INTRODUCTION

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RENIN SECRETION and renin gene expression in renal juxtaglomerular (JG) cells is under the control of both systemic and local factors. Among the intrarenally formed factors potentially involved in the control of the renin system, nitric oxide (NO) has attracted considerable interest, since JG cells are surrounded by cells with a high capacity of NO formation, such as endothelial cells and macula densa cells (1, 35). A series of studies have investigated the effects of NO donors and of NO inhibitors on renin secretion and renin gene expression in vivo and in vitro. All these studies agreed that NO influences renin secretion and renin gene expression. The findings obtained and the conclusions drawn about the particular effect of NO on the renin system, however, are remarkably controversial.

Thus NO has been suggested to stimulate renin secretion in vivo from conscious (9, 12, 22, 27, 32) and anesthetized animals (4, 21, 34). Also, in vitro NO was claimed to stimulate renin secretion from isolated perfused rat kidneys (14, 31) from dispersed renal cells (26) and from cultured JG cells (33). In isolated JG cells, a dual effect of NO on renin secretion was observed: the stimulatory effect of NO on renin secretion occurred with a significant delay, and, in short-term experiments with isolated JG cells, NO was found to inhibit renin secretion (17, 23, 33). Similarly, NO has also been reported to inhibit renin secretion from kidney slices (3, 20) and in anesthetized rats (34) and dogs (28).

A main intracellular signaling pathway of NO comprises the stimulation of soluble guanylate cyclase, leading to an increase of intracellular cGMP levels and the induction of cGMP-induced reactions (25). The results from studies obtained about the effect of cGMP on renin secretion are conflicting, as are the effects of NO itself. Thus membrane-permeable cGMP analogs were reported to stimulate renin secretion from dispersed renal cells (26) or were seen to inhibit renin secretion from kidney slices (20) and cultured JG cells (20, 33).

Because reduplication of experiments with a given experimental model produced consistent results, it is likely that the controversial findings about the effect of NO and cGMP on renin secretion resulted mainly from the different experimental models and conditions used. Consequently, the effect of NO on renin secretion appears to be rather variable, depending on additional modulating conditions. To determine the physiological effect of NO on renin secretion, it was of interest to us to thoroughly characterize the effect of intrarenal NO on renin secretion and to define the pathways through which NO could act on renin secretion. In view of the large number of experimental approaches used so far to investigate the effect of NO on renin secretion, it appeared reasonable to us to use an experimental model that is close to the kidney in vivo and that can be well controlled and pharmacologically manipulated. To this end, we chose the isolated perfused rat kidney model. In this model, we have characterized the effects of NO donors and of cGMP analogs on renin secretion. Moreover, we investigated the involvement of classic second messenger pathways, such as calcium or cAMP, in the effect of NO on renin secretion.

	MATERIALS AND METHODS

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Isolated perfused rat kidney. Male Sprague-Dawley rats (250-300 g body wt), having free access to commercial pellet chow and tap water, were obtained from Charles River-Wiga (Sulzfeld, Germany). Kidney perfusion was performed in a recycling system (30). In brief, the animals were anesthetized with 150 mg/kg of thiobarbituric acid (Inactin; Byk-Gulden, Constance, Germany). Volume loss during the preparation was replaced by intermittent injections of physiological saline via a catheter inserted into the jugular vein. After the abdominal cavity was opened by a midline incision, the right kidney was exposed and placed in a thermoregulated metal chamber. The right ureter was cannulated with a small polypropylene tube (PP-10), which was connected to a larger polyethylene catheter (PE-50). After intravenous heparin injection (2 U/g), the aorta was clamped distal to the right renal artery, and the large vessels branching off the abdominal aorta were ligated. A double-barrel cannula was inserted into the abdominal aorta and placed close to the origin of the right renal artery. After ligation of the aorta proximal to the right renal artery, the aortic clamp was quickly removed, and perfusion was started in situ with an initial flow rate of 8 ml/min. The right kidney was excised, and perfusion at constant pressure (100 mmHg) was established. The renal artery pressure was monitored through the inner part of the perfusion cannula (Statham Transducer P-10 EZ), and the pressure signal was used for feedback control of a peristaltic pump. The perfusion circuit was closed by draining the venous effluent via a metal cannula back into a reservoir (200-220 ml). The basic perfusion medium, maintained at 37°C, consisted of a modified Krebs-Henseleit solution containing (in mM) all physiological amino acids in concentrations between 0.2 and 2.0 mM, 8.7 glucose, 0.3 pyruvate, 2.0 L-lactate, 1.0 -ketoglutarate, 1.0 L-malate, and 6.0 urea. The perfusate was supplemented with 6 g/100 ml bovine serum albumin, 1 mU/100 ml vasopressin 8-lysine, and freshly washed human red blood cells (10% hematocrit). Ampicillin (3 mg/100 ml) and floxacillin (3 mg/100 ml) were added to inhibit possible bacterial growth in the medium. To improve the functional preservation of the preparation, the perfusate was continuously dialyzed against a 25-fold volume of the same composition, but lacking erythrocytes and albumin. For oxygenation of the perfusion medium, the dialysate was gassed with a 95% oxygen-5% carbon dioxide mixture. Under these conditions, both glomerular filtration and filtration fraction remain stable for at least 90 min at values of ~1 ml · min¹ · g¹ and 7%, respectively (29).

