Elevated membrane cholesterol concentrations inhibit glucagon-stimulated adenylate cyclase.

A method was devised which increases the cholesterol concentration of rat liver plasma membranes by exchange from cholesterol-rich liposomes at low temperature (4 degrees C). When the cholesterol concentration of liver plasma membranes is increased, there is an increase in lipid order as detected by a decrease in mobility of an incorporated fatty acid spin probe. This is accompanied by an inhibition of adenylate cyclase activity. The various ligand-stimulated adenylate cyclase activities exhibit different sensitivities to inhibition by cholesterol, with inhibition of glucagon-stimulated greater than fluoride-stimulated greater than basal activity. The bilayer-fluidizing agent benzyl alcohol is able to reverse the inhibitory effect of cholesterol on adenylate cyclase activity in full. The thermostability of fluoride-stimulated cyclase is increased in the cholesterol-rich membranes. Elevated cholesterol concentrations abolish the lipid-phase separation occurring at 28 degrees C in native membranes as detected by an incorporated fatty acid spin probe. This causes Arrhenius plots of glucagon-stimulated adenylate cyclase activity to become linear, rather than exhibiting a break at 28 degrees C. It is suggested that the cholesterol contents of both halves of the bilayer are increased by the method used and that inhibition of adenylate cyclase ensues, owing to the increase in lipid order and promotion of protein-protein and specific cholesterol-phospholipid interactions.     

Facilitation of adenylate cyclase stimulation in macrophages by lectins.

Preincubation of guinea pig peritoneal macrophages with concanavalin A (Con A) markedly enhanced the accumulation of 3′,5′-cyclic-adenosine monophosphate (cAMP) in response to the adenylate cyclase (AC) stimulators prostaglandin E₁ (PGE₁) and isoproterenol (IP). Basal cAMP levels were not altered. Maximal enhancement of cAMP accumulation was induced by preincubation with 50–100 μg/ml Con A for 10 min at 37 °C. Con A-induced facilitation of macrophage responsiveness was prevented by α-methyl-d-mannoside (αMM). No facilitation was induced by the divalent derivative, succinyl-Con A or by Con A immobilized on Sepharose beads. Con A-induced facilitation developed normally in macrophages treated with the microfilament blocking agent, cytochalasin B. The responsiveness of macrophages to PGE₁ and IP was also augmented by phytohemagglutinin (PHA) but wheat germ agglutinin (WGA), soy bean agglutinin (SBA), pokeweed mitogen (PWM), and Lotus tetragonolobus lectin (LL) showed no enhancing effect. The effect of Con A on cAMP levels was the result of augmented cAMP synthesis and not of reduced degradation or a block in cAMP egress from the cells. Lectin-induced facilitation of AC stimulation could be mediated via one of the following mechanisms: (i) induction of receptor clustering; (ii) causing a conformational change in the receptors; (iii) inhibition of negative cooperativity; (iv) causing an increase in membrane fluidity; (v) disruption of microtubules by acting as a Ca²⁺ ionophore; or (vi) inactivation of a sugar-containing inhibitor of AC.     

Endothelins inhibit adenylate cyclase in brain capillary endothelial cells.

The action of endothelins (Et) on cAMP formation was studied in endothelial cells from rat brain microvessels. Et-1 and Et-3 had no action by themselves. They both inhibited cholera toxin stimulated adenylate cyclase by about 50%. K0.5 values were observed at 2 nM and 40 nM for Et-1 and Et-3 respectively, indicating an involvement of a low affinity Et-3 receptor. Coupling to adenylate cyclase was achieved by a pertussis toxin sensitive mechanism. Another action of endothelins in brain capillary endothelial cells was to stimulate phospholipase C. This action involved a low affinity Et-3 receptor and a pertussis toxin insensitive mechanism. It is concluded that in brain capillary endothelial cells, ETA like receptors are coupled to phospholipase C and to adenylate cyclase via two different mechanisms.     

