Characterization of nicotinic receptors in chick retina using a snake venom neurotoxin that blocks neuronal nicotinic receptor function.

Nicotinic receptor function has been described in the retinas of a variety of vertebrate species. Neuronal bungarotoxin (NBT, also known as bungarotoxin 3.1, toxin F, or kappa-bungarotoxin) blocks nicotinic receptors in several neuronal preparations, while the neuromuscular antagonist alpha-bungarotoxin (BGT) fails to block most of these receptors. NBT (100 nM), but not BGT (10 microM), substantially blocks nicotinic function on ganglion cells in intact chick retina. 125I-NBT binds to 2 sites in homogenates of chick retina; one site that is shared with BGT (Kd = 5-7 nM, Bmax approximately 500 fmol/retina) and one which is not (Kd = 2-3 nM, Bmax approximately 100 fmol/retina). 125I-NBT binding to the NBT-specific site (binding in the presence of 1 microM unlabeled BGT) is localized to 2 bands in the inner plexiform layer, corresponding to regions richly innervated by neurons containing immunoreactivity for choline acetyltransferase. Furthermore, this binding is blocked by competitive nicotinic agonists and antagonists, but nicotine or other nicotinic agonists do not displace 125I-NBT binding with very high affinity relative to the displacement of 3H-nicotine reported by others in brain. Thus, of the 2 NBT binding sites, the site not recognized by BGT most likely represents functional nicotinic receptors in the chick retina, but these receptors have relatively low affinity for nicotinic agonists, similar to nicotinic receptors found in autonomic ganglia.

Nicotinic and muscarinic agonists stimulate rapid protein kinase C translocation in PC12 cells.

Phosphoinositide hydrolysis, a major mechanism for signal transduction in neural cells, generates diacylglycerol, which can in turn activate protein kinase C (PKC). Although cholinergic agonists elicit phosphoinositide hydrolysis in neural tissues, little is known about activation of PKC by cholinergic agonists. PKC requires phosphatidylserine for activation, and in intact cells this lipid requirement is satisfied by binding of the enzyme to cell membranes. Therefore, in intact cells, activation of PKC is often associated with a decrease in cytosolic PKC activity accompanied by an increase in membrane-associated activity. We studied cholinergic-induced activation of PKC by examining changes in the subcellular distribution of the enzyme in PC12 cells treated with cholinergic drugs. Carbachol (1 mM) induced large and rapid increases in membrane-associated PKC activity; a maximal increase of 460% occurred after 5 sec of incubation. Carbachol-induced PKC translocation was concentration-dependent, with a biphasic dose-response curve yielding approximate EC50 values of 10(-6) M and 10(-4) M for the high- and low-affinity components, respectively. Experiments with selective cholinergic agents demonstrated that both muscarinic and nicotinic receptors are involved in carbachol-induced PKC translocation, but the response is predominantly mediated by nicotinic receptor stimulation. Muscarinic-induced association of PKC with cell membrane fractions was resistant to extraction by chelators, whereas nicotinic-mediated membrane binding was partially reduced by homogenization of cells in the presence of EGTA. Omission of calcium from the incubation medium or chelation of calcium with EGTA completely blocked muscarinic- and nicotinic-induced translocation. In addition, the calcium channel blocker nifedipine reduced the nicotinic response by 60%. (ABSTRACT TRUNCATED AT 250 WORDS)

Kappa-bungarotoxin blocks nicotinic transmission at an identified invertebrate central synapse.

A comparison was made between the effects of kappa-bungarotoxin and alpha-bungarotoxin upon nicotinic cholinergic transmission at an identified synapse (the cereal afferent, giant interneurone 2 synapse) in the central nervous system of the cockroach (Periplaneta americana). kappa-Bungarotoxin, a snake venom kappa-neurotoxin, completely blocked nicotinic unitary excitatory postsynaptic potentials (EPSPs) and evoked composite EPSPs when applied at a concentration of 1.0 x 10(-7) moll-1. No recovery was observed after a 2h wash in normal saline. kappa-Bungarotoxin produced a decrease in acetylcholine-induced nicotinic responses which paralleled decreases in nicotinic synaptic potentials and currents, indicating that kappa-bungarotoxin blocked postsynaptic nicotinic receptors. This blockade appeared to be specific as resting membrane potential, input resistance and the ability to elicit an action potential in response to direct stimulation of giant interneurone 2 were unchanged following prolonged toxin exposures. Samples of alpha-bungarotoxin which were free from kappa-neurotoxin contamination were also found to be potent antagonists of cockroach neuronal nicotinic receptors. It is concluded that the cockroach receptor is the first reported example of a neuronal nicotinic receptor which is sensitive to blockade by both kappa-neurotoxins and alpha-neurotoxins.

