INTRODUCTION

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THE ASPARTYL PROTEINASE renin is part of the renin-angiotensin-aldosterone system and has an important role in the regulation of the electrolyte and water homeostasis of the body as well as in arterial blood pressure regulation (3, 10). Renin is synthesized and stored in granulated modified vascular smooth muscle cells, the juxtaglomerular cells (JGC), which are located in the media of the afferent arterioles close to the entrance into the glomeruli of the kidney (10). Renin catalyzes the formation of ANG I, which is then converted by angiotensin converting enzyme to ANG II, a potent vasoconstrictor of the vasculature. Several studies have suggested that adenosine, in addition to other autacoids like NO (9, 11, 26) and prostaglandins (3, 6), serves as a mediator link between macula densa cells and JGC and is therefore involved in the regulation of renin release (12, 13, 15, 20, 21, 23, 31). In fact, intra-arterial infusion of adenosine to sodium-restricted rats with high plasma renin activity has been shown to reduce renin release (23). Although numerous in vivo and in vitro experiments have been carried out to elucidate the process of renin secretion from JGC, the underlying regulatory mechanisms at the cellular level are still incompletely understood. Studies on renin secretion were performed with isolated JGC in short-term primary cultures (7, 16, 26) and long-term cultures of JGC transformed with a temperature-sensitive SV40 large T antigen gene (25). To investigate the renin release of freshly isolated JGC from rat kidneys, we established a new superfusion system for continuous monitoring of renin release.

	MATERIALS AND METHODS

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Isolation of rat JGC. All animal experiments were conducted in accordance with the German Law on Protection of Animals. Three groups of male Sprague-Dawley rats were used for JGC preparation as follows: group 1, rats under standard diet with mean body weight of 154 ± 8 g; group 2, rats under standard diet with mean body weight of 308 ± 9 g; and group 3, rats kept on low-sodium diet with mean body weight of 283 ± 11 g. The animals were kept under standard conditions with a 12:12-h light-dark rhythm at 20-22°C room temperature and relative humidity of ~60%. Food and tap water were allowed ad libitum. Standard diet (Altromin C 1324) contained 2.5 g/kg sodium and 3.6 g/kg chloride. For sodium restriction, animals were fed low-sodium diet (Altromin C 1036; 150 mg/kg sodium, 3 mg/kg chloride) for at least 2 wk following a single dose of furosemide (10 mg/kg ip). For one preparation, two to four kidneys were used. The animals were killed by cervical dislocation and bled, and the kidneys were removed, decapsulated, and minced with a razor blade. JGC were isolated according to the method previously described by Della Bruna et al. (7). In brief, the minced tissue was incubated at 37°C for 1 h under gentle stirring in buffer A (in mM: 130 NaCl, 5 KCl, 2 CaCl₂, 10 glucose, 20 sucrose, 10 HEPES, pH 7.4), supplemented with 0.25% trypsin type III (Sigma Chemicals, St. Louis, MO) and 0.1% collagenase A (Boehringer, Mannheim, Germany). The cell suspension was sieved through a 22.4-µm nylon mesh (Verseidag Techfab, Walbeck, Germany). Cells passing the mesh were collected, washed with buffer A without trypsin and collagenase, and resuspended in a final volume of 6-10 ml RPMI-1640 medium (GIBCO-BRL, Life Technologies). Two milliliters of the cell suspension were overlaid on 20 ml of 30% isosmotic Percoll (Pharmacia, Uppsala, Sweden) solution, then centrifuged for 25 min at 4°C and 27,000 g in a 50.2 TI rotor using a Beckman ultracentrifuge L-70. Four apparent bands were obtained. Renin activity was detected in bands I-III. Band IV contained only erythrocytes. In cells from band III, the highest renin activity was found. We therefore used cells from this band for further experiments.

