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The fine structure of the hepatic artery of dogs was studied at electron microscopic level. The majority of nerve fibres innervating the artery was found at the adventitial-medial border of the vessel, but some of them entered the middle part of the media. The average distance of nerve to muscle fibre was found to measure 450 nm. Three types of nerve profile could be observed, viz. adrenergic, cholinergic and cholinergic-peptidergic as suggested by the morphology of their vesicles. It is suggested that the cholinergic and cholinergic-peptidergic nerve endings probably modulate the release and the constrictor effect of the main neurotransmitter noradrenaline in the hepatic artery.

Synapse is the most common and generally accepted structural basis for the interaction between neurons. It provides a "one-to-one" communication system between neurons. However, there is another possibility for interneuronal communication: when one neuron communicates with many others without making synaptic contact. In the past few years neurochemical, morphological and pharmacological evidence has been obtained that some neurotransmitters may be released from non-synaptic sites, for diffusion to target cells more distant than those seen in conventional synaptic transmission. The non-synaptic interneuronal communication between neurons plays a physiological role in the presynaptic modulation of chemical neurotransmission. This would be a transitional form between the classical neurotransmission and the broadcasting of neuroendocrine secretion.

The influence of alpha 2-adrenoceptor agonists and antagonists on the release of noradrenaline was studied in human gall bladder (cystic) artery preparations, in which transmitter stores were labelled with [3H]noradrenaline. The preparations were stimulated at 2 Hz for 3 min (360 shocks each of 1 ms duration two times (S1 and S2)). Both the L-noradrenaline and alpha-methylnoradrenaline (10(-6) M) significantly reduced (S2/S1 = 0.27 +/- 0.05; 0.43 +/- 0.04, respectively), whilst clonidine, xylazine and guanfacine at 10(-6) M failed to affect the stimulation evoked release of [3H]noradrenaline. Yohimbine (10(-6) M), CH-38083, which is a new, selective alpha 2-adrenoceptor antagonist (10(-7) M) and prazosin (10(-6) M) enhanced the evoked release of radioactivity, where S2/S1 were 2.50 +/- 0.19; 2.99 +/- 0.32; 1.48 +/- 0.05, respectively. Administering the alpha 2-antagonists and prazosin together, we were unable to demonstrate an additive effect. Yohimbine and CH-38083 prevented, while prazosin reduced, the inhibitory effects of L-noradrenaline or alpha-methylnoradrenaline on the release of radioactivity. Our results suggest that one type of presynaptic alpha 2-adrenoceptor modulates the release of noradrenaline evoked by electrical stimulation of the human cystic artery. This receptor is sensitive to alpha 2-adrenoceptor agonists which have a phenylethylamine structure, but is insensitive to imidazolines and guanfacine.

The involvement of heterogeneous dopamine (DA) receptors in the regulation of striatal cholinergic neurotransmission was investigated by measuring the release of acetylcholine (ACh) from isolated striatum of the rat. The DA agonists apomorphine (3.7 X 10(-7)-7.4 X 10(-4) mol/l) and piribedil (3.3 X 10(-6)-9.9 X 10(-5) mol/l) exerted a biphasic action: in low concentrations they enhanced, while in high concentrations they inhibited, the ouabain-induced release of ACh. The enhancement of ACh release elicited by low concentrations of DA agonists was reversed by a noncataleptogenic dose of pimozide. 6-hydroxydopamine (6-OHDA) pretreatment also prevented the stimulatory action of apomorphine or piribedil on ACh release. In 6-OHDA pretreated striatum, apomorphine caused a biphasic inhibition of ACh release in a concentration dependent manner. DPI (3,4-dihydroxyphenylamino/-2-imidazoline, 2.5 X 10(-5)-2.5 X 10(-4) mol/l) increased the release of ACh in a monophasic manner and did not induce inhibition of transmitter output in 6-OHDA pretreated striatum. Pretreatment of rats with increasing doses of haloperidol or pimozide (0.037-15 mg/kg) exerted a biphasic action on striatal ACh release; low doses decreased and high doses enhanced the transmitter output and the elevation was accompanied by the appearance of catalepsy. From these experiments it is concluded that multiple DA receptors are involved in the regulation of striatal cholinergic neurotransmission. Among these, the presynaptic DA autoreceptors located on nigrostriatal nerve endings are the most sensitive to both agonists and antagonists. DA receptors located on cholinergic interneurons of the striatum, which are postsynaptic in relation to nigrostriatal neurons, possess high and low affinity to apomorphine. Stimulation of DA autoreceptors enhances, while that of postsynaptic DA receptors leads to inhibition of ACh release.

