Stimulation of the hypothalamo-pituitary-adrenal axis in the rat by the type 4 phosphodiesterase (PDE-4) inhibitor, denbufylline.

1. Otilonium, a clinically useful spasmolytic, behaves as a potent blocker of neuronal nicotinic acetylcholine receptors (AChR) as well as a mild wide-spectrum Ca2+ channel blocker in bovine adrenal chromaffin cells. 2. 45Ca2+ uptake into chromaffin cells stimulated with high K+ (70 mM, 1 min) was blocked by otilonium with an IC50 of 7.6 microM. The drug inhibited the 45Ca2+ uptake stimulated by the nicotinic AChR agonist, dimethylphenylpiperazinium (DMPP) with a 79 fold higher potency (IC50 = 0.096 microM). 3. Whole-cell Ba2+ currents (IBa) through Ca2+ channels of voltage-clamped chromaffin cells were blocked by otilonium with an IC50 of 6.4 microM, very close to that of K(+)-evoked 45Ca2+ uptake. Blockade developed in 10-20 s, almost as a single step and was rapidly and almost fully reversible. 4. Whole-cell nicotinic AChR-mediated currents (250 ms pulses of 100 microM DMPP) applied at 30 s intervals were blocked by otilonium in a concentration-dependent manner, showing an IC50 of 0.36 microM. Blockade was induced in a step-wise manner. Wash out of otilonium allowed a slow recovery of the current, also in discrete steps. 5. In experiments with recordings in the same cells of whole-cell IDMPP, Na+ currents (INa) and Ca2+ currents (ICa), 1 microM otilonium blocked 87% IDMPP, 7% INa and 13% ICa. 6. Otilonium inhibited the K(+)-evoked catecholamine secretory response of superfused bovine chromaffin cells with an IC50 of 10 microM, very close to the IC50 for blockade of K(+)-induced 45Ca2+ uptake and IBa. 7. Otilonium inhibited the secretory responses induced by 10 s pulses of 50 microM DMPP with an IC50 of 7.4 nM. Hexamethonium blocked the DMPP-evoked responses with an IC50 of 29.8 microM, 4,000 fold higher than that of otilonium. 8. In conclusion, otilonium is a potent blocker of nicotinic AChR-mediated responses. The drugs also blocked various subtypes of neuronal voltage-dependent Ca2+ channels at a considerably lower potency. Na+ channels were unaffected by otilonium. This extraordinary potency of otilonium in blocking nicotinic AChR, unrecognised until now, might account in part for its well known spasmolytic effects.     

Inhibition by SEA0400, a selective inhibitor of Na+/Ca2+ exchanger, of Na+ -dependent Ca2+ uptake and catecholamine release in bovine adrenal chromaffin cells

The effects of SEA0400, a selective inhibitor of the Na(+)/Ca(2+) exchanger (NCX), on Na(+)-dependent Ca(2+) uptake and catecholamine (CA) release were examined in bovine adrenal chromaffin cells that were loaded with Na(+) by treatment with ouabain and veratridine. SEA0400 inhibited Na(+)-dependent (45)Ca(2+) uptake and CA release, with the IC(50) values of 40 and 100 nM, respectively. The IC(50) values of another NCX inhibitor KB-R7943 were 1.8 and 3.7 microM, respectively. These results indicate that SEA0400 is about 40 times more potent than KB-R7943 in inhibiting NCX working in the reverse mode. In intact cells, SEA0400 and KB-R7943 inhibited CA release induced by acetylcholine and DMPP. The IC(50) values of SEA0400 were 5.1 and 4.5 microM and the values of KB-R7943 were 2.6 and 2.1 microM against the release induced by acetylcholine and DMPP, respectively, indicating that the potency of SEA0400 is about a half of that of KB-R7943 in inhibiting the nicotinic receptor-mediated CA release. The binding of [(3)H]nicotine with nicotinic receptors was inhibited by SEA0400 (IC(50) = 90 microM) and KB-R7943 (IC(50) = 12 microM). From these results, it is concluded that unlike KB-R7943, SEA0400 has a potent and selective action on NCX in bovine adrenal chromaffin cells.     

