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Am J Physiol Regul Integr Comp Physiol 276: R1241-R1248, 1999;
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Vol. 276, Issue 5, R1241-R1248, May 1999

PGE2 increases substance P release from renal pelvic sensory nerves via activation of N-type calcium channels

Ulla C. Kopp and Michael Z. Cicha

Department of Internal Medicine, Department of Veterans Affairs Medical Center and University of Iowa College of Medicine, Iowa City, Iowa 52242


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of renal pelvic sensory nerves by increased pelvic pressure results in a renal pelvic release of substance P that is dependent on intact prostaglandin synthesis. An isolated renal pelvic wall preparation was used to examine whether PGE2 increases the release of substance P from renal pelvic sensory nerves and by what mechanisms. The validity of the model was tested by examining whether 50 mM KCl increased substance P release from the pelvic wall. Fifty millimolar KCl produced an increase in substance P release, from 9.6 ± 1.6 to 26.8 ± 4.0 pg/min, P < 0.01, that was blocked by the L-type calcium blocker verapamil (10 µM). PGE2 (0.14 µM) increased the release of substance P from the pelvic wall from 8.9 ± 0.9 to 20.6 ± 3.3 pg/min, P < 0.01. PGE2 failed to increase substance P release in a calcium-free medium. The PGE2-induced substance P release was blocked by the N-type calcium blocker omega -conotoxin (0.1 µM) but was unaffected by verapamil. In conclusion, PGE2 increases the release of substance P from renal pelvic sensory nerves by a calcium-dependent mechanism that requires influx of calcium via N-type calcium channels.

omega -conotoxin; verapamil; potassium chloride-mediated neuropeptide release; prostaglandin E2


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN THE KIDNEY, THE MAJORITY of the afferent renal sensory nerves are located in the renal pelvic wall (18, 32). Our previous studies have demonstrated that activation of these sensory nerves by increasing renal pelvic pressure results in an increase in afferent renal nerve activity (ARNA), with the activation threshold being <5 mmHg (17), a pressure well below sensation of pain (33). Renal pelvic afferent nerves may play a role in the renal control of body water and sodium because activation of these afferent nerves results in a reflex-mediated increase in contralateral urinary sodium excretion (10-17).

Prostaglandins are importantly involved in the activation of renal sensory nerves by increased renal pelvic pressure and bradykinin (10-12, 14). Activation of renal sensory neurons by these stimuli increases the release of PGE2 into the renal pelvic effluent (10, 11). The ARNA response to renal sensory receptor activation is impaired in rats fed an essential fatty acid-deficient diet (EFAD) to produce depletion of endogenous arachidonic acid (12) or by renal pelvic perfusion with the prostaglandin synthesis inhibitor indomethacin (14). Administration of PGE2 to the renal pelvis restored the ARNA response to increased renal pelvic pressure in EFAD rats and in rats with indomethacin-treated kidneys (12, 14).

Substance P is another important mediator of the ARNA response to increased renal pelvic pressure and bradykinin (10, 11, 15, 16). Activation of renal sensory neurons by these stimuli increases the release of substance P into the renal pelvic effluent (10, 11). Administration of substance P receptor antagonists into the renal pelvis blocks the ARNA response to increased renal pelvic pressure and bradykinin (11, 16). So, what is the link between PGE2 and substance P in the activation of renal pelvic sensory neurons? Our studies showing that renal pelvic administration of indomethacin blocked the increase in the renal pelvic release of substance P and ARNA (10, 11) would suggest that PGE2 increases the release of substance P, which in turn activates renal sensory neurons. The present study was performed to examine the interaction between PGE2 and substance P in the activation of renal sensory neurons in greater detail.

There is evidence from studies in cultured sensory neurons and spinal cord sections for PGE2 having an effect on the release of substance P (21, 27, 30). PGE2 increases the release of substance P in conjunction with an increase in calcium current (21). The increase in intracellular calcium concentration elicited by PGE2 is dependent on extracellular calcium (4). It is well known that influx of calcium into neurons via high-voltage-activated channels promotes the release of neurotransmitters (e.g., Refs. 1, 22, 24, 28). Multiple types of high-voltage-activated calcium channels have been described in sensory neurons, including the L- and N-type calcium channels (1, 22, 24, 28). The relative role of L- and N-type calcium channels controlling transmitter release is determined by the stimuli evoking membrane depolarization.

