Angiotensin blocks substance P release from renal sensory nerves by inhibiting PGE2-mediated activation of cAMP

Ulla C. Kopp, Michael Z. Cicha, and Lori A. Smith

Departments of Internal Medicine and Pharmacology, Department of Veterans Affairs, Medical Center, and University of Iowa Roy J. and Lucille Carver College of Medicine, Iowa City, Iowa 52242

Submitted 11 November 2002 ; accepted in final form 6 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of renal sensory nerves involves PGE2-mediated release of substance P (SP) via activation of the cAMP-PKA pathway. The PGE2-mediated SP release is suppressed by a low- and enhanced by a high-sodium (Na+) diet, suggesting an inhibitory effect of ANG. We now examined whether ANG II is present in the pelvic wall and inhibits PGE2-mediated SP release by blocking PGE2-mediated increases in cAMP. ANG II levels in renal pelvic tissue were 710 ± 95 and 260 ± 30 fmol/g tissue in rats fed a low- and high-Na+ diet, respectively. In a renal pelvic preparation from high-Na+-diet rats, 0.14 µM PGE2 produced an increase in SP release from 7 ± 1 to 19 ± 3 pg/min that was blocked by 15 nM ANG II. Treating pelvises with pertussis toxin (PTX) abolished the effects of ANG II. In pelvises from low-Na+ rats, neither basal nor bradykinin-mediated SP release was altered by PGE2. However, the bradykinin-mediated release of SP was enhanced by the permeable cAMP analog CPT-cAMP, from 4 ± 1 to 11 ± 2 pg/min, a response similar to that in normal-Na+-diet rats. In vivo, renal pelvic administration of PGE2 enhanced the afferent renal nerve activity (ARNA) response to bradykinin in normal- but not in low-Na+ diet rats. CPT-cAMP produced similar enhancement of the ARNA responses to bradykinin in normal- and low-Na+-diet rats, 1,670 ± 490 and 1,760 ± 400%·s (area under the curve of ARNA vs. time). Similarly, the ARNA responses to increases in renal pelvic pressure were similarly enhanced by CPT-cAMP in normal- and low-Na+-diet rats. In conclusion, renal pelvic ANG II modulates the responsiveness of renal sensory nerves by suppressing PGE2-mediated activation of adenylyl cyclase via a PTX-sensitive mechanism.

afferent renal nerves; high-sodium diet; low-sodium diet; pertussis toxin; Gi protein; bradykinin


IN THE KIDNEY, the majority of the renal sensory nerves containing substance P are located in the renal pelvic wall (22, 32, 51). The renal sensory nerves are activated by increases in renal pelvic pressure within the physiological range, >=3 mmHg (18, 30). The increase in afferent renal nerve activity (ARNA) produced by the increased renal pelvic pressure leads to a reflex decrease in efferent renal nerve activity and a diuresis and natriuresis, a renorenal reflex response (26).

The natriuretic nature of the renorenal reflexes implies that activation of these reflexes may contribute to the spectrum of renal mechanisms involved in the renal control of water and sodium homeostasis. This theory is supported by our previous studies showing that the responsiveness of the renal mechanosensory nerves is modulated by dietary sodium (18). The ARNA and natriuretic responses to increased renal pelvic pressure are suppressed in rats fed a low-sodium diet and enhanced in rats fed a high-sodium diet. Administration of the ANG type 1 (AT1)-receptor antagonist losartan into the renal pelvis enhanced the ARNA and natriuretic responses to increased renal pelvic pressure in rats fed a low-sodium diet but not in rats fed a normal-sodium diet. Conversely, renal pelvic administration of ANG II suppressed the ARNA and natriuretic responses to increased renal pelvic pressure in rats fed a high-sodium diet. Taken together, these studies suggested an important role for endogenous ANG II in modulating the activation of the renorenal reflexes. The current studies were undertaken to examine the mechanisms involved in the inhibitory effect of ANG II on the responsiveness of the renal mechanosensory nerves.

Activation of renal mechanosensory nerves by increased renal pelvic pressure involves bradykinin activating bradykinin-2 receptors with a resultant activation of the phosphoinositide system and cyclooxygenase-2 (COX-2) (21, 23, 24, 27). Activation of COX-2 leads to increased PGE2 synthesis in the renal pelvic wall (21, 24, 25). PGE2 causes a release of substance P via activation of the cAMP-PKA transduction pathway (20). The PGE2-mediated release of substance P requires influx of calcium (Ca2+) via N-type Ca2+ channels (17). Substance P increases ARNA by activating substance P receptors in the renal pelvic area (4, 22, 24, 28).

The modulatory effects of endogenous ANG II on the activation of renal mechanosensory nerves are paralleled by the PGE2-mediated release of substance P from the renal pelvic nerves, being suppressed in rats fed a low-sodium diet and enhanced in rats fed a high-sodium diet (18). Losartan enhanced the PGE2-mediated release of substance P from rats fed a low- but not from rats fed a normal- or high-sodium diet, suggesting that ANG II suppresses the release of substance P via activation of AT1 receptors. Because these findings were derived from studies using an isolated renal pelvic wall preparation, these data suggest the presence of ANG II in renal pelvic tissue. Although the presence of AT1 receptor binding sites in the renal pelvic wall is well established (11, 12, 35, 52), little is known about ANG II levels in renal pelvic tissue. There is considerable evidence for ANG II being produced and modulated by dietary sodium in renal cortical and medullary tissue (10, 16, 36, 40). Therefore, we compared the ANG II levels in renal pelvic and cortical tissue from rats fed a low- and high-sodium diet.

