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Since a significant proportion of diabetic patients have clinical or subclinical neuropathy, there may be concerns about the use of local anaesthetics. The present study was designed to determine and compare the effects of articaine, a widely used anaesthetic in dental practice, and lidocaine on the resting and axonal stimulation-evoked release of [

Intestinal microbiota are critical for health with changes associated with diverse human diseases. Research suggests that altered intestinal microbiota can profoundly affect brain function. However, whether altering brain function directly affects the microbiota is unknown. Since it is currently unclear how brain injury induces clinical complications such as infections or paralytic ileus, key contributors to prolonged hospitalization and death post-stroke, we tested in mice the hypothesis that brain damage induced changes in the intestinal microbiota. Experimental stroke altered the composition of caecal microbiota, with specific changes in Peptococcaceae and Prevotellaceae correlating with the extent of injury. These effects are mediated by noradrenaline release from the autonomic nervous system with altered caecal mucoprotein production and goblet cell numbers. Traumatic brain injury also caused changes in the gut microbiota, confirming brain injury effects gut microbiota. Changes in intestinal microbiota after brain injury may affect recovery and treatment of patients should appreciate such changes.

In this study we investigated the effect of CGS 21680 (2-p-(2-Carboxyethyl)phenethylamino-5-N-ethylcarboxamidoadenosine hydrochloride), an adenosine A2A receptor agonist, in a model of dextran sulphate sodium (DSS)-induced colitis.NMRI mice were fed 5 % (w/v) DSS, and were treated intraperitoneally with 0.5 mg/kg CGS 21680 or vehicle for 10 days. Changes of bodyweight, colon length, the incidence of rectal bleeding, levels of macrophage inflammatory protein (MIP)-1alpha, MIP-2, interferon gamma, interleukin (IL)-1beta, IL-12 and tumour necrosis factor-alpha from homogenates of colon biopsies, and the release of [3H]acetylcholine (ACh) from longitudinal muscle strip were determined.DSS significantly decreased bodyweight, colon length, and it increased the incidence of rectal bleeding and levels of MIP-1alpha, MIP-2 and IL-1beta compared to DSS-untreated animals. CGS 21680 had no effect on these changes. No change could be observed in release of ACh in DSS-induced colitis with or without CGS 21680.In summary, CGS 21680 is ineffective in ameliorating DSS-induced colitis in mice.

The modulation of noradrenaline (NA) release from rat spinal cord slices was investigated and the subtype of presynaptic alpha 2-adrenoceptors involved in the negative feedback modulation was characterized using in vitro perfusion experiments. Rat spinal cord slices were loaded with [3H]NA and the release of radioactivity at rest and in response to field stimulation was determined. The alpha 2-adrenoceptor agonists, clonidine and dexmedetomidine inhibited the stimulation-evoked release of NA from spinal cord slices, whereas alpha 2-adrenoceptor antagonists yohimbine and CH-38083 (7,8-(methylenedioxy)-14-alpha-hydroxyalloberbane HCI), enhanced it. ARC 239, a selective alpha 2B-antagonist, had no effect on the release. In contrast, BRL 44408 a selective alpha 2A-antagonist increased the release of NA. Our results indicate that the negative feedback modulation of NA release from noradrenergic fibres in the spinal cord is mediated via alpha 2A subtype of alpha 2-adrenoceptors.

Objective: The aim was to determine the presynaptic modulation of noradrenaline (NA) release from the sympathetic nerve terminals in human isolated papillary muscle. Methods: Papillary muscle and the right atrial appendage were obtained from operations on 22 patients (10 men and 12 women). The papillary muscle preparations were preincubated with [3H]NA and the release of [3H] at rest and in response to field stimulation was measured. Results: Using an immunohistochemical method dopamine-β-hydroxylase-positive neurones were found in the papillary muscle and right atrial appendage sample. The release of noradrenaline from the papillary muscle, associated with axonal activity, was enhanced by 7,8(methylenedioxy)-14-α-hydroxyalloberbane HC1 (CH-38083). a selective α2 adrenoceptor antagonist, and inhibited by xylazine, an α2 adrenoceptor agonist, indicating that negative feedback modulation was functioning. In addition, the release of [3H]NA was enhanced by atropine, pancuronium, and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), a selective M3muscarinic receptor antagonist, and reduced by oxotremorine, a selective muscarinic receptor agonist, indicating that acetylcholine released from the parasympathetic nerve ending was able to reach the varicose noradrenergic axon terminals that are equipped with inhibitory M3 muscarinic receptors. Conclusions: These findings, obtained for the first time in human papillary muscle, indicate that the release of noradrenaline is modulated by α2 autoreceptors activated by noradrenaline and M, muscarinic heteroreceptors. Thus during parasympathetic stimulation the release of noradrenaline from the sympathetic axon terminals is presynaptically controlled through muscarinic receptors. Cardiovascular Research 1994; 28 :700-704

Rat vas deferens was prepared, loaded with [3H]noradrenaline, and superfused to measure the release of tritium in resting conditions and in response to electrical field stimulation. The α2-adrenoceptor antagonists yohimbine, CH-38083 (7,8-(methylenedioxi)-14α-hydroxyalloberbane HCl), and idazoxan increased the electrically induced release of tritium in a concentration-dependent manner, whereas noradrenaline and the α2-adrenoceptor agonist xylazine exerted opposite effects. The inhibitory effect of noradrenaline on electrically induced tritium release was antagonized by yohimbine, CH-38083, and idazoxan. Of the α2-adrenoceptor antagonists tested, yohimbine and CH-38083 reversed the xylazine-induced inhibition of tritium release, and idazoxan was found to be completely ineffective against xylazine. Idazoxan, yohimbine and CH-38083 antagonized the inhibitory effect of xylazine on electrical stimulation-induced contractions of the vas deferens, as was evidenced by the apparent pA2 values. We conclude from the present experiments that noradrenaline and xylazine inhibit noradrenaline release by acting on distinct prejunctional α2-adrenoceptors and that the receptor subtype that responds to xylazine is insensitive to idazoxan. In addition, inhibition by xylazine of contractility but not of noradrenaline release was antagonized by idazoxan, suggesting that besides noradrenergic neurotransmission, other motor transmitter systems (purinergic) may also be involved in the inhibition by α2-adrenoceptor antagonists of mechanical responses in the rat vas deferens.

