Enhanced cAMP accumulation after termination of cholinergic action in the heart

Cholinergic agents decrease myocardial contractility in part by inhibiting adenylate cyclase (EC 4.6.1.1) activity. We have found that after a prolonged preincubation period (greater than 6 h), washout of cholinergic agents from embryonic chick hearts or cultured heart cells results in a persistent increase in their basal and catecholamine-stimulated cAMP content. Membranes prepared from pretreated cells have elevated basal, forskolin-, and catecholamine-stimulated adenylate cyclase activities. This myocardial adaptation to cholinergic agents is analogous to changes in nerve cells and other cell types after prolonged exposures to narcotics or other inhibitors of adenylate cyclase, respectively. A rapid (less than 5 min) adaptation response to cholinergic agents can also be demonstrated in heart cells by quickly blocking agonist action with atropine. Atropine alone has no effect, but after a brief preincubation period with agonists (methacholine or oxotremorine), the addition of atropine transiently enhances catecholamine-stimulated cAMP accumulation by 2.5-fold. These responses are absent in heart cells pretreated with pertussis toxin. The data indicate that the response is not mediated by the phosphoinositide pathway, which has been demonstrated to be insensitive to pertussis toxin in chick heart. Enhanced cAMP accumulation after termination of muscarinic agonist action may provide an explanation for the observation that acetylcholine sometimes produces biphasic contractile responses.     

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 occurring at 20 mM. Dimethylnitrosamine also elevated the temperature at which the lipid phase separation occurred in rat liver plasma membranes, from 28 degrees C to 31 degrees C. The non-carcinogenic but structurally similar compound, dimethylamine hydrochloride neither inhibited adenylate cyclase nor decreased plasma membrane fluidity. It is suggested that the decrease in membrane fluidity, induced by dimethylnitrosamine, via its effects on membrane fluidity, could influence plasma membrane function and cellular regulation.     

Mechanisms of heart adenylate cyclase activation by epinephrine and fluoride ions

The effects of epinephrine and NaF on the membrane preparations of adenylate cyclase from rabbit heart were studied. After preincubation with epinephrine or NaF at 37 degrees C and subsequent washing of the membranes at 4 degrees C from the effectors, adenylate cyclase passes into the activated state and loses its sensitivity to epinephrine and NaF. The effect may be "reversed" by preincubation of the membranes at 37 degrees C. The addition of ATP to the preincubation media does not affect the regulatory and catalytic properties of the enzyme. It is assumed that adenylate cyclase regulation by hormones and fluoride ions may occur without hypothetical processes of phosphorylation and dephosphorylation of the enzyme. The effect of preincubation is probably due to the temperature-dependent association and dissociation of the enzyme-receptor complex in the membrane. Epinephrine and NaF partially protect the cyclase against trypsin-induced inactivation, which is indicative of structural or conformational changes of the adenylate cyclase complex during its interaction with activators.     

Platelet release products modulate some aspects of polymorphonuclear leukocyte activation.

The aim of this research was to evaluate in vitro interactions between platelets and polymorphonuclear leukocytes. The effects of supernatant from thrombin-activated platelets and two platelet release products (adenosine triphosphate and beta-thromboglobulin) were tested on the following features of polymorphonuclear leukocytes activation: opsonized zymosan and phorbol myristate acetate stimulated chemiluminescence, release of membrane bound calcium, NADPH-oxidase activity, and membrane fluidity (fluorescent polarization). The results showed that the addition of platelet supernatant to polymorphonuclear leukocytes induces a significant activation of cells. On the other hand, after three hours of preincubation of polymorphonuclear leukocytes with platelet supernatant, a decreased response of polymorphonuclear leukocytes to stimulation with phorbol myristate acetate, a significant decrease in NADPH-oxidase activity, and a lowered membrane fluidity were observed. Adenosine triphosphate modulated only opsonized zymosan stimulated chemiluminescence, with and without preincubation with polymorphonuclear leukocytes. Beta-thromboglobulin caused a decrease of the chemiluminescent response of polymorphonuclear leukocytes, using both agonists, with and without preincubation with polymorphonuclear leukocytes. Moreover beta-thromboglobulin only caused a decrease of the polymorphonuclear leukocytes membrane fluidity without preincubation with the cells. These results support the thesis that platelets have a "time-related" modulating activity on polymorphonuclear leukocytes.     