AT1 receptors are coupled to various guanine (G) nucleotide-binding proteins, resulting in activation of multiple signaling pathways (37, 42, 45, 47). In the central nervous system, there are numerous studies showing that ANG II exerts its effects via AT1 receptors coupled to the Gq protein, resulting in activation of the phosphoinositide system and increases in intracellular Ca2+ (1, 6). However, our findings showing that ANG II suppressed the PGE2-mediated release of substance P (18) would argue against an involvement of the Gq protein-phosphoinositidase C pathway. Rather, the important role for the cAMP-PKA pathway in PGE2-mediated release of substance P (3, 14, 20, 44) suggests that ANG II may exert its inhibitory effects on substance P release via activation of AT1 receptors coupled to the Gi protein. Previous studies in nonneural renal tissue (33) provide evidence for ANG II decreasing cAMP activity in renal proximal tubular cells at concentrations similar to those found to decrease PGE2-mediated release of substance P. To examine the involvement of the Gi protein in the effects of ANG II on substance P release, renal pelvic tissue was treated with pertussis toxin (PTX) to deactivate the Gi protein (13). Because these studies showed that PTX treatment abolished the inhibitory effects of ANG II on the PGE2-mediated release of substance P from renal sensory nerves, we pursued the notion that ANG II inhibits the PGE2-mediated activation of adenylyl cyclase. This hypothesis was tested by comparing the effects of PGE2 and the membrane-permeable cAMP analog 8-(4-chlorophenylthio) (CPT)-cAMP on the activation of renal mechanosensory nerves. Increases in cAMP activity sensitize the responsiveness of sensory nerves to other stimuli, including bradykinin and capsaicin, without altering resting membrane potential, afferent discharge, or substance P release (3, 5, 7, 9, 14, 34, 44). Therefore, we compared the sensitization produced by PGE2 and CPT-cAMP on the increase in substance P release and ARNA produced by bradykinin in rats fed a low- and normal-sodium diet.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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" by the American Physiological Society.

The study was performed in male Sprague-Dawley rats weighing 213-414 g (mean 298 ± 3 g). Two weeks before the study, rats were placed on either sodium-deficient pellets (ICN, 1.6 meq/kg Na+) with tap water drinking fluid (low-sodium diet, n = 70), normal-sodium pellets (Teklad, 163 meq/kg Na+) with tap water drinking fluid (normal-sodium diet, n = 22), or normal-sodium pellets with 0.9% NaCl drinking fluid (high-sodium diet, n = 54) (18).

In Vitro Studies

Effects of low- and high-sodium diets on renal pelvic ANG II concentration. Anesthesia was induced with pentobarbital sodium (0.2 mmol/kg ip, Abbott Laboratories). The renal pelvis and renal cortex were dissected from kidneys of rats fed low (n = 15)- and high (n = 15)-sodium diets and immediately placed on dry ice. The renal tissue was stored at -80°C for later analysis of ANG II concentration.

Substance P Release from an Isolated Renal Pelvic Wall Preparation

The procedures for stimulating the release of substance P from an isolated rat renal pelvic wall preparation have been previously described in detail (17-20). In brief, after anesthesia, renal pelvises dissected from the kidneys were placed in wells containing 400 µl HEPES (25 mM HEPES, 135 mM NaCl, 3.5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 3.3 mM D-glucose, 0.1 mM ascorbic acid, 0.1% BSA, 10 µM DL-thiorphan, 1 mM Phe-Ala, 50 µM p-chloromercuriphenylsulfonic acid, pH 7.4) maintained at 37°C. Each well contained the pelvic wall from one kidney.

In all experiments, except those involving pretreatment, for 18 h with PTX or PTX vehicle, the renal pelvic walls were allowed to equilibrate for 130 min. The incubation medium was gently aspirated every 10 min for the first 120 min and every 5 min thereafter. The medium was immediately replaced with fresh HEPES to maintain PO2 of the medium at 160-170 mmHg. The experimental protocol consisted of four 5-min control periods, one 5-min experimental period, and four 5-min recovery periods. The incubation medium, aspirated every 5 min, was placed in siliconized vials and stored at -80°C for later analysis of substance P.

Effects of ANG II on PGE2-mediated release of substance P. Indomethacin (0.14 mM) was present in the incubation bath to minimize the influence of endogenous PGE2 on substance P release. One group of rats (n = 13) fed a high-sodium diet was studied. Throughout the control, experimental, and recovery periods, the ipsilateral pelvis was incubated in HEPES/indomethacin buffer containing 15 nM ANG II and the contralateral pelvis in HEPES/indomethacin buffer containing ANG II-vehicle (0.15 M NaCl). During the experimental period, both pelvises were exposed to 0.14 µM PGE2 dissolved in the incubation medium.

Effects of ANG II on PGE2-mediated release of substance P from PTX-treated renal pelvises. Two groups fed a high-sodium diet were studied. In the first group (n = 13), the ipsilateral and contralateral pelvises were exposed for 18 h to circulating HEPES buffer containing 200 ng/ml PTX, 100 U/ml penicillin, and 100 µg/ml streptomycin. In the second group (n = 13), the ipsilateral and contralateral pelvises were similarly treated, except PTX-vehicle (0.15 M NaCl) was present in the circulating medium instead of PTX. After the 18-h treatment with PTX or vehicle, the pelvises from both groups were rinsed three times with regular HEPES buffer. The pelvises from both groups were then allowed to equilibrate in HEPES/indomethacin buffer for 70 min before the 20-min control period was started. The ipsilateral pelvises from both groups were incubated in HEPES/indomethacin buffer containing ANG II (15 nM), and the contralateral pelvis was incubated in HEPES/indomethacin buffer containing ANG II-vehicle according to the protocol described above. During the experimental period, the ipsilateral and contralateral renal pelvises from both groups were exposed to 0.28 µM PGE2 dissolved in the incubation medium.

Effects of PGE2 vs. CPT-cAMP on bradykinin-mediated release of substance P. Five groups were studied. In the first two groups, we compared the effects of PGE2 at 0.03 and 0.14 µM on the bradykinin-mediated renal pelvic release of substance P in rats fed a low-sodium diet. In the third group fed a low-sodium diet, we compared the effects of PGE2 and CPT-cAMP on the bradykinin-mediated renal pelvic release of substance P from the same rat. In the fourth and fifth groups, we compared the effects of CPT-cAMP on the bradykinin-mediated renal pelvic release of substance P from rats fed low- and normal-sodium diets. In all groups, the ipsilateral and contralateral pelvises from each group were incubated in regular HEPES buffer during the first 10 min of the control period. In the first two groups, the ipsilateral pelvis was exposed to PGE2 at 0.03 or 0.14 µM and the contralateral pelvis to PGE2-vehicle (0.15 M NaCl) during the last 10 min of the control period and the 5-min experimental period. In the third group, the ipsilateral pelvis was exposed to 0.14 µM PGE2 and the contralateral pelvis to 100 µM CPT-cAMP during the last 10 min of the control period and the 5-min experimental period. In the fourth and fifth groups, the ipsilateral pelvis was exposed to 100 µM CPT-cAMP (5, 9, 34) and the contralateral pelvis to CPT-cAMP-vehicle (0.15 M NaCl) during the last 10 min of the control period and the 5-min experimental period. In all groups, 19 µM bradykinin was added to the incubation media of the ipsilateral and contralateral pelvises during the experimental period. During the recovery periods, all pelvises were exposed to HEPES buffer only.

