Effects of prostaglandin H2, prostaglandin E2, and arachidonic acid on parathyroid hormone and antidiuretic hormone activation of rat kidney adenylate cyclase

These experiments examine interactions of arachidonic acid; the substrate for prostaglandin cyclooxygenase, prostaglandin (PG)H₂, a key endoperoxide intermediate in prostaglandin synthesis; and prostaglandin (PG)E₂, an important prostaglandin produced within the kidney; with adenylate cyclase activity in renal cortex, outer medulla, and inner medulla. In addition, the effects of arachidonic acid, PGH₂, and PGE₂ on parathyroid hormone (PTH) activation of adenylate cyclase in cortex, and of antidiuretic hormone (ADH) activation of that enzyme in outer and inner medulla are examined. Arachidonic acid elicited a concentration-dependent inhibition of basal and PTH-stimulated adenylate cyclase activity in renal cortex. Concentration-dependent inhibition by arachidonic acid of basal and ADH-stimulated adenylate cyclase activity was observed in outer and inner medulla. PGH₂ inhibited basal activity in all three areas of the kidney. There was also inhibition by PGH₂ of medullary ADH and cortical PTH stimulation. PGE₂ stimulated adenylate cyclase in all three areas. PGE₂ had no effect upon PTH stimulation in cortex and was additive with ADH in outer and inner medulla. PGE₂ stimulation was inhibited by arachidonic acid, and this inhibition seemed competitive. Inhibition by both arachidonic acid and PGH₂ was not destructive. Experiments with [1-¹⁴C]arachidonic acid and indomethacin suggest that the inhibition by arachidonic acid was actually mediated by arachidonic acid and not a metabolite. Both PGH₂ and arachidonic acid inhibition was independent of phosphodiesterase. This activation by product, PGE₂, and inhibition by its precursors, arachidonic acid and PGH₂, provide a possible mechanism by which the prostaglandin system could modulate adenylate cyclase responsiveness to hormonal activation.     

Epinephrine stimulation of fat cell adenylate cyclase: regulation by guanosine-5'-triphosphate and magnesium ion

Guanosine-5′-triphosphate (GTP) affects the activity and responsiveness of the fat cell adenylate cyclase to epinephrine. In the absence of free magnesium ion, the presence of GTP is an absolute requirement for epinephrine stimulation. Half-maximal activation of both basal and stimulated adenylate cyclase activity occurs at a GTP concentration of 0.6 mM. In the presence of 5 mM MgCl₂, GTP is no longer required but enhances epinephrine stimulation. The half-maximal concentration for this effect occurs at approximately 10 μM GTP. At high (5 mM) magnesium ion concentrations GTP inhibits basal adenylate cyclase activity.GTP lowers the apparent affinity of adenylate cyclase for ATP while simultaneously increasing the velocity of the catalytic reaction, possibly by competing with ATP at the active site as well as by binding at a regulatory site. This effect is observed on both basal and epinephrine-stimulated activities. Epinephrine itself raises the apparent K_M for ATP and the V_max of adenylate cyclase. The interdependence of these effects suggests that transient changes in the levels of GTP, ATP, and magnesium ions in the fat cell may directly regulate the responsiveness of adenylate cyclase to epinephrine.     

Regulation of hormone(PTH and PGE1)-stimulated adenylate cyclase by renal cytosolic factors