We studied the effect of local administration of nicotine on the release of monoamines in striatum, substantia nigra, cerebellum, hippocampus, cortex (frontal, cingulate), and pontine nucleus and on the release of glutamic acid in striatum of rats in vivo, using microdialysis for nicotine administration and for measuring extracellular amine and glutamic acid levels. Following nicotine administration the extracellular concentration of dopamine, increased in all regions except cerebellum; serotonin increased in cingulate and frontal cortex; and norepinephrine increased in substantia nigra, cingulate cortex, and pontine nucleus. Cotinine, the major nicotine metabolite, had no effect at similar concentrations. The cholinergic antagonists mecamylamine and atropine, the dopaminergic antagonists haloperidol and sulpiride, and the excitatory amino acid antagonist kynurenic acid all inhibited the nicotine-induced increase of extracellular dopamine in the striatum. The fact that kynurenic acid almost completely prevented the effects of nicotine, and nicotine at this concentration produced a 6-fold increase of glutamic acid release, suggests that the effect of nicotine is mainly mediated via glutamic acid release.

Because ATP is degraded to adenosine, its effect could be mediated by both P1 and P2 receptors. Hence, the actions of an ATP analogue, resistant to enzymatic breakdown (alpha, beta-methylene ATP), were studied on the resting and electrically evoked release of radioactivity from longitudinal muscle strips of guinea pig ileum, preloaded either with [3H]choline or with [3H]noradrenaline. Their effects were compared with the actions of adenosine and ATP. Although adenosine and ATP markedly decreased the [3H]acetylcholine release evoked by field stimulation, alpha,beta-methylene-ATP, a potent and selective agonist of P2x receptors, enhanced this release. However, 2-methyl-2-thio-ATP, an agonist of the P2y receptors, neither enhanced nor inhibited the [3H]-acetylcholine release. 8-Phenyltheophylline, an antagonist of P1 receptors, increased the stimulation-evoked release of acetylcholine, indicating that the release of acetylcholine is tonically controlled by endogenous adenosine via P1 receptors. When alpha,beta-methylene-ATP and 8-phenyltheophylline were added together, their potentiating effect on the acetylcholine release proved to be additive. Because alpha,beta-methylene-ATP failed to antagonize the presynaptic effect of adenosine on P1 purinoceptors, it seems very likely that its effect to enhance transmitter release is mediated via separate receptors, i.e., via P2x receptors, located on the axon terminals. Similarly, the stimulation-evoked release of [3H]noradrenaline was enhanced slightly by alpha,beta-methylene-ATP. Our results suggest that both cholinergic and noradrenergic axon terminals are equipped with P2 receptors through which the stimulation-evoked release of transmitter can be modulated by ATP in a positive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of 2-(4-phenylpiperidino)cyclohexanol (AH5183 or vesamicol), a compound known to block the uptake of acetylcholine (ACh) into cholinergic synaptic vesicles, on the release of endogenous and [14C]ACh from slices of rat striatum was investigated. ACh release was evoked either by electrical stimulation or by veratridine. The effect of electrical stimulation was entirely dependent on external Ca2+. By contrast, veratridine (40 microM) also enhanced ACh release in the absence of Ca2+. Indeed, with veratridine two components were clearly distinguished: one dependent on external Ca2+ and the other not. Vesamicol inhibited [14C]ACh release evoked by both veratridine and electrical stimulation in the presence of external Ca2+, provided it was added to the tissue prior to loading with [14C]choline. With the same treatment vesamicol only slightly affected the release of endogenous ACh. Under the same conditions the Ca2(+)-independent [14C]ACh release evoked by veratridine was not prevented by vesamicol. The differential responsiveness to vesamicol suggests that ACh pools involved in Ca2+o-dependent ACh release are different from those mobilized during Ca2+o-independent ACh release.

The aim of this study was to verify the presence of presynaptic nicotinic acetylcholine receptors (nAChRs) at the terminals of myenteric motoneurons using a potent and highly selective nicotinic agonist, epibatidine. We examined contraction, and release of [3H]ACh on a guinea-pig longitudinal muscle strip preparation. First, we compared the ability of epibatidine and nicotine to induce isometric contraction and found epibatidine (EC50 = 23.1 nM) to be 300-fold more potent than nicotine (EC50 = 7.09 microM). The release and contraction induced by 30 nM epibatidine were inhibited by the nicotinic antagonist mecamylamine (3 microM) and the Na(+)-channel blocker TTX (1 microM), indicating that the effects are mediated via nAChRs and are fully dependent on the propagation of action potentials. Atropine (0.1 microM) significantly increased the [3H]ACh release but could not block contraction suggesting that a substantial part of the response develops via a noncholinergic mechanism. Epibatidine at a higher concentration (300 nM) induced contraction, which was only partly (45%) inhibited by TTX (1 microM). The TTX-resistant contraction, however, was completely blocked by mecamylamine (3 microM). Our data provide functional neurochemical evidence for the existence of presynaptic nAChRs at myenteric motoneuron terminals and suggest that these receptors can be activated only/by a higher concentration of agonists.

In this study the regulation of cardiac sympathetic outflow by presynaptic P(2X) receptor-gated ion channels was examined. ATP (30 microM - 1 mM) and other P2-receptor agonists elicited [(3)H]-noradrenaline ([(3)H]-NA) outflow from the isolated guinea-pig right atrium with the potency order of ATP2-methyl-thioATPalpha,beta-methylene-ATP=ADP, whereas ss, gamma-methylene-L-ATP was inactive. Ca(2+)-free conditions abolished both electrical field stimulation (EFS)- and ATP-evoked release of tritium. Unlike from EFS-induced outflow, ATP-induced [(3)H]-NA outflow was not reduced by omega-Conotoxin-GVIA (100 nM), Cd(2+) (100 microM) and tetrodotoxin (1 microM). The rapid extracellular decomposition of ATP was revealed by HPLC analysis. However, the effect of ATP to promote [(3)H]-NA release was not prevented by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 250 nM), 3, 7-dimethyl-1-propargylxanthine (DMPX, 250 nM), or by reactive blue 2 (RB2, 10 microM), antagonists of A(1)-, A(2)- and inhibitory P(2) receptors. Zn(2+) (50 microM), the P(2X)-receptor modulator potentiated, and P(2X) receptor antagonists, i.e. suramin (300 microM), pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM) and 2'-o-(trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP, 30 microM) antagonized the ATP (1 mM)-evoked response. RT - PCR study revealed the expression of P(2X2) and P(2X3) receptor mRNAs in guinea-pig superior cervical ganglion. PPADS (30 microM) significantly reduced the EFS-induced [(3)H]-NA outflow in the presence DPCPX (250 nM) and RB2 (10 microM). In summary a P(2X)-type purinoceptor regulates noradrenaline release from the isolated right atrium of the guinea-pig. The pharmacological profile of the receptor resemble to homo-oligomeric P(2X3) or hetero-oligomeric P(2X2)/P(2X3) complexes, and provide a new target to intervene on sympathetic neuroeffector transmission at the presynaptic site.