In Vivo Studies

After induction of anesthesia, an intravenous infusion of pentobarbital sodium (0.04 mmol·kg-1·h-1) at 50 µl/min into the femoral vein was started and continued throughout the course of the experiment. Arterial pressure was recorded from a catheter in the femoral artery. The procedures for stimulating and recording ARNA have been previously described in detail (18-30). In brief, the left kidney was approached by a flank incision, and a PE-60 catheter was placed in the left ureter with its tip in the renal pelvis. The left renal pelvis was perfused, via a PE-10 catheter placed inside the PE-60 catheter, throughout the experiment at 20 µl/min with vehicle or various renal perfusate administered as described in Experimental Protocol. In two groups of rats, renal pelvic pressure was increased by elevating the fluid-filled catheter above the level of the kidney. ARNA was recorded from the peripheral portion of the cut end of one renal nerve branch placed on a bipolar silver wire electrode. ARNA was integrated over 1-s intervals, the unit of measure being microvolts per second per 1 second. Postmortem renal nerve activity, which was assessed by crushing the decentralized renal nerve bundle peripheral to the recording electrode, was subtracted from all values of renal nerve activity. ARNA was expressed as the percentage of its baseline value during the control period (18-30).

Experimental Protocol

Approximately 1.5 h elapsed after the end of surgery and the start of the experiment to allow the rat to stabilize as evidenced by 30 min of steady-state urine collections and ARNA recordings.

Effects of PGE2 vs. CPT-cAMP on the ARNA responses to bradykinin. Two groups were studied. One group (n = 10) was fed a normal-sodium diet and one group (n = 13) a low-sodium diet. In each rat, we compared the effects of PGE2, CPT-cAMP, and vehicle on the ARNA responses to bradykinin. The experiment was divided into four parts. The renal pelvis was perfused throughout the experiment, during the first and third parts with vehicle (0.15 M NaCl), and during the second and fourth parts with PGE2 or CPT-cAMP. The order of PGE2 and CPT-cAMP perfusions was randomized. Each part consisted of a 10-min control, 5-min experimental, and 10-min recovery period. Bradykinin (3.8 µM) was added to the renal pelvic perfusate during each of the four experimental periods. There was a 20-min interval between the second and third parts of the experiment during which the renal pelvic perfusate was switched from PGE2 or CPT-cAMP back to vehicle. PGE2 was administered into the renal pelvis at 0.03 µM in rats fed a normal-sodium diet and at 0.14 µM in rats fed a low-sodium diet. CPT-cAMP was administered at 100 µM to both groups of rats.

Effects of PGE2 vs. CPT-cAMP on the ARNA responses to increased renal pelvic pressure. Two groups were studied. One group (n = 6) was fed a normal-sodium diet and one group (n = 6) a low-sodium diet. The experimental protocol was similar to that in the previous two groups, except renal pelvic pressure was increased during each of the four experimental periods. Thus renal pelvic pressure was increased in the presence of renal pelvic perfusion with vehicle, PGE2, and vehicle and CPT-cAMP. Renal pelvic pressure was increased 3.2 ± 0 mmHg in rats fed a normal-NaCl diet and 5.5 ± 0.1 mmHg in rats fed a low-sodium diet. The magnitude of the increases in renal pelvic pressure is subthreshold for activation of renal sensory nerves in rats fed normal- and low-NaCl diets, respectively (18, 30).

Drugs. Substance P antibody (IHC 7451) was acquired from Peninsula Laboratories (San Carlos, CA) and PGE2 from Cayman Chemicals (Ann Arbor, MI). All other agents were from Sigma (St. Louis, MO) unless otherwise stated. Indomethacin was dissolved together with Na2CO3 (2:1 weight ratio) in HEPES buffer and all other agents in incubation buffer (In Vitro Studies) or 0.15 M NaCl (In Vivo Studies).

Analytic Procedures

Substance P in the incubation medium was measured by ELISA, as previously described in detail (17-20, 22, 23). The rabbit substance P antibody demonstrated 100% cross-reactivity with fragments 2-11, 3-11, 4-11, and 5-11, <5% with fragment 6-11, and <0.01% with fragment 7-11, neurokinin A and B, neuropeptide K, and somatostatin.

Renal tissue ANG II concentration was measured by ELISA (Cayman Chemicals) using a slightly modified method from that described by the manufacturer. In short, renal tissue was homogenized in methanol and centrifuged for 10 min at 3,000 g. The supernatants were lyophilized and reconstituted in assay buffer and transferred to phenyl-bonded SPE columns (Varian Chromotography, Walnut Creek, CA). After being rinsed with water, hexane, and chloroform, ANG II was eluted with 90% methanol. The eluant was lyophilized and stored at -80°C. It was reconstituted in assay buffer at the time of analyses. The mouse ANG II antibody demonstrated 100% cross-reactivity with ANG II, 4% cross-reactivity with ANG I, and 36, 33, and <0.01% cross-reactivity with ANG III, ANG 3-8, and ANG 1-7, respectively.

Statistical Analysis

In vitro, the release of substance P during the experimental period was compared with that during the control and recovery periods using Friedman two-way analysis of variance and shortcut analysis of variance. The Wilcoxon matched-pairs signed-rank test was used to compare the increase in substance P release from ipsilateral and contralateral renal pelvises and the Mann-Whitney U-test to compare the increase in substance P release between groups, the increase in substance P release being calculated as the difference between the value in the experimental period with the average value of the control and recovery periods. In vivo, systemic hemodynamics were measured and averaged over each period. The ARNA responses to bradykinin and renal pelvic pressure were calculated as the area under the curve of ARNA vs. time, where ARNA was expressed as the percentage of its baseline value during the bracketing control and recovery periods. Friedman's two-way analysis of variance and shortcut analysis of variance were used to determine the effects of the various treatments on the ARNA responses within each rat. A significance level of 5% was chosen. Data in text and figures are expressed as means ± SE (43, 46).


