A novel fungal metabolite NG-061 enhances and mimics neurotrophic effect of nerve growth factor (NGF) on neurite outgrowth in PC12 cells.

During the course of our screening program for low molecular natural products with their ability to potentiate and/or mimic neurotrophic effect of NGF, a novel fungal metabolite, phenylacetic acid hydrazide derivative NG-061 was isolated from the fermentation broth of Penicillium minioluteum F-4627. NG-061 enhanced and mimicked neurotrophic effect of NGF on neurite outgrowth in a rat pheochromocytoma cell line PC12.

Stachybotrin C and parvisporin, novel neuritogenic compounds. I. Taxonomy, isolation, physico-chemical and biological properties.

Stachybotrin C and parvisporin, novel neuritogenic compounds, were isolated from the culture broth of Stachybotrys parvispora F4708. Stachybotrin C induced significant neurite outgrowth in PC12 cells and showed cell survival activity in the primary culture of cerebral cortical neurons. Parvisporin demonstrated only weak neuritogenic activity.

Effects of a M1 muscarinic receptor agonist on the central cholinergic system, evaluated by brain microdialysis.

The effects of a novel M1-receptor agonist, AF102B (FKS-508; cis-2-methylspiro(1,3-oxathiolane-5,3')quinuclidine), on the central cholinergic system in vivo were evaluated by determination of acetylcholine (ACh) content in the rat brain after microwave irradiation and by measurement of ACh release with microdialysis perfusion in freely moving rats. Intraperitoneal administration of AF102B resulted in a significant decrease of ACh content in the brain, while AF102B produced an increase of in vivo ACh release. The present results suggest that ACh content in the brain after treatment with muscarinic agents may be related to the changes of ACh release, in which both M1 and M2 muscarinic receptors may be involved.

Central muscarinic activities of an M1-selective agonist: preferential effect on reversal of amnesia.

Effects of FKS-508 (AF102B; cis-2-methylspiro (1,3-oxathiolane-5,3')-quinuclidine), a novel M1-selective agonist, on central muscarinic responses in mice were examined in comparison with oxotremorine. FKS-508 was slightly less potent (6 times) in reversal of scopolamine-induced amnesia (passive avoidance failure), but far less potent (260 and 55 times) in producing hypothermia and tremor than oxotremorine. These results show that the selective M1 agonist FKS-508 differentiates highly between the central muscarinic effects.

Beneficial effects of FKS-508 (AF102B), a selective M1 agonist, on the impaired working memory in AF64A-treated rats.

The effects of FKS-508 [AF102B; cis-2-methylspiro(1,3-oxathiolane-5,3')quinuclidine], a selective M1 muscarinic receptor agonist, were examined to predict the possible activity on memory disorders using a T-maze and radial-arm maze task in experimental amnesia models. The amnesia models were produced by bilateral intracerebroventricular injection of ethylcholine aziridinium ion (AF64A), a selective cholinotoxin, in rats. Repeated administrations of FKS-508 (5 mg/kg/day, i.p.) for 5 weeks significantly ameliorated impaired performance of AF64A-treated rats (AF64A-rats) in a delayed alternation task in the T-maze. Repeated administrations of FKS-508 (1 and 5 mg/kg/day, p.o.) for 5 weeks significantly ameliorated acquisition failures of AF64A-rats in a radial-arm maze task. Single administration of FKS-508 (1 and 5 mg/kg, p.o.) significantly reduced the incorrect choices of AF64A-rats in a radial-arm maze task with 6 hr-delay time. No abnormalities in general behaviors, such as loss of appetite and ataxia, were observed in rats treated with FKS-508 repeatedly during 5 weeks. Our present results showed that FKS-508 can ameliorate memory impairments in AF64A-rats with central cholinergic hypofunction without causing any behavioral abnormalities. FKS-508 may be considered as a candidate for the clinical examination of the cholinergic hypothesis of senile dementia of the Alzheimer type.

Differential effects of M1- and M2-muscarinic drugs on striatal dopamine release and metabolism in freely moving rats.