Superfusion of freshly isolated JGC. The cells from band III were collected by centrifugation, washed twice with buffer A at room temperature to remove Percoll, resuspended in 1,800 to 2,200 µl of buffer A, and incubated for 20 min at 37°C in a humidified atmosphere containing 5% CO₂. Fifty microliters of this suspension (3-4 × 10⁵ cells) were transferred to each superfusion chamber equipped with a nylon mesh and a DE-81 cellulose filter (see Fig. 1). The chamber was placed in a heating block with a servo-controlled temperature of 37°C. The superfusion buffer (buffer B) had the following composition (mM): 91.0 NaCl, 17.5 NaHCO₃, 7.0 KCl, 2.0 CaCl₂, 1.2 MgSO₄, 1.2 NaH₂PO₄, 11.0 glucose, and 49.0 sucrose, pregassed for 1 h with 95% O₂-5% CO₂ at 37°C, pH 7.4 (2). One preparation of JGC was used for simultaneous superfusion of 10-20 superfusion chambers. The superfusion rate with buffer B was set at 7.5 µl/min via syringes and an infusion pump (Braun Melsungen). The volume of the superfusion chamber was 50 µl, and the dead space from the syringe to the collecting tube was 150 µl. Agents to be tested were added to the superfusion medium. After a 20-min equilibration period, the collection of the effluate started for five periods of 20 min. Effluates were collected in vials precooled on ice and stored at 20°C until measurement of renin activity.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Schematic drawing of the superfusion chamber. Juxtaglomerular cells (JGC) are retained on the DE-81 cellulose filter (solid line). Nylon mesh (dashed line) serves to prevent the obstruction of the system. Superfusion fluid is collected after passing the superfusion chamber at different time intervals.

Superfusion of short-term cultured JGC. JGC were isolated and collected as mentioned above. To allow the JGC to recover for a longer period of time than for 20 min from the isolation procedure (see above), cells were cultured according to Della Bruna et al. (7). In brief, 6-7 × 10⁶ cells were suspended in 20 ml RPMI-1640 medium (GIBCO) supplemented with 25 mmol/l HEPES, 15% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml amphotericin B, and 0.66 U/ml insulin-transferrin-sodium selenite supplement and incubated at 37°C in a humidified atmosphere with 5% CO₂ in air. After 24 h of primary culture, the medium was removed, and the cells were harvested using a cell scraper to avoid enzymatic treatment and used for superfusion studies.

Measurement of renin. Renin activity was determined by its capacity to generate ANG I using a specific radioimmunoassay for ANG I. Plasma of bilaterally nephrectomized rats was used as renin substrate. Four microliters of renin substrate and 10 µl of enzyme inhibitors (2,3-dimercaptopropanol, 80 µl/100 ml, plus 8-hydroxyquinoline, 132 mg/100 ml) were added to 10 µl of each sample of collected effluate. The mixture was incubated for 1.5 h at 37°C. The reaction was stopped on ice. Eighty microliters (~2 nCi) of ¹²⁵I-labeled ANG I (NEN Life Science Products, Boston, MA), with a specific activity of 2,200 Ci/mmol, and 80 µl of a specific polyclonal rat ANG I antibody from rabbit (gift from Prof. E. Hackenthal, Heidelberg, Germany) was added and thoroughly vortexed. After equilibration for 18 h at 4°C, free and bound ¹²⁵I-ANG I were separated with bovine -globulin (Cohn Fraction IV-1, Sigma Chemicals)-coated charcoal (Norit A; Serva, Heidelberg, Germany). The radioactivity was measured in aliquots of supernatants using a gamma counter (COBRA, Canberra-Packard) with 82% counting efficiency. Renin activity was calculated using ANG I standards and expressed as nanograms of ANG I per milliliter per hour per milligram cellular protein (ng ANG I · ml¹ · h¹ · mg protein¹). The detection limit of the assay was 5 pg ANG I/10 µl effluate.

Preparation of cellular extracts. Intracellular renin activity was determined in cellular extracts. For that purpose, 50 µl of cell suspension was centrifuged for 2.5 min in a Beckman Microfuge B at 11,000 g. The supernatant was removed, and 100 µl PBS (in mM: 137 NaCl, 2.7 KCl, 9.2 Na₂HPO₄, and 1.5 KH₂PO₄, pH 7.4) containing 0.1% Triton X-100 was added to the pellet, thoroughly mixed, and kept for 30 min at room temperature with repeated vortexing. The mixture was centrifuged again for 2.5 min at 11,000 g, and supernatants were stored at 20°C until further processing and determination of renin activity as described above.

