Adenosine analogs inhibit acetylcholine release and cyclic AMP synthesis in the guinea-pig superior cervical ganglion.

The ability of adenosine agonists to modulate the electrically evoked release of acetylcholine (ACh) from [3H]choline preloaded guinea-pig superior cervical ganglia (SCG) was investigated. The adenosine A1-receptor selective agonist N6-cyclohexyladenosine (CHA) and 2-chloroadenosine (2-CADO) inhibited the evoked transmitter release, the effect being reversed by the A1-receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and by sulmazole (SUL), which blocks both the A1-receptor and the adenylate cyclase inhibitory regulator Gi. In whole ganglia, CHA decreased both the basal and the forskolin (FSK)-stimulated cyclic AMP synthesis. The latter effect was again prevented by the A1 antagonist DPCPX. These results are compatible with the existence, in the guinea-pig SCG, of adenosine A1-receptors, part of which are located on the presynaptic nerve terminals mediating an inhibition of ACh release.

Sulmazole effects on PGE2 and D-Ala2-Met-enkephalinamide modulation of cyclic AMP synthesis and neurotransmitter release in a sympathetic ganglion.

In the guinea-pig superior cervical ganglion, the Gi blocking agent sulmazole enhanced the basal and prostaglandin E2-induced stimulation of cyclic AMP synthesis but had no effect on the prostaglandin-dependent inhibition of acetylcholine release. On the contrary sulmazole counteracted the inhibitory effect of D-Ala2-Met-enkephalinamide both on cyclic AMP formation and acetylcholine release. Moreover sulmazole eliminated the supra-additive effect of the combination of prostaglandin + opiate on cyclic AMP synthesis. The presence of a Gi-coupled opiate receptor at the pre-and postsynaptic levels is suggested.

Receptor-mediated supra-additive activation of guinea pig superior cervical ganglion adenylate cyclase: role of Mn2+ ions and calmodulin.

The effects of Mn2+ and calmodulin were studied on the basal and agonist-modulated adenylate cyclase activity of the guinea pig superior cervical ganglion. The divalent cation strongly stimulates the basal and agonist-modulated enzyme in a concentration-dependent manner. Moreover, in the presence of Mn2+ the inhibitory effects of "high" GTP concentrations and of D-Ala2-Met-enkephalinamide on adenylate cyclase are eliminated, while the stimulation exerted by prostaglandin E2 and the supra-additive activation of the enzyme by the combination of the two drugs are unaffected. In EGTA-washed, calmodulin-depleted membrane preparations, Mn2+ still activates the cyclase but the enkephalin inhibition and the superactivation of the enzyme induced by the combination of opiate and prostaglandin are lost, both in the absence and in the presence of the cation. Reconstituting the depleted membranes with exogenous Ca2+/calmodulin fully restored the enzyme responsivity to the combination and, partially, to the enkephalin. The findings suggest the existence in the guinea pig superior cervical ganglion of both the calmodulin-sensitive and differently regulated calmodulin-insensitive adenylate cyclase.

Supra-additive activation of guinea-pig superior cervical ganglion adenylate cyclase by PGE2 and D-Ala2-Met-enkephalinamide: role of GTP.

The effects of guanine nucleotides were tested on basal and agonist-modulated adenylate cyclase in guinea-pig superior cervical ganglion crude membrane preparations. GTP gamma S and Gpp(NH)p dose-dependently stimulate, while GDP beta S inhibits, both the basal and the prostaglandin E2-stimulated enzyme activity. Low GTP doses, up to 10(-5) M, stimulate, while higher doses inhibit, the ganglionic adenylate cyclase. The GTP-induced diphasic pattern is maintained also in the presence of prostaglandin E2, D-Ala2-Met-enkephalinamide, or a combination of the two drugs. However, the opioid inhibits the enzyme activity, but only at high GTP doses, while the prostaglandin stimulates the enzyme at all GTP concentrations. The effect is potentiated by a combination of prostaglandin and enkephalin. The enhancing effect of the prostaglandin and of the combination with enkephalin is maximally expressed at high, almost physiological, GTP doses.

Supra-additive stimulation of adenylate cyclase activity by prostaglandin E2 and D-Ala2-met-enkephalinamide in the guinea-pig superior cervical ganglion: role of Mg2+ions.