Chronic treatment with amitriptyline produces supersensitivity to nicotine.

The authors used a thermoregulation paradigm to evaluate effects of amitriptyline (AMI) on the sensitivity of a nicotinic mechanism involved in the regulation of core temperature in rats. Treatment with this tricyclic was associated with a significant increase in the hypothermic response to nicotine. Supersensitivity persisted for a minimum of 7.5 days following the last dose of AMI, and a significant proportion of animals displayed increased sensitivity after 14.5 days of abstinence. Implications for the mechanism of action of AMI are highlighted.

Cholinergic receptors mediating secretion in guinea pig colon.

This study examined the role of cholinergic receptors in mediating the chloride secretory response evoked by stimulation of enteric neurons in muscle-stripped segments of mucosa from guinea pig distal colon set up in flux chambers. Basal short-circuit current was not altered by tetrodotoxin, hexamethonium or muscarinic antagonists. The neurally evoked response was not altered by hexamethonium, but was reduced by muscarinic antagonists with a rank order of potency: atropine = 4-diphenyl acetoxy-N-methylpiperidine greater than pirenzepine. 1,1-Dimethyl-4-phenyl-piperazinium iodide increased resting short-circuit current above basal levels. Carbachol and bethanechol evoked a concentration-dependent increase in short-circuit current that was reduced by tetrodotoxin. The effects of carbachol were antagonized by hexamethonium, 4-diphenyl acetoxy-N-methylpiperidine and pirenzepine. Competition for [3H]quinuclidinyl benzilate binding indicated a rank order of potency of 4-diphenyl acetoxy-N-methylpiperidine greater than pirenzepine greater than carbachol greater than bethanechol. These studies indicate that cholinergic and noncholinergic transmission play a significant role in regulation of chloride transport in the guinea pig distal colon. Both nicotinic and muscarinic receptors on neurons that project to the mucosa as well as epithelial muscarinic receptors are mediators of the chloride secretory response.

Acetylcholine causes rapid nicotinic excitation in the medial habenular nucleus of guinea pig, in vitro.

The actions of ACh in the medial habenular nucleus (MHb) were investigated using extra- and intracellular recording techniques in guinea pig thalamic slice maintained in vitro. Applications of ACh to MHb neurons resulted in rapid excitation followed by inhibition. Neither of these responses was abolished by blockade of synaptic transmission, indicating that they are consequences of ACh action directly on MHb cells. Local applications of the nicotinic agonists nicotine and cytisine caused long-lasting excitation, while applications of another nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium caused both the excitatory and inhibitory responses. Applications of the muscarinic agonists DL-muscarine and acetyl-beta-methylcholine did not consistently cause either the excitatory or inhibitory response. Adding the nicotinic antagonist hexamethonium to the bathing medium blocked both the excitatory and inhibitory ACh responses, while addition of the muscarinic antagonists atropine or scopolamine had no effect. These results indicate that the effects of ACh on MHb neurons are mediated by nicotinic receptors. Intracellular recordings revealed that ACh or nicotine cause an increase in membrane conductance associated with depolarizations that had an average reversal potential of -16 to -11 mV. These results indicate that the ACh-induced excitation is due to an increase in membrane cation conductance. The inhibitory response that follows ACh-induced depolarization and repetitive firing was associated with a hyperpolarization and an increase in membrane conductance. Similar postexcitatory inhibition could also be elicited by direct depolarization or by applications of glutamate, indicating that the hyperpolarizing response to ACh may be an endogenous postexcitatory potential that is not directly coupled to activation of nicotinic receptors. These results suggest that cholinergic transmission in the MHb may be largely of the nicotinic type. This nucleus may be of one of the major regions of the nervous system through which nicotine mediates its central effects.

[Pharmacological modifications of neuroendocrine ontogenesis. Development of receptors, nicotine and catecholamines]. / Modifications pharmacologiques de l'ontogenèse neuroendocrine. D'eveloppement de récepteurs, nicotine et catécholamines.

The prenatal ontogeny of several receptor sites is reviewed. Binding sites for nicotine are found in fetal rat brain from gestational day 14; their regional development is described. Prenatal nicotine exposure interferes with the organization of the gonadal axis of the male rat fetus and is followed by permanent alterations in catecholamine metabolite levels. Cannabis, ethanol, barbiturates and opiates are also capable of disturbing the sexual differentiation of the central nervous system. These observations indicate that interactions with the ontogeny of neuroendocrine systems represent an important pathogenetic mechanism through which drugs can affect brain development. Recent observations on receptor development in the human fetus suggest the existence of a comparable sensitive period.

Is a nicotinic influence involved in denervation-induced depolarization of muscle?

