Articles in PresS. Am J Physiol Regul Integr Comp Physiol (November 24, 2010). doi:10.1152/ajpregu.00469.2010
Dietary sodium modulates the interaction between efferent and afferent renal nerve activity by
altering activation of α2-adrenoceptors on renal sensory nerves.
Ulla C. Kopp1, Michael Z. Cicha1, Lori A. Smith1, Saku Ruohonen2,
Mika Scheinin2, Nicolas Fritz3and Tomas Hökfelt3
1
Departments of Internal Medicine and Pharmacology, Department of Veterans Affairs Medical
Center and University of Iowa Carver College of Medicine, Iowa City, IA, 2Department of
Pharmacology, Drug Development and Therapeutics, University of Turku and University
Hospital, Turku, Finland, and 3Departments of Medical Biochemistry and Biophysics and,
Neuroscience, Karolinska Institutet, Stockholm, Sweden
Running Head: Norepinephrine mediated activation of renal α2-adrenoceptors.
Address of Correspondence:
Ulla C. Kopp, Ph.D.
Department of Internal Medicine ,
VA Medical Center, Bldg. 41, Highway 6W
Iowa City, IA 52246
phone: (319) 338-0581, ext. 7608, FAX 319 339-7162
email: [email protected]
1
Copyright © 2010 by the American Physiological Society.
ABSTRACT
Activation of efferent renal sympathetic nerve activity (ERSNA) increases afferent renal nerve
activity (ARNA) which then reflexively decreases ERSNA via activation of the renorenal
reflexes to maintain low ERSNA. The ERSNA-ARNA interaction is mediated by norepinephrine
(NE) that increases and decreases ARNA by activation of renal α1-and α2-adrenoceptors (AR),
respectively. The ERSNA-induced increases in ARNA are suppressed during low sodium, 2470±
770%⋅sec, and enhanced during high sodium diet, 5670±1260%⋅sec. We examined the role of α2AR in modulating the responsiveness of renal sensory nerves during low and high sodium diets.
Immunohistochemical analysis suggested the presence of α2A-AR and α2C-AR subtypes on renal
sensory nerves. During low sodium diet, renal pelvic administration of the α2-AR antagonist
rauwolscine or the AT1 receptor antagonist losartan alone failed to alter the ARNA responses to
reflex increases in ERSNA. Likewise, renal pelvic release of substance P produced by 250 pM
NE, from 8.0±1.3 to 8.5±1.6 pg/min, was not affected by rauwolscine or losartan alone.
However, rauwolscine+losartan enhanced the ARNA responses to reflex increases in ERSNA,
4680±1240 %⋅sec, and renal pelvic release of substance P by 250 pM NE, from 8.3±0.6 to
14.2±0.8 pg/min. During a high sodium diet, rauwolscine had no effect on the ARNA response
to reflex increases in ERSNA or renal pelvic release of substance P produced by NE. Losartan
was not examined because of low endogenous ANG II levels in renal pelvic tissue during high
sodium diet. Conclusion: Increased activation of α2-AR contributes to the reduced interaction
between ERSNA and ARNA during low sodium intake whereas no/minimal activation of α2-AR
contributes to the enhanced ERSNA-ARNA interaction in conditions of high sodium intake.
Key words: kidney, sensory nerves, sympathetic nerves, substance P, PGE2, angiotensin
2
INTRODUCTION
The kidney has a rich supply of sympathetic nerves which innervate all parts of the
kidney (5). The kidney also has abundant afferent innervation - the fibers proceed from the
kidney to the neuraxis and contain substance P and calcitonin gene-related peptide (CGRP) as
primary sensory neurotransmitters. The sensory nerve endings are located primarily in the renal
pelvic wall (15,21,22,24,29). Sympathetic efferent nerve fibers and afferent sensory nerve fibers
often run separately but intertwined in the same nerve bundles in the renal pelvic smooth muscle
layer (22), providing anatomical support for a possible functional interaction between efferent
renal sympathetic nerve activity (ERSNA) and afferent renal nerve activity (ARNA). Indeed,
increasing ARNA by increasing renal pelvic pressure leads to a reflex decrease in ERSNA and
increased natriuresis, i.e., a renorenal reflex response (26). The responsiveness of the renal
sensory nerves is also modulated by ERSNA. Reflex increases and decreases in ERSNA increase
and decrease ARNA, respectively (22,24,27). Taken together, there is strong evidence for a
negative feedback system, where increases in ERSNA increase ARNA which in turn would
decrease ERSNA via activation of the renorenal reflex.
Changes in ERSNA modulate ARNA by the release of the neurotransmitter
norepinephrine (NE) which activates α1-adrenoceptors (AR) and α2-AR on renal sensory nerves
leading to increases and decreases in ARNA, respectively (22). The physiological importance of
the ERSNA-induced increases in ARNA is underlined by the interaction being modulated by
dietary sodium. High sodium diet enhances and low sodium diet reduces the ERSNA-induced
increases in ARNA (24). In high sodium dietary condition, enhancement of the ERSNA-induced
increases in ARNA would increase the inhibitory renorenal reflex control of ERSNA, resulting
3
in reduction of ERSNA to prevent/limit sodium retention. Conversely, in low sodium dietary
conditions, suppression of the ERSNA-induced increases in ARNA would result in increased
ERSNA by reducing the renorenal reflex inhibition of ERSNA eventually leading to sodium
retention. The importance of the renorenal reflex-induced inhibition of ERSNA in the control of
body fluid and sodium homeostasis was demonstrated in our previous studies showing that rats
lacking intact afferent renal innervation are characterized by increased ERSNA, increased
responsiveness of ERSNA to various sympathetic stimuli and increased arterial pressure when
fed a high sodium diet (17,25).
The current studies were performed to examine the mechanisms involved in the altered
responsiveness of the renal sensory nerves to increases in ERSNA produced by varying sodium
dietary intake. The renin-angiotensin (ANG) system is one of the possible mechanisms involved
in the diet-dependent modulation of the interaction between ERSNA and ARNA. Our previous
studies examining mechanisms involved in the activation of the renal mechanosensory nerves
showed that stretching the renal pelvic wall leads to induction of COX-2 and increased renal
pelvic synthesis of PGE2 (19,20,23). PGE2 increases the release of substance P which increases
ARNA via activation of the cAMP - protein kinase A (PKA) signal transduction pathway. In low
sodium diet, the reduced responsiveness of the renal mechanosensory nerves to increased renal
pelvic pressure involves increased activation of ANG II type 1 (AT1) receptors. Activation of
AT1 receptors inhibits the PGE2-mediated activation of adenylyl cyclase by a pertussis toxin
(PTX)-sensitive mechanism (16). Because the ERSNA-induced increases in ARNA are
dependent on intact PG synthesis (22), we reasoned that a similar mechanism(s), i.e., increased
activation of AT1-receptors, would be involved in the reduced ARNA responses to increases in
4
ERSNA in low sodium diet rats. However in our initial studies, the AT1 receptor antagonist
losartan failed to enhance the responsiveness of the renal sensory nerves to increases in ERSNA.