In this study we explored the effect of the stimulation of nicotinic acetylcholine receptors located on interneurons by measuring 4-amino-n-[2,3-(3)H]butyric acid ([(3)H]GABA) release and monitoring [Ca (2+)](i) in superfused hippocampal slices. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione, (+/-)-2-amino-5-phosphonopentanoic acid, and atropine, i.e., under the blockade of N-methyl-D-aspartate and non-N-methyl-D-aspartate glutamate and muscarinic receptors, nicotine did not alter the spontaneous outflow of [(3)H]GABA, but significantly increased the stimulation-evoked [(3)H]GABA efflux. This effect of nicotine depended on the time interval between nicotine treatment and electrical stimulus, the concentration of nicotine (1-100 microM), and the parameters of electrical depolarization. Acetylcholine (0.03-3 mM), and the alpha 7 subtype-selective agonist choline (0.1-10 mM), also potentiated stimulus-evoked release of [(3)H]GABA, whereas 1,1-dimethyl-4-phenilpiperazinium iodide failed to increase the tritium outflow significantly. The effect of nicotine treatment was prevented by tetrodotoxin (1 microM) and by the nicotinic acetylcholine receptor antagonist mecamylamine (10 microM), and the alpha 7 subtype-selective antagonists alpha-bungarotoxin (100 nM) and methyllycaconitine (10 nM), whereas dihidro-beta-erythroidine (20 nM) was without effect. Perfusion of 100 microM nicotine caused a [Ca(2+)](i) transient in about one-third of the tested interneurons; however, the response to subsequent electrical stimulation remained unchanged. Inhibition of the GABA transporter system by nipecotic acid (1 mM) or by decreasing the bath temperature to 12 degrees C abolished completely the effect of nicotine to potentiate the stimulation-evoked release of GABA. These findings indicate that the activation of alpha 7-type nicotinic receptors of hippocampal interneurons results in a long-lasting ability of these cells to respond to depolarization with an increased release of GABA mediated by the transporter system.

The complex effect of lobeline on [(3)H]norepinephrine ([(3)H]NE) release was investigated in this study. Lobeline-induced release of [(3)H]NE from the vas deferens was strictly concentration-dependent. In contrast, electrical stimulation-evoked release was characterized by diverse effects of lobeline depending on the concentration used: at lower concentration (10 microM), it increased the release and at high concentration (100 and 300 microM), the evoked release of [(3)H]NE was abolished. The effect of lobeline on the basal release was [Ca(2+)]-independent, insensitive to mecamylamine, a nicotinic acetylcholine receptor antagonist, and to desipramine, a noradrenaline uptake inhibitor. However, lobeline-induced release was temperature-dependent: at low temperature (12 degrees C), at which the membrane carrier proteins are inhibited, lobeline failed to increase the basal release. Lobeline dose dependently inhibited the uptake of [(3)H]NE into rat hippocampal synaptic vesicles and purified synaptosomes with IC(50) values of 1.19 +/- 0.11 and 6.53 +/- 1.37 microM, respectively. Lobeline also inhibited Ca(2+) influx induced by KCl depolarization in sympathetic neurons measured with the Fura-2 technique. In addition, phenylephrine, an alpha(1)-adrenoceptor agonist, contracted the smooth muscle of the vas deferens and enhanced stimulation-evoked contraction. Both effects were inhibited by lobeline. Our results can be best explained as a reversal of the monoamine uptake by lobeline that is facilitated by the increased intracellular NE level after lobeline blocks vesicular uptake. At high concentrations, lobeline acts as a nonselective Ca(2+) channel antagonist blocking pre- and postjunctional Ca(2+) channels serving as a counterbalance for the multiple transmitter releasing actions.

To determine whether adenosine 5'-triphosphate (ATP) is released from the superior cervical ganglion (SCG) of rats and whether the release is regulated by presynaptic mechanism.Using the luciferin-luciferase technique.Electric stimulation evoked the release of ATP from the rat SCG. Adenosine (100 mumol.L-1), P1(A1) purinoceptor agonist N6-cyclopentyladenosine (0.1 mumol.L-1), the muscarinic agonist oxotremorine (1 mumol.L-1), and 5-hydroxytryptamine (100 mumol.L-1) decreased the evoked release of ATP from the rat SCG. On the contrary, P1(A1) purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (10 nmol.L-1), P2 purinoceptor antagonist pyridoxal-5-phosphate-6-azophenyl-2',4-disulphonic acid (10 mumol.L-1), muscarinic antagonist atropine (1 mumol.L-1), alpha 2 adrenoceptor antagonist yohimbine (3 mumol.L-1), D2 dopamine receptor antagonist sulpiride (20 mumol.L-1), and histamine (100 mumol.L-1) increased the evoked release of ATP from the rat SCG.ATP is released from the rat SCG and the release of ATP can be presynaptically modulated by P1(A1), P2, muscarinic, alpha adrenergic, D2, 5-HT, and H1 receptor agonists and antagonists.

In the present study we investigated the effects of NMDA and non-NMDA glutamate receptor antagonists on the ischemia-evoked release of [3H]noradrenaline from rat spinal cord slices. An in vitro ischemia model (oxygen and glucose deprivation) was used to simulate the ischemic conditions known to cause neuronal injury. Spinal cord slices were loaded with [3H]noradrenaline and superfused with Krebs solution in a micro-organ bath. Both axonal stimulation and ischemia increased the release of [3H]noradrenaline, but the release in response to glucose and oxygen deprivation was [Ca2+]o independent. Dizocilpine (MK-801), an NMDA receptor antagonist, suppressed the release of [3H]noradrenaline produced by ischemia, while it enhanced the release of [3H]noradrenaline evoked by electrical field stimulation. In contrast, LY300168 (GYKI-53655) [(+/-)-3-N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylen e-dioxy-5H-2.3-benzodiazepine] and its (-)isomer LY303070 (GYKI-53784) [(-)-3-N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylene- dioxy-5H-2.3-benzodiazepine] AMPA receptor antagonists, had no effect on the release of [3H]noradrenaline evoked by either electrical stimulation or ischemia. Desipramine, a noradrenaline uptake inhibitor, potentiated the release of [3H]noradrenaline evoked by ischemia, while in the absence of [Ca2+]o but under conditions when [3H]noradrenaline release was further increased, it reduced the release. Dizocilpine also decreased glutamate and aspartate release, measured by high performance liquid chromatography, during ischemia. It is concluded that glutamate release and NMDA receptors, but not AMPA receptors, are involved in the acute effect of oxygen and glucose deprivation on the excessive release of noradrenaline and that this release is not related to physiological axonal conduction.