Modulation of parathyroid hormone-sensitive adenylate cyclase and arginine vasopressin-sensitive adenylate cyclase by calcium and GTP

Arginine vasopressin (AVP)- and parathyroid hormone (PTH)-sensitive adenylate cyclase were studied in the renal tissue of thyroparathyroidectomized dogs. The results indicate that AVP-sensitive adenylate cyclase activity was highest in the inner medulla followed by the middle medulla, outer medulla, and cortex, in declining order. In contrast, PTH-sensitive adenylate cyclase was absent in the inner medulla, and the highest stimulation was found in the cortex with lesser activity in outer and middle medulla. When 1 mm EGTA was included in the incubation mixture, the addition of both AVP- and PTH to the middle medullary homogenate resulted in additive responses suggesting two separate receptors for each hormone. This EGTA-induced additive effect was eliminated by the addition of calcium into the system, indicating that calcium concentration may be critical in modulating the interaction of AVP and PTH-sensitive adenylate cyclase. In contrast to some previous reports, a particulate fraction prepared from the middle medullary tissue was completely insensitive to either AVP or PTH. Hormonal sensitivity was restored by the addition of GTP or the supernatant.     

The control of adenylate cyclase by calcium in turkey erythrocyte ghosts.

The adenylate cyclase of turkey erythrocytes is inhibited by low concentrations of calcium. Calcium binds to the enzyme system so tightly that the enzyme can compete with ethylene glycol bis(beta-aminoethyl ether)-N, N1-tetraacetic acid (EGTA) for the metal. The calcium binding site is shown to be distinct from the magnesium binding sites required for activity. Thus Ca2+ functions as a negative allosteric effector. Calcium decreases dramatically the V max of the catecholamine-stimulated activity without affecting the affinity for the hormone or for the substrate ATP. The cooperativity in the response toward Mg2+ dependence (Hill coefficient, nH equals 3) is also unaffected by Ca2+ where as the S0.5 (concentration yielding one-half V max) for Mg2+ is affected only slightly. The Ca2+ effect is cooperative (nH equals 2) and therefore brought about by a cluster of Ca2+ binding sites. Mn2+ can substitute for Mg2+ as the enzyme activator but the Mn2+-activated enzyme is no longer inhibited by Ca2+. The possible physiological significance of the Ca2+ effect is discussed.     

Inhibition of adenylate cyclase in human blood platelets by 9-substituted adenine derivatives.

A series of 9-substituted adenine derivatives inhibited adenylate cyclase activity (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) of a particulate preparation of human blood platelets. A 3--6 fold elevation of adenylate cyclase activity by prostaglandin E1 (PGE1) was inhibited in a concentration-related manner by 9-(tetrahydro-5-methyl-2-furyl) adenine (SQ 22,538), 9-(tetrahydro-2-furyl) adenine (SQ 22,536), 9-cyclopentyladenine (SQ 22,534), 9-furfuryladenine (sQ 4647) and 9-benzyladenine (SQ 218611). The I50 values ranged from 21 microM for SQ 22,538 to 140 microM for SQ 21,611. These same adenine derivatives reversed the inhibition by PGE1 of ADP-induced aggregation and the PGE1-stimulated elevation of adenosine 3':5'-monophosphate (cyclic AMP). The reversal of platelet aggregation inhibition by SQ 22,536 and SQ 4647 was concentration-related with I50 values of 30 microM in each case, whereas SQ 22,534 and SQ 21,611 reversed inhibition by 30% at 100 microM. SQ 22,536, SQ 22,534 and SQ 21,611 also blocked the increase in cyclic AMP levels in a concentration-related manner with I50 values of 1, 4 and 60 microM, respectively. SQ 4647 inhibited the elevation of cyclic AMP by more than 85% at 1000 microM. The adenine derivatives had no effect on platelet aggregation or on cyclic AMP levels in the absence of PGE1. These results provide additional evidence that the inhibition of platelet aggregation by PGE1 is mediated by cyclic AMP.     

Brevibacterium liquefaciens adenylate cyclase and its in vivo stimulation by pyruvate.

Adenylate cyclase of Brevibacterium liquefaciens depends on pyruvate for activity. Growing in a simple medium containing glucose and DL-alanine, the microorganism excreted pyruvate, which reached 20 mM in the medium at stationary phase. Using [3H]adenosine to label the adenosine 5'-triphosphate pool, we showed that pyruvate in the medium stimulated adenylate cyclase of B. liquefaciens in vivo, in a manner similar to the stimulation observed in vitro. Adenylate cyclase in cells harvested at different phases of growth was equally responsive to exogenous pyruvate, indicating that the allosteric site for pyruvate was present in the enzyme throughout the various phases of cell growth. The specific activity of adenylate cyclase was highest in cells harvested at early log phase; thereafter it declined and was substantially lower at stationary phase. Although adenylate cyclase appears to be activated by pyruvate throughout the life span of the cell, the activity appears not to be critical to cell growth, which was comparable whether the medium contained high or low pyruvate.     