The novel Na(+)/Ca(2+) exchange inhibitor KB-R7943 also blocks native and expressed neuronal nicotinic receptors

We studied the effects of the novel Na(+)/Ca(2+) exchange inhibitor KB-R7943, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate, on the native nicotinic receptors present at the bovine adrenal chromaffin cells, as well as on rat brain alpha(3)beta(4) and alpha(7) nicotinic acetylcholine receptors (AChRs) expressed in XENOPUS: oocytes. As expected, KB-R7943 blocked the Na(+)-gradient dependent (45)Ca(2+) uptake into chromaffin cells (IC(50) of 5.5 microM); but in addition, the compound also inhibited the (45)Ca(2+) entry and the increase of cytosolic Ca(2+) concentration, [Ca(2+)](c), stimulated by 5 s pulses of ACh (IC(50) of 6.5 and 1.7 microM, respectively). In oocytes expressing alpha(3)beta(4) and alpha(7) nicotinic AChRs, voltage-clamped at -60 mV, inward currents elicited by 1 s pulses of 100 microM ACh (I(ACh)) were blocked by KB-R7943 with an IC(50) of 0.4 microM and a Hill coefficient of 0.9. Blockade of alpha(3)beta(4) currents by KB-R7943 was noncompetitive; moreover, the blocker (0.3 microM) became more active as the ACh concentration increased (34 versus 66% blockade at 30 microM and 1 mM ACh, respectively). Inhibition of alpha(3)beta(4) currents by 0.3 microM KB-R7943 was more pronounced at hyperpolarized potentials. If given within the ACh pulse (10 microM), the inhibition amounted to 33, 64 and 80% in oocytes voltage-clamped at -40, -60 and -100 mV, respectively. The onset of blockade was faster and the recovery slower at -100 mV; the reverse was true at -40 mV. In conclusion, KB-R7943 is a potent blocker of nicotinic AChRs; moreover, it displays many features of an open-channel blocker at the rat brain alpha(3)beta(4) AChR. These results should be considered when KB-R7943 is to be used to study Ca(2+) homeostasis in cells expressing nicotinic AChRs and the Na(+)/Ca(2+) exchanger.     

Blockade by agmatine of catecholamine release from chromaffin cells is unrelated to imidazoline receptors

The blockade of exocytosis induced by the putative endogenous ligand for imidazoline receptors, agmatine, was studied by using on-line measurement of catecholamine release in bovine adrenal medullary chromaffin cells. Agmatine inhibited the acetylcholine-evoked release of catecholamines in a concentration-dependent manner (IC(50)=366 microM); the K(+)-evoked release of catecholamines was unaffected. Clonidine (100 microM) and moxonidine (100 microM) also inhibited by 75% and 50%, respectively, the acetylcholine-evoked response. In cells voltage-clamped at -80 mV, the intermittent application of acetylcholine pulses elicited whole-cell inward currents (I(ACh)) that were blocked 63% by 1 mM agmatine. The onset of blockade was very fast (tau(on) = 31 ms); the recovery of the current after washout of agmatine also occurred very rapidly (tau(off = 39 ms). Efaroxan (10 microM) did not affect the inhibition of I(ACh) elicited by 1 mM agmatine. I(ACh) was blocked 90% by 100 microM clonidine and 50% by 100 microM moxonidine. The concentration-response curve for acetylcholine to elicit inward currents was shifted to the right in a non-parallel manner by 300 microM agmatine. The blockade of I(ACh) caused by agmatine (100 microM) was similar at various holding potentials, around 50%. When intracellularly applied, agmatine did not block I(ACh). At 1 mM, agmatine blocked I(Na) by 23%, I(Ba) by 14%, I(K(Ca)) by 16%, and I(K(VD)) by 18%. In conclusion, agmatine blocks exocytosis in chromaffin cells by blocking nicotinic acetylcholine receptor currents. In contrast to previous views, these effects seem to be unrelated to imidazoline receptors.     

Nicotinic acetylcholine receptors in the rat stomach: I. (-)-[3H]nicotine binding

The nicotinic acetylcholine receptors in the rat stomach were characterized by means of a radioligand binding assay with (-)-[3H]nicotine as ligand. Saturation binding studies on the gastric fundus membranes revealed the presence of two binding sites with dissociation constant (KD) values of 3.1 and 289 nM, and maximum binding capacity (Bmax) values of 3.6 and 76 fmol/mg protein, respectively. The Bmax of the high affinity binding site was greatest in the cardia, followed by fundal mucosa, fundal muscle, and, finally antrum. The IC50 values of cholinergic drugs to inhibit (-)-[3H]nicotine binding in fundus membranes were as follows: (-)nicotine, 0.12 nM; cytosine, 9.3 nM; acetylcholine, 17.7 nM; carbachol, 700 nM; hexamethonium, 2270 nM. The IC50 values of alpha-bungarotoxin, d-tubocurarine and atropine were greater than 100 microM. The muscarinic acetylcholine receptors were also characterized with [3H]quinuclidinyl benzilate and the choline acetyltransferase activity was measured. These results suggest that nicotinic acetylcholine receptors as well as muscarinic acetylcholine receptors are present in the rat stomach and that the regional distribution of these receptors is uneven.     