THE effect of the cholinesterase inhibitor neostigmine on hippocampal noradrenaline (NA) release was studied using in vivo microdialysis. Local application of neostigmine significantly increased the release of NA. The effect was potentiated by coperfusion of the nicotinic antagonist mecamylamine but was completely blocked by the muscarinic antagonist atropine. The neostigmine-evoked NA release was not affected by the M2-selective muscarinic antagonist gallamine but was completely blocked by the M1-selective muscarinic antagonist pirenzepine. While muscarinic antagonists had no effect on the resting release of NA, mecamylamine increased it. Our data indicate that acetylcholine can stimulate the hippocampal NA release via M1 muscarinic receptors and that a population of nicotinic receptors mediate inhibitory tone on hippocampal NA release. The fact that neostigmine is able to enhance both cholinergic and noradrenergic neurotransmission may help to understand the beneficial effect of cholinesterase inhibitors in Alzheimer's disease. (C) 1999 Lippincott Williams & Wilkins.

1. Intracellular calcium has been suggested to be an important mediator of the cellular response in endotoxaemia and shock. Dantrolene is an agent that interferes with intracellular calcium fluxes resulting in a decreased availability of calcium in the cytoplasm. Here we have investigated the effect of dantrolene on lipopolysaccharide (LPS)-induced production of interleukin-10 (IL-10), tumour necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) in mice and in cultured RAW 264.7 macrophages in vitro. 2. In BALB/c mice, LPS-induced plasma IL-10 levels were significantly enhanced by pretreatment with dantrolene (20 mg kg(-1), i.p.) (P0.005 at the 90 min time-point). On the other hand, dantrolene pretreatment suppressed circulating TNF-alpha and nitrite/nitrate (breakdown products of NO) concentrations. However, dantrolene had no effect on LPS-induced plasma interleukin-6 (IL-6) levels (67.22+/-5.51 ng ml(-1) in vehicle-pretreated mice and 62.22+/-3.66 ng ml(-1) in dantrolene-pretreated mice, n = 9). 3. Dantrolene inhibited TNF-alpha and NO production in C57BL/6 IL-10+/+ mice, as well as in their IL-10 deficient counterparts (C57BL/6 IL-10(0/0)). 4. In RAW 264.7 macrophages, dantrolene (10-300 microM) reduced IL-10, TNF-alpha, and nitrite (breakdown product of NO) production elicited by LPS (10 microg ml(-1)). Dantrolene (300 microM) did not affect the LPS-induced nuclear translocation of transcription factor nuclear factor kappaB in these cells. 5. Although LPS failed to alter the intracellullar concentration of calcium in single macrophages loaded with Fura-2, dantrolene caused a significant decrease of the basal calcium level as determined 30 min after dantrolene treatment (P0.005). ATP (1 mM) caused a rapid rise in intracellular calcium levels in both dantrolene-pretreated and vehicle-pretreated cells. 6. These results indicate that unlike the secretion of TNF-alpha and NO, IL-10 production is differentially regulated in vitro and in vivo. The decrease of plasma levels of the pro-inflammatory mediators TNF-alpha and NO, and increase in circulating IL-10 concentrations by dantrolene suggest that this drug might offer a new therapeutic approach in inflammatory diseases and septic shock.

Phosphodiesterase (PDE) inhibitors are potent regulators of various immune processes. Immune cells contain type IV and type III PDE. Here we studied in mice the effects of rolipram, a selective PDE IV inhibitor, and amrinone, a selective PDE III blocker, on plasma levels of IL-12 (p70), IFN-gamma, IL-1, TNF-alpha, and nitric oxide (NO) induced by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (80 mg/kg). Pretreatment of BALB/c mice with both rolipram (1-25 mg/kg) and amrinone (10-100 mg/kg) decreased plasma IL-12 levels in a dose-dependent manner. Similarly, LPS-elicited plasma IFN-gamma concentrations were suppressed by both rolipram and amrinone. However, LPS-induced plasma IL-1alpha levels were not affected by either of these compounds. In addition, rolipram inhibited IL-12, IFN-gamma, TNF-alpha and nitrite/nitrate (breakdown products of NO) production in C57BL/6 IL-10(+/+) mice as well as in their IL-10-deficient counterparts (C57BL/6 IL-10(-/-)). Our results suggest that rolipram and amrinone decrease the immune activation in endotoxemia through inhibition of the production of pro-inflammatory mediators IL-12, IFN-gamma, TNF-alpha and NO. These effects are not the consequences of the increase in IL-10 production by PDE inhibition.

The effects of selective nicotinic acetylcholine (ACh) receptor (nAChR) agonists and antagonists on the stimulation-evoked release of [3H]ACh were studied in rat isolated superior cervical ganglion loaded with [3H]choline and superfused in a 2-ml chamber. Nicotine and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), but not cytisine, increased the stimulation (2 Hz)-evoked release of [3H]ACh in a concentration-dependent manner. The rank order of potency to increase stimulation-evoked release for the nAChR agonists (nicotineDMPPcytisine) suggests that the beta4 subunit of nAChRs is not involved in the release. The finding that alpha-bungarotoxin was effective in preventing the effect of DMPP and itself significantly reduced the release indicates that the alpha7 subunit is located presynaptically and may be involved in the positive feedback modulation. Hexamethonium inhibited the effect of DMPP with an apparent dissociation constant (Kd) of 11.5 +/- 1.5 microM. Hexamethonium and other nAChR antagonists, i.e., (+)-tubocurarine (100 microM), mecamylamine (3 microM), dihydro-beta-erythroidine (3 microM), pancuronium (10 microM) and alpha-bungarotoxin (2 microM), also decreased the stimulation-evoked release of [3H]ACh. The effect of hexamethonium was independent of stimulation frequency (2, 10 and 30 Hz) applied. Atropine enhanced the stimulation-evoked release of ACh, indicating that there is negative feedback modulation of ACh release associated with neuronal activity. In contrast, when the nicotinic positive feedback was prevented by hexamethonium, atropine failed to enhance the release. These findings indicate that muscarinic receptor-mediated inhibition of ACh release functions in cases in which the release is enhanced by ACh via stimulation of presynaptic nAChRs. A similar interaction was found between A1 receptor-mediated reduction and nAChR-mediated positive feedback modulation of [3H]ACh release. The results suggest the presence of positive feedback modulation of ACh release via presynaptic nAChRs in rat superior cervical ganglion.