In view of the inhibitory effects of NE-mediated activation of α2-AR on ARNA in normal
sodium diet rats (22), we then hypothesized that the reduced responsiveness of the renal sensory
nerves to increases in ERSNA involves increased NE-mediated activation of renal pelvic α2-AR
in rats fed low sodium diet. If so, we reasoned that the enhanced responsiveness of the renal
sensory nerves to increases in ERSNA in high sodium diet would involve decreased NEmediated activation of renal α2-AR. We studied this hypothesis by examining the effects of renal
pelvic administration of the α2-AR antagonist rauwolscine (2) on the increases in ARNA
produced by reflex increases in ERSNA by thermal cutaneous stimulation. Because the reflexmediated increases in ERSNA were associated with increases in arterial pressure (AP) and heart
rate (HR), these studies were complemented with studies in which we examined the ARNA
responses to renal pelvic administration of NE at a concentration that did not alter systemic
hemodynamics. To examine whether the effects produced by reflex increases in ARNA and NE
on ARNA involved pre- or postsynaptic effects of NE, additional experiments were performed in
an isolated renal pelvic wall preparation from high and low sodium diet rats.
We also carried out immunohistochemical studies to determine whether α2-AR are
located on or close to the sensory nerves and/or sympathetic nerves in the renal pelvic wall. We
focused on the α2A- and α2C-AR subtypes because of their well known expression on primary
afferent neurons (7).
METHODS
5
The study was performed on male Sprague-Dawley rats weighing 202-464g (mean 284±5
g). Two weeks before the study, rats were placed on either sodium-deficient pellets (ICN,
Na+=1.6 meq/kg) and tap water as drinking fluid (low sodium diet, n=115) or normal sodium
pellets with 0.9% NaCl solution as drinking fluid (high sodium diet, n=16) (20).
The experimental protocols were approved by the Institutional Animal Care and Use
Committee and performed according to the "Guide for the Care and Use of Laboratory Animals"
from the National Institutes of Health.
Anesthesia was induced with pentobarbital sodium (0.2 mmol/kg i.p.; Abbott
Laboratories, Abbott Park, IL).
In vivo studies
After induction of anesthesia, an intravenous infusion of pentobarbital sodium (0.04
mmol⋅kg-1⋅hr-1) at 50 µl/min into the femoral vein was started and maintained throughout the
course of the experiment. Arterial pressure was recorded from a catheter in the femoral artery.
The left renal pelvis was perfused with vehicle or various perfusates, described below,
throughout the experiment at 20 :l/min via a PE-10 catheter placed inside a PE-60 catheter
located in the ureter. ERSNA and ARNA were recorded from the central and peripheral portions,
respectively, of the cut ends of two adjacent left renal nerve branches which were placed on
bipolar silver wire electrodes. ERSNA and ARNA were integrated over 1-second intervals, the
unit of measure being microvolts per second per 1 second. All data were collected at 500 Hz and
averaged over 2 seconds. Postmortem renal nerve activity, assessed by crushing the renal nerve
bundles proximal or distal to the recording electrode, was subtracted from all values of ERSNA
and ARNA, respectively. Renal nerve activity was expressed in percentage of its baseline value
6
during the control period (15-27).
Stimulation of renal sensory nerves. Renal sensory nerves were stimulated by reflexmediated increases in ERSNA which were produced by placing the rat's tail in 47°C water
(22,24) or renal pelvic administration of NE. In all experimental groups, a 10-min control and a
10-min recovery period bracketed the experimental period.
Group I: Low sodium diet. Effects of an AT1 receptor antagonist on the ARNA responses
to reflex increases in ERSNA. The experiments were divided into two parts. During each part, the
rat's tail was placed in 47°C water during two 3-min experimental periods. Following each
experimental period, the rat's tail was immediately placed in room temperature water to quickly
terminate the heat stimulation. Twenty minutes after the first recovery period, the renal pelvic
perfusate was switched from vehicle to losartan, 0.44 mM (16,18)(n=8). Ten minutes later, the
control, experimental and recovery periods were repeated.
Group II: Low sodium diet. Effects of an α2-AR antagonist on the ARNA responses to
reflex increases in ERSNA. These experiments used a similar protocol as Group I except
rauwolscine, 0.1 μM (22), was administered into the renal pelvis (n=14).
Group III: Low sodium diet. Effects of an AT1 receptor antagonist plus an α2-AR
antagonist on the ARNA responses to reflex increases in ERSNA. These experiments used a
similar protocol as Groups I and II except losartan+rauwolscine were administered into the renal
pelvis (n=12).
Group IV: High sodium diet. Effects of an α2-AR antagonist on the ARNA responses to
reflex increases in ERSNA. The experiments performed in high sodium diet used a similar
protocol as in Group II (n=8).
7
Groups V, VI and VII: Low sodium diet. Effects of an AT1 receptor antagonist, an α2-AR
antagonist, and an AT1 receptor antagonist plus an α2-AR antagonist on the ARNA responses to
renal pelvic administration of NE. The experiments were divided into three parts. During each
part 10 pM NE, subthreshold concentration of NE for activation of renal sensory nerves in low
sodium diet rats (24), was administered into the renal pelvis during three 5-min experimental
periods. In Group V (n=7), the renal pelvic perfusate was switched from vehicle to losartan 0.44
μM at the end of the first recovery period. Five minutes later, the control, experimental and
recovery periods were repeated. At the end of the second recovery period, the renal pelvic
perfusate was switched from losartan to losartan+rauwolscine. Five minutes later, the control,
experimental and recovery periods were repeated once more. In Group VI (n=8), the
experimental protocol was similar except rauwolscine was administered instead of losartan at the
end of the first recovery period. In Group VII (n=5), only two control, experimental and recovery
period were performed, the first part in the presence of vehicle and the second part in the
presence of losartan+rauwolscine.
In vitro studies
To study whether the mechanisms involved in the altered responsiveness of the afferent
renal nerves to NE in low and high sodium diet involved pre- or postsynaptic mechanisms, we
examined the mechanisms of the NE-mediated release of substance P in an isolated renal pelvic
wall preparation (14-24). NE increases substance P release by a PG-dependent mechanism (22).
Therefore, we also examined whether the altered responsiveness of the renal sensory nerves to
NE in rats fed low and high sodium diets were associated with changes in NE-induced PGE2
release.
8
The isolated renal pelvic wall preparation has previously been described in detail (14-24).
