]i. Application of hypo-osmotic solution
(150 mOsm/kg) hypo, of pranlukast, of pranlukast plus candesartan, of 100 µmol/L zileuton, and of 100 µmol/L zileuton in the presence of
100 nmol/L neutralizing leukotriene antibody (neAB) is indicated. C, Summary of the basal [Ca2+]i after incubation with 50 nmol/L pranlukast for 10 minutes (blue and black striated bars) and of [Ca2+]i before basal (black bars) and during application of hypo-osmotic solution
in the absence (red bars) and the presence of 50 nmol/L pranlukast (blue bars), of 50 nmol/L pranlukast and 1 µmol/L candesartan (violet
bar; C). E, Summary of the [Ca2+]i before (black bars), and during application of hypo-osmotic solution in the absence (red bars) and presence of 100 µmol/L zileuton (orange bar) and of 100 µmol/L zileuton plus neAB (gray and orange striated bar). Numbers over bars indicate
the number of measured cells from 3 to 5 independent preparations. F and G, Fluorimetric calcium measurements with fura-2–loaded
untransfected human embryonic kidney 293 (HEK293) cells and with HEK293 cells overexpressing human CysLT1 receptor. F, Exemplary
time courses of the [Ca2+]i. Application of hypo-osmotic solution (150 mOsm/kg) hypo and of pranlukast is indicated. G, Summary of the
[Ca2+]i before basal (black bars), and during application of hypo-osmotic solution (red bars) and application of 50 nmol/L pranlukast (blue
bars). Numbers over bars indicate the number of measured cells from 3 to 6 independent experiments. HEK293 indicates human embryonic kidney 293.
Storch et al AT1 and CysLT1 Receptors Mediate Myogenic Tone 125
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≥30 minutes to prevent leukotriene production. In the presence of 100 µmol/L zileuton, VSMCs still responded to hypoosmotic membrane stretch with [Ca2+]i increases that were not
different from untreated cells (Figure 2D and 2E). To exclude
an involvement of preformed leukotrienes in mechanically
induced [Ca2+]i increases, we incubated VSMCs with zileuton
together with the neutralizing monoclonal antibody (Figure 2D
and 2E) resulting in unaltered mechanically induced [Ca2+]i
increases, suggesting that endogenously produced leukotrienes are not involved in mechanical VSMC activation.
Moreover, calcium imaging experiments were performed
with human embryonic kidney 293 cells transiently overexpressing human CysLT1Rs. Untransfected human embryonic
kidney 293 cells were unresponsive to hypo-osmotic membrane stretch and to pranlukast applications (Figure 2F and
2G). Hypo-osmotic cell swelling caused calcium transients
in CysLT1Rs overexpressing cells that were significantly suppressed by pranlukast (Figure 2F and 2G). Because human
embryonic kidney 293 cells do not produce leukotrienes, these
findings suggest that stretch-induced CysLT1R activation was
agonist independent, arguing in favor of intrinsic mechanosensitivity. Altogether, our findings provide evidence for a
hitherto unknown function of CysLT1Rs as potential mechanosensors in resistance arteries involved in the regulation of
vascular tone.
Discussion
AT1A/1B−/− arteries exhibited an increased myogenic tone at low
and a reduced myogenic tone at high intraluminal pressures.
This is an uncovering novel vascular phenotype of AT1A/1B−/−
mice because until now, AT1A/1B−/− mice have been known
only for reduced blood pressure15,16 and reduced angiotensin II response15 similar to AT1A−/− mice. Moreover, growth,
body weight, and survival rate were significantly reduced, and
an abnormal kidney morphology was observed in AT1A/1B−/−
mice,15 suggesting an essential role of AT1Rs in viability and
health. Because AT1A/1B−/− vessels did not exhibit a complete
suppression of myogenic responsiveness, myogenic vasoconstriction seems to be of such pivotal importance for the
regulation of blood flow that several distinct safety strategies
may have evolved for compensation purposes. In the case of
AT1A/1B−/− mice, Gq/11-protein–coupled CysLT1Rs were upregulated as an essential backup strategy. However, CysLT1Rs
could not completely compensate the loss of AT1Rs. Probably,
they possess mechanosensory properties distinct from AT1Rs
with a higher sensitivity for mechanical stimuli at low pressure ranges resulting in hyper-reactivity of AT1A/1B−/− arteries.