The present study was undertaken to examine whether PGE2 increased the release of substance P from the renal pelvic wall. In view of the difficulties in precisely regulating the chemical composition of the renal pelvic wall environment in vivo, the current experiments were performed using an isolated renal pelvic wall preparation. The validity of this preparation was initially tested by examining whether a high concentration of KCl depolarized the renal sensory nerves with a subsequent release of substance P. Because a high KCl concentration evoked a reversible release of substance P from the renal pelvic wall, we then examined whether PGE2 elicited a release of substance P and whether this release was inhibited in a calcium-free medium and by blockers of the L- and N-type calcium channels.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolated Renal Pelvic Wall Preparation

Male Sprague-Dawley rats weighing 225-350 g were anesthetized with pentobarbital sodium (50 mg/kg iv). Both kidneys were removed and placed in ice-cold phosphate-buffered NaCl. The renal pelvic wall was dissected from the kidneys and placed in wells containing 400 µl HEPES buffer (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, and 50 µM p-chloromercuriphenylsulfonic acid, pH 7.4) maintained at 37°C. Each well contained the pelvic wall from one kidney. Verapamil, omega -conotoxin, indomethacin, KCl, and PGE2 were added to the HEPES buffer as outlined in the experimental protocols.

The renal pelvic walls were allowed to equilibrate for 140 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 buffer to maintain PO2 of the medium at 160-170 mmHg throughout the equilibration and experimental periods. In all experimental groups, the protocol consisted of four 5-min control, one 5-min experimental, 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.

Experimental Protocols

Effects of KCl on substance P release in the absence and presence of verapamil. One pelvis from each rat (n = 8) was incubated in regular HEPES buffer throughout the experiment. The contralateral pelvis was incubated for 60 min in HEPES buffer and thereafter in HEPES buffer containing 10 µM verapamil, a known blocker of L-type calcium channels (1, 24, 28). Thus the pelvises were treated with verapamil for 70 min before the control periods were started. During the experimental period, both pelvises were exposed to HEPES buffer containing 50 mM KCl (substituted for equimolar sodium). One pelvis from six additional rats was incubated in regular HEPES throughout the experiment and served as time control. Thus these pelvises were not exposed to verapamil or KCl.

Effects of PGE2 on substance P release in the absence and presence of calcium. One pelvis from each rat (n = 10) was incubated in regular HEPES buffer. The contralateral pelvis was incubated in calcium-free HEPES buffer containing 3 mM EGTA. During the experimental period, both pelvises were exposed to 2.8 µM PGE2, dissolved in the incubation medium.

Effects of PGE2 on substance P release in the absence and presence of verapamil. Our previous studies have shown that indomethacin lowers the increased release of PGE2 into the incubation bath produced by mechanical stimulation of the isolated renal pelvic wall, suggesting endogenous PGE2 synthesis in the renal pelvic wall (10). To examine whether endogenous PGE2 synthesis interfered with the effects of exogenously administered PGE2, pilot experiments were performed in which the effects of PGE2 at 0.028, 0.14, and 2.8 µM were examined on substance P release in the presence of 0.14 µM indomethacin. Whereas PGE2 at 0.028 µM failed to increase substance P release, from 6 ± 2 to 4 ± 1 pg/min (NS, n = 8), substance P release was increased in a concentration-dependent fashion by PGE2 at 0.14 and 2.8 µM, from 8 ± 1 to 13 ± 3 pg/min by 0.14 µM PGE2 (P < 0.01, n = 12) and from 7 ± 2 to 19 ± 4 pg/min by 2.8 µM PGE2 (P < 0.01, n = 8). Thus, in the presence of indomethacin, PGE2 at submicromolar concentration increased substance P release. In subsequent experiments all pelvises were incubated in a HEPES buffer containing 0.14 µM indomethacin throughout the experiment, and PGE2 was administered at 0.14 µM.