Measurement of protein. Protein of cellular extracts was determined according to the method of Lowry et al. (19) using bovine serum albumin as standard.

Statistical analysis. Data are presented as means ± SE. Number of observations (n) in renin release experiments are expressed as number of JGC preparations/superfusion chambers. Statistical comparison between different treatment groups was made using Student's t-test for unpaired data. P < 0.05 were considered to be statistically significant.

Substances. The following substances were purchased from the sources indicated. Two different preparations of adenosine deaminase derived from calf intestinal mucosa, ADA type II (activity, 1-5 U/mg protein) and ADA type VII (activity, 150-250 U/mg protein), were from Sigma Chemicals; bovine albumin standard grade was from Serva Heidelberg; 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and N⁶-cyclohexyladenosine (CHA) were from Research Biochemicals International. All other chemicals were of standard laboratory quality.

	RESULTS

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Cellular renin activity. The total renin activity determined in cellular extracts from JGC from young rats on standard diet (mean body wt 154 g) after 20-min preincubation at 37°C was 57.0 ± 4.4 µg ANG I · ml¹ · h¹ · mg protein¹ (n = 30). Cellular renin activity from older control rats (mean body wt 308 g) was 178 ± 18 µg ANG I · ml¹ · h¹ · mg protein¹ (n = 26). The cellular renin activity of JGC from rats on low-sodium diet (mean body wt 283 g) was 214 ± 22.6 µg ANG I · ml¹ · h¹ · mg protein¹ (n = 47).

Spontaneous renin secretion. Cell suspensions (3-4 × 10⁵ cells in 50 µl, 30-40 µg protein) were applied to each superfusion chamber. To adapt the cells to the superfusion buffer and to flush the renin present in the incubation buffer, JGC were equilibrated for 20 min in the chamber by running superfusion (see Fig. 2B). The spontaneous renin secretory rate (RSR) from superfused JGC was 118 ± 30 ng ANG I · ml¹ · h¹ · mg protein¹ · min¹ in the first 20-min collection period and decreased continuously to 3.6 ± 1.7 ng ANG I · ml¹ · h¹ · mg protein¹ · min¹ (n = 9/18) during the following 100 min of superfusion as shown in Fig. 2C. The cell viability at the end of the superfusion was found to be more than 90% tested by trypan blue exclusion.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Time-dependent decrease of spontaneous renin secretion from superfused rat JGC was inhibited by adenosine deaminase type II (ADA II, 3 U/ml). Period A: 20-min preincubation of JGC at 37°C after cell isolation. Period B: equilibration of JGC in the superfusion chamber. Period C: collection periods. Values are means ± SE; n = 9/18. ** P < 0.01.

Effects of ADA II on renin secretion from freshly isolated JGC. One possibility to account for the fall of spontaneous RSR within 100 min of superfusion to the detection limit of ANG I could be that endogenous adenosine occupied its receptors to reduce the RSR. To test this hypothesis, ADA II was added to the superfusion medium to remove extracellular adenosine. When ADA II was present during the superfusion in a concentration of 3 U/ml (~1,400 µg protein), the decrease in RSR as observed in the untreated controls was partially prevented. RSR was about fourfold higher, with 402 ± 100 ng ANG I · ml¹ · h¹ · mg protein¹ · min¹ (n = 9/18) in the first collection period (Fig. 2C), and dropped to 237.5 ± 67 ng ANG I · ml¹ · h¹ · mg protein¹ · min¹ within 100 min.

To test whether the ADA II effect was reversible, the superfusion protocol was changed. ADA II was added to the superfusion medium twice for only 10 min, at which time spontaneous RSR was expected to have reached a low level. The time course of the ADA II effect is shown in Fig. 3. Because of the dead space of 150 µl in the superfusion system, the ADA II effect on RSR could be detected only after a delay of 20 min. The response of the JGC to ADA II was therefore almost immediate and rapidly reversible. After a rechallenge with ADA II, the same response could be elicited and is consistent with the assumption that endogenous adenosine could have contributed to the observed decline in spontaneous RSR.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Reversible effect of ADA II (3 U/ml) added to superfusion fluid (bars) on renin secretion from JGCs. Values are means ± SE; n = 3/6.

