Adenylate Cyclase Activity In Response Research Articles

Physiological and Clinical Implications of Adrenergic Pathways at High Altitude.

The adrenergic system is part of a full array of mechanisms allowing the human body to adapt to the hypoxic environment. Triggered by the stimulation of peripheral chemoreceptors, the adrenergic centers in the medulla are activated in acute hypoxia and augment the adrenergic drive to the organs, especially to the heart, leading to tachycardia. With prolonged exposure to altitude hypoxia, the adrenergic drive persists, as witnessed by elevated blood concentrations of catecholamines and nerve activity in adrenergic fibers. In response to this persistent stimulation, the pathways leading to the activation of adenylate cyclase are modified. A downregulation of β-adrenergic and adenosinergic receptors is observed, while muscarinic receptors are upregulated. The expression and activity of Gi and Gs proteins are modified, leading to a decreased response of adenylate cyclase activity to adrenergic stimulation. The clinical consequences of these cellular and molecular changes are of importance, especially for exercise performance and protection of heart function. The decrease in maximal exercise heart rate in prolonged hypoxia is fully accounted for the observed changes in adrenergic and muscarinic pathways. The decreased heart rate response to isoproterenol infusion is another marker of the desensitization of adrenergic pathways. These changes can be considered as mechanisms protecting the heart from a too high oxygen consumption in conditions where the oxygen availability is severely reduced. Similarly, intermittent exposure to hypoxia has been shown to protect the heart from an ischemic insult with similar mechanisms involving G proteins and downregulation of β receptors. Other pathways with G proteins are concerned in adaptation to hypoxia, such as lactate release by the muscles and renal handling of calcium. Altogether, the activation of the adrenergic system is useful for the acute physiological response to hypoxia. With prolonged exposure to hypoxia, the autonomous nervous system adapts to protect vital organs, especially the heart, against a too high energetic state, via a purely local autoregulation mechanism necessary for the preservation of overall homeostasis.

Autocrine proliferative effect of ghrelin on leukemic HL-60 and THP-1 cells

Ghrelin is a 28 amino acid peptide hormone that is mainly produced by the stomach, but also by several tissues and tumors. Ghrelin is octanoylated on the Ser(3), but is also detected as a des-acylated form. Only the acylated ghrelin activates the GH secretagogue receptor (GHS-R) type 1a to stimulate GH release, and regulate food intake and energy metabolism. For the first time, we report that ghrelin and des-acyl ghrelin are present in human promyelocytic HL-60, monocytic THP-1 and lymphoblastic SupT1 cell lines. The human leukemic cell lines did not express the functional GHS-R 1a, whereas they expressed GHS-R 1b, a truncated variant of the receptor. Leukemic cell proliferation was not modified by the addition of octanoylated or des-acyl ghrelins. However, THP-1 and HL-60 cell proliferations were inhibited by SB801, an antibody directed against the N-terminal octanoylated portion of ghrelin, suggesting that octanoylated ghrelin stimulates cell proliferation via an autocrine pathway involving an as yet unidentified ghrelin receptor. Both octanoylated and des-acyl ghrelins did not alter the basal adenylate cyclase activity. Treatments of THP-1 and SupT1 cells by both octanoylated and des-acyl ghrelins did not modify the adenylate cyclase activity in response to vasoactive intestinal peptide, suggesting that ghrelin is unlikely to modulate the anti-inflammatory and differentiating properties of vasoactive intestinal peptide.

Down-regulation of beta3-adrenergic receptor expression in rat adipose tissue during the fasted/fed transition: evidence for a role of insulin.