Refractoriness of ovarian adenylate cyclase to continued hormonal stimulation.

Culture of preovulatory rat follicles with luteinizing hormone, follicle-stimulating hormone or prostaglandin E2 for 24 h reduced the subsequent response of adenylate cyclase to the homologous by 80, 50 and 90%, respectively; yet follicles refractory to luteinizing hormone fully responded to follicle-stimulating hormone responded to luteinizing hormone and prostaglandin E2, and those refractory to prostaglandin E2 could be stimulated by either gonadotropin. Desensitization of the adenylate cyclase system by luteinizing hormone was achieved by hormone concentrations of 0.8--2.0 mug/ml in the medium; a lower dose of luteinizing hormone (0.4 mug/ml), though effective in stimulating adenylate cyclase, did not induce refractoriness. Prostaglandin E2 caused partial refractoriness at dose levels of 0.1--0.25 mug/ml; higher dose levels were more effective. These findings suggest that continued exposure to the preovulatory follicle to elevated levels of hormones may cause perturbations in either the interaction between the hormone and its specific receptor or in a subsequent step essential for activation of adenylate cyclase.     

Uncoupling by proteolysis of alpha-adrenergic receptor-mediated inhibition of adenylate cyclase in human platelets

Ethyl 2{5(4-chlorophenyl)pentyl}oxiran-2-carboxylate (POCA) is a new hypoglycaemic compound. The POCA-CoA ester strongly inhibits β-oxidation at carnitine palmitoyltransferase I. Chronic administration of POCA to rats decreases plasma concentrations of cholesterol and triacylglycerol and increases the number of hepatic peroxisomes similarly to hypolipidaemic drugs related to clofibrate. Peroxisomal fractions from rats fed a diet containing 0.2% of POCA for 4 weeks were prepared on self-generated Percoll gradients. POCA induced a 4-fold increase in catalase activity and peroxisomal β-oxidation, agreeing with the morphological data. The increase in peroxisomal β-oxidation caused by POCA feeding does not prevent accumulation of lipid following the inhibition of mitochondrial β-oxidation.     

The stimulation of hepatic adenylate cyclase by prostaglandin E1

Adenylate cyclase activity associated with particulate preparations from rat, mouse, rabbit, and dog liver is stimulated 2-to 5-fold by prostaglandin E₁ (PGE₁). This stimulation is dependent upon the presence of guanosine-5′-triphosphate (GTP). Prostaglandins F_1a and F_2a do not alter the enzymatic activity under these same conditions. Optimal concentrations of PGE₁ + GTP stimulate rat liver adenylate cyclase more than glucagon alone, but less than glucagon + GTP. Activity measured with glucagon + GTP is not affected by addition of PGE₁. Stimulation from PGE₁ + GTP is increased by glucagon to the same level measured with glucagon + GTP.     

Glycogen synthesis stimulation by adenylate cyclase inhibition in rat epididymal adipocytes

The effects of adenylate cyclase inhibition on the transport of glucose and fructose and their incorporation into glycogen were investigated in order to assess the extent to which lowered cAMP levels can take part in the various components of glycogen synthesis regulation in isolated rat epididymal adipocytes. The dose-response characteristics of (R)-N-(2-phenylisopropyl)adenosine (PIA), a potent and specific adenylate cyclase inhibitor, on glycogen synthesis were compared with those effectively inhibiting lipolysis, a measure of functional cAMP levels. PIA had no effect on basal glucose or fructose transport but stimulated glucose and fructose incorporation into glycogen. Their respective incorporation was 10 and 69% of that achieved in the presence of insulin. These effects of PIA were shown to be in part the result of increased glycogen synthase I activity. PIA was 20% as effective as insulin in this action. Thus, were insulin to lower cAMP levels and/or inhibit cAMP-dependent protein kinase, this action would be irrelevant to glucose transport but would contribute to the stimulation of glycogen metabolism. However, an additional mechanism(s) involving neither increased glucose transport nor lowered cAMP levels is required to account for the full action of insulin. Fat cells in the absence of medium glucose and in the presence of 10(-7) M PIA and adenosine deaminase constitute a system functionally depleted of cAMP where this mechanism can be studied in isolation.     

