Date of Award

2009

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Todd L. Green

Second Advisor

Robert T. Harris

Third Advisor

Elsa I. Mangiarua

Fourth Advisor

William D. McCumbee

Fifth Advisor

Gary L. Wright

Abstract

Two isoforms of actin have been found to be present in A7r5 smooth muscle cells, α- and β-actin. This body of work sets out to examine the different regulatory factors of the two actin isoforms during smooth muscle contraction. The response of the actin isoforms to phorbol 12, 13-dibutyrate (PDBu) is markedly different. α-Actin remodels to podosomes around the periphery of the cell, while β-actin merely shortens. One protein involved in smooth muscle contraction induced by PDBu is protein kinase C (PKC). Two inhibitors of PKC, staurosporine and bisindolymaleimide, were used prior to PDBu stimulation and after PDBu stimulation to observe the effects of PKC on α- and β-actin stress fiber structure and remodeling. α-Actin showed a decrease in podosome formation when the inhibitors were added prior to or after PDBu stimulation. β-actin demonstrated a loss in stress fiber structure in response to PKC inhibitors. PKC appears to regulate α- and β- actin differently and could play a role in the differential remodeling seen by these two actin isoforms. Myosin light chain kinase (MLCK) is another enzyme involved in smooth muscle contraction. Even though it does not play a major role in PDBu induced contraction of smooth muscle, it has been found to interact with actin at two different actin binding sites, allowing crosslinking of actin filaments. Inhibition of the kinase domain of MLCK does not seem to have an effect on the crosslinking abilities of MLCK. These two key enzymes of smooth muscle contraction appear to not only be able to initiate smooth muscle contraction, but also regulate reorganization of the two actin isoforms during smooth muscle contraction.

Subject(s)

Smooth muscle - Contraction - Research.

Smooth muscle.

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