Recombinant human 5-HT3A receptors in outside-out patches of HEK 293 cells: basic properties and barbiturate effects.
The patch-clamp technique was used on excised (outside-out) patches to characterize h5-HT3A receptors stably transfected in HEK 293 cells and to compare the effects of the barbiturate anaesthetics methohexital and pentobarbital on this ligand-gated cation channel. At negative membrane potentials 5-HT induced inward currents in a concentration-dependent manner (EC50=8.6 microM, Hill coefficient =1.5). The mean peak current induced by 30 microM 5-HT was -110 pA at -100 mV. The 5-HT3A receptor antagonist ondansetron (0.3 nM) reversibly inhibited the 5-HT (30 microM) signal by 70% and at 3 nM it abolished the response. Methohexital and pentobarbital inhibited 5-HT-induced (30 microM) currents in a concentration-dependent manner. The maximal inhibition with a given methohexital or pentobarbital concentration was reached when the respective drug was applied 45 s prior to and during the 2-s 5-HT pulse (IC50 values=95 microM and 127 microM, Hill coefficient = -1.0 and -1.6, respectively). Although the barbiturates were, thus, equipotent, their effects differed substantially with respect to the dependence on the time schedule of application to the patches: the potency of methohexital was virtually maximal when the drug was applied exclusively 45 s before the agonist pulse, but its inhibitory potency decreased considerably when it was exclusively applied during the 2-s 5-HT pulse (IC50=380 microM). Conversely, pentobarbital was almost maximally potent in inhibiting the 5-HT signal when it was exclusively coapplied with this agonist, but its inhibitory potency was considerably lower (IC50 approximately 500 microM) when applied exclusively 45 s before 5-HT. Another difference between both barbiturates involves the rate of inactivation of 5-HT3 receptor-mediated currents: whereas high concentrations of methohexital (> or = 300 microM) were necessary to induce moderate (< or = twofold) acceleration of this parameter, pentobarbital produced such an effect at all concentrations and the extent of acceleration increased with increasing concentration (1.5- to fivefold). In conclusion, two barbiturates, chemically closely related but of different lipophilicity, clearly differ with respect to the kinetics of their effect on 5-HT3 receptor channels; one possible explanation involves drug access to an amphipathic site of action via both an aqueous and a hydrophobic pathway. Pentobarbital, in contrast to methohexital, inhibits hS-HT3A receptor-mediated currents at anaesthetic concentrations (approximately 90 microM).
Barann
M
M
Institut für Pharmakologie und Toxikologie, Universität Bonn, Germany. barann@mailer.meb.uni-bonn.de
Meder
W
W
Dorner
Z
Z
Brüss
M
M
Bönisch
H
H
Göthert
M
M
Urban
B W
BW
D018686:Anesthetics, Intravenous / Q000494:pharmacology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation; D004594:Electrophysiology; D005071:Evoked Potentials / Q000187:drug effects; D006801:Humans; D006993:Hypnotics and Sedatives / Q000494:pharmacology*; D008723:Methohexital / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D010424:Pentobarbital / Q000494:pharmacology*; D011985:Receptors, Serotonin / Q000187:drug effects*; D044406:Receptors, Serotonin, 5-HT3; D014162:Transfection
D018686:Anesthetics, Intravenous; D006993:Hypnotics and Sedatives; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D008723:Methohexital; D010424:Pentobarbital