Observations on the pharmacology of cholinoceptive neurones in the rat brain stem.

The pharmacology of spontaneously active cholinoceptive neurones in the brain stem of rats anaesthetized with urethane has been investigated using microiontophoresis to administer muscarinic and nicotinic agonists and antagonists. 2. Acetylcholine (ACh) excited most cells but occasionally depressed their activity. Muscarine, and the muscarinic agonists methacholine and bethanechol produced prolonged excitation or inhibition of cells whereas nicotine produced prolonged excitations but no inhibitions. 3 Atropine selectively antagonized ACh excitations and both excitation and inhibition of neuronal activity produced by muscarine and muscarinic agonists, but not the excitations produced by nicotine, glutamate or DL-homocysteic acid. 4 Dihydro-beta-erythroidine (DHBE) and tubocurarine antagonized both ACh and nicotine excitations but not those induced by glutamate or DL-homocysteic acid. Inhibitions by ACh or muscarine were not affected. 5 It is concluded that excitations of cholinoceptive neurones in the rat brain stem may be mediated by activation of both muscarinic and nicotinic receptors whereas inhibitions are mediated by activation of a muscarinic receptor.     

An electrophysiological study of muscarinic and nicotinic receptors of rat paratracheal ganglion neurons and their inhibition by Z-338

1. To study the mechanisms involved in the action of Z-338, a newly synthesized gastroprokinetic agent, experiments were performed with the paratracheal ganglion cells acutely dissociated from 2-week-old Wistar rats. The effects of Z-338 on both nicotinic and muscarinic responses of the ganglion cells were studied by nystatin perforated patch recording configuration under the current- and voltage-clamp conditions. 2. Acetylcholine (ACh) or nicotine, and muscarine or oxotremorine-M (OX-M) induced membrane depolarization with rapid and slow time courses respectively, followed by repetitive generation of action potentials in the ganglion cell. Corresponding to the membrane depolarization induced by cholinergic agents, ACh induced biphasic inward currents with rapid and slow time courses under the voltage-clamp condition. Nicotine and muscarine or OX-M evoked inward currents with rapid and slow time courses, respectively. The rapid and slow inward currents were accompanied by increase and decrease in the membrane conductance, respectively. In addition, OX-M dose-dependently suppressed the M-type K(+) current evoked in response to hyperpolarizing voltage-steps from V(H) of -25 mV to -50 mV, indicating that the activation of muscarinic acetylcholine receptors inhibits M-type K(+) current, thus inducing inward current in the ganglion cell. 3. Z-338 competitively suppressed the inward currents induced by OX-M through M(1) ACh receptor, and uncompetitively suppressed the currents induced by nicotine. 4. The inhibitory actions of Z-338 on the membrane depolarization and corresponding inward currents mediated by M(1)-muscarinic and neuronal nicotinic ACh receptors in the isolated ganglion cells were discussed in relation to the inhibitory actions on autoreceptors in the parasympathetic nerve terminals, which would explain the gastroprokinetic actions of Z-338.     

The nature of the acetylcholine receptor in a Buccinum proboscis muscle examined by the sucrose-gap voltage clamp technique.

1. ACh dose-response curves for the radicular retractor muscle of Buccinum showed maximum force and membrane depolarisation of 3.3 mV at 50 mumol l-1 ACh. 2. PCh was found to be almost a full agonist for force and induced higher membrane depolarisations than ACh while BCh was only a partial agonist of very low potency. This suggests an AChR neither muscarinic nor nicotinic in mammalian terminology. 3. Neither muscarine nor nicotine had any direct agonistic effects on the muscle but pre-exposure to nicotine inhibited both force and membrane depolarisation induced by a subsequent dose of ACh. 4. The specific muscarinic and nicotinic antagonists atropine, d-tubocurarine and gallamine all inhibited ACh responses in a dose-dependent manner. 5. Single sucrose-gap recording showed that ACh induced a depolarisation resulting in a contracture. Double sucrose-gap voltage clamp recording showed that 10 mumol l-1 ACh induced an inward transmembrane current of ca 2 microA. Both ACh-induced depolarisation and inward current were abolished in Na-free media. 6. When clamped at a series of membrane voltages between natural Em and positive potentials the ACh-induced Na-dependent inward current declined as Em was reduced and was abolished at -10 mV. This current showed no reversal even at strong positive membrane voltages. 7. The AChR of this muscle appears to be neither exclusively nicotinic nor muscarinic but a hybrid and shows characteristics of voltage inactivation.     

