Adenylate Cyclase Activity In Plasma Membranes Research Articles

Potent and cooperative feedback inhibition of adenylate cyclase activity by calcium in pituitary-derived GH 3 cells

Calcium (Ca 2+) ion concentrations that are achieved intracellularly upon membrane depolarization or activation of phospholipase C stimulate adenylate cyclase via calmodulin (CaM) in brain tissue. In the present study, this range of Ca 2+ concentrations produced unanticipated inhibitory effects on the plasma membrane adenylate cyclase activity of GH 3 cells. Ca 2+ concentrations ranging from 0.1 to 0.8 μM exerted an increasing inhibition on enzyme activity, which reached a plateau (35–45% inhibition) at around 1 μM. This inhibitory effect was highly cooperative for Ca 2+ ions, but was neither enhanced nor dependent upon the addition of CaM (1 μM) to EGTA-washed membranes. The inhibition was greatly enhanced upon stimulation of the enzyme by vasoactive intestinal peptide (VIP) and/or GTP. Prior exposure of cultured cells to pertussis toxin did not affect the inhibition of plasma membrane adenylate cyclase activity by Ca 2+, although in these membranes, hormonal (somatostatin) inhibition was significantly attenuated. Maximally effective concentrations of Ca 2+ and somatostatin produced additive inhibitory effects on adenylate cyclase. The addition of phosphodiesterase inhibitors demonstrated that inhibitory effects of Ca 2+ were not mediated by Ca 2+-dependent stimulation of a phosphodiesterase activity. These observations provide a mechanism for the feedback inhibition by elevated intracellular Ca 2+ levels on cAMP-facilitated Ca 2+ entry into GH 3 cells, as well as inhibitory crosstalk between Ca 2+-mobilizing signals and adenylate cyclase activity.

Evaluation of methods for the isolation of plasma membranes displaying guanosine 5′-triphosphate-dependence for the regulation of adenylate cyclase activity: Potential application to the study of other guanosine 5′-triphosphate-dependent transduction systems

The GTP-dependence for stimulatory and inhibitory regulation of plasma membrane adenylate cyclase activity was measured in plasma membrane fractions isolated from a variety of cell types (platelets, lymphocytes, PC12 cells, GH 3 cells, NBP 2 cells, and hepatocytes). This report shows that the isolation of plasma membranes for the study of GTP-dependent adenylate cyclase activity was, for some cells, enhanced by the exposure of the cells to glycerol prior to cell lysis. The isolation of plasma membranes from other cells, which did not appear to be sensitive to glycerol pretreatment, was enhanced by the removal of heavy particulate matter prior to fractionation of the cell lysate. The regulation of enzyme activity by various agents was found to be dependent upon the presence of (exogenous) GTP to varying degrees, indicating variable contamination of membrane preparations with GTP. It is concluded that (i) exposure of platelets and lymphocytes to glycerol prior to cell lysis decreases subsequent contamination of the plasma membrane preparation with GTP, and (ii) although glycerol pretreatment of other cells does not ensure the subsequent isolation of plasma membrane adenylate cyclase activity displaying high requirements for (exogenous) GTP, it is a reasonable first approach to be used during the development of procedures for the isolation of plasma membranes.

Exogenous steroids alter steroidogenesis in cultured Y-1 adrenal tumor cells by actions preceding cyclic AMP

Using cultured Y-1 mouse adrenal tumor cells which produce 20 alpha-hydroxy-4-pregnen-3-one (20-DHP), it was found that 0.01 mM corticosterone and deoxycorticosterone increased basal and inhibited ACTH-induced 20-DHP production during consecutive 30 and 120 min incubations. Steroid effects were concentration-dependent and reversible. Six other steroids tested did not stimulate 20-DHP production and varied in ability to inhibit ACTH-stimulated steroidogenesis. Experiments demonstrated that 20-DHP production following treatment with cholera toxin, N, 0'-dibutyryl cyclic AMP (dbcAMP), or pregnenolone was not inhibited by exogenous steroids. Corticosterone (0.01 mM) increased basal and inhibited ACTH-induced intracellular cyclic AMP (cAMP) production. Cytochalasin D, a microfilament perturbing agent, inhibited steroid-stimulated 20-DHP production, suggesting that ACTH and steroid stimulation mechanisms were similar. These findings taken together suggest that exogenous steroids can alter steroidogenesis by modifying plasma membrane adenylate cyclase activity.

Does the guanine nucleotide regulatory protein Ni mediate progesterone inhibition of Xenopus oocyte adenylate cyclase?