The beta3-adrenergic receptor (beta3-AR) exerts a central role in the transduction of catecholamine effects in white and brown adipose tissue (WAT and BAT). A recent report has documented that insulin strongly down-regulates beta3-AR expression and catecholamine responsiveness in 3T3-F442A adipocytes [Fève, El Hadri, Quignard-Boulangé and Pairault (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5677-5681]. In the present report we show that the rise in plasma insulin levels elicited by the fasted/fed transition is associated with a reduction in beta3-AR mRNA levels and beta-adrenergic responsiveness in WAT and BAT. beta3-AR transcripts are also decreased in adipose tissue from animals subjected for 6 h to euglycaemic hyperinsulinaemic glucose clamps. Moreover, insulin acts directly on cultured rat white and brown adipocytes to decrease beta3-AR gene expression and adenylate cyclase activity in response to beta3-AR-selective agonists. These results suggest that there is a close relationship between food intake, plasma insulin levels and beta3-AR expression.

Dual bradykinin B2 receptor signalling in A431 human epidermoid carcinoma cells: activation of protein kinase C is counteracted by a GS-mediated stimulation of the cyclic AMP pathway.

Cell membranes of the human epidermoid cell line A431 express classical bradykinin (BK) B2 receptors, as assessed by [3H]BK binding studies. Furthermore, stimulation by BK induced a time-dependent modulation of protein kinase C (PKC) activity in A431 cells: a rapid activation (t1/2 approximately 1 min) is followed by a slow inhibition (t1/2 approximately 20 min) of PKC translocation measured by [3H]phorbol 12,13-dibutyrate binding. In addition, BK stimulated both adenylate cyclase activity in A431 membranes and accumulation of intracellular cyclic AMP (cAMP) in intact cells in a retarded manner. A possible BK-induced activation of the cAMP pathway mediated via PKC, phospholipase D, prostaglandins or Ca2+/calmodulin was excluded. A 35 kDa protein was found in A431 membranes to be specifically phosphorylated in the presence of both BK and protein kinase A (PKA). An anti-alpha s-antibody, AS 348, abolished stimulation of adenylate cyclase activity in response to BK, cholera toxin and isoprenaline, strongly suggesting the involvement of Gs proteins in the BK action. The BK-activated cAMP signalling system might be important for the observed inactivation of PKC slowly evoked by BK: the BK-induced rapid activation of PKC is decreased by dibutyryl cAMP, and the slow inhibition of PKC is prevented by an inhibitor of PKA, adenosine 3':5'-monophosphothioate (cyclic, Rp isomer). The inhibition of PKC translocation might be exerted directly at the level of PKC activation, since stimulation of phosphoinositide hydrolysis by BK was affected by neither dibutyryl cAMP nor forskolin. Thus our results provide the first evidence that A431 cells BK is able to activate two independent signal-transduction pathways via a single class of B2 receptors but two different G proteins. The lagging stimulation of the cAMP signalling pathway via Gs might serve to switch off PKC, which is rapidly activated via Gq-mediated stimulation of phosphoinositide hydrolysis.

Stimulation of rat parietal cell function by histamine and GLP-1-(7-36) amide is mediated by Gs alpha

It was the aim of the present study to determine in rat parietal cells whether Gs alpha, the stimulatory subunit of adenylate cyclase, mediates adenosine 3',5'-cyclic monophosphate (cAMP)-dependent parietal cell function in response to histamine and glucagon-like peptide 1 (GLP-1)-(7-36) amide. Cytoplasmic membrane from enriched (83 +/- 5%) rat parietal cells were incubated for 30 min with 30 microCi/ml [32P]NAD+ and 40 micrograms/ml preactivated cholera toxin (CT), a pharmacological tool for activation of Gs alpha. Subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography revealed [32P]ADP ribosylation of Gs alpha represented by three proteins with molecular masses ranging from 42 to 45 kDa. In intact parietal cells, CT (10(-12)-10(-8) M) caused marked stimulation of [14C]aminopyrine accumulation and cAMP production confirming the functional importance of Gs alpha in regulation of H+ production. Identical membrane preparations were preincubated (2 h, 4 degrees C) in parallel with and without RM/1, a rabbit polyclonal anti-Gs alpha-antibody. Subsequently, adenylate cyclase was stimulated by histamine, GLP-1-(7-36) amide, CT, or forskolin. At a 1:10 dilution, the antiserum completely abolished adenylate cyclase activity in response to maximal concentrations of histamine, GLP-1-(7-36) amide, and CT while reducing forskolin stimulation by only 22.0 +/- 4.9%. At 1:50, RM/1 reduced responses to histamine, GLP-1-(7-36) amide and CT by 20-30% but failed to inhibit forskolin-stimulated enzyme activity. At 1:100, the antiserum was ineffective versus all stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptations of myocardial beta-adrenergic receptor complex in hibernating marmots

