Dimethylphenylpiperazinium Research Articles

Maturation of porcine tracheal cholinergic and inhibitory non-adrenergic, non-cholinergic innervation.

It is known that both excitatory cholinergic neural activity and nitric oxide (NO) release from inhibitory non-adrenergic, non-cholinergic (iNANC) nerves are important determinants of adult human airway smooth muscle tone. However, the level of cholinergic and iNANC activity in the newborn is unknown. Therefore, isolated trachealis muscle strips were obtained from neonatal, 10-day-old, and adult pigs. To quantify cholinergic nerve activity, isometric contractions were elicited by stimulating the nicotinic receptors of the cholinergic intramural ganglia with dimethyl-phenyl-piperazinium (DMPP). After ganglionic blockade with hexamethonium, the post-ganglionic cholinergic nerves were subjected to electrical field stimulation (EFS). To assess whether DMPP and EFS were also stimulating nitric oxide release from iNANC nerves, the stimulations were repeated after NO-synthase inhibition with 3 x 10(-5) M NG-monomethyl-L-arginine acetate (LNMMA). All responses were expressed as a percentage of maximal response to 10(-4) M acetylcholine. Although no significant age-related differences in the contractile responses to DMPP were demonstrated between the three age groups, the EFS responses were significantly different. At EFS frequencies of 1 and 2 Hz the responses of all three groups were different (P < 0.05, unpaired t-test). At frequencies of 0.125 and 0.5 Hz the contractile responses in the neonatal and 10-day-old groups were less than in the adult animal tissues (P < 0.05, unpaired t-test). At 4 Hz and above, the responses of the 10-day-old and adult groups became greater than the neonatal (P < 0.05, unpaired t-test). LNMMA did not significantly increase the contractile responses to DMPP at any age. Only in the neonatal rat and 10-day-old groups was a significant increase of the EFS frequency responses by LNMMA demonstrated (140.4 +/- 38.2% and 144.2 +/- 41.2%, respectively, at 0.5 Hz, ANOVA, P < 0.001). In conclusion, the DMPP responses suggest that the neonatal porcine cholinergic innervation is mature at birth. This contrasts with a frequency-dependent increase in EFS responses from birth. This may be due in part to a relative increase in iNANC modulation of post-ganglionic cholinergic activity at birth. This mechanism is not responsible for the increase in higher EFS frequency responses with post-natal maturation. Inhibitory iNANC activity may serve a protective role in the newborn, given the reported hyper-responsiveness of airway smooth muscle, small airway caliber, and high chest wall compliance at this age.

Differential effects of omega-conotoxin GVIA on cholinergic and non-cholinergic secretomotor neurones in the guinea-pig small intestine.

1. Ussing chambers were used to study the effects of the specific N-type Ca2+ channel antagonist, omega-conotoxin GVIA, on neurally evoked secretion across isolated submucosa/mucosa preparations from the small intestine of the guinea-pig. 2. Cholinergic and non-cholinergic neurones were stimulated with 10 microM dimethylphenylpiperazinium (DMPP). Non-cholinergic secretomotor neurones were preferentially stimulated with 100 nM 5-hydroxytryptamine (5-HT), while cholinergic secretomotor neurones were preferentially stimulated with 3 microM 5-HT in the presence of the 5-HT2 receptor antagonist ketanserin (30 nM). 3. omega-Conotoxin GVIA (1 nM-1 microM) depressed the secretion evoked by DMPP in a concentration-dependent manner, but a substantial residual response was observed. Hyoscine (100 nM) significantly depressed secretion evoked by DMPP, but did not prevent further depression of secretion by omega-conotoxin GVIA. 4. The toxin was substantially more effective when the non-cholinergic secretomotor neurones were preferentially activated with 100 nM 5-HT, with a decrease in the response of more than 75% of the control value in the presence of 1 microM omega-conotoxin GVIA. 5. omega-Conotoxin GVIA (1 microM) was relatively ineffective against secretion evoked by preferential activation of cholinergic secretomotor nuerones with 3 microM 5-HT in the presence of 30 nM ketanserin, inhibiting the response by less than 33%. However, this inhibition was significant. Both 100 nM hyoscine and 300 nM tetrodotoxin abolished this effect of omega-conotoxin GVIA. 6. It is concluded that N-type Ca2+ channels play a major role in transmitter release from non-cholinergic secretomotor neurones, but are not important for release from cholinergic secretomotor neurones in the guinea-pig small intestine.