In Xenopus laevis oocytes progesterone is able to inhibit directly the plasma membrane adenylate cyclase activity and induce reinitiation of meiotic maturation. To determine whether progesterone inhibition is mediated by the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase, Ni, the effect of the Bordetella pertussis toxin (IAP) and limited proteolysis on progesterone action in oocytes was investigated. Treatment of oocyte membranes with islet activating protein (IAP) in the presence of [32P]NAD led to incorporation of radiolabel into a 41 000-dalton membrane protein. However, exposure of isolated oocytes to 100 ng/ml IAP for up to 24 h, or oocyte membranes with concentrations of toxin as high as 100 micrograms/ml, had no effect on either progesterone inhibition of adenylate cyclase or induction of maturation. Similarly, limited alpha-chymotrypsin proteolysis of oocyte membranes failed to modify progesterone-induced inhibition of adenylate cyclase. In contrast, inhibition of human platelet adenylate cyclase by epinephrine, acting via a GTP-dependent, alpha 2-adrenergic receptor-mediated pathway, is almost completely abolished by both IAP treatment and limited proteolysis of platelet membranes. These data indicate that unlike attenuation of platelet enzyme activity, the inhibition of adenylate cyclase in oocyte membranes by progesterone does not occur via a classical Ni-mediated pathway.

Dimethylnitrosamine inhibits the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes and decreases plasma membrane fluidity

The effect of the hepatocarcinogen dimethylnitrosamine on rat liver plasma membrane adenylate cyclase activity and lipid fluidity was assessed. Glucagon-stimulated adenylate cyclase activity exhibited a complex response to increasing concentrations of dimethylnitrosamine, whereas fluoride-stimulated adenylate cyclase activity was progressively inhibited. Maximal inhibitory effects were observed at a concentration of 15 mM in both cases. The activity of detergent-solubilized adenylate cyclase was unaffected by dimethylnitrosamine. ESR analysis using a fatty acid spin probe showed that dimethylnitrosamine produced a marked, dose-dependent reduction in the fluidity of the plasma membrane with a maximal effect occuring at 20 mM. Dimethylnitrosamine also elevated the temperature at which the lipid phase separation occured in rat liver plasma membranes, from 28°C to 31°C. The non-carcinogenic but structurally similar compound, dimethylamine hydrochloride neither inhibited adenylate cyclase nor decreased plasma membrain fluidity. It is suggested that the decrease in membrane fluidity, induced by dimethylnitrosamine, is responsible for the observed inhibition of adenylate cyclase and that dimethylnitrosamine, via its effects on membrane fluidity, could influence plasma membrane function and cellular regulation.

Hepatic adenylate cyclase and phosphodiesterase activity during acute ethionine intoxication

Four hours after the administration of ethionine and in the face of an 80–90% depletion of hepatic ATP there is a twofold increase in cAMP. This increase in cAMP is shown not to be effected by a reduction in phosphodiesterase activity. Both the low K m and high K m forms of the enzyme retain their activity. The purification of liver plasma membranes is not affected by ethionine. There is a significant 20% increase in plasma membrane adenylate cyclase activity following acute ethionine intoxication. This was demonstrable using either ATP or 5′-adenylyl-imidodiphosphate as substrates. The plasma membranes, isolated in the presence of Ca 2+, have a 20-fold higher basal adenylate cyclase activity then previously reported and are not further stimulated by GTP or NaF. A modified protocol for isolating cAMP from the adenylate cyclase reaction is described.

Endogenous biologically active human parathyroid hormone: measurement by a guanyl nucleotide-amplified renal adenylate cyclase assay.

The purpose of the present study was to establish and validate a routine procedure for the measurement of endogenous biologically active human parathyroid hormome (PTH). To accomplish this, we measured the activation of canine renal cortical plasma membrane adenylate cyclase activity produced by PTH standards or test sera in the presence of the hydrolysis-resistant GTP analog, 5′-guanylimidodiphosphate (100 [M). In this system, as little as 3 pg (=0 3 fmol) human PTH-(1–U≃84)′ assay tube produced significant adenylate cyclase activation. Glucagon, human calcitonin, ACTH-(l–), arginine vasopressin, epinephrine, and prostaglandin E1 had little or no activity. A competitive PTH antagonist, [8Nle, 18Nle, 34Tyr]bovine PTH(3–34) amide, completely inhibited enzyme activation produced by exogenous PTH, parathyroid venous effluent serum, or peripheral chronic renal failure serum. Thus, the activity in serum was due specifically to biologically active PTH.The concentrations of biologically active PTH and immunoreac...

Down-regulation in vivo of PGE receptors and adenylate cyclase stimulation.