However, at high intraluminal pressure levels, myogenic tone
was reduced reflecting the lack of AT1Rs.
The discovery of the novel role of CysLT1Rs in a genedeficient mouse model gave rise to the even more important
and novel finding that CysLT1Rs possess an important biological function as mechanosensors mediating myogenic
vasoconstriction even of WT arteries. Most strikingly, pranlukast and montelukast also reduced myogenic responsiveness of WT vessels revealing an uncovering novel function of
CysLT1Rs as possible mechanosensors involved in myogenic
vasoconstriction.
CysLT1Rs are known as constrictor receptors in smooth
muscle. In particular, they play a dominant role for bronchoconstriction in the lung in asthma.17,18 However, an
involvement in myogenic vasoconstriction and in particular a mechanosensitive function of CysLT1Rs has not been
described before. Our findings with heterologously expressed
CysLT1Rs clearly demonstrate that these receptors are mechanosensitive per se without the involvement of endogenous
agonists. In the recombinant system, 50 nmol/L pranlukast
significantly suppressed cell swelling–induced calcium transients. The mild effect of pranlukast on stretch-induced receptor activation might at least partially be caused by the lack
of negative intrinsic activity of this compound. Moreover, the
5-lipoxygenase inhibitor zileuton administered to VSMCs
to prevent endogenous leukotriene production and the additional application of a neutralizing leukotriene antibody had
no effect, suggesting that leukotrienes are not involved in
mechanical VSMC activation. However, in the isolated artery
segment, zileuton slightly suppressed myogenic tone at high
intraluminal pressures. This effect was endothelium mediated
because de-endothelialized arteries showed normal myogenic
tone indicating that endothelium-derived leukotrienes only
fine-tune myogenic responsiveness. However, a neutralizing
leukotriene antibody did not significantly reduce myogenic
vasoconstriction, demonstrating that leukotrienes are not
essentially involved in mechanical CysLT1Rs activation during myogenic vasoconstriction. Moreover, the lack of detectable leukotrienes in the bath solution superfusing pressurized
arteries during myogenic vasoconstriction strongly argues for
a leukotriene-independent CysLT1Rs activation.
Interestingly, simultaneous blockade of AT1 and CysLT1
receptors caused a similar reduction of myogenic tone as specific inhibition of the Gq/11-protein pathway, indicating that
G-protein–coupled receptors determine ≈60% of myogenic
vasoconstriction via Gq/11-proteins implying that 40% of myogenic tone is mediated by other mechanosensory elements.
Altogether our findings suggest an uncovering novel role of
CysLT1Rs cooperating with AT1Rs as mechanosensors initiating myogenic vasoconstriction via the Gq/11-protein signaling
pathway. Because myogenic vasoconstriction is of such vital
interest and is found to be impaired in various pathological
states, such as systemic hypertension, diabetes mellitus and
stroke, our findings may help identify new and verify known
pharmacological targets.
Acknowledgments
We thank Laura Danner and Joanna Zaisserer for excellent technical assistance. We are grateful to Thomas Coffman for providing
AT1A/1B−/− mice, and to Natsuko Kayakiri (Taiho Pharmaceutical) to
provide YM 254890 and to Yoshitaka Takahashi for the neutralizing
monoclonal leukotriene antibody.
Sources of Funding
This work was supported by grants from the Deutsche
Forschungsgemeinschaft.
Disclosures
None.
126 Arterioscler Thromb Vasc Biol January 2015
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Significance
Myogenic vasoconstriction or Bayliss effect is essential for the maintenance of vascular tone, peripheral resistance, and organ perfusion.
Therefore, a better understanding of mechanosensors mediating myogenic vasoconstriction may help identify new and evaluate known
targets for the treatment of common diseases, such as stroke, hypertension, and diabetes mellitus, characterized by impaired myogenic
vasoconstriction. In this study, we identified cysteinyl leukotriene 1 receptors as novel mechanosensors in the vasculature cooperating with
angiotensin II type 1 receptors as mechanosensitive elements thereby determining >60% of myogenic tone via the Gq/11-protein signaling
pathway.