A dialysis loop cannula was implanted into rat striatum under anesthetized condition, and the area was perfused with Ringer's solution under freely moving condition after 3 days for surgical recovery. Dopamine (DA) and 3,4-dihydroxyphenylacetic acid recovered in the dialysate were measured by high-performance liquid chromatography with electrochemical detection. The effects of M1- and M2-muscarinic receptor agents, which were perfused continuously into the striatum through the dialysis membrane, were investigated. Continuous perfusion of AF102B, an M1-selective agonist, and oxotremorine, a non-selective agonist, resulted in a dose-dependent increase in the striatal DA release. Pirenzepine (10(-5) and 10(-7) M), an M1-selective antagonist, decreased the release of DA, and the stimulatory effect of AF102B (10(-5) M) was completely inhibited by 10(-5) and 10(-7) M pirenzepine, while the stimulatory effect of oxotremorine (10(-4) M) was only partly inhibited by 10(-5) M pirenzepine. AF-DX116 (10(-5) M), an M2-selective antagonist, increased the DA release, and showed an additive effect on the DA release evoked by AF102B (10(-5) M), whereas it produced no significant effect on oxotremorine (10(-5) M)-evoked DA release. These results suggest that in vivo DA release in the rat striatum is modulated by different subtypes of muscarinic receptors; i.e., the stimulatory effect is mainly mediated by M1-sites and inhibitory effect is mainly mediated by M2-sites. The changes in the DA release induced by the various drugs were prevented by pretreatment with tetrodotoxin (TTX). Since action potential-dependent DA release (exocytosis) is blocked by the pretreatment with TTX, those drugs affect DA release by means of action potential-dependent processes.

Amelioration of experimental amnesia (passive avoidance failure) in rodents by the selective M1 agonist AF102B.

Effect of AF102B (cis-2-methylspiro-(1,3-oxathiolane-5,3')-quinuclidine) on experimental amnesia was examined using a passive avoidance task in rodents. The amnesia was produced by anti-cholinergic agents, AF64A (intracerebroventricularly) and scopolamine (subcutaneously). AF102B ameliorated the memory deficits in AF64A-treated rats at 0.1-1 mg/kg, i.p. and at 1-5 mg/kg p.o. and in scopolamine-treated mice at 1-10 mg/kg, i.p. These results suggest that AF102B may compensate for central cholinergic defects and could be developed as a possible therapeutic drug for senile dementia of the Alzheimer type.

Heterogeneity of muscarinic autoreceptors and heteroreceptors in the rat brain: effects of a novel M1 agonist, AF102B.

The effects of oxotremorine and AF102B (cis-2-methylspiro-(1,3-oxathiolane-5,3')-quinuclidine), a novel M1-selective muscarinic agonist, on acetylcholine (ACh) and dopamine (DA) release from superfused rat hippocampal and striatal synaptosomes were investigated. Synaptosomes that had been prelabeled with [3H]choline or [3H]DA were depolarized by high K+. Oxotremorine and AF102B decreased the K+-evoked [3H]ACh release from hippocampal synaptosomes and increased the K+-evoked [3H]DA release from striatal synaptosomes. The dose-response curves showed that AF102B was far less potent than oxotremorine at the hippocampal presynaptic muscarinic receptors (autoreceptors). On the other hand, AF102B was more potent than oxotremorine at the muscarinic receptors on the striatal dopaminergic terminals (heteroreceptors). Pirenzepine, a selective M1 antagonist, counteracted the effects of oxotremorine on [3H]DA release more potently than it did the effects of oxotremorine on [3H]ACh release. Our results suggest that AF102B and pirenzepine discriminate pharmacologically between muscarinic autoreceptors and heteroreceptors.

Effects of intracerebroventricular injection of AF64A on learning behaviors in rats.

The effects of intracerebroventricular (ICV) injection of ethylcholine aziridinium ion (AF64A) (3 nmole/2 microliter, each lateral ventricule), a putative selective cholinotoxin, on learning behaviors and choline acetyltransferase (ChAT) activity were studied in rats. AF64A-treated rats (AF64A-rat) exhibited deficient performance in a passive avoidance task and a delayed alternation task in the T-maze, but demonstrated superior avoidance response in a two-way shuttle avoidance task. These changes in learning behaviors were associated with the selective decrease of hippocampal ChAT activity. Physostigmine (0.1 mg/kg, i.p.) significantly improved the retention latency of AF64A-rats in the passive avoidance task. AF64A-rats receiving physostigmine (0.2 mg/kg, i.p.) exhibited a slight but not significant improvement of performance in the delayed alternation task in the T-maze. These findings suggested that ICV injection of AF64A may be useful for producing an experimental amnesia model with hippocampal cholinergic hypofunction like Senile dementia of the Alzheimer type (SDAT), if appropriate learning tests are selected.

Dual synaptic effects of activating M1-muscarinic receptors, in superior cervical ganglia of rabbits.

