Modulation of the pressor response elicited by carbachol and electrical stimulation of the amygdala by muscarinic antagonists in conscious rats.

1. The nature of the muscarinic receptor involved in mediating cardiovascular changes caused by unilateral microinjection of carbachol (5 nmol) into, and electrical stimulation (200-300 microA) of, the amygdaloid complex was investigated in conscious, unrestrained female Sprague-Dawley rats. 2. Unilateral microinjection of carbachol (5 nmol; n = 6) and electrical stimulation (200-300 microA, 80 Hz, 30 s; n = 4) caused a significant rise in blood pressure of 21 +/- 4 mmHg and 25 +/- 5 mmHg, respectively. These changes were associated with no overall effect on heart rate. The effects of electrical stimulation were found to be repeatable. 3. Pretreatment i.c.v. with pirenzepine (5-20 mmol; n = 6-7 for each dose), dose-dependently inhibited the rise in blood pressure induced by carbachol, whereas AF-DX 116 (100 nmol; n = 6) failed to have any effect on the carbachol-induced pressure response. Neither antagonist alone had any effect on resting baseline variables. 4. Unilateral microinjections of atropine sulphate (1-100 nmol; n = 4-6 for each dose), pirenzepine (0.03-10 nmol; n = 4 for each dose) or AF-DX 116 (10-60 nmol; n = 4-5 for each dose), into the amygdala, dose-dependently inhibited the rise in blood pressure caused by electrical stimulation (200-300 microA). The ID50 values were 1.05, 0.23 and 39.5 nmol, respectively. Although pirenzepine seemed to be more potent than atropine, this difference was not significant. 5. It is concluded that the rise in blood pressure elicited by unilateral microinjection of carbachol into, or electrical stimulation of, the amygdaloid complex is mediated by M1-muscarinic receptors.

Pathways mediating CRF-induced inhibition of gastric emptying in rats.

The corticotrophin-releasing factor (CRF) is shown to be released during stress suggesting that CRF has a physiological role in the mediation of central nervous system (CNS) response to stress, including an inhibitory effect on gastric emptying. In the present study, we have examined the pathways by which intracerebroventricularly (i.c.v.) administered CRF and central CRF activation during stress alter the gastric emptying rate of saline (0.14 M), acid (50 mM), peptone (4.5%) and peptone after preload. The emptying rates of all these test meals were significantly (p < 0.05-0.001) delayed with increasing doses of i.c.v. CRF (0.001, 0.003, 0.01, 0.1, 0.3 and 1 nmol/10 microl), when compared with their i.c.v. saline-treated controls. The 1-nmol dose of CRF inhibited the emptying of acid, peptone and peptone after a preload by 43.8%, 64.1% and 81.1%, respectively. Twenty-minute swim stress delayed gastric emptying rate of saline, acid and peptone solutions significantly (p < 0.001) and the CRF receptor antagonist, alpha-helical CRF (8 nmol/10 microl, i.c.v.), applied before the swim stress, abolished the inhibitory effect of stress on the emptying rate of these solutions. Acute intragastric administration of capsaicin (2 mg/rat) and systemic capsaicin (125 mg kg(-1)) treatment facilitated the gastric emptying rate of acid, peptone and peptone after preload significantly, almost abolishing the inhibitory effect of central CRF (p < 0.001). However, either capsaicin treatment had no effect on stress-induced inhibition of the gastric emptying of none of the solutions, except peptone after a preload. Our findings demonstrate that the gastric inhibitory response induced by swimming as a stress-producing stimulus is mediated by the endogenous release of CRF. They also suggest that CRF exerts its CNS actions on the gastrointestinal tract via vago-vagal, capsaicin-sensitive pathways, probably involving the central cholecystokinin (CCK) mechanisms.

The role of brain acetylcholine in GABAA receptor antagonist-induced blood-pressure changes in rat.