Cytosolic factors in a 50–75% (NH₄)₂SO₄ fraction of the 105 000 × g supernatant of the renal cortex modulated adenylate cyclase activity in membrane preparations enriched in renal tubular cell basal-lateral membranes. The crude factor preparation had no effect on basal activity but it contained components that augmented the stimulation of the enzyme by NaF, parathyroid hormone (PTH), prostaglandin e₁ (PGE₁), and inhibited the activation of the enzyme by GMP-PNP. The factor(s) potentiating the stimulation by the hormones was partially purified (13-fold) by DEAE-cellulose and Sephadex G-75 chromatography. During purification, the component(s) that increased hormone-stimulated adenylate cyclase was separated from those affecting the activity in the presence of NaF and GMP-PNP. The factor(s) enhanced the PTH- and PGE₁-stimulated enzyme at all concentrations of hormone, suggesting that the affinity for the hormone was not affected. The factor(s) was heat-stable. Partial proteolysis with chymotrypsin greatly reduced the ability of the factor(s) to enhance hormonal responsive adenylate cyclase. However, the factor(s) was resistant to trypsin digestion. The effect of the factor was not due to GTP, nor was GTP necessary for its action. Ca²⁺ was not needed for the enhancing activity of the factor(s). These findings suggest the presence in the cytosol of the kidney cortex of a protein(s) that regulates the response of renal adenylate cyclase to hormones. The relationship between this kidney cytosolic factor and those reported in other tissues remains to be established.     

Catecholamine and divalent cation effects on frog liver adenylate cyclase

Frog liver adenylate cyclase was characterized with respect to divalent cation interaction and hormonally stimulated activities. The enzyme catalyzed the synthesis of cyclic [³²P]3′,5′-AMP from α-³²P-labeled ATP. The activity of the enzyme was linear with time and00 protein concentration. The K_m for ATP was 0.5 mM, in the presence or absence of stimulators. The temperature optimum was 25°. GTP (10^?4M) increased the stimulation of adenylate cyclase by epinephrine. Similar activities were obtained using 5 mM Mg²⁺ or Mn²⁺. At higher concentrations, both ions inhibited epinephrine-stimulated, but not basal or fluoride-stimulated activities. Approximately equivalent hormonal stimulation was obtained with maximal stimulating concentrations of epinephrine, isoproterenol, glucagon, and prostaglandin E₁. Norepinephrine was less stimulatory. Only catecholamine-stimulated activities were inhibited by propranolol (10^?5M). The data suggest that catecholamines stimulate frog liver adenylate cyclase through interactions with β adrenergic receptors. The adenylate cyclase in frog liver differs from its mammalian counterpart in its response to temperature and maximally stimulatory concentrations of hormones.     

Catecholamine stimulated myocardial adenylate cyclase: Effects of nucleotides

The effects of several nucleotides on canine myocardial adenylate cyclase have been investigated. Basal, fluoride and isoproterenol stimulated enzyme activities were studied. With adenosine 5′ triphosphate at ? 1.5 mm guanosine 5′-triphosphate increased basal and isoproterenol stimulated cyclase activity severalfold. Flouride stimulated activity was inhibited. Significant stimulation occurred at 10 nm guanosine 5′-triphosphate and effects were maximal by 1 μm. In order of decreasing potency the nucleoside 5′-triphosphates were guanosine > deoxyguanosine > uridine > thymidine > cytidine. Insosine 5′-triphosphate and xanthosine 5′-triphosphate were inactive. Although guanosine 5′-triphosphate was most potent, all other guanyl nucleotides tested, including guanosine 5′-diphosphate, guanosine 5′-monophosphate and cyclic guanosine, 3′,5′-monophosphate shared this effect. Guanosine was inert. The stimulatory action of guanosine 5′-triphosphate was exhibited after a lag period of several minutes. When substrate (adenosine 5′-triphosphate) concentrations were lowered to 0.05 mm, stimulation by isoproterenol was virtually dependent on the presence of guanosine 5′-triphosphate. Nucleotides did not affect the apparent K_m for enzyme stimulation by isoproterenol which was 1–2 × 10^?6m with or without guanosine 5′-triphosphate. Propranolol effectively blocked the augmented stimulation by isoproterenol observed in the presence of guanosine 5′-triphosphate. The data indicate that guanyl nucleotides are capable of markedly altering the sensitivity of myocardial adenylate cyclase to catecholamine stimulation.     

