Angiotensin blocks substance P release from renal sensory nerves by inhibiting P - 《American Journal of Physiology》美国生理学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 27 下一页

返回列表

查看: 581945\|回复: 260	go Angiotensin blocks substance P release from renal sensory nerves by inhibiting P [复制链接]

轻羽

新手上路

Rank: 1

积分: 0
威望: 0
包包: 0

楼主

发表于 2009-4-21 13:44 |只看该作者 |倒序浏览 |打印

作者：Ulla C. Kopp, Michael Z. Cicha, and Lori A. Smith作者单位：Departments of Internal Medicine and Pharmacology, Department of VeteransAffairs, Medical Center, and University of Iowa Roy J. and Lucille CarverCollege of Medicine, Iowa City, Iowa 52242 ' O" J' X1 F) l* r

   7 I' p. k: U+ `


         5 J# O; V+ K9 c2 y" m" @0 q1 B

         & u; _; d6 B0 I6 h0 ^




      ( J# D8 k5 O: E% o/ u5 M% h9 k6 v
      【摘要】
   Activation of renal sensory nerves involves PGE 2 -mediatedrelease of substance P (SP) via activation of the cAMP-PKA pathway. ThePGE 2 -mediated SP release is suppressed by a low- and enhanced by ahigh-sodium (Na ) diet, suggesting an inhibitory effect of ANG. Wenow examined whether ANG II is present in the pelvic wall and inhibitsPGE 2 -mediated SP release by blocking PGE 2 -mediated increases in cAMP. ANG II levels in renal pelvic tissue were 710 ± 95and 260 ± 30 fmol/g tissue in rats fed a low- and high-Na diet, respectively. In a renal pelvic preparation fromhigh-Na -diet rats, 0.14 µM PGE 2 produced an increase in SP release from 7 ± 1 to 19 ± 3 pg/min that was blocked by 15nM ANG II. Treating pelvises with pertussis toxin (PTX) abolished the effectsof ANG II. In pelvises from low-Na rats, neither basal norbradykinin-mediated SP release was altered by PGE 2. However, thebradykinin-mediated release of SP was enhanced by the permeable cAMP analogCPT-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 PGE 2 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 ARNAresponses to increases in renal pelvic pressure were similarly enhanced byCPT-cAMP in normal- and low-Na -diet rats. In conclusion, renalpelvic ANG II modulates the responsiveness of renal sensory nerves bysuppressing PGE 2 -mediated activation of adenylyl cyclase via aPTX-sensitive mechanism.
      【关键词】 afferent renal nerves highsodium diet lowsodium diet pertussis toxin G i protein bradykinin
   ----------

IN THE KIDNEY, the majority of the renal sensory nervescontaining substance P are located in the renal pelvic wall( 22, 32, 51 ). The renal sensory nervesare activated by increases in renal pelvic pressure within the physiologicalrange, 3 mmHg ( 18, 30 ). The increase in afferentrenal nerve activity (ARNA) produced by the increased renal pelvic pressureleads to a reflex decrease in efferent renal nerve activity and a diuresis andnatriuresis, a renorenal reflex response( 26 ).
  g! K. ?9 x$ ~+ X) k' T
The natriuretic nature of the renorenal reflexes implies that activation ofthese reflexes may contribute to the spectrum of renal mechanisms involved inthe renal control of water and sodium homeostasis. This theory is supported byour previous studies showing that the responsiveness of the renalmechanosensory nerves is modulated by dietary sodium( 18 ). The ARNA and natriuretic responses to increased renal pelvic pressure are suppressed in rats fed alow-sodium diet and enhanced in rats fed a high-sodium diet. Administration ofthe ANG type 1 (AT 1 )-receptor antagonist losartan into the renalpelvis enhanced the ARNA and natriuretic responses to increased renal pelvicpressure in rats fed a low-sodium diet but not in rats fed a normal-sodiumdiet. Conversely, renal pelvic administration of ANG II suppressed the ARNAand natriuretic responses to increased renal pelvic pressure in rats fed ahigh-sodium diet. Taken together, these studies suggested an important rolefor endogenous ANG II in modulating the activation of the renorenal reflexes.The current studies were undertaken to examine the mechanisms involved in theinhibitory effect of ANG II on the responsiveness of the renal mechanosensory nerves.
3 ^+ s2 T8 C8 U% ]4 v
Activation of renal mechanosensory nerves by increased renal pelvicpressure involves bradykinin activating bradykinin-2 receptors with aresultant activation of the phosphoinositide system and cyclooxygenase-2(COX-2) ( 21, 23, 24, 27 ). Activation of COX-2 leadsto increased PGE 2 synthesis in the renal pelvic wall( 21, 24, 25 ). PGE 2 causes arelease of substance P via activation of the cAMP-PKA transduction pathway( 20 ). The PGE 2 -mediated release of substance P requires influx of calcium (Ca 2 ) via N-type Ca 2 channels( 17 ). Substance P increases ARNA by activating substance P receptors in the renal pelvic area( 4, 22, 24, 28 ).
; q! R- S9 H" f! Q; c  v* O, i$ e8 t
The modulatory effects of endogenous ANG II on the activation of renalmechanosensory nerves are paralleled by the PGE 2 -mediated releaseof substance P from the renal pelvic nerves, being suppressed in rats fed alow-sodium diet and enhanced in rats fed a high-sodium diet( 18 ). Losartan enhanced thePGE 2 -mediated release of substance P from rats fed a low- but notfrom rats fed a normal- or high-sodium diet, suggesting that ANG II suppresses the release of substance P via activation of AT 1 receptors. Becausethese findings were derived from studies using an isolated renal pelvic wallpreparation, these data suggest the presence of ANG II in renal pelvic tissue.Although the presence of AT 1 receptor binding sites in the renalpelvic 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 IIbeing produced and modulated by dietary sodium in renal cortical and medullarytissue ( 10, 16, 36, 40 ). Therefore, we comparedthe ANG II levels in renal pelvic and cortical tissue from rats fed a low- andhigh-sodium diet.
1 G( I& F: X  v2 \5 C9 _9 X
AT 1 receptors are coupled to various guanine (G)nucleotide-binding proteins, resulting in activation of multiple signalingpathways ( 37, 42, 45, 47 ). In the central nervoussystem, there are numerous studies showing that ANG II exerts its effects via AT 1 receptors coupled to the G q protein, resulting inactivation of the phosphoinositide system and increases in intracellular Ca 2 ( 1, 6 ). However, our findingsshowing that ANG II suppressed the PGE 2 -mediated release ofsubstance P ( 18 ) would argue against an involvement of the G q protein-phosphoinositidase Cpathway. Rather, the important role for the cAMP-PKA pathway inPGE 2 -mediated release of substance P( 3, 14, 20, 44 ) suggests that ANG II mayexert its inhibitory effects on substance P release via activation ofAT 1 receptors coupled to the G i protein. Previousstudies in nonneural renal tissue( 33 ) provide evidence for ANGII decreasing cAMP activity in renal proximal tubular cells at concentrationssimilar to those found to decrease PGE 2 -mediated release ofsubstance P. To examine the involvement of the G i protein in theeffects of ANG II on substance P release, renal pelvic tissue was treated withpertussis toxin (PTX) to deactivate the G i protein( 13 ). Because these studiesshowed that PTX treatment abolished the inhibitory effects of ANG II on thePGE 2 -mediated release of substance P from renal sensory nerves, wepursued the notion that ANG II inhibits the PGE 2 -mediated activation of adenylyl cyclase. This hypothesis was tested by comparing theeffects of PGE 2 and the membrane-permeable cAMP analog8-(4-chlorophenylthio) (CPT)-cAMP on the activation of renal mechanosensorynerves. Increases in cAMP activity sensitize the responsiveness of sensorynerves to other stimuli, including bradykinin and capsaicin, without alteringresting membrane potential, afferent discharge, or substance P release ( 3, 5, 7, 9, 14, 34, 44 ). Therefore, we comparedthe sensitization produced by PGE 2 and CPT-cAMP on the increase insubstance P release and ARNA produced by bradykinin in rats fed a low- andnormal-sodium diet.