Heterologous desensitization of adenylate cyclase with prostaglandin E1 alters sensitivity to inhibitory as well as stimulatory agonists

Adenylate cyclase in cultured human fibroblasts is activated by prostaglandin E1 (PGE1) or beta-adrenergic agonists, e.g., isoproterenol, and inhibited by muscarinic agonists. Incubation with PGE1 reduced adenylate cyclase responsiveness to both PGE1 and isoproterenol; this so-called heterologous desensitization is believed to result from impaired function of the stimulatory guanyl nucleotide-binding protein of the cyclase complex. The effect of heterologous desensitization by PGE1 on inhibition of adenylate cyclase by the muscarinic agonist oxotremorine was examined. Muscarinic inhibition of basal and isoproterenol-stimulated cAMP accumulation was attenuated following exposure to PGE1; the concentration of oxotremorine required for half-maximal inhibition of cAMP accumulation was increased. In both intact cells and membrane preparations the number of binding sites for [3H]scopolamine, a muscarinic antagonist, was unaltered by desensitization. Following exposure to PGE1, receptor affinity for oxotremorine, assessed by competition with [3H] scopolamine, and the guanyl nucleotide sensitivity of agonist binding were reduced. The amount of inhibitory guanyl nucleotide-binding regulatory protein available for [32P]ADP-ribosylation by pertussis toxin was unaltered by desensitization. Thus, heterologous desensitization of adenylate cyclase with the stimulatory agonist PGE1 alters sensitivity to inhibitory as well as stimulatory ligands.     

Ceratitis capitata brain adenylate cyclase and its membrane environment

Adenylate cyclase activation by GTP and octopamine as well as basal activity (in the presence of Mg2+) have been studied as a function of membrane structure in plasma membranes from brain of the dipterous Ceratitis capitata. Benzyl alcohol and lidocaine, but not phenobarbital, inhibited the three activities to the same extent. Triton X-100-solubilized adenylate cyclase was also inhibited by benzyl alcohol and lidocaine, but not by phenobarbital. Results could be explained by an effect on the catalytic unit lipid environment, which would be maintained after solubilization, counteracting the effect of these drugs to facilitate lateral diffusion and coupling of adenylate cyclase components in the lipid bilayer. The observation that the insect adenylate cyclase is relatively insensitive to changes in bulk bilayer fluidity is strengthened by the absence of effect of phenobarbital on enzyme activities. Indeed, this compound was as active as lidocaine or benzyl alcohol in increasing bulk membrane fluidity. The response of C. capitata adenylate cyclase to changes in membrane fluidity is different from that recorded in mammalian systems. This may be functionally important and result from the fact that insects are not warm-blooded.     

Ontogenetic study of the regulatory and coupling function of guanyl nucleotide-binding proteins in the hormone-sensitive adenylate cyclase system

Reconstitution of catecholamine-sensitive adenylate cyclase from chick embryonic muscle membranes and guanyl nucleotide-binding proteins of mature rabbit muscle makes it possible to reveal the coupling (potentiating) effect of these nucleotides 1 week earlier than in the native condition. The effective insertion of guanyl-nucleotide-binding proteins into the embryonic membrane coincides with the onset of a pronounced increase in membrane lipid fluidity during the course of embryogenesis. The different ontogenetic time-courses for the origination of the two guanyl nucleotide effects, on catalytic adenylate cyclase activity (in early embryogenesis) and on the coupling process (in postembryonic life), suggest the existence in this system of two separate guanyl-nucleotide-binding proteins performing regulatory and coupling functions, respectively.     

