Pier Giorgio Borasio

Electrophysiological evidence for a PGE-mediated presynaptic control of acetylcholine output in the guinea-pig superior cervical ganglion

Abstract Intracellular recordings from single ganglion neurons show that 10 −8 −10 −7 M PGE 1 reversibly blocks synaptic transmission in isolated preparations of the guinea-pig superior cervical ganglion (SCG), when added to the superfusing medium. Neither resting potential nor membrane resistance of the impaled neurons appear to be affected by PGE 1 . Quantal analyses of transmitter release demonstrate that the number of quanta liberated per volley is sharply reduced by PGE 1 treatment whereas the amplitude of the elementary event does not appear to be significantly changed.

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.

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.

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.

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

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 andd-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 (10−4M) and by the opiate pentapeptide (10−4M), 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 (10−7–10−6M) or at very high doses of the opiate (10−3M), only the inhibitory effect ofd-ala2-met-enkephalinamide was evidenced.

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.

Biochemical and pharmacological characterization of adenosine A1 receptors in eel (Anguilla anguilla) brain

N6-cyclohexyl[3H]adenosine ([3H]CHA) was used to label adenosine A1 receptors in membranes prepared from male and female eel whole brain. The A1 receptor agonist [3H]CHA bound saturably, reversibly and with high affinity (Kd = 0.91 ± 0.12 nM; Bmax = 120.36 ± 5.2 fmol mg−1 protein). In equilibrium competition experiments, the adenosine agonists and antagonists all displaced [3H]CHA from high-affinity binding sites with the rank order of potency in displacing, characteristics of an A1 adenosine receptor. Mg2+ dramatically increased the affinity of [3H]CHA without modifying the maximal binding capacity. The specific binding was inhibited by guanosine 5′-triphosphate (Ki = 2.54 ± 0.98 μM). The [3H]CHA binding sites are ubiquitously distributed with a maximum in cerebellum and a minimum in olfactory bulb. No difference was observed between male and female brain. In eel brain, synaptosomes (P2), stimulation of adenosine 3′,5′-monophosphate (cyclic AMP) accumulation with 10−5 M forskolin was markedly reduced (45.5%) by treatment with the adenosine A1 receptor agonist CHA (10−4 M), and the reduction was reversed in presence of the selective A1 receptor antagonist 8-cyclopentyltheophylline (10−5 M). In superfused eel cerebellar synaptosomes, K+ stimulated the release of adenosine in a partially Ca2+-dependent manner. The findings, taken together, suggest the hypothesis that adenosine A1 receptors present in eel brain could modulate synaptic transmission, as A1 receptors do in other vertebrates.

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.

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. TheKm values were about 1 and 110 μM respectively. Imidazole slightly but constantly stimulated the former enzyme form over a wide range of concentrations and l-methyl-3-isobutylxanthine was a weak competitive inhibitor with aKi value of 90 μM. 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.