Role of central nicotinic and beta-adrenergic receptors in the onset and further development of tail-tremor induced by repeated nicotine administration to rats.

The effects of nicotinic and beta-adrenergic receptor antagonists on tail-tremor induced by repeated nicotine administration were investigated in rats. The daily administration of nicotine (0.5 mg/kg/day, s.c.) for 8 days resulted in an augmentation of tail-tremor. However, repeated administration of dimethyl phenyl piperazinium iodide (1 mg/kg/day, s.c.) for 8 days did not cause tail-tremor. Mecamylamine (0.5 mg/kg, i.p), administered before the nicotine injection on each day, abolished the tail-tremor. After discontinuation of the mecamylamine treatment, nicotine injections caused tail-tremor augmentation. Propranolol (20 mg/kg, i.p.), administered before the nicotine on each day, suppressed the appearance of tail-tremor. After the discontinuation of propranolol treatment, the degree of tail-tremor induced by a single injection of nicotine on day 9 was much greater in the propranolol-treated group than in the saline-treated control group. Neither carteolol (20 mg/kg, i.p.) nor metoprolol (20 mg/kg, i.p.) treatment showed such effects. Intraspinal injection of 6-hydroxydopamine markedly enhanced the tail-tremor induced on the first day of nicotine injection. This effect became more intense on subsequent administration of nicotine. The enhanced tail-tremor following 6-hydroxydopamine treatment was abolished by mecamylamine (0.5 and 1 mg/kg, i.p.), and was suppressed by propranolol (5-20 mg/kg, s.c.) in a dose-dependent manner. These results suggest that central nicotinic receptors are essential for the onset and for the further development of tail-tremor induced by the repeated administration of nicotine, and that beta 2-adrenoceptors are associated with the tremor mechanism. Moreover, spinal noradrenergic mechanisms may be involved in the manifestation of this phenomenon.

Potency of agonists and competitive antagonists on adult- and fetal-type nicotinic acetylcholine receptors.

1. The potency of agonists and competitive antagonists on the two expressed forms of the nicotinic acetylcholine receptor (adult or junctional subtype, epsilon-AChR; fetal or extrajunctional subtype, gamma-AChR) have not previously been compared systematically in homogeneous receptor preparations. 2. Each subtype of the receptor was expressed separately in Xenopus oocytes by cytoplasmic injection of combinations of RNA transcribed in vitro. The presence of each type of receptor was confirmed by single-channel recordings. Expressing oocytes were assayed using discontinuous, single-electrode voltage clamp by measuring peak currents in response to test compounds. 3. The extrajunctional subtype was more potently activated by the nicotinic agonist dimethylphenyl piperazinium iodide (DMPP) than was the junctional form. There was no statistically significant difference in potency between the two subtypes for other nicotinic agonists (nicotine, cytisine and succinylcholine). The rank order of potency for epsilon-AChR was succinylcholine > cytisine > DMPP > nicotine, and that for gamma-AChR was DMPP > cytisine > succinylcholine > nicotine. 4. Two agonists (cytisine and succinylcholine) displayed six- to eight-fold greater intrinsic activity in activating epsilon-AChR over gamma-AChR. There was no difference between the two forms of receptor in efficacy for nicotine. 5. The extrajunctional form was much more potently inhibited by the steroidal competitive antagonist pancuronium than was the junctional receptor. However, there was no significant difference in potency of inhibition by the curariform drug atracurium. 6. Contrary to previous reports, there is no consistent relation between the effect of agonists and antagonists and the subtype of receptor. These data suggest that the resistance or sensitivity to these agents seen in various clinical settings are related to other cellular factors.

Inhibition of neuronal nitric oxide synthase potentiates the dimethylphenylpiperazinium-evoked carrier-mediated release of noradrenaline from rat hippocampal slices

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 μM) to increase the basal release of [3H]NA was significantly potentiated by 7-NI (40 μM). In our previous study we showed that the response to DMPP has two components, a nicotinic receptor-mediated, [Ca 2+]-dependent exocytosis followed by a [Ca 2+]-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 μM) significantly increased the basal release of [ 3H]NA but this release was not affected, whereas in Ca2+-free medium the response to DMPP (20 μM) was still potentiated by 7-NI (40 μM). In the presence of the NA uptake blocker desipramine (10 AM) DMPP (20 μM) was unable to provoke NA release independently from the presence or absence of 7-NI (40 μM). 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.

