Effect of atriopeptin III on renin release in vitro

The ability of atriopeptin III (AP) to directly inhibit renal renin release has not been resolved. This issue was examined in a series of experiments performed in a system of rat renal cortical slices (dry weight 1.91 mg) in which the goal was to explore the effects of AP on renin release induced by cyclic AMP (cAMP)-coupled stimuli or by agents which are believed to decrease intracellular calcium (Cai). Concentration response relationships were initially established for all test agents. The cAMP stimuli utilized were isoproterenol (10(-5) M), forskolin (10(-5) M), and dibutyryl cAMP (3 X 10(-4) M); each of these agents produced a significant increase in renin release in the system (with isoproterenol a 59% increase, with forskolin 37%, and with dibutyryl cAMP 52%). The addition of AP (2.09 X 10(-8) M, a minimum inhibitory concentration derived from preliminary studies) significantly blunted these increases; in the case of the dibutyryl cAMP-stimulated renin release, the inhibition was partial as a significant 25% increase in renin occurred in the presence of AP. The addition of the calcium channel blocking agent diltiazem (10(-4) M) resulted in a significant increase in renin release (364 to 567 ng X mg-1, p less than .05) which was not blocked by the addition of AP. Similarly, TMB-8 (0.6 X 10(-4) M), another agent thought to lower Cai, also resulted in increased renin release (455 to 810 ng X mg-1), p less than .01) which was also unaffected by the addition of the AP. In summary, these results show that AP is capable of partially inhibiting renin release in vitro, particularly renin release coupled to cAMP action. In contrast, renin release induced by a decline in Cai appears to be unaffected by the addition of AP.     

The role of beta receptors in the regulation of renin release.

The effect of the primarily beta 2 type adrenergic receptor stimulating terbutaline (10(-7)--10(-5) M) and of the beta 1 and beta 2 type adrenergic receptor stimulating isoproterenol (10(-7)--10(-5) M) was studied on renin release from incubated slices of renal cortex. Renin release and cAMP content of the slices were significantly higher in the presence of both terbutaline and isoproterenol. A logarithmic dose--response relationship was shown to be present between the beta mimetics and the renin concentration in the medium, and the cAMP content of tissue slices. In equal doses isoproterenol was about 1.5 times more potent than terbutaline. No change was seen in the renin content of the tissue slices. The results supports the view that beside the beta 1 type adrenergic receptors of the renal cortex--even if to a lesser extent--the beta 2 type adrenergic receptors, too, are involved in the regulation of renin release.     

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.     

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.     

Direct inhibitory effect of atriopeptin III on renin release in primate kidney

Patterns of in vitro renal renin release and the ability of atriopeptin to directly inhibit renin release have been examined in the rat, rabbit, and dog, but have been unstudied in the primate kidney. Accordingly, we examined renin release from superficial renal cortical slices of the squirrel monkey (Samiri sciuresus). The average age of the 5 animals was 10.2 +/- 2.5 yr at the time of study. Renin release was stimulated significantly by the beta-adrenergic agonist isoproterenol in concentrations of 10(-5) M (1.67-fold) and 10(-4) M (1.84-fold). Isoproterenol-induced renin release was inhibited by atriopeptin III (ANP, 2 X 10(-8) M) and the adenylate cyclase inhibitor dideoxadenosine (DDA, 10(-5) M). Similarly, the incubation of the superficial cortical slices with arachidonic acid (10(-3) M) resulted in a 4-fold increase in tissue renin release which was blocked by the calcium ionophore A23187 (17 X 10(-6) M) and ANP; interestingly, DDA did not block arachidonic acid-induced renin release. These results suggest that ANP exerts a direct inhibitory effect on B-adrenergic and arachidonic acid-induced renin release in the primate kidney. Further, the inhibitory action of A23187 on renin release suggests, as in other species, an integral role for intracellular calcium in the renin release process. These patterns of renin release in primate kidney are similar to those observed in the rodent kidney in vitro.     

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.     

