Calcium independent PLA2 beta and STIM1 as new molecular determinants of calcium influx and contraction in vascular smooth muscle cells of cerebral arteries

Store-operated Calcium (Ca2+) entry (SOCE) is known to play an important role in the function of vascular smooth muscle cells (SMC), but little is known about the role of Ca2+-independent phospholipase A2 beta (iPLA2beta) and stromal interaction molecule 1 (STIM1), two newly identified components of SOLE, in agonist-induced constriction of cerebral arteries. The two main objectives of these studies were (A) to examine the role of membrane-associated iPLA2beta and SOCE in agonist-induced constriction in cerebral arteries, and (B) to gain new insights into the localization and contribution of STIM1 and iPLA2beta to SOCE in SMC and model cell lines.; The main hypotheses of this proposal are (1) iPLA2beta is crucial for SOCE and agonist-induced constriction in cerebral vessels, and (2) STIM1 association with the plasma membrane variant of iPLA 2beta may be involved in SOCE.; Studies in intact cerebral arteries confirmed an integral role for iPLA 2beta-dependent SOCE in constriction of cerebral arteries, and knock-out mouse studies identified a specific variant of iPLA2beta that is essential for this function. These studies also established that iPLA 2beta-dependent activation of store operated channels (SOC) may be a trigger for secondary activation of voltage-gated L-type calcium channels (Ca2+L), making both channels equally important for Ca2+ influx and constriction of cerebral arteries.; STIM1 has been primarily studied in over-expression systems, and very little is known about endogenous STIM1 in primary SMC. To visualize and assess interactions of endogenous STIM1 and iPLA2beta, co-immunofluorescence experiments using commercial and custom-made antibodies were performed in primary SMC. As an additional approach, live imaging of fluorescently-labeled STIM1 and its accumulation along plasma membrane were evaluated in cell lines. These complementary studies provided evidence for the role of iPLA2beta in STIM1 function in SMC, and to assess the role of STIM1 glycosylation sites in its translocation and initiation of SOCE in a model cell line.; In conclusion, a plasma membrane-associated variant of iPLA2beta was found to play a central role in agonist-induced constriction of cerebral arteries, and new evidence was found for the role of iPLA2beta in STIM1 function in vascular SMC.