Perfusate flow rates were obtained from the revolutions of the peristaltic pump, which was calibrated before and after each experiment. Renal flow rate and perfusion pressure were continuously monitored by a potentiometric recorder. Stock solutions of the drugs to be tested were dissolved in freshly prepared perfusate and infused into the arterial limb of the perfusion circuit directly before the kidneys at 3% of the rate of perfusate flow. For determination of perfusate renin activity, aliquots (~0.1 ml) were drawn from the arterial limb of the circulation and the renal venous effluent, respectively. The samples were centrifuged at 1,500 g for 15 min, and the supernatants were stored at 20°C until assayed for renin activity.

Experiments on renin secretion from isolated perfused kidneys and presentation of results. After reperfusion was established, loop-perfusate flow rates usually stabilized within 15 min. Samples for the determination of renin activity were taken in 5-min intervals. Renin secretion rates were calculated from the arteriovenous differences of renin activity and the perfusate flow rate. The perfusate samples were diluted 1:5 in buffer and were incubated for 1.5 h at 37°C with plasma from bilaterally nephrectomized male rats as renin substrate. The generated angiotensin I was determined by radioimmunoassay (Sorin Biomedica, Düsseldorf, Germany). Five kidneys were used for each experimental protocol.

Experiments with cultured JG cells. Mouse JG cells were isolated, as described previously (10). For 3 ml of final cell suspension, one C57BL6 mouse (4-6 wk old) that had free access to normal food (Altromin, Lage, Germany) was killed by decapitation. The kidneys were removed, decapsulated, and minced with a scalpel blade. The minced tissue was incubated with gentle stirring in buffer 1 [(in mM) 130 NaCl, 5 KCl, 2 CaCl₂, 10 glucose, 20 sucrose, and 10 tris(hydroxymethyl)aminomethane hydrochloride, pH 7.4] supplemented with 0.25% trypsin (Sigma, Deisenhofen, Germany) and 0.1% collagenase (0.5 U/mg, type A; Boehringer, Mannheim, Germany) at 37°C for 70 min. After enzymatic dissociation, the tissue was sieved over a 22-µm screen. Single cells passing the screen were collected, washed, and resuspended in 4 ml of buffer 1 and then further separated using Percoll (Pharmacia, Uppsala, Sweden) density gradients. The cell suspension obtained was added to two tubes each containing 30 ml of 30% isosmotic Percoll in buffer 1. After 25 min of centrifugation at 4°C and 27,000 g, four cell layers with different specific renin activity were obtained. The cellular layer (density, 1.07 g/ml) with the highest specific renin activity was used for cell culture.

These cells were washed in buffer 1 and resuspended in RPMI 1640 medium (Biochrom, Berlin, Germany) containing 0.66 U/ml insulin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2% fetal calf serum (FCS). The cultures were distributed in 100-µl aliquots into 96-well plates. The cultures were incubated at 37°C in humidified atmosphere containing 5% CO₂ in air.