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.

The reversal of experimental hemorrhagic shock induced by nicotine and dimethylphenylpiperazinium is adrenal-dependent

In a rat model of volume-controlled hemorrhagic shock causing the death of all control animals within 30 min, the intravenous injection of either nicotine (50 μg/kg) or dimethylphenylpiperazinium (DMPP) (0.5 μg/kg) produced a rapid and sustained reversal of the shock condition, with 100% survival 2 h after treatment. Bilateral adrenalectomy completely prevented the anti-shock effect of the two drugs, even though administered at higher doses (150 μg/kg in the case of nicotine; 10 μg/kg in the case of DMPP). It is concluded that stimulation of adrenaline release plays a fundamental role in the mechanism of action of nicotine- and DMPP-induced shock reversal.

Otilonium: a potent blocker of neuronal nicotinic ACh receptors in bovine chromaffin cells.

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.

Prostaglandin E2 modulates neurally induced nonadrenergic, noncholinergic gastric relaxations in the rabbit in vivo

BACKGROUND & AIMS: Prostaglandin (PG) E2 has been shown to modulate adrenergic and cholinergic neurotransmission in the gut. This study investigated PGE2 influence on vagally induced, nonadrenergic, noncholinergic (NANC) gastric relaxations. METHODS: Mechanical activity of the stomach was recorded in anesthetized rabbits. RESULTS: In atropine-treated animals, electrical vagal stimulation or arterial bolus injection of the ganglion stimulant dimethyl phenylpiperazinium iodide (DMPP) evoked inhibitory responses that varied from a brisk relaxation, interrupted by a poststimulus excitatory motility (biphasic response), to a long-lasting relaxation (monophasic response). PGE2 reduced and, at the highest doses, abolished the neurally induced relaxant responses elicited either by vagal stimulation or DMPP administration but did not affect the gastric relaxation caused by adenosine triphosphate (ATP), sodium nitroprusside (SNP), or vasoactive intestinal polypeptide (VIP). ATP or 2-methylthioadenosine triphosphate (2-MeSATP) reduced and then suppressed vagally induced inhibitory motility; the relaxant responses elicited by SNP, VIP, and ATP itself were not influenced. After administration of the prostaglandin synthesis inhibitor suprofen, ATP and 2-MeSATP failed to block vagally induced inhibitory responses. Arterial infusion of adenosine at the highest rates did not influence the amplitude of the vagally induced relaxant responses. Following theophylline administration, ATP still blocked the relaxation elicited by vagal stimulation. CONCLUSIONS: PGE2 may modulate NANC inhibitory neurotransmission in the stomach. The effects of ATP on the neurally induced NANC gastric relaxation may be caused by PGE2. (Gastroenterology 1996 Jan;110(1):129-38)

Ionotropic Mechanisms Involved in Postsynaptic Inhibition by the Endothelins of Ganglionic Transmission in Dog Cardiac Sympathetic Ganglia

We investigated the effects of endothelin-1 and endothelin-3 (ET-1, ET-3) on the ganglionic transmission of cardiac sympathetic ganglia in vivo by the direct administration of agents to the ganglia through the right subclavian artery while monitoring the heart rate (HR) as an indicator of the ganglionic function in spinal dogs. The positive chronotropic responses to dimethylphenylpiperazinium (DMPP) and McN-A-343 administered to the ganglia were similarly inhibited by ET-1 (0.05-0.2 microg) and ET-3 (0.5-2 microg), but ET-1 was approximately 10 times more potent than ET-3. The inhibition induced by ETs was antagonized by endothelin ETA receptor antagonist BQ-123 (20 microg). This inhibition was unaffected by pretreatment with indomethacin given intravenously (i.v.), ruling out the possible involvement of endogenous prostaglandins production. The voltage-sensitive Ca2+ channel antagonist nifedipine had no effect on inhibition. However, the inhibition was antagonized by pretreatment with the low conductance Ca2+-activated potassium channel antagonists, such as apamin (20 microg intraarterially, i.a.), scyllatoxin (10 mug i.a.) and D-tubocurarine (0.6 mg i.a.). On the other hand, the voltage-sensitive K+ channel antagonist 4-aminopyridine (4-AP), ATP-dependent K+ channel antagonist, glibenclamide, and high-conductance Ca2+-activated K+ channel antagonists iberiotoxin and charybdotoxin failed to affect the inhibition by ETs. The results suggest that ETs inhibit the nicotinic and muscarinic ganglionic transmission through the ETA receptor-operated low-conductance Ca2+-activated potassium channel at postganglionic sites.