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In rats fed low-, normal-, and high-sodium diets for >=2 wk, urinary sodium excretion averaged 245 ± 20; 2,870 ± 310; and 6,270 ± 410 µmol/24 h, respectively, in the conscious state.

In Vitro Studies

Effects of low- and high-sodium diets on renal pelvic ANG II concentration. The PGE2-mediated release of substance P from the isolated renal pelvis is suppressed in rats fed a low-sodium diet and enhanced in rats fed a high-sodium diet (18). Acute administration of losartan to the incubation medium enhances the PGE2-mediated renal pelvic release of substance P in rats fed a low-sodium diet but not in rats fed a normal- or high-sodium diet. On the basis of these results, we hypothesized that ANG II is present in renal pelvic tissue and modulated by dietary sodium. We tested this idea by measuring ANG II concentrations in renal pelvic and cortical tissue from rats fed low- and high-sodium diets. ANG II levels in renal pelvic tissue from rats fed a low-sodium diet were significantly higher than those in rats fed a high-sodium diet (P < 0.01; Fig. 1). Similarly, renal cortical ANG II concentrations were higher in rats fed a low-sodium diet vs. a high-sodium diet (P < 0.02).



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Fig. 1. Angiotensin II concentrations in renal pelvic and cortical tissue from rats fed a low (filled bars)- and high-sodium (open bars) diet. **P < 0.01, *P < 0.02 low- vs. high-sodium diet.

 

Effects of ANG II on PGE2-mediated release of substance P. Renal pelvic administration of ANG II suppressed the ARNA responses to increased renal pelvic pressure in rats fed a high-sodium diet in vivo (18). Because the PGE2-mediated release of substance P contributes importantly to the ARNA response (23-25, 29), we tested the hypothesis that acute administration of ANG II to the incubation medium suppresses the PGE2-mediated release of substance P from the renal pelvis in rats fed a high-sodium diet. In the absence of ANG II in the incubation bath, 0.14 µM PGE2 resulted in a significant reversible release of substance P from the isolated renal pelvic wall (Table 1). However, in the presence of 15 nM ANG II, in the incubation bath, the PGE2-mediated release of substance P was significantly suppressed (P < 0.01).


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Table 1. Effects of 0.14 µM PGE2 on substance P release in the presence of vehicle and 15 nM ANG II in the incubation bath containing an isolated renal pelvic wall preparation from rats fed a high-sodium diet

 

Effects of ANG II on PGE2-mediated release of substance P from renal pelvises treated with PTX. PGE2 increases the renal pelvic release of substance P and activates renal mechanosensory nerves via activation of the cAMP-PKA transduction pathway (20). Our studies suggest that ANG II suppresses the responsiveness of renal sensory nerves by activating AT1 receptors in the renal pelvic wall (18; Table 1). Because of the inhibitory effect of ANG II on substance P release, we tested the idea that ANG II reduced the PGE2-mediated release of substance P by a mechanism involving activation of the Gi protein. Renal pelvises from high-sodium diet rats were treated with PTX for 18 h to deactivate the Gi proteins (13). We reasoned that if ANG II activated renal pelvic AT1 receptors coupled to the Gi protein, PTX pretreatment would blunt the inhibitory effect of ANG II on the PGE2-mediated release of substance P.

Perfusing isolated renal pelvises for 18 h with 37°C HEPES buffer containing penicillin and streptomycin did not alter baseline substance P release or reduce the sensitivity of the renal sensory nerves to respond to PGE2 (Table 1 and Fig. 2A). Similar to the nonperfused pelvises (Table 1), ANG II blocked the PGE2-mediated release of substance P in the vehicle-perfused pelvises (Fig. 2A). In the absence of ANG II in the bath, the PGE2-mediated release of substance P was similar in the PTX-perfused and vehicle-perfused renal pelvises (Fig. 2). In the PTX-perfused pelvises, ANG II failed to inhibit the PGE2-mediated release of substance P, the increases in substance P release being 190 ± 40 and 150 ± 29% in the absence and presence of ANG II, respectively (Fig. 2B).



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Fig. 2. Effects of 18-h pretreatment with vehicle (A) and pertussis toxin (B; 200 ng) on the effects of 0.28 µM PGE2 on substance P release in the absence ({bullet}) and presence of 15 nM ANG II ({circ}) from an isolated renal pelvic wall preparation from rats fed a high-sodium diet. **P < 0.01 vs. control and recovery. {ddagger}P < 0.01 PGE2-mediated substance P release in the absence vs. presence of ANG II.

 

Effects of PGE2 vs. CPT-cAMP on bradykinin-mediated release of substance P. Our results suggest that ANG II suppresses the PGE2-mediated release of substance P by activating AT1 receptors coupled to the Gi protein. To examine whether ANG II exerts its inhibitory effects via Gi protein-mediated effects on adenylyl cyclase, we compared the effects of PGE2 and CPT-cAMP on substance P release in rats fed a low-sodium diet. Because increases in cAMP by forskolin or membrane-permeable analogs enhance the substance P release produced by bradykinin but have no effects on baseline substance P release in normal-sodium-diet rats (14, 20), we compared the effects of PGE2 and CPT-cAMP on the bradykinin-mediated release of substance P. We reasoned that if ANG II suppresses the PGE2-mediated activation of the renal sensory nerves by inhibiting adenylyl cyclase, the bradykinin-mediated release of substance P would be enhanced by CPT-cAMP but not by PGE2. Our previous studies in rats fed a normal-sodium diet showed that basal substance P release is increased by 0.14 µM PGE2 (17, 18, 20) but not by 0.03 µM PGE2 (17, 20). However, 0.03 µM PGE2 produces a marked enhancement of the bradykinin-mediated release of substance P in normal-sodium-diet rats (20). Therefore, in our initial studies in rats fed a low-sodium diet, we compared the effects of PGE2 at 0.03 and 0.14 µM on the bradykinin-mediated release of substance P. Bradykinin was administered at a concentration (19 µM) that produces no or minimal increases in basal substance P release (20). Basal substance P release was not altered by PGE2 at 0.03 and 0.14 µM, from 5.7 ± 1.1 to 5.1 ± 1.1 pg/min and from 3.8 ± 0.8 to 3.2 ± 0.7 pg/min, respectively. Similarly, the bradykinin-mediated release of substance P was unaffected by PGE2 at 0.03 and 0.14 µM (Table 2). Having shown that PGE2 at 0.14 µM failed to enhance the bradykinin-mediated release of substance P in rats fed a low-sodium diet, we used this concentration of PGE2 to compare the effects of PGE2 and CPT-cAMP on the bradykinin-mediated release of substance P from ipsilateral and contralateral pelvises of the same rat. Although PGE2 failed to alter the bradykinin-mediated release of substance P from the ipsilateral pelvis, 100 µM CPT-cAMP (5, 9) produced a marked enhancement of the bradykinin-mediated release of substance P from the contralateral pelvis (Fig. 3). Comparing the effects of CPT-cAMP on the bradykinin-mediated release of substance P in rats fed low- and normal-sodium diets showed that the enhancement of the bradykinin-mediated release of substance P was similar in rats fed low- and normal-sodium diets (Fig. 3 and Table 3). CPT-cAMP did not affect baseline substance P release in rats fed low- and normal-sodium diets, from 4.5 ± 0.5 to 3.8 ± 0.5 pg/min and from 4.6 ± 0.8 to 4.0 ± 0.5 pg/min, respectively.