One pelvis from each rat (n = 10) was incubated in regular HEPES-indomethacin buffer. The contralateral pelvis was incubated in HEPES-indomethacin buffer containing 10 µM verapamil, added to the incubation buffer 70 min before the start of the control periods, as outlined above. During the experimental period, both pelvises were exposed to 0.14 µM PGE2, dissolved in the incubation medium.

Effects of PGE2 on substance P release in the absence and presence of omega -conotoxin. All pelvises were incubated in a HEPES buffer containing indomethacin, as outlined above. One pelvis from each rat (n = 11) was incubated in a regular HEPES-indomethacin buffer. The contralateral pelvis was incubated for 60 min in HEPES-indomethacin buffer and thereafter in HEPES-indomethacin buffer containing 0.1 µM omega -conotoxin, a known blocker of the N-type calcium channels (1, 22, 24, 28). During the experimental period, both pelvises were exposed to 0.14 µM PGE2 dissolved in the incubation medium.

Drugs and Chemicals

Indomethacin was dissolved together with Na2CO3 (2:1 weight ratio) in HEPES buffer. PGE2, verapamil, and omega -conotoxin were dissolved in HEPES buffer. PGE2 was acquired from Cayman Chemicals (Ann Arbor, MI). Indomethacin, verapamil, omega -conotoxin, and all chemicals used in the ELISA assay were acquired from Sigma Chemical (St. Louis, MO), unless otherwise stated.

Analytic Procedure

ELISA for substance P. The assay for substance P was a modification of the ELISA assay for steroids as described by Munro and Lasley (20) and Kasson et al. (7). The rabbit substance P antibody (IHC 7451; Peninsula Laboratories, San Carlos, CA) 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, neurokinins A and B, neuropeptide K, and somatostatin. The substance P antibody was purified by repeated precipitations by saturated ammonium sulfate. ELISA plates (Costar 3590) were coated with 100 µl of the substance P antibody, diluted 1:30,000 in coating buffer (50 mM Na2CO3, pH 9.6), and incubated overnight at 4°C. The plates were then washed three times with 200 µl of washing solution (150 mM NaCl, 0.05% vol/vol Tween-20). To prevent nonspecific binding, 100 µl of a phosphate buffer (150 mM NaH2PO4, 150 mM NaCl, 0.1% BSA) containing excess BSA (50 mg/ml) was added to the plates, which were then incubated for 30 min at 40°C. After the plates were washed three times with washing solution, 50 µl of sample or standard were pipetted into the wells, along with 50 µl of 150 pM biotinylated substance P (Peninsula Laboratories). Standards and biotinylated substance P were diluted in the HEPES buffer. Samples and standards were assayed in triplicates. After a 3-h incubation at 40°C, bound and free substance P were separated by washing the plates three times in washing solution. Then, 100 µl of 500 ng/ml streptavidin-labeled peroxidase was added to the plates. The plates were incubated for 30 min at 40°C and then washed three times with washing solution. Thereafter, 100 µl of substrate solution [0.6 mM 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), 10 mM sodium citrate, 0.006% vol/vol H2O2; pH 4.0] was added to the wells. The reaction was stopped by adding 100 µl of 1% wt/vol sodium dodecyl sulfate to each well when total absorbance had an optical density of 1.000. Absorbance of the wells was read at 405 nM on a Bio-Rad plate reader. The substance P concentrations of the samples were calculated by entering the optical densities into a curve-fitting software (GraphPad-in-Plot; GraphPad Software, San Diego, CA).

Statistical analysis. The Friedman two-way analysis of variance together with the shortcut analysis of variance were used to determine whether KCl and PGE2 elicited a release of substance P into the incubation bath that was different from that seen during the control and recovery periods. The Wilcoxon signed rank test was used to determine differences in basal substance P release between the ipsilateral and contralateral pelvises. A significance level of 5% was chosen (25, 26). Data are expressed as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of KCl on Substance P Release in the Absence and Presence of Verapamil

The majority of substance P-containing neurons are located in the renal pelvic wall (18, 32). Depolarization of cultured rat dorsal root ganglion neurons by high potassium results in an increase in the release of substance P that is blocked by various dihydropyridines (3, 23), known blockers of the L-type calcium channels (1, 24, 28). To examine whether an isolated renal pelvic wall preparation was a valid experimental model for studying mechanisms involved in the release of substance P, isolated renal pelvises were incubated in HEPES buffer containing 50 mM KCl to depolarize the sensory neurons. After 140 min of equilibration with regular replacement of the incubation bath, the release of substance P into the incubation bath had reached a steady state (Fig. 1). Exposing the isolated renal pelvic wall to 50 mM KCl resulted in a threefold reversible increase in the release of substance P (Fig. 2). Pretreating the renal pelvic wall with the L-type calcium blocker verapamil at 10 µM prevented the potassium-induced release of substance P. 


