Calcitonin gene-related peptide rapidly downregulates nicotinic receptor function and slowly raises intracellular Ca2+ in rat chromaffin cells in vitro. (25/4103)

Although calcitonin gene-related peptide (CGRP) modulates muscle-type nicotinic acetylcholine receptors (nAChRs) via intracellular second messenger-mediated phosphorylation, the action of this peptide on neuronal-type nAChRs remains unknown. Using neuronal nAChRs of rat chromaffin cells in vitro we studied the effect of CGRP, which is physiologically present in adrenal medulla, on membrane currents and [Ca2+]i transients elicited by nicotine. Our main novel observation was that CGRP (either bath-applied or focally applied for a few seconds or even co-applied with nicotine for a few milliseconds) selectively and rapidly blocked nAChRs (a phenomenon unlikely caused by intracellular messengers in view of its speed) without affecting GABA receptors. The inhibitory effect of CGRP was independent of [Ca2+]i or membrane potential and not accompanied by baseline current changes. Like the competitive antagonist N,N,N-trimethyl-1-(4-trans-stilbenoxy)-2-propilammonium, CGRP induced a rightward, parallel shift of the nicotine dose-response curve; during co-application of these blockers the nicotine dose-ratio value was the sum of the values obtained with each antagonist alone. The block by CGRP was insensitive to the receptor antagonist hCGRP8-37 but mimicked by CGRP1-7. Persistent application of CGRP slowly increased [Ca2+]i, a phenomenon independent from external Ca2+, thus implying Ca2+ release from internal stores, and suppressed by hCGRP8-37. CGRP1-7 had no significant effect on [Ca2+]i. We propose that the 1-7 amino acid sequence of CGRP was responsible for the direct, rapid block of nAChRs, whereas the full-length peptide molecule was necessary for the delayed rise in internal Ca2+ potentially able to trigger phosphorylation-dependent modulation of nicotinic receptor function.  (+info)

Cholinergic and GABAergic inputs drive patterned spontaneous motoneuron activity before target contact. (26/4103)

Patterned spontaneous electrical activity has been demonstrated in a number of developing neural circuits and has been proposed to play a role in refining connectivity once axons reach their targets. Using an isolated spinal cord preparation, we have found that chick lumbosacral motor axons exhibit highly regular bursts of activity from embryonic day 4 (E4) (stage 24-25), shortly after they exit the spinal cord and while still en route toward their target muscles. Similar bursts could be evoked by stimulating descending pathways at cervical or thoracic levels. Unlike older embryonic cord circuits, the major excitatory transmitter driving activity was not glutamate but acetylcholine, acting primarily though nicotinic non-alpha7 receptors. The circuit driving bursting was surprisingly robust and plastic, because bursting was only transiently blocked by cholinergic antagonists, and following recovery, was now driven by GABAergic inputs. Permanent blockade of spontaneous activity was only achieved by a combination of cholinergic antagonists and bicuculline, a GABAA antagonist. The early occurrence of patterned motor activity suggests that it could be playing a role in either peripheral pathfinding or spinal cord circuit formation and maturation. Finally, the characteristic differences in burst parameters already evident between different motoneuron pools at E4 would require that the combination of transcription factors responsible for specifying pool identity to have acted even earlier.  (+info)

Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus. (27/4103)

Multiple Site Optical Recording of Transmembrane Voltage (MSORTV) has been used to measure, continuously and simultaneously, the spontaneous electrical activity from all of the neurons in individual ganglia or up to five interconnected ganglia of the submucous plexus of the guinea pig small intestine. These are the first optical recordings of electrical activity with single-cell resolution from a mammalian nervous system. They are used to investigate the effects of acute and chronic application of nicotine on the firing patterns of this neural network containing important cholinergic components. After washout of acutely applied nicotine, the firing rates of selected neurons were dramatically elevated. These results suggest that nAChRs that reversibly desensitize after exposure to nicotine may be responsible for the enhancement of activity that is observed after a brief application of this agonist. In addition, immunostaining with monoclonal antibodies was used to localize alpha3/alpha5, alpha7, and beta2 nAChR subunits, and the results demonstrate the prevalence of alpha3/alpha5. It is this alpha3-containing nAChR subtype that probably accounts for most of the excess activity elicited by nicotine application.  (+info)

