Antioxotremorine action of propranolol.

1. The influence of propranolol on the neuromuscular, tremor-producing and muscarinic effects of oxotremorine was examined. 2. In the isolated rat phrenic nerve-diaphragm preparation the neuromuscular blocking effect of oxotremorine was inhibited by propranolol in a dose-dependent manner. 3. Propranolol intensified the paralytic effect of tubocurarine in the rat diaphragm and prevented antagonism of tubocurarine paralysis by tetraethylammonium. 4. Propranolol was devoid of any curare-like effect in the isolated rectus abdominis muscle of the frog. 5. Vasodepressor responses to oxotremorine in rats and spasmogenic responses to oxotremorine in guinea-pig ileum were not antagonized by propranolol. 6. A dose-dependent antagonism of oxotremorine-induced tremor in mice was observed after pretreatment with propranolol and it is suggested that this effect is due to an antagonism of a presynaptic effect of oxotremorine at skeletal neuromuscular junctions.

Release of prostaglandin E from the isolated urinary bladder of the guinea-pig.

1 Release of prostaglandin E (PGE) from guinea-pig urinary bladder in vitro has been demonstrated both in the resting state and during electrical stimulation. 2 The electrically evoked release of PGE was significantly higher than the resting release and was frequency-dependent. 3 The released substance was characterized as PGE pharmacologically by (a) blockade of its response by SC-19220 on guinea-pig ileum, (b) reduction of the amount of the released substance by indomethacin and (c) the inhibitory effect of the released substance on adrenergic neurotransmission in guinea-pig vas deferens. 4 The prostaglandin seemed to originate from the muscle since tetrodotoxin treatment did not abolish the release during direct muscle stimulation; however, concomitant release from neuronal tissue could not be excluded in the present experiments. 5 Indomethacin failed to inhibit the mechanical responses of the bladder to transmural stimulation. 6 The present experiments suggest that PGE is not involved in mediating the non-cholinergic non-adrenergic neurotransmission in the guinea-pig urinary bladder.

Anti-tremor action of C10Dichol, a peripheral acetylcholine synthesis inhibitor.

1 Anti-tremor action of decamethylene bis-(hydroxyethyl)-dimethylammonium bromide (C10Dichol), a peripheral acetylcholine synthesis inhibitor, was investigated. 2 C10Dichol inhibited tremor induced by oxotremorine, nicotine and physostigmine and afforded partial protection from physostigmine-induced mortality in mice. 3 In non-paralysing doses, C10Dichol antagonized the neuromuscular effects of oxotremorine, nicotine and physostigmine. 4 Prior administration of C10Dichol failed to prevent tremor and neuromuscular paralysis induced by harmine and arecoline. 5 In the absence of any antimuscarinic property of C10Dichol, its neuromuscular effects appeared to be casually related to its anti-tremor action. 6 This study reveals a possibility for the development of peripherally acting anti-Parkinson drugs.

Enhancement by oxotremorine of acetylcholine release from the rat phrenic nerve.

Oxotremorine (10.5 micron) produced a paralytic effect on twitch responses of rat diaphragm in vitro to direct and indirect stimulation. 2 The paralytic effect of oxotremorine was absent when the diaphragm was stimulated directly in the presence of hemicholinium-3 (0.42 mM), at a time when twitch responses to indirect stimulation ceased completely. 3 Oxotremorine, at two different pharmacologically active doses, strikingly increased the resting as well as electrically evoked release of acetylcholine into the bathing fluid from the phrenic nerve-diaphragm preparation. 4 This presynaptic effect of oxotremorine may explain its pharmacological effects at the cholinergic synapses studied so far.

Rat isolated phrenic nerve-diaphragm preparation for pharmacological study of muscle spindle afferent activity: effect of oxotremorine.

1. Muscle spindle afferent discharges exhibiting an approximately linear length-frequency relation could be recorded from the phrenic nerve in the isolated phrenic nerve-diaphragm preparation of the rat. 2. Muscle spindle afferent discharges could be identified by their characteristic "spindle pause" during muscle contraction and by their response to succinylcholine. 3. Cholinergic influence on spontaneous and stretch-induced afferent discharges was indicated by the augmentation produced by physostigmine and acetylcholine. (+)-Tubocurarine, but not atropine, prevented this augmentation indicating the presence of curariform cholinoceptors in muscle spindles. 4. Acetylcholine did not appear to be involved in the genesis of spindle afferent discharges as incubation with hemicholinium-3 and (+)-tubocurarine failed to affect the rate of spontaneous and stretch-induced spindle discharges. 5. Oxotremorine markedly increased the rate of spontaneous and stretch-induced spindle afferent discharges and this effect was prevented in the presence of hemicholinium-3 and (+)-tubocurarine. 6. These results with oxotremorine are of interest in connection with the observation that muscle spindle afferents and hyperactive in Parkinsonian patients.