Agonist and toxin sensitivities of ACh-evoked currents on neurons expressing multiple nicotinic ACh receptor subunits.

1. We have investigated the pharmacological properties of functional nicotinic acetylcholine receptors (nAChRs) on neonatal rat sympathetic neurons from the superior cervical ganglion (SCG) to learn more about the subunit composition of these receptors. These neurons express five nAChR transcripts: alpha 3, alpha 5, alpha 7, beta 2, and beta 4; this finding suggests that SCG neurons may express several different, physiologically distinct, subtypes of nAChRs. 2. To identify potential subtypes, we have characterized currents evoked by different nicotinic agonists and determined their sensitivity to blockade by alpha-bungarotoxin (alpha-BTX) and by neuronal bungarotoxin (n-BTX). From dose-response curves, we find that the ED50 for both cytisine and dimethylphenylpiperazinium (DMPP) is 20 microM and for ACh is 52 microM. n-BTX blocks the ACh-gated currents rapidly, but the kinetics for n-BTX removal is dependent on the duration of the application: brief applications were quickly reversible, whereas prolonged applications took orders of magnitude longer to reverse. 3. Using fast (ms) agonist application, we observed no rapidly desensitizing currents despite the high levels of alpha 7 in these neurons, nor did we observe any currents that could be blocked by alpha-BTX. 4. Using Xenopus oocytes expressing alpha 7 receptors, we show that choline evokes a significant current that is blocked by alpha-BTX. In contrast, choline is much less potent on alpha 3 beta 4 receptors expressed in Xenopus oocytes. Choline can also act as a weak agonist for nAChRs on rat SCG neurons, but its evoked current is not blocked by alpha-BTX. 5. Our results indicate that, when measured at the macroscopic level, most functional nAChRs on SCG neurons behave as a uniform population of receptors, at least with respect to agonist activation and toxin blockade. In comparison with known receptors expressed in heterologous systems, the physiological properties of ACh-evoked currents on SCG neurons are most similar to receptors that have coassembled with both beta 2 and beta 4.

Comparison of the pharmacodynamics of ketamine in the isolated neonatal and adult porcine airway

Ketamine inhibits contractions of adult airway smooth muscle by actions both within the smooth muscle cell and the vagal intramural ganglia. Its effects in the neonate are unknown, as physiological changes occur after birth in both of these structures. Isolated trachealis muscle strips from neonatal and adult pigs were contracted selectively by stimulation of the vagal intramural ganglia using dimethyl-phenyl-piperazinium (DMPP), the post-ganglionic vagus nerve using electrical field stimulation (EFS) and the smooth muscle cell itself using acetylcholine (ACh) in the presence of tetrodotoxin. The inhibitory effects of clinical concentrations of ketamine at each site were compared both within and between age groups. At both ages, ketamine 10(-4) mol litre-1 abolished (P < or = 0.001) and ketamine 10(-5) mol litre-1 attenuated (P < or = 0.001) the contractile responses to DMPP at the intramural ganglia. The inhibitory effect of ketamine 10(-4) mol litre-1 on ACh responses at the smooth muscle cell was greater in neonatal preparations (P = 0.025). Furthermore, only neonatal EFS and ACh responses were attenuated by ketamine 10(-5) mol litre-1 (P = 0.04 and P = 0.03). Ketamine 10(-4) mol litre-1 attenuated EFS responses in both age groups (P < or = 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

The transient nicotinic stimulation of tyrosine hydroxylase gene transcription in bovine adrenal chromaffin cells is independent of c-fos gene activation.