After 20 h of primary culture, the culture medium was removed, and the cultures were washed once with 100 µl RPMI 1640 medium containing 2% FCS. Then 100 µl of fresh and prewarmed culture medium with the chemicals to be tested were added.

Experiments on renin secretion were performed for 20 h of incubation. At the end of experiments, supernatants were collected and centrifuged at 1,000 g at room temperature to remove cellular debris. The supernatants were then stored at 20°C until assayed for renin activity. Cells were lysed by adding to each culture well 100 µl PBS containing 0.1% of Triton X-100 and shaking for 45 min at room temperature. The lysed cells were stored at 20°C until further processing.

Renin secretion rates were estimated from the appearance rate of renin in the culture medium. To minimize differences among different cell culture preparations, renin secretion rates were calculated as fractional release of total renin [i.e., renin activity released/(renin activity released + renin activity remaining in the cells)].

Determination of cAMP content in cultured mouse JG cells. cAMP levels were measured in JG cells grown in 96-well plates. After removal of the culture medium, cAMP was extracted by incubating the cells for 20 h in 95% ethanol-20 mM HCl at 20°C. Ethanol was evaporated, and succinilated samples were analyzed by radioimmunoassay (Amersham International).

Protein determination. Protein concentration in cellular lysates was determined using the Bio-Rad protein assay kit.

Chemicals. 8-Bromo-cGMP, 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP), Rp-8-CPT-cGMPS and Rp-8-CPT-cAMPS were purchased from BIOLOG (Life Science Institute, Bremen, Germany).

Statistics. For evaluation of significance of a certain experimental maneuver on renin secretion, all renin secretion rates obtained within this experimental period (normally four values) were averaged and compared with the average values of renin secretion of an adjoining experimental period. Student's paired t-test was used to calculate levels of significance within individual kidney cell or tissue preparations. For comparisons among kidneys or different cell preparations, Student's unpaired t-test with Bonferroni's correction for multiple comparisons was used. P < 0.05 was considered significant.

	RESULTS

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It was the aim of the study to characterize the cellular pathways along which NO could influence renin secretion. We first assessed the effect of exogenous NO on renin secretion. To this end, the influence of the NO donor sodium nitroprusside (SNP) on renin secretion was examined by adding graded concentrations (1, 10, and 30 µmol/l) of SNP to the perfusate of isolated perfused rat kidneys. The kidneys were exposed to each SNP concentration for consecutive 20-min intervals. Within each interval, renin secretion rate was assayed every 5 min. As shown in Fig. 1, SNP at 1 µmol/l induced a prompt, approximately threefold increase of renin secretion. Further increase of the SNP concentration to 30 µmol/l increased renin secretion rates ~4.5-fold of the basal release rate (Fig. 1).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Effect of graded concentrations of sodium nitroprusside (SNP) on vascular resistance (A) and on renin secretion (B) from isolated rat kidneys perfused at 100 mmHg (); , time controls without addition of SNP. Samples were taken in 5-min intervals during the different experimental periods, duration of which is indicated in A. For determination of significant differences, renin secretion rates obtained within a certain experimental period of each kidney were taken together and averaged. Experimental protocols were run with 5 different kidneys. Data are means ± SE.  P < 0.05. NS, not significant.

To test for the general stimulability of renin secretion in these kidney preparations, the perfusion pressure was lowered to 40 mmHg in the presence of 30 µmol/l SNP at the end of the experiments. This maneuver increased renin secretion rates ~12-fold of the basal value (not shown).

