Allosteric interaction of the anticholinergic drug [N-(4-phenyl)-phenacyl-l-hyoscyamine] (Phenthonium) with nicotinic receptors of post-ganglionic sympathetic neurons of the rat vas deferens.

Phenthonium (Phen), a quaternary analog of hyoscyamine, is a blocker of muscarinic activity and an allosteric blocker of alpha(1)2betagammaepsilon nicotinic receptors. Specifically, Phenthonium increases the spontaneous release of acetylcholine at the motor endplate without depolarizing the muscle or inhibiting cholinesterase activity. This paper compares Phenthonium's effects on sympathetic transmission and on ganglionic nicotinic receptor activation. Neurotransmitter release and twitch of the rat vas deferens were induced either by electrical stimulation or by 1,1-dimethyl-4-phenylpiperazine (DMPP) activation of nicotinic receptors. Contractions independent of transmitter release were induced by noradrenaline and adenosine 5'-triphosphate (ATP). Phenthonium inhibited transmitter release and depressed twitch without changing the responsiveness to noradrenaline or ATP. Twitch depression did not occur after K(+)-channel blockade with 4-aminopyridine (4-AP) or charybdotoxin. DMPP had a similar effect, but high concentrations induced contraction of non-stimulated organs. Incubation of Phenthonium inhibited further DMPP twitch depression and non-competitively depressed the contractile responses elicited by DMPP. Furthermore, mecamylamine, but neither methyllycaconitine nor atropine, blocked the contraction elicited by DMPP. Phenthonium and DMPP are K(+)-channel openers that primarily inhibit sympathetic transmission. Contraction induced by DMPP was probably mediated by neuronal nicotinic receptor other than the alpha7 subtype. The blockade of DMPP contractile response was unrelated to Phenthonium's antimuscarinic or K(+)-channel opening activities. Since Phenthonium's quaternary chemical structure limits its membrane diffusion, the non-competitive inhibition of DMPP excitatory responses should be linked to allosteric interaction with neuronal nicotinic receptors that putatively qualify Phenthonium as a novel modulator of cholinergic synapses.

Modulation of carbachol-induced antinociception from the rat periaqueductal gray.

The tail-flick latency (TFL) and the vocalisation test (VT) thresholds were all increased by microinjecting CCh into the dorsal periaqueductal gray (dPAG) of rats. The effects on the TFL were mimicked by dimethyl-phenylpiperazinium, and inhibited by local mecamylamine or intraperitoneal (i.p.) phenoxybenzamine. The effects on the VT were mimicked by bethanechol and inhibited by local mecamylamine, atropine or naloxone. The effects on the thresholds for motor defence reaction were inhibited by i.p. methysergide or naloxone, and prolonged by i.p. phenoxybenzamine. The effects on the threshold for vocalisation during the stimulation were blocked by i. p. methysergide and shortened by i.p. phenoxybenzamine or naloxone. No significant effect of CCh was found on open arm exploration of rats in the elevated plus maze paradigm. We conclude that the effects of CCh from the dPAG is not due to an anxiolytic effect, and depends on the activation of local cholinergic and opioid sites for the supraspinal modulation of "affective" component of pain response, and nicotinic sites for the activation of descending pain pathways.

Melatonin modulation of presynaptic nicotinic acetylcholine receptors in the rat vas deferens.

Melatonin, the pineal hormone produced during the dark phase of the light-dark cycle, modulates neuronal acetylcholine receptors located presynaptically on the sympathetic nerve terminals of the rat vas deferens. The pD2 values for nicotine were significantly higher at night (4.20 +/- 0.01) than in the afternoon (3.80 +/- 0.07). Exogenous melatonin shifted the concentration-response curves for nicotine to the left, mimicking the effect of darkness. Melatonin modifies both the displacement and the saturation curves of [3H](-)nicotine binding. In membranes from animals killed at 15:00 h, the binding of [3H](-)nicotine (5-6 nM) was first potentiated and then inhibited by sequential concentrations of (-)nicotine. Higher concentrations of [3H](-)nicotine (50-60 nM) were displaced by all concentrations of nonlabeled ligand. However, when membranes from tissues exposed to melatonin (exogenous or endogenous) were tested, the lower [3H](-)nicotine concentration was displaced progressively by increasing concentrations of nonlabeled ligand. Equilibrium binding studies show a single class of high-affinity nicotinic binding sites with an apparent Kd value of 16 nM and an average maximal number of binding sites of 66 fmol mg-1 protein when animals were killed at the afternoon. Melatonin, although it did not change the properties of high-affinity binding sites, induced the appearance of a second population of lower apparent affinity (Kd = 36.7 nM; Bmax = 185.4 fmol/mg). Melatonin does not modify the functional response and the displacement of [3H](-)nicotine by dimethylphenylpiperazinium. The data suggest that nicotinic neuronal acetylcholine receptors stimulated by dimethylphenylpiperazinium do not change between the light and dark phases. Rather, the higher sensitivity to nicotine in prostatic portions incubated with exogenous melatonin, and in organs from animals killed at night, after the rise of endogenous melatonin, is probably due to the appearance of low-affinity neuronal nicotinic ACh binding sites.

