Induction of prostacyclin receptor expression in human erythroleukemia cells

We have identified both high-affinity (KD = 36 +/- 3 nM) and low-affinity (KD = 2.1 +/- 0.8 microM) prostacyclin (PGI2)-receptor sites on human erythroleukemia (HEL) cells using the radiolabelled prostacyclin analogue. [3H]iloprost. The addition of the phorbol ester, TPA, to the culture medium caused a 5-10-fold increase in the number of both the low- and the high-affinity sites, without any change in their affinity constants. Iloprost stimulated HEL cell membrane adenylate cyclase activity 5-fold. This stimulation was potentiated in the presence of GTP, indicating a conventional PGI2 receptor-G2-adenylate cyclase system. HEL cells represent a source of prostacyclin receptor mRNA which may be of value in expression cloning of this receptor.     

Inhibitory action of forskolin on adenylate cyclase activity and cyclic AMP generation

In testicular Leydig cells, forskolin causes the expected stimulation of cAMP and testosterone production and potentiates gonadotropin-induced responses, when present in concentrations of 1-10 microM. In addition, when added at lower doses that did not affect cAMP generation and testosterone responses (100 nM), forskolin caused an increase in sensitivity to hormonal stimulation for all cAMP pools (extracellular, intracellular, and receptor-bound) and a 70% reduction in the ED50 for human chorionic gonadotropin (hCG) stimulation of testosterone production. Forskolin-induced increases in receptor-bound cAMP were less effective than those elicited by hCG in stimulating steroidogenesis. In contrast to the well-known stimulatory actions of forskolin, low doses of the diterpene (in the picomolar to nanomolar range) markedly inhibited the production of cAMP and testosterone. Such inhibitory actions of low-dose forskolin were prevented by preincubation of Leydig cells with pertussis toxin before addition of forskolin and/or hCG. Low concentrations of forskolin also inhibited adenylate cyclase activation by GTP and luteinizing hormone, and this effect was prevented by pretreatment of cell membranes with pertussis toxin. These studies have defined the stimulatory effects of forskolin on Leydig-cell cAMP pools, including potentiation of the hormonal increase in receptor-bound cyclic AMP by forskolin, and have provided additional evidence for the functional importance of cAMP compartmentalization during hormonal stimulation of steroidogenesis. We have also demonstrated a novel, high-affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation, an effect that appears to be mediated by the Ni guanine nucleotide regulatory subunit of adenylate cyclase.     

Binding to prostaglandin (PG) receptors and activation of adenyl cyclase by 20-isopropylidene-PGS in rabbit platelet membranes

20-Isopropylidene-PGE1 (Isop-PGE1) was about 10 times more potent than PGE1 in inhibition of thrombin-induced aggregation of rabbit washed platelets. Likewise, 20-isopropylidene-17(R)-methyl-carbacyclin (CS-570), a stable PGI2 analogue, was more potent than carbacyclin in the anti-aggregatory activity. In order to define the platelet-prostaglandin interactions, a binding assay was done using platelet membranes with [3H]-PGE1 as a radioligand. Isop-PGE1 (IC50 = 0.18 microM) bound to the PG receptors more potently than PGE1 (IC50 = 2.1 microM). CS-570 (IC50 = 0.39 microM) was more potent than carbacyclin (IC50 = 1.9 microM). These indicate that introduction of an isopropylidene group to the carbon 20 of PGs increases the binding ability to the receptors. These PGE1 and PGI2 analogues activated platelet membrane adenyl cyclase and increased intracellular cAMP levels with the same potency series obtained in the binding experiments. All these results suggest that the binding to the receptors by these PGs is coupled to the activation of adenyl cyclase, followed by the increase in cAMP levels in platelets and the inhibition of platelet aggregation. Thus, the increased anti-aggregatory activity of 20-isop-PGs may be explained by their increased affinity for the PG receptors and stimulation of adenyl cyclase. 15-Epimeric-20-isopropylidene-PGE1 (15-Epi-isop-PGE1), which has an unnatural configuration of the 15-hydroxyl group, was much less potent than isop-PGE1 in the binding experiment and the other three investigations. This indicates that the configuration of the 15-hydroxyl group is important for the binding to the PG receptors and the consequent activities in platelets.     