Adenosine released into the extracellular space by immunologic and nonimmunologic stimuli has been shown to regulate various immune functions. In this study we report that i.p. pretreatment of mice with CGS-21680 HCl (CGS), a selective agonist of A2 adenosine receptors, at 0.2 to 2 mg/kg caused an augmentation of plasma IL-10 levels induced by i.p. injection of LPS, but decreased plasma levels of LPS-induced TNF-alpha. 2-Chloro-N6-cyclopentyladenosine (CCPA), an agonist of A1 adenosine receptors, at 0.5 mg/kg diminished LPS-induced plasma TNF-alpha concentrations, but enhanced LPS-induced IL-10 levels only at the highest dose used (2 mg/kg). The specific A3 adenosine receptor agonist 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-beta- D-ribofuranuronamide, at 0.2 and 0.5 mg/kg potentiated LPS-stimulated IL-10 production and inhibited LPS-induced TNF-alpha production. LPS-induced plasma nitrite and nitrate levels (the breakdown products of nitric oxide (NO)) were suppressed by CGS and CCPA. In the RAW 264.7 macrophage cell line, pretreatment of the cells with both CGS and CCPA inhibited LPS-induced IL-10, TNF-alpha, and NO production, each in a concentration-dependent manner. The inhibitory effect of these drugs on cytokine and NO production was associated with improved mitochondrial respiration. Neither CGS nor CCPA affected the LPS-induced nuclear translocation of transcription factor nuclear factor-kappaB in these cells. These results demonstrate that adenosine receptor stimulation differentially modulates the LPS-induced production of IL-10, TNF-alpha, and NO in vitro and in vivo. The increase in LPS-induced IL-10 production and suppression of LPS-induced TNF-alpha and NO production caused by adenosine receptor activation may explain some of the immunomodulatory actions of adenosine released in excess during inflammatory and/or ischemic insult.

The effect of 7-nitroindazole (7-NI), an inhibitor of neuronal nitric oxide synthase (nNOS) on the dimethylphenylpiperazinium(DMPP)-evoked release of [3H]noradrenaline ([3H]NA) from rat hippocampal slices was studied. The effect of DMPP (20 microM) to increase the basal release of [3H]NA was significantly potentiated by 7-NI (40 microM). In our previous study we showed that the response to DMPP has two components, a nicotinic receptor-mediated, [Ca2+]-dependent exocytosis followed by a [Ca2+]-independent, uptake blocker-sensitive carrier-mediated release. To clarify which part of the response was affected by the inhibition of nNOS, we investigated the effect of 7-NI on the nicotine-evoked NA release (nicotine has only receptor-mediated effect) and on the DMPP-evoked NA release in Ca(2+)-free medium where the receptor-mediated component is abolished. Nicotine (100 microM) significantly increased the basal release of [3H]NA but this release was not affected, whereas in Ca(2+)-free medium the response to DMPP (20 microM) was still potentiated by 7-NI (40 microM). In the presence of the NA uptake blocker desipramine (10 microM) DMPP (20 microM) was unable to provoke NA release independently from the presence or absence of 7-NI (40 microM). Our data show that 7-NI influences the carrier-mediated component of DMPP-evoked [3H]NA release, which indicates that nitric oxide produced by nNOS may play a role in the regulation of the NA uptake carrier.

Release of [3H]dopamine ([3H]DA) from rat striatal slices kept under hypoxic or/and glucose-free conditions was measured using a microvolume perfusion method. The corresponding changes in nucleotide content were determined by reverse-phase high-performance liquid chromatography (RPHPLC). The resting release of [3H]DA was not affected by hypoxia, but under glucose-free conditions massive [Ca2+]o-independent release of [3H]DA was observed. Hypoxia reduced the energy charge (E.C.) and the total purine content from 19.36 +/- 4.15 to 6.98 +/- 1.83 nmol/mg protein. Glucose deprivation by itself, or in combination with hypoxia, markedly reduced the levels of adenosine 5'-triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). The E.C under glucose-free conditions was significantly reduced from 0.73 +/- 0.04 to 0.44 +/- 0.20. When the tissue was exposed to hypoxic and glucose-free conditions for 18 min the level of ATP was reduced to 3.15 +/- 0.11 nmol/mg protein. However, when the exposure time was 30 min the ATP level was further reduced to 1.11 +/- 0.37 nmol/mg protein. The resting release was enhanced in a [Ca2+]o-independent manner, but there was no release in response to stimulation, and tetrodotoxin did not affect the enhanced resting release, indicating that the release was not associated with axonal activity. Similarly, 50 microM ouabain, inhibitor of Na+/K(+)-activated ATPase, enhanced the release of [3H]DA at rest in a [Ca2+]o-independent manner. It seems very likely that the reduced ATP level under glucose-free conditions leads to an inhibition of the activity of Na+/K(+)-ATPase that results in reversal of the uptake processes and in [Ca2+]o-independent [3H]DA release from the axon terminals.

The effects of 5 mM NaF + 10 microM AlCl3, a direct activator of guanine nucleotide-binding proteins (G proteins), on the release of [3H]dopamine ([3H]DA), [3H]gamma-aminobutyric acid ([3H]GABA), and [3H]acethylcholine ([3H]ACh) were investigated in slices of rat striatum. When the tissue was exposed to NaF + AlCl3 the release of [3H]DA, [3H]GABA, and [3H]ACh was enhanced significantly. In a calcium-free solution the release of [3H]GABA and [3H]DA was increased by NaF+AlCl3 much more than in the presence of [Ca2+]o. In slice preparations taken from reserpinized animals, in which the vesicular storage of [3H]DA was therefore prevented, NaF + AlCl3 had no effect on [3H]DA release. HPLC analysis of the radioactivity of the perfusate showed that, in the presence of NaF + AlCl3, the content of dihydroxyphenylacetic acid (DOPAC) in perfusate samples increased significantly, while in pargyline-treated animals only the DA content was increased. Inhibition of DA carriers by nomifensine or low temperature prevented the effect of NaF + AlCl3. N-ethylmaleimide (NEM) preincubation did not modify the effect of NaF + AlCl3 on [3H]DA release Neomycin (0.1 mM), a phospholipase C (PLC) inhibitor, significantly decreased the effect of NaF + AlCl3 on [3H]DA and [3H]GABA release. The internal concentration of Ca2+ in synaptosomes was enhanced by NaF + AlCl3 in normal solution. However, [Ca2+]i was not influenced by NaF + AlCl3 in Ca(2+)-free medium. It is concluded that a non-receptor-mediated activation, by NaF + AlCl3, of the alpha-subunit of a G protein, results in a [Ca2+]o-independent release of DA and GABA, but not that of ACh.