Effects of ADA II on renin secretion from short-term JGC. To assess the effect of cell culture conditions on RSR, JGC were first cultured under standard conditions for 24 h and then used for superfusion experiments. Spontaneous renin secretion from 24 h cultured JGC showed a similar rundown as from freshly isolated JGC. Addition of 3 U/ml of ADA II to the superfusion medium prevented the decline during 100 min of superfusion. Renin secretion within this time interval was 0.9 ± 0.26 (n = 3/11) compared with 3.6 ± 0.56 µg ANG I/mg protein from freshly prepared cells. In the presence of ADA II, renin secretion within 100 min was 4.92 ± 1.44 compared with 48.5 ± 7.2 µg ANG I/mg protein seen in freshly prepared JGC. Thus renin secretion from cultured compared with fresh JGC amounted only to 25% and 10%, respectively.

Effects of adenosine receptor modulators. To further test the assumption that adenosine A₁-receptor activation by endogenous adenosine could contribute to the decline in spontaneous renin release in these superfused JGC, experiments with the A₁-receptor antagonist DPCPX and the A₁-receptor agonist CHA were performed. Concentration response curves were established for DPCPX and CHA in the range from 10¹¹ to 10⁶ mol/l. The effects of DPCPX were studied in the absence of ADA II, and those of CHA were studied in the presence of ADA II. The EC₅₀ value for DPCPX was 30 ± 5 pmol/l (Fig. 4) as calculated by nonlinear regression analysis. DPCPX enhanced renin secretion during 100 min superfusion from 3.6 ± 0.8 (controls) to 10.1 ± 1.4 µg ANG I/mg protein (n = 4/8). CHA inhibited renin secretion. The highest effective concentration was 30 nmol/l, which reduced renin secretion from 48.5 ± 7.2 to 29.7 ± 6.6 µg ANG I · mg protein¹ 100 min¹ (n = 4/8). The IC₅₀ value for CHA was calculated to be 0.11 ± 0.09 nmol/l (Fig. 4). These experiments clearly support the assumption that adenosine receptors are involved in the regulation of renin secretion. However, the stimulatory effects of DPCPX and the inhibitory effects of CHA on renin secretion cannot fully account for the effects of the absence and presence of ADA II, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Concentration-dependent effects of N6-cyclohexyladenosine (CHA; , n = 4/8) in presence of ADA II (3 U/ml) and of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; , n = 4/8) in absence of ADA II on renin secretion within 100 min of superfusion from rat JGC. Inset: renin secretion within 100 min of superfusion under control conditions (n = 9/18), in presence of 30 nmol/l DPCPX (n = 4/8), in presence of ADA II (n = 9/18), and in presence of ADA II plus 30 nmol/l CHA (n = 4/8). *** P < 0.001 vs. control. Values are means ± SE.

Unspecific effect of protein on renin release. It has been suggested that an unspecific protein effect of ADA could be responsible for the elevated renin release compared with controls. Therefore the effects of increasing concentrations of albumin (3-3,000 µg/ml) on spontaneous renin release were studied. Albumin elevated renin release by about fourfold, with a maximal effect at 1,000 µg/ml. The EC₅₀ value was calculated to be 223 ± 29 µg/ml (Fig. 5).

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Concentration-dependent effects of bovine serum albumin on spontaneous renin secretion from superfused JGC. Renin secretion was calculated as cumulative release over 60 min and is given as percentage of control (100%). Values are means ± SE; n = 3/6.

To differentiate whether the specific adenosine deaminating effect was the result of impurities in the ADA II preparation that might exert an unspecific protein effect, we repeated the superfusion experiments with ADA VII, which has a 40-fold higher specific activity than ADA II. Renin secretion by JGC in the presence of ADA VII at a concentration of 17 U/ml (100 µg protein/ml) was about twofold higher compared with controls. Increasing ADA VII concentrations up to 170 U/ml, corresponding to 1,000 µg protein/ml, had no further stimulating effect (Fig. 6).

View larger version (9K): [in this window] [in a new window]   Fig. 6.   Effects of increasing concentrations of ADA VII on spontaneous renin secretion from superfused JGC. Renin secretion was calculated as cumulative release over 60 min and is given as percentage of control (100%). Values are means ± SE; n = 3-11/6-40.

