The initiation of calcium release following muscarinic stimulation in rat lacrimal glands.
1. Acinar cells were isolated from rat lacrimal glands, and the Ca2+ release response of these cells was studied using two experimental approaches. In one approach, changes in Ca2+ concentration, Cai2+, were monitored by measuring Ca2(+)-dependent Cl- currents using tight-seal whole-cell recording. Alternatively, such changes were measured as a fluorescence signal in cells loaded with Fura-2. 2. Following bath application of ACh (0.5 microM), the cell current recorded at -60 mV was unchanged for ca 0.8 s, then rose in a biphasic manner. The initial phase of the current rise ('hump') took different appearances depending on the cell studied, and it sometimes stood out from the main part of the response as a partially isolated transient. 3. In cells which had been loaded with Fura-2, Cai2+ was found to rise abruptly following a silent period. The delay was larger if ACh (0.2-0.5 microM) was applied in a depolarizing isotonic K+ saline than if it was applied in the normal saline. In addition, the maximum of the Cai2+ response was reduced with depolarizing stimulating solutions. This indicates that membrane potential modulates the Cai2+ response. 4. Responses to 5 microM-ACh, a saturating agonist concentration, were almost identical in K+ saline and in normal saline. 5. If the cell potential was hyperpolarized, the delay of the ACh-induced current became shorter. 6. Breaking into an acinar cell with a pipette containing an elevated Ca2+ concentration (0.1-1 mM) led to a transient activation of Ca2(+)-induced currents during the first seconds of whole-cell recording. These transients were obtained more reliably if the transition to the whole-cell mode was achieved by applying a sharp pulse of potential ('zapping') rather than by applying suction to the pipette compartment. At -60 mV, the transients elicited with the former method by 0.5 mM-Ca2+ had a time-to-peak near 0.6s and an amplitude varying between 10 and 600 pA. With 0.1 mM-Ca2+, similar transients were also observed, but a number of cells failed to respond. Calcium-induced transients were blocked if cells were previously loaded with 50 microM-Ruthenium Red. 7. Performing the same experiments with inositol trisphosphate (InsP3, 20 microM) in the pipette solutions also led to early transient Ca2(+)-induced currents. Amplitudes, times-to-peak and 20-80% transition times were similar for 0.5 mM-Ca2+ and 20 microM-InsP3 stimulations.(ABSTRACT TRUNCATED AT 400 WORDS)
Marty
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A
Laboratoire de Neurobiologie, Ecole Normale Supérieure, Paris, France.
Tan
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YP
D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000493:pharmacokinetics*; D002478:Cells, Cultured; D004594:Electrophysiology; D007765:Lacrimal Apparatus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012430:Ruthenium Red / Q000494:pharmacology
D012430:Ruthenium Red; D000109:Acetylcholine; D002118:Calcium