In brief, following anesthesia renal pelvises dissected from the kidneys were placed in wells
containing 400 µl HEPES buffer maintained at 37°C. Each well contained the pelvic wall from
one kidney. Throughout the experiment, the incubation medium was replaced with fresh HEPES
every 5 min. The incubation medium was collected in siliconized vials and stored at -80°C for
later analysis of substance P and PGE2. After a 2-h equilibration period, the experiment was
started with four 5-min control periods followed by one 5-min experimental period and four 5min recovery periods. NE was added to the incubation bath to both pelvises during the
experimental periods.
Group VIII: Low sodium diet. Effects of an AT1 receptor antagonist on the NE-induced
increases in substance P and PGE2. One pelvis was incubated in HEPES buffer and the other
pelvis in HEPES buffer containing losartan, 0.44 mM, throughout the control, experimental and
recovery periods. During the experimental period, both pelvises were exposed to NE at 6250 pM
(n=10) or 1250 pM (n=6), threshold and subthreshold concentrations for substance P release in
low sodium diet rats (24). Renal pelvic release of substance P and PGE2 into the incubation bath
was measured throughout the experiment.
Group IX: Low sodium diet. Effects of an α2-AR antagonist on the NE-induced increases
in substance P and PGE2. These experiments used a similar protocol as Group VIII except one
pelvis was incubated in HEPES buffer containing rauwolscine, 0.1 μM, instead of losartan.
During the experimental period, both pelvises were exposed to NE at 6250 pM (n=4), 1250 pM
(n=8) or 250 pM (n=8).
Groups X and XI: Low sodium diet. Effects of an AT1 receptor antagonist plus an α2-AR
9
antagonist on the NE-induced increases in substance P and PGE2. One pelvis was incubated in
HEPES buffer containing losartan (n=8) or rauwolscine (n=16) and the other pelvis in HEPES
buffer containing losartan+rauwolscine throughout the control, experimental and recovery
periods. During the experimental period, both pelvises were exposed to NE at 250 pM.
Group XII: High sodium diet. Effects of an α2-AR antagonist on the NE-induced
increases in substance P and PGE2. These experiments used a similar protocol as Group IX.
During the experimental period, both pelvises were exposed to NE at 2 pM, a subthreshold
concentration for substance P release in high sodium diet rats (24).
Immunohistochemistry
The immunohistochemical procedures have been previously described in detail
(15,21,22,24). In brief, anesthetized male Sprague Dawley rats fed with low or high sodium diet
were transcardially perfused with calcium-free Tyrode solution followed by phosphate-buffered
(0.1 M, pH 7.4) fixative containing 4% w/v paraformaldehyde and 0.2% w/v picric acid. The
kidneys were quickly dissected, post-fixed in fixative for 90 min and stored in 10% sucrose at
4°C. Fourteen µm thick sections were cut on a cryostat and thaw-mounted onto gelatin-coated
slides. All primary antibodies were incubated for 24 h at 4°C.
The sections were incubated with antisera against α2A-AR or α2C-AR [rabbit 1:2,000
(45)]. Immunoreactivity was visualized using the tyramide signal amplification system (TSAPlus: PerkinElmer Life and Analytical Sciences Inc., Waltham, MA). After completion of the
protocol for TSA for detection of α2A-AR and α2C-AR, the tissue sections were incubated with
antiserum for CGRP (mouse; 1:400; Drs JH Walsh and HC Wong), a marker for sensory nerves,
or the NE transporter (NE-t)(rabbit; 1:500; HPA004063, www.proteinatlas.org, 22), a marker for
10
sympathetic nerves, and processed by the indirect immunofluorescence technique.
The specificity of the antisera for α2A-AR and α2C-AR was tested by preincubation of the
primary antisera with an excess amount (10-5 M) of the fusion protein used as immunogen, and
by labeling cultured Chinese hamster ovary (CHO) cells with antisera against α2A-AR or α2C-AR.
These CHO cells had been transfected to express human α2A-AR or α2C-AR (36) or represented
wild-type (WT) CHO cells devoid of α2-AR expression.
The tissue sections were examined using a Nikon Eclipse E600 fluorescence microscope
(Tokyo, Japan) equipped with epifluorescence and the appropriate filter combinations.
Photographs were taken with a Hamamatsu ORCA-ER C4762-80 digital camera (Hamamatsu
City, Japan) using Hamatsu photonics Wasabi software. For confocal analysis, a Radiance Plus
confocal laser scanning system (Bio-Rad, Hemel Hemstead, UK) installed on a Nikon Eclipse
E600 fluorescence microscope was used. Digital images from the microscopy were optimized for
image resolution, brightness and contrast, and color images were merged using Adobe Photoshop
7.0 (Adobe Systems Incorporated, San Jose, CA).
Drugs and reagents
Substance P antibody (IHC 7451) was acquired from Peninsula Laboratories (San Carlos,
CA) and PGE2 from Cayman Chemicals (Ann Arbor, MI). All other reagents/chemicals were
from Sigma Aldrich (St. Louis, MO) unless otherwise stated. NE was dissolved in 0.1% ascorbic
acid in incubation buffer or 0.15 M NaCl. All other agents were dissolved in incubation buffer
(in vitro studies) or 0.15 M NaCl (in vivo studies).
Analytical procedures
Concentrations of substance P and PGE2 in the incubation medium were measured by
11
ELISA, as previously described in detail (14-24).
Statistical analysis
In vivo: Increases in ERSNA, ARNA, mean AP (MAP) and HR produced by placing the
rat’s tail in warm water were evaluated by calculating the area under the curve of each parameter
vs. time, with baseline being the average value of each control period. Likewise, the ARNA
responses to NE were calculated as the area under the curve of ARNA vs. time. In vitro, the
release of substance P and PGE2 during the experimental period was compared with the
substance P and PGE2 release during the control and recovery periods. Because not all data were
normally distributed (D'Agostino and Pearson omnibus normality tests), non-parametric analysis
methods were used. The data were analyzed by Wilcoxon signed rank test and Friedman oneway analysis of variance with repeated measures followed by Dunn’s multiple comparison test
(GraphPad Prism 5.03, GrapPad Software, Inc., La Jolla, CA). A significance level of 5% was
chosen. Data in the text and figures are expressed as means ± SE.
RESULTS
In vivo studies
Similar to our previous studies (24), placing the rat’s tail in warm water resulted in
reversible increases in MAP, HR, ERSNA and ARNA that were of a greater magnitude in rats
fed high sodium diet than in rats fed low sodium diet, although the differences in the ERSNA
responses to thermal cutaneous stimulation between high and low sodium diet did not reach
statistical significance (p=0.054) in the current studies (Table 1).
Groups I-III: Low sodium diet. Effects of an AT1 receptor antagonist and an α2-AR
12
antagonist alone and in combination on the ARNA responses to reflex increases in ERSNA.