ADA VII at 5 U/ml increased RSR to 178 ± 29% of controls, and albumin at 300 µg/ml increased RSR to 162 ± 4% of controls (n = 1/4). When both stimuli were combined, RSR was increased to 343 ± 21% of controls (n = 1/4), indicating that the ADA VII and albumin effects were additive.

Comparison of the ADA II, ADA VII, and DPCPX effects. Renin release from JGC prepared from rats on low-salt diet was 2.5 ± 0.4 µg ANG I · ml¹ · h¹ · mg protein¹ in controls (n = 12/48) and increased to 8.3 ± 0.7 µg by ADA II at a concentration of 1.5 U/ml (n = 8/24) and to 4.9 ± 0.9 µg (n = 8/32) by ADA VII at a concentration of 5 U/ml within 60 min. These concentrations are sufficiently high to deaminate almost all extracellular adenosine in these preparations. DPCPX at 10⁶ mol/l increased RSR to 5.6 ± 0.9 µg ANG I · ml¹ · h¹ · mg protein¹ (n = 7/28) (Fig. 7). Thus ADA VII, which had a much higher specific enzymatic activity than ADA II, stimulated RSR to approximately the same extent as the specific adenosine A₁-receptor antagonist DPCPX. However, the DPCPX effect did not reach the level of ADA II.

View larger version (33K): [in this window] [in a new window]   Fig. 7.   Effects of DPCPX (106 mol/l, n = 7/28), ADA VII (5 U/ml, n = 8/32), and ADA II (1.5 U/ml, n = 8/24) on spontaneous renin secretion of superfused JGC from sodium-restricted rats during 1 h of superfusion compared with controls (n = 12/48). Values are means ± SE. P < 0.05,  P < 0.01, and P < 0.001 vs. controls. × P < 0.05 and ×× P < 0.01 vs. ADA II.

	DISCUSSION

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Although renin secretion was studied by a number of different methods, the regulatory mechanisms on cellular level are still incompletely understood. When renin secretion was studied from rat glomeruli isolated by an electromechanical method (1, 2), from rat kidney slices by superfusion (27), or by perfusion of the rabbit juxtaglomerular apparatus (18, 31), a time-dependent decline was observed. In short-term cultured mouse JGC, no significant changes in extracellular renin activity during the first days of culture were observed (7). Thus none of these models exhibits a constant RSR. In addition, it is difficult to study renin release in cell culture during short periods, i.e., minute-to-minute intervals.

Therefore, in the present study a superfusion apparatus was developed to investigate renin secretion of isolated JGC from rat kidneys to overcome the above-mentioned methodological limitations. The cells were used after adaptation from 4°C to 37°C for 20 min and then equilibrated in the superfusion chamber for another 20 min, because the responsiveness of the cells could have been impaired by the preparation procedure, i.e., enzymatic and ischemia-mediated membrane and/or receptor damage. JGC were also superfused after a recovery period of 24 h primary culture, thus providing a longer time of recovery from preparation stress. In agreement with the findings of Blendstrup et al. (2), Skøtt and Baumbach (1, 27), and Weihprecht et al. (31), the spontaneous renin release from the freshly isolated as well as the cultured and superfused JGC was characterized by a time-dependent decline. The total amount of renin released from the short-term cultured cells was even less than that from freshly prepared JGC. Thus freshly prepared JGC appear to be better suited for short-term analysis of RSR.

The freshly isolated and superfused JGC responded with an elevated RSR over 2 to 3 h when ADA II was present in the superfusion medium. This ADA II effect could be the result of removal of extracellular adenosine due to enzymatic deamination. From in vivo experiments with sodium-restricted rats and dogs, it is known that intrarenal infusion of adenosine inhibits renin secretion (23, 30). This adenosine effect is thought to be mediated by a direct action on renin-containing cells (17, 28, 29). The major sources for adenosine in the kidney are either an increase in ATP metabolism with elevated hydrolysis of 5'-AMP or hydrolysis of S-adenosylhomocysteine (14). The tissue content of adenosine in the kidney is 5 nmol/g wet wt under normoxic conditions and increases after ischemia (22). Therefore, it can be assumed that the JGC during isolation procedure and superfusion were exposed to impaired energy supply and metabolic stress and thus to a release of adenosine at an enhanced rate.