METHODS& p: _: J7 H9 h! w- I) i9 ~& G7 t4 V  z4 V
$ c, ]5 f  H, u4 r# ?
The experimental protocols were approved by the Institutional Animal Careand Use Committee and performed according to the "Guide for the Care andUse of Laboratory Animals" by the American Physiological Society.* Z; E$ ~8 W9 K4 @. F4 I
! P6 G* e1 f' K5 h; x6 }! C' t8 M
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 oneither sodium-deficient pellets (ICN, 1.6 meq/kg Na ) with tapwater 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% NaCldrinking fluid (high-sodium diet, n = 54)( 18 ).
- Y9 X- c/ `2 G' {
In Vitro Studies

Effects of low- and high-sodium diets on renal pelvic ANG II concentration. Anesthesia was induced with pentobarbital sodium (0.2mmol/kg ip, Abbott Laboratories). The renal pelvis and renal cortex weredissected from kidneys of rats fed low (n = 15)- and high (n = 15)-sodiumdiets and immediately placed on dry ice. The renal tissue was stored at-80°C for later analysis of ANG II concentration.+ F8 g2 F* f; M6 L! J
  S* u+ G. ]& ]+ f' `
Substance P Release from an Isolated Renal Pelvic WallPreparation
( U1 N0 u, q% E- k
The procedures for stimulating the release of substance P from an isolatedrat renal pelvic wall preparation have been previously described in detail( 17 - 20 ).In brief, after anesthesia, renal pelvises dissected from the kidneys wereplaced in wells containing 400 µl HEPES (25 mM HEPES, 135 mM NaCl, 3.5 mMKCl, 2.5 mM CaCl 2, 1 mM MgCl 2, 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.
: C, ^. o- s  q  O5 n* I
In all experiments, except those involving pretreatment, for 18 h with PTXor PTX vehicle, the renal pelvic walls were allowed to equilibrate for 130min. The incubation medium was gently aspirated every 10 min for the first 120min and every 5 min thereafter. The medium was immediately replaced with fresh HEPES to maintain P O 2 of the medium at 160-170 mmHg. Theexperimental 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°Cfor later analysis of substance P.$ L: s# x5 K: f% P. K: P( z- b
# A# }1 p- M# Y4 Y9 ~" c
Effects of ANG II on PGE 2 -mediated releaseof substance P. Indomethacin (0.14 mM) was present in the incubation bathto minimize the influence of endogenous PGE 2 on substance Prelease. One group of rats ( n = 13) fed a high-sodium diet wasstudied. Throughout the control, experimental, and recovery periods, theipsilateral pelvis was incubated in HEPES/indomethacin buffer containing 15 nMANG II and the contralateral pelvis in HEPES/indomethacin buffer containingANG II-vehicle (0.15 M NaCl). During the experimental period, both pelviseswere exposed to 0.14 µM PGE 2 dissolved in the incubationmedium.8 r; @3 x* d; x5 k( a0 f" a
4 K- i. V# Q4 p
Effects of ANG II on PGE 2 -mediated releaseof substance P from PTX-treated renal pelvises. Two groups fed ahigh-sodium diet were studied. In the first group ( n = 13), theipsilateral and contralateral pelvises were exposed for 18 h to circulating HEPES buffer containing 200 ng/ml PTX, 100 U/ml penicillin, and 100 µg/mlstreptomycin. In the second group ( n = 13), the ipsilateral andcontralateral pelvises were similarly treated, except PTX-vehicle (0.15 MNaCl) was present in the circulating medium instead of PTX. After the 18-htreatment with PTX or vehicle, the pelvises from both groups were rinsed threetimes with regular HEPES buffer. The pelvises from both groups were thenallowed to equilibrate in HEPES/indomethacin buffer for 70 min before the20-min control period was started. The ipsilateral pelvises from both groupswere incubated in HEPES/indomethacin buffer containing ANG II (15 nM), and the contralateral pelvis was incubated in HEPES/indomethacin buffer containing ANGII-vehicle according to the protocol described above. During the experimentalperiod, the ipsilateral and contralateral renal pelvises from both groups wereexposed to 0.28 µM PGE 2 dissolved in the incubation medium.7 S! o3 J6 c* w8 }# q
6 v* m7 D5 ]6 i/ n! w
Effects of PGE 2 vs. CPT-cAMP onbradykinin-mediated release of substance P. Five groups were studied. Inthe first two groups, we compared the effects of PGE 2 at 0.03 and0.14 µM on the bradykinin-mediated renal pelvic release of substance P inrats fed a low-sodium diet. In the third group fed a low-sodium diet, wecompared the effects of PGE 2 and CPT-cAMP on the bradykinin-mediated renal pelvic release of substance P from the same rat. Inthe fourth and fifth groups, we compared the effects of CPT-cAMP on thebradykinin-mediated renal pelvic release of substance P from rats fed low- andnormal-sodium diets. In all groups, the ipsilateral and contralateral pelvises from each group were incubated in regular HEPES buffer during the first 10 minof the control period. In the first two groups, the ipsilateral pelvis wasexposed to PGE 2 at 0.03 or 0.14 µM and the contralateral pelvisto PGE 2 -vehicle (0.15 M NaCl) during the last 10 min of the controlperiod and the 5-min experimental period. In the third group, the ipsilateral pelvis was exposed to 0.14 µM PGE 2 and the contralateral pelvisto 100 µM CPT-cAMP during the last 10 min of the control period and the5-min experimental period. In the fourth and fifth groups, the ipsilateralpelvis was exposed to 100 µM CPT-cAMP( 5, 9, 34 ) and the contralateralpelvis to CPT-cAMP-vehicle (0.15 M NaCl) during the last 10 min of the controlperiod and the 5-min experimental period. In all groups, 19 µM bradykininwas added to the incubation media of the ipsilateral and contralateralpelvises during the experimental period. During the recovery periods, allpelvises were exposed to HEPES buffer only.
  A1 z6 \- c# i
In Vivo Studies0 L4 |3 a% D* {& d8 h$ ]2 _
0 m2 q1 j9 ~: w: t8 P- ]
After induction of anesthesia, an intravenous infusion of pentobarbital sodium (0.04mmol·kg - 1 ·h - 1 ) at 50µl/min into the femoral vein was started and continued throughout the course of the experiment. Arterial pressure was recorded from a catheter inthe femoral artery. The procedures for stimulating and recording ARNA havebeen previously described in detail ( 18 - 30 ).In brief, the left kidney was approached by a flank incision, and a PE-60catheter was placed in the left ureter with its tip in the renal pelvis. Theleft renal pelvis was perfused, via a PE-10 catheter placed inside the PE-60catheter, throughout the experiment at 20 µl/min with vehicle or variousrenal perfusate administered as described in Experimental Protocol.In two groups of rats, renal pelvic pressure was increased by elevating thefluid-filled catheter above the level of the kidney. ARNA was recorded fromthe 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, theunit of measure being microvolts per second per 1 second. Postmortem renalnerve activity, which was assessed by crushing the decentralized renal nervebundle peripheral to the recording electrode, was subtracted from all values of renal nerve activity. ARNA was expressed as the percentage of its baselinevalue during the control period( 18 - 30 ).

