HYPOXIA DECREASES INTRACELLULAR CALCIUM IN ADULT-RAT CAROTID-BODY GLOMUS CELLS

1. Carotid body chemoreceptors were removed intact from adult rats and subjected to protease and collagenase enzymatic digestion of connective tissue. 2. Recordings from the sinus nerve demonstrated that chemotransduction remains intact for at least 2-3 h after isolation, enzyme exposure, and suspension in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline at room P(o2). 3. After mechanical dissociation, the interrelationship between changes in extracellular P(o2) and pH and relative changes in intracellular calcium (Ca(i)2+) were observed in glomus cells with the use of fluo-3 and confocal microscopy. 4. Brief (60-s) decreases in P(o2) froM 150 mmHg to near 0 mmHg, at nadir, caused a marked reduction in Ca(i)2+ (peak DELTA-F/F0 = -32 +/- 3%, mean +/- SE, n = 43), which rapidly recovered after reoxygenation. The decrease was reproducible from trial to trial and was also observed in HCO3--buffered Ringer solution. 5. Superfusion with Ca2+-free HEPES saline with 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) blocked the hypoxia-induced increase in afferent chemoreceptor activity in vitro. Superfusion of the same solution over isolated cells for 15 min caused a large decrease in Ca(i)2+ (-34 +/- 7%, n = 16). 6. In the presence of Ca2+-free HEPES, reoxygenation caused calcium fluorescence to increase. This suggests that the Ca2+ decrease during hypoxia is due, at least partially, to binding to an intracellular site. 7. Extracellular cobalt (1 mM, 15 min) also reversibly blocked the chemoreceptor response to hypoxia, in vitro, and caused a reduction in Ca(i)2+ (DELTA-F/F0 = -37 +/- 8%, n = 11). Hypoxia caused a further, reversible decrease in Ca(i)2+. 8. In contrast to the effects of hypoxia, extracellular acidity (pH = 6.9) caused a relatively small increase in afferent activity and slight, but significant, decrease in Ca(i)2+ (-6 +/- 2%, n = 12). 9. We conclude that the following: 1) Hypoxia causes a reduction in rat glomus cell Ca(i)2+. 2) Extracellular calcium availability is important for Ca(i)2+ homeostasis. 3) Factors that reduce Ca(i)2+ before a hypoxia exposure cause a large reduction in chemoreceptor hypoxia sensitivity. We speculate that a hypoxia-induced increase in glomus cell Ca(i)2+ is not the primary signal leading to enhanced secretion. Rather, binding of free calcium to an intracellular site may regulate glomus cell secretion.