The calcium channel blocker, nimodipine (Bay e 9736), inhibited the evoked (1 Hz, 1 msec, 180 pulses) release of [14C]acetylcholine ([14C]ACh) while it enhanced the release of [3H]noradrenaline ([3H]NA) measured concomitantly from the same longitudinal muscle strip of guinea-pig ileum loaded with [14C]choline and [3H]noradrenaline. These effects of nimodipine were concentration-dependent: in 0.1 microM concentration yielding maximal inhibition of [14C]ACh release and in 0.5 microM concentration yielding half maximal stimulation of [3H]NA-release. Enhancement of [3H]NA-release by nimodipine was strongly dependent on stimulation frequency while the inhibitory effect of nimodipine on release of [14C]ACh did not depend on stimulation frequency in the 0.5-10 Hz range. In addition, in the presence of nimodipine when the release of [3H]NA was enhanced, i.e. when a significant negative feedback operation could have been expected, alpha 2-adrenoceptor blocking agents (yohimbine and idazoxane) failed to enhance [3H]NA-release. It is proposed that nimodipine somehow exerts a presynaptic alpha 2-adrenoceptor blocking property at noradrenergic axon terminals through an effect on calcium entry, which may be an essential step in events linking alpha 2-adrenoceptor activation and inhibition of noradrenaline release.

Noradrenaline and adrenaline inhibit acetylcholine release due to nerve activity, through α-adrenoceptors. The inhibition of acetylcholine release by the adrenergic transmitters is, in fact, a rather economical way for the adrenergic (orthosympathetic) nervous system to counteract the parasympathetic nervous system. It is shown that there is a permanent adrenergic control of acetylcholine output. The increase in acetylcholine output after catecholamine depletion or α-blockade of nervous tissue only represents partial removal of an adrenergic restraint. The modulatory role of catecholamines on acetylcholine release mechanism seems to be of physiological importance. Furthermore, it is also shown that acetylcholine may regulate its own release by a negative feedback mechanism. Although this mechanism operates in brain cortex, no such phenomenon, however, was found in the Auerbach plexus.

Cerebral ventricular perfusion with prostaglandin E1 (PGE1, 1.4 X 10(-8) M) reduced the release of acetylcholine (ACh) into the ventriculo-cisternal perfusate of the unanaesthetized cat's brain. The samples of effluent were assayed on the isolated guinea-pig ileum. Neither a halving (1.07 X 10(-3) M) nor a threefold rise (6.45 X 10(-3) M) in the calcium concentration of the perfusate had any effect on the spontaneous release of the cholinergic transmitter. The action of PGE1 was counteracted by decreasing and potentiated by increasing the extracellular calcium concentration. Polyphloretin phosphate (PPP, 10(-4) g/ml) in itself did not significantly affect the ACh outflow. Preperfusion however, with this drug readily antagonized the depressant effect of PGE1. Ouabain (2.0 X 10(-5) M) added to the perfusion fluid increased the ACh output from the cat brain, and PGE1 prevented this effect of ouabain. A single intraventricular injection of 6-hydroxydopamine (1 mg) counteracted the inhibitory effect of PGE1 on spontaneous ACh release. A perfusion with the potent PG synthesis inhibitor suprofen (3.8 X 10(-7) M) did not influence change the mean output of the transmitter. Our data indicate that PGE1 reduces ACh release in the CNS via a pre- or postsynaptic modification of monoaminergic neurotransmission.

