Potassium-stimulated catecholamine release from superfused bovine adrenal chromaffin cells (70 mM K' in the presence of 2 mM Ca2+ for 10 s, applied at 5-min intervals) was inhibited by the dihydropyridine furnidipine (3 PM) by 50%. o-Conotoxin MVIIC (CTx-MVIIC, 3 PM) also reduced the secretory response by about half. Combined CTx-MVIIC plus furnidipine blocked 100% catecholamine release. "sCa*+ uptake and cytosolic Car' concentrations ([Ca"],) in K'-depolarized cells were partially blocked by furnidipine or CTx-MVIIC, and completely inhibited by both agents. The whole cell current through Ca" channels carried by Ba*' (Ia.) was partially blocked by CTx-MVIIC. Although w-conotoxin GVIA (CTx-GVIA, I PM) and w-agatoxin IVA (Aga-IVA, 0.2 PM) partially inhibited 45CaZ' entry, I,, and the increase in [Ca"],, the combination of both toxins did not affect the K'-evoked secretory response. The results are compatible with the presence in bovine chromaffin cells of a Q-like Ca*' channel which has a prominent role in controlling exocytosis. They also suggest that Q-and L-type Car' channels, but not N-or P-types are localized near exocytotic active sites in the plasmalemma.

No abstract

This study uses a new strategy to investigate the hypothesis that, of the various Ca2+ channels expressed by a neurosecretory cell, a given channel subtype is coupled more tightly to the exocytotic apparatus than others. The approach is based on the prediction that the degree of inhibition of the secretory response by various Ca2+ channel blockers will differ at low (0.5 mM) and high (5 mM) extracellular Ca2+ concentrations ([Ca2+]o). So, at low [Ca2+]o the K+-evoked catecholamine release from superfused bovine chromaffin cells was depressed 60-70% by 2 microM omega-agatoxin IVA (P/Q-type Ca2+ channel blockade), by 3 microM omega-conotoxin MVIIC (N/P/Q-type Ca2+ channel blockade), or by 3 microM lubeluzole (N/P/Q-type Ca2+ channel blockade); in high [Ca2+]o these blockers inhibited the responses by only 20-35%. At 1-3 microM omega-conotoxin GVIA (N-type Ca2+ channel blockade) or 3 microM furnidipine (L-type Ca2+ channel blockade), secretion was inhibited by 30 and 50%, respectively; such inhibitory effects were similar in low or high [Ca2+]o. Combined furnidipine plus omega-conotoxin MVIIC, omega-agatoxin IVA or omega-conotoxin GVIA exhibited additive blocking effects at both Ca2+ concentrations. The results suggest that Q-type Ca2+ channels are coupled more tightly to exocytotic active sites, as compared to L-type channels. This hypothesis if founded in the fact that external Ca2+ that enters the cell through a Ca2+ channel located near to chromaffin vesicles will saturate the K+ secretory response at both [Ca2+]o, i.e. 0.5 mM and 5 mM. In contrast, Ca2+ ions entering through more distant channels will be sequestered by intracellular buffers and, thus, will not saturate the secretory machinery at lower [Ca2+]o.

Catecholamine release from bovine adrenal medulla chromaffin cells superfused with a Krebs-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid solution was monitored on-line with an electrochemical detector. Caffeine (10 mM) progressively depressed the magnitude of secretory responses to depolarizing pulses of 70 mM K+ and 2 mM Ca2+ (70 K+/2 Ca2+) in cells superfused with a Krebs-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid solution containing 0 mM Ca2+ + 0.5 mM EGTA; blockade reached 80% at the third 70 K+/2 Ca2+ challenge given in the presence of caffeine. A similar effect was obtained when, instead of continuous superfusion, prepulses of caffeine were applied (10 mM for 60 s). The blocking effects of caffeine on K+-induced secretion depended on the time of exposure to the drug: the longer the exposure time the greater the blockade. The recovery of the K+ secretory responses previously impaired by caffeine was always gradual and followed a staircase mode. This contrasts with the effects of caffeine on various parameters measuring Ca2+ entry through Ca2+ channels, which did not parallel its effects on K+-evoked secretion. The secretion data, however, are compatible with the disappearance and recovery of an intracellular Ca2+ concentration signal triggered by K+ in single chromaffin cells loaded with fura 2 and treated with 10 mM caffeine. Thus, contrary to previous views, the depression of secretion by caffeine does not seem to be associated with inhibition of extracellular Ca2+ entry through Ca2+ channels. These functional data are, rather, compatible with the view that the degree of filling of a caffeine-sensitive intracellular Ca2+ store might regulate the extent of exocytosis. When emptied, such a store might act as a sink for the external Ca2+ entering through Ca2+ channels during cell depolarization, thus decreasing the intracellular Ca2+ concentration available for exocytosis.