Down-regulation in vivo of liver plasma membrane receptors for prostaglandin E (PGE) was investigated in Sprague-Dawley rats using the 16,16-dimethyl analogue of PGE2, This analogue was used for subcutaneous injections because it escapes the rapid pulmonic degradation characteristic of PGE and was recognized well by liver plasma membrane receptors. Following treatment with the analogue, the concentration of PGE receptors was significantly decreased (-37%, P less than 0.001), but the binding affinity was not altered. There was no evidence for carry-through of the analogue into the isolated plasma membrane preparation. It was also demonstrated that GTP decreased the binding affinity between PGE and its receptor. Down-regulation of receptor concentration was associated with a significant decrease (P less than 0.001) in PGE1-stimulated plasma membrane adenylate cyclase activity. These data provide the novel demonstration that rat liver plasma membrane receptor for PGE can be down-regulated in vivo and that this causes a corresponding decrease in PGE-induced plasma membrane adenylate cyclase activity.

Change in the cation dependence of rat liver adenylate cyclase after proteolytic activation

We recently reported that rat liver plasma membrane adenylate cyclase activity could be increased after treatment of the membranes by a variety of proteases, with the exception of trypsin [l-3]. This effect was due to an increase in the maximal velocity

Regulations of thyroid decarboxylase by the polyamines Induction of a protein inhibitor of ornithine decarboxylase by the end-products of the reaction

When spermdine, putrescine or 1,3-diaminopropane was injected (12.5 μmol/100 g body weight) into rats i h before thyrotropin, ornithine decarboxylase activity was increased by 75–150% over control levels. However, when ⩾75 μmol polyamine/100 g body weight was injected, thyrotropin-activated activity was inhibited by 70–95%. Multiple polyamine injections inhibited goitrogen-induced activity and gland weight increase by approx. 35%. The polyamines also inhibited thyrotrophin-activated rat thyroid ornithine decarboxylase in vitro in a dose-related fashion, with 50% inhibition occurring at 2–5 · 10 −4 M. The inhibition was not due to a direct effect on the enzyme. No stimulation was seen with low concentration of polyamine. The polyamines had no effect on in vitro thyroid protein/RNA synthesis or glucose oxidation but had a biphasic effect on plasma membrane adenylate cyclase activity. A protein inhibitor to thyroid ornithine decarboxylase was generated in vivo by multiple injections of the polyamines into rats, and in vitro by incubating bovine thyroid slices with 2–10 mM polyamine. The inhibitor was non-dialyzable, destroyed by boiling, and its formation was blocked in a dose-related fashion by cycloheximide. We conclude that: (1) thyroid ornithine decarboxylase is subject not only to positive control, but is also negatively regulated by its end-products, the polyamines, which induce a protein inhibitor to ornithine decarboxylase; (2) since gland growth is also inhibited under these conditions, the polyamine effect on thyroid ornithine decarboxylase may be biologically significant.

Inhibition of Thyroid Adenylate Cyclase by Thyroid Hormone: A Possible Locus for the “Short-Loop” Negative Feedback Phenomenon1

The effect of thyroid hormone on bovine thyroid plasma membrane adenylate cyclase activity was studied in order to determine if the locus of the previously observed “short-loop” negative feedback in the thyroid (J Clin Invest 57: 745, 1976) is the adenylate cyclase enzyme system. Adenylate cyclase activity was measured in bovine thyroid plasma membrane. This preparation was responsive to TSH (100 mU/ml), PGE2 (5 × 10−M) and F− (10−2M) with increases in activity of 70%, 50%, and 1,000% over basal (1.3 ± 0.5 pmol cAMP formed/10 min/mg protein), respectively. L-Thyroxine (T4), 3,5,3′-triiodothyronine (T3), D-thyroxine, 3,5,3′-triiodothyroacetic acid, and 3,3′,5′-triiodothyronine inhibited basal adenylate cyclase activity by 25-35% in contrast, only T4, T3, and D-T4 inhibited TSH- and PGE2-stimulated adenylate cyclase activity. F− stimulation was unaffected. Lubrol-PX, non-ionic detergent, abolished TSH-induced adenylate cyclase activity, but plasma membrane so treated was still responsive to PGE2 and F−. Thy...

Stimulation of adenylate cyclase by Vibrio cholerae toxin and its active subunit.