Because cGMP is the best established mediator of NO effects, it was obviously necessary to consider a possible involvement of the cGMP pathway in mediating the stimulatory effect of SNP on renin secretion. Therefore, the influence of two different membrane-permeable cGMP analogs (8-bromo-cGMP and 8-pCPT-cGMP) on renin secretion was examined in the concentration range between 5 and 50 µmol/l. As shown in Figs. 2 and 3, neither cGMP analog mimicked the effect of SNP on renin secretion. Conversely, there was a trend to inhibit renin secretion, which became significant with 10 µmol/l of 8-bromo-cGMP (Fig. 2). In view of the discrepant effects of SNP and of the cGMP analogs on renin secretion, we wondered whether it was a combination of cGMP and a yet unknown side effect of SNP that produced the stimulation of renin secretion by SNP.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Effect of graded concentrations of 8-bromo-cGMP on vascular resistance (A) and on renin secretion (B) from isolated rat kidneys perfused at 100 mmHg. Samples were taken in 5-min intervals during the different experimental periods, duration of which is indicated in A. For statistical analysis, values on renin secretion obtained within a certain experimental period of each kidney were taken together and averaged. Experimental protocol was run with 5 different kidneys. Data are means ± SE.  P < 0.05.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Effect of graded concentrations of 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) on vascular resistance (A) and on renin secretion (B) from isolated rat kidneys perfused at 100 mmHg. Samples were taken in 5-min intervals during the different experimental periods, duration of which is indicated in A. For statistical analysis, renin secretion values obtained within a certain experimental period of each kidney were taken together and averaged. Experimental protocol was run with 5 different kidneys. Data are means ± SE.  P < 0.05.

This was tested in experiments in which renin secretion was prestimulated by 10 µmol/l SNP followed by addition of 8-bromo-cGMP (30 µmol/l) to the perfusate in the presence of SNP (Fig. 4). In this set of experiments, SNP (10 µmol/l) increased basal renin secretion rates about threefold. Addition of 8-bromo-cGMP (30 µmol/l) in the presence of SNP led to a marked decline of renin secretion rates to almost basal levels (Fig. 4).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effect of 8-bromo-cGMP on vascular resistance (A) and on renin secretion (B) stimulated by SNP from isolated rat kidneys perfused at 100 mmHg. Samples were taken in 5-min intervals during the different experimental periods, duration of which is indicated in A. For statistical analysis, values obtained within a certain experimental period of each kidney were taken together and averaged. Experimental protocol was run with 5 different kidneys. Data are means ± SE.  P < 0.05.

In view of the oppositely directed effects of SNP and of cGMP analogs, which are established activators of cGMP-dependent protein kinase (G kinase), we further examined the effect of SNP on renin secretion in the presence of the G kinase inhibitor Rp-8-CPT-cGMPS (6). As shown in Fig. 5, Rp-8-CPT-cGMPS at 10 µmol/l caused a moderate increase of basal renin secretion. Addition of SNP (10 µmol/l) in the presence of Rp-8-CPT-cGMPS-stimulated renin secretion about sevenfold of basal (Fig. 5). This degree of stimulation achieved with SNP in the presence of the G kinase inhibitor was significantly (P < 0.05) higher than that found with SNP alone (Fig. 9).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effect of SNP in the presence of G kinase inhibitor Rp-8-pCPT-cGMPS on vascular resistance (A) and on renin secretion (B) from isolated rat kidneys perfused at 100 mmHg. Samples were taken in 5-min intervals during the different experimental periods, duration of which is indicated in A. For statistical analysis, values for renin secretion obtained within a certain experimental period of each kidney were taken together and averaged. Experimental protocol was run with 5 different kidneys. Data are means ± SE.  P < 0.05.

Because these experiments rendered a mediator role of the cGMP G kinase pathway for SNP to stimulate renin secretion less likely, we next considered a possible interference of SNP with the regulation of renin by calcium as an explanation for the stimulation of renin secretion. This idea was tested, because NO has been reported to lower the cytosolic calcium concentration (5, 15, 25) and because a lowering of the cytosolic calcium concentration in JG cells is considered to cause potent stimulation of renin secretion (19).