Activation of adenylate cyclase in Acanthamoeba palestinensis

Preincubation of Acanthamoeba palestinensis homogenates in 0.25M sucrose-TM (2mM MgSO4 and 5mM Tris-HCl, pH 7.4) at 0 degree C for increasing periods of time up to 3 h, leads to a progressive increase in the activity of adenylated cyclase. In contrast, preincubation of isolated membrane fractions enriched in enzyme activity in the same medium results in no activation. However, preincubation of membrane fractions in medium containing a high density of sugars (sucrose, glucose or fructose) mimics the activation obtained with homogenates. The high density sugar activation is time and temperature dependent, and reversible upon return to a low density medium. The high osmotic pressure of the sugars utilized may be a factor, since high concentrations of the sucrose polymer, Ficoll, which has low osmotic activity, causes not activation. Soluble activators, protein synthesis and changes in cyclic nucleotide phosphodiesterase activity were all eliminated as possible effectors of the apparent activation of adenylate cyclase. In contrast to mammalian adenylate cyclase, the endoplasmic reticulum localized enzyme of Acanthamoeba is inhibited by NaF and is unaffected by GTP, adenosine, epinephrine, prostaglandin E1, propranolol, and meclofenamic acid. These data indicate that the adenylate cyclase of Acanthamoeba is structurally different from that of most mammalian cells.     

Increase in membrane fluidity and opening of tight junctions have similar effects on sodium-coupled uptakes in renal epithelial cells

Apical membranes of renal epithelial cells were shown to be more rigid than other plasma membranes, due in part to the abundance of sphingomyelin among their constituent phospholipids. Tight junctions play a key role in maintaining differences between the apical and the basolateral domains of the plasma membrane with respect to their lipid composition and fluidity. To evaluate the influence of alterations of membrane fluidity on the activity of two apically located transport systems, we compared the effect of opening of tight junctions, by a preincubation period in calcium-deprived medium and of increasing fluidity, with benzyl alcohol, on Na-dependent uptakes of Pi and alpha-methyl-D-glucopyranoside (MGP) in intact, confluent LLC-PK1 cells and MDCK cells. Benzyl alcohol, at 10 mM, increased the Vmax of Pi uptake by 55 and 42% in LLC-PK1 cells and MDCK cells, respectively, but decreased the Vmax of MGP uptake in LLC -PK1 cells by 23%. Similarly to 10 mM benzyl alcohol, opening of tight junctions also increased the Vmax of Pi uptake by 45 and 46% in LLC-PK1 cells and MDCK cells, respectively, and depressed MGP uptake in LLC-PK1 cells by inducing a 15% decrease of the Vmax. None of the two maneuvers (i.e. addition of benzyl alcohol or opening of tight junctions) affected the Km values of the transport systems. From these results it is concluded that (i) the increase in membrane fluidity, achieved either by benzyl alcohol or by opening of tight junctions, affects Na-Pi and Na-glucose cotransports differently, reflecting differences in the lipid environments of the two transport systems, and (ii) membrane fluidity might play a physiological role in the modulation of the activity of transport systems.     

Alterations in cerebral β-adrenergic receptor-adenylate cyclase system induced by halothane,ketamine and ethanol

The effect of halothane, ketamine and ethanol on β-adrenergic receptor adenylate cyclase system was studied in the brain of rats. An anesthetic concentration of halothane and ketamine added in vitro decreased the stimulatory effect of norepinephrine on cyclic AMP formation in slices from the cerebral cortex. On the other hand, ethanol increased the basal activity of cerebral adenylate cyclase without affecting on the norepinephrine-stimulated activity. The increase of the basal activity induced by ethanol was not antagonized by propranolol, a β-adrenergic antagonist. In the crude synaptosomal (P₂) fraction, these drugs had no significant effect on the basal adenylate cyclase activity, binding of [³H]dihydroalprenolol to β-receptor, and binding of [³H]guanylylimido diphosphate ([³H]Gpp(NH)p) to guanyl nucleotide binding site. In contrast, the adenylate cyclase activity stimulated by Gpp(NH)p or NaF was significantly inhibited by an anesthetic concentration of these drugs. An anesthetic concentration of these drugs increased the membrane fluidity of P₂ fraction monitored by the fluorescence polarization technique. The addition of linoleic acid (more than 500 μM) also induced not only the increase of fluidity, but also the decrease of Gpp(NH)p- or NaF-stimulated adenylate cyclase activity in the cerebral P₂ fraction. The present results suggest that general anesthetics may interfere with the guanyl nucleotide binding regulatory protein-mediated activation of cerebral adenylate cyclase by disturbing the lipid region of synaptic membrane.     