Sensitized guinea-pig lung: altered adenylate cyclase stimulation by epinephrine

Adenylate cyclase (AC) was measured in healthy and sensitized quinea-pig lungs. Basal activities were 24.49 ± 2.50 and 26.73 ± 3.03 pmols cyclic AMP mg protein/minute, respectively. NaF produced about threefold activity increase in both groups. Low concentrations of epinephrine (EPI) 10^?9 ? 10^?6M, maximally stimulated the enzyme in sensitized lungs. In contrast, these concentrations had no effect in healthy lungs. Higher EPI concentrations, 10^?5 ? 10^?2M, while progressively stimulating less the AC in sensitized lungs, increased the response in the healthy lungs. The maximal increase in AC activity, about 200%, was achieved with 10^?6 and 10^?3M EPI in sensitized and healthy lungs, respectively. Propranolol blocked the effect of EPI in both groups. The results indicate that sensitization altered the AC system in guinea-pig lungs.     

Acidic phospholipid species inhibit adenylate cyclase activity in rat liver plasma membranes.

Incubation of rat liver plasma membranes with liposomes of dioleoyl phosphatidic acid (dioleoyl-PA) led to an inhibition of adenylate cyclase activity which was more pronounced when fluoride-stimulated activity was followed than when glucagon-stimulated activity was followed. If Mn2+ (5 mM) replaced low (5 mM) [Mg2+] in adenylate cyclase assays, or if high (20 mM) [Mg2+] were employed, then the perceived inhibitory effect of phosphatidic acid was markedly reduced when the fluoride-stimulated activity was followed but was enhanced for the glucagon-stimulated activity. The inhibition of adenylate cyclase activity observed correlated with the association of dioleoyl-PA with the plasma membranes. Adenylate cyclase activity in dioleoyl-PA-treated membranes, however, responded differently to changes in [Mg2+] than did the enzyme in native liver plasma membranes. Benzyl alcohol, which increases membrane fluidity, had similar stimulatory effects on the fluoride- and glucagon-stimulated adenylate cyclase activities in both native and dioleoyl-PA-treated membranes. Incubation of the plasma membranes with phosphatidylserine also led to similar inhibitory effects on adenylate cyclase and responses to Mg2+. Arrhenius plots of both glucagon- and fluoride-stimulated adenylate cyclase activity were different in dioleoyl-PA-treated plasma membranes, compared with native membranes, with a new 'break' occurring at around 16 degrees C, indicating that dioleoyl-PA had become incorporated into the bilayer. E.s.r. analysis of dioleoyl-PA-treated plasma membranes with a nitroxide-labelled fatty acid spin probe identified a new lipid phase separation occurring at around 16 degrees C with also a lipid phase separation occurring at around 28 degrees C as in native liver plasma membranes. It is suggested that acidic phospholipids inhibit adenylate cyclase by virtue of a direct headgroup specific interaction and that this perturbation may be centred at the level of regulation of this enzyme by the stimulatory guanine nucleotide regulatory protein NS.     

Calmodulin stimulation of adenylate cyclase of intestinal epithelium

The effect of dicyclohexylcarbodiimide (DCCD) on the proton pumping two-subunit cytochrome c oxidase from Paracoccus denitrificans was investigated. Purified Paracoccus oxidase was reconstituted into phospholipid vesicles by cholate dialysis. Following incubation with increasing amounts of DCCD, proton ejection was recorded in response to reductant pulses with reduced cytochrome c. Concentrations of DCCD which greatly reduced proton pumping by bovine cytochrome c oxidase used as a control were found to exert only a minor effect on proton translocation by Paracoccus oxidase. Similarly, incubation of the bacterial enzyme with [14C]DCCD failed to reveal the specific covalent interaction previously demonstrated to occur with bovine cytochrome c oxidase, and here also shown for the oxidase of yeast. Thus, Paracoccus oxidase differs in its interaction with DCCD from the functionally analogous eukaryotic enzymes.     