Dehydroepiandrosterone sulfate (DHEA-S) and 3', 5'-cyclic adenosine monophosphate (cAMP) production in a cultured human adrenocortical carcinoma cell line (SW-13)

Very little has been known of the biochemical function of a human adrenocortical carcinoma cell line, SW-13. In this study, the production of several adrenal steroids and 3', 5'-cyclic adenosine monophosphate (cAMP) were investigated in this cell line. The cells were incubated in L-15 medium containing 0.1% bovine serum albumin with several reagents in an atmosphere of 5% CO2 and 95% air for 2 hours at 37 degrees C. Aldosterone (Ald), corticosterone (B), cortisol (F), dehydroepiandrosterone sulfate (DHEA-S) and cAMP were simultaneously assayed by specific radioimmunoassays in the medium and cells. Significant increases in cAMP production were observed by cholera toxin (10 ng/ml) and forskolin (10 nM), both direct stimulators of adenylate cyclase, in the cAMP concentration without an increase in the steroids. The DHEA-S concentration in the medium was significantly increased by angiotensin-II (10(-7)M), noradrenalin (3 X 10(-5) M), adrenalin (3 X 10(-5) M) or alpha-melanocyte-stimulating hormone (alpha-MSH, 10(-7) M), none of which was associated with cAMP production. Neither adrenocorticotropin (10(-10) M) nor human chorionic gonadotropin (500 mIU/ml) stimulated the release of the steroids or cAMP production. A calcium ionophore, A23187 (10(-7) M), and 12-O-tetradecanoylphorbol-13-acetate (10(-8) M), a direct stimulator of protein kinase C, stimulated the release of DHEA-S, but not those of Ald, B and F. The results suggest that SW-13 retains functioning adenylate cyclase which, however, is not linked with steroidogenesis and that DHEA-S is produced probably by the mechanisms which involve protein kinase C system or calcium ion. This report provides the first demonstration of cAMP and DHEA-S production in SW-13 and suggests that this cell line is potentially useful for investigating the mechanisms of steroidogenesis in the human adrenal cortex.     

Head-to-head association in bovine spermatozoa induced by catecholamines

Noradrenaline, adrenaline, and isoproterenol induce head-to-head association in bovine spermatozoa in a Tris-HCl-buffered medium. Noradrenaline was the most and isoproterenol the least efficient. This effect was stimulated by Ca2+, Mg2+ and Mn2+, Ca2+ being more efficient than the other two ions. At 2 X 10(-6) M Ca2+, oxidation products of adrenaline dissociated spermatozoa associated by washing; at 2 X 10(-5) M Ca2+, the dissociating effect was transformed into association. The induction of association by adrenaline was blocked by both alpha- and beta-adrenergic blockers at low concentrations (2 X 10(-7) M). Both cAMP and dibutyryl substituted cAMP (db-cAMP) induced association in the Tris-buffered medium at 2 X 10(-6) M Ca2+. Further increase in association was brought about by increasing the Ca2+ concentration to 2 X 10(-5) M. Prolongation of the treatment with cAMP increased association. When combined with cAMP under the same conditions as used in the combination with adrenaline, L-propranolol did not inhibit association induced by cAMP. In an identical experiment, performed in Tyrode solution, L-propranolol inhibited association induced by cAMP. At 2 X 10(-5) M, theophylline, caffeine, and papaverine induced association in the presence of 2 X 10(-5) M Ca2+. The results are compatible with the hypothesis that catecholamines act via receptors and formation of cAMP.     

Forskolin-induced bone resorption in neonatal mouse calvaria in vitro

The role of cyclic adenosine 3',5'-monophosphate (cAMP) in inducing bone resorption was studied in neonatal mouse calvaria in vitro. Forskolin, a stimulator of adenylate cyclase, increased the medium calcium concentration at 96 hr of incubation, indicating enhanced bone resorption. Bone resorption was observed between 1 X 10(-4) and 1 X 10(-6) M forskolin; the maximal effect was at 1 X 10(-5) M and there was no effect at 1 X 10(-7) M. Lactic acid release was increased during the 96 hr of incubation in proportion to the calcium release in the media. The bone acid phosphatase activity was increased and the alkaline phosphatase activity was decreased. Bone carbonic anhydrase activity was increased more than twofold. Forskolin-induced bone resorption was significantly but incompletely inhibited by 10(-4) M acetazolamide, a carbonic acid anhydrase inhibitor. These findings support the concept that carbonic anhydrase plays a significant role in bone resorption.     