Because endogenous ANG plays an inhibitory role in the activation of renal mechanosensory
nerves by increased renal pelvic pressure (18) in rats fed low sodium diet, we reasoned that
losartan would enhance the suppressed ARNA responses to increases in ERSNA in low sodium
diet rats. However, renal pelvic perfusion with losartan failed to enhance the ARNA responses to
increases in ERSNA, as shown in Fig. 1A. In view of the inhibitory effects of the NE-mediated
activation of α2-AR on ARNA in rats fed normal sodium diet (22), we then hypothesized that an
α2-AR antagonist would enhance the ARNA responses to increases in ERSNA. In contrast to our
previous findings in normal sodium diet rats (22), renal pelvic perfusion with rauwolscine had no
effect on the ARNA responses to increases in ERSNA (Fig. 1B). Because normal sodium diet
rats are characterized by low endogenous ANG (4,12), we then examined whether rauwolscine
would increase the responsiveness of the afferent renal nerves in the presence of losartan. As
shown in Fig. 1C, renal pelvic perfusion with a combination of losartan and rauwolscine
enhanced the increases in ARNA produced by placing the rat’s tail in 47°C water without
affecting the increases in ERSNA.
Group IV: High sodium diet. Effects of an α2-AR antagonist on the ARNA responses to
reflex increases in ERSNA. Because our findings in low sodium diet rats suggested important
inhibitory roles for both α2-AR and AT1 receptors in the ERSNA induced increases in ARNA,
we next examined the role of α2-AR in the activation of renal sensory nerves in rats fed high
sodium diet, a physiological condition of low endogenous renal ANG levels (5,12,16). In
contrast to our findings in low sodium diet rats treated with losartan, rauwolscine had no effect
on the ERSNA-induced increases in ARNA (Fig. 2).
13
Groups V-VII: Low sodium diet. Effects of an AT1 receptor antagonist and an α2-AR
antagonist alone and in combination on the ARNA responses to renal pelvic administration of
NE. To minimize possible central and hemodynamic effects produced by reflex ERSNA
modulating on the responsiveness of the renal sensory nerves, we examined the effects of NE
administered directly into the renal pelvis at concentrations that did not alter MAP. In the
presence of either renal pelvic perfusion with losartan or rauwolscine alone, renal pelvic
perfusion with 10 pM NE increased ARNA (Fig. 3). The increase in ARNA produced by 10 pM
NE in the presence of vehicle did not reach statistical significance. The increases in ARNA
produced by NE in the presence of rauwolscine or losartan were not different from the ARNA
responses to NE in the presence of vehicle. In contrast in the presence of renal pelvic perfusion
with losartan+rauwolscine, NE resulted in increases in ARNA that were greater than those in the
presence of vehicle. A similar enhancement of the ARNA responses to 10 pM NE by
losartan+rauwolscine was observed in the experiments in which losartan or rauwolscine did not
precede the losartan+rauwolscine perfusion (Group VII). In these rats, the ARNA responses to
NE in the presence of vehicle and losartan+rauwolscine in the renal pelvis were 274±137 and
1859±614 %⋅sec, respectively (P<0.05 vs. vehicle, data not included in Fig. 3). MAP and HR
were not affected by the renal pelvic administration of NE in any of groups.
In vitro studies
Group VIII-XI: Low sodium diet. Effects of an AT1 receptor antagonist and an α2-AR
antagonist alone and in combination on the NE-induced increases in substance P and PGE2. Our
in vivo studies suggested an important inhibitory role for NE-mediated activation of α2-AR in the
reduced activation of renal sensory nerves in rats fed low sodium diet in the presence of AT1
14
receptor blockade. To examine whether this mechanism involved pre- or postsynaptic activation
of α2-AR, we used the isolated renal pelvic wall preparation. Our previous in vitro studies
showed that the threshold concentration of NE for substance P release is 6250 pM in rats fed low
sodium and 250 pM in rats fed normal sodium diet (22,24), suggesting that the reduced
responsiveness of the renal sensory nerves to NE in rats fed low sodium diet involves
mechanism(s) at the peripheral renal sensory nerve endings. Our initial studies showed that
losartan failed to enhance renal pelvic release of substance P and PGE2 produced by NE at 6250
and 1250 pM (Table 2). In contrast, rauwolscine enhanced the increases in renal pelvic release of
substance P produced by 6250 pM NE, from 4.3±0.5 to 9.8±1.0 pg/min in the absence and from
6.2±1.3 to 19.1±2.8 pg/min (n=4) in the presence of rauwolscine in the incubation bath. Further
studies showed that rauwolscine also enhanced the increases in renal pelvic release of substance
P and PGE2 produced by 1250 pM NE (Fig. 4A). However, 250 pM NE failed to increase
substance P and PGE2 in the presence of rauwolscine in the bath (Fig. 4B, Table 3). Adding both
rauwolscine and losartan to the incubation bath normalized the responsiveness of the renal
sensory nerves to NE. As shown in Fig. 4B and Table 4, 250 pM NE resulted in significant
increases in substance P and PGE2 in the presence of rauwolscine plus losartan in the bath.
Group XII: High sodium diet. Effects of an α2-AR antagonist on the NE-induced
increases in substance P and PGE2. Adding rauwolscine to the incubation bath failed to enhance
the increases in renal pelvic release of substance P and PGE2 produced by 2 pM NE,
subthreshold concentration of NE for activation of renal sensory nerves in rats fed high sodium
diet (Fig. 5).
Immunohistochemistry
15
Localization of α2A- and α2C-AR in renal pelvis. Renal tissues were double labeled with
antibodies against α2A-AR, α2C-AR and CGRP or NE-t. As shown in Fig. 6(a-c & e-g), the
antibodies against α2A-AR and α2C-AR labeled fibers that were on or close to CGRPimmunoreactive (ir) nerve fibers in the renal pelvic wall. Higher magnification suggested the
presence of the α2-AR being located on the renal sensory nerve fibers [Fig. 6(d,h)]. The α2C-AR
antibody also labeled fibers on or close to NE-t-ir nerve fibers in the pelvic wall [Fig. 7(a-c)],
renal fat tissue (not shown) and blood vessels [Fig. 7(d)]. No such labeling was observed with
the α2A-AR antibody.
The α2A-AR and α2C-AR antibody labeling in the kidney was blocked by adsorption with
the peptides used as immunogens for the generation of the α2A-AR and α2C-AR antibodies [Fig.
8A (a-d)]. α2A-AR-ir was only seen in CHO cells transfected to express α2A-AR [Fig. 8B (a-c)].
Likewise, α2C-AR-ir was only seen in CHO cells transfected to express α2C-AR [Fig. 8B (d-f)].
