Effects of diltiazem on human nicotinic acetylcholine and GABAA receptors
Introduction
The molecular cloning of ligand-gated ion channels in the last 20 years has permitted the study of the pharmacology of these proteins in isolation in expression systems, thus avoiding the ambiguities often encountered in native cells where multiple ion channels co-exist. Several of these studies have revealed that many of the drugs currently in use as therapeutic agents or pharmacological probes have a wider spectrum of action than previously known. Thus, for example, strychnine, a selective antagonist of glycine-gated Cl− channels, inhibits several types of nicotinic acetylcholine receptors (AChR). These include chick native α-bungarotoxin sensitive receptors (Anand et al., 1993), human recombinant neuronal α2β2 and α4β2 receptors (Garcı́a-Colunga and Miledi, 1999), α7 homomers (Peng et al., 1994) and muscle α1β1γδ, α1β1δ and α1β1γ receptors (Garcı́a-Colunga and Miledi, 1999). Other drugs such as the intravenous and volatile anaesthetics have been shown to potentiate GABA-gated Cl− channels but inhibit acetylcholine (ACh)-gated cationic channels (Franks and Lieb, 1994).
The importance of these studies is that they raise the possibility that many ligand-gated ion channels may play roles hitherto unsuspected in physiological processes as well as in drug therapy. The nicotinic AChR of adrenal chromaffin cells illustrate most noticeably this development. Adrenal chromaffin cells secrete catecholamines when stimulated by ACh released from presynaptic terminals at the splanchnic nerve–chromaffin cell synapse. The released catecholamines provide a relative small, slow-acting, but effective adjunct to the autonomic innervation of the heart, increasing myocardial vigour and heart rate. Several studies have shown that L-type Ca2+ antagonists such as diltiazem, verapamil and the dihydropyridines inhibit the nicotinic AChR of chromaffin cells (Gandı́a et al., 1991, Lopéz et al., 1993). Diltiazem is of particular interest because it is more potent on nAChR than on L-type Ca2+ channels. Thus, 10 μM diltiazem inhibits whole-cell Ba2+ currents through high voltage activated Ca2+ channels by only 20–25%, whilst the same concentration of the drug caused full blockade of whole-cell currents elicited by 1,1-dimethyl-4-phenyl-piperazinium (Gandı́a et al., 1996). This finding has prompted the suggestion that the nicotinic effects of diltiazem may contribute to the antihypertensive and cardioprotective effects of the drug (Gandı́a et al., 1996).
Nicotinic AChR present on chromaffin cells include those that contain α3 and β4 subunits (α3β4 nicotinic AChR) or those appearing to assemble as homomers of α7 subunits (α7 nicotinic AChR) (Garcı́a-Guzmán et al., 1995, Campos-Caro et al., 1997). Although both α3β4 and α7 nicotinic AChR contribute to cathecolamine release (Lopéz et al., 1998), studies of rat recombinant α3β4 and α7 receptors have shown that only α3β4 receptors are inhibited by clinically relevant concentrations of L-type Ca2+ channel antagonists (Herrero et al., 1999). However, since species differences may affect the pharmacology of receptors, we have examined the effect of diltiazem on human recombinant α7 nicotinic AChR expressed in Xenopus oocytes. We have also investigated the effects of diltiazem on other human variants of members of the ligand-gated ion channel family, including α1β2γ2s GABAA receptors, muscle nicotinic α1β1δγ and α1β1δε AChR, and the neuronal nicotinic α4β2 AChR. The results presented here show that diltiazem inhibits nicotinic AChR but has no effects on GABAA receptors. We also present here a detailed study of the effects of diltiazem on human α7 receptors. We show that inhibition of α7 receptors is non-competitive and voltage-independent and that the diltiazem site may be distinct from the site at which another L-type Ca2+ channel antagonist, verapamil, interacts with nicotinic AChR. Our results open the possibility that the nicotinic α7 receptor could become a drug target in therapy of cardiovascular diseases.
Section snippets
Preparation of RNA transcripts and Xenopus oocyte injection
cDNAs encoding the human α1, β2 and γ2S GABAA receptor subunits engineered into the expression vector pCDM8 or pcDNAI/Amp were provided by Dr Paul Whiting (Merck, Sharp and Dohme, Harlow, UK). Human neuronal nicotinic ACh receptor subunit cDNAs were provided by Professor Jon Lindstrom (University of Pennsylvania, USA) ligated into plasmid vectors pSP64 (α4, β2) and pMXT (α7). cDNAs encoding the human foetal and adult muscle nAChR were supplied by Dr David Beeson (Institute of Molecular
Effects of diltiazem on currents mediated by human GABAA and nAChR
The benzothiazepine L-type Ca2+ channel inhibitor diltiazem has been reported to inhibit rat recombinant α3β4 and α7 nicotinic AChR (Herrero et al., 1999) and 5HT3 receptors (Hargreaves et al., 1996). To investigate the effects of diltiazem on human members of the four transmembrane ligand-gated ion channel family we investigated the action of diltiazem on Xenopus oocytes expressing either human α1β2γ2S GABAA receptors or neuronal (α7 or α4β2) or muscle (α1β1δγ or α1β1δε) nicotinic AChR. We
Discussion
The results presented here show that the L-type Ca2+ channel antagonist diltiazem reversibly inhibits human nicotinic ACh receptors at μM concentrations. The findings, therefore, support the view that in addition to its better known effects on Ca2+ channels, diltiazem interacts with specific sites on ligand-gated ion channels (Lopéz et al., 1993, Hargreaves et al., 1996, Lopéz et al., 1998, Herrero et al., 1999). The effect of diltiazem on ligand-gated ion channels is, however, restricted to
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2006, NeuropharmacologyCitation Excerpt :At this holding potential, nicotine elicited a considerably larger inward current than at −60 mV (Fig. 6), yet 5 μM nimodipine still blocked 96.2 ± 0.4% of the current through nAChRs (Fig. 6B,C), virtually the same degree of inhibition as at −60 mV (p > 0.4, unpaired two-tailed Student's t-test). These results suggest that LTCC ligands inhibit nAChRs independent of their actions on LTCCs, consistent with previous studies of recombinant nAChRs (Herrero et al., 1999; Houlihan et al., 2000; Sala et al., 2002). As shown above (Fig. 1C,D), induction of nuclear pCREB by either nicotine or 40 mM K+ is completely inhibited by the DHP nimodipine.