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Table 2. Effects of PGE2 and vehicle on the bradykinin-mediated release of substance P from an isolated renal pelvic wall preparation from rats fed a low-sodium diet

 


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Fig. 3. Effects of 100 µM CPT-cAMP ({triangleup}) and 0.14 µM PGE2 ({blacktriangleup}) on the bradykinin-mediated release of substance P from isolated ipsilateral and contralateral pelvises from rats fed a low-sodium diet. Bradykinin was administered at 19 µM. **P < 0.01 vs. control and recovery. {ddagger}P < 0.01 CPT-cAMP-mediated vs. PGE2-mediated enhancement of bradykinin-induced substance P release.

 

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Table 3. Effects of CPT-cAMP and vehicle on the bradykinin-mediated release of substance P from an isolated renal pelvic wall preparation from rats fed normal- and low-sodium diets

 

In Vivo Studies

Effects of PGE2 vs. CPT-cAMP on the ARNA response to bradykinin. Our in vitro studies suggest that ANG II modulates the PGE2-mediated release of substance P from renal pelvic nerves by a mechanism involving suppression of adenylyl cyclase. To verify the importance of this mechanism of ANG II in vivo, we compared the effects of PGE2 and CPT-cAMP on the bradykinin-mediated activation of renal mechanosensory nerves in rats fed a normal- and low-sodium diet. Renal pelvises were perfused with bradykinin at a concentration (3.8 µM) that is the subthreshold for activation of renal mechanosensory nerves in vivo (29). In rats fed a normal-sodium diet, 0.03 µM PGE2 and 100 µM CPT-cAMP enhanced the ARNA responses to bradykinin (Fig. 4), the duration of the ARNA responses being 49 ± 17 and 80 ± 18 s, respectively. In rats fed a low-sodium diet, PGE2 at a fivefold higher concentration, 0.14 µM, failed to enhance the ARNA response to bradykinin. On the other hand, CPT-cAMP produced an enhancement of the ARNA response to bradykinin that was of a similar magnitude and duration, 105 ± 27 s, as that in normal-sodium-diet rats (Fig. 4). Baseline ARNA was unaltered by renal pelvic perfusion with PGE2 and CPT-cAMP in normal-sodium-diet rats, from 1,730 ± 180 to 1,790 ± 190 and from 1,710 ± 140 to 1,690 ± 140 µV·s-1·1 s-1, respectively, and low-sodium diet rats, from 1,390 ± 130 to 1,410 ± 130 and from 1,440 ± 130 to 1,470 ± 140 µV·s-1·1 s-1, respectively. Mean arterial pressure, 112 ± 3 and 114 ± 2 mmHg, and heart rate, 342 ± 10 and 325 ± 12 beats/min, were similar in the two groups of rats and remained unaltered throughout the experiment.



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Fig. 4. Effects of renal pelvic perfusion with PGE2 (open bars) and CPT-cAMP (filled bars) on the afferent renal nerve activity (ARNA) responses in rats fed normal (A)- and low-sodium (B) diets. PGE2 was administered at 0.03 and 0.14 µM to rats fed normal- and low-sodium diets, respectively. CPT-cAMP was administered at 100 µM and bradykinin at 3.8 µM to both groups. AUC, area under the curve of ARNA vs. time; *P < 0.02, **P < 0.01 vs. baseline ARNA.

 

Effects of PGE2 vs. CPT-cAMP on the ARNA response to increased renal pelvic pressure. Activation of bradykinin-2 receptors contributes to the increase in ARNA produced by increased renal pelvic pressure (23). Therefore, our current in vitro and in vivo data would suggest that ANG II suppresses the ARNA responses to increased renal pelvic pressure in rats fed a low-sodium diet (18) by a mechanism involving inhibition of adenylyl cyclase. Renal pelvic pressure was increased 3.3 ± 0 and 5.5 ± 0.1 mmHg in rats fed normal- and low-sodium diets, respectively. The magnitude of these renal pelvic pressure increases is the subthreshold for activation of renal mechanosensory nerves in normal- and low-sodium diet rats in control conditions (18, 30) (Table 4). In rats fed a normal-sodium diet, renal pelvic perfusion with 0.03 µM PGE2 and 100 µM CPT-cAMP produced a similar enhancement of the ARNA responses to increasing renal pelvic pressure (Table 4). In rats fed a low-sodium diet, a fivefold higher concentration of PGE2, 0.14 µM, produced an enhancement of the ARNA response to increased renal pelvic pressure that was significantly suppressed (P < 0.01) compared with that produced in normal-sodium-diet rats. On the other hand, 100 µM CPT-cAMP produced a similar enhancement of the ARNA response to increased renal pelvic pressure as in normal-sodium-diet rats. Similar to the previous study, renal pelvic perfusion with PGE2 and CPT-cAMP did not affect baseline ARNA in either group, baseline ARNA being 1,765 ± 164 and 1,317 ± 174 µV·s-1·1s-1 in rats fed normal- and low-sodium diets, respectively. Mean arterial pressure, 109 ± 3 and 105 ± 3 mmHg, and heart rate, 304 ± 12 and 367 ± 13 beats/min, remained unaltered throughout the experiment in the two groups of rats.