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  		Fig. 1.   Release of substance P from isolated renal pelvic wall preparation into incubation medium, time control.



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  		Fig. 2.   Effects of 50 mM KCl on release of substance P from isolated renal pelvic wall preparation in absence and presence of 10 µM verapamil in incubation medium. ** P < 0.01.

Effects of PGE2 on Substance P Release in the Absence and Presence of Calcium

Stimulation of renal pelvic sensory nerves results in an increase in PGE2 and substance P, the substance P release being dependent on intact prostaglandin synthesis (10, 11) in vivo. Isolated renal pelvises were exposed to PGE2 in the presence and absence of calcium in the incubation medium to examine whether PGE2 released substance P from the renal sensory nerves by physiological mechanisms. PGE2 at 2.8 µM resulted in a twofold reversible increase in the release of substance P into the incubation medium containing 2.5 mM calcium (Fig. 3). Incubating the contralateral renal pelvis in a calcium-free medium reduced basal substance P release and prevented the PGE2-mediated release of substance P, suggesting that the release of substance P from the renal pelvic wall is calcium dependent.


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  		Fig. 3.   Effects of 2.8 µM PGE2 on release of substance P from isolated renal pelvic wall preparation incubated in calcium-containing and calcium-free media. ** P < 0.01.

Effects of PGE2 on Substance P Release in the Absence and Presence of Verapamil

The isolated renal pelvic wall has the capability to synthesize PGE2 (10). Because pilot experiments showed that a lower, more physiological concentration of PGE2, 0.14 µM, resulted in a submaximal increase in the release of substance P in the presence of cyclooxygenase blockade, this concentration of PGE2 was used and indomethacin was added to the incubation medium in subsequent experiments. As shown in Fig. 4, PGE2 at 0.14 µM resulted in a reversible increase in substance P release in the presence of indomethacin. Because the PGE2-mediated release of substance P was dependent on extracellular calcium (Fig. 3), we examined whether the calcium influx occurred via activation of L-type calcium channels, involved in potassium-induced substance P release (Fig. 2). Pretreating the renal pelvic wall with 10 µM verapamil had no effect on the PGE2-mediated increase in substance P release (Fig. 4).


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  		Fig. 4.   Effects of 0.14 µM PGE2 on release of substance P from indomethacin-treated isolated renal pelvic wall preparation in absence and presence of 10 µM verapamil. ** P < 0.01.

Effects of PGE2 on Substance P Release in the Absence and Presence of omega -Conotoxin

Sensory neurons contain both L- and N-type calcium channels (1, 22, 24, 28). Because the PGE2-induced release of substance P was unaffected by verapamil (Fig. 4), we examined whether the PGE2-mediated substance P release was blocked by omega -conotoxin, a selective blocker of the N-type calcium channels (1, 22). omega -Conotoxin has been shown to block voltage step-induced calcium current (3) and PGE2-mediated substance P release in cultured sensory neurons (30). In the absence of omega -conotoxin, 0.14 µM PGE2 resulted in a similar increase in substance P release (Fig. 5) as in the group of pelvises depicted in Fig. 4. Pretreating renal pelvises with 0.1 µM omega -conotoxin blocked the PGE2-induced substance P release. Basal substance P release was unaltered (Fig. 5).