The neurotrophic role of acetylcholine (ACh) in the denervation-dependent decline of muscle resting membrane potential (RMP) was evaluated. Freshly dissected rat hemidiaphragms with short or long (2 cm) nerve stumps attached ("-N" and "+N" preparations, respectively) were incubated in organ culture in the presence or absence of the nicotinic blockers, alpha-bungarotoxin (alpha-BTX) or d-tubocurarine (curare). Subsequently, RMPs and miniature endplate potentials (MEPPs) of the junctional region were measured. Spontaneous MEPPs disappeared with a half-life of 12 and 20 hr in -N and +N preparations, respectively. A 10- to 15-mV depolarization of RMP was observed between 15 and 20 hr in -N muscles and between 24 and 28 hr in +N muscles. This time course of disappearance of spontaneous potentials and of membrane depolarization agrees well with that observed in vivo. Although nicotinic transmission was blocked from the initiation of the incubation period in alpha-BTX- or curare-treated muscles, no acceleration of RMP decline in -N muscles in vitro was observed. Moreover, in +N preparations the effect of the nerve stump in delaying RMP depolarization persisted despite the continuous presence of alpha-BTX or curare. Since excess ACh triggers a lysosomal proteolytic response at the nerve-muscle junction and since this may occur early in denervation, the possible role of a nicotinic-induced proteolytic mechanism was tested in vitro with the potent protease inhibitor leupeptin. This inhibitor did not delay or prevent the denervation-dependent alterations.(ABSTRACT TRUNCATED AT 250 WORDS)

Rates and equilibria for a photoisomerizable antagonist at the acetylcholine receptor of Electrophorus electroplaques.

Voltage-jump and light-flash experiments have been performed on isolated Electrophorus electroplaques exposed simultaneously to nicotinic agonists and to the photoisomerizable compound 2,2'-bis-[alpha-(trimethylammonium)methyl]-azobenzene (2BQ). Dose-response curves are shifted to the right in a nearly parallel fashion by 2BQ, which suggests competitive antagonism; dose-ratio analyses show apparent dissociation constants of 0.3 and 1 microM for the cis and trans isomers, respectively. Flash-induced trans----cis concentration jumps produce the expected decrease in agonist-induced conductance; the time constant is several tens of milliseconds. From the concentration dependence of these rates, we conclude that the association and dissociation rate constants for the cis-2BQ-receptor binding are approximately 10(8) M-1 s-1 and 60 s-1 at 20 degrees C; the Q10 is 3. Flash-induced cis----trans photoisomerizations produce molecular rearrangements of the ligand-receptor complex, but the resulting relaxations probably reflect the kinetics of buffered diffusion rather than of the interaction between trans-2BQ and the receptor. Antagonists seem to bind about an order of magnitude more slowly than agonists at nicotinic receptors.

Neuroregulators and the reinforcement of smoking: towards a biobehavioral explanation.

This paper examines findings on the neuroregulatory effects of nicotine in an effort to formulate a unified hypothesis that can explain the remarkable persistence of smoking behavior. Because nicotine alters the bioavailability of several behaviorally active neuroregulators, including acetylcholine, norepinephrine, dopamine, beta-endorphin, and vasopressin, we propose that nicotine is "used" by smokers to produce temporary improvements in performance or affect. Under this formulation, a potential large number of exteroceptive and interoceptive cues unrelated to the nicotine-dependence cycle may serve as discriminative stimuli for smoking, over and above smoking to terminate or avoid withdrawal. The rapid action of nicotine, and its diverse neuroregulatory effects, render it particularly effective as a "coping response" to the demands of daily living. Of special interest in this regard is a biphasic pattern of arousal/catecholaminergic activation followed by cholinergic blockade or beta-endorphin release. Apparently smokers can adjust nicotine intake to enhance these effects selectively, which may add considerably to the appeal of smoking.

Acetylcholine-induced electrical responses in neuroblastoma cells.

The response to iontophoretic application of acetylcholine in the mouse neuroblastoma cell line N1E-115 was composed of three phases. The initial fast depolarizing phase was blocked by 10 microM d-tubocurarine, but not by 0.1 microM atropine. This phase was followed by a transient hyperpolarization which in turn was followed by a secondary slow depolarization. Both the hyperpolarization and slow depolarization were blocked by atropine (0.1 microM), but not by d-tubocurarine (10 microM). The hyperpolarization and slow depolarization were also evoked by iontophoretic application of the muscarinic agonist methacholine. Under voltage-clamp conditions, an initial fast inward current, a transient outward current, and a secondary slow inward current were recorded in response to acetylcholine application. These three phases of current correspond to the three phases of the membrane potential response. The initial fast inward current increased in amplitude by hyperpolarization of the membrane, and decreased by depolarization. The mean reversal potential was estimated to be -1 mV. The outward current increased in amplitude by depolarization, decreased by hyperpolarization, and reversed its polarity at -67 mV. Alteration of external K+ concentration shifted the reversal potential in the manner expected for an increase in potassium permeability. The slow inward current increased in amplitude by hyperpolarization, decreased by depolarization, and reversed its polarity at +20 mV. It is concluded that the initial fast inward current is mediated by a nicotinic receptor similar to that in muscle end-plate membranes and in postsynaptic membranes of the sympathetic ganglia. Both the outward current and the slow inward current are mediated by muscarinic receptors. The outward current results from an increase in the membrane permeability to K+, and the slow current appears to be carried, at least in part, by Na+.