Activation and blockade by choline of bovine alpha7 and alpha3beta4 nicotinic receptors expressed in oocytes

Choline, the precursor and the metabolite of acetylcholine, is reputed as a selective alpha7 nicotinic receptor agonist. In this study, however, we have seen that choline exerted a dual effect on bovine nicotinic receptors expressed in Xenopus oocytes. On the one hand, choline behaved as a weak full agonist on bovine alpha7-mediated inward currents, with an EC50 of 0.43 mM. On the other, choline blocked bovine alpha3beta4 currents, with an IC50 of 0.97 mM. The blockade by choline was fast (tau(on), 0.36 s), fully reversible (tau(off), 1.23 s), exhibited voltage-dependence (60% blockade at -100 mV and 30% blockade at -40 mV), and was of a non-competitive nature, suggesting an open-channel type of alpha3beta4 receptor blockade. Thus, choline by activating alpha7 receptors and/or blocking alpha3beta4 receptors might play a physiological role in the control of neurotransmission at cholinergic synapses where alpha7 and alpha3beta4 receptor are expressed.     

Evidence for muscarinic receptors in the adrenal medulla of the dog

1. The release of catecholamines from the adrenal medulla by the cholinergic drugs was monitored by the perfusion pressure rise in autoperfused and sympathetically denervated hindlimbs of the dog.2. Acetylcholine, dimethylphenylpiperazinium (DMPP) and methacholine, given in relatively small doses into the aorta proximal to the blood supply to the adrenal glands, caused a marked rise in perfusion pressure which was due to the release of catecholamines from the adrenal medulla.3. Atropine (1 mg/kg intravenously) blocked the pressor response to methacholine only. The ganglion-blocking agents (mecamylamine 4-5 mg/kg or hexamethonium 10 mg/kg) given subsequently blocked the pressor responses to the other two cholinergic drugs. The ganglion-blocking agents, when given before atropine, blocked the pressor action only of DMPP. These agents partly depressed, rather than potentiated, the pressor response to methacholine.4. The results suggest that muscarinic as well as nicotinic receptors are present in the adrenal medulla of the dog.5. Neither atropine nor ganglion-blocking drugs alone reduced the pressor response to acetylcholine. It is postulated that the blockade of one set of receptors makes more acetylcholine available for the other set of receptors and so inactivation of the former receptors are compensated by the increased release through the activation of the latter's.     

Differential blockade of rat alpha3beta4 and alpha7 neuronal nicotinic receptors by omega-conotoxin MVIIC, omega-conotoxin GVIA and diltiazem.

Rat alpha3beta4 or alpha7 neuronal nicotinic acetylcholine receptors (AChRs) were expressed in Xenopus laevis oocytes, and the effects of various toxins and non-toxin Ca2+ channel blockers studied. Nicotinic AChR currents were elicited by 1 s pulses of dimethylphenylpiperazinium (DMPP, 100 microM) applied at regular intervals. The N/P/Q-type Ca2+ channel blocker omega-conotoxin MVIIC inhibited alpha3beta4 currents with an IC50 of 1.3 microM; the blockade was non-competitive and reversible. The alpha7 currents were unaffected. At 1 microM, omega-conotoxin GVIA (N-type Ca2+ channel blocker) inhibited by 24 and 20% alpha3beta4 and alpha7 currents, respectively. At 1 microM, omega-agatoxin IVA (a P/Q-type Ca2+ channel blocker) did not affect alpha7 currents and inhibited alpha3beta4 currents by only 15%. L-type Ca2+ channel blockers furnidipine, verapamil and, particularly, diltiazem exhibited a preferential blocking activity on alpha3beta4 nicotinic AChRs. The mechanism of alpha3beta4 currents blockade by omega-conotoxins and diltiazem differed in the following aspects: (i) the onset and reversal of the blockade was faster for toxins; (ii) the blockade by the peptides was voltage-dependent, while that exerted by diltiazem was not; (iii) diltiazem promoted the inactivation of the current while omega-toxins did not. These data show that, at concentrations currently employed as Ca2+ channel blockers, some of these compounds also inhibit certain subtypes of nicotinic AChR currents. Our data calls for caution when interpreting many of the results obtained in neurons and other cell types, where nicotinic receptor and Ca2+ channels coexist.     