Characterization of a serotonin transporter and an adenylate cyclase-linked serotonin receptor in the cestode Hymenolepis diminuta

[3H]5-HT exhibited specific binding in membrane preparations of Hymenolepis diminuta. The specific binding was saturable, reversible and temperature dependent. A non-linear Scatchard plot was obtained in a concentration range of 11 nM - 1000 nM [3H]5-HT, which could be resolved into sites having apparent dissociation constants (KD) of 0.10 microM and 6.25 microM for the high-affinity and low-affinity components, respectively. The latter could be selectively eliminated by binding [3H]5-HT to H. diminuta membranes in the presence of 10(-3) M nitroimipramine. Drug displacement studies, using 0.20 microM and 2.0 microM [3H]5-HT, revealed that while low-affinity [3H]5-HT binding was displaced by unlabelled 5-HT and inhibitors of 5-HT uptake, high affinity [3H]5-HT binding was affected only by tryptamine derivatives and, to a lesser extent, methysergide. In addition, high-affinity binding was stimulated by MgCl2 while low-affinity binding showed sodium-dependency. The data implicate the low-affinity site as a putative 5-HT transporter and the high-affinity site as a putative 5-HT 1 receptor. Exposure of H. diminuta membranes to 5-HT resulted in a 3-4 fold stimulation of cAMP levels. The EC 50 for the 5-HT-induced activation of adenylate cyclase (0.76 microM) was of the same order of magnitude as the apparent KD for high-affinity binding. Furthermore, the order of drug potency for the elevation of cAMP levels by 5-HT agonists and reversal by 5-HT antagonists was identical to the order of drug potency for the inhibition of high-affinity binding, suggesting linkage of the putative 5-HT 1 receptor to adenylate cyclase in H. diminuta.     

Direct anesthetic-like effects of forskolin on the nicotinic acetylcholine receptors of PC12 cells

Forskolin is thought to be a highly specific activator of adenyl cyclase. However, when applied to rat pheochromocytoma (PC12) cells at concentrations of 1 microM or higher it caused an immediate, concentration-dependent inhibition of carbachol-stimulated uptake of 86Rb+ through the nicotinic receptors, which did not appear to be related to activation of adenyl cyclase. The inhibition of receptor activation occurred instantaneously whereas cellular cAMP content did not increase for a measureable period of time. Normal receptor function was recovered rapidly upon removal of forskolin. Additional evidence that this effect of forskolin was not related to cAMP was obtained when 1,9-dideoxyforskolin (an analog of forskolin which does not activate adenyl cyclase) also caused a rapid, concentration-dependent, rapidly reversible inhibition of receptor-mediated influx of 86Rb+ into the cells. An examination of the effect of forskolin on 86Rb+ uptake at various concentrations of carbachol showed that forskolin was not acting by competing with carbachol for the receptor activation site. Given the lipophilic nature of forskolin, it probably acts like a general anesthetic to perturb the plasma membrane lipid structure and alter the function of the nicotinic acetylcholine receptors, possibly by increasing the rate of closure of open channels.     

Possible Involvement of Pertussis Toxin-Sensitive G Proteins and D2 Dopamine Receptors in the A1 Adenosine Receptor-Adenylate Cyclase System in Rat Cerebral Cortex

