Measurements of intracellular Ca2+ in dissociated type I cells of the rabbit carotid body.
1. The carotid body chemoreceptors are stimulated in situ by cyanide (CN-), which mimics the effect of hypoxia. We have shown that CN- increases a calcium-dependent potassium conductance (gK(Ca)) in single type I cells dissociated from the carotid body of the rabbit. We have now used the Ca2(+)-sensitive fluorophore, Fura-2, to measure intracellular Ca2+ directly in single type I cells. 2. CN- reversibly increased [Ca2+]i from approximately 90 nM to a mean of approximately 200 nM. Some of this Ca2+ originated from an intracellular store, which was depleted by exposure to Ca2(+)-free solutions. Prolonged application of CN- caused a sustained increase in [Ca2+]i, suggesting that CN- impairs the removal or sequestration of Ca2+. 3. pHi measured with the dye BCECF (2,7-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein) did not change consistently in response to CN-, although pHi changed predictably in response to both ammonium chloride and to acidification of the superfusate with CO2. 4. Potassium-induced depolarization (35 mM-K+) caused a large, cadmium-sensitive rise in [Ca2+]i. The K(+)-induced Ca2+ load was used to study the regulation of [Ca2+]i. 5. The clearance of a Ca2+ load was slowed either by removal of [Na+]o or by application of CN-. This shows that both a Na+-Ca2+ exchange and an energy-dependent process or processes contribute to the regulation of [Ca2+]i. 6. Carbachol (CCh, 10-100 microM), which also hyperpolarizes type I cells, caused a small transient rise in [Ca2+]i, indicating release from an exhaustible intracellular pool. The response to CN- was unaffected by prior or continued exposure to CCh, suggesting that the two stimuli operate by distinct mechanisms. 7. The increased gK(Ca) seen in type I cells in response to CN- thus reflects a change in cellular Ca2+ homeostasis. The rise in [Ca2+]i presumably underlies the documented increase in transmitter release from the carotid body in response to CN-. If chemotransduction is a consequence of the release of transmitters from the type I cell, the response of the carotid body to CN-, and possibly also to hypoxia, is thus a direct consequence of the energy dependence of Ca2+ homeostasis in the type I cell.
Biscoe
T J
TJ
Department of Physiology, University College London.
Duchen
M R
MR
Eisner
D A
DA
O'Neill
S C
SC
Valdeolmillos
M
M
D000818:Animals; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002344:Carotid Body / Q000378:metabolism*; D003486:Cyanides / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects; D011188:Potassium / Q000493:pharmacokinetics; D011817:Rabbits; D012964:Sodium / Q000378:metabolism
D001572:Benzofurans; D003486:Cyanides; D005456:Fluorescent Dyes; D002217:Carbachol; D012964:Sodium; D011188:Potassium; D002118:Calcium; D016257:Fura-2