Evidence for parathyroid hormone sensitive adenylate cyclase in rat glomeruli

Isolated rat glomeruli contain an adenylate cyclase system. The amount of cyclic AMP formed increased progressively with incubation time. The rate of cyclic AMP formation increased linearly with glomerular protein concentration. This adenylate cyclase system was temperature and pH dependent. There was no evidence for saturation of the enzyme with substrate up to 10^?2 M ATP. Adenylate cyclase was strikingly activated by fluoride (10^?2 M). Purified bovine parathyroid hormone (PTH) and synthetic 1–34 bovine PTH fragment both stimulated adenylate cyclase activity: maximum activity was 3.9 to 5.4 basal activity and k_m close to 10^?7 M. Epinephrine and isoproterenol produced a slight stimulation whereas salmon calcitonin, glucagon, norepinephrine and antidiuretic hormone were inactive. The demonstration of PTH activated adenylate cyclase in glomeruli raises the possibility of a role for this hormone in regulation of glomerular activity.     

Adenylate cyclase activity of avian granulosa: Effect of gonadotropin-releasing hormone

The activity of adenylate cyclase in crude and purified preparations of hen granulosa was investigated by measuring the production of cyclic AMP during a 20-min incubation at 30°. Both NaF and guanosine 5′-β,γ-imidotriphosphate (Gpp(NH)p), the nonhydrolyzable analog of GTP, stimulated enzyme activity in a dose-related manner. Ovine LH and, to a lesser extent, ovine FSH also activated adenylate cyclase in the presence of half-maximally stimulating concentrations of Gpp(NH)p (10^?7M). Gonadotropin-releasing hormone (10^?10–10^?4M) failed to significantly affect basal- or gonadotropin-promoted adenylate cyclase activity or the production of cyclic AMP by intact granulosa cells. Progesterone production, on the other hand, was enhanced by gonadotropin-releasing hormone (GnRH) (10^?8–10^?6M). It is suggested that in chicken granulosa cells, as in the mammalian pituitary cells, the adenylate cyclase/cyclic AMP system is not a mediator of GnRH action.     

Inhibition of histamine-sensitive adenylate cyclase from guinea pig fundic gastric mucosa by arachidonic acid and by an analog of prostaglandin endoperoxide PGH2

The effects of prostaglandin precursors, namely an analog of prostaglandin endoperoxide PGH₂ [(15S)-hydroxy-9α,11α-(epoxymethano)prosta-5, 13-dienoic acid] and arachidonic acid, were assessed on gastric adenylate cyclase activity from cell-free preparations of guinea pig fundic mucosa. The two precursors were tested against basal adenylate cyclase activity and that stimulated by histamine (10^?4M), by PGE₂(10^?4M), by 5′-guanylylimidodiphosphate [Gpp(NH)p] (10^?4M) and by NaF(10^?2M). PGH₂ analog (10^?4M) and arachidonic acid (10^?4M) both inhibited to a similar extent adenylate cyclase stimulated by histamine or by NaF, but not that stimulated by PGE₂ or by Gpp(NH)p. Neither agent significantly affected basal adenylate cyclase levels. In the presence of indomethacin (10^?4M), basal adenylate cyclase activity remained unchanged but the inhibitory effect of arachidonic acid was almost entirely abolished, suggesting that such inhibitory effect may be caused by prostaglandin endoperoxides generated from arachidonic acid in the course of assay. Moreover, indomethacin did not attenuate PGH₂ inhibition of histamine action. Unlike arachidonic acid, which is a natural metabolic precursor of PGE₂, arachidic acid did not significantly influence histamine-stimulated adenylate cyclase activity. These results suggest that the prostaglandin endoperoxides may have an inhibitory effect on histamine-sensitive ciclic AMP generation in gastric mucosa.     

Cardiac adenylate cyclase: Kinetics of synergistic activation by guanosine-5′-triphosphate (GTP) and glucagon