To identify the involvement of dopamine receptors in the transmembrane signaling of the adenosine receptor-G protein-adenylate cyclase system in the CNS, we examined the effects of pertussis toxin (islet-activating protein, IAP) and apomorphine on A1 adenosine agonist (-)N6-R-[3H]phenylisopropyladenosine ([3H]PIA) and antagonist [3H]xanthine amine congener ([3H]XAC) binding activity and adenylate cyclase activity in cerebral cortex membranes of the rat brain. Specific binding to a single class of sites for [3H]XAC with a dissociation constant (KD) of 6.0 +/- 1.3 nM was observed. The number of maximal binding sites (Bmax) was 1.21 +/- 0.13 pmol/mg protein. Studies of the inhibition of [3H]XAC binding by PIA revealed the presence of two classes of PIA binding states, a high-affinity state (KD = 2.30 +/- 1.16 nM) and a low-affinity state (KD = 1.220 +/- 230 nM). Guanosine 5'-(3-O-thio)triphosphate or IAP treatment reduced the number of the high-affinity state binding sites without altering the KD for PIA. Apomorphine (100 microM) increased the KD value 10-fold and decreased Bmax by approximately 20% for [3H]PIA. The effect of apomorphine on the KD value increase was irreversible and due to a conversion from high-affinity to low-affinity states for PIA. The effect was dose dependent and was mediated via D2 dopamine receptors, since the D2 antagonist sulpiride blocked the phenomenon. The inhibitory effect of PIA on adenylate cyclase activity was abolished by apomorphine treatment. There was no effect of apomorphine on displacement of [3H]quinuclidinyl benzilate (muscarinic ligand) binding by carbachol. These data suggest that A1 adenosine receptor binding and function are selectively modified by D2 dopaminergic agents.     

Prostaglandins and muscarinic agonists induce cyclic AMP attenuation by two distinct mechanisms in the pregnant-rat myometrium. Interaction between cyclic AMP and Ca2+ signals.

In pregnant-rat myometrium (day 21 of gestation), isoprenaline-induced cyclic AMP accumulation, resulting from receptor-mediated activation of adenylate cyclase, was negatively regulated by prostaglandins [PGE2, PGF2 alpha; EC50 (concn. giving 50% of maximal response) = 2 nM] and by the muscarinic agonist carbachol (EC50 = 2 microM). PG-induced inhibition was prevented by pertussis-toxin treatment, supporting the idea that it was mediated by the inhibitory G-protein Gi through the inhibitory pathway of the adenylate cyclase. Both isoprenaline-induced stimulation and PG-evoked inhibition of cyclic AMP were insensitive to Ca2+ depletion. By contrast, carbachol-evoked attenuation of cyclic AMP accumulation was dependent on Ca2+ and was insensitive to pertussis toxin. The inhibitory effect of carbachol was mimicked by ionomycin. Indirect evidence was thus provided for the enhancement of cyclic AMP degradation by a Ca2(+)-dependent phosphodiesterase activity in the muscarinic-mediated effect. The attenuation of cyclic AMP elicited by carbachol coincided with carbachol-stimulated inositol phosphate (InsP3, InsP2 and InsP) generation, which displayed an almost identical EC50 (3 microM) and was similarly unaffected by pertussis toxin. Both carbachol effects were reproduced by oxotremorine, whereas pilocarpine (a partial muscarinic agonist) failed to induce any decrease in cyclic AMP accumulation and concurrently was unable to stimulate the generation of inositol phosphates. These data support our proposal for a carbachol-mediated enhancement of a Ca2(+)-dependent phosphodiesterase activity, compatible with the rises in Ca2+ associated with muscarinic-induced increased generation of inositol phosphates. They further illustrate that a cross-talk between the two major transmembrane signalling systems contributed to an ultimate decrease in cyclic AMP in the pregnant-rat myometrium near term.     

A cAMP independent inhibitory action of high doses of forskolin in rat Leydig cells