The first part of this study described continuous superfusion of JGC with ADA II. The effect of ADA II to prevent the spontaneous decline in RSR was reversible. Since adenosine-induced reduction of renin secretion is thought to be mediated by A₁-receptor activation (5, 24), a selective nonmetabolizable adenosine A₁-receptor agonist should antagonize the ADA II effect. CHA reduced RSR in a concentration-dependent manner. From the literature, it is known that in rat kidney slices CHA inhibited renin release in nanomolar concentrations, whereas in micromolar concentrations renin release was stimulated. This stimulatory effect was suggested to be due to A₂-receptor activation (24). In the present study the inhibitory effect was not reversed even at a concentration of 1 µmol/l CHA. These findings suggest that isolated JGC do not exhibit functional A₂ receptors which can stimulate renin secretion (5). In fact, it has been reported that stimulation of renin secretion by activation of A₂ receptors requires intact innervation of the kidney (24).

Interestingly, CHA inhibited renin secretion only by 50% at 30 nmol/l in the presence of ADA II. This result was somewhat unexpected. One can conclude that a significant part of the action of ADA II may be independent from its enzymatic activity and thus from removal of adenosine from the receptors.

ADA II contained ~70% of unidentified natural proteins. Proteins might have an effect on their own on spontaneous renin release from isolated superfused JGC. One candidate for unspecific protein action is albumin. Bovine albumin stimulated renin release in a concentration-dependent manner. The underlying mechanism for this stimulatory effect of albumin remains speculative at present. One possibility could be a reduction of cytosolic Ca²⁺ concentration, which has been observed in human endothelial cells exposed to albumin (8). It might therefore be assumed that the incomplete response of JGC to the adenosine A₁-receptor agonist CHA could be due to an additional unspecific effect of protein which is independent from adenosine receptor stimulation.

As CHA was expected to antagonize the ADA action, the selective antagonist of A₁-receptors, DPCPX, should mimic the ADA effects. DPCPX increased RSR in a concentration-dependent manner. The calculated IC₅₀ value was similar to the K_D value reported from binding studies (4, 29). The maximal observed increase in RSR was about fourfold at 10⁶ mol/l compared with controls. The expected level of RSR as observed in the presence of ADA II, however, was not reached. Therefore, the same argument as proposed above, that the ADA II preparation effect cannot fully account for adenosine receptor deactivation, may apply also to the DPCPX effect.

To analyze the obviously unspecific action of ADA II, additional experiments were performed with the purified ADA VII of high specific activity. Superfusion of JGC with ADA VII resulted in a maximal twofold increase in RSR at ~5 U/ml and did not increase further even at 100 U/ml, corresponding to ~1,000 µg protein/ml. It is therefore suggested that ADA VII mainly acted by deaminating adenosine and had no unspecific effects as seen with ADA II.

From the literature it is known that in in situ autoperfused kidneys the A₁-antagonist 1,3-dipropyl-8-(p-sulfophenyl)xanthine (DPSPX) increased RSR significantly more in rats kept under sodium restriction compared with rats fed a normal sodium diet (15). This effect was independent from sympathetic innervation.

We have included studies on renin release from superfused JGC of rats fed a low-sodium diet and found that 10⁶ mol/l DPCPX stimulated renin release to the same extent as ADA VII, indicating that low-sodium diet did not change the JGC response to DPCPX or ADA VII. These results support the assumption that the effect of ADA VII was indeed due to inactivation of adenosine and not due to additional unspecific protein stimulation.

In summary, we conclude that the superfusion apparatus is a suitable method to study renin secretion at the cellular level. Renin secretion from isolated superfused JGC remains at an elevated rate in the presence of adenosine deaminase and can be measured for several hours. The magnitude of the response depends on the type of ADA used. The purified ADA VII with high specific activity stimulates RSR by removing endogenous adenosine. The less purified ADA II elicits, in addition to its enzymatic activity, stimulation of RSR by unidentified proteins present in this preparation. Our present findings confirm that adenosine reduces renin secretion by stimulation of adenosine A₁ receptors in isolated JGC.