Thermodynamic studies with acetylthiocholine on nicotinic receptors of mammalian skeletal muscle in vitro.

The temperature dependency of binding of acetylthiocholine, a specific nicotinic agonist, to the nicotinic receptor of mammalian skeletal muscle was studied using isotonic contractions of the rat denervated diaphragm preparation in vitro. The dissociation constants at different temperatures (22-39 degrees) were determined by the Furchgott method using alpha-bungarotoxin as an irreversible antagonist. Both free energy of association (delta G zero = -22.93 kJ/mol at 37 degrees) and enthalpy of binding (delta H zero = -58.35 kJ/mol) calculated from Kd (dissociation constant) and slope of lnKd versus 1/T (van't Hoff plot) respectively were found to be negative. The negative entropy value (delta S zero = -0.113 kJ/mol/deg) obtained from the intercept of this van't Hoff plot differs from the large positive value obtained earlier employing radioligand binding studies of the nicotinic receptor of Electrophorus electricus.

Action of oxotremorine on the sub-cellular distribution of glycine in the rat spinal cord.

Intraperitoneal (i.p.) injection of 250 micrograms/kg of oxotremorine (OT) caused a 50% decrease in the glycine content of the synaptosomal-mitochondrial fraction of spinal cord homogenates prepared from rats killed 15 min after treatment. The glycine content of the supernatant fraction was correspondingly raised. In synaptosomes isolated from the spinal cord of OT-treated rats, the decrease in glycine content was 30%. Prior administration of atropine, but not of methylatropine, abolished this effect of OT on synaptosomal glycine content. Eserine exerted a potentiating effect on the action of OT in lowering the glycine content of spinal synaptosomes. Prior administration of L-DOPA, apomorphine, haloperidol, muscarine or mecamylamine had no significant effect on the action of OT on synaptosomal glycine content. OT alone or in combination with eserine, and acetylcholine (ACh) in combination with eserine, did not alter the rate of release of glycine from spinal synaptosomes of untreated rats incubated under appropriate conditions. OT was also without effect on the rate of release of glycine from normal spinal synaptosomes subjected to electrical stimulation, as well as on the eventual glycine content of the synaptosomes. On the basis of the present findings it has been suggested that (i) glycine may be released from Renshaw cells at their synapses with motoneurones in response to the muscarinic action of OT; (ii) dopaminergic modulation may not be involved in the OT-induced glycine release from Renshaw cells; and (iii) excessive release of glycine onto motoneurones may be the causative factor of the akinesia observed in OT-induced experimental Parkinsonism.

Behavioral and neurochemical alterations following intracerebroventricular administration of anti-serotonin antibodies in adult Balb/c mice.

The effects of intracerebroventricular injections of serotonin (5-HT) antibodies were studied for changes in 5-HT, dopamine (DA), their metabolites and norepinephrine (NE) as well as 5-HT mediated behavior in adult mice. While nociceptive thresholds (tail-flick latency) were inhibited in antibody treated animals, tremor response to 5-methoxy-N,N-dimethyl tryptamine administration was increased. 5-HT and DA in the nucleus raphe dorsalis (NRD), substantia nigra (SN), nucleus caudatus putamen (NCP) and in the substantia grisea centralis, and NE in the former two nuclei were significantly decreased in these animals. 5-Hydroxyindoleacetic acid was unaffected in all nuclei except NRD, where it was inhibited. Homovanillic acid and 3,4-dihydroxyphenylacetic acid were inhibited in all nuclei except in NCP. The brunt of insult was more evident in NRD and SN where all neurotransmitters were inhibited for a longer period. 5-HT turnover was increased in all the nuclei, however only SN showed increased DA turnover. It may be assumed that the observed neurochemical and behavioral changes were the consequence of the antibodies binding to 5-HT, which in turn influenced the anatomically and functionally connected neurotransmitters. While the study contributes to the existing understanding of central neurotransmitter control on behavior, it fails to delineate the underlying mechanism. The possibility of developing a useful, drug-free 5-HT deficient animal model for studying clinical disorders, as well as for solving some of the basic questions related to the physiological functions of 5-HT in adult animals are envisaged from the study.

Neonatal treatment with 5-HT antiserum alters 5-HT metabolism and function in adult rats.