In addition to well known direct stimulatory and potentiatory actions of forskolin, we have previously reported that low doses of this diterpene (10(-9), 10(-12) M) markedly inhibit the production of cAMP and testosterone in rat Leydig cells through a pertussis toxin sensitive G-protein (A. Khanum and M. L. Dufau, J. Biol. Chem. 261, 1986). A different type of inhibitory effect of forskolin is described in this study. Forskolin (10(-5) M) markedly stimulates basal adenylate cyclase activity (about 200%) in rat Leydig cell membranes and potentiates the stimulatory effect of gonadotropin (10(-9), 10(-7) M) on adenylate cyclase in presence or in absence of GTP (10(-5) M). Similarly a time-dependent stimulation of forskolin (10(-5) M) alone is noted on all cAMP pools and testosterone production. Using a supramaximal steroidogenic dose of hCG (0.26 nM) or choleragen (0.1 microM), forskolin potentiates the gonadotrophin and toxin-induced responses of all cAMP pools significantly while inhibiting testosterone production. Moreover, forskolin also inhibits 8-Bromo-cAMP stimulated steroidogenesis. In contrast, pregnenolone synthesis was not altered by the diterpene. We have demonstrated in this study that the inhibitory effect of high doses of forskolin on steroidogenesis is distal to cAMP generation, and resulted from a steroidogenic block residing beyond pregnenolone synthesis.     

Mechanisms of leptin secretion from white adipocytes

The mechanisms regulating leptin secretion were investigated in isolated rat white adipocytes. Insulin (1-100 nM) linearly stimulated leptin secretion from incubated adipocytes for at least 2 h. The adrenergic agonists norepinephrine, isoproterenol (two nonselective beta-agonists), or CL-316243 (potent beta3) all inhibited insulin (10 nM)-stimulated leptin release. The inhibitory effects of norepinephrine and isoproterenol could be reversed not only by the nonselective antagonist propranolol but also by the selective antagonists ICI-89406 (beta1) or ICI-118551 (beta2), the beta2-antagonist being less effective than the beta1. Insulin-stimulated leptin secretion could also be inhibited by a series of agents increasing intracellular cAMP levels, such as lipolytic hormones (ACTH and thyrotropin-stimulating hormone), various nonhydrolyzable cAMP analogs, pertussis toxin, forskolin, methylxanthines (caffeine, theophylline, IBMX), and specific inhibitors of phosphodiesterase III (imazodan, milrinone, and amrinone). Significantly, antilipolytic agents other than insulin (adenosine, nicotinic acid, acipimox, and orthovanadate) did not mimic the acute stimulatory effects of insulin on leptin secretion under these conditions. We conclude that norepinephrine specifically inhibits insulin-stimulated leptin secretion not only via the low-affinity beta3-adrenoceptors but also via the high-affinity beta1/beta2-adrenoceptors. Moreover, it is suggested that 1) activation of phosphodiesterase III by insulin represents an important metabolic step in stimulation of leptin secretion, and 2) lipolytic hormones competitively counterregulate the stimulatory effects of insulin by activating the adenylate cyclase system.     

Evidence that changes in platelet cyclic AMP levels regulate the fibrinogen receptor on human platelets

Fibrinogen binds to human platelets after specific receptor sites are exposed by thrombin, ADP, epinephrine, and other stimuli. Since prostaglandin I2 (PGI2), a potent activator of platelet adenylate cyclase, prevents mobilization of the fibrinogen receptor by aggregating agents, we investigated the relationship between platelet cAMP levels and fibrinogen receptor status in thrombin-stimulated human platelets. A dose-dependent rise in platelet cAMP in response to two adenylate cyclase agonists, PGI2 and forskolin, correlated with progressive inhibition of fibrinogen binding. Moreover, the receptor inhibition produced by either agonist was sustained up to 2 h and was associated with a persistent increase in cAMP levels. The phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine, in the presence of a subthreshold concentration of PGI2 also raised cAMP and inhibited fibrinogen binding. In contrast, the effects of PGI2 on both cAMP and fibrinogen binding were markedly attenuated by 9-(tetrahydro-2-furyl) adenine, an adenylate cyclase inhibitor. These results indicate that the inhibition of fibrinogen binding by PgI2 is linked to its effect on cAMP levels and suggest that elevation of platelet cAMP levels from any cause prevents exposure of the fibrinogen receptor.     