Experimental Protocol

Approximately 1.5 h elapsed after the end of surgery and the start of theexperiment to allow the rat to stabilize as evidenced by 30 min ofsteady-state urine collections and ARNA recordings.6 T9 S" n* J/ a. _1 m

Effects of PGE 2 vs. CPT-cAMP on the ARNAresponses to bradykinin. Two groups were studied. One group ( n =10) was fed a normal-sodium diet and one group ( n = 13) a low-sodiumdiet. In each rat, we compared the effects of PGE 2, CPT-cAMP, andvehicle on the ARNA responses to bradykinin. The experiment was divided into four parts. The renal pelvis was perfused throughout the experiment, duringthe first and third parts with vehicle (0.15 M NaCl), and during the secondand fourth parts with PGE 2 or CPT-cAMP. The order ofPGE 2 and CPT-cAMP perfusions was randomized. Each part consisted ofa 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 fourexperimental periods. There was a 20-min interval between the second and thirdparts of the experiment during which the renal pelvic perfusate was switchedfrom PGE 2 or CPT-cAMP back to vehicle. PGE 2 wasadministered into the renal pelvis at 0.03 µM in rats fed a normal-sodiumdiet and at 0.14 µM in rats fed a low-sodium diet. CPT-cAMP wasadministered at 100 µM to both groups of rats.
8 Q+ g: V  U7 ^) w4 e1 l! S
Effects of PGE 2 vs. CPT-cAMP on the ARNAresponses 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 tothat in the previous two groups, except renal pelvic pressure was increased during each of the four experimental periods. Thus renal pelvic pressure wasincreased in the presence of renal pelvic perfusion with vehicle,PGE 2, and vehicle and CPT-cAMP. Renal pelvic pressure was increased3.2 ± 0 mmHg in rats fed a normal-NaCl diet and 5.5 ± 0.1 mmHgin rats fed a low-sodium diet. The magnitude of the increases in renal pelvicpressure is subthreshold for activation of renal sensory nerves in rats fednormal- and low-NaCl diets, respectively( 18, 30 ).

Drugs. Substance P antibody (IHC 7451) was acquired from Peninsula Laboratories (San Carlos, CA) and PGE 2 from Cayman Chemicals (AnnArbor, MI). All other agents were from Sigma (St. Louis, MO) unless otherwisestated. Indomethacin was dissolved together with Na 2 CO 3 (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* H& ~7 d* {; g+ l+ h* ~
  x/ `7 B0 M) ]  g5 H
Substance P in the incubation medium was measured by ELISA, as previouslydescribed in detail( 17 - 20, 22, 23 ). The rabbit substance Pantibody demonstrated 100% cross-reactivity with fragments 2 - 11,3 - 11, 4 - 11, and 5 - 11, 6 ~. T" E! k3 f9 k3 p, o6 G+ ^8 }
: [# s- `4 P" {) H3 c1 K% X3 W
Renal tissue ANG II concentration was measured by ELISA (Cayman Chemicals)using a slightly modified method from that described by the manufacturer. Inshort, renal tissue was homogenized in methanol and centrifuged for 10 min at3,000 g. The supernatants were lyophilized and reconstituted in assaybuffer and transferred to phenyl-bonded SPE columns (Varian Chromotography,Walnut Creek, CA). After being rinsed with water, hexane, and chloroform, ANGII 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. Themouse ANG II antibody demonstrated 100% cross-reactivity with ANG II, 4%cross-reactivity with ANG I, and 36, 33, and % w  \+ X3 v' X7 w, }  n) G+ e! v
$ ~+ P" [7 S9 J  }$ Q8 f
Statistical Analysis6 E5 c4 A1 K/ V9 a  `, L( w, I& E

In vitro, the release of substance P during the experimental period wascompared with that during the control and recovery periods using Friedmantwo-way analysis of variance and shortcut analysis of variance. The Wilcoxonmatched-pairs signed-rank test was used to compare the increase in substance Prelease 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 betweenthe value in the experimental period with the average value of the control andrecovery periods. In vivo, systemic hemodynamics were measured and averagedover each period. The ARNA responses to bradykinin and renal pelvic pressurewere calculated as the area under the curve of ARNA vs. time, where ARNA wasexpressed as the percentage of its baseline value during the bracketingcontrol and recovery periods. Friedman's two-way analysis of variance andshortcut analysis of variance were used to determine the effects of thevarious treatments on the ARNA responses within each rat. A significance levelof 5% was chosen. Data in text and figures are expressed as means ± SE( 43, 46 ).3 J* ^$ B+ T+ Q; J: C4 N, F
. q5 S% i( [: E! \5 A8 T
RESULTS  M+ n, E4 e+ f) k- c

In rats fed low-, normal-, and high-sodium diets for 2 wk, urinarysodium excretion averaged 245 ± 20; 2,870 ± 310; and 6,270± 410 µmol/24 h, respectively, in the conscious state.' {' F: k2 O. w7 U% w
6 z: e, r$ q( g& s! N! V' h
In Vitro Studies: ~2 x3 Z7 @( S6 f+ |! u

Effects of low- and high-sodium diets on renal pelvic ANG II concentration. The PGE 2 -mediated release of substance P from the isolated renal pelvis is suppressed in rats fed a low-sodium diet andenhanced in rats fed a high-sodium diet( 18 ). Acute administration oflosartan to the incubation medium enhances the PGE 2 -mediated renalpelvic release of substance P in rats fed a low-sodium diet but not in ratsfed a normal- or high-sodium diet. On the basis of these results, wehypothesized that ANG II is present in renal pelvic tissue and modulated bydietary sodium. We tested this idea by measuring ANG II concentrations inrenal pelvic and cortical tissue from rats fed low- and high-sodium diets. ANGII levels in renal pelvic tissue from rats fed a low-sodium diet weresignificantly higher than those in rats fed a high-sodium diet ( P 8 z3 K' _1 i/ \: \8 \; N
. q' }2 G6 c4 `5 p
Fig. 1. Angiotensin II concentrations in renal pelvic and cortical tissue from ratsfed a low (filled bars)- and high-sodium (open bars) diet. ** P
0 d' |1 z. D0 `: P, C( D$ H
Effects of ANG II on PGE 2 -mediated releaseof substance P. Renal pelvic administration of ANG II suppressed the ARNAresponses to increased renal pelvic pressure in rats fed a high-sodium diet invivo ( 18 ). Because thePGE 2 -mediated release of substance P contributes importantly to theARNA response( 23 - 25, 29 ), we tested the hypothesisthat acute administration of ANG II to the incubation medium suppresses thePGE 2 -mediated release of substance P from the renal pelvis in ratsfed a high-sodium diet. In the absence of ANG II in the incubation bath, 0.14 µM PGE 2 resulted in a significant reversible release of substance P from the isolated renal pelvic wall( Table 1 ). However, in thepresence of 15 nM ANG II, in the incubation bath, the PGE 2 -mediatedrelease of substance P was significantly suppressed ( P

Table 1. Effects of 0.14 µM PGE 2 on substance P release in thepresence of vehicle and 15 nM ANG II in the incubation bath containing anisolated renal pelvic wall preparation from rats fed a high-sodiumdiet
/ V5 M0 s% F. R% P+ k
Effects of ANG II on PGE 2 -mediated releaseof substance P from renal pelvises treated with PTX. PGE 2 increases the renal pelvic release of substance P and activates renalmechanosensory nerves via activation of the cAMP-PKA transduction pathway( 20 ). Our studies suggest thatANG II suppresses the responsiveness of renal sensory nerves by activatingAT 1 receptors in the renal pelvic wall( 18; Table 1 ). Because of theinhibitory effect of ANG II on substance P release, we tested the idea thatANG II reduced the PGE 2 -mediated release of substance P by amechanism involving activation of the G i protein. Renal pelvisesfrom high-sodium diet rats were treated with PTX for 18 h to deactivate theG i proteins ( 13 ).We reasoned that if ANG II activated renal pelvic AT 1 receptorscoupled to the G i protein, PTX pretreatment would blunt theinhibitory effect of ANG II on the PGE 2 -mediated release ofsubstance P.