Properties of marmot (Marmota flaviventris) myocardial beta-adrenergic receptor complex (beta-AR) were evaluated during hibernation (H), in summer (S) animals, and in animals aroused from hibernation (C). The results obtained for S and C animals were identical, and only the results for C animals are shown. In H-animal myocardial membrane preparations assayed at 37 degrees C, isoproterenol-dependent adenylate cyclase activity (ACA) was consistently higher, whereas the synergistic contribution of 5'-guanylylimidodiphosphate [Gpp(NH)p] in this reaction was reduced. When assayed at 10 degrees C, only the ACA in H animals responded to the combination of isoproterenol and Gpp(NH)p. In contrast, at 10 degrees C, ACA in response to Gpp(NH)p alone is essentially equal in H and C animals. Hibernation did not change myocardial beta-AR receptor density or affinity. In contrast, analysis of isoproterenol displacement of [125I]iodocyanopindolol revealed that the proportion of beta-AR in the high-affinity state was substantially greater in H than in C animals, and this relationship was retained even in the presence of Gpp-(NH)p. In an evaluation of the role of the GTP binding proteins that couple the beta-AR to the effector adenyl cyclase, we determined that there was no change in the cholera toxin- or pertussis toxin-dependent ADP ribosylation patterns. Immunochemical detection of the individual GTP binding proteins revealed no change in the levels of G alpha i1, G alpha i2, or G alpha i3. In contrast, we observed a hibernation-associated decrease in G alpha o associated with the plasma membrane-enriched particulate fraction. (ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the small cardioactive peptide B (SCP B) on adenylate cyclase of the central nervous system and peripheral organs of the freshwater snail Planorbarius corneus

The effect of the small cardioactive peptide B (SCP B) on adenylate cyclase activity of the central nervous system (CNS) and some peripheral organs of the freshwater snail Planorbarius corneus was investigated. This peptide stimulates enzyme activity in a dose-dependent manner in all the tested ganglia, in the salivary glands, the buccal mass and the oesophagus. The amount required for half-maximal stimulation is approx 0.1 μM. Moreover, the response of adenylate cyclase to SCP B progressively increases with time, without desensitizing. On the other hand, the peptide is ineffective on heart adenylate cyclase. The responsiveness of adenylate cyclase activity to the peptide and the wide distribution of SCP B-related immunoreactivity in all the tested ganglia suggest that this substance may exert an important role as neurotransmitter as well as neuromodulator in the CNS of the snail. Since SCP B is consistently present in the buccal and cerebral ganglia, associated nerves, salivary glands, buccal mass and oesophagus, and in the light of the appreciable effects exerted by SCP B on adenylate cyclase activity in the examined peripheral tissues, we suggest that the peptide is strongly involved in the control of feeding behaviour of P. corneus.

Recovery of β-receptors and adenylate cyclase from desensitization induced by short term heat exposure in rat parotid glands

1. 1. The recovery of rat parotid β-adrenergic receptors (β-AR) and adenylate cyclase (AC) from heat (33°C)-induced desensitization was studied. 2. 2. Down-regulated cell surface β-AR and AC activity in response to isoprenaline (IPR) returned to the control level 120 hr after the termination of heat exposure. 3. 3. However, β-AR in parotid crude membranes increased over the control level for 48–120 hr. 4. 4. Coupling between β-AR and G protein(s) was attenuated at 120 hr. 5. 5. These data suggest that β-AR on the cell surface, but not those internalized, can transduce biological responses.

Modulation by GTP of basal and agonist-stimulated striatal adenylate cyclase activity following chronic blockade of D1 and D2 dopamine receptors: involvement of G proteins in the development of receptor supersensitivity.