The effect of Met-enkephalin on the release of radioactivity (14C) from a myenteric plexus-longitudinal muscle preparation loaded with [14C]choline has been investigated under different conditions, when the muscarinic receptor mediated negative feedback inhibition was operative or when it was completely excluded by atropine. Separation of the [14C]acetylcholine (ACh) and [14C] choline components of the released radioactivity revealed that during 45-min incubation periods about 3.2% of the ACh store became labeled and that during stimulation only the release of [14C]ACh increased above resting level. The fractional release at rest measured in the 5-min collection period was 1.07 +/- 0.09 X 10(-2) in the absence and 1.56 +/- 0.07 X 10(-2) in the presence of physostigmine. Met-enkephalin had no effect on the release of ACh evoked by a 2-Hz stimulation when cholinesterase was inhibited by physostigmine. However, in the presence of atropine or in the absence of cholinesterase inhibition, the release by stimulation was significantly higher and subject to inhibition by Met-enkephalin. The present results indicate that Met-enkephalin is able to reduce ACh release only under those conditions in which the negative feedback modulation is negligible and the release is not yet reduced completely. These findings also suggest that in the myenteric plexus there is no independent population of cholinergic neurons exclusively sensitive to either ACh or to Met-enkephalin; cholinergic varicosities are equipped with at least both types of receptors we studied. When the effect of Met-enkephalin on twitch tension and on the release of radioactivity was studied simultaneously, a positive correlation was found.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of 2-, and 4-aminopyridine (4-APYR) on the release mechanism of acetylcholine (ACh) from the nerve terminals of the Auerbach plexus-longitudinal muscle preparation of the guinea-pig ileum, suspended in eserinized Krebs' solution, was investigated. 2- and 4-APYR increased the release of ACh from the nerve terminals at rest and at both low and high frequency stimulation. The enhanced ACh release was found to be due to increased volley output. At lower frequency of stimulation, the potentiation of ACh release was much higher than at higher rate of stimulation. 4-APYR was able to increase ACh release in the absence of [Ca2+]0. However, when a Ca-chelating agent, EDTA, was also added to the Ca-free Krebs' solution, 4-APYR was entirely ineffective. The depression of ACh release induced by Mg-excess was completely antagonized by 4-APYR. Tetrodotoxin (TTX) prevented augmentation of ACh release by 4-APYR. It is suggested that 4-APYR lowers the demand of nerve terminals for [Ca2+]0 required for the excitation-secretion coupling process. The presence of a low concentration [Ca2+]0, however, is essential for the action of 4-APYR.

The influence of 4-aminopyridine (4-APYR) on parasympathetic transmission was investigated on the myenteric plexus - longitudinal muscle preparation of the guinea-pig ileum. 4-APYR increased the force of contraction of the longitudinal muscle elicited by electrical field stimulation or by the addition of nicotine to the bath. It had little effect on the contraction caused by the addition of acetylcholine (ACh) to the bath. The decrease of the force of electrical, ACh or nicotine contraction caused by decreasing the [Ca] to 0.5×10 −3 M was antagonized by 4-APYR. Inhibition of the electrical contraction by homatropine methylbromide, normorphine, enkephalins or 1 -epinephrine was antagonized by 4-APYR. Similarly, the inhibition of the nicotine contraction by normorphine was also antagonized by 4-APYR. The site of the antagonist effect of 4-APYR on the inhibition of the electrical and nicotine contraction by normorphine appears to be different from that of naloxone. 4-APYR increased both the spontaneous and stimulated ACh release and the volley output of ACh in this preparation and also antagonized the inhibition of ACh release caused by normorphine and [ d -Ala 2 -]-Met 5 - enkephalin. The facilitation of parasympathetic transmission, in this preparation, by 4-APYR can be explained by its effect on stimulated ACh release.

Direct evidence has been obtained that the neurogenic responses of the hepatic and pancreatico-duodenal arteries of the dog are mainly due to norepinephrine released from varicosities and that this effect is mediated via alpha 1-adrenoceptors. In addition, there is a prazosin-resistant response to nerve stimulation that is certainly not mediated via alpha 2-adrenoceptors. These vessels are 10-100 times less sensitive to applied norepinephrine than the great majority of peripheral arteries; however, the pA2 value for prazosin (7.5) is the same as in other systems. The varicose terminal plexus is located deep in the media, as shown by electron microscopic study. Findings indicate that these gastrointestinal arteries are mainly controlled by adrenergic innervation, that their density is as high as that of any other vessel, and that these arteries might be much less influenced by the circulating catecholamines than others. The neuroeffector transmission of hepatic and pancreatico-duodenal arteries is subject to presynaptic modulation. Muscarinic (oxotremorine) and P1 (adenosine) receptor agonists are effective inhibitors of transmission, whereas xylazine surprisingly has no effect.