Perfusing isolated renal pelvises for 18 h with 37°C HEPES buffercontaining penicillin and streptomycin did not alter baseline substance Prelease or reduce the sensitivity of the renal sensory nerves to respond toPGE 2 ( Table 1 and Fig. 2 A ). Similar tothe nonperfused pelvises ( Table1 ), ANG II blocked the PGE 2 -mediated release ofsubstance P in the vehicle-perfused pelvises( Fig. 2 A ). In theabsence of ANG II in the bath, the PGE 2 -mediated release ofsubstance P was similar in the PTX-perfused and vehicle-perfused renalpelvises ( Fig. 2 ). In thePTX-perfused pelvises, ANG II failed to inhibit the PGE 2 -mediatedrelease 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.2 B ).2 E/ Y7 T3 _+ N4 {8 N

Fig. 2. Effects of 18-h pretreatment with vehicle ( A ) and pertussis toxin( B; 200 ng) on the effects of 0.28 µM PGE 2 on substanceP release in the absence ( ) and presence of 15 nM ANG II ( ) from anisolated renal pelvic wall preparation from rats fed a high-sodium diet. ** P " I& f) P# s2 c2 G' m4 s
/ R/ X% B* i& O2 C( Y' s
Effects of PGE 2 vs. CPT-cAMP onbradykinin-mediated release of substance P. Our results suggest that ANGII suppresses the PGE 2 -mediated release of substance P byactivating AT 1 receptors coupled to the G i protein. Toexamine whether ANG II exerts its inhibitory effects via G i protein-mediated effects on adenylyl cyclase, we compared the effects ofPGE 2 and CPT-cAMP on substance P release in rats fed a low-sodiumdiet. Because increases in cAMP by forskolin or membrane-permeable analogs enhance the substance P release produced by bradykinin but have no effects onbaseline substance P release in normal-sodium-diet rats( 14, 20 ), we compared the effectsof PGE 2 and CPT-cAMP on the bradykinin-mediated release ofsubstance P. We reasoned that if ANG II suppresses thePGE 2 -mediated activation of the renal sensory nerves by inhibitingadenylyl cyclase, the bradykinin-mediated release of substance P would beenhanced by CPT-cAMP but not by PGE 2. Our previous studies in ratsfed a normal-sodium diet showed that basal substance P release is increased by0.14 µM PGE 2 ( 17, 18, 20 ) but not by 0.03 µMPGE 2 ( 17, 20 ). However, 0.03 µMPGE 2 produces a marked enhancement of the bradykinin-mediatedrelease of substance P in normal-sodium-diet rats( 20 ). Therefore, in ourinitial studies in rats fed a low-sodium diet, we compared the effects ofPGE 2 at 0.03 and 0.14 µM on the bradykinin-mediated release ofsubstance P. Bradykinin was administered at a concentration (19 µM) thatproduces no or minimal increases in basal substance P release( 20 ). Basal substance Prelease was not altered by PGE 2 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 releaseof substance P was unaffected by PGE 2 at 0.03 and 0.14 µM( Table 2 ). Having shown thatPGE 2 at 0.14 µM failed to enhance the bradykinin-mediatedrelease of substance P in rats fed a low-sodium diet, we used thisconcentration of PGE 2 to compare the effects of PGE 2 andCPT-cAMP on the bradykinin-mediated release of substance P from ipsilateral and contralateral pelvises of the same rat. Although PGE 2 failed toalter the bradykinin-mediated release of substance P from the ipsilateralpelvis, 100 µM CPT-cAMP ( 5, 9 ) produced a markedenhancement of the bradykinin-mediated release of substance P from thecontralateral 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 theenhancement of the bradykinin-mediated release of substance P was similar inrats fed low- and normal-sodium diets ( Fig.3 and Table 3 ).CPT-cAMP did not affect baseline substance P release in rats fed low- andnormal-sodium diets, from 4.5 ± 0.5 to 3.8 ± 0.5 pg/min and from4.6 ± 0.8 to 4.0 ± 0.5 pg/min, respectively.6 @2 U3 M" m6 W% [; ^; N

Table 2. Effects of PGE 2 and vehicle on the bradykinin-mediatedrelease of substance P from an isolated renal pelvic wall preparation fromrats fed a low-sodium diet' w, X. n$ G: H8 P; |. f
1 \# D% w% }- S6 ~7 \6 Y3 `
Fig. 3. Effects of 100 µM CPT-cAMP ( ) and 0.14 µM PGE 2 ( ) on the bradykinin-mediated release of substance P from isolatedipsilateral and contralateral pelvises from rats fed a low-sodium diet.Bradykinin was administered at 19 µM. ** P ( K6 \" V+ k8 @% Y6 {7 D5 p

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

In Vivo Studies: F2 x. b7 D$ {' Z  D+ e

Effects of PGE 2 vs. CPT-cAMP on the ARNAresponse to bradykinin. Our in vitro studies suggest that ANG IImodulates the PGE 2 -mediated release of substance P from renalpelvic nerves by a mechanism involving suppression of adenylyl cyclase. Toverify the importance of this mechanism of ANG II in vivo, we compared theeffects of PGE 2 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 anormal-sodium diet, 0.03 µM PGE 2 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, PGE 2 ata fivefold higher concentration, 0.14 µM, failed to enhance the ARNA response to bradykinin. On the other hand, CPT-cAMP produced an enhancement ofthe 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 PGE 2 and CPT-cAMP in normal-sodium-diet rats, from 1,730 ± 180 to 1,790 ± 190 andfrom 1,710 ± 140 to 1,690 ± 140µV·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 ± 140µV·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 inthe two groups of rats and remained unaltered throughout the experiment.

Fig. 4. Effects of renal pelvic perfusion with PGE 2 (open bars) andCPT-cAMP (filled bars) on the afferent renal nerve activity (ARNA) responsesin rats fed normal ( A )- and low-sodium ( B ) diets.PGE 2 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 andbradykinin at 3.8 µM to both groups. AUC, area under the curve of ARNA vs.time; * P 3 f% S& e# t9 c# M% v% ~

Effects of PGE 2 vs. CPT-cAMP on the ARNAresponse to increased renal pelvic pressure. Activation of bradykinin-2receptors 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 IIsuppresses the ARNA responses to increased renal pelvic pressure in rats fed alow-sodium diet ( 18 ) by amechanism involving inhibition of adenylyl cyclase. Renal pelvic pressure wasincreased 3.3 ± 0 and 5.5 ± 0.1 mmHg in rats fed normal- andlow-sodium diets, respectively. The magnitude of these renal pelvic pressure increases is the subthreshold for activation of renal mechanosensory nerves innormal- and low-sodium diet rats in control conditions ( 18, 30 )( Table 4 ). In rats fed anormal-sodium diet, renal pelvic perfusion with 0.03 µM PGE 2 and100 µM CPT-cAMP produced a similar enhancement of the ARNA responses toincreasing renal pelvic pressure ( Table4 ). In rats fed a low-sodium diet, a fivefold higher concentrationof PGE 2, 0.14 µM, produced an enhancement of the ARNA responseto increased renal pelvic pressure that was significantly suppressed( P 7 s4 @6 j# {5 o' f5 s9 A9 t

Table 4. Effects of renal pelvic perfusion with vehicle, PGE 2, andCPT-cAMP on the ARNA responses to increased renal pelvic pressure in rats fednormal- and low-sodium diets, C) X2 P" P( v9 ]

DISCUSSION

The results of our experiments show that ANG II is present in renal pelvictissue and modulated by dietary sodium. ANG II suppressedPGE 2 -mediated release of substance P from an isolated renal pelvicwall preparation. Treating renal pelvic tissue with PTX blocked the inhibitoryeffect of ANG II on PGE 2 -mediated release of substance P.PGE 2 failed to enhance bradykinin-mediated release of substance Pfrom 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 substanceP that was of similar magnitude in rats fed low- and normal-sodium diets. Ourin vivo studies showed that renal pelvic perfusion with PGE 2 enhanced the ARNA response to bradykinin in rats fed a normal-sodium diet butnot in rats fed a low-sodium diet. However, renal pelvic perfusion withCPT-cAMP produced a similar enhancement of the ARNA response to bradykinin inrats fed low- and normal-sodium diets. Similarly, the ARNA responses toincreases in renal pelvic pressure were similarly enhanced by CPT-cAMP innormal- and low- sodium diet rats. Taken together, these findings suggest thatANG II modulates the responsiveness of the renal mechanosensory nerves bysuppressing the PGE 2 -mediated activation of adenylyl cyclase via amechanism that involves the G i protein.