Identification and role of muscarinic receptor subtypes expressed in rat adrenal medullary cells

The muscarinic receptor is known to be involved in the acetylcholine (ACh)-induced secretion of catecholamines in the adrenal medullary (AM) cells of various mammals. The muscarinic receptor subtype involved and its physiological role, however, have not been elucidated yet. Thus, we investigated these issues in acutely isolated rat AM cells and perfused rat adrenal medulla. The RT-PCR analysis revealed the presence of M(2), M(3), M(4), and M(5) mRNAs. Immunocytochemistry with specific antibodies showed that M(5)-like immunoreactivities (IRs) were detected at half the cell membrane area, which was much larger than that with M(3)- or M(4)-like IRs. Muscarine produced inward currents in a dose-dependent manner. Pilocarpine, McN-A-343, and oxotremorine were less efficient than muscarine; and RS-86, which has no action on the M(5) receptor, produced no current. Electrical stimulation of nerve fibers produced a frequency-dependent increase in the Ca(2+) signal in perfused adrenal medullae. Muscarinic receptors were found to be involved in neuronal transmission in AM cells in the presence of a cholinesterase inhibitor, which suppresses ACh degradation. We concluded that the M(5) receptor is the major muscarinic receptor subtype in rat AM cells and may be involved in neuronal transmission under conditions where ACh spills over the synapse.     

Inhibitory effects of caffeine on secretagogue-induced catecholamine secretion from adrenal chromaffin cells of the guinea-pig.

1. The inhibitory action of caffeine on catecholamine secretion induced by secretagogues was investigated in perfused adrenal glands and dispersed chromaffin cells of the guinea-pig. 2. Caffeine (10 mM) caused a reversible inhibition of catecholamine secretion evoked by acetylcholine (ACh, 50 microM), KCl (56 mM, high K+) and veratridine (100 microM) and that induced by muscarinic receptor activation in the absence of extracellular Ca2+ in perfused adrenal glands. 3. In dispersed chromaffin cells, caffeine caused a dose-dependent inhibition of the secretory responses to 100 microM ACh and veratridine. Forskolin (30 microM), dibutyryl cyclic AMP (1 mM) and 8-bromo cyclic AMP (1 mM) did not mimic the action of caffeine. 4. In the voltage-clamp, whole-cell recording mode (at a holding potential of -60 mV or -70 mV), ACh (100 microM) evoked an inward current, and depolarizing pulses elicited inward Na+, Ca2+ and outward K+ currents. All these responses were partially inhibited by caffeine (20 mM). 5. ACh rapidly increased the intracellular concentration of Ca2+ ([Ca2+]i) in fura-2-loaded cells in either the presence or the absence of external Ca2+, though its magnitude was decreased by about 50% in Ca(2+)-free conditions. Caffeine (20 mM) inhibited these ACh-induced increases in [Ca2+]i. 6. In permeabilized chromaffin cells, caffeine (20 mM) caused an inhibition of catecholamine secretion evoked by Ca2+ (10 microM). 7. These results suggest that caffeine inhibits evoked catecholamine secretion through mechanisms such as the blockade of voltage-dependent Na+ and Ca2+ currents and ACh receptor current, and reduction of the release of intracellularly stored Ca2+ and/or Ca(2+)-sensitivity of the secretory apparatus.     

Muscarinic acetylcholine response in pyramidal neurones of rat cerebral cortex.

1. The effects of acetylcholine (ACh) on pyramidal neurons acutely dissociated from the rat cerebral cortex were studied in the whole-cell mode, by use of the nystatin-perforated patch recording configuration. 2. ACh induced a net inward current (IACh) accompanied by a membrane conductance decrease at a holding potential (VH) of -40 mV. IACh increased in a concentration-dependent manner with a half-maximum concentration (EC50) of 8.7 x 10(-7) M. 3. IACh mainly resulted from the suppression of the voltage- and time-dependent K+ current (M-current). 4. Muscarine and muscarinic agonists such as McN-A-343, oxotremorine and oxotremorine-M mimicked the ACh response. The potency was in the order of oxotremorine-M > McN-A-343 > or = muscarine > oxotremorine. 5. Pirenzepine shifted the concentration-response curve for ACh to the right and the corresponding Schild plot yielded a pA2 value of 7.81. Other muscarinic antagonists also reversibly blocked IACh in a concentration-dependent manner. The inhibitory potency was in the order of atropine > 4-DAMP > pirenzepine > AF-DX-116. 6. IACh could be induced normally even after pre-incubation of dissociated neurones in external solution with 200 ng ml-1 pertussis toxin (PTX) for 8 h, whereas the inhibitory effect of ACh on high-voltage-activated Ca2+ channels was completely abolished by the PTX treatment.     