Agonists modulation of Mg2(+)-dependent adenylate cyclase activity has been studied in guinea-pig superior cervical ganglion crude membrane preparations. In the absence of receptors ligands, Mg2+ stimulates the enzyme in a concentration-dependent manner. The dose-activation curve shows heterogeneity and two components with "higher" and "lower" apparent affinity states, are extrapolated. In the presence of D-Ala2-met-enkephalinamide only one component is present and the apparent affinity of the ganglionic adenylate cyclase system for the divalent cation as well as Vmax are inhibited. On the contrary, prostaglandin E2 increases affinity and Vmax values of the lower and, to a lesser extent, of the higher Km component. When the two drugs are tested in combination, not only the inhibitory effect of the opiate is overcome, but a large increase of the apparent affinities and Vmax values for both components is obtained, suggesting the involvement of the Mg2(+)-regulated subunits of the adenylate cyclase system in the supra-additive stimulation mechanism of the enzyme.

Presynaptic muscarinic receptors in guinea pig superior cervical ganglion.

This study characterizes the presynaptic muscarinic cholinergic receptors associated with the modulation of the electrically-evoked acetylcholine output from guinea pig superior cervical ganglion preincubated with [3H]choline. The M1-selective agonist pilocarpine had no effect while carbachol and oxotremorine strongly decreased the evoked outflow of tritium. Atropine increased such evoked release of [3H]acetylcholine whereas the M1-selective antagonist pirenzepine was ineffective. Moreover, atropine but not pirenzepine antagonized the inhibitory effect of carbachol. These results suggest that the guinea-pig superior cervical ganglion is equipped with presynaptic inhibitory muscarinic receptors of the M2 subtype.

Diazepam effects on frog skin cyclic nucleotide phosphodiesterase.

1. The activities of cAMP phosphodiesterases (cAMP-PDE) have been measured in the homogenate of the skin of Rana esculenta. 2. The tissue possesses two distinct enzymes: a "low" Km PDE (Km = 0.42 x 10(-6) M; Vmax = 16 pmol/mg protein/min) and a "high" Km PDE (Km = 180 x 10(-6) M; Vmax = 2853 pmol/mg prot/min). Only the "high" Km form is stimulated by calcium. 3. Diazepam (1-0.5 mM) significantly inhibits both enzymes, the inhibition being of competitive type.

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.

Interactions between prostaglandin E2 and D-ala2-met-enkephalinamide on adenylate cyclase activity in the guinea-pig superior cervical ganglion.

Crude membrane fractions, obtained from superior cervical ganglia of normal and sympathectomized guinea-pigs, have been used to investigate the role of prostaglandin E2 and D-ala2-met-enkephalinamide in the modulation of ganglionic adenylate cyclase as well as their functional interrelationship. In ganglia from normal animals the enzyme activity was stimulated and inhibited, respectively, by the prostaglandin (10(-4)M) and by the opiate pentapeptide (10(-4)M), while little or no effects were observed in denervated preparations. When the two substances were tested in combination, a supra-additive stimulation of adenylate cyclase activity was obtained both in normal and denervated ganglia. In the latter preparation the opiate increased prostaglandin E2 specific binding, suggesting that the mechanism of supra-additivity could involve interactions at receptors level. Furthermore, the supra-additive stimulation of adenylate cyclase activity by the combination of the two drugs was obtained in a narrow range of concentrations since at low prostaglandin E2 doses (10(-7)-10(-6)M) or at very high doses of the opiate (10(-3)M), only the inhibitory effect of D-ala2-met-enkephalinamide was evidenced.

Some properties of adenosine 3',5'-cyclic monophosphate phosphodiesterase in the superior cervical ganglion of the guinea pig.

Adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase activity in crude guinea-pig superior cervical ganglion homogenates was assayed under a variety of experimental conditions. Two forms of cAMP phosphodiesterase were found, one with high and the other with low affinity for the substrate. The Km values were about 1 and 110 microM respectively. Imidazole slightly but constantly stimulated the former enzyme form over a wide range of concentrations and 1-methyl-3-isobutylxanthine was a weak competitive inhibitor with a Ki value of 90 microM. Low affinity cAMP phosphodiesterase activity was increased by calmodulin and Ca2+. This stimulation was not observed in the presence of trifluoperazine, a specific inhibitor of calmodulin. On the other hand, neither [D-Ala2]met-enkephalinamide nor prostaglandin E2, alone or in combination, influenced high affinity cAMP phosphodiesterase.

Opiates modulation of cAMP levels and PGE2 binding in mammalian sympathetic ganglia.

The effects of opiates on cyclic adenosine monophosphate (cAMP) levels have been studied in sympathetic ganglia of guinea-pig, rat and rabbit. D-[Ala2]-Met-enkephalinamide inhibits cAMP synthesis in guinea pig and rat but not in rabbit ganglia, while morphine is always ineffective. In the presence of the enkephalin plus prostaglandin E2 (PGE2) a synergistic increase of the nucleotide levels is observed in guinea pig. This effect is not induced by morphine plus PGE2 and is not shared by rat and rabbit. In guinea pig alpha-endorphin inhibits both basal and PGE2-stimulated cAMP synthesis. In the same preparation the enkephalin increases [3H]PGE2 binding. In guinea pig ganglia a cooperative effect of the enkephalin and PGE2 on the cAMP system is suggested.