The nicotinic agonist dimethylphenylpiperazinium (DMPP) transiently stimulates tyrosine hydroxylase (TH) gene transcription in cultured bovine adrenal chromaffin cells (Craviso et al., J. Neurochem., 59 (1992) 2285-2296). The present studies examined the mechanism of this stimulation, exploring the hypothesis that c-fos- and/or cyclic AMP-related mechanisms are involved. As determined by nuclear run-on assay, exposure of chromaffin cells to DMPP (1 microM) induced c-fos and TH gene transcription fivefold and twofold, respectively. Nitrendipine (20 microM) blocked both responses, indicating a similar dependency of each on extracellular calcium. Both c-fos and TH gene transcription rates were also elevated by entry of calcium due to the presence of the calcium ionophore A23187 (5 microM). Comparison of the time dependence of the DMPP stimulation of c-fos and TH gene transcription revealed similar time courses. Both were rapid and transient, peaking within 10-30 min of nicotinic receptor occupancy and returning to control values by 1 h. This simultaneous activation of the TH and c-fos genes indicates that Fos induction cannot be responsible for the stimulation of TH gene transcription. This conclusion was further supported by a failure of anisomycin (100 microM) pretreatment of chromaffin cells, which blocked protein synthesis 99%, to have any effect on either the rapid stimulation of TH gene transcription or the length of time that the TH gene was activated by DMPP. Thus, neither Fos nor other high turnover-rate transcription factors appear to be responsible for the stimulation, or return to control level, of TH gene activity following nicotinic stimulation of chromaffin cells. In other experiments, treating chromaffin cells with a combination of maximally effective concentrations of DMPP and forskolin was found to produce no greater stimulation of TH gene transcription than either agent alone, suggesting that DMPP acts through the same mechanism as forskolin. Taken together, these results support the conclusion that the mechanism of TH gene activation in chromaffin cells by DMPP involves a cyclic AMP-dependent process and not the induction of transcription factors such as Fos.

Evidence of the existence of cholinergic muscarinic receptors in human placenta

The action of some nicotinic acetylcholine receptor agonists was re-examined on the surface field potentials (N-waves) evoked by electrical stimulation of the lateral olfactory tract in guinea-pig olfactory cortical brain slices. Bath superfusion of nicotine or the nicotinic stimulants dimethylphenylpiperazinium (DMPP), lobeline, cytisine, tetramethylammonium or suberyldicholine (up to 100 μM) had little or no effect on the extracellular N-wave amplitude, or the membrane potential, input resistance or excitability of olfactory neurones recorded intracellularly. In contrast, the muscarinic agonists, carbachol or oxotremorine-M consistently depressed the field in a reversible dose-dependent manner. Interestingly, in the presence of the ganglionic stimulants DMPP (n = 6 slices) or lobeline (n = 5 slices) (10–50 μM), the effects of carbachol or oxotremorine-M were antagonized in a week competitive-type manner (pA2 values = 5.58 and 5.63 respectively, estimated from Schild plots, constrained to unity slope). This anti-muscarinic action was unaffected by d-tubocurarine or hexamethonium. Nicotine, cytisine, tetramethylammonium and suberyldicholine showed much weaker and inconsistent carbachol-blocking effects. Combination of DMPP with atropine produced dose ratio shifts close to those predicted for a common-site interaction of two competitive antagonists. In conclusion, consistent pre- or postsynaptic nicotinic agonist actions could not be detected in olfactory cortex slices; however, some ganglionic nicotinic agonists were shown to exhibit significant anti-muscatinic effects on this preparation. We suggest this action might be due to a direct atropine-like mechanism.

Neurally maintained hypersecretion in undernourished rat intestine activated by E. coli STa enterotoxin and cyclic nucleotides in vitro.

1. The electrogenic secretory responses of stripped jejuna and ilea from chronically undernourished rats (50% control diet for 21 days) to the bacterial enterotoxin Escherichia coli STa, measured as the short-circuit current in vitro, show an enhanced maximum secretion (ISC, max) with a prolonged duration compared with fed intestine. 2. The ISC, max is unaffected by pretreatment of the intestine in vitro with hexamethonium, atropine, procaine or indomethacin, or by desensitization to 5-hydroxytryptamine (5-HT), while the prolonged duration is unaffected by atropine, indomethacin or 5-HT desensitization but is reduced by hexamethonium and procaine. 3. Both 8-bromo-cyclic GMP and dibutyryl cyclic AMP added serosally activate the enhanced ISC, max and its maintenance. Pretreatment with tetrodotoxin had no effect on the initial ISC, max but prevented its maintenance. 4. Bethanechol, dimethyl phenyl piperazinium, vasoactive intestinal polypeptide, 5-HT and luminal propionate all induced the characteristic hypersecretory activity in the undernourished intestine compared with the fed state, but none could activate the maintenance circuit to prolong their transient responses. 5. Maintenance of the induced hypersecretory activity is the first example of induction of the neural control of intestinal secretion by the dietary intake level and illustrates the plasticity of the enteric nervous system.