TEI-9063, a stable and highly specific prostacyclin analogue for the prostacyclin receptor in mastocytoma P-815 cells.

The prostacyclin (PGI2) analogues, TEI-9063 and its methyl ester, TEI-1324, have been compared with another stable analogue, iloprost, with respect to binding to the PGI2 receptor, stimulation of adenylate cyclase activity and inhibition of thrombin-induced Ca2+ mobilization in mastocytoma P-815 cells. TEI-9063 displaced the [3H]iloprost binding to the membrane fraction, the IC50 value being 3 nM, but showed very low affinity for the PGE receptor. TEI-9063 dose dependently stimulated cAMP formation in the cells and GTP-dependent adenylate cyclase activity in the membrane fraction, the EC50 value being 50 and 10 nM, respectively. Furthermore, TEI-9063 prevented the thrombin-induced increase in the intracellular Ca2+ concentration, the IC50 value being 50 nM. These IC50 and EC50 values are lower than those obtained for iloprost. On the other hand, those of TEI-1324 were about two-orders higher. Although PGI2 lost its ability to stimulate cAMP formation by preincubation for 20 min at 37 degrees C, TEI-9063 completely retained its ability after 60-min preincubation. These results demonstrate that TEI-9063 is a stable and stronger agonist for the PGI2 receptor than iloprost, and that it prevents thrombin-induced Ca2+ mobilization through stimulation of the adenylate cyclase system in mastocytoma cells.     

Effects of oxotremorine and RMI 12330 A on [3H]acetylcholine release and adenylate cyclase activity in guinea pig superior cervical ganglion

There is considerable evidence that adenosine 3, 5-cyclic monophosphate (cAMP) is involved in the modulation of synaptic transmission in the guinea pig superior cervical ganglion (SCG). Presynaptic muscarinic receptors are known to attenuate, when activated, acetylcholine (ACh) release in the periphery as well as in the brain. Thus, the possible relationship between ganglionic adenylate cyclase activity and the output of ACh from electrically stimulated ganglia, preloaded with [³H]choline, was investigated. The muscarinic agonist oxotremorine significantly reduced in a dose-dependent manner the electrically evoked neurotransmitter release. The adenylate cyclase inhibitor N-(cis-2-phenylcyclopentyl)azacyclotridecan-2-imine hydrochloride (RMI 12330 A) also decreased ACh output. The inhibitory effects of these two drugs were additive. In crude ganglion membrane fractions oxotremorine significantly inhibited adenylate cyclase activity. The results indicate that drugs capable of inhibiting adenylate cyclase, significantly decrease ACh output from preganglionic nerve terminals in guinea pig SCG.     

Differential effects of chronic agonist administration on mu-opioid receptor- and muscarinic receptor-regulated adenylate cyclase in rat striatal neurons.

In cultured rat striatal neurons exposed to 10 microM morphine or oxotremorine for 24 hours, we observed an increased (about 30%) dopamine D1 receptor-stimulated cyclic AMP production, whereas no desensitization of mu-opioid receptor or muscarinic cholinergic receptor was found. However, whereas upregulation of dopamine D1 receptor-stimulated adenylate cyclase activity upon 7 days morphine exposure was at least as pronounced as observed after 24 hours of exposure to the opioid, this adaptive phenomenon was virtually absent following one week of oxotremorine treatment. This reduced adenylate cyclase sensitization upon 7 days oxotremorine exposure appeared to coincide with desensitization of muscarinic cholinergic receptors. A possible role of the resistance of mu receptors to desensitization and the (resulting) upregulation of the neuronal adenylate cyclase system upon chronic receptor activation in the development of opiate tolerance and dependence is suggested.     