No labeling with either α2A-AR or α2C-AR antibodies was observed in non-transfected CHO
cells.
DISCUSSION
The interaction between ERSNA and ARNA is modulated by dietary sodium (24). The
present results show that reduced activation of ARNA by reflex increases in ERSNA in rats fed
low sodium diet is not affected by renal pelvic perfusion with losartan or rauwolscine alone but
is enhanced by the combined administration of these two drugs. Likewise, losartan+rauwolscine,
but not either agent alone, enhanced the ARNA responses to renal pelvic administration of NE.
In vitro studies in isolated renal pelvic wall preparations from rats fed low sodium diet showed
16
that rauwolscine but not losartan, alone, enhanced the release of substance P and PGE2 produced
by 1,250 pM NE. At 250 pM, NE failed to increase substance P or PGE2 release in the presence
of either losartan or rauwolscine alone but increased substance P and PGE2 significantly in the
presence of both losartan and rauwolscine in the bath. In vivo and in vitro studies in rats fed high
sodium diet showed that rauwolscine failed to enhance the responsiveness of the renal sensory
nerves to reflex increases in ERSNA or NE. Immunohistochemical analysis suggested the
presence of α2A-AR and α2C-AR on/close to the sensory nerves in the renal pelvic wall. Taken
together, our studies support the notion that dietary sodium modulates the NE-mediated
activation of α2-AR on renal pelvic sensory nerve endings resulting in altered responsiveness of
the renal sensory nerves to increases in ERSNA. Thus, in addition to endogenous ANG II
inhibiting the responsiveness of the renal sensory nerves, increased activation of renal α2-AR
appears to play an important inhibitory role in the interaction between ERSNA and ARNA in
conditions of low sodium intake. Conversely, no or minimal activation of renal α2-AR facilitates
the enhanced interaction between ERSNA and ARNA in high sodium conditions.
Interaction between ERSNA and ARNA.
Our previous studies in normal sodium diet rats suggested that ERSNA increases ARNA
by NE-mediated activation of α1-AR and decreases ARNA by NE-mediated activation of α2-AR
(22).
Our current functional studies showed that thermal cutaneous stimulation results in a
general increase in sympathetic nerve activity as evidenced by increases in MAP, HR and
ERSNA. The increases in these parameters were greater in high than in low sodium diet. The
lack of statistically significant differences in the ERSNA responses to thermal cutaneous
stimulation (p=0.054) between rats fed high and low sodium diet in the current study is likely
17
related to the magnitude of the ERSNA response being the result of both stimulatory reflexes
(heating of the tail, per se) and inhibitory reflexes (activation of the renorenal and baroreceptor
reflexes)(5). Nevertheless, the ERSNA-induced increases in ARNA differed in magnitude
between the high and low sodium diet rats.
Mechanisms involved in the reduced responsiveness of renal sensory nerves in low
sodium diet. Activation of endothelin (ET)A receptors contributes to the reduced ERSNA-ARNA
interaction (24). However, the ETA-receptor antagonist did not normalize the responsiveness of
the renal sensory nerves to NE in low sodium diet, suggesting that additional mechanisms
contribute to the reduced ERSNA-ARNA interaction. Because increased ANG II levels in the
renal pelvic wall (16) reduced the ARNA responses to increased renal pelvic pressure in low
sodium diet rats (16,20), we reasoned that increased activation of AT1 receptors would
contribute to the reduced interaction between ERSNA and ARNA. AT1 receptors are located on
renal vascular and tubular structures (32,50) and, most importantly, in the renal pelvic wall
(8,10,31,50). However, our initial studies showed no effects of renal pelvic administration of
losartan on the increases in ARNA produced by reflex increases in ERSNA or on the substance P
release produced by 1250 or 6250 pM NE, supramaximal concentrations of NE for substance P
release in normal sodium diet rats. The threshold concentration of NE for substance P release is
250 pM in rats fed a normal sodium diet (22). These studies suggested that additional
mechanisms were involved in suppressing the responsiveness of renal sensory nerves to changes
in ERSNA and NE.
In view of our studies in rats fed a normal sodium diet which showed that rauwolscine
enhanced the interaction between ERSNA and ARNA (22), we reasoned that among other
18
possible inhibitory mechanisms that may contribute to the reduced ERSNA-ARNA interaction
during low sodium intake would be increased activation of renal pelvic α2-AR. The involvement
of α2-AR in the central nervous system in cardiovascular regulation has long been known (35,
37) and activation of α2-AR on primary afferent neurons has antinociceptive effects (34). Three
subtypes of α2-AR have been cloned, α2A, α2B and α2C (2). There is wide-spread distribution of
α2A-AR and α2C-AR in the central nervous system (33). NE is suggested to have higher affinity
for α2C-AR which are activated at lower action potential frequencies (11,35). Both α2A-AR and
α2C-AR are expressed on primary afferent neurons in the spinal cord and dorsal root ganglia
(DRG) (42,43). In the central nervous system, available evidence indicates a limited distribution
of α2B-AR (6,11,42,43). Studies examining the presence of α2B-AR on DRG have shown
conflicting results, with one study showing a majority of the neurons to express α2B-AR (9), and
other studies showing only few neurons expressing α2B-AR (3,42). Whereas many neurons in
lumbar DRG showed co-expression of α2A-AR and α2C-AR mRNA with CGRP mRNA, only
very few neurons showed co-expression of α2B-AR with CGRP (42). In the kidney, the α2A-AR
transcript is present in the outer and inner medulla and the renal pelvic wall (30). A similar
distribution, albeit somewhat with lower intensity, was observed for α2C-AR mRNA. The
distribution of mRNA for α2B-AR is quite different, with intense expression in the renal cortex
and outer medulla but no expression in the inner medulla or renal pelvic wall (30). There is
limited immunohistochemical evidence for α2-AR on peripheral sensory nerve endings. A recent
study by Riedl et al. (40) demonstrated colocalization of α2A-AR-ir and substance P-ir in skin
sensory nerves.
The results of the present studies showed that in contrast to losartan, rauwolscine
19
enhanced the responsiveness of the renal sensory nerves to relatively high concentrations of NE,
1250 and 6250 pM in vitro. These findings demonstrated important suppressive effects of α2-AR
on renal sensory nerve activation in rats fed low sodium diet and suggested that activation of α2AR by pharmacological concentrations of NE can suppress the responsiveness of the renal
sensory nerves to physiological activation of AT1 receptors produced by low sodium diet. The
lack of an effect of rauwolscine alone on the responsiveness of the renal sensory nerves to
stimuli of a more physiological nature, e.g. thermal cutaneous stimulation in vivo and/or 250 pM
NE in vitro in low sodium diet rats would support this hypothesis.