Possible muscarinic regulation of catecholamine secretion mediated by cyclic GMP in isolated bovine adrenal chromaffin cells

Catecholamine secretion and cyclic GMP levels were measured in chromaffin cells isolated from bovine adrenal medulla. Acetylcholine (ACh) and nicotine, but not muscarine, induced 8- to 10-fold increases in catecholamine secretion, with respective ED₅₀ values of 10 and 2 M. Cyclic GMP levels were also increased from 3- to 5-fold in the presence of ACh, and this stimulation was mimicked by muscarine but not by nicotine. Half-maximum stimulations of cyclic GMP levels with ACh and muscarine were observed at 0.1 and 0.3 M respectively. The order of potency of various cholinergic drugs for cyclic GMP stimulation was as follows: ACh > oxotremorine > methacholine > muscarine > carbamylcholine > furthretonium > arecholine > bethanechol. Pilocarpine, McN-A-343, and AHR-602 were inactive at concentrations between 10^?8 and 10^?3 M. Isobutylmethylxanthine (1 mM), a specific phosphodiesterase inhibitor, caused a 7-fold increase in cyclic GMP and potentiated 3-fold the stimulation of cyclic GMP by ACh. The nicotine-induced catecholamine secretion was inhibited 19 and 33 per cent by the co-stimulation of the muscarinic receptor with 0.2 and 0.5 M ACh, respectively. Isobutylmethylxanthine (1 mM) also caused a 44 per cent inhibition of nicotine-induced catecholamine secretion, and its effect was additive to that of ACh. Atropine (0.1 M) selectively abolished the inhibition caused by ACh. Similar inhibitions were also obtained in the presence of exogenous dibutyryl cyclic GMP or 8-bromo cyclic GMP. These data indicate that the nicotinic stimulation of catecholamine secretion from bovine adrenal chromaffin cells may be regulated by cyclic GMP via the stimulation of a muscarinic receptor.     

Identification of muscarinic receptor subtypes involved in catecholamine secretion in adrenal medullary chromaffin cells by genetic deletion

Background and Purpose

Activation of muscarinic receptors results in catecholamine secretion in adrenal chromaffin cells in many mammals, and muscarinic receptors partly mediate synaptic transmission from the splanchnic nerve, at least in guinea pigs. To elucidate the physiological functions of muscarinic receptors in chromaffin cells, it is necessary to identify the muscarinic receptor subtypes involved in excitation.

Experimental Approach

To identify muscarinic receptors, pharmacological tools and strains of mice where one or several muscarinic receptor subtypes were genetically deleted were used. Cellular responses to muscarinic stimulation in isolated chromaffin cells were studied with the patch clamp technique and amperometry.

Key Results

Muscarinic M₁, M₄ and M₅ receptors were immunologically detected in mouse chromaffin cells, and these receptors disappeared after the appropriate gene deletion. Mouse cells secreted catecholamines in response to muscarinic agonists, angiotensin II and a decrease in external pH. Genetic deletion of M₁, but not M₃, M₄ or M₅, receptors in mice abolished secretion in response to muscarine, but not to other stimuli. The muscarine-induced secretion was suppressed by MT7, a snake peptide toxin specific for M₁ receptors. Similarly, muscarine failed to induce an inward current in the presence of MT7 in mouse and rat chromaffin cells. The binding affinity of VU0255035 for the inhibition of muscarine-induced currents agreed with that for the M₁ receptor.

Conclusions and Implications

Based upon the effects of genetic deletion of muscarinic receptors and MT7, it is concluded that the M₁ receptor alone is responsible for muscarine-induced catecholamine secretion.     

