Forskolin-stimulated Adenylate Cyclase Activity Research Articles

Effect of alpha tocopheryl succinate on adenylate cyclase activity in murine neuroblastoma cells in culture.

Alpha tocopheryl succinate treatment (6-8 micrograms/ml), which inhibited the growth of murine neuroblastoma (NBP2) cells (46 +/- 3%), reduced basal and prostaglandin (PG)E1- and PGA2-stimulated adenylate cyclase (AC) activity in vitro. It also inhibited sodium fluoride (NaF)- and forskolin-stimulated AC activity, suggesting that the effect of vitamin E succinate on AC activity is mediated via stimulatory GTP-binding protein (Gs) and catalytic subunit. Vitamin E succinate-induced reduction of AC activity is not strictly related to inhibition of cell growth. This is substantiated by the finding that, although retinoic acid and butylated hydroxyanisole reduced the growth by over 50%, they did not inhibit AC activity. On the other hand, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (R020-1724, 200 micrograms/ml), which inhibited growth (73 +/- 3%) and induced differentiation in NB cells, increased basal and PGE1-stimulated AC activity. Vitamin E succinate treatment also reduced PGE1- and PGA2-AC activity in murine fibroblasts (L-cells) without inhibiting growth.

Glucocorticoids modulate beta-adrenoceptor subtypes and adenylate cyclase in brown fat.

Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the beta-adrenergic receptor. Glucocorticoids potentiate the action of catecholamines in some tissues by increasing the expression of beta-adrenergic receptors. Paradoxically, glucocorticoids suppress and adrenalectomy enhances BAT thermogenesis. To further study the reasons for this discrepancy, we assessed the effects of methylprednisolone administration, adrenalectomy, and adrenalectomy with corticosterone replacement on adenylate cyclase activity in BAT and on beta-adrenergic receptor density in lungs and BAT of rats. In lungs, the density of the beta 2-adrenergic receptor subtype increases after methylprednisolone administration and decreases after adrenalectomy. There was no change in BAT receptor density, but isoproterenol-, NaF-, and forskolin-stimulated adenylate cyclase activity was reduced by 20-35% after methylprednisolone treatment. There was a two- to threefold increase in adenylate cyclase activity after adrenalectomy, which was reversed by corticosterone administration. These data suggest that one mechanism by which glucocorticoids regulate BAT thermogenesis is by modulating the beta-adrenergic pathway at the level of adenylate cyclase activation.

Effect of high doses of somatostatin on adenylate cyclase activity in peripheral mononuclear leukocytes from normal subjects and from acute leukemia patients.

In normal lymphocytes somatostatin non-competitively inhibited basal (ID50 5 x 10(-4) M) and isoproterenol- and forskolin-stimulated adenylate cyclase activity (Ac). In acute leukemia blasts, non-responsive to isoproterenol, forskolin, which activates the catalytic subunit, stimulated and somatostatin inhibited Ac, thus indicating the leukemic enzyme, though defective, retains the inhibitory pathway and catalyst function.

A novel site of action of a high affinity A 1 adenosine receptor antagonist

XAC, a high affinity antagonist of the A1 adenosine receptor, enhances adenylate cyclase activity by 1.3-2 fold with an EC50 of approximately 47 nM in adipocyte membranes pretreated with adenosine deaminase to eliminate adenosine and in the presence of total phosphodiesterase inhibition by 100 microM papaverine. This effect of XAC is observed only at concentrations of GTP sufficient to activate Gi (approximately 5 x 10(-6) M GTP) and is not evident in the absence or presence of lower GTP concentrations. ADP ribosylation of Gi by pertussis toxin treatment also abolishes this stimulatory action of XAC. Furthermore, in the presence of GTP activation of inhibitory prostaglandin E1 receptors diminishes the stimulatory effect of XAC on adenylate cyclase. In addition, XAC interferes with GTP-mediated inhibition of forskolin-stimulated adenylate cyclase activity in a noncompetitive manner. Finally, XAC is only a weak inhibitor of the low Km cyclic AMP phosphodiesterase, producing approximately 40% inhibition of phosphodiesterase activity at a concentration of 100 microM. These data suggest that XAC increases adenylate cyclase activity in absence of endogenous adenosine by inhibiting tonic Gi activity in a reversible manner.

The 5-hydroxytryptamine 5-HT1D receptor subtype is negatively coupled to adenylate cyclase in calf substantia nigra.