Previous experimental studies have shown that intracerebroventricular (i.c.v.) injection of the GABAA receptor antagonist, bicuculline methiodide, results in marked increases in blood pressure due to an increase in sympathetic nervous system activity. It is well recognized that the central cholinergic system is also involved in the regulation of blood pressure. In the present study, we examined the role of brain acetylcholine in the pressor response induced by bicuculline methiodide in conscious Sprague-Dawley rats. I.c.v. (0.05, 0.3 and 0.5 nmol) and intrahypothalamic (40 pmol) administration of bicuculline methiodide produced blood-pressure increases in a dose-dependent manner. Hemicholinium-3 was given i.c.v. 1 h prior to bicuculline methiodide. The depletion of brain acetylcholine was demonstrated by the suppression of physostigmine-induced pressor responses, but blood pressure increases in response to carbachol remained unchanged. The pressor responses to bicuculline methiodide in animals pre-treated with hemicholinium-3 were significantly higher than those seen in saline-pre-treated groups. Likewise, bicuculline methiodide, at a dose that did not alter blood pressure alone, caused pressor responses in rats pre-treated with the nicotinic receptor antagonist, mecamylamine, whereas the muscarinic receptor antagonist, atropine, was ineffective in this respect. In conclusion, it seems likely that endogenous brain acetylcholine has a modulator role on GABAA receptor-mediated blood-pressure control via nicotinic receptors.

A-4, a tertiary amine analog of HC-3, lowers arterial pressure in spontaneously hypertensive rats.

The 4-methyl piperidine analog (A-4) of hemicholinium-3 is a tertiary amine. A-4, like hemicholinium-3, inhibits sodium-dependent, high-affinity choline transport. The present study examined whether central cholinergic systems are involved in the expression of genetic hypertension. We examined the effects of i.v. and i.c.v. administration of A-4 in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY; control). Basal mean arterial pressure and heart rate values were 108 +/- 4 mm Hg and 370 +/- 5 bpm in WKY and 167 +/- 5 mm Hg and 337 +/- 13 bpm in SHR. The i.v. injection of A-4 (5, 10 and 20 mumol/kg) evoked a dose-dependent decrease in MAP in SHR, but not in WKY. The maximal decrease in MAP was 18 +/- 6 mm Hg (P < .01) in SHR. Depressor responses appeared within 1 min and reached maximum within 10 min. The reductions in MAP were not associated with reductions in peripheral vascular resistances, suggesting that the hypotension was due to a reduction in cardiac output. The i.c.v. injection of A-4 (100 nmol/rat) significantly decreased MAP in SHR (-23.8 +/- 2.4 mm Hg, P < .01), but not in WKY. The maximal decrease in MAP appeared within 1 min and reached maximum 10 min later. These falls in MAP were associated with falls in vascular resistances, suggesting that the hypotension was due to peripheral vasodilation. This dose was ineffective when given i.v. in either strain. A-4-induced decreases in MAP were accompanied by significant tachycardia, which was maximal within 3 min of injection. These studies demonstrate that A-4 lowers MAP in SHR, but not in WKY. The rapid onset of hypotension in SHR after A-4 administration suggests that there is rapid turnover of brain acetylcholine which may be directly involved in maintaining elevated arterial pressure in SHR.

A-ring ortho-disubstituted aporphine derivatives as potential agonists or antagonists at serotonergic 5-HT1A receptors.

(R)- And (S)-11-hydroxy-10-methylaporphine 1 and 2 are, respectively, a potent, highly specific serotonergic (5-HT1A) agonist and antagonist. In an ongoing structure-activity study, racemates of the positional isomers 8-hydroxy-9-methyl- and 8-methyl-9-hydroxyaporphine were prepared by modifications of literature methods and were resolved. The methyl ethers of the target compounds were also evaluated pharmacologically. All of the free phenolic derivatives [(+)- and (-)-8 and 10] were inert in an assay for 5-HT1A receptor activity. All of the methyl ethers [(+)- and (-)-9 and 11] demonstrated quantitatively similar low potency stimulant effect at 5-HT1A receptors. The agonist or antagonist activity exhibited by 1 and 2 reflects the high degree of structural specificity required of aporphine derivatives for action at 5-HT1A receptors.

