Graduation Year

2007

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Molecular Pharmacology and Physiology

Major Professor

Kay-Pong Yip, Ph.D.

Keywords

Calcium sparks, Paramagnetic beads, Myogenic response, Fluorescence confocal microscopy, Impedance sensing

Abstract

Integrins are transmembrane heterodimeric proteins that link extracellular matrix (ECM) to cytoskeleton and have been shown to function as mechanotransducers in non-muscle cells. Synthetic integrin-binding peptide triggers Ca2+ mobilization and contraction in vascular smooth muscle cells (VSMCs) from rat afferent arteriole, indicating that interactions between ECM and integrins modulate vascular tone. RGD, an integrin binding peptide, triggered contraction in cultured VSMCs as observed by Electric Cell-Substrate Impedance Sensing technique. To examine whether integrins transduce extracellular mechanical stress into intracellular Ca2+ signaling events in VSMCs, unidirectional mechanical force was applied to freshly isolated renal VSMCs through paramagnetic beads coated with fibronectin (FN, natural ligand of alpha5beta1 integrin in VSMCs). Pulling of fibronectin-coated beads with electromagnet triggered Ca2+ sparks, followed by global Ca2+ mobilization.

Paramagnetic beads coated with low-density lipoprotein (LDL), whose receptors are not linked to cytoskeleton, were minimally effective in triggering Ca2+ sparks and global Ca2+ mobilization. Pre-incubation with ryanodine, cytochalasin-D, or colchicine substantially reduced the occurrence of Ca2+ sparks triggered by fibronectin-coated beads. Binding of VSMCs with antibodies specific to the extracellular domains of alpha5 and beta1 integrins triggered Ca2+ sparks simulating the effects of fibronectin-coated beads. Anti-beta2- integrin antibody served as the negative control. Traction force microscopy studies showed that only the force transduced via integrins could potentially trigger cytoskeletal remodeling in cultured VSMCs. Atomic force microscopy revealed a significant increase in surface roughness in VSMCs when treated with RGD peptide though there was no difference in the maximum deflection of the force curves.

Pre-incubation of microperfused afferent arterioles with ryanodine or integrin specific binding peptide inhibited pressure-induced myogenic constriction. In conclusion, integrins transduce mechanical force into intracellular Ca2+ signaling events in renal VSMCs. Integrin-mediated mechanotransduction is probably involved in myogenic response of afferent arterioles. Thus, integrins can potentially act as sensors for myogenic response phenomenon and affect the autoregulatory mechanism in the vasculature.

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