Document Details

Document Type:   Dissertation
Title:   PKCa Translocation and Actin Remodeling in Contracting A7r5 Smooth Muscle Cells
Author:   Chenwei Li
College:   Joan C. Edwards School of Medicine
Degree Program:   Biomedical Sciences, Ph.D.
Degree:   Doctor of Philosophy
Committee Director:   Gary L. Wright
Document Availability:   Document available for World-Wide access.
Date of Defense:   03/22/2002

PKC activation and specificity of substrate phosphorylation is believed to be associated with the relocalization of the enzyme to specific cell sites. PKCα was diffusely distributed throughout the cytosol in the unstimulated A7r5 cell. Upon stimulation with phorbol 12, 13 dibutyrate (PDBu), PKCα was translocated primarily to either the perinuclear region (10-5 M to10-6 M PDBu) of the cell or to subplasmalemmal sites (10-7 M to10-8M PDBu). Translocation of PKCα to the perinucleus was blocked by the use of colchicine to disrupt cell microtubules. cytochalasin B disruption of actin microfilaments had no significant effect on PKCα translocation to either the plasmalemma or the perinucleus. This suggests that multiple pathways are available for the redistribution of PKCα. PDBu (10-8 M) induced a slow but robust and irreversible relocation of the PKCα -EGFP fusion protein from the cytosol to the plasmalemma. Thapsigargin (10-5 M) and A23187 (2 X 10-5 M) induced a rapidly transient translocation to the cell membrane. The results show that the spatial and temporal characteristics of individual PKC isoform translocation may differ markedly, depending on the stimulating agent. The elevation of intracellular calcium ([Ca2+]i) is thought to be the initiating event of smooth muscle cell contraction to the majority of physiological agonists, we examined the effect of increasing [Ca2+]i by A23187 and thapsigargin on α- and β-actin remodeling. During the interval of contraction induced by A23187 and thapsigargin, β-actin cables shortened without evidence of disassembly. In marked contrast, elevation of intracellular calcium resulted in the partial or complete dissolution of α-actin cables and blocked further α-actin remodeling in response to PDBu. Incubation of the cells in the calcium-free medium prevented α-actin dissolution by A23187/thapsigargin and blocked PDBu-mediated α-actin remodeling. The results suggest that extracellular calcium is necessary for α-actin remodeling. We have further observed that of six kinase inhibitors investigated only ML-7, a myosin light chain kinase (MLCK) inhibitor, blocked the dissolution of α-actin cables induced by increased [Ca2+]i. This finding may be suggesting a novel role of MLCK in destabilizing α-actin structure in the [Ca2+]i activated smooth muscle cell.  

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