The mechanism by which Vibrio cholerae toxin and its active subunit A stimulate adenylate cyclase of rat liver plasma membrane after in vivo or in vitro exposure was investigated. Stimulation of adenylate cyclase was more efficient with cholera toxin than with subunit A in whole animals and in "soluble" preparations of adenylate cyclase. Toxin-stimulated plasma membrane adenylate cyclase activity persisted after solubilization from the membrane by detergent, while subunit A stimulation was destroyed by detergent treatment. Detergent-free "soluble" enzyme could be stimulated in vitro by cholera toxin, but such stimulation by subunit A was very weak. Binding studies with iodotoxin or iodosubunit A confirmed that the interaction of toxin with adenylate cyclase in "soluble" preparations could be the result of specific binding to the enzyme. The uncertainty as to whether G M1 ganglioside (galactosyl-N-acetylgalactosaminyl [sialosyl] lactosyl ceramide; GGnSLC) is present in these fractions limited the interpretation of the data on binding. A biological modification of subunit A prior to the activation of adenylate cyclase is inferred from these data.

An Impaired Response of Adenylate Cyclase to Stimulation by Epinephrine in Adipocyte Plasma Membranes from Genetically Obese Mice (ob/ob)

The present studies have established that there is an impaired response to epinephrine of the adenylate system in adipocyte preparations from obese hyperglycemic mice as compared to their thin littermates. In contrast, membrane preparations from both groups of animals were found to exhibit a similar response to fluoride ion. The response of adenylate cyclase to epinephrine was enhanced to a similar extent by increasing the ATP concentration in adipocyte plasma membranes from the two groups of animals. While GTP (0.1 muM) elicited an ATP-like response of similar magnitude in adenylate cyclase activity in both membrane preparations, it did not therefore abolish the impaired response to epinephrine of adenylate cyclase activity in membranes of obese mice. The response of adenylate cyclase activity to (--)-epinephrine in membrane preparations from obese mice progressively diminished with the age of these animals. In contrast, the concentration of (--)-epinephrine required for half-maximal stimulation of adenylate cyclase was similar and remained unchanged with the age for both membrane preparations. These data suggest that a perturbation may occur in the coupling step between the hormone receptor and the catalytic site of the adenylate cyclase system in obese mice. While a 15-day restrictive diet or a 72-h period of fasting was found to normalize the hyperinsulinemia of obese animals, neither affected the response of adenylate cyclase to epinephrine in preparations of adipocyte membranes from these mice. These results suggest that the observed defect in the response of plasma membrane adenylate cyclase activity to epinephrine in obese mice does not result from their hyperinsulinism.

The relation between adenylate cyclase activation and camp accumulation in the horse thyroid gland stimulated by thyrotropin

The concentration-response relationships for TSH and plasma membrane adenylate cyclase activity and for TSH and cAMP levels in slices have been compared in horse thyroids: the curves exhibited striking differences. Several models of cAMP metabolism are shown not to account for these discrepancies: notably the existence of one phosphodiesterase or the hypothesis of cAMP sequestration in the cell. However, the existence of two cAMP phosphodiesterases with K m in the 10 −6 M and 10 −3 M ranges allows a good fitting of the experimental date. It is shown that the presence of such phosphodiesterases provides the cell with a powerful amplification device. By comparing the theoretical values with those which could eventually be measured, it would be possible to check if the plasma membrane adenylate cyclase assay is a good experimental model for the enzyme in vivo and to test the hypothesis that the major part of cellular cAMP is in a free form.

Purification of Adenohypophyseal Plasma Membranes and Properties of Associated Adenylate Cyclase

Abstract Plasma membranes were purified from bovine anterior pituitary tissue by differential and isopycnic centrifugation according to a modification of Neville's technique (Biophys. Biochem. Cytol. 8, 413, 1960). Plasma membranes, rough endoplasmic reticulum, and secretory granules sedimented at the interfaces of sucrose layers of different densities and were thus simultaneously obtained in high yield by this technique. The yields of these different fractions were, respectively, 1, 2.5, and 10 mg per g of original pituitary tissue. As shown by electron microscopy, the plasma membrane fraction was not contaminated by other identifiable subcellular components. Growth hormone and prolactin accounted for, respectively, 10 to 30% and 60 to 80% of the total proteins of secretory granules. There was a 7- to 14-fold enrichment of Mg2+-dependent-(Na+-K+)-stimulated ATPase and 5'-nucleotidase activities in the plasma membrane fraction relative to the total homogenate. Adenylate cyclase activity was approximately 10-fold higher in the plasma membrane fraction than in the original homogenate. NaF, 10 mm, gave a 4- to 10-fold stimulation of enzymatic activity in all fractions. Maximal activation of plasma membrane adenylate cyclase activity by Mg2+ and Mn2+ was observed, respectively, at concentrations of 3.5 and 2 mm. Ca2+ inhibited cyclic adenosine 3' : 5'-monophosphate formation at all concentrations studied.