Measurements of intracellular Ca2+ in dissociated type I cells of the rabbit carotid body.

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Abstract
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.

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