Effects of salmeterol on muscarinic inhibition of adenylyl cyclase in bovine trachealis cells

The goal was to assess whether salmeterol, a potent and long-acting beta-2-adrenergic agonist used in the treatment of asthma, also has non-beta-2-adrenergic effects on the stimulation or inhibition of adenylyl cyclase activity. Salmeterol (100 nM) maximally stimulated cAMP accumulation in enzyme dispersed bovine trachealis cells and this was entirely inhibited by propranolol, as expected for beta-adrenergic stimulation. However, the same concentration of salmeterol also antagonized carbachol inhibition of cAMP accumulation and altered binding of carbachol to muscarinic receptors. These effects of salmeterol were sensitive to washing of the cells and this was not consistent with a beta-2-adrenergic mechanism. The findings suggested that the maximal, beta-2-adrenergic stimulation of cAMP accumulation by salmeterol was accompanied by a non-beta-2-adrenergic interaction of salmeterol with muscarinic receptors that attenuated muscarinic inhibition of adenylyl cyclase.     

Regulation by butyrate of the cAMP response to cholera toxin and forskolin in pituitary GH1 cells

In pituitary GH1 cells, a rat growth hormone-producing cell line, butyrate elicited a dose-dependent increase in cholera toxin receptors as measured by an increased binding of 125I-labeled cholera toxin to the intact cells. Butyrate did not alter the affinity of cholera toxin binding, the dissociation constant being 0.4 nM for both control and butyrate-treated cells. Despite the increased binding, the cAMP response to cholera toxin was strongly reduced after exposure to butyrate. This reduction was dose-dependent and with butyrate 1--5 mM, intracellular and extracellular (medium) cAMP levels were decreased by more than 70% in cells incubated for 24 h with 1 nM cholera toxin. Forskolin (30 microM) elicited a cAMP response similar to that found with the toxin, and a similar inhibition of cAMP was also found after incubation of GH1 cells with butyrate. Butyrate also affected basal cAMP levels which were reduced by 40--60% in cells cultured for 24--48 h with the fatty acid. In order to study whether butyrate influenced cAMP synthesis and/or cAMP degradation, adenylyl cyclase and phosphodiesterase activities were determined in control cells and in cells incubated for 24 h with cholera toxin or forskolin. Butyrate had a dual effect since, besides activating phosphodiesterase by more than twofold, it also inhibited the cyclase by 40--50% in all groups. The in vitro response of adenylyl cyclase to stimulatory (NaF) and inhibitory (carbachol and adenosine) effectors was also examined. The absolute activity of the cyclase was always 40--50% lower in the cells incubated with butyrate, but the percentage change of activity obtained in butyrate-treated and untreated cells was unaltered. In addition, ADP-ribosylation of the guanine nucleotide stimulatory component of the cyclase (Gs) was not affected in the cells incubated with butyrate. These results suggest that the catalytic (C) subunit of adenylyl cyclase and/or its interaction with the regulatory components might be altered in butyrate-treated GH1 cells. The inhibition of the cAMP response in GH1 cells was accompanied by an inhibition of a biological action of the nucleotide, namely growth hormone (somatotropin) production which is primarily controlled by thyroid hormones in these cells. Forskolin alone did not affect the somatotropin levels but potentiated the growth hormone response to triiodothyronine. Butyrate produced a dose-dependent inhibition of this response, which was totally abolished at concentrations of butyrate higher than 1 mM.     

Interaction of clonidine and clonidine analogues with alpha-adrenergic receptors of neuroblastoma X glioma hybrid cells and rat brain: comparison of ligand binding with inhibition of adenylate cyclase

