Augmentation of L-type calcium current by hypoxia in rabbit carotid body glomus cells: evidence for a PKC-sensitive pathway.
Previous studies have suggested that voltage-gated Ca(2+) influx in glomus cells plays a critical role in sensory transduction at the carotid body chemoreceptors. The purpose of the present study was to determine the effects of hypoxia on the Ca(2+) current in glomus cells and to elucidate the underlying mechanism(s). Experiments were performed on freshly dissociated glomus cells from rabbit carotid bodies. Ca(2+) current was monitored using the whole cell configuration of the patch-clamp technique, with Ba(2+) as the charge carrier. Hypoxia (pO(2) = 40 mmHg) augmented the Ca(2+) current by 24 +/- 3% (n = 42, at 0 mV) in a voltage-independent manner. This effect was seen in a CO(2)/HCO(3)(-)-, but not in a HEPES-buffered extracellular solution at pH 7.4 (n = 6). When the pH of a HEPES-buffered extracellular solution was lowered from 7.4 to 7. 0, hypoxia augmented the Ca(2+) current by 20 +/- 5% (n = 4, at 0 mV). Nisoldipine, an L-type Ca(2+) channel blocker (2 microM, n = 6), prevented, whereas, omega-conotoxin MVIIC (2 microM, n = 6), an inhibitor of N and P/Q type Ca(2+) channels, did not prevent augmentation of the Ca(2+) current by hypoxia, implying that low oxygen affects L-type Ca(2+) channels in glomus cells. Protein kinase C (PKC) inhibitors, staurosporine (100 nM, n = 6) and bisindolylmaleimide (2 microM, n = 8, at 0 mV), prevented, whereas, a protein kinase A inhibitor (4 nM PKAi, n = 10) did not prevent the hypoxia-induced increase of the Ca(2+) current. Phorbol 12-myristate 13-acetate (PMA, 100 nM), a PKC activator, augmented the Ca(2+) current by 20 +/- 3% (n = 8, at 0 mV). In glomus cells treated with PMA overnight (100 nM), hypoxia did not augment the Ca(2+) current (-3 + 4%, n = 5, at 0 mV). Immunocytochemical analysis revealed PKCdelta-like immunoreactivity in the cytosol of the glomus cells. Following hypoxia (6% O(2) for 5 min), PKCdelta-like immunoreactivity translocated to the plasma membrane in 87 +/- 3% of the cells, indicating PKC activation. These results demonstrate that hypoxia augments Ca(2+) current through L-type Ca(2+) channels via a PKC-sensitive mechanism.
Summers
B A
BA
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Overholt
J L
JL
Prabhakar
N R
NR
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000378:metabolism*; D002344:Carotid Body / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015687:Cell Hypoxia / Q000502:physiology*; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D003600:Cytosol / Q000201:enzymology; D005110:Extracellular Space / Q000378:metabolism; D006531:HEPES / Q000494:pharmacology; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D015737:Nisoldipine / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D015398:Signal Transduction / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001639:Bicarbonates; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D007527:Isoenzymes; D015737:Nisoldipine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D006531:HEPES; D002118:Calcium