Q‐ and L‐type Ca²⁺ channels dominate the control of secretion in bovine chromaffin cells

Abstract

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.

Inhibition of nicotinic receptor‐mediated responses in bovine chromaffin cells by diltiazem

“…Thus, it was not unexpected that diltiazem inhibited only a minor component of the K+-evoked 45Ca2+ uptake (Figures 2 and 3) implying that only the L-type, but not N-P-Q-types of Ca21 channels are blocked by this drug. In fact, in the presence of cw-conotoxin MVIIC, a blocker of N, P and Q channels in these cells (Lopez et al, 1994) diltiazem still inhibited an additional component of K+-evoked 45Ca2+ uptake (Figure 3 IBa, it seems that the effects of diltiazem on neuronal nicotinic receptors are selective and specific in these cells. Concerning the mechanism of blockade of IDMPP, it is interesting that diltiazem inhibits more the integrated IDMPP than the peak IDMPP ( Figure 6).…”

“…The following materials were used: collagenase from Clostridium histolyticum (Boehringer-Manheim); bovine serum albumin fraction V, cytosine arabinoside, fluorodeoxyuridine, 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) and EGTA (Sigma); foetal calf serum, penicillin and streptomycin (GIBCO); scintillation fluid Ready micro (Beckman); 45Ca (specific activity 10-40 mCi mg-' calcium, Amersham (Lopez et al, 1994) and diltiazem could only block the L-component. This possibility was studied in the experiment shown in Figure 3.…”

Fast exocytosis mediated by T- and L-type channels in chromaffin cells: distinct voltage-dependence but similar Ca2+-dependence

“…This slower exocytotic process is primarily controlled by Ca 2+ inXux through multiple isoforms of high-voltage activated Ca 2+ channels, which contribute to secretion proportionally to their density of expression and gating properties, with no speciWc requirements of Ca 2+ channels colocalization to the release sites (Kim et al 1995;Engisch and Nowycky 1996;Klingauf and Neher 1997;Lukyanetz and Neher 1999;Ulate et al 2000;Carabelli et al 2003;Chan et al 2005;Giancippoli et al 2006;Polo-Parada et al 2006). Alternatively, other groups suggest a preferential role of some channel subtype in close proximity to the secretory sites (Lopez et al 1994;Artalejo et al 1994;Lara et al 1998;O'Farrel and Marley 2000;Albillos et al 2000) and a further role of mitochondria in sequestering Ca 2+ entering through Ca 2+ channels (Ales et al 2005). In addition to this, the eYciency of stimulus-secretion coupling can be altered in many ways, some relevant examples are: (1) PKC activation, which enhances exocytosis (Gillis et al 1996), (2) rapid and sustained increase of intracellular cAMP, which potentiates Ltype channel densities and secretion at diVerent proportions , (3) transdiVerentiation of chromaYn cells with astrocyte-conditioned medium, which promotes the acquisition of a neuronal phenotype and accelerates the exocytotic kinetics, by a better coupling between secretory vesicles and Ca 2+ channels (Ardiles et al 2006) and (4) -adrenergic stimulation, which enhances the stimulus-secretion coupling of Ltype Ca 2+ channels by repeated action potential-like stimulations (Polo-Parada et al 2006).…”

Neurite extensions in chromaffin cells: study of the influence of the cytoskeletal structure on calcium dynamics and secretion

“…As can be seen, the secretory response is both faster and steeper in neurites, in agreement with the experimental observations. In any case, this mathematical approach gives theoretical support to the concept that preferential localization of specific calcium channel subtypes in neuronal cells facilitates a coordinated strong exocytotic response in the neurite terminals (Catterall 1998) or a more efficient coupling of channel subtypes with the secretory response in neuroendocrine cells (Lopez et al 1994;Garcia et al 1998). In addition, even if there is secretory response that is proportional to the overall calcium elevation rather than the presence of specific channels subtypes (Lukyanetz & Neher 1999), the asymmetric distribution of calcium channel subtypes may influence the voltage dependence of secretory inactivation (Villarroya et al 1999), the posterior process of endocytosis (Rosa et al 2011), the clustering of SNAREs forming the secretory machinery or directly influence the exocytotic machinery (Seagar et al 1999;Atlas et al 2001).…”