Rats receiving injections of specific antagonists of dopamine receptors (SCH 23390 for D1, haloperidol for D2, and haloperidol+SCH 23390) once daily for 21 days develop a selective supersensitivity of the blocked receptors. To study the molecular correlates of these adaptive changes, we evaluated the involvement of GTP-binding proteins in the development of supersensitivity of dopamine receptors. By means of adenylate cyclase studies, we tested whether any of the treatments modified the functional response to GTP in striata dissected from control and treated rats. Our data show that the chronic blockade of D1 and/or D2 receptors potentiates both basal and dopamine receptor-stimulated adenylate cyclase activity in response to GTP. D1 receptor up-regulation correlates with an increased adenylate cyclase response to GTP, whereas D2 receptor up-regulation is accompanied by an enhanced GTP-induced inhibition of enzyme activity, in both basal and receptor-activated conditions. This potentiation does not seem to match the changes in mRNA content of Gs and Gi alpha subunits. Unexpectedly, however, a significant increase in Gi alpha subunit mRNA was found after the chronic blockade of D1 receptors; this result could be explained by cross-regulation between GTP-binding protein-mediated pathways. This cross-regulation could serve as a protective mechanism whereby cells exposing up-regulated receptors protect themselves from a condition of hyperactivity of the adenylate cyclase enzyme.

Inhibition of platelet aggregation by prostacyclin is attenuated after exercise in patients with angina pectoris

We tested whether alteration of platelet sensitivity to prostacyclin (PGI 2) is involved in the activation of platelets induced by exercise in patients with stable angina. Twenty patients and 20 control subjects underwent treadmill testing. Blood samples were obtained before and immediately after exercise for plasma thromboxane B 2 (TXB 2) and 6-keto-PGF 1α (6kP) assays and platelet aggregation studies. Dose-response curves for platelet aggregation to coliagen were obtained in the presence and absence of 1 nmol/L PGI 2 to quantify the antiaggregation effects of PGI 2. At rest, platelet aggregation by collagen was enhanced in the patients. However, platelets were more sensitive to exogenous PGI 2, apparently associated with lower plasma 6kP levels in the patients. After exercise, plasma TXB 2 levels increased in the patients but not in the control subjects. Plasma 6kP levels remained unchanged and platelet sensitivity to PGI 2 decreased in the patients whereas these values increased in the control subjects. The exercise-induced changes in platelet sensitivity to PGI 2 correlated with those of platelet adenylate cyclase activity in response to 1 nmol/L PGI 2 ( r = 0.787, p < 0.01). Thus impaired sensitivity of platelets to PGI 2, in addition to the reduced response of prostanoid secretion, might be relevant to the platelet activation associated with exercise in patients with stable angina.

Adrenergic receptors and adenylate cyclase activity in hepatocytes of the streptozotocin-diabetic rat.

Effects of short and long exposure to the diabetic state induced by an injection of streptozotocin to young female rats on glucagon- and catecholamine-sensitive adenylate cyclase activity and adrenergic receptors of hepatic membranes have been studied. The short period of exposure to the diabetic state exhibited an increase in the sensitivity of the enzyme to isoproterenol without changes in the affinity and the number of beta-adrenergic receptors. The increased response of adenylate cyclase activity to isoproterenol was accompanied with a greater GTP-induced lowering of the affinity to the beta-adrenergic agonist in diabetic membranes than in the controls. The chronic diabetic state produced a decrease in the adenylate cyclase activity to hormonal or non-hormonal stimuli with a fall in the number of alpha- and beta-adrenergic receptors. These results suggest that the observed effects of the diabetic state on hormonally sensitive adenylate cyclase activities and their receptor binding sites of the hepatic membranes would vary depending on the duration and/or severity of the diabetic state experimentally induced.