ANG II in Renal Pelvic Tissue) U+ a5 X6 d8 ~5 U: \9 P+ W

There is extensive evidence for ANG II synthesis in renal cortical andmedullary 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 studiesshowed that the responsiveness of the renal mechanosensory nerves is altered by endogenous ANG II modulating the release of substance P from renal pelvicsensory nerves. The PGE 2 -mediated release of substance P from anisolated renal pelvic wall preparation was impaired in rats fed a low-sodiumdiet and rats with congestive heart failure (CHF) fed a normal-sodium diet( 18, 19 ), conditions of high ANG IIlevels in renal tissue and plasma( 10, 15, 16, 36, 40 ). Conversely, thePGE 2 -mediated release of substance P was enhanced in rats fed ahigh-sodium diet ( 18 ) wherein renal tissue and plasma ANG II levels are suppressed( 10, 15, 16, 36 ). Also, the current studyshowed that baseline substance P release from the isolated renal pelvises waslower 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

Role of the G i Protein in the ANG II-Mediated Suppressionof PGE 2 -Evoked Release of Substance P

AT 1 receptors are widely distributed in the central andperipheral nervous system, including sensory neurons in dorsal root ganglia (DRG) and nodose ganglia ( 1 ).In the kidney, AT 1 receptors are located on vascular and tubularstructures ( 36, 52 ) and, most importantly, inthe renal pelvic wall ( 11, 12, 35, 52 ). Our current and previousstudies provide evidence for a role of renal pelvic AT 1 receptorsmediating the suppressive effects of locally generated ANG II on theresponsiveness of renal mechanosensory nerves. AT 1 receptors arecoupled to various G proteins, resulting in activation of multiple pathwaysvia different regions of the AT 1 receptor( 42 ). There are numerous reports of ANG II-activating AT 1 receptors coupled to theG q protein, resulting in increased intracellularCa 2 and facilitation of neurotransmitter release( 1, 6 ). However, our studies showing that ANG II in renal pelvic tissue suppressed the release of substanceP would argue against a role for the G q protein in the ANGII-mediated effects in the current studies. There is evidence for ANGII-activating AT 1 receptors coupled to the G i protein inneural and nonneural cells (e.g., 1, 2, 33 ). Similarly, renalmicroperfusion studies showed that ANG II exerts its physiological effects ontubular transport via a PTX-sensitive mechanism( 33 ). To test the notion thatANG II suppressed the activation of renal mechanosensory nerves via activationof AT 1 receptors coupled to the G i protein, isolatedrenal pelvises from rats fed a high-sodium diet were treated for 18 h with PTXto deactivate the G i protein( 13 ). Importantly, perfusingthe renal pelvic tissue for 18 h with HEPES buffer containing antibiotics toprevent growth in the incubation medium did not reduce the responsiveness ofthe renal sensory nerves to PGE 2. The increase in substance Prelease produced by 0.28 µM PGE 2 from renal pelvises perfused for 18 h was greater than that produced by 0.14 µM PGE 2 in thenonperfused pelvises (18 ± 2 vs. 12 ± 2 pg/min, P % Z1 _+ P  |/ I0 H6 b  \
6 m& m# B% B; J& I
Role of the cAMP-PKA Transduction Cascade in the Effects of ANG II onthe PGE 2 -Mediated Release of Substance P. k; M0 F- A- i, m

Our previous studies showed that the PGE 2 -mediated release of substance P from renal pelvic sensory nerves is markedly suppressed in ratsfed a low-sodium diet ( 18 ).Compared with rats fed a normal-sodium diet, a 25-fold higher concentration ofPGE 2 is required to produce a significant, albeit suppressed,increase in the release of substance P from renal sensory nerves in rats fed alow-sodium diet. Activation of AT 1 receptors coupled to theG i protein may decrease the release of substance P by decreasingcAMP activity ( 33 ) andCa 2 influx by an effect on the N-typeCa 2 channels( 2, 47 ). Because of the important role for the cAMP-PKA transduction pathway in the PGE 2 -mediated release of substance P from various sensory nerves( 3, 14, 44 ), including the renalsensory nerves ( 20 ), wehypothesized that the impaired PGE 2 -mediated renal pelvic releaseof substance P in rats fed a low-sodium diet was due to ANG II suppressing PGE 2 -mediated activation of adenylyl cyclase. To test this theory, we compared the effects of PGE 2 and the membrane-permeable cAMP analog CPT-cAMP on substance P release in rats fed a low-sodium diet. Wereasoned that if ANG II suppressed the PGE 2 -mediated release ofsubstance P by inhibiting adenylyl cyclase, direct activation of cAMP wouldlead 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 Prelease from the renal pelvises of either normal- or low-sodium diet rats,suggesting that increases in cAMP, per se, do not alter resting levels ofsubstance P release. These findings are in agreement with our previous studiesin normal rats in which we showed that forskolin did not alter baselinesubstance P release ( 20 ).Furthermore, our findings are in accord with studies in cultured dorsal rootganglion (DRG) neurons and isolated jejunum, which showed that similarconcentrations of cAMP analogs and forskolin as used in our present andprevious 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 nottested in our studies. Studies in cultured DRG showed that 10, 100, and 1,000µM of 8-bromo-cAMP, a cAMP analog equipotent to CPT-cAMP( 5 ), produced similar peakenhancement of the capsaicin-induced current( 34 ). However, the duration ofthe sensitization was inversely correlated to the concentration of the cAMPanalog. Similar findings were observed with 10 and 100 µM CPT-cAMP. Thesestudies may argue against the idea that a higher concentration of the cAMP analog would have resulted in an increase in baseline substance P release. Inaddition, there is extensive evidence for cAMP analogs increasing theexcitability of sensory neurons without causing membrane depolarization( 3, 5, 7, 9, 14, 34 ), i.e., sensitizing thesensory nerve responses to other stimuli. Similarly, our previous studies innormal rats showed that forskolin and PGE 2, at a subthresholdconcentration for substance P release, enhanced the substance P releaseproduced by bradykinin ( 20 ).Therefore, we compared the effects of PGE 2 and CPT-cAMP on thebradykinin-mediated release of substance P in rats fed a low-sodium diet. Ourresults showed that PGE 2 at 0.03 and 0.14 µM failed to enhancethe bradykinin-mediated release of substance P in rats fed a low-sodium diet.It is important to note that PGE 2 at 0.03 and 0.14 µM enhancesbradykinin-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. Theincrease in substance P release produced by CPT-cAMP bradykinin was of asimilar magnitude as that produced in renal pelvises from normal-sodium dietrats. In this context, it is interesting to note that the enhancement producedby the membrane-permeable cAMP analog db-cAMP of the bradykinin-mediated increases in afferent discharges from an isolated ileum preparation isindependent of prostaglandin synthesis( 3 ).