Noncompetitive inhibition by camphor of nicotinic acetylcholine receptors

The effect of camphor, a monoterpenoid, on catecholamine secretion was investigated in bovine adrenal chromaffin cells. Camphor inhibited [3H]norepinephrine ([3H]NE) secretion induced by a nicotinic acetylcholine receptor (nAChR) agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), with a half-maximal inhibitory concentration (IC50) of 70 +/- 12 microM. In addition, camphor inhibited the rise in cytosolic calcium ([Ca2+]i) and sodium ([Na+]i) induced by DMPP with IC50 values of 88 +/- 32 and 19 +/- 2 microM, respectively, suggesting that the activity of nAChRs is also inhibited by camphor. On the other hand, binding of [3H]nicotine to nAChRs was not affected by camphor. [Ca2+]i increases induced by high K+, veratridine, and bradykinin were not affected by camphor. The data suggest that camphor specifically inhibits catecholamine secretion by blocking nAChRs without affecting agonist binding.     

Effects of dichlorobenzene on acetylcholine receptors in human neuroblastoma SH-SY5Y cells

para-Dichlorobenzene (DCB), a deodorant and an industrial chemical, is a highly volatile compound and is known to be an indoor air contaminant. Because of its widespread use and volatility, the toxicity of DCB presents a concern to industrial workers and public. Some toxic aspects of DCB have already been focused but its effects on neuronal signal transduction have been hitherto unknown. The effects of DCB on the cytosolic calcium homeostasis are investigated in human neuroblastoma SH-SY5Y cells in this study. DCB, above 200 microM, was found to induce a rise in cytosolic calcium concentration that could not be counteracted by nicotinic acetylcholine receptor (nAChR) and muscarinic acetylcholine receptor (mAChR) antagonists but was partially inhibited by thapsigargin. To understand the actions of DCB on the acetylcholine receptors, we investigated its effects on the changes of cytosolic calcium concentration following nicotinic AChR stimulation with epibatidine and muscarinic AChR stimulation with methacholine in human neuroblastoma SH-SY5Y cells. DCB inhibited the cytosolic calcium concentration rise induced by epibatidine and methacholine with respective IC(50)s of 34 and 294 microM. The inhibitions of DCB were not the same as thapsigargin's inhibition. In the electrophysiological observations, DCB blocked the influx currents induced by epibatidine. Our findings suggest that DCB interferes with the functional activities of AChR, including its coupling influx currents and cytosolic calcium elevations.     

Inhibition of nicotinic receptor-mediated responses in bovine chromaffin cells by diltiazem.