In vivo adaptive control of β-receptors and adenylate cyclase during short-term heat exposure in rat parotid glands

1. Adaptation of β-adrenergic receptors (β-AR) and adenylate cyclase (AC) in rat parotid glands during short-term heat exposure (33°C) were studied. 2. Heat exposure reduced AC activity in response to isoproterenol (IPR). 3. The number of β-AR on the cell surface significantly increased after 24 hr but returned to control level after 48 hr. 4. IPR-induced [ 3H]GDP release was significantly reduced throughout exposure. 5. The data suggest that the major factor which results in the desensitization of AC during short-term heat exposure is a blunted coupling between β-AR and GTP binding protein(s).

Central dopaminergic transmission is selectively increased in the limbic system of rats chronically exposed to antidepressants

Repeated electroconvulsive shock (ECS) exposure produced a decrease of [ 3H]SCH 23390 binding sites and a reduced response of adenylate cyclase activity to dopamine D-1 receptor stimulation in the rat limbic area analogous to that previously observed in rats chronically treated with imipramine. These effects were completely prevented by the repeated administration of a small dose of α-methyl-p-tyrosine (α-MPT), associated with the tricyclic compound. Increased dopaminergic transmission seems to be involved in the mechanism of antidepressant action. Rats chronically treated with imipramine showed a decrease of dihydroxyphenylacetic acid (DOPAC) concentration restricted to the limbic area. Finally, both imipramine and desipramine blocked the uptake of [ 3H]dopamine in the limbic system with a 100-fold greater potency than that observed in the basal ganglia.

The Role of Calmodulin in the Responses to Adrenocorticotropin of Plasma Membranes from Adrenal Cells*

Inhibitors of calmodulin [trifluoperazine, chlorpromazine, pimozide, and calmidazolium N-(6-aminohexyl)5-chloro-1-napthalenesulphonamide (W7)] and calmodulin antibodies were used to investigate the role of calmodulin in the response of Y-1 mouse adrenal cells to ACTH, with particular reference to events in the plasma membrane. In whole cells it was found that two responses (production of steroids and cAMP) to two stimulating agents (ACTH and forskolin) were inhibited by trifluoperazine at concentrations consistent with those involved in binding of the inhibitor to pure calmodulin (10-25 microM). The steroidogenic responses were also inhibited by the three other inhibitors of calmodulin (chlorpromazine, calmidazolium, and W-7). Trifluoperazine and pimozide (1-500 microM) did not inhibit binding of an [125I]ACTH analog to highly purified plasma membranes of Y-1 cells or to the cells themselves. With Y-1 plasma membranes it was found that trifluoperazine, pimozide, W-7, and calmodulin antibodies inhibited the increase in adenylate cyclase activity in response to ACTH, but not the cyclase responses to cholera toxin or forskolin. Moreover, the effect of cholera toxin on the ADP-ribosylation of specific membrane substrates was independent of the presence or absence of endogenous and/or exogenous Ca2+/calmodulin. The response of adenylate cyclase to ACTH was also decreased in plasma membranes from which calmodulin was removed by washing, and exogenous calmodulin partly reversed this decrease. Anti-calmodulin immunoglobulin inhibited the stimulation of adenylate cyclase produced in plasma membrane by ACTH, but was without effect on the responses to cholera toxin and forskolin. Exogenous calmodulin partly reversed the inhibition of stimulation by ACTH of adenylate cyclase produced by the antibody. It is concluded that calmodulin influences the events taking place in the plasma membrane in response to ACTH, after the binding of the hormone to its receptor and before the action of the G protein (Gs). That is, calmodulin is involved in coupling the occupied receptor to Gs. The effects of inhibitors of calmodulin in whole cells must involve some additional effect(s) requiring the intact cell.

Testosterone synthesis and adenylate cyclase activity in the early human fetal testis appear to be independent of human chorionic gonadotropin control.