Adult rats were studied for serotonin and its metabolite in basal ganglia, nociceptive responses to electric current and tremor induced by 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) to assess the effects of a single systemic injection of serotonin antiserum given at 1 day old. Antiserum-treated animals showed no overt behavioural abnormalities, but tremor induced by 5-MeODMT was augmented. Basal nociceptive thresholds for tail withdrawal and vocalization were unaffected, whereas vocalization after-discharge was significantly reduced. Significantly decreased serotonin levels and increased turnover were found in nucleus caudatus putamen, nucleus accumbens and tuberculum olfactorium of adult rats treated as neonates with serotonin antiserum. These results demonstrate long-lasting functional alterations and neurochemical suppression of the central serotoninergic system following neonatal administration of serotonin antiserum.

Involvement of the presynaptic dopamine D2 receptor in the depression of spinal reflex by apomorphine.

The relative roles of D1 and D2 dopamine (DA) receptors in mediating apomorphine (APO)-induced changes in the spinal reflex was investigated. Low doses of APO, a DA receptor agonist (0.2 mg kg-1, i.v.), depressed the monosynaptic mass reflex (MMR) in spinalized rats. Pretreatment with the D2-specific antagonist, spiperone, 10 min before APO prevented the APO-induced MMR depression. Pretreatment with the D1 antagonist SCH 23390 failed to prevent the APO-induced depression. Interestingly, SCH 23390 pretreatment preferentially antagonized the depression induced by a high dose of APO (3 mg kg-1, i.v.). Pretreatment with SKF 38393, a selective D1 agonist, completely prevented the APO-induced MMR depression. These results suggest that inhibition of spinal transmission by low dose of APO may be mediated through its action on presynaptic D2 receptors and that D1 and D2 receptors are functionally coupled at the spinal level in modulating the spinal motor output.

Desensitization of spinal 5-HT1A receptors to 8-OH-DPAT: an in vivo spinal reflex study.

Serotonergic influence on spinal monosynaptic transmission and the desensitization of spinal 5-HT1A receptors following a single pretreatment with a 5-HT1A ligand were examined in vivo in acutely spinalized adult rats. Administration of a selective 5-HT1A agonist, 8-OH-DPAT (0.1 mg kg-1) significantly depressed the monosynaptic mass reflex (MMR) amplitude, which was prevented effectively by S(-)-propranolol, a 5-HT1A antagonist. The inhibitory effect of 8-OH-DPAT on MMR amplitude was significantly attenuated with a single dose of 8-OH-DPAT (1 mg kg-1, s.c.) administered 24 h before the experiments, indicating a marked desensitization of spinal 5-HT1A receptors. Desensitization of 5-HT1A receptors could be reversed by treatment of spiperone (1 mg kg-1, i.p.) 3 h before 8-OH-DPAT pretreatment. These results demonstrate that 5-HT1A receptor functionally modulates the spinal motor output and confirms the ability of 8-OH-DPAT to desensitize presynaptic 5-HT1A receptors as observed for the first time in rat spinal cord.

Thermodynamics of the interaction of d-tubocurarine with nicotinic receptors of mammalian skeletal muscle in vitro.

Thermodynamic study is important for defining drug receptor interactions, and denervated rat hemidiaphragm is a unique preparation for such a study on nicotinic receptors. As a continuation of our earlier study with acetylthiocholine on the same preparation, we now report on the characteristics of temperature-dependent binding of d-tubocurarine, a reversible antagonist. The O. Arunlakshana and H.O. Schild (1959, Br. J. Pharmacol. 14, 48) equation, as improved by D.R. Woud and R.B. Parker (1971, J. Pharmacol. Exp. Ther. 177, 13), was used to calculate the dissociation constant of d-tubocurarine at various temperatures (10-37 degrees C) from the parallel shift of the acetylcholine dose-response curve to the right by effective doses of d-tubocurarine. It was observed that the values of the dissociation constant increased with a decrease in temperature. Both the enthalpy (delta H degree) and entropy (delta S degree) changes as evaluated from the van't Hoff plot (In Kd vs. 1/T) were found to be positive and their relative value (delta H degree - T delta S degree) produced a negative free energy change which characterises the binding of d-tubocurarine as an entropy-controlled process. This finding is in agreement with the neurotoxin binding reported earlier. The present finding and earlier observations with acetylthiocholine reveal that agonist and antagonist binding to the nicotinic receptor may differ depending on the experimental conditions.

Effect of prostaglandin E2 on acetylcholine release from some peripheral cholinergic nerve terminals.