Cotinine inhibits catecholamine release evoked by cholinergic stimulation from the rat adrenal medulla

The aim of the present study was to clarify whether cotinine affects the release of catecholamines (CA) from the isolated perfused rat adrenal gland, and to establish the mechanism of its action, in comparison with the response of nicotine. Cotinine (0.3-3 mM), when perfused into an adrenal vein for 60 min, inhibited CA secretory responses evoked by ACh (5.32 mM), DMPP (a selective neuronal nicotinic agonist, 100 microM for 2 min) and McN-A-343 (a selective muscarinic M1-agonist, 100 microM for 2 min) in dose- and time-dependent manners. However, cotinine did not affect CA secretion by high K+ (56 mM). Cotinine itself also failed to affect basal CA output. Furthermore, in the presence of cotinine (1 mM), CA secretory responses evoked by Bay-K-8644 (an activator of L-type Ca2+ channels, 10 microM) and cyclopiazonic acid (an inhibitor of cytoplasmic Ca2+-ATPase, 10 microM) were relative time-dependently attenuated. However, nicotine (30 microM), given into the adrenal gland for 60 min, initially rather enhanced CA secretory responses evoked by ACh and high K+, followed by the inhibition later, while it time-dependently depressed the CA release evoked by McN-A-343 and DMPP. Taken together, these results suggest that cotinine inhibits greatly CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors, but does fail to affect that by the direct membrane-depolarization. It seems that this inhibitory effect of cotinine may be exerted by the cholinergic blockade, which is associated with blocking both the calcium influx into the rat adrenal medullary chromaffin cells and Ca2+ release from the cytoplasmic calcium store. It also seems that there is a big difference in the mode of action between cotinine and nicotine in the rat adrenomedullary CA secretion.     

Inhibitory effects of cortical steroids and adrenocorticotropic hormone on catecholamine secretion in guinea-pig perfused adrenal glands

1 We investigated the effects of exogenously applied steroids and endogenously released cortisol on catecholamine (CA) secretion induced by cholinergic agonists in perfused guinea-pig adrenal glands. 2 Acetylcholine (ACh) and electrical stimulation induced CA secretion, which was reversibly inhibited by cortisol. Adrenocorticotropic hormone (ACTH) increased the concentration of cortisol in the perfusion effluent and partly inhibited the secretory response to ACh. 3 Cortisol or aldosterone dose-dependently inhibited secretory responses to nicotine and muscarine. These inhibitory effects were not antagonized by mifepristone and spironolactone, respective cortisol and aldosterone receptor blockers. 4 Dexamethasone, cortisone, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, prednisolone and cholesterol inhibited nicotine-evoked CA secretion. The secretory response to muscarine was inhibited by these compounds except for dexamethasone and prednisolone. 5 Dexamethasone, cortisol and aldosterone had no effect on CA secretion induced by high KCl. 6 These results suggest that steroids affect nicotinic and muscarinic ACh receptor-mediated responses through distinct mechanisms, and that cortisol released from the adrenal cortex inhibits CA secretion from the adrenal medulla.     

Effects of genistein and herbimycin, tyrosine kinase inhibitors, on catecholamine release in bovine adrenal chromaffin cells

1 The effects of genistein and herbimycin, tyrosine kinase inhibitors, on catecholamine (CA) release were examined in bovine adrenal chromaffin cells. 2 In intact cells, genistein (10-100 microm) and herbimycin (3-30 microm) inhibited CA release induced by acetylcholine (ACh; 100 microm) or the nicotinic receptor stimulant 1,1-dimethyl-4-phenyl-piperazinium (DMPP; 10 microm), but did not affect CA release induced by high K+ (40 mm). 3 Genistein and herbimycin inhibited (45)Ca2+ uptake induced by ACh (100 microm). 4 Neither genistein nor herbimycin affected [(3)H]nicotine binding with nicotinic receptors. 5 In beta-escin-permeabilized cells, neither genistein nor herbimycin affected CA release induced by Ca2+ (1 microm). 6 These results suggest that protein tyrosine kinase plays the facilitatory role in the regulation of CA release induced by nicotinic receptor stimulation in stimulus-secretion coupling of bovine adrenal chromaffin cells.     