The role of sulfhydryl groups in functioning of components of adenylate cyclase signal system in smooth muscles of the mollusk Anodonta cygnea (effect of N-ethylmaleimide and p-chloromercuribenzoic acid)]

The alkylating agent N-ethylameimide and the sulfhydryl group blocker p-chloromercuribenzoic acid (CPMA) inhibited in dose-dependent manner both basal activity of adenylyl cyclase (AC) and its activity stimulated by non-hormonal substances (forskolin, sodium fluoride, guanylilimidodiphosphate) in smooth muscles of the freshwater bivalve mollusk Anodonta cygnea. The double increase (from 30 to 60 min) in the time of preincubation of a sarcolemmal membrane fraction with ethylmaleimide and CPMA led to an essential increase in enzyme inhibition (especially for CPMA). 50 mM SH-containing reagent beta-mercaptoethanol (ME) partially restored the AC activity, inhibited by N-ethylmaleimide and CPMA, except when these two latter reagents were in high concentrations (1-10 and 0.5 mM, respectively). The data obtained point to the key role of cysteine SH-groups in regulation of the functional activity of proteins, components of the adenylyl cyclase system--AC and heterotrimeric G-proteins.     

Hepatic plasma membrane fluidity and dietary proteins.

Effect of protein deficient diet on hepatic plasma membrane fluidity has been studied in rats using (i) steady state fluorescence polarization and anisotropy, (ii) phospholipid and cholesterol contents, (iii) phospholipid fatty acid composition, (iv) turnover of phosphatidyl choline (PC), and (v) activities of membrane-bound enzymes as parameters and rats fed casein (20%) diet as standard group. A significant increase in steady state fluorescence and anisotropy values was registered in the deficient group, indicating increased resistance and hence decrease in fluidity of the plasma membrane. Supplementation of the diet with lysine and threonine improved these values, thereby suggesting the significance of diet for membrane fluidity. Simultaneous significant alterations in other parameters, viz. (i) decrease in PC, PE and free cholesterol and increase in esterified cholesterol contents, (ii) decrease in unsaturation of fatty acids of PC, (iii) decrease in incorporation of NaH2 32PO4, [CH3-14C]choline and [CH3-14C]methionine into plasma membrane PC, and (iv) decrease in activities of plasma membrane 5'-nucleotidase and phosphodiesterase along with increase of (Na(+)-K+)ATPase and adenyl cyclase, were observed in the deficient group which on supplementation with lysine and threonine showed improvement over alterations.     

Attenuation of chick heart adenylate cyclase by muscarinic receptors after pertussis toxin treatment

Pertussis toxin selectively modifies the function of Ni, the inhibitory guanine nucleotide binding protein of the adenylate cyclase complex. In chick heart membranes, guanine nucleotide activation of Ni resulted in a decrease in the apparent affinity of the muscarinic receptor for the agonist oxotremorine, inhibition of basal adenylate cyclase activity, and the attenuation of adenylate cyclase by oxotremorine. Treatment of chicks with pertussis toxin caused the covalent modification of 80-85% of cardiac Ni. After this treatment Gpp(NH)p had no effect on muscarinic receptor affinity and GTP stimulated basal adenylate cyclase activity. In contrast, the GTP-dependent attenuation of adenylate cyclase caused by muscarinic receptors was unaffected.     

Inhibition of adenylate cyclase by transglutaminase-catalyzed reactions in pigeon erythrocyte ghosts

We report the occurrence in pigeon erythrocytes of a soluble Ca2+-dependent transglutaminase (TGase) activity. The effect of the erythrocyte ghost protein modifications, determined by TGase-catalyzed reactions, on adenylate cyclase, phospholipid methyltransferase I and II activities and on the lipidic matrix fluidity of the membrane was investigated by using a purified guinea pig liver TGase preparation. The results showed a significant inhibitory effect of such modifications both on the basal and on the variously stimulated (by NaF, Gpp(NH)p alone or in the presence of 1-isoproterenol) adenylate cyclase activity. By contrast, both the phospholipid methylation and the fluidity of the lipidic matrix of the membrane were unaffected by TGase-mediated reactions. These data suggest a new possible inhibitory mechanism of the cyclic AMP synthesis which might be triggered by the enhancement of the cytosolic Ca2+ concentration.     

