In vitro evidence for a blood-borne renin-releasing factor

The present studies using kidney slices were designed to test whether serotonergic stimulation of renin secretion is mediated via an endocrine signal. Previous in vivo studies have indicated that central serotonergic neurons regulate renin secretion. Administration of the serotonin releaser dl-p-chloroamphetamine-HCl (PCA) to rats causes dose-dependent increases in renin secretion that can be blocked by serotonin depletion with p-chlorophenylalanine (PCPA), injections of 5,7-dihydroxytryptamine into the dorsal raphe nucleus or ablation of the mediobasal hypothalamus. The renin-releasing substance was obtained from nephrectomized male donor rats which were sacrificed 1 hour after receiving an injection of PCA intraperitoneally. Plasma from rats that received saline injections was used as control. The plasma was collected and separated by ultrafiltration into fractions containing solutes with molecular weights between 500-10,000 daltons. The renin-releasing ability of this substance was studied in vitro using rat renal cortical slices. The plasma fraction (M.W. = 500 - 10,000) from rats treated with PCA caused dose-dependent increases in renin release from the kidney slices. Heating of the plasma factor at 100 degrees C for 30 minutes did not reduce the ability of this substance to release renin from the kidney slices. PCA alone (66 X 10(-6)M) did not increase renin release from the kidney slices. These data suggest that stimulation of serotonergic receptors in the brain triggers the release of an endocrine factor that is capable of directly stimulating renin release from the kidneys.     

Renin release in anesthetized rats is enhanced by the calmodulin antagonist W-7

In foregoing work, we found that the release of renin from rat kidney cortical slices was stimulated by the calmodulin antagonist W-7. The present work was done to determine whether W-7 would stimulate renin release in vivo. W-7 and its control agent, W-5 were directly infused into the renal artery of anesthetized rats. W-7 and W-5, at 50 micrograms/kg/min, produced no significant effects on renin release. Infusion of W-7 at 100 micrograms/kg/min resulted in a marked stimulation of renin release, but there was no significant alteration in the release when the same dose of W-5 was infused. Both compounds elicited a slight decrease in renal blood flow. The alterations in renin release and renal blood flow seen with W-7 were not affected by pretreatment with phentolamine or propranolol. As W-7 stimulates renin release in vivo, the hypothesis that Ca2+-calmodulin plays an inhibitory role in renin release from the kidney is given added support.     

Quantitative changes in rat renin secretory granules after acute and chronic stimulation of the renin system

In order to study the cellular mode of renin secretion, stereological methods were used to estimate number and volume of rat renin secretory granules during stimulation of the renin system. An acute decrease in renal perfusion pressure to 40 mmHg for 5 min increased plasma renin concentration (PRC) twofold, but did not significantly change the number of renin granules per arteriole or the renin-containing volume of the arteriole. Chronic stimulation was achieved by a combination of low-salt diet and inhibition of angiotensin-converting enzyme (ACE) for 14 days, and resulted in a 36-fold increase in PRC, a 20-fold increase in the number of granules per arteriole, and a 17-fold increase in the arteriolar volume that contained renin. An acute decrease in renal perfusion pressure to 40 mmHg for 5 min in the chronically stimulated rats increased PRC further (1.6-fold), and significantly reduced the number of granules per arteriole by 4000 (45% reduction), but did not change the renin-containing arteriolar volume significantly. The average renin granule size was 0.35 μm³ with no significant differences among the groups. We conclude that recruited granular cells contribute significantly to renin release, and that all granular cells along the arteriole participate in secretory responses. The reduced number of renin granules after acute stimulation is compatible with exocytosis as the dominating mechanism of renin release.     

Role of beta-1 and beta-2 adrenoceptors in isoproterenol-induced renin release in conscious dogs.

The present experiments were designed to classify the ß-adrenoceptors pertaining to the renin release induced by isoproterenol in conscious dog. Atenolol (ß-1 adrenoceptor antagonist), in oral dose of 6 mg/kg, produced a significant inhibition of renin release caused by isoproterenol. This dose of atenolol suppressed effectively the tachycardia of isoproterenol. On the other hand, the renin release produced by isoproterenol was not modified significantly by a ß-2 adrenoceptor antagonist, IPS-339, at a oral dose of 3 mg/kg which fully antagonized hypotensive response to isoproterenol. These results strongly suggest that the renin release induced by isoproterenol is largely due to stimulation of ß-1 type adrenoceptors.     