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  		Fig. 5.   Effects of 0.14 µM PGE2 on release of substance P from indomethacin-treated isolated renal pelvic wall preparation in absence and presence of 0.1 µM omega -conotoxin. ** P < 0.01.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results of these experiments show that PGE2 elicits an increase in the release of substance P from the renal pelvic wall that is blocked in a calcium-free medium or by omega -conotoxin but not affected by verapamil. Exposing the renal pelvic wall to high KCl concentration results in an increase in substance P release that is blocked by verapamil. These studies suggest that PGE2 increases the release of substance P from the renal pelvic sensory nerves by a calcium-dependent mechanism that requires the influx of calcium via N-type calcium channels. The potassium-induced substance P release involves activation of the L-type calcium channel.

Immunohistochemical studies using antibodies against the neuropeptides substance P and calcitonin gene-related peptide have localized the neuropeptides to nerve fiber structures in the renal pelvic wall (18, 31). The current study used an isolated renal pelvic wall preparation to study the mechanisms involved in the release of substance P from the renal pelvic sensory nerves. The in vitro preparation allows a precise control of the chemical composition of the solution bathing the renal pelvis, which is not possible in vivo. Our initial studies, performed to test the validity of the isolated renal pelvic wall preparation for studying the release of substance P from renal sensory nerves, used KCl to depolarize the neurons. Studies in cultured dorsal root ganglion neurons and spinal cord sections have shown that high concentrations of KCl increase the release of substance P (3, 21, 23, 27). In the present study, 50 mM KCl resulted in a reversible release of substance P from the renal pelvic wall into the incubation medium. Calcium influx is essential for release of neuropeptides and transmitters from neurons (1, 22, 24, 28). Comparing the substance P release from renal pelvises incubated in a calcium-containing medium with that from contralateral pelvises incubated in a calcium-free medium showed that removing calcium from the incubation medium reduced basal release of substance P. Basal release of substance P from the isolated renal pelvic wall incubated in a calcium-containing medium was in a similar range as that in the renal pelvic effluent in vivo (10, 11). Taken together, these findings suggest that the release of substance P reflects a physiological release and that the isolated renal pelvic wall preparation is a valid experimental model to study substance P release mechanisms from renal sensory nerves.

Multiple high-voltage-sensitive calcium channels have been characterized in sensory neurons (1, 23, 24). The increased substance P release from dorsal root ganglion neurons produced by high KCl concentrations is reduced in calcium-free medium (21, 27) and blocked by dihydropyridine calcium antagonists (3, 23), demonstrating the involvement of L-type calcium channels in potassium-induced substance P release. In agreement with these studies in cultured sensory nerves, the results of the present study show that the substance P release from renal pelvic sensory nerves induced by 50 mM KCl was inhibited by the L-type channel blocker verapamil.

Mechanical stimulation of an isolated renal pelvic wall preparation results in a release of PGE2 into the incubation bath that is abolished by indomethacin (10). These studies suggest that the renal pelvic wall, which contains the majority of the sensory neurons in the kidney (18, 32), has the capability of synthesizing PGE2. Activation of renal sensory nerves by increasing renal pelvic pressure or bradykinin results in an increase in the release of substance P into the renal pelvic effluent that is dependent on intact prostaglandin synthesis (10, 11). The present data show that PGE2 resulted in a reversible increase in the release of substance P from the renal pelvic wall. The concentration of PGE2 increasing substance P release in indomethacin-treated pelvises is in the range of (30) or even lower than (27) that required to increase substance P release in rat dorsal root ganglion neurons. Removing calcium from the incubation medium blocked the PGE2-induced increase in substance P release from the renal pelvic wall, suggesting that the increase in substance P release produced by PGE2 requires an influx of calcium. In this context, it is of interest that PGE2 has been shown to increase calcium current elicited by voltage steps (21) and intracellular calcium concentration (4) in cultured sensory neurons. The PGE2-mediated increase in substance P release from the renal pelvic wall was not affected by pretreatment with the L-type calcium channel blocker verapamil, applied at the same concentration, 10 µM, that blocked the potassium-induced substance P release. These findings are in agreement with previous studies showing that the L-type calcium blocker nifedipine failed to inhibit the PGE2-mediated substance P release from dorsal root ganglion neurons (30).

