1 The effects of ethanol, chloral hydrate and trichloroethanol upon the 5-HT3 receptor have been investigated by use of electrophysiological techniques applied to recombinant 5-HT3 receptor subunits (5-HT(3)R-A or 5-HT(3)R-A(S)) expressed in Xenopus laevis oocytes. Additionally, the influence of trichloroethanol upon the specific binding of [H-3]-granisetron to membrane preparations of HEK 293 cells stably transfected with the murine 5-HT(3)R-A(S) subunit and 5-HT3 receptors endogenous to NG 108-15 cell membranes was assessed. 2 Ethanol (30-300 mM), chloral hydrate (1-30 mM) and trichloroethanol (0.3-10 mM), produced a reversible, concentration-dependent, enhancement of 5-HT-mediated currents recorded from oocytes expressing either the 5-HT(3)R-A, or the 5-HT(3)R-A(S) subunit. 3 Trichloroethanol (5 mM) produced a parallel leftward shift of the 5-HT concentration-response curve, reducing the EC(50) for 5-HT from 1 +/- 0.04 mu M (n = 4) to 0.5 +/- 0.01 mu M (n = 4) for oocytes expressing the 5-HT(3)R-A. A similar shift, from 2.1 +/- 0.05 mu M (n = 11) to 1.3 +/- 0.1 mu M (n = 4), was observed in oocytes expressing the 5-HT(3)R-A(S) subunit. Trichloroethanol (5 mM) had little or no effect upon the maximum current produced by 5-HT for either recombinant receptor. 4 Trichloroethanol (5 mM) similarly reduced the EC(50) for 2-methyl-5-HT from 13 +/- 0.4 mu M (n = 4) to 4.6 +/- 0.2 mu M (n = 4) and from 15 +/- 2 mu M (n = 4) to 5 +/- 0.4 mu M (n = 4) for oocytes expressing the 5-HT(3)R-A and 5-HT(3)R-A(S), subunit respectively. Additionally, trichloroethanol (5 mM) produced a clear enhancement of the maximal current to 2-methyl-5-HT (expressed as a percentage of the maximal current to 5-HT) from 63 +/- 0.7% (n = 4) to 101 +/- 1.6% (n = 4) and from 9 +/- 0.2% (n = 4) to 74 +/- 2% (n = 4) for oocytes expressing the 5-HT(3)R-A and 5-HT(3)R-A(S) subunit respectively. 5 Trichloroethanol (2.5 mM) had no effect upon the K-d, or B-max, of specific [H-3]-granisetron binding to membrane homogenates of NG 108-15 cells or HEK 293 cells. Similarly, competition for [H-3]-granisetron binding by the 5-HT3 receptor antagonists ondansetron and tropisetron was unaffected. However, competition for [H-3]-granisetron binding by the 5-HT3 receptor agonists, 5-HT, 2-methyl-5-HT and phenylbiguanide was enhanced by trichloroethanol (2.5 mM). 6 Unexpectedly, the competition for [H-3]-granisetron binding by the 5-HT3 receptor antagonist, quipazine, was enhanced by 2.5 mM trichloroethanol. Quipazine (1 nM-0.3 mu M) antagonized 5-HT-evoked currents recorded from oocytes expressing the 5-HT(3)R-A subunit with an IC50 of 18 +/- 3 nM (n = 4). Additionally, quipazine (30 nM-0.3 mu M) produced a small inward current which was greatly enhanced by 5 mM trichloroethanol and antagonized by 100 nM ondansetron. Collectively, these observations suggest that quipazine may act as a partial agonist. 7 The demonstration that a recombinant homo-oligomeric receptor, expressed in a foreign membrane, retains a sensitivity to alcohols, together with the sequencing of alcohol-insensitive 5-HT3 receptor subunits, may lead to a better definition of the alcohol binding site(s).