Kinetic analysis of cardiac adenylate cyclase activation by GTP and glucagon has been performed. The maximal stimulation produced by 1 mm GTP or 10 μm glucagon alone was approximately 2-fold over basal activity; with both GTP and glucagon, activity was synergistically increased 5-fold over basal.The apparent K_D for activation of cardiac adenylate cyclase by glucagon was 0.16 μm and was not altered by maximal or submaximal concentrations of GTP. The apparent K_D for activation by GTP was 4.18 μm; this value was not altered by maximal or submaximal concentrations of glucagon. The apparent K_m values for MgATP and apparent V_m values were determined in the basal state and in the presence of 10 μm glucagon and various fixed levels of GTP (none, 1, 10, and 100 μm) and in the presence of 1 mm GTP and various fixed concentrations of glucagon (none, 0.1, 1, 10 μm). No difference in the apparent K_m for the substrate was determined under these various conditions (mean of all determination = 0.280 ± 0.01 mm). The apparent V_m values, however, increased greatly in the presence of the two effectors. The V_m values determined under basal conditions or with GTP alone or glucagon alone were 51 ± 3, 67 ± 3, and 79 ± 3 pmol/mg protein/min, respectively. At saturating levels of both GTP and glucagon the V_m value was 255 ± 11 pmol/mg protein/min.The kinetic data for the activation of the cardiac adenylate cyclase by GTP and glucagon were analyzed further using a rate equation which expresses the high degree of synergism between GTP and glucagon in terms of interaction constants.     

Characterization of the adenylate cyclase of the rat corpus luteum during luteolysis induced by a prostaglandin F2α analogue

Rats with superluteinized ovaries were injected with the prostaglandin F_2α (PGF_2α) analogue cloprostenol to induce luteolysis. The treatment led to decreased adenylyl cyclase response to hCG and isoproterenol in ovarian homogenates while the response to forskolin remained unchanged indicating that the catalytic unit of the enzyme was not affected by the treatment. The activation of adenylyl cyclase by Mg²⁺ or the non-hydrolysable guanosine triphosphate (GTP) analogue guanosine-5′-(β,γ-imido)-triphosphate (GMP-P(NH)P) was not altered by the treatment with cloprostenol. Both basal and hormone-stimulated adenylyl cyclase activity increased with increasing concentration of GMP-P(NH)P. Unstimulated adenylyl cyclase continuously increased with increasing concentrations of Mg²⁺. The same applied to forskolin. The dependence of the adenylyl cyclase stimulation by hCG and isoproterenol on Mg²⁺ was complex. It is postulated that PGF_2α induces the attenuation of ovarian adenylyl cyclase by a modification in the coupling of the hormone-receptor to the Nβ-component of the adenylyl cyclase complex while the catalytic unit remains unchanged.     

Hormone-sensitive adenylate-cyclase in isolated rabbit glomeruli

Adenylate cyclase activity and its hormonal control was measured in glomeruli isolated from collagenase treated rabbit kidneys. Experiments were performed on glycerol treated frozen glomeruli since the freezing procedure was shown to greatly enhance adenyl-cyclase activity. It was found that cAMP generation was linear 1) as a function of time up to 60 min and 2) as a function of the number of glomeruli up to 50 per sample. All further determinations were made using samples of ten glomeruli each, incubated for 30 min at 30 °C. Under such conditions, the mean control adenylate cyclase activity was found to be equal to 0.65 ± 0.04 SEM pmoles/10 gl/30 min. Glomerular adenylate cyclase was stimulated by PTH. From the mean dose response curve, an apparent k_m value (2.3 × 10^?7 M) and a maximal stimulation ratio (V_max equal to 272% of the control) were calculated. PTH stimulation up to 5-fold could be observed in the most sensitive preparations. Low concentrations of arginine vasopressin produced a small but significant stimulatory effect with a mean value equal to 139 per cent of that measured under the control conditions. No effect on adenylate cyclase activity was obtained either with angiotensin II or isoproterenol.     

Epinephrine-sensitive adenylate cyclase of human fat cell ghosts: Stabilization of enzyme activity by fluoride and nucleotides

The effects of fluoride and nucleotides on the stability of the human fat cell adenylate cyclase were studied. Preincubation of fat cell ghosts in 1 mM KHCO₃ at 30°C for 20 min resulted in a substantial loss of basal, epinephrine- and NaF-stimulated enzyme activities. Inactivation was almost completely prevented by nucleotides (ATP, GTP and GMP-P(NH)P). ¹ Epinephrine was without influence on the time course and degree of inactivation. NaF (20 mM), however, like the nucleotides protected from inactivation. In contrast to epinephrine NaF failed to stimulate enzyme activity in membranes pretreated with GMP-P(NH)P (0.1 mM). Our results show that NaF exerts two distinct effects on the human fat cell adenylate cyclase: stimulation of enzyme activity and protection from inactivation. Both effects appear to be related to the binding of nucleotides.     

