Drastic facilitation by alpha-latrotoxin of bovine chromaffin cell exocytosis without measurable enhancement of Ca2+ entry or [Ca2+]i.
1. Latrotoxin (LTX, 1-3 nM) caused a gradual increase of the spontaneous catecholamine release rate in bovine adrenal chromaffin cells superfused with normal Krebs-Hepes solution containing 2.5 mM Ca2+. Ca2+ removal abolished this effect. LTX enhanced also the secretory responses to high K+ (35 or 70 mM) and to acetylcholine (ACh, 30 microM). 2. The application of Ca2+ pulses to cells previously superfused with a 0 Ca2+ solution (Krebs-Hepes deprived of CaCl2) induced secretory responses that gradually reached 400-800 nA of catecholamines, provided that LTX was present. The responses to ACh or 35 mM K+ pulses (in the presence of Ca2+) were also enhanced by LTX, from around 100-200 nA to over 1000 nA. Though such enhancement remained in the presence of Ca2+ channel blockers, it disappeared upon the lowering of [Na+]o or in electroporated cells. 3. Using protocols similar to those of secretion, LTX did not enhance basal 45Ca2+ uptake, whole-cell Ca2+ currents or basal [Ca2+]i. In fact, LTX attenuated the K(+)- or ACh-evoked increases in 45Ca2+ uptake and [Ca2+]i. 4. It is proposed that the secretory response to brief periods of Ca2+ reintroductions is triggered by local subplasmalemmal Ca2+i transients, produced by the Na(+)-Ca2+ exchanger of the plasma membrane working in the reverse mode. This situation might be physiologically reproduced during ACh stimulation of chromaffin cells, which is followed by the firing of Na(+)-dependent action potentials.
Michelena
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P
Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Spain. pmichelena@mvax.fmed.uam.es
de la Fuente
M T
MT
Vega
T
T
Lara
B
B
López
M G
MG
Gandía
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L
García
A G
AG
D000109:Acetylcholine / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000313:Adrenal Medulla / Q000166:cytology; D000818:Animals; D002021:Buffers; D002118:Calcium / Q000378:metabolism / Q000493:pharmacokinetics*; D015220:Calcium Channels / Q000378:metabolism; D002132:Calcium Radioisotopes; D002395:Catecholamines / Q000502:physiology; D002417:Cattle; D019439:Chromaffin Cells / Q000737:chemistry / Q000166:cytology* / Q000201:enzymology; D018274:Electroporation; D005089:Exocytosis / Q000187:drug effects*; D006531:HEPES; D015640:Ion Channel Gating / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000494:pharmacology; D012636:Secretory Rate / Q000187:drug effects; D015398:Signal Transduction / Q000502:physiology; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D013111:Spider Venoms / Q000494:pharmacology*; D013153:Splanchnic Nerves / Q000502:physiology
D002021:Buffers; D015220:Calcium Channels; D002132:Calcium Radioisotopes; D002395:Catecholamines; D013111:Spider Venoms; C020763:alpha-latrotoxin; D012964:Sodium; D000251:Adenosine Triphosphatases; C034688:sodium calcium ATPase; D000109:Acetylcholine; D011188:Potassium; D006531:HEPES; D002118:Calcium