1. The effects of alpha-1 adrenoceptor agonists on 3H-acetylcholine release from mouse phrenic nerve-hemidiaphragm preparation were studied. 2. In preparation which had been incubated with 3H-choline, electrical stimulation (50 Hz, 0.8 s trains every 10 s) released 3H-acetylcholine. Neurochemical evidence was obtained that noradrenaline, adrenaline and phenylephrine facilitated the stimulation evoked release of 3H-acetylcholine. This effect was much more marked when the prejunctional nicotinic receptors mediating positive feedback modulation were blocked by (+)-tubocurarine. In the presence of this drug, when the release of 3H-acetylcholine was reduced, alpha-1 adrenoceptor agonists enhanced the release in a prazosin-sensitive manner. 3. The rank order of potency of alpha-1 adrenoceptor agonists was adrenaline greater than noradrenaline greater than phenylephrine. By contrast, methoxamine had no effect on the release of 3H-acetylcholine. It is suggested, therefore, that circulating catecholamines may be able to affect neuromuscular transmission through stimulation of presynaptic alpha-1 adrenoceptors.

Cerebral cortex slices from the rat were loaded with [3H]norepinephrine ([3H]NE) and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. The (-)-isomer and the (+)-isomer of CH-38083 ([7,8-(methylenedioxy)-14- alpha-hydroxyalloberbane HCl), a selective alpha-2-adrenoceptor antagonist with an alloberbane skeleton, increased the electrically induced release of [3H]NE in a concentration-dependent manner, and a similar effect was observed with racemic CH-38083 and idazoxan. The stereoisomers of CH-38083 applied in a concentration range of 10(-8) to 10(-6) mol/l were equipotent in facilitating stimulation-evoked [3H]NE release: concentrations needed to enhance tritium outflow by 50% were 1.3 X 10(-7) mol/l for (-)-CH-38083 and 1.4 X 10(-7) mol/l for (+)-CH-38083. Exogenous NE decreased the electrically stimulated release of [3H]NE, and the stereoisomers of CH-38083 antagonized this inhibition with different potencies: the dissociation constant (KB) values for (-)-isomer and for (+)-isomer of CH-38083 were 14.29 and 97.18 nmol/l. These data indicate that presynaptic alpha-2 adrenoceptors that are available for NE released from axon terminals do not show stereospecificity toward enantiomers of CH-38083, whereas those that are occupied by exogenous NE are much more sensitive toward (-)-CH-38083. The alpha-1 adrenoceptor antagonist prazosin also differentiated between the alpha-2 adrenoceptor subtypes: prazosin (10(-6) mol/l) did not alter the increase of electrically induced [3H]NE release evoked by (-)- and (+)-CH-38083; however, in its presence, the stereoisomers of CH-38083 failed to antagonize the inhibitory effect of exogenous NE on its own release.(ABSTRACT TRUNCATED AT 250 WORDS)

While cholinergic nervous control of pancreatic enzyme secretion is well documented, data concerning adrenergic regulation of the exocrine pancreas are contradictory. In the present study we attempted to elucidate the direct action of adrenergic stimulation on pancreatic enzyme secretion. Rat pancreatic segments were set up in an organ bath and superfused with modified Krebs-Henseleit solution. Electrical field stimulation (EFS) stimulated amylase release from the segments. This stimulation was subject to inhibition with atropine up to 80%. Atropine-resistant enzyme discharge in response to EFS could be blocked by propranolol. Cholinergic agonist urecholine-induced amylase release was completely blocked by atropine. Noradrenaline (NA) exhibited a biphasic effect on amylase release. It inhibited the urecholine-induced amylase release in lower concentrations (10(-8)-10(-7) M), while it stimulated basal enzyme secretion in higher concentrations (10(-5)-10(-4) M). The inhibitory effect was mimicked by phenylephrine and completely prevented by prazosin. Isoprenaline concentration dependently enhanced, while clonidine and guanfacine did not affect amylase discharge. In conclusion, in rat pancreatic acinar tissue it seems likely that acetylcholine is the main neurotransmitter. Adrenergic action can be dual, inhibitory via alpha 1-adrenoceptors or stimulatory via beta-adrenoceptors on amylase secretion.

The effect of supramaximal electric field stimulation on [3H]noradrenaline (NA) release and hormone production by rat adrenal capsule-glomerulosa preparations was studied using a microvolume perfusion system. A substantial proportion (about 20%) of nerve endings (varicosities) were observed close to zona glomerulosa cells, and about half of them appeared to be catecholaminergic, as judged by the chromaffin reaction of the synaptic vesicles studied at electron microscopic level. In tissue, preloaded with [3H]NA, the release of NA in response to electrical stimulation was frequency-dependent. Reserpinization, calcium removal or inhibition of Na+ influx by tetrodotoxin completely blocked NA release by field stimulation, indicating that the release resulted from axonal activity and is of vesicular origin. Neither the α2-adrenoceptor agonist xylazine nor the muscarine-receptor agonist oxotremorine affected the stimulation-evoked release of [3H]NA, suggesting that, in contrast with other neurones present in the central nervous system or in the peripheral autonomic nervous system but like those in the median eminence, these axon terminals contained few presynaptic modulatory receptors. The NA (10·20 ± 1·79 (s.e.m.) μg/g, n = 9), adrenaline (24·38 ± 5·50 μg/g, n = 9) and dopamine (0·35 ± 0·09 μg/g, n = 6) contents of the preparations were high, as determined by high-performance liquid chromatography. Our observations that the release and content of NA is high, and that a substantial proportion of catecholaminergic axon terminals lie in close proximity to zona glomerulosa cells (median value of the distance 300 nm) or to smooth muscle cells of the vessels, suggest that NA released from local adrenergic neurones without being presynaptically modulated may play an important role in finetuning both steroid production and/or blood flow through the gland, itself a powerful modulator of the adrenocortical response. This local modulating effect of NA may be especially significant when sympathetic activity is enhanced. Journal of Endocrinology (1992) 135, 551–561

The ability of somatostatin to inhibit the muscular twitches elicited by field stimulation of rat vas deferens and guinea pig ileum was studied. Evidence should be summarized which would suggest that the presynaptic inhibitory effect of somatostatin on chemical neurotransmission is indirect. Its effect on vas deferens is partly mediated via alpha 2-adrenoceptors and due to the released noradrenaline, in guinea pig ileum, however, it seems likely that it releases an unidentified inhibitory substance which, in fact, inhibits the release of acetylcholine, and thereby twitches of the ileum. The fast tachyphylaxis seen following repeated administration of the peptide is likely due to its indirect action.