Prevention of organophosphate-induced toxicity in mice.

bis-Quaternary amines, which are acetal analogues of hemicholinium-3, were synthesized and several compounds were potent chemicals to antagonize toxicity induced by the organophosphate, paraoxon. Structural requirements were specific and included two oxygen atoms (bis-acetal substitution) within 6 or 7 atom heterocyclic rings, oxygen atoms spaced 2-carbon atoms from the quaternary nitrogen, and carbonyl substitutions adjacent to the spacing moieties, either bicyclohexyl or biphenyl. Biological testing showed a positive potency correlation between the chemicals when data from the following tests were compared: antagonism in mice of paraoxon-induced motor impairment using the incline screen and toxicity, and ability to induce contractions of guinea-pig isolated ilea. The compounds were compared with the often used protective antagonist of organophosphate-induced toxicity, pyridostigmine. One compound, MFS-3, was seven times more efficacious and possessed a much higher therapeutic index. Possible mechanisms of action for these chemicals are discussed.

Cardiovascular effects of centrally active cholinomimetics in conscious and anesthetized rats: the role of amygdala.

Central cardiovascular effects of cholinergic agonists depend on the dose, site and mode of administration, species, and to the state of the animal. Intravenous injection of physostigmine and intracerebroventricular injection of carbachol produced pressor and tachycardic responses in urethane-anesthetized rats. Both agents also elicited pressor responses in conscious rats, but bradycardia occurred in the presence of anesthesia. Additionally, pressor responses to physostigmine, but not to carbachol, were significantly exaggerated by urethane anesthesia. These results demonstrate that anesthesia depresses cardiovascular reflexes and the inhibitory control mechanisms on acetylcholine release from the nerve endings involved in cardiovascular regulation. The role of the central nucleus of the amygdala (CNA) was also investigated in this study. The pressor effects of intracerebroventricular injection of carbachol were significantly attenuated by electrolytic ablation of the CNA, but heart rate changes were not altered both in anesthetized and conscious rats. These results indicate that the CNA plays a role in cholinergic control of blood pressure, but not in the regulation of heart rate.

Reversal of hemorrhagic shock in rats by oxotremorine: the role of muscarinic and nicotinic receptors, and AV3V region.

In an experimental model of hemorrhagic shock resulting in the death of almost all rats within 20-30 min, centrally active cholinomimetic drugs are reported to induce a prompt, sustained and dose-dependent improvement in blood pressure and survival rate claimed to be due to nicotinic, but not muscarinic actions. In the present study, cholinergic receptor agonist, oxotremorine (50 micrograms/kg, i.v.) increased mean arterial pressure (from 22 +/- 1 to 123 +/- 3 mm Hg) and 60 min-survival rate (from 0% to 92%) in rats bled to hypovolemic shock. Atropine (2 mg/kg, i.v.) pretreatment inhibited the pressor effect of oxotremorine significantly, but did not modify its effect on survival rate. On the other hand, pretreatment with mecamylamine (50 micrograms, i.c.v.) almost abolished the reduction in mortality rate, but inhibited the pressor effect of oxotremorine, partially. These results indicate that oxotremorine-induced pressor response and decrease in mortality in rats with severe hemorrhagic shock are primarily mediated via central muscarinic and nicotinic receptors, respectively. AV3V region was previously reported to be involved in pressor and natriuretic effects of i.c.v. carbachol in normotensive rats. In the present study, the electrolytic lesions of AV3V region significantly inhibited oxotremorine-induced increases in both blood pressure and survival rate in rats subjected to hemorrhagic shock. These findings indicate that AV3V region plays a major role in cholinergic cardiovascular control in hypotensive animals as well as normotensives.