Effect of L‐glutamic acid on acid secretion and immunohistochemical localization of glutamatergic neurons in the rat stomach

Glutamatergic neurons in the rat stomach were localized immunohistochemically using antibodies against L-glutamate (L-Glu) as well as glutamate synthesizing enzyme, glutaminase (GLNase). Myenteric ganglia and nerve bundles in the circular muscle and the longitudinal muscle were found to contain GLU- and GLNase-positive nerve fibers, while submucosa and mucosa were devoid of glutamatergic innervation. The distribution of glutamatergic neurons and their processes in both myenteric ganglia and circular muscle is heterogeneous within the stomach. The effect of L-Glu on gastric acid secretion was investigated on an everted preparation of isolated rat stomach. L-Glu at 10(-7) and 10(-8) M alone had no effect on acid secretion. It was found that the oxotremorine-, histamine-, or gastrin-stimulated acid secretion was markedly reduced by L-Glu at 10(-8) M, whereas L-Glu had little effect on the acid secretion stimulated by dimethyl-phenylpiperazinium (DMPP) at this concentration. However, at higher concentration, e.g., 10(-7) M, L-Glu also markedly reduced DMPP-induced acid secretion. Among L-Glu receptor agonists tested, quisqualic acid (QA) is most potent, followed by kainic acid (KA) and N-methyl-D-aspartic acid (NMDA) in inhibiting oxotremorine-stimulated acid secretion. Furthermore, this inhibitory effect of L-Glu on oxotremorine-stimulated acid secretion is blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a specific non-NMDA receptor antagonist. All these results suggest that glutamatergic neurons are involved in the modulation of gastric acid secretion via ionotropic QA/KA receptors, probably through openings of Ca2+ channels.

The cholinergic pharmacology of the frog saccule

Stimulation of the efferent nerves to the vestibular organs of the frog's inner ear produces either facilitation or inhibition of afferent firing. Similarly, application of acetylcholine (ACH), the major transmitter of the cfferents, can produce both facilitation and/or inhibition as previously reported [Guth et al. (1986) Acta Otolaryngol. 102, 194–204; Norris et al. (1988) Hear. Res. 32. 197–206]. The firing rates of afferent neurons of the semicircular canal (SCC using multiunit recordings are generally facilitated by ACH. Conversely, the firing rates of afferent units innervating the saccule are generally inhibited by ACH. This latter inhibition is antagonized by strychnine more potently than by curare, which is more potent than atropine. When inhibition is antagonized by strychnine or curare an underlying facilitation is revealed. The inhibition of saccular afferents by ACH shows desensitization requiring about 20 min to recover. The ACH-induced inhibition is mimicked by nicotine at very high concentrations but not by dimethyl phenylpiperazinium or cytisine. The fact that multiunit afferent firing from the SCC is generally facilitated while that from the saccule is generally inhibited by ACH suggests a different distribution of ACH receptors and receptor types (i.e. muscarinic or nicotinic and their subtypes) in the two organs and demonstrates the usefulness of recording from multiple units simultaneously. The difference in distribution of ACH receptors may be important for understanding the physiology of vestibular efferents.

Clinical concentrations of edrophonium enhance desensitization of the nicotinic acetylcholine receptor.

The principal acetylcholinesterase inhibitors used in clinical practice, edrophonium, neostigmine, and pyridostigmine, differ in their abilities to reverse profound neuromuscular block. This difference may reflect differential inhibition of the nicotinic acetylcholine receptor (nAChR) itself. To investigate this possibility, we studied the effects of these drugs on the function of nAChR (alpha 2 beta gamma delta subtype expressed in Xenopus laevis oocytes) using a whole-cell voltage clamp technique. All three drugs produced concentration-dependent inhibition of nAChR currents induced by the nicotinic agonist dimethylphenyl piperazinium iodide (DMPP). However, only with edrophonium did the effective inhibitory concentration overlap with the clinical range, producing 47% inhibition of nAChR current at the peak serum concentration (60 microM) obtained from a 1 mg/kg dose. The inhibition by edrophonium was voltage-dependent, being more potent at hyperpolarized membrane potentials [IC50(-60 mV) = 82.1 +/- 5.0 microM; IC50(-90 mV) = 50.8 +/- 2.7 microM; IC50(-120 mV) = 41.1 +/- 1.3 microM] and implying some degree of channel block within the ion-conducting pore. Edrophonium also enhanced desensitization of the nAChR within the clinically observed range. Edrophonium desensitization of the nAChR was further increased by simultaneous exposure to other drugs known to promote desensitization of the receptor. These two mechanisms, channel block and enhanced desensitization, may provide molecular explanations for the lesser capacity of edrophonium to promote complete reversal of profound neuromuscular block.