Effects of opioid compounds on basal and muscarinic induced accumulation of inositol phosphates in cultured bovine chromaffin cells

The mammalian adrenal medulla expresses a variety of both opioid peptides and opioid receptors. The function of this adrenal opioid system is, however, largely unknown. We have examined the ability of a number of opioid compounds to influence basal and muscarinic stimulated accumulation of inositol phosphates in cultured bovine chromaffin cells. Muscarine produced a dose-dependent 1.5-fold increase in total inositol phosphates. This response was sensitive to atropine inhibition. The ten opioid compounds examined were chosen because between them they possess selectivity for all of the identified opioid receptor subtypes. However, none of these opioids in the concentration range 10nM-10 microM had any significant effect on either basal or muscarinic induced total inositol phosphate accumulation. We conclude that it is unlikely that opioid peptides released from either the chromaffin cells themselves or the splanchnic nerve can modulate the inositol phosphate second messenger system within the adrenal chromaffin cells.     

Effect of muscarine on release of catecholamines from the perfused adrenal gland of the cat.

1 The secretory effect of muscarine was studied in the perfused adrenal gland of the cat. During perfusion of the adrenal gland with Krebs-bicarbonate solution containing muscarine 480 microM, the rate of catecholamine (CA) secretion was 2.02 +/- 0.43 micrograms/2 min in the first 2 min; thereafter, CA output declined only moderately, to reach about 70% of the initial value after 10 min. Secretory responses to brief infusions of muscarine remained reproducible for at least the first 3 infusions. 2 When the adrenal gland was perfused with muscarine (480 microM), infusions of high K+, nicotine, or veratridine produced their usual responses. A 100 fold lower dose of muscarine also failed to modify these responses. 3 During perfusion with high K+, muscarine evoked a secretory response that was only slightly smaller than the response to muscarine alone. 4 It is concluded that muscarine and nicotine activate CA secretion in the cat adrenal gland by independent mechanisms and that the muscarinic response, unlike the nicotinic response, is not readily desensitized.     

Effects of collagenase on the release of [3H]-noradrenaline from bovine cultured adrenal chromaffin cells.

Bovine isolated adrenal chromaffin cells maintained in culture at 37 degrees C for 1-7 days become polygonal and bipolar, with typical varicosity-like extensions. Catecholamine levels and dopamine beta-hydroxylase activity decreased after 24-48 h of culture, but recovered to normal levels 3-7 days later. Incubation of 1-7 day-old cells in the presence of increasing concentrations of [3H]-noradrenaline (3.91 to 125 nM) resulted in the retention by the cells of amounts of radioactivity directly proportional to the amine present in the media. One day-old cells took up and retained only one third of the radioactivity found in 2-7 day-old cells. The addition of collagenase to cultured cells caused a decrease in the uptake of tritium. However, the enzyme treatment did not affect the amine taken up by the cell before collagenase treatment. Release of tritium from cultured cells evoked by nicotine, acetylcholine (ACh) or 59 mM K+ was very poor in 24 h-old cells; the secretory response to nicotine, ACh or K+ was dramatically increased after 2-7 days of culture. Bethanecol did not cause any secretory response. When treated with collagenase, cultured cells which had recovered fully their secretory response, lost again the ability to release tritium evoked by ACh or nicotine. However, the responses to high K+, veratridine or ionophore X537A were not affected. The nicotinic response was recovered two days after collagenase treatment. The data suggest that the use of collagenase to disperse the adrenomedullary tissue during the isolation procedure might be responsible for the lost secretory response of young cultured chromaffin cells. Since collagenase specifically impairs the nicotinic cholinoceptor-mediated catecholamine release, it seems likely that the enzyme is exerting its action on the ACh receptor complex. It is unlikely that either voltage-sensitive Na+ or Ca2+ channels are affected by collagenase as the responses induced by high K+ or veratridine were unaffected by this enzyme.     