Effects of PGE2, misoprostol,and enprostil on guinea pig enterocyte adenylate cyclase

Prostaglandins of the E series (PGE) are known to stimulate intestinal water and electrolyte secretions via the activation of the enterocyte adenylate cyclase. Their methylated synthetic analogs misoprostol and enprostil induced diarrhea in 5–13% of the patients in most clinical studies. In order to elucidate the role of PGE-adenylate cyclase interaction in these phenomena, we studied the stimulation of adenylate cyclase by native prostaglandin E₂ (PGE₂) and synthetic PGE analogs on isolated guinea pig intestinal epithelial cells. PGE₂ stimulation of adenylate cyclase was dose-dependent, reaching a maximum for 3×10^–4 M, with an EC₅₀ of 3.7×10^–6 M. The Hill analysis of the concentration-response curve gave a straight line, with a slope close to 1. The effect of PGE₂ was strictly additive to that of 10^–5 M forskolin, whereas it was decreased in terms of potency by 10^–9 M cholera toxin. Somatostatin-14 markedly inhibited PGE₂ stimulation by 37% and 45% with 10^–9 M and 10^–6 M, respectively. The two PGE methylated analogs misoprostol and enprostil were less potent than PGE₂ in stimulating adenylate cyclase in our model. We conclude that (1) the mechanism of PGE₂-mediated intestinal water and electrolyte secretions involves the activation of one single class of specific receptors coupled to enterocyte adenylate cyclase via a cholera toxin-sensitive regulatory subunit (Gs); (2) somatostatin is a potent inhibitor of PGE-stimulated adenylate cyclase and thus could be a interest in the treatment of certain PGE-induced secretory diarrheas; and (3) the diarrheogenic effects of misoprostol and enprostil are unlikely to be mediated primarily by adenylate cyclase activation.     

Properties of LH-sensitive adenylate cyclase in purified plasma membranes from rat ovary.

A procedure for the purification of ovarian plasma membranes (PM) is described and applied to ovaries from immature (25-day-old) rats stimulated with pregnant mare serum gonadotropin (PMSG). Luteinizing hormone (LH)-sensitive adenylate cyclase, 5′-nucleotidase and the binding of ¹²⁵I-labeled human chorionic gonadotropin (hCG) served as PM markers. Judged by these three criteria, 8–15-fold purification of PM was achieved, with a yield of 30–40% of the activity present in the crude homogenate. Optimal conditions for the response of rat ovarian adenylate cyclase to LH and hCG were defined with respect to time, pH and the concentrations of Mg²⁺, ATP, GTP and β,γ-imidoguanosine-5′-triphosphate [Gpp(NH)p].     

Effects of hormone treatment and age on the stimulation of rat anterior pituitary adenylate cyclase and cyclic adenosine 3',5' monophosphate by hypothalamic extract

Crude ovine hypothalamic extract in vitro significantly stimulated adenylate cyclase activity and LH release by pituitary gland obtained from newborn rats (0.25–0.5 days old) and increased cyclic adenosine 3′,5′-monophosphate (cyclic AMP) in 2-day-old rats. In adult rats, the stimulation of adenylate cyclase activity and cyclic AMP formation, but not LH release, induced by hypothalamic extract was inhibited by adrenalectomy. In vivo administration of pharmacologic doses of 1 mg dexamethasone asd 100 μg thyroxine for 1 day markedly reduced the effect of 0.2 mg/ml but not 1 mg/ml hypothalamic extract on cyclic AMP concentrations. The action of the latter dose of hypothalamic extract on cyclic AMP was inhibited by injection of dexamethasone and thyroxine for 3 days, while the stimulation of LH release was preserved. Such dissociation between activation of the adenylate cyclase-cyclic AMP system and LH release suggests that cyclic AMP formation may not be essential for increased release of LH. Adrenalectomy did not inhibit stimulation of adenylate cyclase activity or cyclic AMP formation caused by prostaglandin E₁ (PGE₁). Hypothalamic extract or PGE₁ added to intact pituitary in vitro increased protein kinase activity and decreased the protein binding of exogenous cyclic AMP, reflecting the increased generation of endogenous cyclic AMP.     

