Studies on the Role of Ca^+ Channel Subtypes in Catecholamine Release from Rat Adrenal Chromaffin Cells

Hdl Handle:
http://hdl.handle.net/10675.2/346170
Title:
Studies on the Role of Ca^+ Channel Subtypes in Catecholamine Release from Rat Adrenal Chromaffin Cells
Authors:
Hollins, Bettye
Abstract:
Intracellular Ca2+ functions in many cellular processes including the exocytotic release of neurotransmitters and hormones, gene expression, growth and differentiation. Voltage-dependent Ca^+ channels (VDCC) are the major routes of Ca^+ 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 Ca?+ 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 Ca^+-channel antagonists showed the presence of an N-type channel and an L-type channel. A cadmium-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 showed that the N-type, L-type and R-type comprised 25±13, 45±10 and 28±12 percent of the Ca2+ current, respectively (n=25 cells). The application of antagonists to other VDCC subtypes (such as P- and Q-type) were ineffective on the Ca^+ currents. Release dynamics were evaluated in single chromaffin cells voltage-clamped at the resting potential (-60 mV) and monitored with a carbon-fiber electrode placed next to the cell membrane. The first electrochemical signal arising from the carbon-fiber electrode (a measure of catecholamine released from single vesicles) following a 50 ms pulse to 0 mV, showed a mean release latency of 43±2 ms and 46±3 ms in cells studied 4 to 8 hr and 24 to 36 hr following cell isolation, respectively. In contrast, the probability of release declined by 50 percent over this period. Increasing the total intracellular [Ca^+J via the patch pipette over the range of 20 to 1000 pM produced a concentration dependent increase in secretion in the absence of membrane depolarization. Release was further augmented by depolarizing the cell to 0 mV only in the range of 20 to 100 pM intracellular Ca^+ 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 0 mV. Both blockers produced significant decreases in the number of release events in response to the two stimuli. The data suggest, however, that the inhibition of release in the presence of the N-type channel blocker, © -conotoxin GVIA occurred by a different mechanism than a simple block of Ca^+ current. The L-type channel blocker, nimodipine 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 Ca^+\ 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 channels function in docking of these granules to release sites.
Affiliation:
Not Listed
Issue Date:
Jul-1996
URI:
http://hdl.handle.net/10675.2/346170
Additional Links:
http://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/304326377?accountid=12365
Type:
Dissertation
Appears in Collections:
Theses and Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.authorHollins, Bettyeen
dc.date.accessioned2015-03-05T00:46:40Zen
dc.date.available2015-03-05T00:46:40Zen
dc.date.issued1996-07en
dc.identifier.urihttp://hdl.handle.net/10675.2/346170en
dc.description.abstractIntracellular Ca2+ functions in many cellular processes including the exocytotic release of neurotransmitters and hormones, gene expression, growth and differentiation. Voltage-dependent Ca^+ channels (VDCC) are the major routes of Ca^+ 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 Ca?+ 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 Ca^+-channel antagonists showed the presence of an N-type channel and an L-type channel. A cadmium-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 showed that the N-type, L-type and R-type comprised 25±13, 45±10 and 28±12 percent of the Ca2+ current, respectively (n=25 cells). The application of antagonists to other VDCC subtypes (such as P- and Q-type) were ineffective on the Ca^+ currents. Release dynamics were evaluated in single chromaffin cells voltage-clamped at the resting potential (-60 mV) and monitored with a carbon-fiber electrode placed next to the cell membrane. The first electrochemical signal arising from the carbon-fiber electrode (a measure of catecholamine released from single vesicles) following a 50 ms pulse to 0 mV, showed a mean release latency of 43±2 ms and 46±3 ms in cells studied 4 to 8 hr and 24 to 36 hr following cell isolation, respectively. In contrast, the probability of release declined by 50 percent over this period. Increasing the total intracellular [Ca^+J via the patch pipette over the range of 20 to 1000 pM produced a concentration dependent increase in secretion in the absence of membrane depolarization. Release was further augmented by depolarizing the cell to 0 mV only in the range of 20 to 100 pM intracellular Ca^+ 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 0 mV. Both blockers produced significant decreases in the number of release events in response to the two stimuli. The data suggest, however, that the inhibition of release in the presence of the N-type channel blocker, © -conotoxin GVIA occurred by a different mechanism than a simple block of Ca^+ current. The L-type channel blocker, nimodipine 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 Ca^+\ 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 channels function in docking of these granules to release sites.en
dc.relation.urlhttp://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/304326377?accountid=12365en
dc.rightsCopyright protected. Unauthorized reproduction or use beyond the exceptions granted by the Fair Use clause of U.S. Copyright law may violate federal law.en
dc.subjectCa2+ Channelsen
dc.subjectExocytosisen
dc.subjectrat chromaffin cellsen
dc.subjectNimodipineen
dc.subjectoo-conotoxinen
dc.subjectcarbon-fiber electrodeen
dc.titleStudies on the Role of Ca^+ Channel Subtypes in Catecholamine Release from Rat Adrenal Chromaffin Cellsen
dc.typeDissertationen
dc.contributor.departmentNot Listeden
dc.description.advisorIkeda, Stephen R.en
dc.description.committeeLewis, Deborah; Creazzo, Tony; Caldwell, William; Carrier, Jerryen
dc.description.degreeDoctor of Philosophy (Ph.D.)en
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