The selectivity and specificity of CH-38083 [7,8-(methylenedioxi)-14-alpha-hydroxyalloberbane HCl], a berbane derivative for alpha adrenoceptors has been studied and compared with yohimbine and idazoxan in peripheral tissues and in the central nervous system. In isolated tissue experiments CH-38083 was a competitive antagonist at presynaptic alpha-2 adrenoceptors on the axon terminals of the rat vas deferens (pA2 against xylazine = 8.17 +/- 0.06) and of the longitudinal muscle strip of guinea pig ileum (pA2 against xylazine = 8.07 +/- 0.20). As far as its postsynaptic alpha-2 adrenoceptor antagonistic activity is concerned its affinity in rat vas deferens (pA2 = 4.95 +/- 0.11) against l-phenylephrine and in rabbit pulmonary artery (pA2 = 5.38 +/- 0.33 against l-norepinephrine) was markedly less than that displayed for presynaptic sites. From pA2 values obtained in rat vas deferens the calculated alpha-1/alpha-2 adrenoceptor selectivity ratios for yohimbine, idazoxan and CH-38083 were 4.7, 117.5 and 1659, respectively. CH-38083 failed to show any affinity for histamine and muscarinic receptors and it even potentiated the effect of serotonin on atropinized longitudinal muscle strip of guinea pig ileum. It enhanced the release of [3H]norepinephrine from electrically stimulated mouse vas deferens loaded previously with labeled [3H]norepinephrine. In binding studies carried out in rat brain membrane preparations using [3H]prazosin and [3H]idazoxan, the selectivity ratios (Ki alpha-1/Ki alpha-2) proved to be 32.5, 289.5 and 1368 for yohimbine, idazoxan and CH-38083, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The postsynaptic antimuscarinic properties of different nondepolarizing muscle relaxants were compared with their postsynaptic antinicotinic effect. d-Tubocurarine, pipecuronium and vecuronium were the most selective antagonists on postsynaptic nicotinic receptors. Gallamine, diadonium and Duador (RGH-4201) had relatively greater effect on postsynaptic muscarinic receptors. Therefore, much less side effect is expected to occur when pipecuronium, d-tubocurarine or vecuronium are used.

1. Acetylcholine (ACh) released from mouse diaphragm was gel filtrated and estimated by bio-assay and compared with electrophysiologically measured quantal release, expressed either as frequency of miniature end-plate potentials or quantum content of end-plate potentials. 2. Activation of Na+-K+-dependent membrane ATPase (membrane ATPase) in Na+-loaded muscles lowered the total amount of ACh released at rest to one tenth of the control value, but quantal release remained unchanged. 3. Inhibition of membrane ATPase by 2 X 10(-5) M-ouabain or by K-free solution led to an increase in total release and to a delayed progressive increase in quantal release. When Ca2+ was removed only the total release was enhanced. 4. Depolarization of the diaphragm by 8, 11 and 14 mM-K increased both total and quantal release only in the presence of Ca2+ in the perfusion medium. When Ca2+ was removed, no significant increase in release was observed. 5. The total and quantal release in response to 2 Hz stimulation of the preparation was increased 1.4 and 45 times, respectively. It is concluded that the total amount of ACh released at rest consists of two fractions, quantal and non-quantal, the former representing about 1% of the total release.

The kinetic parameters of azidomor phine (6-deoxy-6-dihydro-azido-isomorphine), a new highly potent semisynthetic derivative of morphine was studied on the longitudinal muscle strip of guinea-pig ileum and with the hot-plate method. Azidomorphine reduced the neuro-effector transmission in the longitudinal muscle strip and this effect was antagonized by naloxone in a competitive manner. In contrast to morphine, the agonist concentration (ID50) of azidomorphine failed to exert an antagonistic effect in this test. The analgetic effect of azidomorphine was much more easily antagonized by naloxone than that of morphine. However, the opposite was observed in the hot-plate test with azidomorphine and morphine when nalorphine was used as antagonist. The significant differences in the kinetics between morphine and azidomorphine indicate that some important differences in their interaction with the receptor might exist between the two structurally related substances.

Glycogenolysis in the liver produced by isoprenaline was inhibited by β-receptor blocking agents using the perfusion technique. Dichloroisoprenaline also inhibited spontaneous glucose release, as did α-receptor stimulation by noradrenaline.

1. The relationship between the number of shocks delivered, the frequency of stimulation and the acetylcholine output per volley from nerve terminals of the longitudinal muscle strip of the guinea-pig ileum was studies.2. There was an inverse correlation between acetylcholine output per volley and the frequency of stimulation when the same number of shocks was applied in each train.3. With sustained stimulation, the volley output declined more rapidly the higher the frequency of stimulation. There was no decrease in volley output (11.7 (ng/g)/volley) when the frequency applied was 0.1 Hz or less.4. Noradrenaline (10(-6) g/ml) reduced the acetylcholine output per volley to the level produced by sustained stimulation at 10 Hz (1.4-1.9 (ng/g)/volley). The acetylcholine output following application of the first shocks of a train at high frequency stimulation was much reduced by noradrenaline as the output was higher than 1.4-1.9 (ng/g)/volley. This action of noradrenaline was antagonized by phentolamine (2 mug/ml for 20 min).5. Amphetamine and methylamphetamine, which release NA from sympathetic nerve terminals, were active in reducing the acetylcholine output to low frequency parasympathetic nerve stimulation. Reserpine and alpha-methyl-p-tyrosine pretreatment prevented the effect of amphetamine and reduced that of methylamphetamine.6. The fact that addition or release of noradrenaline, reduced acetylcholine output when the firing rate was high but short in duration, suggests that noradrenaline plays a general modulator role in controlling the output of acetylcholine from the parasympathetic nerve terminals.