It is unlikely that the lack of effects of renal pelvic administration of rauwolscine on the
activation of the renal sensory nerves in vivo and in vitro were due rauwolscine not producing
significant blockade of renal pelvic α2-AR. Rauwolscine was administered at a concentration, 0.1
µM, which is 25 and 500 fold higher than its Ki for α2A-AR and α2C-AR, respectively (2).
Furthermore, the concentration of rauwolscine used in the current study is 10 fold higher than
that shown to block renal vasoconstrictor responses to the α2-AR agonists clonidine and
guanabenz (6). In addition, our previous studies showed that 0.1 µM rauwolscine enhances the
activation of renal sensory nerves by increases in ERSNA in vivo and NE in vitro in rats fed
normal sodium diet (22), a dietary condition characterized by low renal tissue levels of ANG II
levels (12). Rather, we reasoned that suppression of the ARNA responses to sympathetic stimuli
of a magnitude within the physiological range may involve increased activation of both α2-AR
and AT1 receptors. If so, concurrent blockade of α2-AR and AT1 receptors would enhance the
responsiveness of the renal sensory nerves to increases in ERSNA and NE. The results confirm
our hypothesis. Renal pelvic administration of losartan + rauwolscine enhanced the ARNA
20
responses to increases in ERSNA in vivo and lowered the responsiveness of the renal sensory
nerves to NE for substance P release to 250 pM in vitro. The similar findings in vivo and in vitro
suggested a mechanism(s) at the sensory nerve endings, the isolated renal pelvic wall preparation
being sympathetically denervated.
The mechanisms by which losartan plus rauwolscine enhanced the interaction between
ERSNA and ARNA are currently not known. Activation of the renal sensory nerves by increases
in ERSNA and NE is dependent on intact PG synthesis (22). Our previous studies showed that
increased activation of AT1 receptors reduced the ARNA response to increased renal pelvic
pressure by inhibiting the PGE2-mediated activation of adenylyl cyclase by a PTX-sensitive
mechanism (16). These findings together with the α2-AR being coupled to PTX-sensitive Gi/o
proteins (11,48) suggest a similar mechanism(s) being involved in the ANG II and NE-mediated
suppression of the NE-mediated activation of renal sensory nerves in low sodium dietary
conditions. However, this reasoning does not explain the increases in NE-induced PGE2 release
in the presence of rauwolscine and losartan. The increased PGE2 release may be related to crosstalk among the second messenger systems activated by Gq, Gi and/or Gs protein coupled
receptors. Activation of Gi-coupled receptors has been shown to affect PLC and/or PKC activity
either directly or via inhibition of the AC/cAMP transduction pathway in neuronal and nonneuronal cells (1,28,38,47). The relevance of these findings to the current studies relates to our
previous studies showing that activation of renal mechanosensory nerves involves activation of
the PKC and cAMP/PKA transduction cascades (16,23). Whether the interaction between the
activation of α2-AR and AT-1 receptors occurs at the level of the receptors or beyond is currently
unknown.
21
Mechanisms involved in the enhanced responsiveness of renal sensory nerves in high
sodium diet. High sodium diet enhances the responsiveness of the renal sensory nerves to NE as
evidenced by the greater ARNA responses to increases in ERSNA and the threshold
concentration of NE for substance P release being 10 pM in high vs. 6250 pM in low sodium
dietary conditions (24). Our previous studies suggested an important role for ET-mediated
activation of ETB-receptors in the enhanced ARNA response to increases in ERSNA involving
an interaction between α1-AR and ETB receptors (24). The present findings showed that
rauwolscine has no effect on the ARNA responses to increases in ERSNA or the substance P
release produced by 2 pM NE, a subthreshold concentration of NE for substance P release in
high sodium diet rats (24). It is unlikely that adding losartan to the renal pelvic perfusate
containing rauwolscine would have resulted in increased responsiveness of the renal sensory
nerves to increases in ERSNA and/or 2 pM NE in rats fed high sodium diet. Our previous studies
showed that rats fed high sodium diet had markedly reduced endogenous ANG II concentrations
in renal pelvic tissue compared to rats fed low sodium diet (16). It is highly likely that the
responsiveness of the renal sensory nerves is modulated by renal pelvic tissue concentration and
not urinary sodium concentration of ANG II because dietary sodium modulates the activation of
the renal sensory nerves in a similar fashion in vivo and in vitro, the incubation bath being same
for pelvises derived from high and low sodium diet rats. Furthermore, renal pelvic administration
of losartan had no effect on the responsiveness of the renal sensory nerves to increases in renal
pelvic pressure or PGE2 in rats fed normal or high sodium diet (18). In view of the results from
these previous studies, the findings from our current studies suggest that NE-mediated activation
of α2-AR plays no or only a minimal role in the ERSNA-induced activation of the renal sensory
22
nerves in high sodium diet rats.
Taken together, our findings in rats fed high and low sodium diet suggest an important
role for α2-AR in modulating the responsiveness of renal sensory nerves to increases in ERSNA
during various dietary sodium intakes. The lack of effects of rauwolscine on the activation of the
renal sensory nerves in rats fed high sodium diet is most likely not related to low level of
endogenous ANG II in renal pelvic tissue since our previous studies showed that rauwolscine
enhanced the responsiveness of the renal sensory nerves to similar stimuli in rats fed normal
sodium diet (22) which is also characterized by low endogenous ANG II in renal tissue (12).
Rather the lack of effects of rauwolscine on the activation of the renal sensory nerves in rats fed
high sodium diet is most likely related to the high sodium diet, per se. Whether high sodium diet
decreases the affinity of the α2-AR to NE and/or reduces the density of α2-AR on the renal
sensory nerve fibers is currently unknown. Of interest in this context are studies in platelets and
renal cortical tissue which showed decreased affinity of α2-AR with increasing Na+
concentrations (46, 49). The current studies showed no apparent differences in the intensity of
labeling or number of nerve fibers in kidney labeled with either antibody against the α2A-AR or
α2C-AR between the high sodium and low sodium diet rats. It is well recognized that only large
differences in antibody labeling can be detected by immunohistochemical analyses. Thus, these
studies do not allow a firm conclusion whether there is a difference in the labeling of the α2-AR
antibodies of the renal sensory nerves between rats fed high and low sodium diet.
Immunohistochemical analysis of α2-AR in renal pelvic tissue. Because of the lack of
truly subtype-selective agonists or antagonists of the α2-AR subtypes, the subtype involved in the
modulation of the responsiveness of the peripheral renal sensory nerves to changes in ERSNA
23
could not be evaluated in our functional studies. Previous in situ hybridization studies suggested
the presence of α2A-AR and α2C-AR in renal pelvic tissue, without any evidence for α2B-AR (30).
