Author

Chenwei Li

Date of Award

2002

Degree Name

Doctor of Philosophy

College

College of Liberal Arts

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Gary L. Wright

Second Advisor

Patrick I. Brown

Third Advisor

Todd L. Green

Fourth Advisor

William D. McCumbee

Fifth Advisor

Michael L. Norton

Abstract

Recent research indicates that protein kinase C (PKC) plays an important role in smooth muscle contraction. Because PKC activation and specificity of substrate phosphorylation is believed to be associated with the relocalization of the enzyme to specific cell sites, we first investigated the subcellular translocation of PKCα in A7r5 smooth muscle cells by confocal microscopy through use of standard immunohistologic staining and PKCα - enhanced green fluorescent protein (PKCα -EGFP) fusion protein expression. 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 of the cell or to subplasmalemmal sites depending on the concentration of the stimulating agent. Specifically, PKCα was translocated to the perinuclear area in response to high PDBu concentrations (10-5 M to10-6 M) and relocated to the plasma membrane at lower PDBu concentrations (10-7 M to10-8 M). Translocation of PKCα to the perinucleus but not the plasmalemma was blocked by the use of colchicine to disrupt cell microtubules. By comparison, cytochalasin B disruption of actin microfilaments had no significant effect on PKCα translocation to either the plasmalemma or the perinucleus. The results indicate that the target site of PKCα translocation may vary with activating stimulus strength in A7r5 cells and that the translocation of the isoform to the perinuclear region of the cell is dependent on an intact microtubular cytoskeleton. This suggests that multiple pathways are available for the redistribution of PKCα that may employ different mechanisms to regulate the movement and/or docking of the isoform at specific target sites. We next compared the spatial and temporal pattern of PKCα translocation in response to different stimulating agents in live A7r5 smooth muscle cell preparations utilizing cells transfected with PKCα-EGFP. PDBu (10-8 M) induced a slow but robust and irreversible relocation of the PKCα -EGFP fusion protein from the cytosol to the plasmalemma. By comparison, thapsigargin (10-5 M) and A23187 (2 X 10-5 M) induced a rapidly transient translocation to the cell membrane, which was completed within 4 minutes. In contrast to these agents, angiotensin II (Ang II, 10-6 M) caused only partial relocalization of cytosolic PKCα-EGFP to brightly fluorescing patches at the cell periphery. Moreover, the translocation of the kinase to peripheral patches was completed within seconds and the fusion protein returned to the cytosol within 2 minutes. The PKC inhibitor staurosporine blocked cellular contraction to PDBu but not to A23187 and had no effect on PKCα- EGFP translocation. By comparison, the calcium chelators EDTA and BAPTA-AM blocked the contraction to A23187, attenuated the contraction to PDBu, and abolished the translocation of PKCα-EGFP by both agents. The results show that A7r5 cells retain the ability to respond to several types of contractile agents and the spatial and temporal characteristics of individual PKC isoform translocation may differ markedly, depending on the stimulating agent.

Subject(s)

Smooth muscle.

Cells - Contraction.