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Articles in PresS, published online ahead of print August 30, 2002
J Appl Physiol, 10.1152/jap.00988.2001
Submitted on September 26, 2001
Accepted on August 3, 2002
1 Departments of Chemical Engineering and Bioengineering, Biomolecular Transport Dynamics Laboratory, Pennsylvania State University, University Park, PA, USA
* To whom correspondence should be addressed. E-mail: jmt{at}psu.edu.
Smooth muscle cells (SMCs) are exposed to substantial levels of fluid shear stress due to interstitial flow driven by the transmural pressure gradient across the arterial wall. This shear stress may play a role in the myogenic response and flow-mediated vasomotion. We, therefore, examined the effects of fluid flow on contraction of rat aortic smooth muscle cells, intracellular calcium concentration and the mediating signal transduction pathways of this contraction response. SMCs that had been serum-starved to induce a contractile phenotype were plated on quartz slides and exposed to controlled shear stress levels of 1, 11 and 25 dynes/cm2 in a parallel plate flow chamber. The area of the cells was quantified and changes in the cell area in response to shear stress were reported as a contraction response. At 25 dynes/cm2, significant area reduction was apparent 3 minutes after the onset of flow. The area reduction relative to the pre-shear area exceeded 30% at 30 minutes, and when the shear was turned off after 30 minutes of exposure, area reduction continued to increase to 46% after an additional 30 minutes. At 1 dyne/cm2 shear stress, no significant contraction was observed after 30 minutes of exposure. The threshold for significant shear-induced contraction appeared to be 11 dynes/cm2. The signal transduction mechanism of shear-induced contraction was studied at 25 dynes/cm2. Intracellular calcium ([Ca2+]i) was imaged under shear stress conditions using the calcium sensitive fluorescent dye fura 2-AM. There was no detectable change in [Ca2+]i during 10 minutes of exposure to shear stress even though the cells displayed a significant calcium response to 10 µM thapsigargin, 2 µM calcium ionophore and 51 mM KCl. Further studies using pathway inhibitors provided evidence that the most important signal transduction pathway mediating calcium-independent contraction in response to fluid flow is the Rho- kinase pathway although there was a suggestion that protein kinase C plays a secondary role.
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