Calcium-dependent proteolytic stimulation of adenylate cyclase in platelets from spontaneously hypertensive rats

Abnormalities of platelet aggregation and cyclic nucleotide metabolism are present in hypertension. We observed a greater increase in the level of cyclic adenosine monophosphate (AMP) after prostaglandin E₁ (PGE₁) stimulation and a lack of decrease of this cyclic nucleotide by epinephrine in platelets from spontaneously hypertensive rats (SHR) as compared to normotensive rats. The difference in cyclic AMP production between SHR and control rats in response to PGE₁ is dependent upon platelet exposure to calcium. Since calcium and cyclic AMP are closely related and are both abnormally regulated in hypertension, we have studied the effect of calcium on adenylate cyclase activity. We show here that two forms of endogenous calcium-dependent proteases (membrane-bound and soluble) stimulate the basal activity and the hormonal responsiveness of adenylate cyclase. The sensitivity of calcium-dependent proteolytic control of adenylate cyclase to very-low concentrations of calcium indicates that the regulation may be physiologically important. Furthermore, calcium exerts a greater influence on platelet adenylate cyclase from SHR than on that from normotensive rats. The adenylate cyclase defect seems to be located in the membrane fraction and may, therefore, result from an increase in the activity of the membrane-bound calcium-protease or may be intrinsic to adenylate cyclase itself. The exact site that is sensitive to proteolysis remains to be established.     

Characteristics of an adenylate cyclase enhancing factor from mouse pancreatic islet cytosol

Summary The role of cytosolic components in the regulation of mouse pancreatic islet adenylate cyclase activity was studied. Addition of mouse islet cytosol (27000 g supernatant of mouse islet sonicate), devoid of adenylate cyclase activity itself, increased adenylate cyclase activity by 93±17% (n = 9) in the 27000 g total particulate fraction of mouse islets. Addition of GTP stimulated adenylate cyclase activity by 91±11% (n = 13) or to the same degree as cytosol. Like GTP, the substance causing the enhancing activity of the cytosol was found to be dialysable, resistant to heat, sensitive to charcoal treatment and alkaline phosphatase and insensitive to digestion with trypsin. However, in contrast to the stimulation by GTP, the stimulation by cytosol was not inhibited by guanosine 5-0-(2-thiodiphosphate), and furthermore, the effects of cytosol and GTP were additive. Neither NAD nor phosphoenolpyruvate stimulated adenylate cyclase activity. The cytosolic factor did not confer sensitivity towards glucose, Ca²⁺ or Ca²⁺-calmodulin on adenylate cyclase. The results demonstrate that mouse pancreatic islets contain a phosphocompound (or several compounds) distinct from GTP and capable of markedly stimulating adenylate cyclase. The identity of the compound and its physiological significance remain to be established.     

Antioxidant inhibition of prostaglandin production by rat renal medulla.