1) The possibility was explored that the recently defined 5-HT1D binding sites could be negatively coupled to adenylate cyclase in calf substantia nigra. 2) 5-HT inhibited forskolin-stimulated adenylate cyclase activity in a concentration-dependent manner (EC50 value = 24.0 nmol/l, Emax = 22.7% inhibition) in the presence of GTP (10 mumol/l), which was required for this inhibitory effect. 3) The following 5-HT receptor agonists inhibited adenylate cyclase activity (in decreasing order of potency): 5-carboxamidotryptamine greater than 5-HT greater than 5-methoxytryptamine greater than 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H indole (RU 24969) greater than or equal to N,N-dipropyl-5-carboxamidotryptamine greater than 8-hydroxy-2(di-n-propylamino)-tetralin (8-OH-DPAT) greater than buspirone greater than ipsapirone; the latter two compounds apparently behaved as partial agonists. 4) Other compounds displaying agonist activity in this system were: metergoline greater than methysergide greater than or equal to rauwolscine greater than or equal to cyanopindolol greater than or equal to yohimbine greater than (+/-)-4(3-tert-butyl-amino-2-hydroxypropoxy)-indol-2 carbonic acid isopropylester (21-009) greater than corynanthine. 5) Methiothepin, mianserin and spiperone displaced the concentration-effect curve of 5-HT to the right without depressing the Emax value. The same held true for the partial agonists ipsapirone, buspirone and corynanthine. 6) The rank order of potency of agonists as well as of antagonists in this system was in full agreement with their affinities at 5-HT1D binding site. A highly significant correlation was found between both parameters (r = 0.94, P = 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Modulation of adenylate cyclase activity of mouse spinal cord-ganglion explants by opioids, serotonin and pertussis toxin

Organotypic cultures of fetal mouse spinal cord-ganglion explants (2–4 weeks in vitro) contain forskolin-stimulated adenylate cyclase (AC) activity that is inhibited by levorphanol and other opioid agonists in a dose-dependent manner. Inhibition by levorphanol no longer occurs if sodium is omitted from the incubation and the levorphanol inhibition is blocked by the opioid antagonist, naloxone. These findings together with the ineffectiveness of dextrophan indicate that the opioid inhibition of forskolin-stimulated AC is receptor mediated. Both the δ- and κ-receptor subtypes appear to be involved since the selective δ-opioid agonist, [ d-Pen 2. d-Pen 5]enkephalin, and the selective κ-opioid agonist, t-3,4-dichloro- N-methyl- N[2-(1-pyrrolidinyl)cyclohexyl]-benzene acetamide (U-50,488H) are both effective at nanomolar concentrations. In contrast, the selective μ-opioid agonists, Tyr- d-Ala-Gly -N- MePhe-Gly-ol , has no significant effect even at micromolar concentrations. Both cord and ganglion components of the explants contain opioid-sensitive AC. Forskolin-stimulated AC of the explants is also inhibited by serotonin and carbachol. The serotonin effect appears to be mediated by 5-HT 1A receptors, based on relative agoonist and antagonist selectivity. Chronic exposure of cultures to morphine results in enhanced basal and forskolin-stimulated AC as well as attenuation of opioid-inhibition of AC assayed in the presence of forskolin: treatment of explants with pertussis toxin causes similar changes in the AC system. The inhibitory effect of serotonin is also attenuated by the pertussis toxins treatment. Basal AC activity of the explants (assayed without forskolin present) is stimulated to a small but significant extent opioids and by serotonin. The opioid stimulatory effect is markedly enhanced following either morphine or pertussis toxin treatment of the explants. The attenuation of opioid- and serotonin-inhibition of AC produced by chronic exposure to pertussis toxin and the attenuation of opioid inhibition produced by exposure to morphine are consonant with the attenuation of opioid and monoaminergic depression of sensory evoked dorsal horn network responses after similar chronic treatments. It is proposed that the inhibitory effects of opioids and seroronin on these neurons are mediated by receptors that are negatively coupled via a pertussis toxin sensitive G 1 protein to AC. Furthermore, alterations of AC with chronic morphine treatment may be involved in the development of physiologic tolerance to opioids.

Neuropeptide Y and peptide YY inhibit adenylate cyclase activity in the rat striatum.

The equilibrium binding of [3H]propionyl neuropeptide Y ([ 3H]pNPY) to receptors in a crude synaptic membrane preparation from the rat striatum was influenced by GTP, which caused an apparent loss of high-affinity binding sites for [3H]pNPY. In the presence of GTP (10(-5) M), NPY and peptide YY (PYY) inhibited basal and forskolin-stimulated adenylate cyclase activity in a concentration-dependent manner in a cell-free preparation from rat striatum. The IC50 values for NPY and PYY were 1 X 10(-8) M and 1.4 x 10(-8) M respectively. The inhibitory action of NPY (10(-6) M) or of PYY (10(-6) M) was additive to that of acetylcholine (10(-4) M). The two peptides together also showed additivity (P less than 0.05) in inhibiting adenylate cyclase.