1. The release of acetylcholine by parasympathetic ganglion stimulants from the nerve terminals of the guinea-pig longitudinal muscle strip was studied. The acetylcholine released was collected in the presence of physostigmine sulphate.2. Nicotine and 1,1-dimethyl-4'-phenylpiperazinium iodide (DMPP) released acetylcholine. Tetrodotoxin (50 ng/ml) and hexamethonium completely prevented their action on acetylcholine release, indicating that their effect is on nicotinic receptors of cell bodies. Noradrenaline (1 mug/ml), excess magnesium, and calcium withdrawal inhibited the acetylcholine release induced by nicotine or by DMPP.3. There was rapid tachyphylaxis to the acetylcholine releasing action of both nicotine and DMPP.4. The octapeptide amide of cholecystokinin and caerulein (10(-9)M) enhanced the acetylcholine release without producing tachyphylaxis. Tetrodotoxin completely inhibited acetylcholine release. However, hexamethonium or desensitization with 5-hydroxytryptamine did not prevent their action. In the early phase of nicotine action the polypeptides studied failed to increase acetylcholine release. However, a few minutes later the tissue became sensitive to polypeptides despite the fact that the tissue was still exposed to nicotine. These data suggest the presence in the parasympathetic ganglion cells of separate gastrointestinal hormone-sensitive receptors.5. Noradrenaline inhibited the acetylcholine releasing action of polypeptides. This effect was mediated via alpha-adrenoceptors since phentolamine prevented its action.6. Excess magnesium (9.3 mM) also reduced the acetylcholine release in response to the polypeptides.

We propose a nonlinear microscope scheme being capable of simultaneous, 3D investigation of the electric activity pattern of neural networks or signal summation rules of individual neurons in a 0.6times0.6times 0.2 mm3 volume with sub-micrometer spatial resolution

The modulation by adenosine analogues and endogenous adenosine of the electrically evoked release of [3H]acetylcholine ([3H]ACh) was compared in subslices of the three areas of the rat hippocampus (CA1, CA3, and dentate gyrus). The mixed A1/A2 agonist 2-chloroadenosine (CADO; 2-10 microM) inhibited, in a concentration-dependent manner, the release of [3H]ACh from the three hippocampal areas, being more potent in the CA1 and CA3 areas than in the dentate gyrus. The inhibitory effect of CADO (5 microM) on [3H]ACh release was prevented by the A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM) in the three hippocampal areas and was converted in an excitatory effect in the CA3 and dentate gyrus areas. The A2A agonist CGS-21680 (30 nM) produced a greater increase of the evoked release of [3H]ACh in the CA3 than in the dentate gyrus areas, whereas no consistent effect was found in the CA1 area or in the whole hippocampal slice. The excitatory effect of CGS-21680 (30 nM) in the CA3 area was prevented by the adenosine receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 microM). Both adenosine deaminase (2 U/ml) and DPCPX (250 nM) increased the evoked release of [3H]ACh in the CA1 and CA3 areas but not in the dentate gyrus. The amplitude of the effect of DPCPX and adenosine deaminase was similar in the CA1 area, but in the CA3 area DPCPX produced a greater effect than adenosine deaminase. It is concluded that the electrically evoked release of [3H]ACh in the three areas of the rat hippocampus can be differentially modulated by adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

We propose a two-photon microscope scheme capable of real time, 3D investigation of the neural activity in a 0.6 x 0.6 x 0.2 mm3 volume with sub-micrometer spatial resolution.

The interaction between phenylalkylamines and reserpine was studied on rats and cats be examination of the pressor and basal metabolic rate enhancing action of amphetamine, the psychostimulant and basal metabolic rate enhancing actions of ortho-bromo-methyl-amphetamine (V-104), and the pressor, psychotomimetic, basal metabolic rate enhancing and EEG-actions of para-bromo-methylamphetamine (V-111) and para-chloro-methylamphetamine(S-33). In all measurements of the effects of the compounds it was observed that if 0·5 mg/kg of reserpine was administered subcutaneously after the end of the phenylalkylamine effects, all central and peripheral effects reappeared with the equal intensity to that which could be expected from a repeated dose of the particular phenylalkylamine derivative. This phenomenon is thoughy to be due to the inactive bonding of the major part of the phenylalkylamine molecules on the receptor area from where they are released by reserpine into the active state. No such phenomenon may be observed after chronic reserpine pretreatment. Thus, it is suggested that chronic reserpine treatment enhances the sensitivity of the central nervous system to phenylalkylamines.