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Table 4. Effects of renal pelvic perfusion with vehicle, PGE2, and CPT-cAMP on the ARNA responses to increased renal pelvic pressure in rats fed normal- and low-sodium diets

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of our experiments show that ANG II is present in renal pelvic tissue and modulated by dietary sodium. ANG II suppressed PGE2-mediated release of substance P from an isolated renal pelvic wall preparation. Treating renal pelvic tissue with PTX blocked the inhibitory effect of ANG II on PGE2-mediated release of substance P. PGE2 failed to enhance bradykinin-mediated release of substance P from the renal pelvic wall in rats fed a low-sodium diet. On the other hand, CPT-cAMP produced an enhancement of the bradykinin-evoked release of substance P that was of similar magnitude in rats fed low- and normal-sodium diets. Our in vivo studies showed that renal pelvic perfusion with PGE2 enhanced the ARNA response to bradykinin in rats fed a normal-sodium diet but not in rats fed a low-sodium diet. However, renal pelvic perfusion with CPT-cAMP produced a similar enhancement of the ARNA response to bradykinin in rats fed low- and normal-sodium diets. Similarly, the ARNA responses to increases in renal pelvic pressure were similarly enhanced by CPT-cAMP in normal- and low- sodium diet rats. Taken together, these findings suggest that ANG II modulates the responsiveness of the renal mechanosensory nerves by suppressing the PGE2-mediated activation of adenylyl cyclase via a mechanism that involves the Gi protein.

ANG II in Renal Pelvic Tissue

There is extensive evidence for ANG II synthesis in renal cortical and medullary tissue (16, 36, 48). Intrarenal ANG II levels are higher than plasma ANG II levels and modulated by dietary sodium (15, 16, 36, 48). Our previous studies showed that the responsiveness of the renal mechanosensory nerves is altered by endogenous ANG II modulating the release of substance P from renal pelvic sensory nerves. The PGE2-mediated release of substance P from an isolated renal pelvic wall preparation was impaired in rats fed a low-sodium diet and rats with congestive heart failure (CHF) fed a normal-sodium diet (18, 19), conditions of high ANG II levels in renal tissue and plasma (10, 15, 16, 36, 40). Conversely, the PGE2-mediated release of substance P was enhanced in rats fed a high-sodium diet (18) wherein renal tissue and plasma ANG II levels are suppressed (10, 15, 16, 36). Also, the current study showed that baseline substance P release from the isolated renal pelvises was lower in rats fed a low-sodium diet than in rats fed a high-sodium diet, 5.4 ± 0.3 vs. 9.1 ± 0.8 pg/min (n = 73 and 78 pelvises, respectively, P < 0.01). Our previous studies further showed that acute administration of losartan restored the impaired PGE2-mediated release of substance P in low-sodium diet rats and CHF rats toward that in normal rats (18, 19). Because these findings were derived from an isolated renal pelvic wall preparation, the data suggest that renal pelvic mechanosensory nerve fibers are modulated by ANG II derived from renal pelvic tissue. To examine this hypothesis, we measured ANG II levels in renal pelvic and cortical tissue from rats fed low- and high-sodium diets. In agreement with previous studies (16, 48), ANG II levels in renal cortical tissue were higher in rats fed a low- than in rats fed a high-sodium diet. Our data further showed the presence of ANG II in renal pelvic tissue. ANG II concentration in pelvic tissue was significantly greater in rats fed a low-sodium diet than in rats fed a high-sodium diet (P < 0.01, n = 15). Interestingly, when ANG II concentrations in renal cortical and pelvic tissue from the same kidneys were compared, it was found that ANG II concentration was higher in renal pelvic tissue than in cortical tissue from rats fed a low-sodium diet, 1,094 ± 172 vs. 516 ± 81 fmol/g tissue (P < 0.05, n = 5), and similar in rats fed a high-sodium diet, 273 ± 65 vs. 230 ± 47 fmol/g tissue (n = 5). Although the significance of these findings may be confounded by the rather limited number of kidneys from which both cortical and pelvic tissues were taken, our data suggest that renal pelvic ANG II concentration is in the range of, or even greater, than that in renal cortical tissue. Overall, these studies suggest that the modulatory effects of ANG II on renal pelvic sensory nerves (18) are exerted by ANG II in renal pelvic tissue. The importance of ANG II in renal pelvic tissue controlling the responsiveness of renal pelvic sensory nerves is further supported by the lack of effects of dietary sodium on urinary ANG II concentration (50).

Role of the Gi Protein in the ANG II-Mediated Suppression of PGE2-Evoked Release of Substance P

AT1 receptors are widely distributed in the central and peripheral nervous system, including sensory neurons in dorsal root ganglia (DRG) and nodose ganglia (1). In the kidney, AT1 receptors are located on vascular and tubular structures (36, 52) and, most importantly, in the renal pelvic wall (11, 12, 35, 52). Our current and previous studies provide evidence for a role of renal pelvic AT1 receptors mediating the suppressive effects of locally generated ANG II on the responsiveness of renal mechanosensory nerves. AT1 receptors are coupled to various G proteins, resulting in activation of multiple pathways via different regions of the AT1 receptor (42). There are numerous reports of ANG II-activating AT1 receptors coupled to the Gq protein, resulting in increased intracellular Ca2+ and facilitation of neurotransmitter release (1, 6). However, our studies showing that ANG II in renal pelvic tissue suppressed the release of substance P would argue against a role for the Gq protein in the ANG II-mediated effects in the current studies. There is evidence for ANG II-activating AT1 receptors coupled to the Gi protein in neural and nonneural cells (e.g., 1, 2, 33). Similarly, renal microperfusion studies showed that ANG II exerts its physiological effects on tubular transport via a PTX-sensitive mechanism (33). To test the notion that ANG II suppressed the activation of renal mechanosensory nerves via activation of AT1 receptors coupled to the Gi protein, isolated renal pelvises from rats fed a high-sodium diet were treated for 18 h with PTX to deactivate the Gi protein (13). Importantly, perfusing the renal pelvic tissue for 18 h with HEPES buffer containing antibiotics to prevent growth in the incubation medium did not reduce the responsiveness of the renal sensory nerves to PGE2. The increase in substance P release produced by 0.28 µM PGE2 from renal pelvises perfused for 18 h was greater than that produced by 0.14 µM PGE2 in the nonperfused pelvises (18 ± 2 vs. 12 ± 2 pg/min, P < 0.05). Comparing the effects of ANG II on the PGE2-mediated release of substance P in vehicle-perfused and nonperfused kidneys showed that ANG II had a similar inhibitory effect on the PGE2-mediated release of substance P in the two groups, the increases in the PGE2-mediated release of substance P being only 3 ± 2 and 3 ± 1 pg/min, which were significantly less (P < 0.01) than that produced by PGE2 in the absence of ANG II in both groups. Perfusing the renal pelvises with PTX for 18 h did not alter the magnitude of the PGE2-mediated release of substance P. However, PTX blocked the inhibitory effects of ANG II on the PGE2-mediated release of substance P. The lack of a possible enhancement of substance P release in response to PGE2 in the PTX-treated pelvises is most likely due to the fact that these pelvises were derived from rats fed a high-sodium diet. Taken together, these findings suggest that ANG II exerts its inhibitory effect on the PGE2-mediated renal pelvic release of substance P via activation of AT1 receptors coupled to the Gi protein.

