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    Studies on the role of calcium channel subtypes in catecholamine release from rat adrenal chromaffin cells

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    Authors
    Hollins, Bettye
    Issue Date
    1996-07
    URI

    http://hdl.handle.net/10675.2/623175
    
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    Abstract
    Intracellular ca2+ functions in many cellular processes including the exocytotic release of neurotransmitters and hormones, gene expression, growth and differentiation. Voltage-dependent Ca2+ channels (VDCC) are the maj~r routes of ca2+ entry into cells · and although most neurons and neuroendocrine cells express multiple VDCC subtypes, it is unclear whether the co-expression of multiple subtypes represents a means of subserving the~various ca2+ functions. The present study examined the VDCC subtypes in isolated rat adrenal chromaffin cells and evaluated individual subtypes for preferential effects on catecholamine release. Pharmacological subtyping of VDCC with ca2+ -channel antagonists showed the presence of ~n :N-type channel .and an L-type channel. A cadt;nium-sensitive residual current (resistant or R-type channel) ·remained in the combined presence of both N- and' L-type channel antagonists. Serial application of the antagonists to the same cell show~d . ~ ·,/"'' ,._-~ that the N-type, L-type and R-type comprised 25±13, 45±10 and 28±12 percent ofthe Ca2+ current, respectively (n=25 cells). The applicatio_n of antag?nists to other VDCC subtypes (such as P- and Q-type) were ineffective on the Ca2+ currents. Release dynamics were evaluated in single chromaffin cells voltage-clamped at th.~_resting potential (-60 mV) and monitored with a carbon-fiber elec~ode.placed next to ,·the cell membrane. The first electrochemical signal arising from the carbon-fiber f1, (· electfode (a measure of catecholamine released from single: vesicles) follo~ing a 50 ms _ pulse to O mV, showed a mean release latency of 43±2 ms ·and 46±3 t)lS in cells studied 4, to 8 hr and 24 to.36 hr following cell isolation, respectively. In contrast, the p~obability·b'f release declined by 50 percent over this period. Increasing the total intracellular. [Ca2+] via the patch pipette over the range of 20 to 1000 μM produced a concentration dependent increase in secretion in the absence of membrane depolarization. Release was further augmented by depolarizing the cell to O m V only in the range of 20 to 100 μM intracellular ca2+ indicating a maximum rate of release above these values. Release in the presence and absence of N- and L-type channel blockers was compared for a pre-recorded acetylcholine-induced action potential command (AP command) and rectangular voltage pulses to O m V. Both blockers produced significant decreases in the number of release events in response to the t~o stimuli. · The data suggest, ·however, that the inhibition of release in the presence of the N-type channel blocker, ro-conotoxin GVIA occurred by a different mechanism than a si.mple block of Ca2+ current. The· L-type channel blocker, nimodipi_ne suppressed release by 87 and 73% in response to the AP command and rectangular voltage pulses, respectively. The percentage block was greater in normal saline than in solutions that isolated Ca2+ currents (50% inhibition) and suggests that K+ channels serve a role -in antagonism of release that is independent of its role in repolarization. The incomplete block of release in the presence . of both inhibitors suggests that other channel subtypes are likely to participate in release, albeit to a lesser extent. A model is proposed in which L-type channels function in recruitment of granules for release while N-type c~annels fu~ction in docking of these granules to release sites.
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    School of Graduate Studies
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