Chymotrypsin selectively decreases forskolin stimulation of adenylate cyclase

We studied the effects of chymotrypsin on turkey erythrocyte membrane adenylate cyclase activity. Proteolysis with chymotrypsin led to a concentration- and time-dependent increase in activation of adenylate cyclase by isoproterenol + guanine nucleotides, and fluoride, and to a decrease in activation by forskolin. Maximal effects (up to 10-fold increases in fluoride- and isoproterenol + guanine nucleotide-stimulated activity, and up to 100% inhibition of forskolin-stimulated activity) occurred under similar conditions (10-20 micrograms/ml chymotrypsin for 10-15 min at 30 degrees C). Augmentation of isoproterenol + guanosine-3'-O-thiotriphosphate (GTP-gamma-S)-stimulated activity by chymotrypsin occurred only if proteolysis preceded stimulation with isoproterenol + GTP-gamma-S. Addition of isoproterenol + GTP-gamma-S to membranes before proteolysis, however, did not prevent chymotrypsin from augmenting subsequent stimulation by these agents. In contrast, addition of forskolin during proteolysis with chymotrypsin prevented the time- and concentration-dependent decline in forskolin stimulation observed with chymotrypsin. Proteolysis decreased the magnitude of stimulation at any concentration of forskolin, but did not alter the concentration dependence of forskolin stimulation (apparent half-maximum = 3 microM). The data are consistent with the existence of a chymotrypsin-sensitive site essential for forskolin stimulation of adenylate cyclase. In view of the simultaneous effect of chymotrypsin to augment fluoride- and isoproterenol + guanine nucleotide-stimulated activities, it is highly unlikely that the site is on the stimulatory guanine nucleotide binding protein. Since forskolin is thought to act directly on the catalytic unit of adenylate cyclase, and since forskolin can protect against the effect of proteolysis with chymotrypsin, the site involved may be on the catalytic unit itself.     

Activation of adenylate cyclase by molybdate.

The effect of molybdate on adenylate cyclase (EC 4.6.1.1) in rat liver plasma membranes has been examined. The apparent K alpha for molybdate activation of the enzyme is 4.5 mM, and maximal, 7-fold stimulation is achieved at 50 mM. The observed increase in cAMP formation in the adenylate cyclase assay is not due to: (a) an inhibition of ATP hydrolysis; (b) a molybdate-catalyzed conversion of ATP to cAMP; (c) an inhibition of cAMP hydrolysis; or (d) an artifact in the isolation of cAMP formed in the reaction. Molybdate activation of adenylate cyclase is a general phenomenon exhibited by the enzyme in brain, cardiac, and renal tissue homogenates and in erythrocyte ghosts. However, like fluoride and guanyl-5'-yl imidodiphosphate (Gpp(NH)p), molybdate does not activate the soluble rat testicular adenylate cyclase. Molybdate is a reversible activator of adenylate cyclase. Activation is not due to an increase in ionic strength and is independent of the salt used to introduce molybdate. Molybdate does not activate adenylate cyclase previously stimulated with Gpp(NH)p or fluoride. At concentration greater than 20 mM, molybdate inhibits fluoride-stimulated adenylate cyclase, and at concentrations greater than 100 mM, molybdate stimulation of basal adenylate cyclase activity is diminished.     

Thromboxane receptor stimulation inhibits adenylate cyclase and reduces cyclic AMP-mediated inhibition of ADP-evoked responses in fura-2-loaded human platelets.

Stimulation of human platelets with the thromboxane A2 analogue, U46619, after treatment with prostaglandin E1 or forskolin, reduced the inhibition of ADP-evoked Mn2+ influx and the release of Ca2+ from intracellular stores. U46619 decreased the elevated concentration of 3',5'-cyclic AMP in platelets that were pretreated with prostaglandin E1. These results suggest that occupation of prostaglandin H2/thromboxane A2 receptors, like those for other agonists, inhibits adenylate cyclase activity, which can contribute to the promotion of platelet activation.     

Y1 receptors for neuropeptide Y are coupled to mobilization of intracellular calcium and inhibition of adenylate cyclase

Two types of binding sites have previously been described for neuropeptide Y (NPY), called Y1 and Y2 receptors. The intracellular events following Y1 receptor activation was studied in the human neuroblastoma cell line SK-N-MC. Both NPY and the specific Y1 receptor ligand, [Leu31,Pro34]-NPY, caused a rapid and transient increase in the concentration of free calcium in the cytoplasm as measured by the fluorescent probe, Fura-2. The effect of both peptides was independent of extracellular calcium as addition of EGTA or manganese neither changed the size nor the shape of the calcium response. The calcium response to NPY was abolished by pretreatment with thapsigargin, which can selectively deplete a calcium store in the endoplasmic reticulum. Y1 receptor stimulation, by both NPY and [Leu31,Pro34]NPY, also inhibited the forskolin-stimulated cAMP production with an EC50 of 3.5 nM. There was a close relation between the receptor binding and the cellular effects as half-maximal displacement of [125I-Tyr36]monoiodoNPY from the receptor was obtained with 2.1 nM NPY. The Y2-specific ligand NPY(16-36)peptide had no effect on either intracellular calcium or cAMP levels in the SK-N-MC cells. It is concluded that Y1 receptor stimulation is associated with both mobilization of intracellular calcium and inhibition of adenylate cyclase activity.