To explore the notion that the inhibitory effects of ANG II on theactivation of renal mechanosensory nerves in vivo were also related toinhibition of cAMP activity, we compared the effects of renal pelvic perfusionwith PGE 2 and CPT-cAMP on the ARNA responses to renal pelvicadministration of bradykinin in rats fed low- and normal-sodium diets. Whereasin rats fed a normal-sodium diet the ARNA responses to bradykinin were enhanced by both PGE 2 and CPT-cAMP, only CPT-cAMP enhanced the ARNAresponses to bradykinin in rats fed a low-sodium diet. PGE 2 had noeffect, despite the fact that PGE 2 was administered at a fivefoldhigher concentration than in rats fed a normal-sodium diet, 0.14 vs. 0.03µM. The CPT-cAMP-mediated enhancement of the ARNA responses was similar inthe two groups of rats. Subsequent studies in which we compared the effects ofrenal pelvic perfusion with PGE 2 and CPT-cAMP on the ARNA responses to increased renal pelvic pressure in rats fed low- and normal-sodium dietsshowed similar results. The ARNA responses to increased renal pelvic pressurewere enhanced to a similar extent by PGE 2 and CPT-cAMP innormal-sodium diet rats and CPT-cAMP in low-sodium diet rats. However, theeffects of PGE 2 on the ARNA response to increased renal pelvicpressure were markedly suppressed in low-sodium-diet rats. Because activationof bradykinin-2 receptors contributes importantly to the increase in ARNAproduced by increased renal pelvic pressure( 23 ), these data provide further support for our in vitro findings.7 c6 Q# c# u, ^, [) z% B/ R
# i" T4 q2 i1 C1 i- x+ b+ [% e
Taken together, our in vitro and in vivo studies suggest that theinhibitory effect of ANG II on PGE 2 -mediated release of substance Pinvolves ANG II suppressing the PGE 2 -mediated activation ofadenylyl cyclase. Whether the renal nerves are modulated by ANG II in therenal sensory nerves, per se, or in the uroepithelium and/or pelvic musclewall surrounding the renal sensory nerves cannot be deduced from our currentfindings. The numerous reports of ANG II-binding sites in the renal pelvicwall ( 11, 12, 35, 52 ) together with the knownlocalization of AT 1 receptors on central sensory nerves( 1, 6 ) may suggest the presence ofrenal sensory nerves containing AT 1 receptors. These receptors maybe activated by ANG II from the surrounding tissue. On the other hand, ANG IIhas shown to be present in neurons in the central nervous system associatedwith cardiovascular control ( 1 ). These findings togetherwith studies in DRG neurons showing that sensory nerves can be one source ofPGE 2 ( 49 ) suggest the intriguing hypothesis that the renal sensory nerves can autoregulate theirsensitivity by producing PGE 2 and ANG II, exerting opposing effectson neuropeptide release.
/ I* ^. s( V. M% S: c- l
The mechanisms involved in the sensitization of the renal sensory nervesproduced by increases in cAMP were not addressed in the current studies.Previous studies in cultured DRG neurons examining the effects ofPGE 2, forskolin, and cAMP analogs on sensitization of sensoryneurons suggested that activation of the cAMP-PKA transduction cascadeincreases the membrane excitability of these neurons by inhibiting the delayedrectifier-like potassium current( 9 ) and/or increasing thetetrodotoxin-resistant sodium current( 7 ). The increased membraneexcitability produced by activation of the cAMP-PKA pathway sensitized thesensory 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 intracellularCa 2 (Ca i 2 ) is notclear. Whereas the findings by Smith et al.( 44 ) would suggest thatdb-cAMP enhances the bradykinin-mediated release of substance P in associationwith increases in Ca i 2  , the study by Evanset al. ( 8 ) showed no effect ofCa 2 channel blockers of the N, L, or T type on the PGE 2 -mediated facilitation of substance P release.9 O3 ~6 K5 A3 `  e; ]5 }

There is evidence for ANG II and bradykinin decreasing phosphodiesterase activity in nonneural cells by mechanisms involving protein kinase C( 31, 38 ). Whether this mechanismcontributed to the increase in substance P release and activation of renal sensory nerves in response to bradykinin CPT-cAMP in the rats fed alow-sodium diet was not addressed in the current studies. However, studies inDRG neurons showing that forskolin does not increase baseline substance Prelease despite a 10-fold increase in cAMP( 14 ) may argue against thismechanism playing an important role in the sensitization of the renalmechanosensory nerves.
+ H( z$ A$ f0 i) A
The present studies were not designed to examine the nature of theCa 2 channels involved in the ANG II-mediated inhibition of the PGE 2 -mediated substance P release from renal sensory nerves.However, our previous studies showing that -conotoxin blocked thePGE 2 -mediated release of substance P( 17 ) may suggest aninvolvement of N-type Ca 2 channels in the ANG II-mediated inhibition of substance P release via the PGE 2 -mediated activation of the cAMP-PKA transduction pathway. This hypothesis is supportedby studies in the nodose ganglia by Bacal and Kunze( 2 ). ANG II at 10 nM, i.e., aconcentration in the range of that used in the current studies, decreasesCa 2 current in isolated nodose ganglia via activationof AT 1 receptors coupled to PTX-sensitive G proteins and involving -conotoxin-sensitive Ca 2 channels( 2 ). However, there are reportsin sympathetic neurons that may argue against a role for PTX-sensitiveCa 2 channels playing a major role in the inhibition ofCa 2 current produced by ANG II( 41 ). The apparent differencesbetween these studies may be explained by the concentrations of ANG II appliedin the two studies, being 10( 2 ) and 500 nM( 41 ). A study in chromaffincells ( 47 ) showed that ANG IIat low concentrations (9 C) S0 w; Y! ?
$ H7 w& b7 T: }7 s' d
In summary, the present study shows that ANG II is present in renal pelvicwall tissue and modulated by dietary sodium. Studies in an isolated renalpelvic wall preparation showed that ANG II suppressed the release of substanceP from renal pelvic sensory nerves in rats fed a high-sodium diet by aPTX-sensitive mechanism. In rats fed a low-sodium diet, CPT-cAMP but not PGE 2 enhanced the bradykinin-mediated release of substance P. Themagnitude of the CPT-cAMP-induced enhancement of bradykinin-mediated releaseof substance P was similar in rats fed low- and normal-sodium diets.Furthermore, the results from the in vitro studies were supported by the invivo findings that showed that renal pelvic perfusion with PGE 2 enhanced the ARNA response to bradykinin in rats fed normal- but notlow-sodium diets. On the other hand, renal pelvic perfusion with CPT-cAMPproduced a similar enhancement of the bradykinin-mediated increase in ARNA in rats fed normal- and low-sodium diets. Similarly, the ARNA responses toincreased renal pelvic pressure were enhanced to a similar extent by CPT-cAMPin the two groups of rats. The PGE 2 -mediated enhancement of theARNA response to increased renal pelvic pressure in normal-sodium-diet ratswas markedly suppressed in rats fed a low-sodium diet. Taken together, thesestudies along with our previous studies( 18 ) suggest that ANG IImodulates the PGE 2 -mediated release of substance P from renalsensory nerves by blocking the PGE 2 -evoked stimulation of adenylyl cyclase via activation of AT 1 receptors coupled to theG i protein.2 p" Z# N  a) Y4 O7 b& K
' b8 M; _. b& @, f% K4 t: @4 T4 F
DISCLOSURES( X% q, V, {! Y6 r! _8 L

This work was supported by grants from the Department of Veterans Affairs,National Heart, Lung, and Blood Institute, R-O1 HL-66068, and SpecializedCenter of Research, HL-55006, and American Heart Association Grant-In-Aid0150024N.

ACKNOWLEDGMENTS

We are indebted to Dr. L. G. Navar and D. M. Seth, Department ofPhysiology, Tulane University School of Medicine, New Orleans, LA, forvaluable advice on the angiotensin assay.
      【参考文献】* l6 o; r  N, }/ ]7 W! F& R8 Y
Allen AM,Moeller I, Jenkins TA, Zhuo J, Aldred GP, Chai SY, and Mendelsohn FAO. Angiotensin receptors in the nervous system. Brain ResBull 47: 17-28,1998.