Androgen secretion by the fetal testis is essential for male phenotypic differentiation. In the human fetus testosterone formation is initiated soon after the differentiation of the testis (approximately 8 weeks of gestation), and the maximal testosterone content in fetal testes is achieved between 10 and 15 weeks of fetal life. The testosterone content of the fetal testis declines at the beginning of the third trimester and remains low until after birth. In an effort to understand the regulation of the onset of testosterone formation in the human fetal testis we measured adenylate cyclase activity in response to hCG stimulation in homogenates of fetal testes obtained from first and second trimester human abortuses. Basal adenylate cyclase activity was 50 pmol/mg protein.min at 10 weeks gestation, the peak activity was 137 pmol/mg protein.min at 12 weeks gestation, and activity declined thereafter to 8 pmol/mg protein.min by 16 weeks gestation. NaF-stimulated (0.6 mmol/L) and forskolin-stimulated (50 mumol/L) activities were 4- to 8-fold greater than basal adenylate cyclase activities. The maximal forskolin-stimulated activity occurred at 11 weeks (803 pmol/mg protein.min), and it fell to 35 pmol/mg protein.min by 17 weeks gestation. In contrast, hCG-stimulated (1 mumol/L) adenylate cyclase activity was only slightly greater than basal rates at all ages examined. In addition, hCG did not stimulate baseline testosterone formation in minces of testes obtained between 12 and 18 weeks of gestation. These findings suggest that the onset of testosterone formation in human fetal testes may be independent of gonadotropin control.

Altered thyroid status regulates the adipocyte A 1 adenosine receptor-adenylate cyclase system

To assess the effect of hyperthyroidism on the adenosine receptor-adenylate cyclase system in adipocytes, membranes from hyperthyroid and control rats were prepared. Rats were rendered hyperthyroid by five days of injection with triiodothyronine (T 3). Basal as well as isoproterenol-sodium fluoride-, forskolin- and manganese (Mn++)-stimulated adenylate cyclase activities are attenuated 20–30% in adipocyte membranes from hyperthyroid animals. There is a greater inhibition of total adenylate cyclase activity in response to R -PIA, A 1 selective inhibitory agonist, in membranes from hyperthyroid animals. However, on a percentage basis, R -PIA is equally effective at inhibiting adenylate cyclase activity in control and treated membranes. Using antagonist radioligands, [ 3H]XAC (A 1 receptor) and [ 125I]CYP (β-adrenergic receptor), no significant alteration in receptor number is observed in hyperthyroidism. In addition, no alteration in G i protein-A 1 receptor coupling is noted as exhibited by R -PIA competition curves. These findings suggest hyperthyroidism most likely results in a decrease of the catalytic moiety of adenylate cyclase either quantitatively or functionally.

Dexamethasone accelerates differentiation of A6 epithelia and increases response to vasopressin.

When seeded heavily on a porous tissue culture dish, A6 cells, derived from the kidney of Xenopus laevis, form a highly differentiated epithelium within 4-6 days. When dexamethasone is added to the culture medium, morphological differentiation is completed by day 2, a time at which the control (untreated) is still a disorganized multilayer of cells. In addition to the morphologically evident monolayer of cuboidal cells, the accelerated differentiation is expressed as high transepithelial electrical resistance, short-circuit current, and adenylate cyclase response to vasopressin. When grown on impermeable plastic tissue culture dishes, A6 epithelia are less differentiated and do not respond to vasopressin. With the addition of dexamethasone at the time of seeding on a plastic tissue culture dish, vasopressin responsive adenylate cyclase activity is expressed, albeit at a slower rate than when grown on a porous surface. In addition, dexamethasone treatment of mature epithelia grown on a porous surface results, in hours, in an increase in the adenylate cyclase response to vasopressin. These results reveal two previously unrecognized interactions between adrenal steroid hormones and vasopressin, namely, accelerated differentiation and increased responsiveness of adenylate cyclase.

Adenylate cyclase activity as a predictor of thyroid tumor aggressiveness.