Electrophysiological studies in aortic baroreceptors isolated from nodose ganglia showed that a major portion of the high-threshold calcium current is sensitive to blockade by omega -conotoxin (19). The N-type calcium channel, known to be blocked by omega -conotoxin, has been shown to be involved in transmitter release from sensory and sympathetic neurons in response to various stimuli (3, 5, 22, 30). In view of the present study, it is of interest that many nerve terminals in the dorsal horn of the spinal cord expressing the N-type calcium channels also contain substance P (29). In the present study, renal pelvises were pretreated with omega -conotoxin at a concentration, 0.1 µM, shown to reduce calcium current in sensory neurons (3). In agreement with studies in cultured sensory neurons (30), omega -conotoxin blocked the PGE2-mediated increase in substance P release from the renal pelvic wall. Our findings are also in concordance with studies in the isolated guinea pig atria that showed that omega -conotoxin blocked the release of the neuropeptide calcitonin gene-related peptide from atrial sensory nerves produced by bradykinin (5), known to release PGE2 in various tissues, including the renal pelvic wall (11).

In accordance with studies in cultured dorsal root ganglion neurons (3, 23, 30), basal substance P release was not affected by verapamil or omega -conotoxin in the present study. Although the mechanisms involved in basal substance P release from renal pelvic neurons are unknown, our data showing markedly reduced basal substance P release in calcium-free medium would suggest an involvement of calcium channels.

Our findings using an in vitro model of the renal pelvic wall suggest that the prostaglandin-dependent release of substance P in response to increased renal pelvic pressure or bradykinin in vivo is due to PGE2 increasing the release of substance P by activating N-type calcium channels. The link between the in vitro and in vivo studies is further supported by previous in vitro studies in cultured sensory neurons. These studies showed that bradykinin stimulates an influx of calcium via a prostaglandin-dependent pathway (4) and that the bradykinin-induced release of substance P is blocked by omega -conotoxin but not by nifedipine (3). Because the N-type calcium channels are most prevalent in neuronal tissue (22), our present findings suggest that PGE2 increases the release of substance P by acting directly on renal sensory nerve endings in the pelvic wall.

The mechanisms involved in the PGE2-mediated activation of calcium channels in renal sensory nerves are currently unknown. Studies in cultured dorsal ganglion neurons would suggest that the cAMP transduction cascade is involved in the PGE2-mediated enhancement of neuropeptide release (6). PGE2 was shown to increase neuronal cAMP content, and inhibition of adenylyl cyclase reduced PGE2-mediated enhancement of bradykinin-mediated release of neuropeptides. It is likely that the PGE2-mediated increase in cAMP leads to phosphorylation of various proteins, including ion channels, by protein kinase A (PKA). Our previous studies have shown an important role for protein kinase C (PKC) in the activation of renal sensory neurons (13). Our data suggest that bradykinin activates PKC in the renal pelvic wall with a subsequent release of PGE2 and substance P (8, 13). Thus it is possible that the release of substance P in response to activation of renal pelvic sensory nerves involves a complex interaction between the phosphoinositide and cAMP transduction cascades, with PKC activation being involved in the mechanisms leading to release of PGE2 and PKA activation in the mechanisms leading to the PGE2-mediated release of substance P.

In summary, the present study shows that high potassium results in an increase in the release of substance P from the renal pelvic wall that is blocked by verapamil. PGE2 increases the release of substance P from the renal pelvic wall. Basal and PGE2-mediated release of substance P is dependent on extracellular calcium. The PGE2-mediated release of substance P is blocked by omega -conotoxin but not by verapamil. These findings suggest that the relative role of L- and N-type calcium channels in controlling the release of substance P from the renal pelvic sensory nerves depends on the stimulus. The release of substance P by potassium involves activation of L-type calcium channels, whereas the release of substance P by PGE2 is mediated via activation of N-type calcium channels.

Perspectives

The present data show that the isolated renal pelvic wall preparation may serve as a valid model for studying neuropeptide release mechanisms from peripheral sensory nerves. The release of substance P is in the range of that seen in the renal pelvic effluent during both basal and stimulated conditions in vivo. Furthermore, the calcium mechanisms involved in the release of substance P mirror those described for cultured central sensory neurons. It is highly likely that the mechanisms involved in the PGE2-induced substance P release from renal sensory nerves are not unique to the renal sensory nerves but are similar to those of other peripheral sensory nerves as well.