1. The effects of diltiazem on various functional parameters were studied in bovine cultured adrenal chromaffin cells stimulated with the nicotinic receptor agonist dimethylphenylpiperazinium (DMPP) or with depolarizing Krebs-HEPES solutions containing high K+ concentrations. 2. The release of [3H]-noradrenaline induced by DMPP (100 microM for 5 min) was gradually and fully inhibited by increasing concentrations of diltiazem (IC50 = 1.3 microM). In contrast, the highest concentration of diltiazem used (10 microM) inhibited the response to high K+ (59 mM for 5 min) by only 25%. 3. 45Ca2+ uptake into cells stimulated with DMPP (100 microM for 1 min) was also blocked by diltiazem in a concentration-dependent manner (IC50 = 0.4 microM). Again, diltiazem blocked the K(+)-evoked 45Ca2+ uptake (70 mM K+ for 1 min) only by 20%. In contrast, the N-P-Q-type Ca2+ channel blocker omega-conotoxin MVIIC depressed the K+ signal by 70%. In the presence of this toxin, diltiazem exhibited an additional small inhibitory effect, indicating that the compound was acting on L-type Ca2+ channels. 4. Whole-cell Ba2+ currents through Ca2+ channels in voltage-clamped chromaffin cells were inhibited by 3-10 microM diltiazem by 20-25%. The inhibition was readily reversed upon washout of the drug. 5. The whole-cell currents elicited by 100 microM DMPP (IDMPP) were inhibited in a concentration-dependent and reversible manner by diltiazem. Maximal effects were found at 10 microM, which reduced the peak IDMPP by 70%. The area of each curve represented by total current (QDMPP) was reduced more than the peak current. At 10 microM, the inhibition amounted to 80%; the IC50 for QDMPP inhibition was 0.73 microM, a figure close to the IC50 for 45Ca2+ uptake (0.4 microM) and [3H]-noradrenaline release (1.3 microM). The blocking effects of diltiazem developed very quickly and did not exhibit use-dependence; thus the drug blocked the channel in its closed state. The blocking effects of 1 microM diltiazem on IDMPP were similar at different holding potentials (inhibition by around 30% at -100, -80 or -50 mV). Diltiazem did not affect the current flow through voltage-dependent Na+ channels. 6. These data are compatible with the idea that diltiazem has little effect on Ca2+ entry through voltage-dependent Ca2+ channels in bovine chromaffin cells. Neither, does diltiazem affect INa. Rather, diltiazem acts directly on the neuronal nicotinic receptor ion channel and blocks ion fluxes, cell depolarization and the subsequent Ca2+ entry and catecholamine release. This novel effect of diltiazem might have clinical relevance since it might reduce the sympathoadrenal drive to the heart and blood vessels, thus contributing to the well established antihypertensive and cardioprotective effects of the drug.     

Neuropharmacology of the muscarinic antagonist telenzepine in myenteric ganglia of the guinea-pig small intestine

Intracellular recording methods were used to investigate the actions of the putative M1 muscarinic receptor antagonist telenzepine on the electrical and synaptic behavior of myenteric neurons. Telenzepine had no effect on resting membrane potential, input resistance, excitability and antidromic potentials in both AH/type 2 and S/type 1 neurons, when applied in concentrations of 0.1-2000 nM, although higher concentrations (10-100 microM) did have a significant non-specific effect on the postsynaptic membrane. Micromolar concentrations of telenpzepine (1-2 microM) had no effect on excitatory responses to substance P, vasoactive intestinal peptide, the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium or the nicotinic action of acetylcholine. Nicotinic fast excitatory postsynaptic potentials were also unaffected by 2 microM telenzepine. In contrast, at submicromolar concentrations (100 nM), telenzepine abolished responses to either muscarine or the muscarinic component of the acetylcholine response. The excitatory effect of muscarine at postsynaptic M1 receptors was dose dependently inhibited by telenzepine (0.1-1000 nM) at concentrations which had no effect on the electrical properties of the cells. This effect was slowly reversible, usually requiring more than 60 min for significant recovery. The threshold dose of telenzepine as an antagonist of the muscarinic depolarization in AH/type 2 neurons was in the range of 0.1-1 nM. The IC50 concentration of telenzepine needed to abolish the response was 8.5 nM. A small proportion of stimulus-evoked slow excitatory postsynaptic potentials in both AH/type 2 and S/type 1 cells were abolished by 1 microM telenzepine, while the majority of them remained unaffected, indicating that some slow excitatory postsynaptic potentials are mediated by the muscarinic action of released acetylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of peripheral vascular resistance in perfused hindquarters of dogs: lack of effect of nicotinic and muscarinic ganglion block.

The effects of nicotinic and muscarinic ganglion blockade were studied on the peripheral resistance of the perfused hindlimbs of the dog. Nicotinic ganglion blockade by chlorisondamine (1 mg/kg) significantly reduced reflex vasomotor responses but did not significantly alter hindlimb peripheral resistance. Combined nicotinic ganglion blockade by chlorisondamine (1 mg/kg) and muscarinic ganglion blockade by atropine (50mug/kg) abolished the reflex vasomotor response but did not alter hindlimb peripheral resistance. It was concluded that autoregulatory mechanisms are capable of maintaining a peripheral resistance after block of both muscarinic and nicotinic ganglion transmission.     

Effect of noncompetitive nicotinic receptor blockers on catecholamine release from cultured adrenal chromaffin cells.

