Date of Award


Degree Name

Biomedical Sciences


Joan C. Edwards School of Medicine

Type of Degree


Document Type


First Advisor

Elsa I. Mangiarua

Second Advisor

William D. McCumbee

Third Advisor

Richard Niles

Fourth Advisor

Donald A. Primerano

Fifth Advisor

Gary L. Wright

Sixth Advisor

Leonard J. Deutsch


The exact nature of the mechanisms and the regulation of vascular smooth muscle contraction is not well understood. To better understand these processes, we examined two systems involved in smooth muscle contraction, the cytoskeleton and the protein kinases. In order to study the role of the cytoskeleton in smooth muscle contraction, we examined the contractile and mechanical effects of cytoskeleton disruption. We found that the relationship between passive tension applied to aortic rings and the resulting increase in tissue length was nearly linear over the range of 1 g to 15 g. However, even with increasing tissue length, within the range of 1 g to 10 g passive tension, the total active force generated upon stimulation was not significantly changed. These observations emphasize the great flexibility of the mechanism(s) underlying the contractile response of vascular smooth muscle with regard to changes in tissue preload and length. Neither the blockade of microtubule polymerization by colchicine nor actin polymerization by cytochalasin B significantly changed the slope of the tissue length-passive tension preload curve indicating no effect on the tissues' capacity to stretch at a given preload. With stimulation of the tissue at different levels of stretch, colchicine caused an increase in the initial fast component of active tension development, but partially blocked the secondary slow rise in tension. Cytochalasin B dramatically reduced the total contractile response at each preload studied, and this effect was confined almost exclusively to the secondary slow increase in tension. When tissues were cooled to cause complete dissolution of the microtubule network and then warmed in the presence of colchicine to prevent repolymerization of both the active and stable populations of microtubules, there was also a significant reduction in the slow component of contraction with no effect on the fast response. The partial blockade of synthesis of the microtubule-associated motor protein kinesin by application of an antisense oligonucleotide to aortae in situ or to aortic rings in tissue culture significantly reduced the contractile response to potassium depolarization. These results suggest that the microtubules and the actin filaments of the cytoskeleton play an active role in slow force development as opposed to a solely passive role based on the effect of the static, structural properties of these filaments on mechanical resistance. We propose that a tension-bearing element of the actin-containing cytoskeleton undergoes remodeling to adjust tension within the system. The microtubules could act through either the direct action of kinesin-mediated intracytoskeletal interactions in force development that involve a remodeling of the tension-bearing elements of the cytoskeleton or through the directed movement of the molecules involved in the transduction process.

Because the cytoskeleton and the protein kinases of smooth muscle are intimately linked, we examined the potential role of protein kinases in vascular smooth muscle contraction. We began by assessing the effects of a panel of specific kinase inhibitors on smooth muscle contraction. We found reductions in contraction with inhibition of myosin light chain kinase (MLCK), calcium-dependent calmodulin kinase (CaMKII), mitogen activated protein (MAP) kinase, and protein kinase C (PKC). Protein kinase C (PKC) is translocated in an isoform-specific manner to distinct subcellular locations after stimulation of cells. It is thought that translocation is essential for PKC activation and that cellular localization underlies the PKC isoform-specific phosphorylation of substrate in the intact cell that is largely absent in in vitro assays. In the present studies, it was shown using Western blot analysis that the ratio of particulate to cytosolic PKC-α was reduced in rat aortic segments treated with colchicine to disrupt microtubular structure prior to stimulation with phorbol 12, 13 dibutyrate (PDB). Subsequent studies using laser confocal microscopy revealed that within thirty seconds after stimulation with PDB, PKC-α in cultured rat aortic smooth muscle cells changed from a diffuse cytoplasmic distribution to a highly structured filamentous pattern of staining. Dual immunostaining further indicated that the stimulation-induced filamentous pattern was due to colocalization of PKC-α with cell microtubules. At longer time intervals after PDB stimulation, PKC-α was observed to translocate to the perinuclear region of the cell. Disruption of the microtubular but not the actin-containing component of the cytoskeleton blocked the translocation of PKC-α to the perinuclear membrane. It was further shown that slow tension development, which has been reported to be selectively blocked by PKC antagonists in vascular smooth muscle, was also blocked by disruption of the cell microtubules. The results provide further evidence for the involvement of PKC in slow tension development by smooth muscle and indicate that PKC translocation may involve microtubular transport.


Smoot muscle – Contraction – Research.