Although basal and stimulated substance P release was reduced by removing extracellular calcium, only the stimulated substance P release was reduced by blockers of high-voltage-activated calcium channels. These findings suggest that different calcium channels are involved in controlling basal and stimulated substance P release. We speculate that basal substance P release may be dependent on activation of low-voltage-activated calcium channels, which are activated at negative membrane potentials close to the resting potential and require only weak depolarization for activation (1, 24).

The inhibitory nature of the renorenal reflexes may suggest that basal and PGE2-mediated substance P release into the renal pelvis may be important contributory mechanisms to the renal control of sodium balance during both basal conditions and conditions of cardiovascular stress when renal prostaglandin synthesis is increased. If so, then the impairment of the renorenal reflexes would lead to increased renal sympathetic nerve activity and sodium retention. This hypothesis is supported by our previous studies in spontaneously hypertensive rats (SHR), a hypertensive model characterized by increased renal sympathetic nerve activity and sodium retention. Our findings show impaired renorenal reflexes in SHR, the impairment being due to a defect in PGE2-mediated release of substance P and impaired responsiveness of renal pelvic substance P receptors (9).

Renal prostaglandins serve a compensatory role in various conditions of circulatory stress to offset renal vasoconstriction and sodium retention. Our data would suggest that the inhibitory renorenal reflexes activated by PGE2-induced substance P release contribute to the modulatory effects of prostaglandins on sodium reabsorption during stress. Consequently, some of the deleterious effects of prostaglandin inhibitors on renal functions observed during conditions of, for example, volume depletion, may partly be related to decreased release of substance P. Substance P not only activates the inhibitory renorenal reflexes but also increases pelvic and ureteral peristalsis, important components of the mechanisms involved in the excretion of urine.

Prostaglandin synthesis inhibitors are commonly used for pain relief in conditions of ureteral obstruction. Our data would suggest that part of the beneficial effects of these agents is related to decreased release of substance P, a known mediator of pain. The inhibitory effect of omega -conotoxin on PGE2-mediated substance P release would suggest that inhibitors of N-type calcium channels would have an analgesic effect on renal colic.

Our findings that 50 mM KCl depolarized renal sensory nerves with a subsequent release of substance P are intriguing considering the fact that urine potassium concentration in humans is in the range of 20-200 mM. Our data would suggest that the afferent renal nerves are activated by urine potassium concentrations within the normal range. Of interest in this context are early studies in rats showing that the inhibitory renorenal reflexes are tonically active (2).


    ACKNOWLEDGEMENTS

This work was supported by grants from the Department of Veterans Affairs, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52617, National Heart, Lung, and Blood Institute Specialized Center of Research Grant HL-55006, and the American Heart Association, Iowa Affiliate.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: U. C. Kopp, Dept. of Internal Medicine, Univ. of Iowa College of Medicine, Iowa City, IA 52242 (E-mail: ukopp{at}blue.weeg.uiowa.edu).

Received 27 October 1998; accepted in final form 15 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bertolino, M., and R. R. Llinas. The central role of voltage-activated and receptor-operated calcium channels in neuronal cells. Annu. Rev. Pharmacol. Toxicol. 32: 399-421, 1992[Medline].

2.   DiBona, G. F., and L. L. Rios. Renal nerves in compensatory renal response to contralateral renal denervation. Am. J. Physiol. 238 (Renal Fluid Electrolyte Physiol. 7): F26-F30, 1980.

3.   Evans, A. R., G. D. Nicol, and M. R. Vasko. Differential regulation of evoked peptide release by voltage-sensitive calcium channels in rat sensory neurons. Brain Res. 712: 265-273, 1996[Medline].

4.   Gelperin, D., D. Mann, J. Del Valle, and J. W. Wiley. Bradykinin increases cytosolic calcium in cultured rat myenteric neurons via BK-2 type receptors coupled to mobilization of extracellular and intracellular sources of calcium: evidence that calcium influx is prostaglandin dependent. J. Pharmacol. Exp. Ther. 271: 507-514, 1994[Abstract/Free Full Text].

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Am J Physiol Regul Integr Compar Physiol 276(5):R1241-R1248
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