Somatic and prejunctional nicotinic receptors in cultured rat sympathetic neurones show different agonist profiles. (28/4103)

1. The release of [3H]-noradrenaline ([3H]-NA) in response to nicotinic acetylcholine receptor (nAChR) agonists was compared with agonist-induced currents in cultured rat superior cervical ganglion (SCG) neurones. 2. [3H]-NA release in response to high concentrations of nicotinic agonists was reduced, but not fully inhibited, by the presence of either tetrodotoxin (TTX) or Cd2+ to block voltage-gated Na+ or Ca2+ channels, respectively. We used the component of transmitter release that remained in the presence of these substances (named TTX- or Cd2+-insensitive release) to pharmacologically characterize nAChRs in proximity to the sites of vesicular exocytosis (prejunctional receptors). Prejunctional nAChRs were activated by nicotinic agonists with a rank order of potency of dimethylphenylpiperazinium iodide (DMPP) > nicotine > cytisine > ACh, and with EC50 values ranging from 22 microM (DMPP) to 110 microM (ACh). 3. [3H]-NA release in response to low concentrations of nAChR agonists was fully inhibited by the presence of either TTX or Cd2+ (named TTX- or Cd2+-sensitive release). TTX-sensitive release was triggered by nicotinic agonists with a rank order of potency of DMPP > cytisine approximately nicotine approximately ACh, which due to its similarity to TTX-insensitive release indicates that it might also be triggered by prejunctional-type nAChRs. The EC50 values for TTX (Cd2+)-sensitive release were less than 10 microM for all four agonists. 4. By contrast to transmitter release, somatic nAChRs as seen by patch clamp recordings were most potently activated by cytisine, with a rank order of potency of cytisine > nicotine approximately DMPP > ACh. EC50 values for the induction of currents exceeded 20 microM for all four agonists. 5. The nicotinic antagonist mecamylamine potently inhibited all transmitter release in response to nicotine. alpha-Bungarotoxin (alpha-BuTX) was, on the other hand, without significant effect on nicotine-induced TTX-insensitive release. The competitive antagonist dihydro-beta-erythroidine (DHbetaE) caused rightward shifts of the dose-response curves for both TTX-sensitive and TTX-insensitive transmitter release as well as for currents in response to nicotine, with pA2 values ranging from 4.03 to 4.58. 6. Due to clear differences in the pharmacology of agonists we propose that nAChRs of distinct subunit composition are differentially targeted to somatic or axonal domains.  (+info)

Structures of the M2 channel-lining segments from nicotinic acetylcholine and NMDA receptors by NMR spectroscopy. (29/4103)

The structures of functional peptides corresponding to the predicted channel-lining M2 segments of the nicotinic acetylcholine receptor (AChR) and of a glutamate receptor of the NMDA subtype (NMDAR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. Both M2 segments form straight transmembrane alpha-helices with no kinks. The AChR M2 peptide inserts in the lipid bilayer at an angle of 12 degrees relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminal side of the membrane, which is assigned to be intracellular. A model built from these solid-state NMR data, and assuming a symmetric pentameric arrangement of M2 helices, results in a funnel-like architecture for the channel, with the wide opening on the N-terminal intracellular side.  (+info)

Dependence of nicotinic acetylcholine receptor recovery from desensitization on the duration of agonist exposure. (30/4103)