Nicotinic acetylcholine receptors are found on both muscle tissue and neuronal tissue, including adrenal chromaffin cells. Certain drugs, such as the noncompetitive ion-channel blockers and the vinca alkaloids, have been demonstrated to interact with muscle-type nicotinic receptor-associated ion channels. The objectives of these studies were: (1) to determine the effects of the noncompetitive blockers of the nicotinic receptor-associated ion-channel drugs on adrenal chromaffin cells, and (2) to establish whether the vinca alkaloids and the ion-channel drugs have similar actions on adrenal nicotinic receptors. The ion-channel drugs, adiphenine, tetracaine, quinacrine, amantadine, lidocaine and procaine, inhibited receptor-stimulated catecholamine release from cultured adrenal chromaffin cells in a concentration-dependent manner (IC50s: 2, 4, 6, 41, 55 and 87 microM, respectively). The inhibitory effects of these drugs appeared to be noncompetitive. These drugs had little or no effects on catecholamine release stimulated by depolarizing concentrations of K+ or on basal release. Our results demonstrate that the ion-channel blockers interact with adrenal nicotinic receptors in a manner similar to their interaction with muscle-type nicotinic receptors and they interfere with adrenal receptor function in a manner similar to the vinca alkaloids.     

N和M胆碱能受体之间的相互调节

N和M胆碱能受体拥有同一种内源性配基ACh ,并且在多种组织中两种受体共存 ,两者之间存在着多种相互作用。如副交感神经节后N受体可通过刺激胆碱能神经纤维末梢释放ACh来调节组织中M受体的功能 ;而胆碱能神经元突触后N受体的活性也可被突触前膜上M和N的自受体通过反馈调控节前纤维ACh的释放来调节 ;另外 ,N受体被阻断或失敏后不同组织中M受体的功能会发生不同的变化 ,以将机体胆碱能神经系统维持在一个稳定状态     

3H]N-methyl-carbamylcholine, a new radioligand specific for nicotinic acetylcholine receptors in brain

The present study characterized the binding of [3H]N-methyl-carbamylcholine ([3H]methyl-carbachol), a new radioligand, to rat cerebral cortex membranes and demonstrated the autoradiographic distribution of these sites in rat brain. With atropine used to block muscarinic acetylcholine sites and nicotine to define non-specific binding, [3H]methyl-carbachol bound specifically, saturably and with high affinity (Kd = 11.0 nM, Bmax = 118.4 fmol/mg protein and Hill coefficient = 0.92) to a population of presumably nicotinic sites in cerebral cortex membranes. When nicotine was used to block nicotinic acetylcholine sites and atropine to define non-specific binding there was no specific binding of [3H]methyl-carbachol (concentrations up to 45 nM) to possible muscarinic sites in cerebral cortex membranes. The binding parameters under non-selective conditions (without blockade of either muscarinic or nicotinic acetylcholine sites) had very similar values to those obtained under nicotinic conditions (Kd = 8.0 nM, Bmax = 125.0 fmol/mg protein and Hill coefficient = 0.98). [3H]Methyl-carbachol binding was potently inhibited by nicotinic agonists and antagonists but only poorly displaced by muscarinic agents. Autoradiographic studies evidenced high densities of [3H]methyl-carbachol binding sites in the interpeduncular nucleus, various thalamic nuclei, superior colliculus and layers III/IV of the cortex. Such a distribution was very similar to those previously reported for nicotinic acetylcholine sites and other radioligands. These results suggest that [3H]methyl-carbachol is a specific radioligand of the neuronal nicotinic receptor. Its stability and high selectivity constitute distinct advantages over previously used nicotinic radioligands such as acetylcholine and nicotine.     

Choline, CDP-choline or phosphocholine increases plasma glucagon in rats: involvement of the peripheral autonomic nervous system