Both somatostatin and the caudal neuropeptide, urotensin II, stimulate lipid mobilization from coho salmon liver incubated in vitro

The direct effects of somatostatin-14 (SRIF; synthetic ovine) and the fish caudal neuropeptide, urotensin II (UII; synthetic Gillichthys), on fatty acid (FA) release and on lipolytic enzyme (triacylglycerol lipase) activity were determined on coho salmon liver slices incubated in vitro. FA release was continuously measured by pH-stat titration. Additionally, gas chromatographic analysis of the incubation medium was performed to determine the type and relative composition of medium fatty acid constituents. SRIF and UII both stimulated FA release in a dose-dependent manner; the two peptides appeared to stimulate FA release in an equimolar manner. Maximal response was obtained at 1 X 10(-5) M; ED50 was approximately 2 X 10(-7) M. SRIF-stimulated FA release did not result in differential secretion of any particular FA type. Tissue triacylglycerol lipase activity was significantly enhanced by addition of UII or SRIF (2 X 10(-6) M). Dibutyryl cAMP and IBMX both stimulated FA release and lipase activity; dbcAMP stimulated FA release in dose-dependent manner. These results indicate that SRIF and UII directly enhance lipid mobilization from salmon liver slices and suggest that SRIF- and UII-stimulated lipid mobilization from salmon liver slices is mediated through cAMP.     

Effect of atrial natriuretic peptide on renin release in rat isolated glomeruli

Effects of atrial natriuretic peptide (ANP) on renin release in isolated rat glomeruli were investigated. ANP suppressed renin release by 25% at 5 x 10(-8) M when glomeruli were incubated in a medium containing 1.26 mM calcium (p = 0.0019). When glomeruli were incubated in a calcium free medium containing 2 mM EGTA, ANP suppressed stimulated renin release significantly at 5 x 10(-8) and 5 x 10(-9) M by 25% (p = 0.0204, and p = 0.0101, respectively). These results indicate that ANP suppresses renin release in a dose dependent manner, probably through a calcium independent process.     

Prostaglandin biosynthesis does not participate in isoproterenol-induced renin release

Rat renal slices were incubated in two different media. One was a normal K, physiological saline solution and the other a high K medium. Renin release was measured every 15 min in the presence and absence of 10(-6) M isoproterenol and also in the presence and absence of aspirin, 0.8 or 1.6 X 10(-5) M. In all experiments renin release was linear during the 75 min of incubation. Isoproterenol increased renin release by approximately 100%. This was the case even in the presence of aspirin which significantly inhibited prostaglandin release (PGE2, PGF2 alpha and 6-keto-PGF1 alpha). Nor was there any reduction in the basal secretory rate by aspirin alone. These data are taken to indicate that aspirin in pharmacological doses does not interfere with either in vitro basal release rates of renin, nor the response to B agonists. It is also suggested that B agonists do not exert their effect by stimulating prostaglandin secretion.     

The inhibitory effects of calmodulin antagonists on TSH-stimulated thyroid hormone release by mouse thyroid lobes

Calcium ions have been shown to play a mojor regulatory role in the release of various hormones from a wide variety of endocrine organs. More recently, in vitro evidence suggests that a calcium-binding protein, calmodulin, is also involved in the release of many hormones. So we examined the effects of several types of calmodulin antagonists on TSH-stimulated thyroid hormone release in vitro. Mouse thyroid lobes (one thyro-tracheal unit/tube) were incubated in Krebs-Ringer bicarbonate buffer at 37 degrees C for 4h. Free thyroxine (fT4) released in the incubation medium, thyroidal cAMP and calmodulin content were measured by RIA. TSH (5 mU/ml) and dibutyryl cAMP (DBC) (200 micrograms/ml) caused a 2-4 fold increase in thyroidal release of fT4. The stimulatory effects of TSH on fT4 release were significantly inhibited by trifluoprazine and prenylamine lactate at the concentration of 5 X 10(-5) M. More specific calmodulin antagonists, W-7 and W-13, were also shown to inhibit TSH stimulation of fT4 release at the concentration of 5 X 10(-5) M. In contrast, TSH stimulation of fT4 release was not depressed by non-specific antagonists, W-5 or W-12, at the same concentration as 5 X 10(-5) M. Further, W-13 also markedly inhibited DBC-stimulated fT4 release. Neither TSH nor PGI2 altered the thyroidal calmodulin content, dissociating with a marked increase in the cAMP concentration. These results suggest that calmodulin plays an important role in TSH-stimulated thyroid hormone release and further that this mechanism exists, at least in part, at the site subsequent to the generation of cAMP.     

