We propose: (i) to clone and characterize the flow-sensitive K+ channel of endothelial cells primarily by employing a combined molecular cloning/electrophysiological strategy utilizing expression cloning of endothelial ion channel mRNA in Xenopus oocytes. The rationale is that the K+ channel may be the mechanosensor, or if not, is a key second messenger mechanism of the cellular response and therefore well situated for intervention studies. The cloned cDNA will be used to generate recombinant protein and for transfection studies of ion channel activity. (ii) Flow responses requiring longer periods to develop will be identified at the transcriptional level by the creation of cDNA libraries from endothelial cells exposed to shear stress. Differentially expressed mRNAs of unknown sequence will be isolated by subtractive hybridization, if necessary after PCR amplification. (iii) to test the hypothesis that flow regulates the mass transport of agonists in the cell boundary layer, thereby limiting or enhancing agonist effectiveness. We will examine the acute intracellular responses of endothelial cells to agonist mass transport as a function of flow; this part of the project is an adjunct to the direct effect of shear stress upon a mechanosensor. (iv) to test the hypothesis that flow-induced electrical (and possibly humoral) changes in the endothelium are transferred to adjacent smooth muscle cells via gap-junctional coupling, i.e. effectively bypassing a secretory (humoral) route of communication such as occurs for relaxing and hyperpolarizing factors. The hypothesis will be tested in electrically and metabolically coupled endothelial-smooth muscle pairs.
| Tsao, R; Jones, S A; Giddens, D P et al. (1995) An automated three-dimensional particle tracking technique for the study of modeled arterial flow fields. J Biomech Eng 117:211-8 |