The nicotinic acetylcholine receptor of the bovine chromaffin cell, a new target for dihydropyridines

The effects of 1,4-dihydropyridine derivatives on divalent cation transients and catecholamine release stimulated by either high K + or the nicotinic receptor agonist dimethyl-phenyl-piperazinium (DMPP) have been compared in bovine adrenal chromaffin cells. The activation of Ca 2+ entry pathways was followed by measuring 45Ca 2+ or Mn 2+ uptake, or by the changes of [Ca 2+] i in fura-2-loaded chromaffin cells. Various dihydropyridine Ca 2+ channel blockers (nimodipine, PCA50938, nifedipine, nitrendipine, furnidipine) abolished the DMPP-mediated effects, but prevented only partially the activation by high [K +] 0 of 45Ca 2+ uptake. The IC 50 for DMPP-induced activation was around 1 μM. The L-type Ca 2+ channel activator Bay K 8644 potentiated the uptake of 45Ca + induced by K ++ depolarization at concentrations between 10 nM and 1 μM, but completely inhibited the uptake of 45Ca 2+ by DMPP (IC 50, 0.9 μM). Both high [K +] 0 and DMPP produced membrane depolarization as measured using bis-oxonol. The DMPP-evoked, but not the K +-evoked membrane depolarization was prevented by Na + removal, suggesting that the depolarization was due to Na + entry through the acetylcholine receptor ionophore. Nimodipine at 10 μM abolished the depolarization induced by DMPP, leaving the K +-evoked depolarization unaffected. Tetrodotoxin (2 μM) did not affect the DMPP- or high K +-mediated cell depolarization. Whole-cell inward current evoked by 100 μM DMPP (I DMPP) was measured in cells voltage-clamped at −80 mV. Nimodipine (10 μM) reduced I DMPP by 36%; Bay K 8644 (10 μM) inhibited I DMPP by 67%. DMPP-evoked catecholamine release from superfused chromaffin cells was reduced by over 90% with 10 μM nimodipine; in contrast, K +-evoked release was decreased by 20%. The results suggest that the nicotinic acetylcholine receptor of bovine chromaffin cells contains a site for dihydropyridines whose occupation blocks Na + entry through the receptor ionophore. This chromaffin cells contains a site for dihydropyridines whose occupation blocks Na + entry through the receptor ionophre. This limits the ensuing membrane depolarization, firing of action potentials, recruitment of Ca 2+ channels and entry of Ca 2+ in the cells, leading to a decrease in catecholamine secretion.

Identification of acetylcholine receptors in adult rat trigeminal ganglion neurons

Nicotinic acetylcholine receptors (nAChRs) were identified in a subpopulation of cultured adult rat trigeminal ganglia (TG) neurons by whole-cell patch-clamp recordings. Dimethylphenylpiperazinium (DMPP), a nAChR agonist, induced inward currents in 2168 of TG neurons having soma diameters greater than 28 μm. These currents were inhibited by hexamethonium, mecamylamine and atropine, indicating the presence of neuronal ganglionic-type nAChRs. This interpretation is consistent with the finding that the nicotine- or DMPP-induced currents were not inhibited by α-bungarotoxin (α-Bng) in 5 of the 9 cells tested with this compound. However, in 2 of the 9 cells tested, the DMPP-induced currents were completely inhibited by α-Bng, and in the remaining two cells tested, the currents were partially inhibited by α-Bng. About 22% of the cells having diameters ≧28μm were specifically labeled with FITC-labeled α-Bng, whereas only 2% of the cells with soma diameters <28μm were labeled. These data taken together suggest that more than one subtype of nAChR is present in TG.