The inhibitory effect of azidomorphine (AM), 14-OH-azidomorphine (OAM), azidocodeine (AC) in comparison to morphine, codein, hydromorphone, oxymorphone, methadone and pethidine on the propulsion of ch

The possible direct action of vasoactive intestinal peptide (VIP) on dog hepatic arterial wall or on the noradrenergic innervation of the artery was investigated in vitro. In addition, VIP-containing nerve fibers and terminals were located in the wall of the artery with immunochemical staining. Direct evidence showed that VIP did not affect the release of [3H]norepinephrine but reduced the response of the isolated hepatic artery to electrical field stimulation and exogenous norepinephrine. This suggests that the effect of VIP is postjunctional on the smooth muscle of the artery. VIP-containing nerve fibers and varicosities were observed in the adventitial and medial layer of the arterial wall. These findings strongly support the hypothesis that vasoactive intestinal peptide is a physiological mediator of vasodilation in the hepatic artery.

The effects of indomethacin (IND) and PGI2 on the tone of human isolated pulmonary arteries were studied. Baseline tone of the vessels was increased by IND, electric stimulation (ES), norepinephrine (NE), prostaglandin F2α (PDF2α) or K+-excess. This high tone was decreased by PGI2 in a concentration dependent manner. IC50 values (molar concentrations producing 50% relaxation) for PGI2 were in the same concentration range (10 to 58.8 nmoles/l). The potency of the relaxant effect of PGI2 was inversely related to the magnitude of tone induced prior to the addition of PGI2 and independent of the type of tone inducer. It is suggested that the relaxant effect of prostacyclin on human pulmonary artery may be of clinical importance in the treatment of conditions associated with a rise in pulmonary vascular resistance.

In the presence of phentolamine, stimulation of the cat splanchnic nerve decreased the resistance of the superior mesenteric arterial bed. This effect was not significantly influenced by sulpiride. Sulpiride, in the presence of phentolamine, did not inhibit the decrease in resistance in animals pretreated with guanethidine but propranolol or ablation of the adrenal glands prevented the effect of stimulation. These results are not compatible with the assumption that dopamine released from dopaminergic nerves is involved in the vasodilatation but do provide evidence for the role of beta-adrenoceptors and circulating catecholamines in the vasodilatation of mesenteric vessels.

Hypoglycaemic sulphonylureas inhibit the increased glycogenosis and glucose release of the rat perfused liver produced by β-adrenergic agonists. The β-receptor antagonist propranolol exerted a similar effect. Dichloroisoprenaline increases the hypoglycaemic effect of chlorpropamide given in doses ineffective in the intact rat. Hypoglycaemic sulphonylureas have no effect on the catecholamine sensitivity of the nictitating membrane and heart auricle of the cat, indicating that their ability to inhibit glycogenosis induced by β-receptor agonists is not due to a β-receptor antagonist activity as is the action of propranolol.

Thick filaments consisting probably of myosin could be identified regularly in both untreated and atropine-pretreated stretched longitudinal smooth muscles of guinea pig ileum. In cells of unstretched muscles thick filaments occurred only sporadically. Our findings suggest the importance of the physiological tonus in the formation of myosin filaments.

The effect of an endogenous ouabain-like compound (OCL) and of ouabain was studied on [3H]noradrenaline release and on the tension of rabbit pulmonary arterial strip. Similarly to ouabain, the OLC enhanced release of [3H] NA in resting and in stimulated condition. Moreover, in the presence of OLC and ouabain, the tension of the rabbit artery increased gradually and the contraction evoked by electrical stimulation was potentiated. It is suggested that this mechanism might be involved in the physiological regulation of blood pressure or in the genesis of hypertension.

The release of [3H]norepinephrine from isolated mouse vas deferens has been measured. 1-Phenylephrine and 1-norepinephrine significantly enhanced the spontaneous release of radioactivity. As shown by a combination of HPLC and scintillation spectrometry, the release of total radioactivity in response to 1-phenylephrine or 1-norepinephrine consisted mainly of [3H]norepinephrine. Evidence has been obtained that the release of endogenous norepinephrine by exogenous norepinephrine and 1-phenylephrine is independent of the external Ca2+ concentration. The released endogenous norepinephrine in turn inhibits the release of norepinephrine in response to electrical field stimulation. In the presence of yohimbine, the enhancement of spontaneous release due to 1-phenylephrine (or to 1-norepinephrine) was not affected, whereas there was a significant superimposed release of [3H]norepinephrine in response to field stimulation, indicating that the inhibition of stimulation-induced norepinephrine release is an alpha 2-adrenoceptor-mediated process. An important consequence of these findings is to question previous interpretations that the effects of administration of 1-norepinephrine or 1-phenylephrine are due exclusively to their direct effects on the effector cells. The Ca-independent release of endogenous norepinephrine might partly initiate their pharmacological responses. It is concluded that this Ca-independent release is of functional importance, since norepinephrine may accumulate in a concentration sufficient to modulate the release of norepinephrine from varicosities in response to electrical stimulation.

Ca2+-dependent release of [3H] noradrenaline ([3H] NA) evoked by electrical stimulation of the isolated mouse vas deferens was subject to negative feedback modulation by idazoxan an alpha 2-adrenoceptor blocking agent. Both the resting release and that evoked by 1-phenylephrine proved to be Ca0-independent and unaffected by idazoxan. Ouabain-evoked release of [3H] acetylcholine from the myenteric plexus of ileal longitudinal muscle strips in the presence of eserine was not affected by atropine, but that evoked by electrical stimulation was enhanced. Since the release of NA or ACh by 1-phenylephrine and ouabain respectively is mainly of cytoplasmic origin, it is concluded that the release of transmitter from the cytoplasm is not subject to negative feedback modulation.

In the rats treated with ACTH or hydrocortisone for 14 days the catalepsy induced by morphine was almost completely inhibited, while the haloperidol induced catalepsy remained unchanged. The morphine induced hypermotility was altered neither by prolonged treatment with ACTH nor by an acute glucocorticoid administration. Inhibition of Na/K ATPase by ouabain led to an increase of striatal acetylcholine (Ach) release, which was enhanced by Met-enkephalin. This effect of the opioid peptide was not demonstrable in the striata of ACTH or hydrocortisone pretreated rats. It is concluded that glucocorticoids are regulatory factors of the striatal opiate neurotransmission possibly via altered receptorial mechanisms.