Postsynaptic potentials elicited by various muscarinic agonists and by preganglionic stimuli in the presence of such agonists were recorded from rabbit superior cervical ganglia using sucrose-gap and air-gap methods. While methacholine and bethanechol (both at 10(-4) M) induced biphasic potential changes, McN-A-343 and a novel synthetic compound AF-102B (10(-7) M-10(-5) M) produced only a depolarizing response which was depressed by the M1-antagonist pirenzepine (10(-7) M), but not by the M2 antagonist AF-DX 116 (same concentration), indicating that these compounds act purely as M1-muscarinic agonists in this system. These agonists selectively depressed the orthodromic slow excitatory postsynaptic potential (EPSP) in a dose-dependent manner without substantially affecting the fast EPSP; this is in accord with the view that their depolarizing action is on the same postsynaptic muscarinic receptor that mediates the slow EPSP. The slow inhibitory post synaptic potential (IPSP), on the other hand, was found potentiated in the presence of these agonists. This potentiation was antagonized not only by pirenzepine but also by yohimbine; the potentiation was itself enlarged by nomifensine (a dopamine-uptake inhibitor). We postulate that M1-muscarinic receptors are present not only on the postganglionic principal cells but also on the interneurons; the former were already known to be responsible for the generation of slow EPSP, but the latter may be on terminals of dopamine-containing small intensely fluorescent cells and regulate the orthodromic release of dopamine and are to be distinguished from the M2-receptors.

Parallel but separate release of catecholamines and acetylcholinesterase from stimulated adrenal chromaffin cells in culture.

A pharmacological study was made of the effects of veratridine and lasalocid on the release of catecholamines, acetylcholinesterase (AChE) and dopamine-beta-hydroxylase (DBH) from cultures of isolated bovine adrenal chromaffin cells. Exposure of the cultures to veratridine resulted in concomitant release of catecholamines and AChE into the external medium in a dose-dependent and Ca2+-dependent manner. A Ca2+ ionophore, lasalocid, also produced a dose-dependent and parallel release of both catecholamines and AChE. The release of the two components was accompanied by release of DBH. The present results provide pharmacological evidence for a parallel release of catecholamines, AChE, and DBH from cultured adrenal chromaffin cells, and the stoichiometry of the release evoked by different secretagogues suggests that AChE and catecholamines are released from different cellular compartments.

Nicotine stimulates secretion of both catecholamines and acetylcholinesterase from cultured adrenal chromaffin cells.

There is conflicting evidence from studies on sympathetic ganglia and the adrenal medulla concerning the morphological and biochemical localization and physiological role(s) of the enzyme acetylcholinesterase (AChE). Furthermore, the origin of the AChE released from the adrenal medulla (whether from chromaffin cells or splanchnic nerve, or both) has not been firmly established. We have examined the efficacy of cholinergic agonists to release endogenous AChE and catecholamines (CA) from monolayer cultures of purified bovine adrenal chromaffin cells. The nicotinic agonist (nicotine), but not the muscarinic agonist (methacholine), released both AChE and CA from the adrenal chromaffin cells. The concomitant release of CA and AChE evoked by nicotine was Ca++ dependent with a correlation coefficient r = 0.82 (p less than 0.001). The results show that adrenal chromaffin cells in vitro, a system free of splanchnic nerve elements, can still release AChE. The finding that concomitant release of AChE and catecholamines occurs on exposure of the cells to nicotinic agonists suggests that released AChE may have a physiological role at neuroeffector junctions.

Use of isolated chromaffin cells to study basic release mechanisms.

An account is given of the authors' work with isolated adrenal chromaffin cells to study the synthesis, storage and release of catecholamines and of a number of neuropeptides endogenous to the adrenal medulla. A review of other studies in the literature with the isolated chromaffin cell system is included. It is seen that the isolated chromaffin cells are a convenient in vitro system well-suited to studies of basic release mechanisms. The isolated adrenal chromaffin cells maintain high levels of catecholamines and opiates and release them by exocytosis. The cells have both nicotinic and muscarinic receptors but only the nicotinic are involved in the agonist-evoked release of catecholamines (EC50 nicotine 5 X 10(-6) M: ACh 5 X 10(-5) M). The cells can synthesize AChE and selectively release the 10S molecular form by a mechanism different from exocytosis. Substance P (SP) modulates the secretion of catecholamines and ATP evoked by ACh or nicotine but not that evoked by K+ or veratridine. SP appears to interact with the nicotinic receptor-ionophore complex to regulate Na+ entry. SP receptors on the chromaffin cells show similar structural requirements to SP receptors in other SP responsive tissues. Binding studies on isolated chromaffin cell membranes with [4-3H-Phe]SP have shown specific binding in the nM range. In addition, at high concentrations of ACh, SP protects against nicotinic receptor desensitization. Since SP is contained in the splanchnic nerve terminals that innervate the medulla, the demonstration of SP action and SP receptors on the chromaffin cells suggests a physiological role for SP in the regulation of secretion from the adrenal medulla. Somatostatin (SS) and a number of SS analogues also inhibit release, but are approximately 15-fold less potent than SP. Leu- and Met-enkephalin, which are co-stored with adrenaline in the bovine adrenal medullary cells produce a non-specific inhibition of the nicotine-evoked release of CA, but enhance the basal release of endogenous catecholamines by a mechanism that is Ca2+-dependent, stereospecific and reversible by naloxone and naltrexone. The implication of these peptide-amine interactions for physiological processes regulating homeostasis in the adrenal are discussed.

