Involvement of voltage-dependent Ca2+ channels in cytosolic Ca2+ increase induced by deprivation of extracellular cations in adrenal chromaffin cells of the guinea pig.
The mechanisms of an increase in cytosolic Ca2+ concentration ([Ca2+]i) due to the removal of extracellular cations were examined using the whole-cell patch-clamp technique in combination with fura-2 microfluorometry in guinea pig adrenal chromaffin cells. Removal of Ca2+ and Mg2+ from sucrose-substituted Na+-free solution (0Ca, 0Mg-sucrose) evoked an increase in [Ca2+]i that was suppressed by the addition of Ca2+, Mg2+ or EGTA extracellularly. When Na+ was replaced by Tris+, NMDG+, Li+, Cs+ or choline+, no comparable increase in [Ca2+]i was evoked in the absence of external Ca2+. In voltage-clamped cells at -60 mV with K+-internal solution, 0Ca, 0Mg-sucrose evoked a transient [Ca2+]i increase concomitant with an outward current. In Cs+-loaded cells, 0Ca, 0Mg-sucrose caused a marked inward current (Iin) accompanied by an increase of [Ca2+]i. Choline+-substituted solution produced only a brief Iin without any increase of [Ca2+]i. Both Iin and [Ca2+]i responses to 0Ca, 0Mg-sucrose occurred with holding potentials more positive than -65 mV. The increase in [Ca2+]i expected from the time-integrated Iin evoked by 0Ca, 0Mg-sucrose was correlated with that in [Ca2+]i measured with fura-2. A voltage-dependent Ca2+ channel (VDC) blocker reversibly suppressed the Iin and [Ca2+]i responses to 0Ca, 0Mg-sucrose. Cyclopiazonic acid, an internal store-emptying agent, did not affect both current and [Ca2+]i responses to 0Ca, 0Mg-sucrose. Brief exposure to external EGTA greatly reduced these responses, suggesting that Ca2+ near and/or bound to the cell membrane plays a role in 0Ca, 0Mg-sucrose-induced responses. In sucrose-substituted solution, current-voltage relations for VDC were shifted to negative potentials by decreasing the external Ca2+. These results suggest that VDC activation, probably due to the change in the surface potentials on the gating mechanism of VDC, is involved in the elevation of [Ca2+]i induced by deprivation of divalent cations from sucrose substituted Na+-free solution in adrenal chromaffin cells.
Ohta
T
T
Laboratory of Pharmacology, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 0600818, Japan.tohta@vetmed.hokudai.ac.jp
Ito
S
S
Kaneko
M
M
Nakazato
Y
Y
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology*; D002412:Cations / Q000378:metabolism; D019439:Chromaffin Cells / Q000378:metabolism*; D003470:Culture Media; D003600:Cytosol / Q000378:metabolism*; D006168:Guinea Pigs; D016014:Linear Models; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D012964:Sodium / Q000502:physiology; D013395:Sucrose / Q000494:pharmacology
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002412:Cations; D003470:Culture Media; D013395:Sucrose; D012964:Sodium; D002118:Calcium