Chronic electroconvulsive shock and desimipramine reduce the degree of inhibition by 5-HT and carbachol of forskolin-stimulated adenylate cyclase in rat hippocampal membranes

The degree of inhibition of forskolin-stimulated adenylate cyclase activity by 5-HT and by carbachol in hippocampal membranes was significantly reduced after administration of either chronic ECS (10 days) or desimipramine (10 mg/kg i.p. for 3 weeks). A single ECS had no effect on the 5-HT response and slightly augmented the carbachol response. These results parallel previous observations on the effects of ECS and antidepressants on behavioral responses to 5-HT 1a agonists and on muscarinic receptor number, and indicate the possible involvement of these receptors in the mechanism of action of antidepressant drugs.

5-HT 1D receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in calf substantia nigra

5-HT 1D receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in calf substantia nigra

Phorbol esters inhibit adenylate cyclase activity in cultured collecting tubular cells.

Activators of protein kinase C, a calcium- and phospholipid-dependent protein kinase, inhibit vasopressin-stimulated water flow in toad bladder. To determine the biochemical mechanisms of this inhibition, we examined the effects of activators of protein kinase C on arginine vasopressin (AVP)-stimulated adenylate cyclase activity in cultured rabbit cortical collecting tubular cells. The phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA), the diacylglycerol, 1-oleyl-2-acetyl glycerol (OAG), and the diacylglycerol kinase inhibitor, R59022, all rapidly activate protein kinase C in collecting tubular cells. Pretreatment with PMA produces a delayed inhibition (greater than or equal to 4 h) of AVP-stimulated adenylate cyclase activity. The 4-h time lag suggests that the effects of protein kinase C are mediated indirectly, possibly as a consequence of stimulating cell proliferation. PMA does not inhibit cholera toxin- or forskolin-stimulated adenylate cyclase activity, suggesting an effect on the vasopressin receptor or coupling of the receptor to the stimulatory guanine nucleotide regulatory protein. Neither prostaglandins nor the inhibitory guanine nucleotide regulatory protein appear to mediate this effect. In contrast, treatment with either OAG or R59022 produces a rapid inhibition of both AVP- and forskolin-stimulated adenylate cyclase activity suggesting a prominent distal site of action, presumably at the catalytic subunit of adenylate cyclase. The results demonstrate that different activators of protein kinase C inhibit AVP-stimulated adenylate cyclase activity by distinctly different mechanisms possibly by altering the substrate specificity or activating multiple forms of the kinase. These results have important implications when using different activators to study the biological effects of protein kinase C.

Somatostatin inhibition of anterior pituitary adenylate cyclase activity: different sensitivity between male and female rats

Somatostatin (SRIF) is a potent inhibitor of growth hormone (GH) secretion. Although cyclic AMP (cAMP) has been suggested as intracellular mediator of SRIF action, a complete characterization of its effect and the different sensitivity between male and female animals, has not yet been carried out. In this study SRIF inhibited basal and GH-releasing factor (GRF) stimulated anterior pituitary adenylate cyclase activity with a greater effectiveness in male than in female glands. Similarly SRIF reduction of forskolin-stimulated anterior pituitary adenylate cyclase activity, was more pronounced in male than in female animals. By using pertussis toxin (PTX), which uncouples inhibitory receptors from adenylate cyclase catalytic subunit, SRIF inhibition of both basal and forskolin-stimulated adenylate cyclase activity was nearly abolished. These results show that anterior pituitary SRIF receptors are coupled in an inhibitory fashion with adenylate cyclase enzyme, and that male rat adenohypophyses are more responsive to SRIF inhibition.

Uncoupling of the platelet α2-adrenoceptor adenylate cyclase system by N-ethylmaleimide and the resulting effect on receptor density and sensitivity

Preincubation of platelet-rich plasma with N-ethylmaleimide (NEM) attenuated the inhibitory effect of the α 2-adrenoceptor agonist UK 14304 on basal and forskolin-stimulated adenylate cyclase activities. NEM also led to concomitant marked reductions of the specific binding of [ 3H]UK 14304 to platelet membranes and of the primary aggregation response induced by UK 14304. These results indicate that uncoupling of the receptor adenylate cyclase system by NEM induces down-regulation of platelet α 2-adrenoceptor density ( 3H-agonist binding sites) and of the associated functional response (platelet aggregation).

Alpha tocopheryl succinate inhibits melanocyte-stimulating hormone (MSH)-sensitive adenylate cyclase activity in melanoma cells.