Reflex activation of postganglionic vasoconstrictor neurones supplying skeletal muscle by stimulation of arterial chemoreceptors via non-nicotinic synaptic mechanisms in sympathetic ganglia.

Postganglionic sympathetic neurones supplying skeletal muscle and skin can be activated from the preganglionic site via cholinergic nicotinic, muscarinic and noncholinergic synaptic mechanisms. The experiments described in this paper were designed in order to show that postganglionic vasoconstrictor neurones supplying skeletal muscle can be activated by the naturally occurring discharge pattern in preganglionic axons when the nicotinic transmission is blocked. For this purpose, the activity was recorded simultaneously from postganglionic vasoconstrictor axons supplying skeletal muscle and vasoconstrictor axons supplying hairy skin. The preganglionic neurones were driven reflexly by stimulation of the arterial chemoreceptors. 1) During blockade of nicotinic transmission muscle vasoconstrictor neurones were activated via the CNS during stimulation of arterial chemoreceptors. This activation is either generated by muscarinic action of released acetylcholine or by a noncholinergic synaptic mechanism. 2) Postganglionic cutaneous vasoconstrictor neurones were inhibited during stimulation of arterial chemoreceptors. During blockade of cholinergic nicotinic transmission these neurones were not activated reflexly by stimulation of arterial chemoreceptors although they received inputs via cholinergic muscarinic and noncholinergic synaptic mechanisms. 3) The results illustrate that postganglionic vasoconstrictor neurones supplying skeletal muscle can not only be activated via non-nicotinic synaptic mechanisms through synchronous repetitive electrical stimulation of preganglionic axons but also by the discharge pattern produced in preganglionic neurones during stimulation of arterial chemoreceptors.

Acyltryptophols reversibly inhibit muscle contractions caused by the actions of acetylcholine and raised potassium ion concentrations.

1. Acetylmethoxytryptophol, originally isolated from the pineal gland, inhibits both the nicotinic and muscarinic receptor stimulatory activities of acetylcholine on frog rectus muscle and guinea pig ileum. 2. Synthetic homologues and analogues, including the acetyl-, propionyl-, butyryl-, and valeryl-methoxytryptophols, tryptophols and alpha-methyl-tryptophols have been prepared and shown to possess similar activity on the frog muscle, with butyryl compounds being the most active. Methoxytryptophol, tryptophol, alpha-methyltryptophol and acetylhydroxytryptophol possess little or no activity. 3. All acyltryptophols tested inhibit the effect of increased potassium concentrations on frog muscle. 4. It is concluded that the acyltryptophols act not at the transmitter receptor level but either at the potassium ion channel or elsewhere in the cell membrane.

Effect of synaptic transmission blockade on morphine action in the guinea-pig myenteric plexus.

Morphine, which inhibits release of acetylcholine from neurons in the myenteric plexus, also inhibits the spontaneous electrical activity of some myenteric neurons. To determine whether morphine acts at a site presynaptic to these neurons, we investigated this morphine effect under conditions of synaptic transmission blockade. Synaptically driven action potentials evoked by point stimulation were recorded extracellularly, and it was shown that all synaptic responses were eliminated or greatly reduced in Ca-free, high-Mg Ringer's with ethylenebis [(oxyethylenenitrilo)]-tetraacetic acid (EGTA), suggesting that synaptic transmission was blocked. Under these conditions, the ability of morphine to inhibit spontaneous electrical activity was virtually unimpaired. Assuming a single locus of narcotic action in the myenteric plexus, it is unlikely, therefore, that the primary action of opiates is to stimulate release of an inhibitory transmitter, to prevent release of an excitatory transmitter or to block the postsynaptic receptor for an excitatory transmitter. Rather, opiates may raise the membrane threshold of a class of neurons. Electric field stimulation activates myenteric neurons, resulting in a morphine-sensitive release of acetylcholine and a contraction of the longitudinal muscle. The ability of field stimulation to induce contractions and of morphine to inhibit these contractions, was virtually unchanged when the only two known excitatory inputs to the cholinergic motor neuron were eliminated by receptor blockade. These observations, taken together, suggest that opiates act directly on the cholinergic motor neuron of the myenteric plexus.