Clonidine and several analogues of clonidine are shown to be useful probes for alpha 2-adrenergic receptors in a comparative study of ligand binding and inhibition of adenylate cyclase. The alpha-adrenergic properties of a new potential probe, N-(4-hydroxyphenacetyl)-4-aminoclonidine hydrochloride, are described. [3H]Clonidine binds to alpha-receptors of NG108-15 neuroblastoma X glioma hybrid cell membranes with Kd values of 1.7 and 33 nM for putative high-affinity and low-affinity sites, respectively. p-Aminoclonidine and hydroxyphenacetyl aminoclonidine displace [3H]clonidine from the high-affinity sites with Kd values of 2.3 and 5.8 nM, respectively. Rat brain alpha 2-receptors also exhibit high affinity toward clonidine, p-aminoclonidine, and hydroxyphenacetyl aminoclonidine, as determined by displacement of specifically bound [3H]clonidine. Clonidine, p-amino-clonidine, and hydroxyphenacetyl aminoclonidine elicit modest inhibition (up to 24%) of NG108-125 adenylate cyclase by interaction with alpha 2-receptors (Kd,app 300, 30, and 130 nM, respectively); these compounds also partially reverse the inhibition elicited by (--)-norepinephrine. Components of the adenylate cyclase assay mixture, particularly ATP, GTP, sodium ions, and a nucleoside-triphosphate-regenerating system, decrease the high-affinity [3H]clonidine binding to NG108-15 membranes; in the presence of these components, alpha-receptors possess only low affinity (Kd 43 nM) for [3H]clonidine. The results are consistent with the concept that certain components required for the receptor-mediated inhibition of adenylate cyclase convert alpha 2-receptors from a high-affinity inactive state to a low-affinity active state.     

Independent and interrelated regulation of ornithine decarboxylase by calcium and cAMP in fetal rat osteoblasts

The present study was undertaken to determine in fetal rat osteoblasts whether and how the intracellular messengers calcium and cAMP are involved in stimulation of ornithine decarboxylase (ODC) activity. For that purpose we used different drugs affecting [Ca2+]i and cAMP concentration. A23187 stimulates ODC activity in a biphasic way, with maximal stimulation at 100 nM A23187. At that concentration no stimulation of cAMP production was observed. Basal and A23187-stimulated (100 nM) ODC activity were inhibited by EGTA and trifluoperazine. Forskolin stimulated dose-dependently both ODC activity and cAMP production. Besides these effects forskolin (1 and 10 microM) increased the [Ca2+]i via an increased calcium influx. Addition of La3+, verapamil or EGTA, but not of trifluoperazine, significantly inhibited the forskolin-stimulated (10 microM) ODC activity. When forskolin (100 nM and 1 microM) was added together with 1 microM A23187, a synergistic stimulation of ODC activity was observed. These results implicate that calcium is involved in basal ODC activity, and that ODC activity can be stimulated via (1) a cAMP-independent calcium pathway, and (2) a calcium-dependent, cAMP pathway. It is proposed that ODC activity can be stimulated via interaction between calcium and cAMP.     

Stimulation and inhibition of rat basophilic leukemia cell adenylate cyclase by forskolin

The influence of the diterpene, forskolin, was studied on adenylate cyclase activity in membranes of rat basophilic leukemia cells. Forskolin increased basal adenylate cyclase activity maximally 2-fold at 100 microM. However, adenylate cyclase activity stimulated via the stimulatory guanine nucleotide-binding protein, Ns, by fluoride and the stable GTP analog, guanosine 5'-O-(3-thiotriphosphate), was inhibited by forskolin. Half-maximal and maximal inhibition occurred at about 1 and 10 microM forskolin, respectively. The inhibition occurred without an apparent lag phase, whereas the enzyme stimulation by forskolin was preceded by a considerable lag period. The inhibition was not affected by treating intact cells or membranes with pertussis toxin and proteolytic enzymes, respectively, which have been shown in other cell types to prevent adenylate cyclase inhibition mediated by the guanine nucleotide-binding regulatory component, Ni. The forskolin inhibition of the stable GTP analog-activated adenylate cyclase was impaired by increasing the Mg2+ concentration and was reversed into a stimulation by Mn2+. Under optimal inhibitory conditions, forskolin even decreased basal adenylate cyclase activity. Finally, forskolin largely reduced the apparent affinity of the rat basophilic leukemia cell adenylate cyclase for its substrate, MgATP, which reduction resulted in an apparent inhibition at low MgATP concentrations and a loss of the inhibition at higher MgATP concentrations. The data indicate that forskolin can cause both stimulation and inhibition of adenylate cyclase and, furthermore, they suggest that the inhibition may not be mediated by the Ni protein, but may be caused by a direct action of forskolin at the adenylate cyclase catalytic moiety.     