Regulation by forskolin of octopamine-stimulated adenylate cyclase from brain of the dipterous Ceratitis capitata

Forskolin, a diterpene that exerts several pharmacological effects, activates adenylate cyclase in brain and in some other mammalian tissues. Properties of forskolin activation of adenylate cyclase from central nervous system of the dipterous Ceratitis capitata are described. The interaction of forskolin with the insect adenylate cyclase system was studied by evaluating its effect on metal-ATP kinetics, protection against thermal inactivation, membrane fluidity and enzyme modulation by fluoride, guanine nucleotides, octopamine, and ADP-ribosylation by cholera toxin. The diterpene stimulated basal enzyme activity both in membranes and Triton X-100-solubilized preparations, apparently devoid of functional regulatory unit, this effect being rapidly reversed by washing the membranes. An increase of Vmax accounts for the activation of soluble and membrane adenylate cyclase preparations by forskolin, whereas the affinity of the enzyme for the substrate was not affected. Forskolin apparently protects the membrane enzyme from thermal inactivation, and at concentrations that promote the enzyme activity the diterpene does not alter membrane microviscosity. Forskolin does not appear to alter the sensitivity of insect adenylate cyclase to sodium fluoride, guanine nucleotide, or regulatory subunit ADP ribosylated by cholera toxin, the combined effect of these factors with the diterpene resulting in a nearly additive enzymatic activation. However, forskolin blocks the octopamine stimulatory input. Results obtained with the insect adenylate cyclase system are discussed and compared to what is known about mammalian systems to propose a mechanism of enzyme activation by forskolin.     

Cholesterol modulation of beta-adrenergic receptor characteristics

Cholesterol, a major structural component of plasma membranes, has a profound influence on cell surface receptor characteristics and on adenylate cyclase activity. beta-Adrenergic receptor number, adenylate cyclase activity, and receptor-cyclase coupling were assessed in rat lung membranes following preincubation with cholesteryl hemisuccinate. beta-Adrenergic receptor number increased by 50% without a change in antagonist affinity. However, beta-adrenergic receptor affinity for isoproterenol increased 2-fold as a result of an increase in the affinity of the isoproterenol high-affinity binding site. The increase in agonist affinity did not potentiate hormone-stimulated adenylate cyclase activity, which decreased 3-fold following cholesterol incorporation. However, the ratio of isoproterenol to GTP-stimulated activity was unchanged with cholesterol. Stimulation distal to the receptor by GTP, NaF, GppNHp, Mn2+ and forskolin also demonstrated 50-80% reduced enzyme activity following cholesterol incorporation. These data suggest that membrane cholesterol incorporation decreases catalytic unit activity without affecting transduction of the hormone signal.     

Muscarinic agents modify kinetics properties of membrane-bound guanylyl cyclase activity

Plasma membranes from bovine tracheal smooth muscle show guanylyl cyclase activity, which can be stimulated by muscarinic agonists such carbamylcholine and oxotremorine and blocked by atropine. This stimulation was observed in the presence of 150 mM NaCl. In the absence of this salt, guanylyl cyclase activity was considerably higher but was not affected by muscarinic agonists. Carbamylcholine decreased the apparent Km but did not change the Vmax of this enzyme. When plasma membrane fractions were extracted with 1% octylglucoside, guanylyl cyclase activity was preserved, however the muscarinic activation was abolished, despite a muscarinic receptor capable of [3H]quinuclidinylbenzilate binding being present in the extract. The detergent extraction changed the affinity of guanylyl cyclase for GTP but the Mn2+ kinetics was unaltered. Based on these findings and on current information in the literature, we propose that another component is required to restore the link between the muscarinic receptor and guanylyl cyclase, however the nature of this component remains to be established.