Alpha-adrenoceptor-mediated inhibition of acetylcholine release in guinea-pig superior cervical ganglion.

The effect of different substances on the electrically evoked acetylcholine (ACh) output from guinea-pig superior cervical ganglion (SCG) preincubated with [3H]choline was investigated. Forskolin significantly reduced neurotransmitter release and dose-dependently enhanced cyclic AMP (cAMP) levels. Phenylephrine and clonidine, alpha 1-and alpha 2-adrenergic agonists respectively, caused a significant inhibition of ACh output from presynaptic nerve terminals. In the presence of RMI 12330A, phenylephrine but not clonidine failed to reduce neurotransmitter release. These results suggest that cAMP and different alpha-adrenergic receptors are involved in the regulation of cholinergic transmission.

cAMP-dependent and cAMP-independent modulation of synaptic transmission in guinea-pig superior cervical ganglion.

RMI 12330A dose-dependently inhibits cAMP accumulation induced by PGE2 in guinea-pig superior cervical ganglion (SCG). Norepinephrine (NE) is unable to modify cAMP concentration in the same preparation. On the other hand, both PGE2 and NE block the firing discharge of guinea-pig SCG neurons elicited by electrical stimulation (1 Hz) of the cervical sympathetic trunk. The effect of PGE2, but not that of NE, is prevented by RMI 12330A, suggesting that NE and PGE2 act through different pathways

Evidence for increased release of prostaglandins of E-type in response to orthodromic stimulation in the guinea-pig superior cervical ganglion.

Prostaglandins of the E type (PGEs) stimulate cyclic adenosine 3',5'-monophosphate (cAMP) biosynthesis both in isolated preparations of rat, guinea-pig and rabbit superior cervical ganglia (SCG) and in calf SCG slices. Electrical stimulation of preganglionic nerve fibers of the guinea-pig SCG remarkably increased PGE release and cAMP biosynthesis. These effects were blocked by reducing the Ca2+ to Mg2+ ratio in the incubation medium. Atropine (1 microM) and phentolamine (10 microM) inhibited PGE biosynthesis and significantly reduced cAMP levels.

Synthesis and prostaglandin-like activity of 2-(trans-3-hydroxy-1-octenyl)-3-indoleheptanoic acid.

The synthesis of 2-(trans-3-hydroxy-1-octenyl)-3-indoleheptanoic acid (1) is described. The title compound appeared to show a weak prostaglandin-like activity in two different systems. It contracted rat stomach fundus strips and guinea-pig ileum preparations only at concentrations about 10(3)- and 10(2)-fold higher, respectively, than PGE1. Moreover, it stimulated adenylate cyclase from rat liver plasma membrane, but the relative potency was 4--5 X 10(2)-fold lower than the natural compound. The title compound showed also a certain degree of PGE1 antagonism.

Inhibition of potassium uptake by low concentrations of norepinephrine and dibutyryl cyclic AMP.

(1) The inhibition of potassium uptake by low concentration of norepinephrine (3 X 10-8 M) and of dibutyryl cyclic AMP (DBcAMP, 10 minus5 M) was studied in cardiac Purkyne fibres. (2) The inhibitory action of DBcAMP on K uptake was abolished by the alpha blocker phentolamine. (3) Norepinephrine alone decreased K uptake and such inhibition was somewhat larger when DBcAMP was added. DBcAMP alone caused the usual decrease in K uptake but addition of norepinephrine abolished it. (4) The inhibition caused by norepinephrine reduced the increase in uptake caused by a high concentration (10 minus 3 M) of DBcAMP. (5) The inhibitory effect of norepinephrine was reversed in the presence of high concentration of magnesium (5.25 mM). (6) The inhibitory effect of norepinephrine was reversed by aminophylline and abolished by caffeine. (7) The inhibitory action of norepinephrine and BCcAMP was reversed or abolished, respectively, by imidazole. (8) It is concluded that the inhibition of potassium uptake by low concentration of DBcAMP is mediated by an alpha receptor mechanism and that possibly the "receptors" for this effect of norepinephrine and DBcAMP are located at different sites. Also it appears that DBcAMP may be acting at the membrane and that the action of methylxanthines and imidazole is not necessarily mediated only by a modification of phosphodiesterase activity.