Cleavage of a 100 kDa membrane protein (aggregin) during thrombin-induced platelet aggregation is mediated by the high affinity thrombin receptors

Thrombin-induced platelet aggregation is accompanied by cleavage of aggregin, a surface membrane protein (Mr = 100 kDa), and is mediated by the intracellular activation of calpain. We now find that agents that increase intracellular levels of platelet cAMP by stimulating adenylate cyclase, also inhibit thrombin binding and platelet activation by destabilizing thrombin receptors on the platelet surface. Iloprost (a stable analog of PGI2) and forskolin each completely inhibited platelet aggregation by 2 nM thrombin and markedly decreased cleavage of aggregin. Thrombin inactivated by D-phenylalanine-L-prolyl-L-arginine chloromethyl ketone (PPACK-thrombin) binds to the highest affinity site for thrombin on the platelet surface, but thrombin modified by N alpha-tosyl-L-lysine chloromethylketone (TLCK-thrombin) does not. We now demonstrate that preincubation of platelets with PPACK-thrombin blocked platelet aggregation and cleavage of aggregin induced by 2 nM thrombin. In contrast, TLCK-thrombin neither blocked platelet aggregation nor the cleavage of aggregin. These results show that a) platelet aggregation and cleavage of aggregin by thrombin (2nm) involves the occupancy of high affinity alpha-thrombin receptors on the platelet surface, and b) stimulators of adenylate cyclase which increase cAMP, inhibit thrombin-induced platelet aggregation and cleavage of aggregin by mechanisms which include inhibiting the binding of thrombin to its receptors.     

Phosphatidic acid inhibition of PGE1-stimulated cAMP accumulation in WI-38 fibroblasts: similarities with carbachol inhibition

To test the hypothesis that phosphatidic acid (PhA) is involved in the carbachol inhibition of hormone stimulated accumulation of cAMP we observed the effects of PhA on PGE1-stimulation of cAMP in WI-38 fibroblasts. PhA inhibited PGE1-stimulated cAMP accumulation of WI-38 fibroblasts; maximum inhibition (approximately 50-80%) occurred at a PhA concentration of 1.0 microM and significant inhibition was observed with a concentration of 0.1 microM. The full effects of PhA were evident within 15 sec after the co-addition of PGE1 and PhA. Addition of PhA to cells which had been pre-stimulated with PGE1 resulted in the rapid decay of cAMP levels to a new steady state level with a t 1/2 of approximately 65 sec. The inhibition produced by PhA did not appear to be simply attributable to a depolarization or increased intracellular Ca2+, since addition of either KCl or the Ca2+ ionophore A23187 did not lower PGE1-stimulated cAMP accumulation. When intact cells were pretreated with PhA then lysed and adenylate cyclase immediately assayed, no detectable changes in broken cell adenylate cyclase activities were observed. Also, PhA added directly to adenylate cyclase assays at concentrations as high as 100 microM produced no detectable inhibition of the membrane fraction adenylate cyclase activities. Nonetheless, our results suggest that adenylate cyclase activity in intact cells may be directly affected by physiological levels of PhA . Further, the similarities of carbachol [Butcher, R. W., Journal of Cyclic Nucleotide Research, 4:411 (1978)] and PhA inhibition support the hypothesis that carbachol (acetylcholine) exerts its effect on adenylate cyclase through alterations of the plasma membrane phospholipid composition.     

