M. Quik

A role for the nicotinic α-bungarotoxin receptor in neurite outgrowth in PC12 cells

Abstract The addition of nicotine decreased neuritic outgrowth in PC12 cells in culture. This effect occurs as early as one day after addition of nicotine to the culture medium in a concentration-dependent manner. The nicotine-induced decline in neunte outgrowth was prevented by d -tubocurarine (10 − M) indicating that the effect was mediated through a nicotinic receptor. α-Bungarotoxin (10 −8 M) was also able to inhibit the nicotine-induced decrease in process formation in a dose-dependent manner. The concentrations of α-bungarotoxin required to affect process outgrowth correlated with those required to inhibit radiolabelled α-bungarotoxin binding. α-Bungarotoxin had no effect on [ 3 H]noradrenaline release, a functional response mediated through the α-bungarotoxin-insensitive neuronal nicotinic acetylcholine receptor, suggesting that α-bungarotoxin specifically interacts with the neuronal α-bungarotoxin receptor. The present results suggest a functional role for the neuronal nicotinic α-bungarotoxin receptor in neurite outgrowth.

α-Bungarotoxin blocks the nicotinic receptor mediated increase in cell number in a neuroendocrine cell line

Exposure of H69 small cell lung carcinoma cells to nicotinic agonists resulted in a significant increase (up to 100%) in cell number after 6 to 12 days. The effect of nicotine (10(-8) M to 10(-4) M) was both dose and time dependent as was that of another nicotinic agonist cytisine (10(-6) M to 10(-4) M). Interestingly, both the nicotine and cytisine induced increases in H69 cell number were blocked by alpha-bungarotoxin, as well as d-tubocurarine a nicotinic blocker which appears to interact with most nicotinic receptors. These results suggest that the nicotine induced increase in cell number is mediated through an interaction at the nicotinic alpha-bungarotoxin receptor. This idea is further supported by experiments which show (1) that H69 cells possess high affinity alpha-bungarotoxin sites (Kd = 25 nM, Bmax = 10.4 fmol/10(6) cells) with the characteristics of a nicotinic alpha-bungarotoxin receptor and (2) that the potencies of nicotinic receptor ligands in the alpha-bungarotoxin binding assay were similar to those observed in the functional studies. Northern analysis showed that mRNA for alpha 7, a putative nicotinic alpha-bungarotoxin binding subunit, and for alpha 5 were present in H69 cells. The present data provide further evidence that nicotine increases cell number in small cell lung carcinoma and are the first to show that this effect is mediated through an interaction at the nicotinic alpha-bungarotoxin receptor population. These results suggest that the alpha-bungarotoxin site may be involved in modulating proliferative responses in neuroendocrine derived SCLC cells.

Receptor-linked cyclic amp systems in rat neostriatum: Differential localization revealed by kainic acid injection

Various receptor-linked cyclic AMP systems were measured in rat neostriatum 2--14 days after selective destruction of neuronal cell bodies and dendrites by micro-injection of 3 microgram of kainic acid. Basal adenylate cyclase activity was reduced by up to 56% in the injected side and the sensitivity to dopamine was abolished. Up to 84% of cyclic nucleotide phosphodiesterase activity, hydrolyzing either cyclic AMP or cyclic GMP, was destroyed by kainic acid injection. Specific binding of [3H]etorphine and [3H]spiroperidol was reduced by up to 62% in the injected side, while non-specific binding was unchanged. All of these changes were time-dependent, and were greatest 7--14 days after kainic acid treatment. On the other hand, intrastriatal kainic acid injection caused no change in the steady-state concentration of cyclic AMP in striatal slices, or in the in vivo cyclic AMP content in the striatum of rats killed by microwave irradiation. Receptor-mediated increases in cyclic AMP accumulation in striatal slices were either unchanged or markedly potentiated by kainic acid treatment. The maximum response to adenosine was unchanged, while the response to isoprenaline was increased up to 3.7-fold, the response to dopamine increased up to 6.7-fold, and the response to PGE1 increased up to 30-fold. The effect of dopamine in kainic acid-treated striatal slices was no longer blocked by fluphenazine, but was blocked by propranolol, suggesting an interaction of dopamine with a beta-adrenoceptor in kainic acid-treated slices. The results suggest differential cellular localizations of the various receptor-linked cyclic AMP systems in rat neostriatum. Some dopamine and opiate receptors, as well as most of the phosphodiesterase activity, are associated with local neuronal elements, while beta-adrenoceptor, adenosine and PGE1 alterations in cyclic AMP are not. The potentiation of the beta-adrenoceptor and PGE1 responses suggests that they may occur in glial cells. In addition, the pool of adenylate cyclase destroyed by kainic acid appears to make little contribution to normal levels of cyclic AMP in the tissue.