AbstractPrior studies in our laboratory have shown that human thyroid neoplasms have a greater adenylate cyclase activity in response to thyroid stimulating hormone (TSH) than does the adjacent histologically normal thyroid tissue. However, there is little information relating activity of the TSH receptor‐adenylate cyclase system to the type of thyroid neoplasm. Thyroid tissue from 67 patients was divided by clinical and histological criteria into 6 categories: normal (59), benign tumors (20), stage 1 carcinoma—intrathyroidal involvement only (25), stage 2 carcinomaregional lymph node involvement (6), stage 3 and 4 carcinoma—tissue invasion or distant metastasis (11), and medullary carcinoma (3). Adenylate cyclase activity in an 8,000 x g thyroid membrane preparation was determined in the basal state and when maximally stimulated with 300 mU/ml TSH. The cyclase responsiveness was the ratio of TSH stimulated adenylate cyclase activity compared to basal adenylate cyclase activity. The cyclase responsiveness by category is: normal, 2.8±0.2 (mean ± SEM); benign, 17.9±2.4; stage 1 carcinoma, 9.2±1.9; stage 2 carcinoma, 4.0±1.0; stage 3 and 4 carcinoma, 1.6±0.4; and medullary carcinoma, 1.05±0.04 (for the neoplasms,p &lt;0.02 by ANOVA). Tumor stage was the only correlate with this trend as other prognostic risk factors (age, sex, a history of neck irradiation, or papillary versus follicular histology) showed no difference in cyclase responsiveness. These studies demonstrate a consistent inverse correlation between adenylate cyclase responsiveness and tumor stage or aggressiveness. Cyclase responsiveness appears to have clinical application for predicting which thyroid tumors will behave aggressively.

Phorbol esters induce two distinct changes in GH 3 pituitary cell adenylate cyclase activity

Phorbol esters alter cyclic AMP levels in a number of tissues, including the anterior pituitary. We report that membrane preparations from GH 3 cells exposed to phorbol esters exhibit decreased vasoactive intestinal peptide (VIP)-stimulated and enhanced forskolin-stimulated adenylate cyclase activity. The responsiveness of adenylate cyclase activity to NaF, guanylyl-imidodiphosphate, and Mn 2+ was also reduced by phorbol ester treatment. The ability of somatostatin to inhibit forskolin-stimulated adenylate cyclase activity was reduced while phorbol ester exposure had no apparent effect on somatostatin inhibition of VIP-stimulated adenylate cyclase activity. We suggest that protein kinase C alters at least two distinct components of the adenylate cyclase system. One modification disrupts hormone receptor-G s interaction (lowering VIP efficacy) and the second perturbation augments the activity of the adenylate cyclase catalytic subunit.

Isolation and characterization of 9-hydroxy-10-trans,12-cis-octadecadienoic acid, a novel regulator of platelet adenylate cyclase from Glechoma hederacea L. Labiatae.

We have identified and characterized a fatty acid, (9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid (9-HODE) as a regulator of adenylate cyclase activity of human platelet membranes. This fatty acid was isolated from a methanolic extract of the plant Glechoma hederacea L. Labiatae (commonly known as 'lierre terrestre', 'ground ivy' or 'creeping Charlie'; it was identified by nuclear magnetic resonance and mass spectroscopy. This compound increased basal adenylate cyclase activity in platelet membranes about threefold and had an EC50 of 10-20 microM. This increase in adenylate cyclase activity occurred without a temporal lag, was reversible, and represented an increase in Vmax without a substantial change in Km for ATP, Mg2+ or Mn2+. In addition, 9-HODE additively or synergistically increased platelet adenylate cyclase activity in response to guanosine 5'-[beta,gamma-imido]triphosphate and forskolin, but the fatty acid failed to alter inhibition of adenylate cyclase activity mediated by epinephrine (alpha 2-adrenergic receptor). Studies of the interaction of 9-HODE with activation of platelet adenylate cyclase activity mediated by prostaglandin E1 (PGE1) and prostaglandin D2 (PGD2) indicated that this fatty acid produced a parallel shift in the concentration/response curve of PGE1 and PGD2 without altering maximal response, which was substantially greater than that observed with 9-HODE alone. From these results, we conclude that 9-HODE appears to be a partial agonist at PGE1 and PGD2 receptors on human platelets. We believe that this is a novel example of a plant-derived fatty acid which acts on cells to regulate adenylate cyclase via prostaglandin receptors.