To lower the calcium concentration, the standard perfusate of the isolated perfused kidneys containing 2 mmol/l calcium was switched to a nominally calcium-free perfusate supplemented with 0.5 mmol/l of the calcium chelator EGTA. As shown in Fig. 6, the low-calcium perfusate led to a fourfold increase of basal renin secretion rates. Addition of SNP (10 µmol/l) to the low-calcium perfusate stimulated renin secretion ~16-fold of basal, suggesting a potentiation of renin stimulatory effect of SNP by low calcium. At the end of the experiments, the effect of isoproterenol was also examined with low-calcium perfusate. With low-calcium perfusate isoproterenol (10 nmol/l) stimulated renin secretion ~30-fold of basal (Fig. 6), whereas, with normal calcium-containing perfusate, the same concentration of isoproterenol stimulated renin secretion ~10-fold of basal (not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Effect of SNP and of isoproterenol (Iso) with low-calcium perfusate on vascular resistance (A) and on renin secretion (B) from isolated rat kidneys perfused at 100 mmHg (); , time controls without addition of SNP and isoproterenol. Samples were taken in 5-min intervals during the different experimental periods, duration of which is indicated in A. For statistical analysis, values for renin secretion obtained within a certain experimental period of each kidney were taken together and averaged. Experimental protocols were run with 5 different kidneys. Data are means ± SE.  P < 0.05.

The strong potentiation of SNP-stimulated renin secretion by low extracellular calcium led us also to consider the effect of SNP during inhibition of renin secretion by elevated cytosolic calcium. We therefore examined the effect of SNP on renin secretion in the presence of ANG II, which is known to increase cytosolic calcium in JG cells and to inhibit renin secretion by a calcium related mechanism (19, 24, 29).

As shown in Fig. 7, ANG II (1 nmol/l) decreased basal renin release to ~75% of the control value. In the presence of ANG II, SNP (10 µmol/l) failed to stimulate renin secretion (Fig. 7). Conversely, it appeared as if SNP would enhance the inhibitory effect of ANG II. For control, we also examined the effect of isoproterenol (10 nmol/l) in the presence of ANG II (1 nmol/l) at the end of the experiments. Although isoproterenol significantly stimulated renin secretion in the presence of ANG II, the degree of stimulation achieved in the presence of ANG II (~1.5-fold of basal) was by far smaller than that observed in the absence of ANG II (~10-fold, not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Effect of SNP and of isoproterenol in the presence of ANG II on vascular resistance (A) and on renin secretion (B) from isolated rat kidneys perfused at 100 mmHg. Samples were taken in 5-min intervals during the different experimental periods, duration of which is indicated in A. For statistical analysis, values for renin secretion obtained within a certain experimental period of each kidney were taken together and averaged. Experimental protocol was run with 5 different kidneys. Data are means ± SE.  P < 0.05.

These findings suggested that lowering of the cytosolic calcium concentration is less likely the essential mechanism by which SNP stimulated renin secretion. At the same time, the experiments revealed an obvious similar response of the effects of SNP and of isoproterenol on renin secretion, with regard to modulations of the cytosolic calcium activity. Because isoproterenol is considered to stimulate renin secretion via the cAMP/A kinase pathway, we therefore further considered a possible relevance of the A kinase pathway for the renin stimulatory effect of SNP. To assess a possible involvement of A kinase in the renin stimulatory effect of SNP, the specific A kinase inhibitor Rp-8-CPT-cAMPS (16) was used. As shown in Fig. 8, Rp-8-CPT-cAMPS at 25 µmol/l had no significant effect on basal renin release. Addition of SNP (10 µmol/l) in the presence of Rp-8-CPT-cAMPS stimulated renin secretion less than twofold (Fig. 8). The stimulation achieved with SNP in the presence of the A kinase inhibitor was significantly (P < 0.05) lower than that found with SNP alone (Fig. 9). To test for the efficacy of Rp-8-CPT-cAMPS to inhibit cAMP-mediated renin secretion, isoproterenol (10 nmol) was added to the perfusate in the presence of Rp-8-CPT-cAMPS, which stimulated renin secretion about threefold over the control value. After removal of Rp-8-CPT-cAMPS from the perfusate, renin secretion markedly increased in the presence of isoproterenol (Fig. 8).

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Effect of SNP in the presence of the A kinase inhibitor Rp-8-pCPT-cAMPS on vascular resistance (A) and on renin secretion (B) from isolated rat kidneys at 100 mmHg. Samples were taken in 5-min intervals during the different experimental periods, duration of which is indicated in A. For statistical analysis, values for renin secretion obtained within a certain experimental period of each kidney were taken together and averaged. Experimental protocol was run with 5 different kidneys. Data are means ± SE.  P < 0.05.