Muscarinic Receptor Stimulated GTPase Activity in Synaptic Membranes from Bovine Retina

GTPase activity has been measured in synaptic membranes from bovine retina, with and without muscarinic receptor stimulation. Maximal stimulation above basal levels was achieved with 5 microM oxotremorine and 100 microM carbachol. (4-Hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbanilate chloride, which is selective for the M1 muscarinic receptor, failed to stimulate GTPase activity. 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) inhibition of oxotremorine stimulation demonstrated the presence of two populations of receptors, a low-affinity site (IC50 +/- SEM, 0.63 +/- 0.18 microM) which accounted for 63% of the inhibition and a high-affinity site (IC50 less than 1 nM) which accounted for the remaining 37%. When carbachol-stimulated GTPase activity was assayed, a single 4-DAMP inhibitory site was apparent (IC50 +/- SEM, 2.0 +/- 0.9 microM). Pirenzepine inhibited GTPase activity at a single site (IC50 values +/- SEM, 46.9 +/- 11 and 25.4 +/- 6.5 microM against oxotremorine and carbachol, respectively). Methoctramine was equipotent against carbachol and oxotremorine stimulation (IC50 values, 4.2 +/- 1.8 and 6.2 +/- 1.5 microM). Inhibition of maximal carbachol and oxotremorine stimulation by muscarinic antagonists at the major site had a rank order of potency of 4-DAMP = methoctramine greater than pirenzepine. Thus, the major site for muscarinic stimulation of GTPase activity in bovine retinal membranes is pharmacologically similar to M2 receptors.     

Role of cAMP in the functional interaction of carbachol with different cAMP elevating agents in rabbit atrium.

The muscarinic agonist carbachol antagonized positive inotropic responses of rabbit left atria to the beta-adrenoceptor agonist isoproterenol, the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor IBMX. Carbachol also reduced cAMP levels elevated by isoproterenol, but had no significant effect on cAMP levels in the presence of either forskolin or IBMX. Pre-treatment of rabbits with a dose of pertussis toxin which completely blocked the reduction by carbachol of isoproterenol-induced increases in cAMP, also blocked the reversal by carbachol of positive inotropic responses to isoproterenol, but only partially attenuated the antagonism by carbachol of inotropic responses to forskolin and IBMX. These data suggest that antagonism by carbachol of forskolin and IBMX-induced increases in cAMP levels does not play an important role in the functional interaction of carbachol with these cAMP-elevating agents.     

Forskolin Stimulation of Cyclic AMP Accumulation in Rat Brain Cortex Slices Is Markedly Enhanced by Endogenous Adenosine

Stimulation of cyclic AMP (cAMP) accumulation in rat cortex slices by 1 microM forskolin (F) was markedly reduced (96%) by treatment with adenosine deaminase (ADA). The effect of ADA was progressively less at higher concentrations of F, but still inhibited the response by 50% at 100 microM F. ADA-mediated inhibition of the cAMP response to 1 microM F was completely reversed by 5 microM 2-chloroadenosine (CA), an ADA-resistant analogue. Stimulation by F (controls) and F plus CA (ADA treated) in cortex slices was significantly inhibited by 200 microM caffeine (CAF) and by 10 microM 8-phenyltheophylline. cAMP accumulation in ADA-treated cortex slices stimulated with CA at concentrations from 5 to 100 microM was markedly enhanced by 1 microM F. Neither ADA treatment nor 200 microM CAF significantly affected cAMP accumulation in slices stimulated by 1 microM vasoactive intestinal polypeptide or adenylate cyclase in membranes stimulated by 1 microM F. CAF (1 mM) did not significantly increase basal cAMP levels in cortex slices, whereas 1 mM 3-isobutyl-1-methylxanthine caused a significant 80% increase and 100 microM rolipram enhanced cAMP levels by 4.5-fold. F-stimulated cAMP accumulation (1 microM) in cortex slices was inhibited 98% by 1 mM CAF and 49% by 1 mM 3-isobutyl-1-methylxanthine, and was enhanced 2.5-fold by 100 microM rolipram. These data have been interpreted to indicate that the stimulation of cAMP accumulation in rat cortex slices by 1 microM F is predominantly due to synergistic interaction with endogenous adenosine and that the inhibition of this response by CAF is largely due to blockade of adenosine receptors.