电刺激兔下丘脑乳头体核上区及内侧视前区对血浆肾素活性的影响

本工作观察了电刺激麻醉兔的乳头体核上区及内侧视前区对血浆肾素活性(Plasmarenin activity,PRA)的影响。电刺激乳头体核上区引起血压升高、心率减慢、呼吸变快变浅、PRA增加120％,双侧去肾神经后或静脉注射心得安后,同样的刺激使PRA增加的现象基本消失。电刺激内侧视前区导致血压下降、心率加快、呼吸加深、PRA降低30.7％,双侧去肾神经后,电刺激内侧视前区使PRA降低的作用明显减弱。上述两组实验表明,电刺激兔下丘脑乳头体核上区及内侧视前区可分别增加或减少肾素释放。     

Prostacyclin-independence in beta-adrenoceptor mediated renin release from dog renal cortical slices

There is some controversy regarding whether stimulation of renin release by the beta-adrenergic system is dependent on prostaglandin (PG) production. We have examined this problem in renal cortical slices of the dog and have obtained the following results: (1) Isoproterenol (4 X 10(-6) M) stimulated renin release, but had no effect on the formation of 6-keto PGF1 alpha, a stable metabolite of PGI2; (2) Indomethacin (2 X 10(-5) M) had no effect on isoproterenol stimulated renin release, but inhibited 6-keto PGF1 alpha formation; (3) Dibutyryl cyclic AMP (10(-3) M) stimulated both renin release, and 6-keto PGF1 alpha release. Indomethacin (2 X 10(-5) M) did not inhibit dibutyryl cyclic AMP-stimulated renin release, but did inhibit the production of 6 keto PGF1 alpha. These results indicate that the beta-adrenoceptor mediated renin release does not depend on the formation of PGI2, but renin release is dependent on cyclic AMP formation.     

The effect of prostaglandin synthesis inhibition on the direct stimulation of renin release from rabbit renal cortical slices

Prostaglandins have been hypothesized to have several mechanistic functions in sympathetically mediated release of renin. The rabbit renal cortical slice system was chosen to examine the prostaglandin dependency of renin release directly stimulated by either a direct adenylate cyclase activator, forskolin, or a beta-agonist, isoproterenol. In this study, we demonstrate that with forskolin (1 X 10(-5) M) or isoproterenol (1 X 10(-6) M), renin release was elevated 2-3 fold above control, and that this increase was shown to accompany a substantial increase in the tissue levels of cAMP (19.5 fold and 3.5 fold respectively). We also demonstrate that the increase in renin release produced by these compounds was not inhibited by cyclooxygenase inhibitors, indomethacin (25 microM) or eicosatetraynoic acid (30 micrograms/ml), nor was it inhibited by the selective prostacyclin synthesis inhibitor, U-51605 (30 micrograms/ml). Each of these inhibitors was demonstrated to block the synthesis of prostaglandins in the cortical slices at the concentrations used. Thus we propose that prostaglandins do not play a role in the induction of renin release resulting from elevated cyclic nucleotide levels or beta-adrenergic stimulation.     

Stimulation of renin secretion from the intact kidney and from isolated glomeruli by the calcium ionophore A23187.

The ionophore A23187 evoked a dose-dependent release of renin from the isolated perfused cat kidney, which was inhibited by calcium deprivation and adrenergic blockade. The latter finding indicates that the effects of A23187 on the intact kidney are mediated mainly by catecholamine release from sympathetic nerve endings. Ionophore also elicited a concentration-dependent enhancement of renin secretion from a pure preparation of glomeruli isolated from cat kidney; this stimulation was still manifest when the glomeruli were superfused with a calcium-free solution. These findings indicate that A23187 evokes renin secretion from juxtaglomerular cells by mobilizing cellular calcium and support the view that an increase in intracellular calcium is intimately involved in the mechanism of renin secretion.     