When subjected to prolonged exposure to nicotinic agonists, nicotinic acetylcholine receptors undergo desensitization, resulting in an inactive receptor that does not allow for the passage of ions. The induction of desensitization of diverse nicotinic acetylcholine receptor subtypes in muscle, ganglia, or brain is likely to play important modulatory roles in synaptic transmission. Furthermore, nicotinic receptor desensitization may contribute to behavioral changes in humans or animals subjected to prolonged nicotine exposure pharmacologically or through the use of tobacco products. We investigated the recovery from desensitization of muscle-type nicotinic acetylcholine receptors in TE671/RD cells induced by exposure to acetylcholine or nicotine. Rates of recovery from desensitization are dependent on the length of agonist exposure and on the agonist used to induce desensitization. Increasing the time of exposure results in an increase in the time constant of recovery for both agonists. The recovery from nicotine-induced desensitization is consistently faster than the recovery from acetylcholine-induced desensitization regardless of whether nicotine or acetylcholine is used to assess levels of desensitization. These findings suggest the existence of more than one state of receptor desensitization and that nicotinic agonists vary in their efficiency of inducing receptors to states of differing depths of desensitization.  (+info)

Effects of ethanol on recombinant human neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes. (31/4103)

Alcohol and tobacco use is highly correlated in humans, and studies with animal models suggest an interaction of alcohol with neuronal nicotinic acetylcholine receptors (nAChRs). The aim of the present study was to characterize the effect of acute ethanol treatment on different combinations of human nAChR (hnAChR) subunits expressed in Xenopus oocytes. Ethanol (75 mM) potentiated ACh-induced currents in alpha2beta4, alpha4beta4, alpha2beta2, and alpha4beta2 receptors. This effect was due to an increase in Emax, without a change in the EC50 or Hill coefficient. hnAChR alpha2beta4 did not develop tolerance to repeated applications of ethanol or continuous exposure (10 min). The alpha3beta2 and alpha3beta4 combinations were insensitive to ethanol. Low concentrations of ethanol (25 and 50 mM) significantly inhibited homomeric alpha7 receptor function, but these receptors showed highly variable responses to ethanol. These results indicate that ethanol effects on hnAChRs depend on the receptor subunit composition. In light of recent evidence indicating that nAChRs mediate and modulate synaptic transmission in the central nervous system, we postulate that acute intoxication might involve ethanol-induced alterations in the function of these receptors.  (+info)

Two pharmacologically distinct components of nicotinic receptor-mediated rubidium efflux in mouse brain require the beta2 subunit. (32/4103)

Nicotinic agonist-stimulated efflux of 86Rb+ from mouse brain synaptosomes was monitored continuously by on-line radioactivity detection. The concentration-effect curve following a 5-s stimulation with acetylcholine was biphasic (EC50 = 7.2 and 550 microM). alpha-Bungarotoxin (100 nM) did not inhibit the response, but dihydro-beta-erythroidine (DHbetaE) blocked both phases with differing potency (average IC50 =.22 and 8.9 microM for responses activated by low and high acetylcholine concentrations, respectively). Differential sensitivity DHbetaE inhibition was used to measure stimulation of 86Rb+ efflux by 17 nicotinic agonists, which differed markedly in potency and efficacy. All agonists were more potent at the DHbetaE-sensitive site. Both components were inhibited by the six antagonists tested. Methyllycaconitine and DHbetaE were more potent for the DHbetaE-sensitive component, whereas hexamethonium was more potent at the DHbetaE-resistant component. Both DHbetaE-sensitive and DHbetaE-resistant responses were reduced more than 95% in beta2-null mutant mice, establishing the requirement for the beta2 subunit for both components. Both components were widely, but not identically, distributed throughout the brain. The DHbetaE-sensitive component appears to be identical with agonist-stimulated 86Rb+ efflux described previously and is likely to be mediated by alpha4beta2 receptors. The DHbetaE-resistant component is a novel, active, and widely distributed response mediated by nicotinic receptor(s) that also require the beta2 subunit.  (+info)