Therefore, our immunohistochemical studies focused on the localization of the α2A-AR and α2CAR subtypes on or close to the peripheral sensory nerves in the renal pelvic wall. We applied the
very same antibodies against α2A-AR and α2C-AR that have that have previously and consistently
been shown to recognize the receptors against which they were generated (e.g., 7,40,45). Our
studies showed that the α2A-AR antibody and α2C-AR antibody labeled fibers in the renal pelvic
wall that were also labeled with the CGRP antibody. These data suggested the presence of both
α2A-AR and α2C-AR on renal sensory nerve fibers. Our studies further showed α2C-AR-ir fibers
on or close to NE-t-ir fibers throughout the kidney suggesting the presence of α2C-AR on renal
sympathetic nerve fibers. It is unlikely that activation of these presynaptic receptors played a
major role in the inhibition of the activation of renal sensory nerves due to the similar effects
produced by rauwolscine in the innervated in vivo and denervated in vitro preparations.
Similar to previous studies in the spinal cord (7,40,45), the α2A-AR and α2C-AR labeling
of the nerve fibers in renal tissue was blocked by preadsorption with the antigens used to
generate the antibodies against the two α2-AR subtypes. Further studies using CHO cells
transfected to express either α2A-AR, α2C-AR or neither, i.e., WT cells, showed a2A-AR-ir only
on cells transfected to express α2A-AR and α2C-AR-ir was observed only on cells transfected to
express α2C-AR in agreement with previous studies using Madin–Darby canine kidney cells (45).
Taken together, these studies suggest that the α2A-AR-ir and α2C-AR-ir observed on/close to the
renal sensory nerves in the pelvic wall represented the α2A-AR and α2C-AR subtypes,
respectively. However, applying the antibodies against α2A-AR and α2C-AR to renal tissue
24
derived from mice deficient in α2A-AR or α2A/2C-AR resulted in similar labeling as in WT mice
(data not shown). The reasons for this apparent discrepancy in our studies examining the
specificity of the two antibodies in labeling α2A-AR and α2C-AR are currently unclear. Although
the α2A-AR and α2A/2C-AR deficient mice lack functional α2A-AR and α2C-AR, we can currently
not discount the possibility that there are remnants of the α2-AR proteins that are recognized by
the two antibodies.
Perspectives
An important role for the renorenal reflex mechanism in determining ERSNA is
suggested by our studies showing that increases in ERSNA increase ARNA (22,24) which in
turn would lead to decreases in ERSNA via activation of the renorenal reflexes, i.e. a negative
feedback mechanism (26). Importantly, high sodium diet enhances the ERSNA-induced
increases in ARNA (24) which would lead to increased inhibitory renorenal reflex control of
ERSNA to minimize any ERSNA-induced sodium retention in high sodium dietary conditions.
Conversely, low sodium diet reduces the ERSNA-induced increases in ARNA which would lead
to decreased inhibitory renorenal reflex control of ERSNA. Because increases in ERSNA are
essential to prevent sodium loss in low sodium dietary conditions (5), the reduced renorenal
reflex control of ERSNA would serve as an important physiological mechanism that will
contribute to the required increases in ERSNA to prevent sodium loss.
The current studies suggest that the altered responsiveness of the renal sensory nerves to
increases in ERSNA produced by changes in dietary sodium involves modulation of the NEmediated activation of α2-AR on the renal pelvic sensory nerves. In high sodium diet, NEmediated activation of the renal sensory nerves is mediated by activation α1-AR with little or no
25
inhibitory effects produced by activation of α2-AR. In low sodium diet, increased NE-mediated
activation of α2-AR together with increased endogenous ANG II suppresses the NE-mediated
activation of α1-AR (Fig. 9). Interestingly, α2-AR density in renal tissue is greater in SHR than in
WKY rats (41) and increases further in conditions of high sodium dietary intake in association
with further increases in arterial pressure (41,44). In this context, it is of interest that the
responsiveness of the renal sensory nerves is impaired in SHR (14). In view of our current
findings, we hypothesize that the impaired responsiveness of the renal mechanosensory nerves in
SHR involves inappropriately increased activation of renal α2-AR, especially in high sodium
diet. Thus, increased NE-mediated activation of α2-AR on the renal sensory nerves may play an
important role in the development of salt-sensitive hypertension.
26
ACKNOWLEDGMENT
This work was supported by grants from the Department of Veterans Affairs, The
National Institutes of Health, Heart, Lung and Blood Institute, RO1 HL66068, Swedish Research
Council (04X-2887), Knut and Alice Wallenberg Foundation, Sweden and from the Academy of
Finland.
We are grateful for the generous supply of CGRP antiserum from the late Dr. J.H. Walsh
and Dr. H.C. Wong, The Center for Ulcer Research and Education of the Veterans Affairs/
University of California Gastroenteric Biology Center, Los Angeles (antibody/RIA Core Grant
#DK41301). We also express our gratitude to Drs. M. Uhlén and J. Mulder, Science for Life
Laboratory, Karolinska Institutet and The Royal Technical High School, Stockholm, Sweden, for
generous donation of antiserum to the NE-transporter.
The content is solely the responsibility of the authors and does not necessarily represent
the official views of the National Heart, Lung and Blood Institute or the National Institutes of
Health.
27
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FIGURE LEGENDS
Figure 1. in vivo low salt diet: Effects of renal pelvic administration of vehicle (open bar)
and (A) losartan, 0.44 mM, (hatched bar) (B) rauwolscine, 0.1 μM, (hatched bar) or (C)
losartan+rauwolscine (cross-hatched bar) on the increases in ERSNA and ARNA produced by
placing the rat’s tail in 47°C water. * P<0.05, **P<0.01 vs. baseline. ‡P<0.01, ARNA responses
to thermal cutaneous stimulation in the absence and presence of renal pelvic perfusion with
losartan+rauwolscine. Δ ERSNA, efferent renal sympathetic nerve activity response; Δ ARNA,
afferent renal nerve activity response; AUC, area under the curve of RNA vs. time.
Figure 2. in vivo high salt diet: Effects of renal pelvic administration of vehicle (open bar)
and rauwolscine (hatched bar) on the increases in ERSNA and ARNA produced by placing the
rat’s tail in 47°C water. **P<0.01 vs. baseline. For abbreviations see legend for Fig. 1.
Figure 3. In vivo low sodium diet: Effects of renal pelvic administration of vehicle (open
bar) and losartan (hatched bar), rauwolscine (hatched bar) or losartan+rauwolscine (crosshatched bar) on the ARNA responses to renal pelvic administration of 10 pM NE. Losartan or
rauwolscine (random order) preceded the administration of rauwolscine+losartan. *P<0.05,
**P<0.01 vs. baseline, †P<0.05, ‡ P<0.01, ARNA responses to NE during renal pelvic
administration of rauwolscine+losartan vs. during vehicle administration. NE, norepinephrine;
for additional abbreviations see legend for Fig. 1.