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Cysteinyl Leukotriene 1 Receptors as Novel Mechanosensors Mediating Myogenic Tone
Together With Angiotensin II Type 1 Receptors−−Brief Report
Ursula Storch, Stephanie Blodow, Thomas Gudermann and Michael Mederos y Schnitzler
Arterioscler Thromb Vasc Biol. 2015;35:121-126; originally published online November 13,
2014;
doi: 10.1161/ATVBAHA.114.304844
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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Supplemental Material and Methods
Ethical approval:
All experiments and procedures were approved by the governmental oversight authority,
District Government of Upper Bavaria (Germany).
Analysis of myogenic tone: After decapitation of anesthetized male mice, mesentery was
removed and transferred to cold (4°C), oxygenated (carbogen: 95% O2 and 5% CO2),
physiological salt solution (PSS) with the following composition (in mM): 119 NaCl, 25
NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.6 CaCl2 and 11.1 glucose (pH 7.6 with NaOH,
and pH 7.4 after purging by carbogen). Mesenteric arteries devoid of surrounding fat and third
and fourth order branches were cannulated on extended borosilicate glass pipettes (GB120T8P, Science Products, Hofheim, Germany) filled with PSS allowing application of intravascular
pressure. Cannulated vessels were mounted onto a pressure myograph (DMT 111P, DMT,
Aarhus, Denmark) and outer vessel diameters were determined performing video microscopy
using MyoView software (DMT, Aarhus). Measurements were performed with continuous
superfusion at 37°C. Viability of the vessels was tested by applying 60 mM potassium chloride
solution with the following composition (in mM): 63.7 NaCl, 25 NaHCO3, 60 KCl, 1.2 KH2PO4,
1.2 MgSO4, 1.6 CaCl2 and 11.1 glucose (pH 7.6 with NaOH). Only arteries showing a
constriction of at least 30% were further investigated. To analyze the vascular response, the
outer vessel diameter was monitored in response to step-wise increases in intravascular
pressure from 5 to 80 mmHg in 2-min steps and from 90 to 120 mmHg in 5-min steps. The first
step-wise pressure increase resulted in vessel sensitization. The second incremental pressure
elevation was used to determine myogenic tone and the third which was performed in calcium
free PSS solution with 3 mM EDTA, served as a reference to determine maximal passive
dilatative diameters which were used for normalization. In the presence or absence of all used
blockers maximal high potassium-induced vasoconstrictions revealed no differences between
the analyzed arteries and before and after application of blockers ruling out toxic side effects
on the contractile apparatus. For some experiments vessels were deendothelialized by
passing an air bubble through the lumen to destroy the endothelium. Absence of functional
endothelium was verified by the lack of acetylcholine (3 µM) induced vasodilation in arteries
preconstricted with 10 µM phenylephrine. As a control for vessel viability 60 mM potassium
chloride solution was applied and only deendothelialized vessels showing a constriction of at
least 30% were further analyzed. The summary of the absolute reduction of myogenic tone
was calculated as total difference of mean values to control arteries at indicated intraluminal
pressures.
Mice used in this study:
The following mice were analyzed in this study: AT1A and AT1B receptor double gene-deficient
(AT1A/1B-/-) mice 1 in C57BL6/J genetic background (from Thomas Coffman, Division of
Nephrology, Department of Medicine, Duke University, and Durham VA Medical Centers,
Durham, North Carolina, USA). C57BL6/J were used as wild-type control mice. Only male mice
older than 80 days were used.
Preparation of vascular smooth muscle cells:
Single mesenteric smooth muscle cells were enzymatically isolated from mesenteric arteries
of mice as described in detail elsewhere 2.
Determination of intracellular calcium concentrations:
Intracellular free calcium was determined with fura-2 acetoxymethyl ester (5 μM; Molecular
Probes Inc., Eugene, OR) loaded mesenteric VSMCs as described in detail elsewhere 3 and
with HEK293 cells over-expressing the human CysLT1 receptor (AY242130). Cells were
continuously superfused with an isotonic bath solution containing (in mM): 55 NaCl, 5 KCl, 2
CaCl2, 10 HEPES, 10 glucose, 1 MgCl2 (pH 7.4 with NaOH) which was supplemented with
1 mannitol to 300 mOsm kg-1. The hypoosmotic solution had the same salt concentration without
added mannitol resulting in an osmolality of 149 to 152 mOsm kg-1.
Quantitative real-time polymerase chain reaction (qPCR) analysis: Total RNA from
indicated vessel segments was isolated using QIAzol lysis reagent (Qiagen, Hilden, Germany).
