Acetylcholine‐evoked currents in cultured neurones dissociated from rat parasympathetic cardiac ganglia.

Abstract

SUMMARY1. The properties of acetylcholine (ACh)-activated ion channels of parasympathetic neurones from neonatal rat cardiac ganglia grown in tissue culture were examined using patch clamp recording techniques. Membrane currents evoked by ACh were mimicked by nicotine, attenuated by neuronal bungarotoxin, and unaffected by atropine, suggesting that the ACh-induced currents are mediated by nicotinic receptor activation.2. The current-voltage (I-V) relationship for whole-cell ACh-evoked currents exhibited strong inward rectification and a reversal (zero current) potential of -3 mV (NaCl outside, CsCl inside). The rectification was not alleviated by changing the main permeant cation or by removal of divalent cations from the intracellular or extracellular solutions. Unitary ACh-activated currents exhibited a linear I-V relationship with slope conductances of 32 pS in cell-attached membrane patches and 38 pS in excised membrane patches with symmetrical CsCl solutions.3. Acetylcholine-induced currents were reversibly inhibited in a dose-dependent manner by the ganglionic antagonists, mecamylamine (Kd = 37 nM) and hexamethonium (IC50 1 UtM), as well as by the neuromuscular relaxant, d-tubocurarine (Kd = 3 /M). Inhibition of ACh-evoked currents by hexamethonium could not be described by a simple blocking model for drug-receptor interaction.4. The amplitude of the ionic current through the open channel was dependent on the extracellular Na+ concentration. The direction of the shift in reversal potential upon replacement of NaCl by mannitol indicates that the neuronal nicotinic receptor channel is cation selective and the magnitude suggests a high cation to anion permeability ratio. The cation permeability (Px/PNa) followed the ionic selectivity sequence Cs+(1 06) > Na+(1P0) > Ca2+(093). Anion substitution experiments showed a relative anion permeability, PCi/pNa 1< 005.5. The nicotinic ACh-activated channels described mediate the responses of postganglionic parasympathetic neurones of the mammalian heart to vagal stimulation.

Nicotinic Acetylcholine Receptors

“…The calcium permeability of ganglion-type nicotinic receptors is reported to be similar to that of adult muscle in experiments on superior cervical ganglion, intracardiac ganglia and chromaffin cells, with values for calcium permeability (relative to sodium or caesium) between 0.5 and 1 and fractional current measurement between 2.5 and 4.7% (Fieber & Adams, 1991;Zhou & Neher, 1993;Vernino et al, 1992;Vernino et al, 1994;Nutter & Adams, 1995;Rogers & Dani, 1995). This is in broad agreement with recombinant work on heteromeric receptors or native receptors likely to be heteromeric (Mulle et al, 1992a;Costa et al, 1994;Ragozzino et al, 1998).…”

“…Heterologous expression data show that mecamylamine is not selective for the different receptor types (see for instance Chavez-Noriega et al, 1997;ChavezNoriega et al, 2000), and is effective at low micromolar concentrations. At concentrations greater than 1 µM mecamylamine is an open channel blocker on recombinant α4β2 receptors (Bertrand et al, 1990), and on nicotinic receptors of intracardiac ganglia (Fieber & Adams, 1991) and chromaffin cells (Nooney et al, 1992b;Giniatullin et al, 2000). Indeed, there is now evidence that, like hexamethonium, mecamylamine gives rise to a persistent block because it is trapped in the channel.…”

“…Thus, recovery is speeded up by combining agonist application with membrane depolarisation: modelling of use dependence and time course of recovery suggests that channels which have trapped blocker open more slowly (Giniatullin et al, 2000). Nevertheless, when mecamylamine is applied at very much lower concentrations to rat parasympathetic ganglion neurones, it is more effective against low agonist concentrations (Ascher et al, 1979;Fieber & Adams, 1991). This observation is inconsistent with channel block and suggests a competitive mechanism at these low concentrations, with a Kd of the order of 25-50 nM.…”

Nicotinic receptor signaling in nonexcitable cells

“…The P Ca /P Na ratios calculated for nAChRs range from about 1 for ganglionic nAChRs (Fieber and Adams, 1991;Rathouz and Berg, 1994) to about 20 for heterologously expressed ␣7 gene product (Seguela et al, 1993). The ␣Bgt-sensitive current from cultured hippocampal neurons exhibit a P Ca /P Na ratio of 5 (Castro and Albuquerque, 1995).…”

A Receptor-Mediated Mechanism of Nicotine Toxicity in Oral Keratinocytes

“…To identify the Nic-induced structural changes of keratinocyte nAChRs, we exposed normal human OKC to pure Nic or whole AEST containing the same concentration of Nic and in both cases found dramatic changes in the subunit composition. We observed increases in the mRNA and/or protein levels of the subunits that form the ACh-gated ion channels that are permeable to Ca 2ϩ (Delbono et al, 1997;Fieber and Adams, 1991;Grando et al, 1996;Quik et al, 1997). A switch in the nAChR subunit composition from predominantly ␣3␤2 and ␣3␤4 channels to ␣3␤2␣5 and ␣3␤4␣5 channels increases Ca 2ϩ influx (Gerzanich et al, 1998), which may launch the keratinocyte differentiation program (Grando et al, 1996).…”