The present study was designed to test the effects of choline, cytidine-5'-diphosphocholine (CDP-choline) and phosphocholine on plasma glucagon concentrations in rats. Intraperitoneal (i.p.) injection of 200-600 micromol/kg of choline, CDP-choline or phosphocholine produced a dose-dependent increase in plasma glucagon and choline concentrations. Pretreatment with hexamethonium (15 mg/kg; i.p.), a peripherally-acting ganglionic nicotinic acetylcholine receptor antagonist, entirely blocked the increases in plasma glucagon by 600 micromol/kg of choline, CDP-choline or phosphocholine. The increases in plasma glucagon by these choline compounds was reduced significantly (P<0.01) by about 25% by pretreatment with atropine methylnitrate (2 mg/kg), a peripherally-acting muscarinic acetylcholine receptor antagonist. Blockade of central acetylcholine receptors did not alter the increase in plasma glucagon induced by i.p. choline (600 micromol/kg). While alpha(2)-adrenoceptor blockade or bilateral adrenalectomy attenuated the increase in plasma glucagon evoked by choline compounds, blockade of alpha(1)- or beta-adrenoceptors or chemical sympathectomy failed to alter this increase. Intracerebroventricular (i.c.v.) choline (1.5 micromol) administration also increased plasma glucagon; the effect was blocked by central pretreatment with a neuronal type nicotinic acetylcholine receptor antagonist, mecamylamine (50 microg; i.c.v.) or the neuronal choline uptake inhibitor, hemicholinium-3 (20 microg; i.c.v.). These data show that choline, CDP-choline or phosphocholine increases plasma glucagon concentrations by increasing peripheral nicotinic and muscarinic cholinergic neurotransmissions. Central choline also increases plasma glucagon by augmenting central nicotinic cholinergic neurotransmission by acting presynaptically. Stimulation of adrenal medullary catecholamine release and subsequent activation of alpha(2)-adrenoceptors are mainly involved in the increase in plasma glucagon induced by choline, CDP-choline or phosphocholine.     

Inhibition of nicotinic acetylcholine receptors and calcium channels by clozapine in bovine adrenal chromaffin cells

The effects of clozapine on the activities of nicotinic acetylcholine receptors (nAChRs) and voltage-sensitive calcium channels (VSCCs) were investigated and compared with those of chlorpromazine (CPZ) in bovine adrenal chromaffin cells. [(3)H]Norepinephrine ([(3)H]NE) secretion induced by activation of nAChRs was inhibited by clozapine and CPZ with half-maximal inhibitory concentrations (IC(50)) of 10.4 +/- 1.1 and 3.9 +/- 0.2 microM, respectively. Both cytosolic calcium increase and inward current in the absence of extracellular calcium induced by nicotinic stimulation were also inhibited by clozapine and CPZ, but the greater inhibition was achieved by CPZ. In addition, [(3)H]nicotine binding to chromaffin cells was inhibited by clozapine and CPZ with IC(50) values of approximately 19 and 2 microM, respectively. On the other hand, [(3)H]NE secretion induced by high K(+) was inhibited by clozapine and CPZ with similar IC(50) values of 15.5 +/- 3.8 and 17.1 +/- 3.9 microM, respectively. Our results suggest that clozapine, as well as CPZ, inhibits nAChRs and VSCCs, thereby causing inhibition of catecholamine secretion, and that clozapine is much less potent than CPZ in inhibiting nAChRs.     

Mechanism of blockade by flunarizine of bovine adrenal catecholamine release.

How flunarizine, a class IV Ca2+ antagonist, affects the secretion of catecholamines in response to nicotinic receptor activation (10-s pulses with 100 microM dimethylphenylpiperazinium, DMPP) or direct depolarization of chromaffin cells (10-s pulses with 100 mM K+ and 2.5 mM Ca2+, 100 K+/2.5 Ca2+) was studied in bovine adrenal glands perfused with an oxygenated Krebs-Tris solution at 37 degrees C at a rate of 20 ml/min. Experimental protocols aimed to test voltage and time dependence of the flunarizine blocking effects on secretion are described. The DMPP pulses released an average of 217 micrograms catecholamines and the K+ pulses, an average of 117 micrograms. These responses were blocked by flunarizine concentration dependently; IC50s were 3.7 microM for DMPP and 1.1 microM for K+. Under polarizing conditions (60-s perfusion with a solution containing 5.9 mM K+ and nominally zero Ca2+), a 10-s pulse with 100 K+/2.5 Ca2+ released 117 +/- 26 micrograms of catecholamines (n = 12). Under depolarizing conditions (60-s perfusion with 118 K+/0 Ca2+ prior to the Ca2+ pulse), the pulse with 118 K+/2.5 Ca2+ released 307 +/- 36 micrograms of catecholamines (n = 14). Flunarizine blocked these secretory responses equally and concentration dependently with an IC50 of 3.4 microM under polarizing conditions and of 3.8 microM under depolarizing conditions. Thus, blockade by flunarizine of secretion was apparently not voltage-dependent. The blockade was, however, clearly dependent on the time of exposure of the adrenal medullary tissue to flunarizine.(ABSTRACT TRUNCATED AT 250 WORDS)