Role of the cAMP-PKA Transduction Cascade in the Effects of ANG II on the PGE2-Mediated Release of Substance P

Our previous studies showed that the PGE2-mediated release of substance P from renal pelvic sensory nerves is markedly suppressed in rats fed a low-sodium diet (18). Compared with rats fed a normal-sodium diet, a 25-fold higher concentration of PGE2 is required to produce a significant, albeit suppressed, increase in the release of substance P from renal sensory nerves in rats fed a low-sodium diet. Activation of AT1 receptors coupled to the Gi protein may decrease the release of substance P by decreasing cAMP activity (33) and Ca2+ influx by an effect on the N-type Ca2+ channels (2, 47). Because of the important role for the cAMP-PKA transduction pathway in the PGE2-mediated release of substance P from various sensory nerves (3, 14, 44), including the renal sensory nerves (20), we hypothesized that the impaired PGE2-mediated renal pelvic release of substance P in rats fed a low-sodium diet was due to ANG II suppressing PGE2-mediated activation of adenylyl cyclase. To test this theory, we compared the effects of PGE2 and the membrane-permeable cAMP analog CPT-cAMP on substance P release in rats fed a low-sodium diet. We reasoned that if ANG II suppressed the PGE2-mediated release of substance P by inhibiting adenylyl cyclase, direct activation of cAMP would lead to an increase in substance P release in rats fed a low-sodium diet. Adding CPT-cAMP to the incubation bath did not alter baseline substance P release from the renal pelvises of either normal- or low-sodium diet rats, suggesting that increases in cAMP, per se, do not alter resting levels of substance P release. These findings are in agreement with our previous studies in normal rats in which we showed that forskolin did not alter baseline substance P release (20). Furthermore, our findings are in accord with studies in cultured dorsal root ganglion (DRG) neurons and isolated jejunum, which showed that similar concentrations of cAMP analogs and forskolin as used in our present and previous studies did not alter resting membrane potential, afferent discharges, or substance P release (3, 5, 14). Whether higher concentrations of CPT-cAMP would alter baseline substance P release was not tested in our studies. Studies in cultured DRG showed that 10, 100, and 1,000 µM of 8-bromo-cAMP, a cAMP analog equipotent to CPT-cAMP (5), produced similar peak enhancement of the capsaicin-induced current (34). However, the duration of the sensitization was inversely correlated to the concentration of the cAMP analog. Similar findings were observed with 10 and 100 µM CPT-cAMP. These studies may argue against the idea that a higher concentration of the cAMP analog would have resulted in an increase in baseline substance P release. In addition, there is extensive evidence for cAMP analogs increasing the excitability of sensory neurons without causing membrane depolarization (3, 5, 7, 9, 14, 34), i.e., sensitizing the sensory nerve responses to other stimuli. Similarly, our previous studies in normal rats showed that forskolin and PGE2, at a subthreshold concentration for substance P release, enhanced the substance P release produced by bradykinin (20). Therefore, we compared the effects of PGE2 and CPT-cAMP on the bradykinin-mediated release of substance P in rats fed a low-sodium diet. Our results showed that PGE2 at 0.03 and 0.14 µM failed to enhance the bradykinin-mediated release of substance P in rats fed a low-sodium diet. It is important to note that PGE2 at 0.03 and 0.14 µM enhances bradykinin-mediated substance P release and basal substance P release, respectively, in rats fed a normal-sodium diet (18). However, our studies further showed that CPT-cAMP produced an enhancement of the bradykinin-mediated release of substance P in rats fed a low-sodium diet. The increase in substance P release produced by CPT-cAMP + bradykinin was of a similar magnitude as that produced in renal pelvises from normal-sodium diet rats. In this context, it is interesting to note that the enhancement produced by the membrane-permeable cAMP analog db-cAMP of the bradykinin-mediated increases in afferent discharges from an isolated ileum preparation is independent of prostaglandin synthesis (3).

To explore the notion that the inhibitory effects of ANG II on the activation of renal mechanosensory nerves in vivo were also related to inhibition of cAMP activity, we compared the effects of renal pelvic perfusion with PGE2 and CPT-cAMP on the ARNA responses to renal pelvic administration of bradykinin in rats fed low- and normal-sodium diets. Whereas in rats fed a normal-sodium diet the ARNA responses to bradykinin were enhanced by both PGE2 and CPT-cAMP, only CPT-cAMP enhanced the ARNA responses to bradykinin in rats fed a low-sodium diet. PGE2 had no effect, despite the fact that PGE2 was administered at a fivefold higher concentration than in rats fed a normal-sodium diet, 0.14 vs. 0.03 µM. The CPT-cAMP-mediated enhancement of the ARNA responses was similar in the two groups of rats. Subsequent studies in which we compared the effects of renal pelvic perfusion with PGE2 and CPT-cAMP on the ARNA responses to increased renal pelvic pressure in rats fed low- and normal-sodium diets showed similar results. The ARNA responses to increased renal pelvic pressure were enhanced to a similar extent by PGE2 and CPT-cAMP in normal-sodium diet rats and CPT-cAMP in low-sodium diet rats. However, the effects of PGE2 on the ARNA response to increased renal pelvic pressure were markedly suppressed in low-sodium-diet rats. Because activation of bradykinin-2 receptors contributes importantly to the increase in ARNA produced by increased renal pelvic pressure (23), these data provide further support for our in vitro findings.

