title: Calibration of agonist concentrations applied by pressure pulses or via rapid solution exchanger.
abstract: Desensitization of neuronal nicotinic acetylcholine receptors (nAChRs) can develop so rapidly to preclude functional studies when agonists are applied under steady-state conditions. To minimize receptor desensitization, we recorded from rat cultured chromaffin cells currents evoked by nicotine applied by pressure (via a fine tipped pipette) or through a multibarreled rapid solution exchanger. By comparing equi-amplitude, non-fading responses on the same cells, we constructed log 'dose'-response curves with the amount of nicotine expressed as either pulse duration (ms) or initial concentration (microM) inside the perfusion tubes. The linear part of these plots (10-50 ms or 20-100 microM) allowed extrapolating the initial nicotine concentration for a certain pulse duration within these limits. Potential dilution of nicotine in the bath was calculated with diffusion equations for a continuous point source: the maximal concentration of nicotine attainable by pressure application from a 100 microM containing pipette was 92 microM. We also calculated how diluted the same nicotine solution became following rapid superfusion and found it to be 46%. These results indicate that the amount of agonist applied by pressure to a relatively close cell could be calibrated in terms of effective concentration at membrane level and, with the present experimental arrangement, underwent limited dilution in the extracellular microenvironment.
authors.affiliation: Biophysics Sector and INFM Unit, International School for Advanced Studies (SISSA), Via Beirut 4, 34014 Trieste, Italy. diangel@sissa.it
authors.forename: S
authors.initials: S
authors.lastname: Di Angelantonio
authors.forename: A
authors.initials: A
authors.lastname: Nistri
mesh_terms: D000399:Air Pressure; D000818:Animals; D002138:Calibration; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism; D015194:Diffusion Chambers, Culture / Q000379:methods; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems / Q000295:instrumentation / Q000379:methods*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008839:Microelectrodes; D008959:Models, Neurological; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D051381:Rats; D011930:Reaction Time / Q000187:drug effects / Q000502:physiology; D011978:Receptors, Nicotinic / Q000187:drug effects* / Q000378:metabolism
chemical_list: D018722:Nicotinic Agonists; D011978:Receptors, Nicotinic; D009538:Nicotine