The pharmacology of the nicotinic antagonist, chlorisondamine, investigated in rat brain and autonomic ganglion

1 A single administration of the ganglion blocker, chlorisondamine (10 mg kg−1, s.c.) is known to produce a quasi‐irreversible blockade of the central actions of nicotine in the rat. The mechanism of this persistent action is not known. It is also unclear whether chlorisondamine can block neuronal responses to excitatory amino acids and whether chronic blockade of nicotinic responses also occurs in the periphery. 2 Acute administration of chlorisondamine (10 mg kg−1, s.c.) to rats resulted in a blockade of central nicotinic effects (ataxia and prostration) when tested 1 to 14 days later, but caused no detectable cell death in tissue sections sampled throughout the rostrocaudal extent of the brain which were stained in order to reveal neuronal degeneration. 3 Long‐term blockade of central nicotinic effects by chlorisondamine was not associated with significant alterations in the density (Bmax) of high‐affinity [3H]‐nicotine binding to forebrain cryostat‐cut sections. 4 In cultured dissociated mesencephalic cells of the foetal rat, chlorisondamine and mecamylamine inhibited [3H]‐dopamine release evoked by N‐methyl‐d‐aspartate (NMDA, 10−4 m), but only at high concentrations (IC50 approx. 600 and 70 μm, respectively). A high concentration of chlorisondamine (10−3 m) had no effect on responses to quisqualate (10−5 m) and only slightly reduced responses to kainate (10−4 m). Mecamylamine (10−3 m) was ineffective against both agonists. 5 In adult rat hippocampal slices, chlorisondamine depressed NMDA receptor‐mediated synaptically‐evoked field potentials, but again only at high concentrations (10−4–10−3 m). Synaptic responses that were mediated by non‐NMDA excitatory amino acid receptors were less affected. 6 In rat isolated superior cervical ganglion, electrically‐evoked synaptic transmission was reduced 1 h after acute in vivo administration of chlorisondamine (0.1 mg kg−1, s.c.). However, in vivo administration of a higher dose (10 mg kg−1, s.c.) did not significantly affect ganglionic transmission when tested two weeks later, despite the continued presence of central nicotinic blockade. 7 These results indicate that the persistent CNS nicotinic blockade by chlorisondamine is not accompanied by changes in nicotinic [3H]‐nicotine binding site density or by neuronal degeneration in the brain; that at doses sufficient to produce nicotinic receptor blockade, chlorisondamine acts in a pharmacologically selective manner; and that chronic central blockade is not accompanied by long‐term peripheral ganglionic blockade.

Selective destruction of the serotonergic fibers of the fornix-fimbria and cingulum bundle increases 5-HT1 but not 5-HT2 receptors in rat midbrain.

Selected and localized lesions of serotonergic (5-HT) neurons were made by microinjection of 5,7-dihydroxytryptamine (5,7-DHT), after pretreatment with desipramine, into the cingulum bundle and fornix-fimbria; these are the major serotonergic hippocampal inputs from the median raphe nucleus. Two weeks after the lesion, the binding of [3H]5-HT (5-HT1 receptor) was determined in the hippocampus which receives the afferent terminals and, in addition, in the septum/hypothalamus and midbrain from where the fibers originate. Scatchard analysis showed there was no significant change in binding parameters in the hippocampus; however, a significant increase was observed in the Bmax in the midbrain (38%) with no change in the KD. The caudate which receives 5-HT inputs via other 5-HT tracts was not affected by the lesion. The changes in 5-HT1 receptor number or affinity were not observed 6 days or 5 weeks after the lesion. The binding of the ligands [3H]spiroperidol and [3H]ketanserin to the 5-HT2 receptor population was also determined in the same brain areas; no changes in receptor binding occurred two weeks after the lesion. These experiments demonstrate that a selective lesion of the serotonergic system can increase 5-HT1 receptors in the midbrain, which contains the serotonin cell bodies. In addition, as 5-HT2 binding is not altered, this further supports the hypothesis that 5-HT1 and 5-HT2 receptors are distinct populations of receptors.