Effect of dopamine and dopamine-receptor blockade on in vitro renin release, tissue renin content and tissue cyclic AMP content in the rat

This study evaluated the in vitro renin release, tissue cyclic AMP content (TcAMPc), and tissue renin content (TRC) changes with time, in response to administration of dopamine (DOP) and of the dopamine-receptor blocking agent pimozide (PIM) to renal cortical slices from sodium deficient (SD) rats. Addition of 10(-3)M DOP to the slice preparation resulted in a gradual stimulation of RR with time, which was significantly different from that seen in control samples after 60 min of incubation. In contrast, TcAMPc of the DOP-treated samples was significantly greater than that of controls after 5 min of incubation. At 60 min, mean TRC of DOP-treated samples was greater than that of controls but not significantly. Two PIM doses (10(-8)M and 10(-6)M, whether added alone or together with 10(-3)M DOP to the cortical slice system, significantly increased RR in each instance while simultaneously depressing TcAMP content markedly below that of unstimulated controls at all incubation times examined. Mean TRC of pimozide-treated samples was also lower than that of controls by 60 min. These in vitro data in the SD rat suggest that: 1) stimulation of renin release by DOP is time-dependent and is mediated by a TcAMP-generating mechanism, and 2) the increase in renin release by PIM administration appears to involve pharmacological inactivation of TcAMP-generating pathways and disruption of membrane permeability, leading to uncontrolled RR.     

Nitrendipine-induced stimulation of renin release by the isolated perfused rat kidney

The direct effects of the organic calcium antagonist nitrendipine upon renin release were assessed using the isolated rat kidney perfused at constant pressure. This model circumvents the indirect actions of vasodilating agents by artificially maintaining perfusion pressure constant, thereby avoiding the hypotensive effects associated with the systemic administration of such agents. Renin release as assessed by radioimmunoassay was stimulated 2.6-fold upon the administration of 10(-6) M nitrendipine. Since this stimulation of renin release occurred in the absence of any alteration in perfusion pressure, we conclude that it represents a direct action of nitrendipine. This finding is in support of the current hypothesis concerning the inverse relationship between cytosolic Ca2+ and renin secretory rate, and suggests that Ca entry into the juxtaglomerular cells of the juxtaglomerular apparatus is sensitive to blockade by organic calcium antagonists such as nitrendipine.     

Modulation of active renin secretion by renin-binding protein (RnBP) in mouse pituitary AtT-20 cells transfected with human renin and RnBP cDNAs.

To investigate the role of renin-binding protein (RnBP) in renin metabolism, RnBP expression plasmid, which was constructed to express human RnBP under the control of mouse mammary tumor virus long terminal repeat, was transfected into mouse pituitary AtT-20 cells together with the expression plasmid encoding human renin. The transfectant secreted prorenin and active renin, whereas RnBP was expressed only in the presence of dexamethasone and without secretion into the medium. The secretion of active renin was stimulated by forskolin, and the stimulation was repressed by dexamethasone. The secretion of prorenin, however, was insensitive to forskolin irrespective of the presence or absence of dexamethasone. Moreover, the forskolin-stimulated release of active renin was hardly repressed by dexamethasone in AtT-20 cells transfected with the renin expression plasmid and a selectable plasmid pMAMneo. Coexistence of RnBP and renin mRNAs in human Wilms' tumor G-401 cells was shown by means of polymerase chain reaction of respective cDNAs from the cells. These results suggest that RnBP modulates the release of active renin in renin-producing cells.     

Quinacrine antagonizes the effects of Na,K-ATPase inhibitors on renal prostaglandin E2 release but not their effects on renin secretion

Previous results have demonstrated that two inhibitors of Na-and-K-activated adenosine triphosphatase (ouabain, vanadate) lead to stimulated prostaglandin E2 release and to inhibited renin secretion in the rat renal cortical slice preparation. It was speculated that stimulation of phospholipase A2 activity accounted for the effect on prostaglandin E2 release. We used the same preparation in the present experiments, and showed that another inhibitor of Na-and-K-activated adenosine triphosphatase (K-free incubation medium) stimulates prostaglandin E2 release and inhibits renin secretion. Quinacrine antagonized the stimulatory effects of ouabain, vanadate, and K-free medium on prostaglandin E2 release (consistent with phospholipase A2 involvement), but did not antagonize their inhibitory effects on renin secretion. Collectively, these observations lend further weight to the argument against a mediatory role of prostaglandin synthesis in the renin secretory process.     