D-alpha tocopheryl succinate (vitamin E succinate), which is known to induce differentiation and growth inhibition in murine B-16 melanoma cells, reduced basal and melanocyte-stimulating hormone (MSH)-stimulated adenylate cyclase (AC) activity in vitro. Vitamin E succinate treatment also reduced sodium fluoride- and forskoline-stimulated AC activity of melanoma cells in vitro. Treatment of cells with vitamin E succinate (6 micrograms/ml] for a period of 24 hours was sufficient to reduce MSH-stimulated AC activity. Other forms of vitamin E, such as d1-alpha tocopheryl nicotinate, d1-alpha tocopheryl acetate, and d1-alpha tocopherol, which did not affect growth or morphology of melanoma cells, were relatively less effective in altering basal and MSH-stimulated AC activity. Retinoic acid, which inhibited the growth of B-16 melanoma cells, also reduced basal and MSH-, NaF-, and forskolin-stimulated AC activity in vitro. Prostaglandin A2, which inhibited growth and altered morphology, did not change basal or MSH-stimulated AC activity. These results show that one of the mechanisms of action of vitamin E succinate and retinoic acid on melanoma cells may involve reduction of basal and MSH-sensitive AC activity, and this vitamin effect is not necessarily related to growth inhibition.

Modulation of receptors and adenylate cyclase activity during sucrose feeding, food deprivation, and cold exposure.

Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the beta-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the beta-adrenergic pathway, adenylate cyclase activity and beta-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. beta-Adrenergic receptors were identified in BAT using [125I]iodocyanopindolol. Binding sites had the characteristics of mixed beta 1- and beta 2-type adrenergic receptors at a ratio of 60/40. After sucrose feeding or cold exposure, there was the expected increase in BAT mitochondrial mass as measured by total cytochrome-c oxidase activity but a decrease in beta-adrenergic receptor density due to a loss of the beta 1-adrenergic subtype. This BAT beta-adrenergic receptor downregulation was tissue specific, since myocardial beta-adrenergic receptors were unchanged with either sucrose feeding or cold exposure. In contrast, food deprivation did not alter BAT beta-adrenergic receptor density. Forskolin-stimulated adenylate cyclase activity increased in BAT after sucrose feeding or cold exposure but not after food deprivation. The ratio of isoproterenol-stimulated to forskolin-stimulated adenylate cyclase activity decreased in the sucrose-fed and cold-exposed rats but not in the food-deprived rats. These data suggest that in BAT, sucrose feeding or cold exposure result in downregulation of beta-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability.

Forskolin-resistant Y1 mutants harbor defects associated with the guanyl nucleotide-binding regulatory protein, Gs.

The properties of the adenylate cyclase from forskolin-resistant mutants of Y1 adrenocortical tumor cells was compared with the properties of the enzyme from parental Y1 cells in order to localize the site of mutation. In parental Y1 cells, forskolin stimulated adenylate cyclase activity with kinetics suggestive of an interaction at two sites; in mutant cells, forskolin resistance was characterized by a decrease in enzymatic activity at both sites. Forskolin potentiated the enzyme's responses to NaF and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) in parent and mutant clones, and the mutant enzyme showed the same requirements for Mg2+ and Mn2+ as did the parent enzyme. The adenylate cyclase associated with forskolin-resistant mutants was insensitive to ACTH and was less responsive to Gpp(NH)p than was the parent enzyme. In parental Y1 cells and in the forskolin-resistant mutants, cholera toxin catalyzed the transfer of [32P]ADP-ribose from [32P]NAD+ into three membrane proteins associated with the alpha subunit of Gs; however, the amount of labeled ADP-ribose incorporated into mutant membranes was reduced by as much as 70%. Both parent and mutant membranes were labeled by pertussis toxin to the same extent. The insensitivity of the mutant adenylate cyclase to ACTH and Gpp(NH)p and the selective resistance of the mutant membranes to cholera toxin-catalyzed ADP-ribosylation suggest that a specific defect associated with Gs is involved in the mutation to forskolin resistance in Y1 cells.

Inhibition by melittin and fluphenazine of melanotropin receptor function and adenylate cyclase in M2R melanoma cell membranes.