Antagonism of nicotinic receptors of rat chromaffin cells by N,N, N-trimethyl-1-(4-trans-stilbenoxy)-2-propylammonium iodide: a patch clamp and ligand binding study

The effect of the oxystilbene derivative F3 was tested on nAChRs of whole-cell patch-clamped rat chromaffin cells in vitro and of rat adrenal gland membranes using (125)I-epibatidine. F3 (30 nM) rapidly and reversibly blocked inward currents generated by pulse applications of nicotine, shifting the dose-response curve to the right in a parallel fashion without changing the maximum response. The action of F3 was voltage insensitive and not due to altered current reversal potential. The R isomer of F3 was more potent (IC(50) = 350+/-30 nM) than its S-enantiomer (IC(50) = 1.5+/-0.3 microM). Nicotine-evoked currents were insensitive to 10 microM alpha-bungarotoxin. Equi-amplitude currents evoked by nicotine or epibatidine were similarly antagonized by R-F3 in a reversible fashion. Epibatidine-evoked currents readily produced receptor desensitization. Adrenal membranes specifically bound (125)I-epibatidine with a single population of binding sites endowed with high affinity (K(D) = 159 pM) and B(max) of 6.5+/-1.3 fmol mg(-1) of protein. (125)I-epibatidine binding was specifically displaced by cytisine (K(i) = 68 nM) or ACh (K(i) = 348 nM). F3 specifically displaced (125)I-epibatidine binding although with lower affinity (K(i) = 29.6 microM) than in electrophysiological experiments. (125)I-epibatidine binding to rat adrenal tissue was insensitive to alpha-bungarotoxin which readily antagonized (125)I-epibatidine binding to bovine adrenal tissue. The present results suggest that F3 is a relatively potent and apparently competitive antagonist of nAChRs on rat chromaffin cells. Since previous studies have indicated that F3 targets different subtypes on chick neuronal tissue, it appears that nAChRs display interspecies differences to be considered for drug development studies.     

Inhibition of the nicotinic acetylcholine response by serotonergic and muscarinic agents in chick ciliary ganglion neurones.

Chick ciliary ganglion neurones were investigated by whole cell voltage clamp recordings. The ACh- or nicotine-induced inward current was partially inhibited by perfusing the neurones with 5-HT. This effect was rapid (< or = 1 min), dose-dependent (50-1000 microM) and quickly reversible. The selective 5-HT1A agonist 8-OH-DPAT (10 microM) reduced the nicotinic ACh response more potently, irrespective of the absence or presence of propranolol (1 microM), a known 5-HT1A antagonist. Other serotonergic antagonists, like ICS 205-930 (1 microM), mianserin (10 microM) and methysergide (10 microM), also failed to antagonize the 5-HT-mediated decrease in the nicotinic response. Muscarine (50 microM) did not affect the nicotine-induced inward current but the muscarinic agonist oxotremorine (10 microM) also decreased the nicotine-induced inward current. Atropine, at small concentrations failed to block this effect but caused some reduction of the ACh response itself at larger (1-10 microM) concentrations. It is suggested that 5-HT may modulate synaptic transmission in ciliary ganglion neurones in vivo. The site of action of 5-HT, oxotremorine and atropine might be at or close to the ACh receptor complex, because of the fast onset and reversibility of the effects and lack of specificity for structurally different drugs.     

In vitro inhibition of adrenal catecholamine secretion by steroidal metabolites of ginseng saponins

We reported previously that the protopanaxatriol saponins in Panax ginseng greatly reduce the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh). However, protopanaxadiol saponins showed only slight inhibitory effects. Recent studies have demonstrated that oligosaccharides connected to the hydroxyl groups of the aglycone in ginseng saponins (ginsenosides) are in turn hydrolyzed in the digestive tract and absorbed into the circulation following oral administration of ginseng. Therefore, the present study was performed to investigate the effects of the major ginsenoside metabolites (M1, M2, M3, M4, M5, M11, and M12) on catecholamine secretion. All of these metabolites were shown to be potent inhibitors of ACh-evoked secretion, and M4 was the most effective. M4 blocked not only the ACh-induced Na(+) influx into the chromaffin cells but also the ACh-induced inward current into Xenopus oocytes expressing human alpha 3 beta 4 neuronal nicotinic ACh receptors. M4 reduced the secretion induced by high K(+), an activator of voltage-sensitive Ca(2+) channels, to a much lesser extent than that evoked by ACh. M1, M2, M3, M5, and M12 are protopanaxadiol saponin-derived metabolites. Therefore, these results imply that the protopanaxadiol saponins are prodrugs, and they show more potent inhibitory activity following metabolism in the digestive tract. The results further suggest that the metabolites act on nicotinic ACh receptors, blocking Na(+) influx through the receptors, and consequently reduce the catecholamine secretion from bovine adrenal chromaffin cells. The inhibitory effect of ginsenoside metabolites is probably one of the mechanisms of action responsible for the pharmacological effects of ginseng.     