1 o4 m( l8 c9 r. ^5 `
3 x. e+ U7 N8 {/ N8 k6 c
Bacal K andKunze DL. Dual effects of angiotensin II on calcium currents in neonatalrat nodose neurons. J Neurosci 14: 7159-7167,1994.
% v6 |' s' Z" F. E5 d6 o

Brunsden AM andGrundy D. Sensitization of visceral afferent to bradykinin in rat jejunumin vitro. J Physiol 521:517-527, 1999.' J' s6 n  T' y8 i$ \9 I

# f5 G+ P  I9 i& s; K
Chen Y andHoover DB. Autoradiographic localization of NK 1 andNK 3 tachykinin receptors in rat kidney. Peptides 16:673-681, 1995./ h, }9 m2 \5 L3 D  H( b
# a, l# ~. J6 ]$ U

Cui M and NicolGD. Cyclic AMP mediates the prostaglandin E 2 -inducedpotentiation of bradykinin excitation in rat sensory neurons. Neuroscience 66:459-466, 1995.' H/ T: q- w7 n/ w  J
* Z; [9 I/ B0 d

2 K, ^% e$ N6 I( ^) S
De Gasparo M,Catt KJ, Inagami T, Wright JW, and Unger TH. International Union ofPharmacology. XIII. The angiotensin II receptors. PharmacolRev 52: 415-472,2000.' n( x1 B$ j6 x3 o

England S,Bevan S, and Docherty. PGE 2 modulates thetetrodotoxin-resistant sodium current in neonatal rat dorsal ganglion neuronsvia the cyclic AMP-protein kinase A cascade. J Physiol 495: 429-440,1996.

4 x3 A5 u1 U( |$ ?1 I9 G
Evans AR, NicolGD, and Vasko MR. Differential regulation of evoked peptide release byvoltage-sensitive calcium channels in rat sensory neurons. BrainRes 712: 265-273,1996.
2 J7 w# V( v6 {( M9 t6 M
% L8 w$ @4 c1 ?3 T! E+ n2 N
% z3 _/ t* n' v8 g
Evans AR, VaskoMR, and Nicol GD. The cAMP transduction cascade mediates thePGE 2 -induced inhibition of potassium currents in rat sensoryneurones. J Physiol 516:163-178, 1999.. v: H/ K$ H! \' r  ^

) w( ?; C8 m3 }4 k+ {4 e3 X! e
/ p3 i& I- _# o  v8 v) q
Fox J, Guan S,Hymel AA, and Navar LG. Dietary Na and ACE inhibition effects on renaltissue angiotensin I and II and ACE activity in rats. Am J PhysiolRenal Fluid Electrolyte Physiol 262:F902-F909, 1992.5 w; A. C; u1 R5 `
" F; U& l. z+ p7 e; @5 }

  S- y  C7 Y# e- I  y6 |. e1 k1 c" N
Gasc JM,Shanmugam S, Sibony M, and Corvol P. Tissue-specific expression of type 1angiotensin II receptor subtypes. An in situ hybridization study. Hypertension 24:531-537, 1994.
; |! Q- Z) E5 R- o# Z* y9 G, n
/ `( O3 N0 s3 E8 E$ \2 L! o

Healy DP, YeMQ, and Troyanovskaya M. Localization of angiotensin II type 1 receptorsubtype mRNA in rat kidney. Am J Physiol Renal Fluid ElectrolytePhysiol 268:F220-F226, 1995.$ p  }6 u) \+ \% ~0 n- O" r

Hildebrandt JD,Sekura RD, Codina J, Iyengar R, Manclark CR, and Birnbaumer L. Stimulationand inhibition of adenylyl cyclases mediated by distinct regulatory proteins. Nature 302:706-709, 1983.
: U; l& ^/ N: `5 T/ n# w2 J

0 {. L- \2 j0 p, j9 r
Hintgen CM,Waite KJ, and Vasko MR. Prostaglandins facilitate peptide release from ratsensory neurons by activating the adenosine 3',5'-cyclicmonophosphate transduction cascade. J Neurosci 15: 5411-5419,1995.* I: G8 |' Z* s# d6 p1 {6 a

Imig JD, NavarGL, Zou LX, O'Reilly KC, Allen PL, Kaysen JH, Hammond TG, and Navar LG. Renal endosomes contain angiotensin peptides, converting enzyme, andAT 1A receptors. Am J Physiol Renal Physiol 277: F303-F311,1999.

Ingert C, GrimaM, Coquard C, Barthelmebs M, and Imbs JL. Effects of dietary salt changeson renal renin-angiotensin system in rats. Am J Physiol RenalPhysiol 283:F995-F1002, 2002.

4 x" M6 {: b8 ~' K+ Z( G
, J+ ]" \) J: w3 L
Kopp UC andCicha MZ. PGE 2 increases substance P release from renal pelvicsensory nerves via activation of N-type calcium channels. Am JPhysiol Renal Physiol 276:F1241-F1248, 1999.
" d; k/ I4 W! W! M
1 Z- ?/ U( w" ~# V$ S; V: z
% Z8 z8 Z5 {/ B7 s" A- d$ I. o
Kopp UC, CichaMZ, and Smith LA. Endogenous angiotensin modulatesPGE 2 -mediated release of substance P from renal mechanosensorynerve fibers. Am J Physiol Regul Integr Comp Physiol 282: R19-R30,2002.

Kopp UC, CichaMZ, and Smith LA. Impaired responsiveness of renal mechanosensory nervesin heart failure: role of endogenous angiotensin. Am J PhysiolRegul Integr Comp Physiol 284:R116-R124, 2003.  e5 |' ]3 B' z

- t4 J4 ^1 @% C  G( i6 ^
/ _3 x# E' l0 q% l/ j
Kopp UC, CichaMZ, and Smith LA. PGE 2 increases release of substance P fromrenal sensory nerves by activating the cAMP-PKA transduction cascade. Am J Physiol Regul Integr Comp Physiol 282: R1618-R1627,2002.4 \  B8 }) U9 k& |6 f

, {/ _) g0 ^" }% X6 Q+ A

Kopp UC, CichaMZ, Smith LA, Haeggström JZ, Samuelsson B, and Hökfelt T. Cyclooxygenase-2 involved in stimulation of renal mechanosensitive neurons. Hypertension 35:373-378, 2000.# ^: x& N8 V; [; U4 o
" q. z9 q! h4 K2 s: ^/ G3 V( N0 Y

Kopp UC, CichaMZ, Smith LA, and Hökfelt T. Nitric oxide modulates the renal sensorynerve fibers by mechanisms related to substance P receptor activation. Am J Physiol Regul Integr Comp Physiol 281: R279-R290,2001.: f; d' O# q. O# G
5 t; N# j% R9 y( @

Kopp UC, FarleyDM, Cicha MZ, and Smith LA. Activation of renal mechanosensitive neuronsinvolves bradykinin, protein kinase C, PGE 2, and substance P. Am J Physiol Regul Integr Comp Physiol 278: R937-R946,2000.5 \* I" a( b9 ]" B9 t  l

0 @( E% B! K2 n* c5 k6 \

Kopp UC, FarleyDM, and Smith LA. Bradykinin-mediated activation of renal sensory neuronsdue to prostaglandin-dependent release of substance P. Am J PhysiolRegul Integr Comp Physiol 272:R2009-R2016, 1997.
# ~5 s7 J+ s* b. Y, _
$ s1 ?* n8 S1 W: \9 h* b' {' o' @3 M
6 v( W( w# r" j  z# @
Kopp UC, FarleyDM, and Smith LA. Renal sensory receptor activation causesprostaglandin-dependent release of substance P. Am J Physiol RegulIntegr Comp Physiol 270:R720-R727, 1996.3 |) Y1 I# ]# \) B3 E
9 F3 ~# |+ f7 W1 R/ N/ \

2 C- v0 {0 N/ B  j' J- N
Kopp UC, OlsonLA, and DiBona GF. Renorenal reflex responses to mechano- andchemoreceptor stimulation in the dog and rat. Am J Physiol RenalFluid Electrolyte Physiol 246:F67-F77, 1984.
( i1 M; J" Y5 a. E, z# l: q

Kopp UC andSmith LA. A role for protein kinase C in bradykinin-mediated activation ofrenal pelvic sensory receptors. Am J Physiol Regul Integr CompPhysiol 269:R331-R338, 1995.0 H$ _( s6 d+ F

9 Y  A( a# j' x& K

Kopp UC andSmith LA. Effects of the substance P receptor antagonist CP-96345 on renalsensory receptor activation. Am J Physiol Regul Integr CompPhysiol 264:R647-R653, 1993.