Evidence against a generalized membrane defect in dystrophic mice platelets.

The response of the membrane-bound enzyme AChE to changes in temperatures was investigated to test the applicability of the "generalized membrane defect" hypothesis proposed for human myotonic and Duchenne muscular dystrophies to the two forms of muscular dystrophy expressed in mice. For intact platelets from homozygous normal and dystrophic mice of both strains, a break (Tc) occurred in the Arrhenius plot of AChE activity at approximately 22 C. Solubilization of membrane-bound AChE by Triton X-100 produced a nonlinear Arrhenius plot over the temperature range (7.7 C to 37 C) in normal and dystrophic mice of both strains. However, in the presence of phospholipase A2 + C and Triton X-100, a linear Arrhenius plot was produced indicating that the membrane-bound enzyme is normally modulated by a bulk lipid domain as well as by a tightly bound (immobilized) phospholipid domain. The temperature response of platelet AChE from normal and dystrophic mice of both strains was not significantly different. These results showing normal temperature kinetics of AChE do not lend support to the theory of a membrane defect in the platelets of dystrophic mice.

An improved technique for the isolation of lymphocytes from small volumes of peripheral mouse blood.

Centrifugation of mouse blood on density gradients of polyvinylpyrrolidone-coated colloidal silica (Percoll) resulted in the simultaneous separation of platelets, lymphocytes, granulocytes and erythrocytes. By this method, we have been successful in isolating a fraction of highly purified and viable lymphocytes from small volumes of peripheral mouse blood with good recovery.

Production and release of acetylcholinesterase by a primary cell culture of bovine adrenal medullary chromaffin cells.

Bovine adrenal chromaffin cells were isolated and maintained as primary culture monolayers. Total acetylcholinesterase (AChE) activity in the cells increased during the culture period, and AChE activity appeared in the culture medium. We have examined the role of the AChE synthesized by the cells on ACh-evoked release of catecholamine from the cells. A progressive decrease in the efficacy of ACh (5 X 10(-5) M) to evoke release of [3H]norepinephrine from day 3-15 cultures suggests that exogenously applied ACh is hydrolyzed by the nascent AChE synthesized by the cells. These findings provide evidence that chromaffin cells produce AChE and release it into their immediate environment.

Pharmacological characterization of adrenal paraneurons: substance P and somatostatin as inhibitory modulators of the nicotinic response.

A pharmacological study was made of the effects of various muscarinic and nicotinic agonists and their antagonists on the release of [3H]noradrenaline ([3H]NA) from cultures of isolated bovine adrenal medullary cells. A study was also made of the effects of substance P and somatostatin on the release of [3H]NA evoked by nicotinic agonists. By 2 days in culture these adrenal 'paraneurons' had developed long varicose processes with growth cones and generally resembled noradrenergic neurons in culture. In the present study, adrenal paraneurons were incubated with [3H]NA which was taken up and stored in reserpine-sensitive sites. Exposure of the cultures to acetylcholine (ACh) resulted in release of [3H]NA into the external medium. High concentrations of K+ (56 mM) also evoked release of [3H]NA. The release of [3H]NA induced by ACh or K+ (56 mM) was Ca2+-dependent. Pharmacological studies with nicotinic (ACh, nicotine) and muscarinic (methacholine, pilocarpine) agonists and their antagonists (mecamylasmine, d-tubocurarine, hexamethonium; and atropine, scopolamine, respectively) showed that the adrenal paraneurons contained only nicotinic receptors. Substance P produced a dose-dependent inhibition of ACh (5 x 10(-5) M) stimulated [3H]NA release in the range of 10(-8) to 5 x 10(-5) M with an ID50 of 10(-6) M. A similar inhibition of NA release by substance P was obtained when nicotine (K X 10(-6) M) was used as the agonist, but not when K+ (50 MM) was used to depolarize the cells. Substance P (10-10) to 5 x 10(-5) M) by itself did not have a significant effect on the basal release rate of [3H]NA from these cells. Somatostatin at relatively high concentrations (10(-6)-10(-3) M; ID50 2 x 10(-5) M) inhibited the release induced by ACh, but not by K+ (56 mM). The present results provide the first direct evidence at a cellular level that substance P and somatostatin act as inhibitory modulators of the nicotinic ACh response, and support a role for these peptides as inhibitory neuromodulators at nicotinic receptor sites in the nervous system.