The release of oxytocin from isolated posterior lobe of the hypophysis of untreated rats and rats pretreated with alpha-methyl-p-tyrosine (alpha-MPT) has been studied. The amount of oxytocin released under resting conditions, in response to ouabain was much higher in those preparations which had been pretreated with alpha-MPT. Dopamine failed to affect the resting release in tissue taken from control rats but it significantly reduced the secretion of oxytocin in tissue dissected from dopamine-deficient rats. Opioid peptides, beta-endorphin or D-Ala2-Pro5-enkephalinamide enhanced the release of oxytocin from isolated neural lobe of the hypophysis dissected from untreated rats, but they failed to enhance significantly the release from posterior lobe of rats which had been pretreated by alpha-MPT. Naloxone prevented the effect of the opioid peptides, and by itself significantly reduced the release of oxytocin. The data suggest that (i) dopamine stored in, and released from, the neural lobe may inhibit the secretion of oxytocin; (ii) the release of oxytocin may be continously controlled by an endogenous opioid peptide: opioid peptides may exert their effect via a disinhibitory phenomenon; they remove the inhibitory effect of dopamine on oxytocin release possibly by inhibiting the release of dopamine.

Histamine was extracted from deproteinized plasma with Amberlite CG 50 weak cation-exchange resin (analytical recovery of [3H]histamine, 60.5%). The eluate was evaporated and histamine in the redissolved sample was condensed with o-phthalaldehyde and 2-mercaptoethanol at pH 11.5. This adduct was separated by liquid chromatography under isocratic conditions and oxidized on a glassy carbon electrode at +0.4 V for electrochemical detection. 3-Methylhistamine was used as internal standard. As little as 0.45 pmol of standard histamine condensate was detected. The histamine concentration in 88 human plasma samples appeared to be normally distributed; its mean value was 7.20 (SD 2.61) nmol/L. Authentic and extracted histamine produced similar hydrodynamic voltammograms, and exogenous and endogenous histamine gave identical chromatographic characteristics with different mobile phases or different chromatographic columns. Standard and extracted histamine had similar degradation rates when samples were incubated with diamine oxidase (EC 1.4.3.6). Analytical recovery of known amounts of histamine added to pooled plasma was 97.7% (SD 22.3%). The inter- and intra-assay CVs for histamine determinations were 9.0% and 8.6%, respectively.

A radioisotope method has been developed for measuring the stimulation-evoked release of acetylcholine without the use of cholinesterase inhibitors from the mouse hemidiaphragm preparation which had been loaded with 3H-choline. Evidence has been obtained that 3H-choline was taken up by and released from both innervated and non-innervated mouse hemidiaphragm preparations. However, it was released in the form of 3H-acetylcholine in response to electrical field stimulation only from the innervated preparations. Long lasting (51 min) S1 stimulation of the preparations exhausted the radioactive acetylcholine stores to the extent that S2 did not evoke any release of 3H. These data suggest that when the labelled acetylcholine stores become exhausted, the labelled choline, still present in the tissue, cannot be released by electrical stimulation. Tetrodotoxin (1 mumol/l) administration and Ca withdrawal inhibited, 20-100 mumol/l 4-aminopyridine enhanced the release of 3H-acetylcholine in response to electrical stimulation. Activation of the presynaptic muscarinic receptors by the agonist oxotremorine (50 mumol/l) decreased the liberation of 3H-acetylcholine. The muscarinic antagonist atropine (1 mumol/l) abolished the inhibitory effect of oxotremorine and by itself increased the evoked release of the newly formed 3H-acetylcholine. Adenosine (50 mumol/l) reduced the evoked release of radioactivity. Theophylline (30 mumol/l) prevented the inhibitory effect of adenosine and itself enhanced the release. Xylazine (1 mumol/l), an alpha 2-adrenoceptor agonist did not affect the release. It is concluded that the stimulation-evoked release of 3H-acetylcholine from the mouse phrenic nerve hemidiaphragm preparation preloaded with 3H-choline is derived from the motor nerves. The release of acetylcholine is modulated by activation of presynaptic muscarinic and adenosine receptors.

The electrical and mechanical activities of guinea pig taenia coli smooth muscle were measured by a "sucrose gap" technique. Under the same experimental conditions the ionic content of smooth muscle was also measured. The mean value of the resting potential was 56.9 +/- 1.1 mV (S.E.M.; n = 46). In normal Krebs solution immediately after dissection intracellular sodium amounted to 30.1, and intracellular calcium to 1.5 mmole x kg-1 wet weight. In response to adrenaline administration there was a Ca-dependent hyperpolarization (peak, 6.8 +/- 0.3 mV S.E.M.; n = 5) and an increased Na efflux with a rate constant (k) of 0.16 min-1 (60'). Removal of adrenaline was followed by so-called "postadrenaline depolarization" i.e. the decrease of the membrane potential was greater than the initial rise, an effect enhanced by ouabain (2 X 10(-5) M). Clonidine (5.3 X 10(-6) M), a selective presynaptic-adrenoceptor (alpha 2-receptor) stimulant failed to produce hyperpolarization, however, phenylephrine (5 X 10(-5) M) a pure postsynaptic alpha-adrenoceptor (alpha 1-receptor) stimulant produced a similar effect as adrenaline. In addition, yohimbine (1.4 X 10(-6) M), a typical presynaptic alpha-adrenoceptor inhibitor failed to affect the action of adrenaline or phenylephrine. These facts indicate that the alpha 1-adrenoceptors present on the smooth muscle are different from those situated presynaptically on the cholinergic nerve terminals modulating the release of acetylcholine. The effect of ouabain to lower membrane potential proved to be Ca2+-dependent. The intracellular sodium content was enhanced by ouabain from 30.1 to 90.9 +/- 4.7 mmole x kg-1 wet weight (60'). On washing out ouabain, hyperpolarization "post-ouabain hyperpolarization" was detected, i.e. the rise of membrane potential was greater than the initial fall. It is suggested that the sodium pump plays a significant role in the post-ouabain hyperpolarization. Direct calculation of sodium movements suggests that the sodium permeability is about 3.7 p-mole x cm-2 x sec-1 and the pump generates a current of 1.06 X 10(-7) A x cm-2 which, in fact, while crossing the smooth muscle membrane with a resistance of 30-60 k omega cm2 would produce a potential difference of 3.2-6.4 mV. It is suggested that the electrogenic sodium pump may contribute to the resting membrane potential about 15 mV, a value obtained from the summation of depolarization induced by ouabain and hyperpolarization produced by adrenaline.

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.

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.

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.

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)