Central muscarinic M2 cholinoceptors involved in cholinergic hypertension.

Cholinomimetic agents increase blood pressure and heart rate via central muscarinic cholinoceptors in various species. It was reported that i.c.v. injection of the muscarinic M1 and M3 cholinoceptor selective antagonist, 4-DAMP (4-diphenylacetoxy-N-methyl-piperidine methiodide), inhibited the pressor response to physostigmine, while the M1 selective antagonist, pirenzepine, was ineffective. In the present study, the involvement of muscarinic M2 cholinoceptors in central cholinergic hypertension and tachycardia was investigated. Physostigmine (10-80 micrograms/kg i.v.), a cholinesterase inhibitor, and oxotremorine (20-40 micrograms/kg i.v.), a direct muscarinic cholinoceptor agonist, caused a dose-dependent increase in blood pressure. Additionally, physostigmine induced dose-dependent tachycardiac responses. I.c.v. administration of the muscarinic M2 cholinoceptor antagonists, AF-DX 116 and methoctramine, inhibited both physostigmine (60 micrograms/kg) and oxotremorine (20 micrograms/kg)-induced pressor responses at their lower doses used in this study (100 nmol/rat and 10 nmol/rat, respectively). These findings indicate the partial involvement of postsynaptic muscarinic M2 cholinoceptors. The higher doses of the antagonists (AF-DX 116,300 nmol/rat and methoctramine 30 nmol/rat) potentiated the blood pressure increase due to physostigmine but did not affect that due to oxotremorine. The physostigmine-induced tachycardiac responses were influenced similarly by these antagonists. These results suggest the presence and tonic influence of presynaptic inhibitory muscarinic M2 cholinoceptors.

The effect of nordihydroguaiaretic acid on leukotriene C4 and prostaglandin E2 production following different reperfusion periods in rat brain after forebrain ischemia correlated with morphological changes.

Leukotriene C4 (LTC4) and prostaglandin E2 (PGE2) are the 5-lipoxygenase and cyclooxygenase metabolites of arachidonic acid (AA). They constrict blood vessels and enhance vascular permeability inducing vasogenic edema that may hurt the ischemic penumbra after cerebral ischemia and reperfusion. Nordihydroguaiaretic acid (NDGA) is known as the most potent inhibitor of 5-lipoxygenase in different tissues. Furthermore, it has considerable inhibitory activity against cyclooxygenase. In this study, after developing a global ischemic model in the rat, the levels of LTC4 and PGE2 in the forebrain were measured, following different reperfusion periods after 10 min ischemia including 8 rats for each reperfused group. Sham operations were performed for each corresponding control group (n = 8). AA metabolites were then correlated with neuropathological findings. In the combined reperfused groups both metabolites increased significantly when compared with 10 min, ischemic group (P < 0.05). In the 8 min reperfused group, PGE2 and LTC4 increased significantly compared with each corresponding control group (P < 0.005). These mediators also increased to high levels compared with the 4 min reperfused group (P < 0.05, P < 0.005). PGE2 and LTC4 were reduced significantly at the 15th and 60th min of reperfusion compared with the 8 min reperfused group (P < 0.05, P < 0.005). NDGA (0.1 mg/kg) reduced both metabolites in the 8 min reperfused group significantly (P < 0.05). Brain cortex specimens were taken for light and electromicroscopical investigations. No significant differences were noted between the structural changes in the 4, 8 and 15 min of reperfusion and NDGA administered groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Are m-cholinoceptors of guinea pig gallbladder smooth muscle of m4 subtype?