View larger version (16K): [in this window] [in a new window]   Fig. 9.   Compilation of effects of nitric oxide (NO) donor SNP of renin secretion rates from isolated rat kidneys perfused at a constant pressure of 100 mmHg in the absence and in the presence of Rp-8-pCPT-cGMPS (10 µmol/l) or Rp-8-pCPT-cAMPS (25 µmol/l). Data are expressed as percentage of the renin secretion rates measured during the control period at the beginning of the experiments. Values for SNP in the absence of Rp compounds were taken from Figs. 1 and 4, those for Rp-8-pCPT-cGMPS were taken from Fig. 7, and those for Rp-8-pCPT-cAMPS were taken from Fig. 8.  P < 0.05 vs. respective condition in the absence of Rp compounds.

In view of the results obtained with the kinase inhibitors in the isolated perfused kidneys, we also tested the effect of the G and A kinase inhibitors on renin secretion in primary cultures of mouse renal JG cells. Within 20 h of incubation, the cells released on average 14% of their initial content of active renin (Fig. 10). This value was not changed if the cells were incubated with the G kinase inhibitor Rp-8-CPT-cGMPS (100 µmol/l). In the presence of the A kinase inhibitor Rp-8-CPT-cAMPS (100 µmol/l), spontaneous renin release tended to be increased to ~18%. SNP (100 µmol/l) increased renin release from 14 to 33% in the absence of kinase inhibitors, as well as in the presence of Rp-8-CPT-cGMPS (Fig. 10). In the presence of Rp-8-CPT-cAMPS, SNP stimulated renin secretion from 18 to 26% (Fig. 10). The stimulation of renin secretion by SNP was therefore significantly attenuated by the A kinase inhibitor (P < 0.05).

View larger version (28K): [in this window] [in a new window]   Fig. 10.   Effects of SNP in the absence or presence of Rp-8-pCPT-cAMPS and Rp-8-pCPT-cGMPS on renin secretion from cultured mouse renal juxtaglomerular cells during a 20-h incubation. Renin release is given as fractional release of the total amount of renin activity. Data are means ± SE of 5 experiments, with each experiment representing the mean of quadruplicate culture wells.  P < 0.05 vs. respective controls without SNP. § P < 0.05 vs. SNP + vehicle. Total renin activity of the cultures was 35 ± 7 µg ANG I · h1 · mg protein1.

In the cultured JG cells, we further examined whether SNP could influence intracellular cAMP levels. Therefore, the cellular cAMP content was determined 0.5, 1, 3, 6, and 24 h after addition of SNP (100 µmol) to the culture medium. Table 1 demonstrates that SNP led to a transient, ~2.5-fold increase of cAMP content in the cells.

	DISCUSSION

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This study aimed to characterize the influence of NO on renin secretion from the kidneys. We found that the NO donor SNP induced a concentration-dependent stimulation of renin secretion, which exerted a rapid onset of action and which reached a maximal stimulation at ~30 µmol/l of SNP. The observation that SNP stimulates of renin secretion from isolated perfused kidneys is in accordance with previous observations (18, 31) and, together with the findings that inhibition of endogenous NO formation decreases renin secretion from isolated kidneys (14, 31), suggests that the overall effect of NO on renin secretion is a stimulatory one in the isolated rat perfused kidney.

Among the possible pathways along which NO could stimulate renin secretion, the cGMP pathway would be an obvious candidate. NO is known as a ubiquitous stimulator of soluble guanylate cyclase (25), and one may therefore assume that SNP elevated cGMP also in the JG cells of the isolated perfused rat kidneys. This assumption is supported by the stimulation of cGMP formation by SNP in primary cultures of rat and mouse JG cells (23, 33).

However, from our experiments, it appears unlikely that the classic cGMP/G kinase pathway mediates the stimulatory effect of NO on renin secretion, since phosphodiesterase (PDE)-resistant cGMP analogs, which are known activators of cGMP-dependent kinases, are potent inhibitors of NO-stimulated renin secretion. It is reasonable to assume, therefore, that the stimulation of renin secretion by the NO donor was not mediated via the G kinases types I or II, which both have been localized in JG cells (13). In contrast, one would predict that activating the cGMP/G kinase pathway by NO counteracts the primary stimulatory effect of NO on renin secretion. This inference is supported by the observation that inhibition of G kinase activity increased basal renin secretion but more clearly enhanced the stimulatory effect of the NO donor on renin secretion.