Figure 4. In vitro-isolated renal pelvises, low sodium diet: (A) Effects of 1250 pM NE on
the release of substance P and PGE2 in the presence of vehicle (solid line) or rauwolscine, 0.1
μM, (dashed line) in the bath. (B) Effects of 250 pM NE on the release of substance P and PGE2
in the presence of rauwolscine (dashed line) or rauwolscine+losartan (dotted line) in the
34
bath.**P<0.01, vs. CNT and REC; ‡ P<0.01 NE-induced substance P and PGE2 release in the
presence of vehicle vs. rauwolscine (A) and rauwolscine vs. rauwolscine+losartan (B). CNT,
control; REC, recovery. For additional abbreviations see legends for Figs. 1&3.
Figure 5. In vitro-isolated renal pelvises, high sodium diet: Effects of 2 pM NE on the
release of substance P and PGE2 in the presence of vehicle (solid line) or rauwolscine, 0.1
μM,(dashed line) in the bath. For abbreviations see legends for Figs. 1, 3 and 4.
Figure 6. Immunofluorescence double-labeling of renal tissue with antibodies against
α2A-AR, α2C-AR (green) and CGRP (red) shows α2A-AR-ir fibers (a) and α2C-AR-ir fibers (e)
close or on CGRP-ir sensory nerve fibers (b,c, & f,g, respectively) (colocalization yellow,
arrows) in the renal pelvic wall. Higher magnification showed α2A-AR-ir (green, d) and α2C-ARir (green, h) on CGRP-ir fibers (red), colocalization yellow (arrows).
Figure 7. Immunofluorescence double-labeling of renal tissue with antibodies against
α2C-AR (green) and NE-t (red) shows α2C-AR-ir fibers (a) close or on NE-t-ir fibers (b,c)
(colocalization yellow, arrows) in the renal pelvic wall. Higher magnification showed α2C-AR-ir
(green, d) on NE-t-ir fibers (red), colocalization yellow (arrows) on a vessel in renal cortical
tissue.
Figure 8. (A) Immunofluorescence labeling of renal tissue shows α2A-AR-ir (a) and α2CAR-ir (c) fibers in the renal pelvic wall (arrows). The α2A-AR and α2C-AR labeling is blocked by
adsorption with the appropriate peptide (b&d). (B). The α2A-AR antibody labeled CHO cells
transfected to express α2A-AR (a) but not cells transfected to express α2C-AR (b) or WT cells (c).
The α2C-AR antibody labeled CHO cells transfected to express α2C-AR (d) but not cells
transfected to express α2A-AR (e) or WT cells (f).
35
Figure 9. Conclusion/Hypothesis: Decreased activation of α2-AR contributes to enhanced
norepinephrine (NE)-mediated activation of renal sensory nerves in high sodium dietary
conditions (left panel). In low sodium dietary conditions, increased activation of α2-AR together
with activation of AT1 receptors contribute to the reduced responsiveness of the renal sensory
nerves to NE (right panel). Modulation of the interaction between ERSNA and ARNA is an
appropriate physiological response to changes in dietary sodium intake in the overall goal of
maintaining water and sodium balance.
36
Figure 9
Activation of Afferent Renal Sensory Nerves by nERSNA
High Sodium Diet -Enhanced
Low Sodium Diet -Impaired
ERSNA
ERSNA
2-R
2-R
PGE2
NE
PGE2
NE
1-R
ARNA
ANG II
1-R
Substance P
ARNA
Substance P
Table 1 Effects of thermal cutaneous stimulation on, MAP, HR, ERSNA and ARNA in rats fed
low and high sodium diet during vehicle administration
MAP, mmHg#s
low sodium diet
high sodium diet
n=33
n=8
3370±260*
5220±920*†
22,010±2390*
35,090±6650*†
ERSNA, %#s
7920±860*
12,910±3780*
ARNA, %#s
2220±450*
HR, beats#min-1#s
5670±1260*‡
MAP, HR, ERSNA and ARNA expressed as area under the curve of MAP, HR, ERSNA,
and ARNA, respectively vs. time; response; MAP, mean arterial rpessure; HR, heart rate;
ERSNA, efferent renal sympathetic nerve activity; ARNA, afferent renal nerve activity; *P<0.01
vs. baseline; † P<0.05; ‡ P<0.01 vs. low sodium diet.
Table 2. Effects of losartan, 0.44 mM, on the norepinephrine-mediated release of substance P
and PGE2 from isolated renal pelvic wall preparations derived from low sodium diet rats.
Substance P, pg/min
PGE2, pg/min
n=10
control
NE 6250 pM
recovery
control
NE 6250 pM
recovery
vehicle
5.7±0.7
8.9±1.4**
5.5±0.6
33.5±3.0
77.9±11.5**
38.0±2.8
losartan
5.2±.5
7.4±0.9*
4.8±0.4
42.6±4.3
77.2±15.7**
25.9±3.3
n=6
control
NE 1250 pM
recovery
control
NE 1250 pM
recovery
vehicle
5.3±0.9
5.5±1.2
5.5±1.1
55.4±5.1
77.7±11.5
55.2±3.4
losartan
5.6±1.3
6.8±1.4
5.3±1.2
63.6±14.5
75.2±12.7
45.0±5.1
NE, norepinephrine; ** P<0.01, *P<0.05 vs. average of control and recovery
Table 3. Effects of norepinephrine, 250 pM, on the release of substance P and PGE2 from
isolated renal pelvic wall preparations derived from low sodium diet rats.
Substance P, pg/min
PGE2, pg/min
control
NE,250 pM
recovery
control
NE, 250 pM
recovery
vehicle
8.0±1.2
8.5±1.6
8.0±1.4
59.6±11.9
77.3±12.3
50.0±10.9
rauwolscine
7.0±1.0
9.4±1.1
7.1±1.0
66.9±8.2
77.4±8.0
63.4±11.5
NE, norepinephrine
Table 4. Effects of losartan alone and in combination with rauwolscine on the norepinephrinemediated release of substance P and PGE2 from isolated renal pelvic wall preparations derived
from low sodium diet rats.
Substance P, pg/min
PGE2, pg/min
control
NE 250 pM
recovery
control
NE 250 pM
recovery
LOS
5.9±0.7
6.0±0.7
5.7±0.6
67.7±8.1
68.7±14.8
41.7±5.3
LOS+RWC
4.5±0.6
4.5±0.7
60.3±10.2
103.7±15.5**
35.4±5.9
10.1±1.4**‡
NE, norepinephrine; ** P<0.01 vs. average of control and recovery, ‡ P<0.01 vs the NE-induced
increase in substance P release during losartan alone.