Intracellular calcium and its sodium-independent regulation in voltage-clamped snail neurones.
1. We have used both Ca(2+)-sensitive microelectrodes and fura-2 to measure the intracellular free calcium ion concentration ([Ca2+]i or its negative log, pCai) of snail neurones voltage clamped to -50 or -60 mV. Using Ca(2+)-sensitive microelectrodes, [Ca2+]i was found to be approximately 174 nM and pCai, 6.76 +/- 0.09 (mean +/- S.E.M.; n = 11); using fura-2, [Ca2+]i was approximately 40 nM and pCai, 7.44 +/- 0.06 (mean +/- S.E.M., n = 10). 2. Depolarizations (1-20 s) caused an increase in [Ca2+]i which was abolished by removal of extracellular Ca2+, indicating that the rise in [Ca2+]i was due to Ca2+ influx through voltage-activated Ca2+ channels. 3. Caffeine (10-20 mM) caused an increase in [Ca2+]i in the presence or absence of extracellular Ca2+. The effects of caffeine on [Ca2+]i could be prevented by ryanodine. 4. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a small increase in resting [Ca2+]i and slowed the rate of recovery from Ca2+ loads following 20 s depolarizations. 5. Neither replacement of extracellular sodium with N-methyl-D-glucamine (NMDG), nor loading the cells with intracellular sodium, had any effect on resting [Ca2+]i or the rate of recovery of [Ca2+]i following depolarizations. 6. The mitochondrial uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCmP) caused a small gradual rise in resting [Ca2+]i. Removal of extracellular sodium during exposure to CCmP had no further effect on [Ca2+]i. 7. Intracellular orthovanadate caused an increase in resting [Ca2+]i and prevented the full recovery of [Ca2+]i following small Ca2+ loads, but removal of extracellular sodium did not cause a rise in [Ca2+]i. We conclude that there is no Na(+)-Ca2+ exchanger present in the cell body of these neurones and that [Ca2+]i is maintained by an ATP-dependent Ca2+ pump.
Kennedy
H J
HJ
Department of Physiology, School of Medical Sciences, Bristol, UK.
Thomas
R C
RC
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D005110:Extracellular Space / Q000378:metabolism; D016257:Fura-2; D006372:Helix, Snails; D007425:Intracellular Membranes / Q000378:metabolism*; D008928:Mitochondria / Q000502:physiology; D009474:Neurons / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D012016:Reference Values; D012433:Ryanodine / Q000494:pharmacology; D012964:Sodium / Q000502:physiology*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014638:Vanadates / Q000378:metabolism
D013729:Terpenes; D012433:Ryanodine; D002110:Caffeine; D014638:Vanadates; D019284:Thapsigargin; D012964:Sodium; D000252:Calcium-Transporting ATPases; D002118:Calcium; D016257:Fura-2