Antioxidant effects upon renal production of both prostaglandins and cAMP were investigated using slices of rat inner medulla. Synthetic antioxidants were more potent inhibitors of prostaglandin production than were naturally occurring antioxidants. Synthetic compounds 2,7-naphthalenediol, and Santoquin^® (Ethoxyquin) caused a 60% inhibition of prostaglandin E₂(PGE₂) synthesis at a concentration of 0.01 mM. Ascorbic acid caused only a 30% inhibition at a concentration of 10 mM. Antioxidant inhibition of prostaglandin production was also observed following arachidonic acid addition. Antioxidants that reduced PGE₂ synthesis also reduced PGF_2α synthesis. Test agents found to reduce prostaglandin synthesis also lowered cAMP content. 2,7-Naphthalenediol elicited a dose-dependent decrease in both prostaglandin synthesis and cAMP content. While PGE₁ did not increase cAMP in control slices, the low cAMP level produced by Santoquin was increased to control values by PGE₁. Furthermore, Santoquin and 2,7-naphthalenediol did not alter arginine vasopressin-stimulated cAMP content. By contrast, inhibition of the arginine vasopressin stimulation by butylated hydroxyanisole suggested additional effects by this agent. These results are consistent with the hypothesis that endogenously produced PGE₂ can exert a hormonelike action in the inner medulla by increasing cAMP content. Advantages of the inner medullary slice system compared to homogenates for investigation of the actions of antioxidants or other agents thought to alter prostaglandin synthesis are discussed.     

Free radicals generated by xanthine oxidase-hypoxanthine damage adenylate cyclase and ATPase in gerbil cerebral cortex

The generation of superoxide radicals from xanthine oxidase-hypoxanthine in a particulate fraction of gerbil cerebral cortex influenced the activity of the synaptic enzyme adenylate cyclase, as well as Mn²⁺- and Na⁺,K⁺-sensitive forms of ATPase. Low concentrations of xanthine oxidase actually elevated the sensitivity of adenylate cyclase to GTP, GTP + norepinephrine (NE), and forskolin but not significantly to Mn²⁺. Higher levels of xanthine oxidase elicited a marked inhibition of these responses. The stimulation of adenylate cyclase mechanisms requiring GTP (GTP, forskolin, and NE) was more susceptible than was Mn²⁺, suggesting that the guanine nucleotide stimulatory protein was more vulnerable to free radical attack than the catalytic site of adenylate cyclase. superoxide dismutase (SOD), but not catalase, partially protected the forskolin-sensitive enzyme from the action of xanthine oxidase-hypoxanthine. A combination of SOD plus catalase preserved enzyme responses to forskolin. In comparison, additions of SOD plus mannitol or catalase plus flunarizine were less effective. The sensitivity of the particulate ATPase to Mn²⁺ was more labile to the consequence of superoxide formation than Na⁺, K⁺-ATPase. In this regard the Ca²⁺, Mg²⁺ sensitivity of the enzyme was reduced only to a marginal extent. The findings might be analogous toin vivo data in which cerebral adenylate cyclase and Na⁺, K⁺-ATPase are damaged following postischemic reperfusion in gerbils, a process thought to be mediated by free radicals.     

Modulation of follicle-stimulating hormone-sensitive rat testicular adenylate cyclase activity by guanyl nucleotides.

We have studied modulation of FSH-sensitive adenylate cyclase activity in testes of immature rats by guanyl nucleotides. Highly purified hFSH alone stimulated adenylate cyclase activity 2.2-fold over basal levels. Addition of the GTP analog, 5'-guanylyl imidodiphosphate [Gpp(NH)p], caused an additional 2.8-fold augmentation of adenylate cyclase activity to 6 times over basal levels and 3.7 times greater than that seen in the presence of Gpp(NH)p alone. GTP did not significantly stimulate basal levels of adenylate cyclase and augmented FSH stimulated activity by 1.4-fold; other nucleotides were without effect. Half-maximum activation of adenylate cyclase in each instance was produced by approximately similar concentrations of either guanyl nucleotide (about 10 microM). The Km for hormone activation of adenylate cyclase was nearly the same in the presence and absence of Gpp(NH)p. Maximum adenylate cyclase stimulation in the presence of nucleotide and/or hRSH was always less than obtained by fluoride alone. Of all nucleotides tested, only GTP and its analog, Gpp(NH)p, significantly augmented FSH stimulation of testicular adenylate cyclase activity. Gpp(NH)p also markedly inhibited binding of radiolabeled hFSH to testicular receptor, but at a concentration 15-fold greater than that required for significant stimulation of testicular adenylate cyclase activity. The results suggest a specific role for guanyl nucleoside triphosphate in regulation of FSH effects on testicular adenylate cyclase activity.