Hydrogen sulfide regulates cAMP homeostasis and renin degranulation in As4.1 and rat renin-rich kidney cells

The present study aims to investigate the regulatory effect of hydrogen sulfide (H(2)S) on cAMP homeostasis and renin degranulation in As4.1 and rat renin-rich kidney cells. It was found in the present study that NaHS at 0.1-10 μM significantly decreased cAMP production in As4.1 cells treated with isoproterenol (a β-adrenoceptor agonist), forskolin (an adenylyl cyclase activator), or 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor). NaHS at 10 μM suppressed adenylate cyclase activity but stimulated phosphodiesterase activity. We continued to study whether H(2)S may mediate cAMP-dependent renin degranulaion in As4.1 cells. It was found that NaHS at 0.1-10 μM significantly increased intracellular renin protein level. Moreover, NaHS reversed the declined renin content within As4.1 cells and normalized the upregulated renin activity in the culture medium of As4.1 cells treated with the above three stimuli. RT-PCR showed that cystathionine-γ-lyase is the main enzyme to produce endogenous H(2)S in As4.1 cells. Overexpression of cystathionine-γ-lyase increased endogenous H(2)S production and suppressed isoproterenol-induced renin release, suggesting that endogenous H(2)S may also inhibit renin release from As4.1 cells. We also tested whether H(2)S has a similar effect in renin-rich kidney cells. It was found that isoproterenol elevated intracellular cAMP level and extracellular renin activity but decreased renin protein level in the renin-rich kidney cells. Pretreatment with NaHS abolished these effects. In conclusion, H(2)S regulates cAMP homeostasis via inhibition of adenylate cyclase and stimulation of phosphodiesterase. Our findings suggest that H(2)S plays a critical role in regulation of renin degranulation in As4.1 and rat renin-rich kidney cells.     

Functional compartmentalization of arginine-vasopressin-activated cyclic AMP in anterior pituitary gland: the presence of a compartment activated by prostaglandin E2

AVP(10(-8)-10(-6)M) increased ACTH as well as PGE2 release from rat anterior pituitary quarters in vitro in a concentration dependent manner. IBMX (0.1 mM), a phosphodiesterase inhibitor, increased the ACTH response to AVP. The cAMP content in pituitary tissue was increased by AVP. Cyclooxygenase inhibition by indomethacin(1.4 X 10(-5) M) or diclofenac (1.8 X 10(-5)M) led to a potentiation of AVP-evoked ACTH secretion and to a decrease in AVP-stimulated cAMP formation. PGE2(10(-6)M) significantly increased pituitary cAMP content and indomethacin did not affect cAMP levels activated by PGE2. PGE2 attenuated the AVP-induced ACTH release. These results indicate that at least two functional compartments of AVP-activated cAMP responses are involved in the AVP-induced ACTH release. One compartment is directly activated by AVP and participates in the propagation of AVP-induced ACTH release. The second compartment is activated by PGE2. The contribution of the second compartment to the regulation of ACTH secretion is not well understood since PGE2 shows an inhibitory effect on AVP-induced ACTH secretion.     

Actin mediated release of ATP from a myosin-ATP complex.

The apparent second-order rate constant, ka-2, of actin binding to a myosin-ATP state (M*.ATP) and releasing ATP to the medium has been determined by two methods. The first was the measurement of the amount of ATP released when actin was added to the intermediate state, M*.ATP; the second was the measurement of oxygen exchange between ATP and HOH. A quantitative treatment of ATP in equilibrium HOH exchange is given to allow extraction of elementary rate constants from the data. Agreement between the two methods was good and at low ionic strength and 23 degrees C, ka-2 is 6 X 10(5) M-1 s-1 which is about one-third the value of the apparent second-order rate constant, ka4, of actin binding to the myosin product state (M**.ADP.Pi). The determination of ka-2 allows a lower limit of 6 s-1 to be placed upon the first-order rate of ATP release from AM.ATP. This is to be compared with a value of less than or equal to 1.5 X 10(-4) s-1 for the equivalent steps of the myosin scheme; thus actin enhances the rate by a factor of 4 X 10(4) or more. A greater proportion of the bound ATP is released to the medium as ATP with increasing actin concentration. This reflects the contribution to rate limitation at saturating actin concentration of steps between myosin states dissociated from actin.     