The effect of prostaglandin E2 (PGE2) on the acetylcholine (ACh) release evoked from rat phrenic nerve terminals and from Auerbach's plexus of the guinea-pig ileum was investigated. PGE2 enhanced the evoked release of ACh from phrenic nerve terminals and from Auerbach's plexus in a concentration-dependent manner. Preincubation with 7-oxa-13-prostynoic acid, the PGE receptor blocker, and indomethacin inhibited the PGE2-induced increase of evoked release of ACh while atropine failed to do so. Whereas a single administration of either 7-oxa-13-prostynoic acid or indomethacin significantly inhibited the control evoked release of ACh from the Auerbach's plexus, they failed to alter the control evoked release of ACh from the phrenic nerve terminals. The study indicates that the PGE2-induced increase in release of ACh from cholinergic nerve terminals is accomplished through activation of prostaglandin receptors and that PGE2 may play a physiological role in ACh liberation from the cholinergic autonomic nerve terminals but not from motor nerve terminals.

Alpha 2-antagonistic effect of N-carbamoyl-2-(2,6-dichlorophenyl) acetamidine hydrochloride (LON-954).

The effect of N-carbamoyl-2-(2,6-dichlorophenyl) acetamidine hydrochloride (LON-954) on alpha 2-adrenoceptors was studied in in vivo and in vitro preparations. Like yohimbine, LON-954 antagonised the clonidine-induced inhibition of twitch responses in the Auerbach's plexus of guinea pig ileum and rat vas deferens preparations. It also reversed the anti-convulsant effect of clonidine on pentylenetetrazol (PTZ)-induced convulsions in the rat, a property shared by yohimbine. However LON-954 failed to prevent the hypothermic response to clonidine in mice. The dissimilarity in action of LON-954 and yohimbine on clonidine-induced hypothermia could be due to the fact that the anticlonidine effect of yohimbine on hypothermia is mediated through its antiserotonin action. The results indicate that LON-954 acts as an alpha 2-antagonist both centrally and peripherally.

Tremorogenesis by physostigmine is unrelated to acetylcholinesterase inhibition: evidence for serotoninergic involvement.

Studies were performed to bring out a serotoninergic involvement in physostigmine tremor, hitherto known to be working via the cholinergic system. 5-Hydroxytryptamine (5-HT) was estimated fluorimetrically after isolation on Sephadex G-10 and acetylcholinesterase (AChE) was assayed spectrophotometrically. The dose-dependent tremor was quantified by a double-blind study. No correlation (r = 0.01) existed between tremor and AChE inhibition since the non-tremoring dose of physostigmine caused the same degree of enzyme inhibition. An increase of 5-HT was found to be correlated (r = 0.59) with the duration and intensity of tremor. Cholinergic antagonists atropine (2 and 5 mg/kg, i.p.), scopolamine (0.5, 1.0, 2.0 mg/kg, i.p.) and mecamylamine (1 mg/kg, i.p.) failed to block the tremor while the 5-HT antagonists methysergide (5 mg/kg, i.v.) and cyproheptadine (10 and 30 mg/kg, s.c.) could afford more than 60% protection. These results suggest a serotoninergic rather than a cholinergic component in the genesis of physostigmine tremor.

5-Hydroxytryptamine in the phrenic nerve diaphragm: evidence for its existence and release.

Evidence is presented for the existence of 5-hydroxytryptamine (5-HT) within the phrenic nerve of the rat and its release following electrical stimulation. Contents of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in the phrenic nerve and the indoleamine released into the bathing fluid were estimated fluorimetrically after isolation on Sephadex G-10 and/or solvent-solvent extraction. Bioassays of 5-HT were done on rat fundus strip. The phrenic nerve and the end-plate zone contains high levels of 5-HT (1.9 micrograms/g wet weight) and 5-HIAA (1.5 micrograms/g wet weight). The resting release of around 1 ng 5-HT/diaphragm/min was enhanced by 50% (1.5 ng 5-HT/diaphragm/min) upon supramaximal (2-4 V) electrical stimulation of 5 Hz. Phrenic nerve diaphragm prepared from the denervated and p-chlorophenylalanine (300 mg/kg/day i.p. for 3 days) treated rats failed to release 5-HT confirming the neuronal origin and the identity of the indoleamine respectively. Furthermore, methysergide, an antagonist of 5-HT in rat fundus strip, blocked the response obtained by the sample on it. A modulatory role of 5-HT in the phrenic nerve diaphragm of the rat is envisaged from the present study.

Evidence for D1 dopamine receptor-mediated modulation of the synaptic transmission from motor axon collaterals to Renshaw cells in the rat spinal cord.