First strand synthesis was carried out with random hexamers as primers, using REVERTAID
reverse transcriptase (Fermentas, Sankt Leon-Roth, Germany). The following primer pairs
were used for the amplification of specific fragments from the first strand synthesis: CysLT1for
(5’-aaggtgctgaggtaccagatagag-3’), CysLT1rev (5’-aatcacagcccttgagaagc-3’) and three
references hypoxanthin phosphoribosyltransferase 1, Hprt1for (5’-tcctcctcagaccgctttt-3’),
Hprt1rev
(5’-cctggttcatcatcgctaatc-3’),
tyrosine
3-monooxygenase/tryptophan
5monooxygenase activation protein, zeta polypeptide, Ywhazfor (5’-taaaaggtctaaggccgcttc-3’),
Ywhazrev (5’-caccacacgcacgatgac-3’) and succinate dehydrogenase complex, subunit A,
Sdhafor (5’-ccctgagcattgcagaatc-3’), Sdharev (5’-tcttctccagcatttgcctta-3’) giving predicted
product sizes of 95 bp for CysLT1, 90 bp for Hprt1, 60 bp for Ywhaz and 70 bp for Sdha. Realtime polymerase chain reaction (RT-PCR) was performed using the master mix from the
Absolute QPCR SYBR Green Mix kit (Abgene, Epsom, UK). Ten picomole of each primer pair
and 0.2 μl of the first strand synthesis was added to the reaction mixture, and PCR was carried
out in a light-cycler apparatus (Light-Cycler 480, Roche, Mannheim, Germany) using the
following conditions: 15 min initial activation and 45 cycles of 12 s at 94°C, 30 s at 50°C, 30 s
at 72°C, and 10 s at 80°C each. Fluorescence intensities were recorded after the extension
step at 80°C after each cycle to exclude fluorescence of primer dimers melting lower than
80°C. All primers were tested by using diluted complementary DNA (cDNA) from the first strand
synthesis (10–1000×) to confirm linearity of the reaction and to determine particular
efficiencies. Data were calculated as percentage of the geometric mean expression of the three
references which showed the highest tissue-independent transcription stability. Samples
containing primer dimers were excluded by melting curve analysis and identification of the
products by agarose gel electrophoresis. Crossing points were determined by the software
program. All experiments were performed in triplets and experiments were repeated at least
three times. Wilcoxon rank-sum test was used for analysis of mRNA expression levels.
Determination of leukotriene concentration: To determine total cysteinyl leukotriene
concentration in samples from bath solution superfusing the pressurized vessel during
myogenic tone measurements, a commercially available enzyme immunoassay with a
monoclonal leukotriene C4, D4 and E4 antibody and a detection limit of approximately 100 pM
was performed following the manufacturer’s instructions (Cysteinyl leukotriene EIA Kit,
Cayman chemical, USA).
Statistical analysis: Data are presented as means±standard error of the mean (s.e.m.). The
myogenic tone measurements were analyzed using the one‐way analysis of variance
(ANOVA) with Bonferroni post hoc means comparison. Unless stated otherwise, all other data
were compared by a paired or unpaired Student's t-test, if a Gaussian distribution was
confirmed by applying a Shapiro-Wilk (normality) test. Significance was accepted at P<0.05.
*P<0.05, **P<0.01, ***P<0.001, n.s. P>0.05. Myogenic tone was fitted using the Hill equation.
Supplemental References
1. 2. Oliverio MI, Kim HS, Ito M, Le T, Audoly L, Best CF, Hiller S, Kluckman K, Maeda N, Smithies O, Coffman TM. Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor‐mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II. Proc Natl Acad Sci U S A. 1998;95:15496‐15501. Dietrich A, Mederos y Schnitzler M, Gollasch M, Gross V, Storch U, Dubrovska G, Obst M, Yildirim E, Salanova B, Kalwa H, Essin K, Pinkenburg O, Luft FC, Gudermann T, Birnbaumer L. Increased vascular smooth muscle contractility in TRPC6‐/‐ mice. Mol Cell Biol. 2005;25:6980‐
6989. 2 3. Storch U, Forst AL, Philipp M, Gudermann T, Mederos y Schnitzler M. Transient receptor potential channel 1 (TRPC1) reduces calcium permeability in heteromeric channel complexes. J Biol Chem. 2012;287:3530‐3540. 3