Antinociceptive effects of carbachol microinjected into different portions of the mesencephalic periaqueductal gray matter of the rat.

The changes in tail-flick latency (TFL) to noxious heating of the skin produced by the microinjection of carbachol (CCh) into the dorsal (dPAG), lateral (lPAG), and ventral (vPAG) portions of the mesencephalic periaqueductal gray matter (PAG) were studied in the rat. A significant increase in TFL was produced by CCh (0.2 microgram/0.5 microliter) microinjected into sites widely distributed within the PAG. The effect of CCh was stronger in the most caudal portion of the DPAG. Smaller effects were obtained after injection of CCh into the aqueduct, indicating that drug diffusion from the injection sites to the aqueduct lumen is unlikely to cause the antinociceptive effect of CCh. Dimethyl-phenyl-piperazinium (0.35 microgram/0.5 microliter), but not bethanechol (0.22 and 0.44 microgram/0.5 microliter), produced effects similar to CCh (0.2 microgram/0.5 microliter), when injected into the dPAG. The effects of CCh were inhibited by the previous administration of mecamylamine (1 microgram/0.5 microliter), but not atropine (1 microgram/0.5 microliter) or naloxone (1 microgram/0.5 microliter), into the dPAG. These results are indicative that antinociception produced by CCh from the dPAG depends on nicotinic, but not muscarinic or opioid mechanisms within the dPAG. The intraperitoneal administration of phenoxybenzamine (1 mg/kg) or mecamylamine (1 mg/kg), but not naloxone (1 mg/kg), methysergide (1 mg/kg), or atropine (1 mg/kg), inhibited the effects of CCh injected into the dPAG. In contrast, a higher dose of intraperitoneal phenoxybenzamine (5 mg/kg) was ineffective against the antinociception evoked by CCh when injected into the vPAG. Therefore, the effects of CCh from the dPAG may depend on the activation of centrifugal pathways involving both nicotinic and alpha-adrenergic mechanisms. In addition, the results indicate that different cholinergic substrates in the PAG may mediate both alpha-adrenergic and non-alpha-adrenergic descending pain mechanisms activated by the dPAG and vPAG, respectively.

Cardiovascular responses to cholinergic agonists in sinoaortic denervated rats.

1. The cardiovascular effect of systemic nicotinic and muscarinic cholinergic stimulation were studied in conscious sham operated and sinoaortic denervated (SAD) rats, 7 days after the corresponding operation. 2. The administration of the nicotinic ganglionic agent, 1,1-dimethyl-4-phenylpiperazinium (DMPP, 50-100 micrograms.kg-1, i.v.) induced a fall of heart rate that was significantly higher in SAD rats than in sham rats. DMPP induced in sham rats an increase of arterial pressure but in SAD animals a biphasic response: an initial hypotension followed by an increase of arterial pressure. 3. Under muscarinic blockade, DMPP only showed a pressor and tachycardic action in both groups of rats without differences between them. 4. The muscarinic agonist, carbachol (0.1-10 micrograms.kg-1, i.v.) showed the same hypotensive and bradycardic action in both groups of rats. 5. Our results suggest that after 7 days of SAD, differences in the response to DMPP between sham and denervated animals could be due to the loss of baroreflex mechanisms. The increased bradycardic effect of DMPP in SAD rats could be mediated by a supersensitivity of parasympathetic ganglionic nicotinic receptors, whilst the sympathetic ganglionic nicotinic receptors remained unaltered. On the other side, the cardiovascular muscarinic responses to carbachol remain unaffected in SAD rats.

Pharmacology of an Indian-snuff obtained from Amazonian Maquira sclerophylla.

The powdered bark of Maquira sclerophylla is consumed as snuff in north Brazil. Both the crude and the purified hydrosoluble extract (WP) injected i.p. in the dose range of 0.05-0.5 g/kg induced hyperexcitability, tremors, motor incoordination, ataxia, quietness and muscle relaxation in rats. The effects were progressive, dose-related and reversed after 30 min. Anesthetized rats, guinea-pigs and dogs injected with the purified extract (10-50 mg/kg, i.v.) showed a biphasic change of carotid blood pressure. The early and transient hypotension was blocked by atropine but not by vagotomy: the secondary hypertension was long lasting and sustained for over 30 min. The hypertension was shortened but not blocked after ganglionic blockade or reserpine treatment. Either pithing or alpha receptor blockade with yohimbine reduced both effects of the extract. Guinea-pigs and dogs were more responsive than rats and died by heart arrest. Incubation of WP (20 micrograms/ml) increased both the rate and force of contraction of isolated guinea-pig right atria by 2 and 5 times, respectively. Propranolol (4 micrograms/ml) blocked the chronotropic effect but did not decrease the inotropic effect. In electrically driven guinea-pig left atria, WP (10 micrograms/ml) increased the force of contraction by 80% and the maximum rate of force development by 60%, but did not change the time to peak tension, the time to 50% relaxation, or the rate of relaxation. These cardiovascular effects resemble those of digitalis-like drugs. Cardenolides were detected in WP by phytochemical screening.