The possible modulatory role of D1 dopamine receptors on the excitability of lumbar spinal Renshaw cells was studied in anesthetized rats spinalized at T4 level. Burst responses elicited by single electrical shocks to ipsilateral ventral roots L6 (frequency 0.5 Hz, stimulus width 0.1 ms) and spontaneous activity were recorded extracellularly using conventional 3 M KCl filled glass micropipettes. The specific D1 agonist SKF 38393 (0.5-1 mg/kg i.v.) enhanced Renshaw cell burst responses by 20-60% (n = 7) and increased their spontaneous discharge rate (n = 3). This effect was clearly antagonized by the specific D1 antagonist SCH 23390 (1 mg/kg i.v.) although SCH 23390 proved ineffective per se. We conclude that SKF 38393 induced facilitation was due to activation of the specific D1 receptors which could be the functional counterpart of the presynaptic D2 receptors described earlier by us in the same synapse.

Triterpenoid saponins from the roots of tea plant (Camellia sinensis var. assamica).

Three olean-12-ene type triterpenoid saponins, named TR-saponins A, B and C, were isolated as methyl esters from tea roots (Camellia sinesis var. assamica) after treatment with diazomethane. Their structures were established as the methyl esters of 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-21, 22-di-O-angeloyl-R1-barrigenol-23-oic acid, 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-21-O-angeloyl-22-O-2-me thylbutanoyl-R1- barrigenol-23-oic acid and 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-16 alpha-O-acetyl-21-O-angeloyl-22-O-2-methylbutanoyl-R1-bar rigenol-23-oic acid, by extensive 1D and 2D-NMR as well as FABMS and HR-MS analyses.

Tremorogenesis by LON-954 [N-carbamoyl-2-(2,6-dichlorophenyl) acetamidine hydrochloride]: evidence for the involvement of 5-hydroxytryptamine.

LON-954 [N-carbamoyl-2-(2,6-dichlorophenyl) acetamidine hydrochloride], a novel tremorogen known to affect the central dopaminergic system, has been investigated in rats for tremor and 5-hydroxytryptamine (5-HT) metabolism. Five, 10 and 20 mg/kg of LON-954 IP caused a reproducible and consistent tremor with a high frequency (16 Hz) within 2 minutes and lasting 30-45 minutes. 5-HT content of the tuberculum olfactorium and basal ganglia was found to be increased significantly at a time when 5-hydroxyindoleacetic acid (5-HIAA) content showed a decrease. 5-HT and 5-HIAA of medulla oblongata showed significant changes only after 15 minutes. The alterations in the levels of the indoleamine in tuberculum olfactorium and its relationship with dosage as well as duration and intensity of LON-954 tremor indicate the involvement of the mesolimbic system in its action. A direct role of 5-HT in LON-tremor was evidenced since the drug failed to produce tremor in rats pretreated with p-chlorophenylalanine (300 mg/kg IP) for 3 days.

Dissociation of serotoninergic and dopaminergic components in acute effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice.

Behavioural and neurochemical effects of acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in mice have been studied in order to determine the change in the neurotransmitter profile of the following areas of the brain: substantia nigra (SN), nucleus caudatus putamen (NCP), limbic system (LS; tuberculum olfactorium and nucleus accumbens), medulla oblongata (MO) and cerebellum (CER). Subcutaneous administration of MPTP (40 mg/kg) caused behavioural syndromes including restlessness, straub tail, hindlimb abduction, tremor, jumping, bradykinesia and akinesia in Balb/c mice. There existed a well-defined biphasic profile of motor activity comprising of an initial excitatory phase followed by an inhibitory phase lasting about two and a half and five hours, respectively. A significant rise in 5-hydroxytryptamine (5-HT) content together with a decreased 5-HT utilization as evidenced by lower 5-hydroxyindole acetic acid (5-HIAA) to 5-HT ratio in the above brain areas demarcated the excitatory phase, whereas the inhibitory phase was distinguished by a significant decrease in dopamine (DA) content along with an increased turnover of the amine as shown by a higher homovanillic acid (HVA) to DA ratio in the functionally important nuclei of the extrapyramidal system like SN, NCP and LS. Methysergide, a nonspecific 5-HT receptor blocker, but not ketanserin, a specific 5-HT2 antagonist, prevented the occurrence of the initial excitatory phase without affecting the depressive phase. Administration of apomorphine, a dopamine agonist, 30 minutes prior to MPTP was ineffective, whereas its application 90 minutes after MPTP prevented the occurrence of bradykinesia and akinesia. Interestingly, treatment with haloperidol, the dopamine (D1/D2) antagonist, before and after MPTP administration caused an early onset and prolongation of the inhibitory phase without affecting the initial hyperexcitement. The results provide direct evidence for the involvement of serotoninergic and dopaminergic mechanisms in the genesis of the early and late syndromes of acute MPTP poisoning respectively.