Heterogeneity of neuronal nicotinic acetylcholine receptors in 5-HT-containing chemoreceptor cells of the chicken aorta

The effects of nicotinic agonists and antagonists on whole-cell currents and 5-hydroxytryptamine (5-HT) release were studied in order to characterize nicotinic ACh receptors on the 5-HT-containing chemoreceptor cells of the chicken aorta. ACh, nicotine and dimethylphenylpiperazinium (DMPP) evoked concentration-dependent inward currents accompanied by increases in current noise at a holding potential of -70 mV. The peak amplitude of the current response to DMPP was 50% larger than that to either nicotine or ACH: Hexamethonium, alpha - bungarotoxin (alpha - BTX) and methyllycaconitine decreased nicotine-induced inward currents in a concentration-dependent manner. Although hexamethonium (0.1 mM) abolished the current response to nicotine (30 microM), a high concentration (1 microM) of alpha - BTX decreased it only by about 30% of the control response. Methyllycaconitine (0.1 microM) decreased the current response to nicotine to the same extent as did alpha - BTX whilst a high concentration (10 microM) abolished the response. ACh, nicotine and DMPP caused concentration-dependent increases in 5-HT output from the thoracic aorta which effect was blocked by hexamethonium (0.1 mM). Pre-treatment with alpha - BTX (1 microM) for 30 min reduced the output of 5-HT induced by ACh to 70% of the control response. It is suggested that neuronal nicotinic ACh receptors, sensitive and insensitive to alpha - BTX, are present on the chemoreceptor cells of the chicken aorta, the activation of which causes the release of 5-HT.     

Muscarinic M(4) receptors regulate GABAergic transmission in rat tuberomammillary nucleus neurons

Histaminergic neurons within the tuberomammillary nucleus (TMN) play an important role in sleep-wakefulness regulation. Here, we report the muscarinic modulation of GABAergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in mechanically dissociated rat histaminergic neurons using a conventional whole-cell patch clamp technique. Muscarine, a nonselective muscarinic acetylcholine (mACh) receptor agonist, reversibly decreased mIPSC frequency without affecting the current amplitude, indicating that muscarine acts presynaptically to decrease the probability of spontaneous GABA release. The muscarine action on GABAergic mIPSC frequency was completely blocked by atropine, a nonselective mACh receptor antagonist, and tropicamide, an M(4) receptor antagonist. The muscarine-induced decrease in mIPSC frequency was completely occluded in the presence of Cd(2+), a general voltage-dependent Ca(2+) channel blocker, or in a Ca(2+)-free external solution. However, pharmacological agents affecting adenylyl cyclase or G-protein coupled inwardly rectifying K(+) channel activity did not prevent the inhibitory action of muscarine on GABAergic mIPSCs. These results suggest that muscarine acts on M(4) receptors on GABAergic nerve terminals projecting to histaminergic neurons to inhibit spontaneous GABA release via the inhibition of Ca(2+) influx from the extracellular space. Muscarine also inhibited action potential-dependent GABA release by activating presynaptic M(4) receptors in more physiological conditions. The M(4) receptor-mediated modulation of GABAergic transmission onto TMN neurons may contribute to the regulation of sleep-wakefulness.     

Characterization of muscarinic receptors of bovine adrenal chromaffin cells: binding, secretion and anti-microtubule drug effects.

1. Binding of [3H]QNB to adrenal membranes is saturable, specific and to a single class of receptors. 2. Tubulozole, and not other microtubule drugs, inhibits [3H]QNB binding. 3. Pretreating cultured chromaffin cells with oxotremorine, a muscarinic receptor agonist, has no effect on either basal, nicotine (10 microM) or K(+)-stimulated catecholamine release and failed to enhance secretion of submaximal concentrations of nicotine (3-5 microM). 4. These results confirm that binding of [3H]QNB is associated with muscarinic receptors on bovine adrenal medullary tissue. 5. These studies also demonstrate that although bovine adrenal chromaffin cells possess muscarinic receptors, these receptors do not appear to be coupled to secretory processes.