Interaction between clonidine and physostigmine in normal rats and in rats after sinoaortic denervation.

Clonidine (3-30 micrograms.kg-1, i.v.) induced a fall in mean arterial pressure in rats after sinoaortic denervation but not in sham-operated animals. Moreover, sinoaortic denervation reduced the bradycardic action of this antihypertensive drug. Pressor and tachycardic response to physostigmine (60 micrograms.kg-1, i.v.) were greater in denervated than in sham-operated rats. The increase of mean arterial pressure was 26.2 +/- 2.2 mm Hg in sham-operated rats (n = 12) and 53.8 +/- 2.0 mm Hg in denervated rats (n = 12, P less than 0.005). Pretreatment with 3 micrograms.kg-1 (i.v.) of clonidine did not alter the pressor response to physostigmine (60 micrograms.kg-1) in either of the two groups; 10 and 30 micrograms.kg-1 of clonidine reduced the physostigmine-induced increase of mean arterial pressure in sham-operated rats but enhanced the pressor response in denervated animals. Furthermore, an ineffective dose of physostigmine (30 micrograms.kg-1, i.v.) induced a pressor response after pretreatment with clonidine (10 micrograms.kg-1) in denervated rats. Clonidine (10 micrograms.kg-1) did not affect the pressor effect of 1,1 dimethyl-4-phenylpiperazinium iodide (DMPP: 50 micrograms.kg-1, i.v.) or phenylephrine (4 micrograms.kg-1, i.v.) in either group. The anticholinergic effect of clonidine in sham-operated rats may be explained by an inhibitory action on the release of acetylcholine in several brain structures but the facilitatory effect of clonidine observed in denervated animals is not clear. The results did not suggest a peripheral involvement in this facilitatory effect.

The relaxing response of the isolated rat duodenum to nicotine.

1. The relaxing responses of the atropine-treated isolated rat duodenum to 12 microM nicotine (N) and transmural electrical stimulation (S) have been compared. 2. The effects of both N and S were unaffected by 3-30 microM guanethidine or previous reserpinization (1 mg/kg, i.p. during 5 days). 3. The effects of N and S were completely blocked by 0.5-2.0 mM procaine, 0.1 mM butacaine or 0.3-1.2 microM tetrodotoxin. 4. The relaxing response to N but not that to S was blocked by 0.3 mM hexamethonium or 3 microM mecamylamine and absent in the nicotine-desensitized preparation. 5. The effects of both N and S were unaffected in the adenosine-5'-triphosphate (ATP)-desensitized preparation. 6. It is suggested that the nonadrenergic and noncholinergic (NANC) relaxation of the rat duodenum in response to N and S is also nonpurinergic.

Lack of cholinergic modulation of tyrosine hydroxylase activation by potassium.

Cholinergic modulation on the regulation of tyrosine hydroxylase was studied in guinea pig atria depolarized with high potassium concentration. In these conditions there was an increment in tyrosine hydroxylase activity as well as in the release of radioactivity from atria preincubated with 3H-noradrenaline. Both effects were concentration-dependent. Incubation in the presence of 10 microM methacholine, 31 microM 1,1 dimethyl-4-phenylpiperazinium iodide (DMPP), 0.15 microM atropine or 83 microM hexamethonium did not affect tyrosine hydroxylase activation by 100 mM K+. The percentage of activation was reduced however, when 7 microM physostigmine (acetylcholinesterase inhibitor) was present during depolarization. This effect is unlikely to be related to the increase of acetylcholine concentration in the interface, but more probably to a direct effect on the enzyme. In conclusion, tyrosine hydroxylase activation induced by 100 mM potassium appears to be unrelated to the stimulation of nicotinic and muscarinic presynaptic receptors.

Supersensitivity induced by the toxin of Brazilian scorpion venom (Tityus serrulatus Lutz e Mello).

The purified toxin of the Brazilian scorpion venom (TsTX) enhances the pharmacological actions of directly and indirectly acting substances on several preparations. The enhancing action of TsTX is more pronounced for the indirectly acting drugs, which is highly suggestive of a prejunctional site of action. Secondarily, TsTX does also produce an unspecific increase of sensitivity at the level of the pharmacological receptor itself. The mechanism by which these changes are brought about remains to be determined.