, Y6 {, U0 Y8 B) |
/ C1 y) k5 p9 W* Q% b/ E' P% K8 ?
Kopp UC andSmith LA. Role of prostaglandins in renal sensory receptor activation bysubstance P and bradykinin. Am J Physiol Regul Integr CompPhysiol 265:R544-R551, 1993.  Z4 R3 K- _8 Y3 a: O, d  R" g0 ?3 j: y

' y+ k# V  _) `/ S
" d- w' I# f, T2 y
Kopp UC, SmithLA, and Pence A. Na -K -ATPase inhibition sensitizesrenal mechanoreceptors activated by physiological increases in renal pelvicpressure. Am J Physiol Regul Integr Comp Physiol 267: R1109-R1117,1994.

, A& l! p2 H$ n: z8 I9 S! W

Liu H, PalmerD, Jimmo SL, Tilley DG, Dunkerley HA, Pang SC, and Maurice DH. Expressionof phosphodiesterase 4D (PDE4D) is regulated by both the cyclic AMP-dependentprotein kinase and mitogen-activated protein kinase signaling pathways. J Biol Chem 275:26615-26624, 2000.

4 H, e$ U. r4 B# _5 r; o* [6 m& l
8 |- ~7 }8 Z8 V2 \/ f, b2 j
Liu L andBarajas L. The rat renal nerves during development. AnatEmbryol (Berl) 188:345-361, 1993.% p8 ]. L$ i7 b; E1 J( e
- I  m' f) Z2 W

Liu PY andCogan MG. Angiotensin II stimulates early proximal bicarbonate absorptionin the rat by decreasing cyclic adenosine monophosphate. J ClinInvest 84: 83-91,1989.
1 X: o* q* u2 i

( i" S! _# y2 l5 Y+ h+ Y! R) Z# y
Lopshire JC andNicol GD. The cAMP transduction cascade mediates the prostaglandinE 2 enhancement of the capsaicin-elicited current in rat sensoryneurons: whole-cell and single-cell studies. JNeurosci 18:6081-6092, 1998.
7 _: b) H9 q. ^" O4 R6 e. P
5 C2 M% }, w  Y- s; ?; k6 i) E

Miyazaki Y,Tsuchida S, Nishimura H, Pope JC, Harris RC, McKanna JM, Inagami T, Hogan BLM,Fogo A, and Ichikawa I. Angiotensin induces the urinary peristalticmachinery during the perinatal period. J Clin Invest 102: 1489-1497,1998.2 b/ C- @, K7 z% I* k& ?$ E1 x
7 g8 e# Q3 [9 J% |( O

Navar LG, ImigJD, and Wang CT. Intrarenal production of angiotensin II. SeminNephrol 17:412-422, 1997.
) N' n8 U' Y6 Q$ {. g

Ohnishi J,Ishido M, Shibata T, Inagami T, Murakami K, and Miyazaki H. The ratangiotensin II AT 1A receptor couples with three different signaltransduction pathways. Biochem Biophys Res Commun 186: 1094-1101,1992.' H! x% h% a  n/ n6 \4 l

/ d! I! h5 J3 D2 E3 K( U8 {- U1 ^$ t
Pyne NJ, TolanD, and Pyne S. Bradykinin stimulates cAMP synthesis via mitogen-activatedprotein kinase-dependent regulation of cytosolic phospholipase A 2 and prostaglandin E 2 release in airway smooth muscle. Biochem J 328:689-694, 1997.
: ^4 z" O% v. i* G
" v3 g' t8 r6 Q! D) t) T
- E3 O' u! y, A+ B  j* O% p
Rich TC, FaganKA, Tse TE, Schaack J, Cooper DMF, and Karpen JW. A uniform extracellularstimulus triggers distinct cAMP signals in different compartments of a simplecell. Proc Natl Acad Sci USA 98: 13049-13054,2001.
6 `1 S( v2 B! r' u- n" K& Z

- d9 \+ T8 N3 F$ _  f
Schunkert H,Tang SS, Litwin SE, Diamant D, Riegger G, Dzau VJ, and Ingelfinger JR. Regulation of intrarenal and circulating renin-angiotensin systems in severeheart failure in the rat. Cardiovasc Res 27: 731-735,1993.

Shapiro MS,Wollmuth LP, and Hille B. Angiotensin II inhibits calcium and M currentchannels in rat sympathetic neurons via G proteins. Neuron 12:1319-1329, 1994.

' Z, U) a& ]8 n# M: r/ q
Shibata T,Suzuki C, Ohnishi J, Murakami K, and Miyazaki H. Identification of regionsin the human angiotensin II receptor type I responsible for G i andG q coupling by mutagenesis. Biochem Biophys ResCommun 218:383-389, 1996.: `0 M* A! a& i/ w

Siegel S andCastellan N Jr. Nonparametric Statistics for the BehavioralSciences (2nd ed.). New York: McGraw-Hill, 1988, p.128-137, 174-183.
1 C% u& m5 n0 g

Smith JA, DavisCL, and Burgess GM. Prostaglandin E 2 -induced sensitization ofbradykinin-evoked responses in rat dorsal root ganglion neurons is mediated bycAMP-dependent protein kinase A. Eur J Neurosci 12: 3250-3258,2000.# O3 w' o, Q3 j+ K7 V
! I& a6 f$ d9 o" h6 B1 H0 `% _4 U7 F- Z

Sumners C,Fleegal MA, and Zhu M. Angiotensin AT 1 receptor signallingpathways in neurons. Clin Exp Pharmacol Physiol 29: 483-490,2002.1 _0 e5 N5 l* L( }' v) O- I
: |1 S) E! _! M2 P
, k# c$ d6 `/ h; A1 a: t; [
8 G/ s5 x( P- w$ w- l# U. _
Tate MW andClelland RC. Nonparametric and Shortcut Statistics in theSocial, Biological Sciences. Danville, IL: Interstate,1957, p. 111-113, 120-121.* O4 q. k! P6 o8 `/ L

7 ]3 n8 D# L+ _! x8 D2 r
. U4 e7 l. Y# z# T
Teschemacher AG and Seward EP. Bidirectional modulation of exocytosis by angiotensin IIinvolves multiple G protein-regulated transduction pathways in chromaffincells. J Neurosci 20:4776-4785, 2000.7 @' d* u# l0 B5 n  y5 |+ `

9 \5 K  I7 s7 l& Z
Van Kats JP,Schalekamp MADH, Verdouw PD, Duncker DJ, and Danser AHJ. Intrarenalangiotensin II: interstitial and cellular levels and site of production. Kidney Int 60:2311-2317, 2001., ~0 c, D+ L/ k& ^; o$ u9 }3 `
# y, t) G! ~) ^/ F) r

/ t. K7 t0 W3 N' g: D! c  M
Vasko MR,Campbell WB, and Waite KJ. Prostaglandin E 2 enhancesbradykinin-stimulated release of neuropeptides from rat sensory neurons inculture. J Neurosci 14:4987-4997, 1994.4 s# @, M/ Q% o/ M

Vos PF, Boer P,Braam B, and Koomans HA. The origin of urinary angiotensin in humans. J Am Soc Nephrol 5:215-223, 1994.  n) K% c& j0 m
  ~& f2 p/ w% M2 W' C

" }* Y, s/ _; |/ `$ J
Zheng F andLawson SN. Neurokinin A in rat renal afferent neurons and in nerve fiberswithin smooth muscle and epithelium of rat and guinea-pig renal pelvis. Neuroscience 76:1245-1255, 1997.: ?) [; @- ?7 ^  A' s. C
$ Q  k# g' }8 g) y

Zhuo JL. Renomedullary interstitial cells: a target for endocrine and paracrine actionsof vasoactive peptides in the renal medulla. Clin Exp PharmacolPhysiol 27:465-473, 2000.

收藏0 分享0 顶0 踩0