Taken together, our in vitro and in vivo studies suggest that the inhibitory effect of ANG II on PGE2-mediated release of substance P involves ANG II suppressing the PGE2-mediated activation of adenylyl cyclase. Whether the renal nerves are modulated by ANG II in the renal sensory nerves, per se, or in the uroepithelium and/or pelvic muscle wall surrounding the renal sensory nerves cannot be deduced from our current findings. The numerous reports of ANG II-binding sites in the renal pelvic wall (11, 12, 35, 52) together with the known localization of AT1 receptors on central sensory nerves (1, 6) may suggest the presence of renal sensory nerves containing AT1 receptors. These receptors may be activated by ANG II from the surrounding tissue. On the other hand, ANG II has shown to be present in neurons in the central nervous system associated with cardiovascular control (1). These findings together with studies in DRG neurons showing that sensory nerves can be one source of PGE2 (49) suggest the intriguing hypothesis that the renal sensory nerves can autoregulate their sensitivity by producing PGE2 and ANG II, exerting opposing effects on neuropeptide release.

The mechanisms involved in the sensitization of the renal sensory nerves produced by increases in cAMP were not addressed in the current studies. Previous studies in cultured DRG neurons examining the effects of PGE2, forskolin, and cAMP analogs on sensitization of sensory neurons suggested that activation of the cAMP-PKA transduction cascade increases the membrane excitability of these neurons by inhibiting the delayed rectifier-like potassium current (9) and/or increasing the tetrodotoxin-resistant sodium current (7). The increased membrane excitability produced by activation of the cAMP-PKA pathway sensitized the sensory neurons to bradykinin, resulting in increased numbers of action potentials and substance P release (5, 14, 44). Whether cAMP-induced sensitization of sensory neurons is associated with increases in intracellular Ca2+ (Cai2+) is not clear. Whereas the findings by Smith et al. (44) would suggest that db-cAMP enhances the bradykinin-mediated release of substance P in association with increases in Cai2+, the study by Evans et al. (8) showed no effect of Ca2+ channel blockers of the N, L, or T type on the PGE2-mediated facilitation of substance P release.

There is evidence for ANG II and bradykinin decreasing phosphodiesterase activity in nonneural cells by mechanisms involving protein kinase C (31, 38). Whether this mechanism contributed to the increase in substance P release and activation of renal sensory nerves in response to bradykinin + CPT-cAMP in the rats fed a low-sodium diet was not addressed in the current studies. However, studies in DRG neurons showing that forskolin does not increase baseline substance P release despite a 10-fold increase in cAMP (14) may argue against this mechanism playing an important role in the sensitization of the renal mechanosensory nerves.

The present studies were not designed to examine the nature of the Ca2+ channels involved in the ANG II-mediated inhibition of the PGE2-mediated substance P release from renal sensory nerves. However, our previous studies showing that {omega}-conotoxin blocked the PGE2-mediated release of substance P (17) may suggest an involvement of N-type Ca2+ channels in the ANG II-mediated inhibition of substance P release via the PGE2-mediated activation of the cAMP-PKA transduction pathway. This hypothesis is supported by studies in the nodose ganglia by Bacal and Kunze (2). ANG II at 10 nM, i.e., a concentration in the range of that used in the current studies, decreases Ca2+ current in isolated nodose ganglia via activation of AT1 receptors coupled to PTX-sensitive G proteins and involving {omega}-conotoxin-sensitive Ca2+ channels (2). However, there are reports in sympathetic neurons that may argue against a role for PTX-sensitive Ca2+ channels playing a major role in the inhibition of Ca2+ current produced by ANG II (41). The apparent differences between these studies may be explained by the concentrations of ANG II applied in the two studies, being 10 (2) and 500 nM (41). A study in chromaffin cells (47) showed that ANG II at low concentrations (<=10 nM) activated AT1 receptors coupled only to Gi proteins, whereas at higher concentrations, such as 100 nM, ANG II-activated AT1 receptors coupled to both Gi and Gq proteins, the Gq protein coupling being dominant. Moreover, it cannot be excluded that ANG II exerts its effect via AT1 receptors being coupled to different G proteins in sensory and sympathetic neurons (2, 41).

In summary, the present study shows that ANG II is present in renal pelvic wall tissue and modulated by dietary sodium. Studies in an isolated renal pelvic wall preparation showed that ANG II suppressed the release of substance P from renal pelvic sensory nerves in rats fed a high-sodium diet by a PTX-sensitive mechanism. In rats fed a low-sodium diet, CPT-cAMP but not PGE2 enhanced the bradykinin-mediated release of substance P. The magnitude of the CPT-cAMP-induced enhancement of bradykinin-mediated release of substance P was similar in rats fed low- and normal-sodium diets. Furthermore, the results from the in vitro studies were supported by the in vivo findings that showed that renal pelvic perfusion with PGE2 enhanced the ARNA response to bradykinin in rats fed normal- but not low-sodium diets. On the other hand, renal pelvic perfusion with CPT-cAMP produced a similar enhancement of the bradykinin-mediated increase in ARNA in rats fed normal- and low-sodium diets. Similarly, the ARNA responses to increased renal pelvic pressure were enhanced to a similar extent by CPT-cAMP in the two groups of rats. The PGE2-mediated enhancement of the ARNA response to increased renal pelvic pressure in normal-sodium-diet rats was markedly suppressed in rats fed a low-sodium diet. Taken together, these studies along with our previous studies (18) suggest that ANG II modulates the PGE2-mediated release of substance P from renal sensory nerves by blocking the PGE2-evoked stimulation of adenylyl cyclase via activation of AT1 receptors coupled to the Gi protein.


    DISCLOSURES
 
This work was supported by grants from the Department of Veterans Affairs, National Heart, Lung, and Blood Institute, R-O1 HL-66068, and Specialized Center of Research, HL-55006, and American Heart Association Grant-In-Aid 0150024N.


    ACKNOWLEDGMENTS
 
We are indebted to Dr. L. G. Navar and D. M. Seth, Department of Physiology, Tulane University School of Medicine, New Orleans, LA, for valuable advice on the angiotensin assay.


    FOOTNOTES
 

Address for reprint requests and other correspondence: U. C. Kopp, Dept. of Internal Medicine, VA Medical Center, Bldg. 3, Rm. 226, Highway 6W, Iowa City, IA 52246 (E-mail: ukopp{at}blue.weeg.uiowa.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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