P1 and P2 purinergic receptor signal transduction in rat type II pneumocytes

Extracellular ATP is a potent agonist of surfactant phosphatidylcholine (PC) exocytosis from type II pneumocytes in culture. We studied P1 and P2 receptor signal transduction in type II pneumocytes. The EC50 for ATP on PC exocytosis was 10(-6) M, whereas the EC50 for ADP, AMP, adenosine, and the nonmetabolizable ATP analogue alpha,beta-methylene ATP was 10(-4) M. The rank order of agonists for PC exocytosis was ATP greater than ADP greater than AMP greater than adenosine greater than alpha,beta-methylene ATP. The rank order of agonists for phosphatidylinositol (PI) hydrolysis was ATP greater than ADP, whereas AMP, adenosine, and alpha,beta-methylene ATP did not stimulate PI hydrolysis. ATP (10(-4) M) caused a 15-fold increase in adenosine 3',5'-cyclic monophosphate (cAMP) production, and the nonmetabolizable adenosine analogue 5'-N-ethylcarboxyamidoadenosine (10(-6) M) increased cAMP production threefold. The effects of both these agonists on cAMP production were completely inhibited by the adenosine antagonist 8-phenyltheophylline (10(-5) M). The effects of ATP (10(-4) M) on PC exocytosis were inhibited 38% by 10(-5) M 8-phenyltheophylline. Thus, ATP regulates PC exocytosis by activating P2 receptors, which stimulate PI hydrolysis to inositol phosphate, as well as by activating P1 receptors, which stimulate cAMP production. Interactions between the P1 and P2 pathways may explain the high potency of extracellular ATP as an agonist of PC exocytosis.     

Cyclic AMP modulation of adrenoreceptor-mediated arterial smooth muscle contraction

We examined the effects of cyclic AMP (cAMP) on the intracellular Ca2+ release in both the intact and skinned arterial smooth muscle. The amount of Ca2+ in the sarcoplasmic reticulum (SR) was estimated indirectly by caffeine-induced contraction of the skinned preparation and directly by caffeine-stimulated 45Ca efflux from the previously labeled skinned preparation. The norepinephrine-induced release contraction was markedly enhanced by dibutyryl cAMP (dbcAMP) and reduced by propranolol. The stimulatory effect of dbcAMP was best observed when the muscle was exposed to 10(-5) M dbcAMP and 2 X 10(-6) M norepinephrine was used to induce the release contraction. 10(-5) M cAMP had no effect on the Ca2+-induced contraction or on the pCa-tension relationship in the skinned preparation. This concentration of cAMP increased Ca2+ uptake into the SR of the skinned preparation when the Ca2+ in the SR was first depleted. 10(-5) M cAMP stimulated Ca2+-induced Ca2+ release from the SR after optimal Ca2+ accumulation by the SR. The results indicate that the stimulatory effect of cAMP on the norepinephrine-induced release contraction could be due to enhancement of the Ca2+-induced Ca2+ release from the SR in arterial smooth muscle.     

Direct and Continuous Detection of ATP Secretion from Primary Monolayer Cultures of Bovine Adrenal Chromaffin Cells

A method was developed for direct and continuous detection of secretion of ATP from primary monolayer cultures of bovine adrenal chromaffin cells. ATP, which is costored with catecholamines within adrenal chromaffin cells, was released into the incubation medium, where it reacted with firefly luciferin-luciferase producing light detected by a photomultiplier located directly below the culture well. Acetylcholine, nicotine, the Ca2+ ionophore A23187, BaCl2, and KCl induced release of ATP. Induction of release of ATP by acetylcholine was dose dependent, with a threshold at 10(-7) M and a maximum at 10(-4) M. The dose-response curve for nicotine was bell shaped, with a threshold at 10(-7) M, a maximum at 10(-5) M, and diminished release at higher concentrations, an observation indicative of desensitization. Investigation of the initial rates of ATP secretion revealed that 10(-4) M nicotine actually induced release of ATP at a faster rate than 10(-5) M nicotine. However, the rate of ATP release evoked by 10(-4) M nicotine began to decline by 6 s, a result indicating the onset of receptor desensitization, whereas release induced by 10(-5) M nicotine continued unabated. Induction of release of ATP by acetylcholine or nicotine was biphasic, with a rapid, initial phase of release followed by a plateau at 0.5-1.5 min and a second phase of release beginning at 1.5-2 min, reaching a maximum by 2-3 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelin inhibits renin release from isolated rat glomeruli

The effect of endothelin on renin release from isolated rat glomeruli was examined. Endothelin inhibited basal renin release in a dose-dependent manner with an IC50 of 1.0 x 10(-9) M. Endothelin also inhibited renin release stimulated by isoproterenol (10(-5) M). Nifedipine (10(-5) M), a calcium channel blocker, induced an increase in renin release. Endothelin did not affect this nifedipine-induced renin release. These results suggest that endothelin inhibits renin release via a calcium entry mechanism and increases intracellular calcium.