Renin release from nonclipped kidneys of 2K-1C hypertensive rats

This study examined renin release and renal function of the nonclipped kidney of 2K-1C rats (HYPER) with hypertension of 3 to 4 weeks duration. Kidneys from uninephrectomized rats (UN) served as controls. The kidneys were removed and perfused in vitro and renin release was compared under basal conditions and then under stimulated conditions using (a) a 50 mm Hg reduction in perfusion pressure (LP), (b) beta-receptor stimulation with isoproterenol (ISOP), or (c) perfusion with a low calcium solution (LCa). Basal perfusate flow, glomerular filtration rate, urine flow, sodium excretion, and basal renin release were all lower in HYPER kidneys than UN kidneys. UN kidneys showed striking increases in renin release with all three stimuli employed. HYPER kidneys showed a significant but attenuated response to ISOP and showed no detectable response to LP or LCa. Renin content of HYPER kidneys was found to be 28% of the renin content of UN kidneys. The results show that chronic hypertension leads to increased renal vascular resistance, reduced glomerular filtration, and reduced solute excretion in the nonclipped kidney. The results suggest that a reduction in renin content plays a major role in the reduced rates of basal renin release and the attenuated renin responses to a number of stimuli observed in this experimental model.     

The effect of prostaglandin synthesis inhibition on the direct stimulation of renin release from rabbit renal cortical slices

Prostaglandins have been hypothesized to have several mechanistic functions in sympathetically mediated release of renin. The rabbit renal cortical slice system was chosen to examine the prostaglandin dependency of renin release directly stimulated by either a direct adenylate cyclase activator, forskolin, or a β-agonist, isoproterenol. In this study, we demonstrate that with forskolin (1 × 10⁻⁵M) or isoproterenol (1 × 10⁻⁶M), renin release was elevated 2–3 fold above control, and that this increase was shown to accompany a substantial increase in the tissue levels f cAMP (19.5 fold and 3.5 fold respectively). We also demonstrate that the increase in renin release produced by these compounds was not inhibited by cyclooxygenase inhibitors, indomethacin (25 uM) or eicosatetraynoic acid (30 ug/ml), nor was it inhibited by the selective prostacyclin synthesis inhibitor, U-51605 (30 ug/ml). Each of these inhibitors was demonstrated to block the synthesis of prostaglandins in the cortical slices at the concentrations used. Thus we propose that prostaglandins do not play a role in the induction of renin release resulting from elevated cyclic nucleotide levels or β-adrenergic stimulation.     

Effect of sodium and osmolarity on renin secretion.

The effect of changes in sodium and osmolarity on renin secretion has been studied in the isolated perfused rat kidney. Perfusion with low sodium buffer (110 mM/l) produced a significant increase in renin secretion compared with control experiments (Na+:135 mM/l). Since the presence of tubules seems necessary for such an effect to take place, it suggests that the high renin secretion stimulated by a low sodium buffer centers in the Macula densa. Perfusion with high sodium buffer (170 mM/l; osmolarity 350 mOs/l) induces a stimulation on renin release. However, a greater rise in renin is achieved in control experiments if choline chloride increases the osmolarity from 300 to 350 mOs/l. All this suggests that high sodium buffer, independently of its osmotic effect, has an inhibitory role on renin release.     

The effect of thyroid hormone on renin production and release by rat kidney slices

The effect of thyroid hormone on renin productiona and release by rat kidney slices was studied. Rat kidney slices were incubated in Warburg flasks containing Krebs-Ringer-Phosphate- Glucose- Dextran solution at 37 C for 5 hours. Renin content, renin released into the incubation media and oxygen consumption were measured. Kidney slices actively secreted renin. Kidney slices of hyperthyroid rats released more renin, and kidney slices of hypothyroid rats released less renin than normal kidneys (p less than 0.001). The addition of 1-thyroxine to the incubation medium increased significantly (p less than 0.001) renin release by kidney slices from normal and hypothyroid rats. Thyroid hormone affects renin release through a mechanism independent of the ouabain-sensitive sodium pump and protein synthesis, since ouabain and cycloheximide did not modify renin release or production. The results of this study suggest that thyroid hormone plays a role in renin release from the juxtaglomerular cells.     