Blockade of transmission in rat sympathetic ganglia by a toxin which co-purifies with α-bungarotoxin

Bungarus multicinctus venom was fractionated into its toxin components using ion-exchange chromatography on CM-Sephadex. According to previous reports, rechromatography of fraction II on a CM cellulose column yields chemically homogenous alpha-bungarotoxin (II2) of molecular weight 9000. However, in our hands, using the identical purification procedure, two discrete proteins of molecular weight 9000 and 15,000 were obtained as demonstrated by SDS gel electrophoresis. Subsequent fractionation of this alpha-bungarotoxin fraction (II2) was achieved on Sephadex G-50. The 9000 weight component (labelled II-S2) was identical to alpha-bungarotoxin; at a concentration of 1 microgram/ml it blocked transmission at the neuromuscular junction but did not block nicotinic responses in rat sympathetic ganglia. Very different properties were exhibited by II-SI, the 15,000 molecular weight component; it inhibited ganglionic transmission but was ineffective at the neuromuscular junction at the same concentration (1 microgram/ml). BGT II-S1 was equipotent in blocking the ganglionic action potential in the presence or absence of eserine; thus, it is not acting as an acetylcholinesterase by increasing acetylcholine breakdown. In the presence of toxin, [3H]choline incorporation into ganglionic acetylcholine during preganglionic stimulation was not altered, suggesting that the toxin did not block transmission by a presynaptic mechanism. Thus, the site of action of the toxin appears to be postsynaptic although it did not affect depolarization of the ganglia induced by carbachol.

Regulation of adrenal tyrosine hydroxylase activity: Neuronal versus local control studied with apomorphine☆

Abstract Administration of apomorphine to rats leads to a temporary decrease in adrenal catecholamines and a long-term increase in adrenal tyrosine hydroxylase activity. The kinetic characteristics of tyrosine hydroxylase of the apomorphine-treated and control rats were determined. A significant increase in V max was observed in the treated group as compared to controls; however, there was no change in the K m for the cofactor DMPH 4 or tyrosine between the two groups in undialyzed or dialyzed preparations, indicating that the increase in tyrosine hydroxylase activity was probably due to an increase in enzyme protein. The temporary decrease in adrenal catecholamines was found to be due to increased secretion after apomorphine treatment, even though the concentration of injected apomorphine appeared to be sufficient to inhibit adrenal tyrosine hydroxylase. This was further substantiated by the fact that the decrease in adrenal catecholamines was prevented by adrenal denervation. The delayed increase in tyrosine hydroxylase activity after apomorphine treatment was observed in hypophysectomized rats; however, it was abolished after splanchnic nerve transection. A relationship was sought between the decrease in adrenal catecholamines and increase in adrenal tyrosine hydroxylase activity. When apomorphine and L -3,4-dihydroxyphenylalanine ( L -DOPA) were administered simultaneously, there was no short-term decrease in adrenal catecholamine content but the increased tyrosine hydroxylase activity was still observed. Also, the administration of α -methyl- p -tyrosine to rats decreased the concentration of adrenal catecholamines and yet did not affect adrenal tyrosine hydroxylase activity. The results suggest that increased nerve activity, and not adrenal catecholamine concentrations, regulates the induction of adrenal tyrosine hydroxylase. Furthermore, regulation would be by way of some central mechanism involving dopamine-sensitive receptors.

Dopamine D2 receptor binding in adrenal medulla: Characterization using [3H]spiperone

The possibility that dopamine may function as a neuromodulator or neurotransmitter in the adrenal gland, and not merely serve as a precursor to the catecholamines, has been suggested. If this hypothesis is correct, receptors for dopamine should be identifiable in the adrenal. The present work demonstrates the existence of a high-affinity receptor in adrenal medulla using [3H]spiperone as the radioligand to label the receptors. [3H]Spiperone bound rapidly, reversibly, and with high affinity to bovine adrenal medullary membranes. Scatchard analysis yielded a Kd of 0.09 nM and a Bmax of 51 fmol/mg protein. In competition binding experiments, dopaminergic antagonists were at least 100 times more potent in displacing [3H]spiperone from its binding sites than adrenergic or serotonergic receptor antagonists. Similarly, agonists at the dopamine receptor more readily competed for [3H]spiperone binding than other receptor agonist drugs tested. Furthermore, D2 selective antagonists and agonists were much more potent than D1 receptor ligands. These results suggest that [3H]spiperone may bind to a high-affinity D2 dopamine receptor in adrenal medulla.