Melanotropin (MSH) receptor activity in the M2R mouse melanoma cell line is tightly controlled by calcium by an unknown mechanism. The possibility that calcium regulation is mediated by calmodulin or a calmodulin-related calcium binding protein has been addressed in this report by studying the effects of two known calmodulin antagonists, fluphenazine and melittin, on MSH receptor function. Stimulation of adenylate cyclase (AC) in M2R plasma membranes by beta MSH was strongly inhibited by both antagonists. The concentrations of fluphenazine and melittin yielding half-maximal inhibition (IC50) of AC were 16 microM and 2.4 microM, respectively. Both fluphenazine and melittin also inhibit prostaglandin E1-, GTP gamma S, and forskolin-stimulated AC activity, as well as that of unstimulated enzyme, although inhibition is shown to occur at significantly higher concentrations of antagonist. We have shown that the calcium-dependent rate-limiting step in MSH stimulation of adenylate cyclase, that of hormone binding, is strongly inhibited by these antagonists at concentrations identical to, if not lower than, those required for the inhibition of AC activity (fluphenazine-IC50, 14 microM; melittin-IC50, 0.7 microM). The actions of these antagonists, furthermore, appear to be calcium insensitive, as melittin affects the stability of both the high affinity (calcium containing) and low affinity (calcium depleted) receptor-MSH complexes. The sensitivity of the MSH receptor to inhibition by calmodulin antagonists resembles that described for purified calmodulin-sensitive enzyme systems, which suggests a possible role for calmodulin in MSH receptor function. Among peptide hormone receptors, this effect by calmodulin antagonists appears to be unique for the MSH receptor.

Expression of rat mRNA coding for hormone‐stimulated adenylate cyclase in Xenopus oocytes 1

Beta-Adrenergic agonist-stimulated hyperpolarization, whole-cell cAMP accumulation, and activity of isoproterenol-stimulated membrane-bound adenylate cyclase (EC 4.6.1.1) in Xenopus laevis ovarian oocytes are entirely dependent on the presence of nascent follicle cells. A method was developed to remove rapidly and completely all extra-oocyte cell types to yield defolliculated oocytes that exhibited normal viability and resting membrane potentials yet lacked beta-adrenergic receptor (beta AR)-stimulated responses. Purified follicle membranes contained beta AR-stimulated adenylate cyclase activity, whereas oocyte cell membranes did not. Purified oocyte membrane preparations from X. laevis oocytes previously microinjected with C6-2B rat astrocytoma mRNA, and subsequently defolliculated, exhibited novel beta AR and forskolin-stimulated adenylate cyclase activity. These experiments demonstrate that oocytes expressed rat C6-2B mRNA coding for the beta-adrenergic receptor and the components necessary for forskolin-stimulated adenylate cyclase activity.

3H]Spiroxatrine labels a serotonin 1A-like site in the rat hippocampus

[ 3H]Spiroxatrine was examined as a potential ligand for the labeling of 5-HT 1A sites in the rat hippocampus. Analysis of the binding of [ 3H]spiroxatrine in the absence and presence of varying concentrations of three monoamine neurotransmitters revealed that serotonin (5-HT) had high affinity (IC 50= 20.7 nM for the [ 3H]spiroxatrine binding sites, consistent with the labeling of 5-HT 1 sites, while dopamine and norepinephrine had very low affinity (IC 50=57600 nM and >10 −4 M respectively). Saturation studies of the binding of [ 3H]spiroxatrine revealed a single population of sites with a K d=2.21 nM. Further pharmacologic characterization with the 5-HT 1A ligands 8-hydroxy-2-(di-n-propylamino) tetralin, ipsapirone, and WB4101 and the butyrophenone compounds spiperone and haloperidol gave results that were consistent with [ 3H]spiroxatrine labeling 5-HT 1A sites. This ligand produced stable, reproducible binding with a good ratio of specific to nonspecific binding. The binding of [ 3H]spiroxatrine was sensitive to GTP, suggesting that this ligand may act as an agonist. This was supported by the finding that spiroxatrine inhibits forskolin-stimulated adenylate cyclase activity (a proposed 5-HT 1A receptor model) in the rat hippocampus. Since [ 3H]spiroxatrine is structurally distinct from other currently available radioligands for the 5-HT 1A site, it should provide new information about the properties of this putative serotonergic receptor.

Differential effects of chronic administration of desipramine on the cyclic AMP response in cortical slices and membranes in the rat

Chronic administration of desipramine resulted in a significant decrease in the degree of stimulation of production of cyclic AMP by noradrenaline, isoproterenol and forskolin in slices of the cerebral cortex of the rat. In preparations of membranes from similarly treated animals, GppNHp- and forskolin-stimulated adenylate cyclase activity was increased, while isoproterenol-, manganese- and sodium fluoride-stimulated activity was unchanged. It is concluded that chronic administration of desipramine exerts effects on the adenylate cyclase system additional to its action on beta-adrenergic receptors, possibly by modifying the catalytic or guanyl nucleotide binding units of the enzyme.