The antagonism of carbachol-induced contractions of guinea pig gallbladder smooth muscle strips via selective antagonists, methoctramine, HHSiD, pf-HHSiD and DABDMA has been investigated in order to find out the m-cholinoceptor subtype(s) of gallbladder smooth muscle. All m-cholinoceptor antagonists examined, displaced the concentration-response curves to the right parallel in a concentration-dependent manner without affecting the maximum response. Schild analysis of data was consistent with competitive antagonism. -log KB values of the antagonists were 7.37 for HHSiD, 7.53 for pf-HHSiD, 6.58 for DABDMA and 7.60 for methoctramine. These results, together with the high affinity of pirenzepine and low affinities of 4-DAMP and AF-DX 116, indicate that the m-cholinoceptors of the guinea pig gallbladder which mediate cholinergic contractions are not of m1-, m2- and m3- subtypes but seem likely to be of m4-subtype.

Effect of methylene blue on blood pressure in rats.

Methylene blue (MB) is a soluble guanylate cyclase inhibitor, and known as an endothelium-derived relaxing factor (EDRF) inhibitor in vitro. In the present study, it was demonstrated that intravenous administration of MB caused a dose-dependent hypertensive effect in rats. The hypertensive responses to the higher doses (10 and 20 mg/kg) of MB was followed by a reflex hypotension which did not appear in pithed rats. Noradrenaline depletion by reserpine pretreatment did not inhibit MB-induced hypertension, but abolished the hypotensive response. Both hypertensive and hypotensive phases were not altered by indometacin. These results may suggest that in vivo guanylate cyclase inhibition leads to an increase in blood pressure; prostaglandins and noradrenaline release from sympathetic nerve endings do not contribute to MB-induced hypertension and it may be due in part to the inhibition of EDRF.

Prostaglandin E2 and leukotriene C4 levels following different reperfusion periods in rat brain correlated with morphological changes.

Prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) are the metabolites of arachidonic acid (AA) that increase in forebrain following global ischemia and reperfusion. These mediators are highly potent vasoconstrictors of cerebral arteries leading to enhanced vascular permeability that induces the formation of vasogenic edema. In this study, after developing an experimental animal model simulating the concept of ischemic penumbra in the rat, the levels of PGE2 and LTC4 produced in the forebrain were measured and the effects of these mediators in short duration and prolonged reperfusion were investigated and then correlated with neuropathological findings. We found statistically significant reduction both in PGE2 and LTC4-like activities after just 10 min ischemia (p less than 0.05, p less than 0.05). PGE2-like activity significantly increased in the 4th and 60th min of reperfusion (p less than 0.05, p less than 0.05). In the 15th min of reperfusion, PGE2 was found to be significantly reduced (p less than 0.005) that may be due to the formation of free oxygen radicals by activation of PG hydroperoxidase reaction that inhibits PGE2 production in the cyclooxygenase pathway. LTs were not significantly increased in any reperfused group. Inhibition of the lipoxygenase pathway of AA metabolism may occur as a result of 15-HPETE (15-hydroperoxyeicosatetraenoic acid) production. Pathologically, edema and degeneration of brain tissue were seen beginning from the 4th min of reperfusion that reached a peak in the 60th min of reperfusion which is in accordance with biochemical changes in the damaged tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic receptor subtypes of guinea-pig common bile duct.

The antagonism of carbachol-induced contractions of guinea-pig common bile duct smooth muscle strips by various antagonists has been investigated in order to find out the muscarinic receptor subtype(s) of common bile duct smooth muscle. Atropine, pirenzepine, 4-DAMP and AF-DX 116 were used as nonselective, M1-selective, M1- and M3-selective and M2-selective muscarinic antagonists, respectively. All muscarinic antagonists examined displaced the concentration-response curves to the right, parallelly and in a concentration-dependent manner, without affecting maximum response. Schild analysis of data was consistent with competitive antagonism. pA2 values of the antagonists were as follows: atropine, 9.59; pirenzepine, 7.32; 4-DAMP, 8.99; AF-DX 116, 6.85. When these pA2 values are compared with those obtained in the ileum, it may be concluded that the muscarinic receptors of the guinea-pig common bile duct mediating cholinomimetic-induced contractions, are of the M3 subtype, but not of the M1 and M2 subtypes.