Presence of an endogenous factor which inhibits binding of α-bungarotoxin 2.2 to its receptor

Abstract Cerebral cortical membranes and supernatant from rat were prepared by centrifugation of tissue homogenates at 45,000 g for 10 min. The supernatant fraction thus obtained was found to significantly inhibit α-bungarotoxin binding to the membrane preparation. After a 3 min incubation period, the supernatant inhibited toxin binding by approximately 65%, while the inhibition declined to about 40% after 30 min of incubation, presumably due to the slow reversility of α-bungarotoxin binding. The choice of buffer was found to be an important determinant of the degree of inhibition observed, with 10 mM Tris pH 7.4 providing the most effective condition. This inhibition of toxin binding to cortical membranes by the 45,000 g supernatant was shown not to be due to adsorption of the radiolabeled compound to soluble or residual particulate material in the supernatant fraction. Specificity of the supernatant for the α-bungarotoxin site was demonstrated; a supernatant fr action could be prepared which inhibited α-bungarotoxin binding by 50% but had no effect on [ 3 H]spiroperidol (DA 2 and 5-HT 2 ), [ 3 H]prazosin, ( α 1 -adrenergic), [ 3 H]5-hydroxytryptamine (5-HT 1 ) and [ 3 H]quinuclidinylbenzilate (muscarinic cholinergic) binding. The inhibition of toxin binding also occurred in several other CNS regions including hippocampus, brainstem, spinal cord and cerebellum with an 80 to 90% inhibition of binding occuring in the latter two regions. In addition, the 45,000 g cortical supernatant completely prevented the binding of α-bungarotoxin to extrajunctional neuromuscular receptors and inhibited the binding to junctional receptors by 50%. Supernatants prepared from heart, liver and kidney or bovine serum albumin, at a concentration similar to the supernatant fraction, did not alter radiolabeled toxin binding to cortical membranes, while supernatant prepared from striated muscle tissue was effective. These results suggest there may be an endogenous ligand for the α-bungarotoxin 2.2 binding site in tissues which receive nicotinic cholinergic innervation.

Inhibition of nicotinic receptor mediated ion fluxes in rat sympathetic ganglia by BGT II-S1 a potent phospholipase

The mechanism of action of the bungarotoxin fraction II-S1 (BGT II-S1), which copurifies with alpha-bungarotoxin (alpha-BGT) and inhibits nicotinic transmission, has been further characterized. BGT II-S1 (1 microM) inhibited the carbachol (100 microM) or nicotine (50 microM) stimulated uptake of [3H]agmatine into rat sympathetic ganglia by 73% and 52%, respectively. These responses were inhibited 90% by D-tubocurarine (100 microM), but unaffected by alpha-BGT (1 microM) or atropine (10 microM), suggesting that BGT II-S1 affects nicotinic function at a postsynaptic site. Binding of physiologically active [125I]BGT II-S1 could be demonstrated to intact sympathetic ganglia; however, the binding could not be displaced by nicotinic agents, suggesting that BGT II-S1 is not interacting at the receptor. Because some neurotoxins produce their effect at the synapse through a phospholytic action, the phospholipase activity of BGT II-S1 was determined. The results demonstrate that BGT II-S1 is a very potent calcium dependent phospholipase. In addition, conditions which abolished the toxins phospholytic activity prevented its effects on nicotinic transmission and on nicotinic receptor mediated ion fluxes. These include irreversible inhibition of enzymic activity by treatment of BGT II-S1 with p-bromophenacylbromide, as well as reversible inhibition of the phospholipase by substitution of Ba2+ or Sr2+ ions for Ca2+ ions in the physiological medium. Thus, in rat sympathetic ganglia, BGT II-S1 blocks the nicotinic receptor mediated movement of ions across the membrane. This is probably not due to a direct interaction at the nicotinic acetylcholine recognition site; rather, it may be an ion channel associated effect which is mediated by alterations in the phospholipid environment of the receptor complex or of the membrane. Although BGT II-S1 also has presynaptic actions, in a cultured system of postsynaptic cells, it could prove a useful tool to study the role of phospholipids in neuronal nicotinic receptor regulation.