We therefore considered another signaling pathway that could account for the stimulation of renin secretion by NO. Renin secretion is thought to be inhibitorily regulated by the cytosolic calcium concentration in renal JG cells (19). Because NO has been found to lower the cytosolic calcium concentration in cells (5, 15, 25), it is conceivable that the stimulatory effect of NO on renin secretion could have resulted from disinhibition from the negative calcium signal. In accordance with previous results, we found that lowering the extracellular calcium concentration caused a prominent stimulation of renin secretion from the isolated perfused kidney (29). At low extracellular calcium, the renin stimulatory effect of NO was greatly enhanced, suggesting that renin secretion activated by NO was still under the inhibitory control of calcium. If NO would have stimulated renin secretion primarily via lowering of the calcium concentration, the combination of NO donor and low extracellular calcium should have exerted an effect even smaller than the sum of the individual effects on renin secretion. Moreover, if NO would have stimulated renin secretion via lowering of the cytosolic calcium concentration, one would expect that NO should have attenuated the calcium-related inhibitory effect of ANG II (19, 24, 29) on renin secretion, which did not occur in our experiments. In summary, it appears less likely that the stimulatory effect of NO on renin secretion was primarily due to an interference with the calcium control of renin secretion.

On the other hand, the stimulatory effect of NO was clearly attenuated in the presence of a competitive inhibitor of A kinase, both in the perfused kidneys and in isolated JG cells, suggesting that the cAMP pathway is involved in the action of NO on renin secretion. In fact, both cAMP-stimulated and NO-stimulated renin secretion behaved very similarly in response to changes of the cytosolic calcium activity. Whether cAMP plays a more permissive or a more causal regulatory role in the effect of NO on renin secretion cannot be clearly answered from our study.

Nonetheless, it is conceivable that NO could stimulate renin secretion via the cAMP/A kinase pathway. Thus one could imagine that elevated cGMP levels induced by NO cause transactivation of A kinase. For example, in intestinal cells, native cGMP but not cGMP analogs can activate cystic fibrosis transmembrane conductance regulator chloride channels (8, 11), and evidence was produced to show that this effect of native cGMP was due to transactivation of A kinase by cGMP. One could further imagine that NO influences the intracellular cAMP levels, as suggested by our findings in cultured JG cells, where SNP led to a transient twofold increase of the cellular cAMP content. Such an increase of cAMP levels could be due to a stimulation of cAMP formation by NO, for which no evidence yet exists in the literature. More obvious would be an influence of NO on the degradation of cAMP, because the existence of cGMP-regulated cAMP PDEs is well known (2). In particular, PDE-III, which is a cGMP-inhibited cAMP PDE (2), could be of interest in this context. PDE-III is only inhibited by native cGMP but not by cGMP analogs such as 8-pCPT-cGMP or 8-bromo-cGMP, which have a very low affinity for PDE-III, in contrast to native cGMP (7).

These different affinities could provide an explanation of why native cGMP but not cGMP analogs stimulated renin secretion. It has in fact already been suggested from in vivo experiments that the stimulatory effect of NO on renin secretion could involve PDE-III activity (9). Certainly, we cannot rule out from our experiments that NO could increase cAMP levels also by a cGMP-independent mechanism.

Together, our findings suggest that NO can in principle exert a dual control on renin secretion: a stimulatory one via the A kinase pathway and an inhibitory one via the G kinase pathway. In the isolated perfused kidney, the stimulatory effect appears to be preponderant. It is conceivable that, once the cAMP pathway is deteriorated (for example, by an increase of the cytosolic calcium activity), an inhibitory effect of NO could become apparent. This phenomenon would thus provide an explanation for the seemingly contradictory findings about effects of NO on renin secretion found in the literature.

This study has opened two further main questions concerning the mechanisms by which G kinase activity inhibits renin secretion and concerning the mechanisms along which NO activates the cAMP pathway in JG cells. It must be a task for future experiments to answer these questions.