Augmentation of L-Type Calcium Current by Hypoxia in Rabbit Carotid Body Glomus Cells: Evidence for a PKC-Sensitive Pathway

Abstract

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.

Multiple targets of chemosensitive signaling in locus coeruleus neurons: role of K+ and Ca2+channels

“…Full blockage required at least 15 min of exposure to nifedipine. These data clearly indicate that HA activates L-type Ca 2ϩ channels in LC neurons, most likely by an indirect pathway as previously observed in glomus cells (56).…”

“…This neuron displayed very few Na ϩ spikes, but note that HA increased the frequency of SROs. Recent work on carotid body glomus cells has shown an augmentation of L-type Ca 2ϩ currents during hypercapnia (56). To test whether the HA-induced Ca 2ϩ spikes in LC neurons were mediated through L-type Ca 2ϩ channel activation, we exposed these neurons to the L-type Ca 2ϩ channel blocker nifedipine.…”

“…In another type of chemosensitive cell, the peripheral chemoreceptors or type-I glomus cells (30), L-type Ca 2ϩ channels were shown to be activated in the presence of hypoxia (55,61). Although hypoxia resulted in increased membrane excitability in these cells through both a membrane depolarization and activation of Ca 2ϩ channels (61), it was shown that the mechanism of activation of Ca 2ϩ channels was not directly linked to V m changes but was attributed to a PKC-mediated pathway (56).…”

Hypoxia. 4. Hypoxia and ion channel function

“…However, the effects of hypoxia on in vitro carotid body preparations are depressed when CO 2 /bicarbonate-free (i.e., HEPES-buffered) extracellular solution is utilized (105,201), which causes a considerable alkaline shift in intracellular pH (30). In contrast to the studies in which HEPES-buffered solutions were used and no excitatory effect of hypoxia was observed (92,134,147,169), in carotid bodies superfused with bicarbonate-based extracellular solution, hypoxia caused an increase in Ca 2ϩ current (I Ca ) that was blocked by inhibitors of protein kinase C (PKC) (213), indicating that hypoxia could increase [Ca 2ϩ ] i by phosphorylation-dependent activation of VGCCs. These results suggest that direct enhancement of VGCCs activation coupled with a K ϩ channel-mediated shift in E m could work in concert to produce Ca 2ϩ influx.…”

H 2 S mediates O 2 sensing in the carotid body

“…Previous studies showed that Ca 2+ influx via high voltage-gated Ca 2+ channels, especially the L-type, is necessary for carotid body stimulation by hypoxia (1). L-type Ca 2+ channels in glomus cells are redox sensitive, activated by hypoxia, and inhibited under normoxia, as well as by gaseous messengers such as NO (22,23). A recent study demonstrated that H 2 S signaling involves covalent modification of redox sensitive cysteine residues in proteins through S-sulfhydration (10).…”