Prostaglandins and renin release: III. Effects of PGE1, E2 F2α and D2 on renin release from rabbit renal cortical slices

We have investigated the direct effects of prostaglandins E₁, E₂, F_2α and D₂ on renin release from rabbit renal cortical slices. Prostaglandin E₁ (PGE₁) was the most potent stimulant of renin release, while PGE₂ was 20–30 fold less active. PGF_2α was found not to be an inhibitor of renin release as reported by others, but rather a weak agonist. PGD₂ up to a concentration of 10 μg/ml had no activity in this system. That the stimulation of renin release by PGE₁ is a direct effect is supported by the finding that PGE₁-induced release is not blocked by L-propranolol or by Δ^5,8,11,14-eicosatetraynoic acid (ETYA), a prostaglandin synthesis is inhibitor. The fatty acid precursor of PGE₁, Δ^8,11,14-eicosatrienoic acid, also stimulated renin release, an effect which was blocked by ETYA. In addition to the above findings, ethanol, a compound frequently used to dissolve prostaglandins, was shown to inhibit renin release.     

Changes of renin isoelectric heterogeneity after acute and chronic stimulation of renin secretion

Changes in the multiple forms of renin secreted or stored in vitro by renal cortical slices were studied in rats made hypertensive with deoxycorticosterone, adrenalectomized rats, and rats fed a high or low salt diet. Renal slices from normal rats were also incubated with angiotensin II, vasopressin, and verapamil. Aliquots of incubation media were subjected to isoelectric focusing, and the six forms of renin were quantified and expressed as a percentage of the total renin activity recovered from the gel. The results showed that chronic and acute stimulation of renin secretion produced a similar modification of the isoelectric focusing profile, consisting of an increased proportion of renin forms with the more acidic isoelectric points. The change in the proportions of the more acidic renin forms was greater with chronic stimulation than that after stimulation with verapamil. However, chronic and acute inhibition or reductions of the rate of renin secretion did not modify the renin profile. We suggest that the progression in the shift of secreted renin forms to those with the more acidic isoelectric points correlates with the intensity or duration of stimulation of renin secretion. These data support the hypothesis that different pools of renin exist and are altered differently by chronic and acute stimulation of renin secretion.     

Active and inactive renin in human forearm of hypertensive patients.

Although many in vitro and animal studies indicate the existence of a local renin--angiotensin system, data regarding its physiological role are quite controversial, and moreover, evidence suggesting inactive and active renin release from vascular tissue in vivo is lacking both in animal and humans. The aim of our study was to evaluate whether beta-adrenoceptor stimulation, a well-known stimulus to renin production, through isoproterenol might cause local renin production from vessels of the forearm of hypertensive patients. Drugs were infused into the brachial artery at systemically ineffective rates, while forearm blood flow (FBF, venous plethysmography), mean intra-arterial pressure, and heart rate were monitored throughout. Active and inactive vessel renin production was measured by calculating venous-arterial (V-A) differences by simultaneous sampling from brachial artery and an ipsilateral deep vein. Active renin (PRA) and total renin (Sepharose bound trypsin activation) were measured by radioimmunoassay while inactive renin was calculated as the difference between total and active renin. V-A differences were corrected for FBF to calculate renin extraction or production. In a group of 10 patients, isoproterenol, which was infused at increasing cumulative rates (0.03, 0.1, 0.3 micrograms.100 mL-1 forearm tissue.min-1 for 5 min each), caused a dose-dependent increment in FBF that was blunted by intra-arterial propranolol (n = 5) pretreatment (10 micrograms.100 mL-1 forearm tissue.min-1 for 10 min). beta-Adrenoceptor stimulation caused a dose-dependent outflow of both active and inactive renin, an effect antagonized by propranolol. In conclusion, our data represent the first evidence in humans of tissue active and inactive renin production in the forearm vascular bed.     

Species differences in the effect of isoproterenol and pindolol on renin release in vitro.

The effects of selective beta adrenergic receptor stimulation with isoproterenol (3 X 10(-8) M) and of beta adrenergic blockade with pindolol (3 X 10(-5) M) on the renin release in vitro were investigated in incubated canine and rat kidney slices. Bioassay was used to measure the renin content of the tissue samples and incubation media; renin content in the canine incubation medium was measured also by radioimmunoassay. Isoproterenol in a concentration of 3 X 10(-8) M brought about a significant increase in the renin content of the incubation media as well as the tissue slices obtained from canine kidney, however, there was no change in these parameters under similar conditions if rat kidneys were incubated. Pindolol, on the other hand, in a concentration of 3 X 10(-5) M caused a significant decrease in the renin release from as well as in the renin content of the rat kidney slices, while canine kidney slices failed to respond to the same dose of the drug. The differences between the two species is suggested to be due to the differences in basal renin levels.