Modulation of BKCa Channels by Protein Kinase C in Idiopathic Pulmonary Arterial Hypertension

Hdl Handle:
http://hdl.handle.net/10675.2/318807
Title:
Modulation of BKCa Channels by Protein Kinase C in Idiopathic Pulmonary Arterial Hypertension
Authors:
Zhu, Shu
Abstract:
Idiopathic pulmonary arterial hypertension (IPAH) is a severe and progressive vascular disease which ultimately leads to right-side heart failure and death. Pulmonary vasoconstriction is thought to be a key factor that leads to IPAH. The large-conductance, voltage- and calcium-activated potassium (BKCa) channel is a key element that controls vascular smooth muscle tone. Inhibition of BKCa channels in pulmonary arterial smooth muscle cells (PASMC) cause membrane depolarization and pulmonary vasoconstriction. We have shown that protein kinase C (PKC) inhibits BKCa channels in PASMC and constricts pulmonary arteries from the pulmonary hypertensive Fawn-Hooded rat (FHR). However, the underlying mechanism is unknown. Patch-clamp studies demonstrated that 100μM IBMX -a non-specific phosphodiesterase (PDE) inhibitor, 10μM milrinone -a specific PDE3 inhibitor, or 10μM zaprinast -a specific PDE5 inhibitor, reversed the inhibitory effect of 100nM thymeleatoxin (a PKC activator) on BKCa channels in FHR PASMC. Additionally, 100nM PMA (a PKC activator) increased PDE activity significantly, and also decreased cyclic nucleotide concentrations significantly. In inside-out patches, application of 2nM purified PKC catalytic subunits blocked the BKCa channel activity significantly. Furthermore, a cell-membrane permeable and PDE-resistant cyclic nucleotide analog, 100μM CPT-cAMP, reversed the inhibitory effect of 100nM thymeleatoxin on BKCa channels. Serine1076 (S1076) is a conserved PKC phosphorylation site on the C-terminus of the human BKCa channel α-subunit, and phospho-null (S1076A) and phospho-mimetic (S1076E) forms of this PKC phosphorylation site were created. Western blot revealed equal BKCa channel expression in wild-type BKCa-α/β1, BKCa-α(S1076A)/β1 and BKCa-α(S1076E)/β1-transfected HEK293 cells. Patch-clamp recordings demonstrated that wild-type BKCa-α/β1 was activated by PKC and PKG, while BKCa-(S1076A)/β1 responded to the stimulation of PKC to a lesser extent, but was unresponsive to PKG. In contrast, BKCa-α(S1076E)/β1 enhanced the stimulatory effect of PKC. Thus, PKC may inhibit BKCa channels in FHR PASMC primarily through PDE activity, and also by directly acting on BKCa channels. In addition, the PKC phosphorylation site S1076 on the human BKCa channel α-subunit is very important for the regulation of channel function. The findings in this study should increase our understanding of how PKC modulates BKCa channels in IPAH, and may contribute to the development of new approaches to treat IPAH.
Affiliation:
Department of Pharmacology and Toxicology
Issue Date:
Oct-2007
URI:
http://hdl.handle.net/10675.2/318807
Additional Links:
http://ezproxy.augusta.edu/login?url=http://search.proquest.com/docview/304775595?accountid=12365
Type:
Dissertation
Language:
en_US
Appears in Collections:
Theses and Dissertations; Department of Pharmacology and Toxicology Theses and Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.authorZhu, Shuen
dc.date.accessioned2014-06-03T20:14:07Z-
dc.date.available2014-06-03T20:14:07Z-
dc.date.issued2007-10-
dc.identifier.urihttp://hdl.handle.net/10675.2/318807-
dc.description.abstractIdiopathic pulmonary arterial hypertension (IPAH) is a severe and progressive vascular disease which ultimately leads to right-side heart failure and death. Pulmonary vasoconstriction is thought to be a key factor that leads to IPAH. The large-conductance, voltage- and calcium-activated potassium (BKCa) channel is a key element that controls vascular smooth muscle tone. Inhibition of BKCa channels in pulmonary arterial smooth muscle cells (PASMC) cause membrane depolarization and pulmonary vasoconstriction. We have shown that protein kinase C (PKC) inhibits BKCa channels in PASMC and constricts pulmonary arteries from the pulmonary hypertensive Fawn-Hooded rat (FHR). However, the underlying mechanism is unknown. Patch-clamp studies demonstrated that 100μM IBMX -a non-specific phosphodiesterase (PDE) inhibitor, 10μM milrinone -a specific PDE3 inhibitor, or 10μM zaprinast -a specific PDE5 inhibitor, reversed the inhibitory effect of 100nM thymeleatoxin (a PKC activator) on BKCa channels in FHR PASMC. Additionally, 100nM PMA (a PKC activator) increased PDE activity significantly, and also decreased cyclic nucleotide concentrations significantly. In inside-out patches, application of 2nM purified PKC catalytic subunits blocked the BKCa channel activity significantly. Furthermore, a cell-membrane permeable and PDE-resistant cyclic nucleotide analog, 100μM CPT-cAMP, reversed the inhibitory effect of 100nM thymeleatoxin on BKCa channels. Serine1076 (S1076) is a conserved PKC phosphorylation site on the C-terminus of the human BKCa channel α-subunit, and phospho-null (S1076A) and phospho-mimetic (S1076E) forms of this PKC phosphorylation site were created. Western blot revealed equal BKCa channel expression in wild-type BKCa-α/β1, BKCa-α(S1076A)/β1 and BKCa-α(S1076E)/β1-transfected HEK293 cells. Patch-clamp recordings demonstrated that wild-type BKCa-α/β1 was activated by PKC and PKG, while BKCa-(S1076A)/β1 responded to the stimulation of PKC to a lesser extent, but was unresponsive to PKG. In contrast, BKCa-α(S1076E)/β1 enhanced the stimulatory effect of PKC. Thus, PKC may inhibit BKCa channels in FHR PASMC primarily through PDE activity, and also by directly acting on BKCa channels. In addition, the PKC phosphorylation site S1076 on the human BKCa channel α-subunit is very important for the regulation of channel function. The findings in this study should increase our understanding of how PKC modulates BKCa channels in IPAH, and may contribute to the development of new approaches to treat IPAH.en
dc.language.isoen_USen
dc.relation.urlhttp://ezproxy.augusta.edu/login?url=http://search.proquest.com/docview/304775595?accountid=12365en
dc.subjectBKCa Channelsen
dc.subjectPKCen
dc.subjectPASMCen
dc.subjectIPAHen
dc.subjectProtein Kinase Cen
dc.subjectIdiopathic Pulmonary Arterial Hypertensionen
dc.subjectPulmonary Arterial Smooth Muscle Cellen
dc.titleModulation of BKCa Channels by Protein Kinase C in Idiopathic Pulmonary Arterial Hypertensionen
dc.typeDissertationen
dc.contributor.departmentDepartment of Pharmacology and Toxicologyen
dc.description.advisorWhite, Richard E.en
dc.description.committeeBrowning, Darren D.; Caldwell, Robert W.; Wang, Mong-heng; Barman, Scott A.-
dc.description.degreeDoctor of Philosophy (Ph.D.)-
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