We have recently demonstrated that calcium/calmodulin-dependent protein kinase II (CaMK II) activation is required for PDGF-directed Boyden chamber chemotaxis (measured at 4 hrs) by proliferating vascular smooth muscle cell (VSMC) in vitro, and that VSMCs induced to differentiate after growth arres fail to migrate largely due to attenuated CaMK II activation. Since chemotaxis can be restored in growth-arrested VSMCs by Ca2+-ionophores, we tested the hypothesis that Ca2+-signalling regulates chemotaxis, and that PDGF-stimulated Ca2+-signalling differs markedly between these two phenotypes in a cultured VSMC model from rat aorta. PDGF-BB (10 ng/ml) causes a large increase in intracellular Ca2+ (Cai) in proliferating-VSMCs owing to intracellular release, followed by a tonic elevation, which is accompanied by parallel changes in CaMK II activation via its phosphorylation. In contrast, this Cai response and CaMK II activation pattern are both markedly attenuated in growth-arrested VSMCs. Ionomycin ( ~M), which restores migration in growth-arrested VSMCs, induces both a biphasic increase in Cai and an immediate activation of CaMK II qualitatively similar to the PDGF response of proliferating-VSMCs. Thus, altered PDGF Ca2+-signalling, which accompanies changes in VSMC phenotype, likely regulates both CaMK II activation and chemotaxis in response to PDGF In addition to differences in chemotaxis between these two differentiation phenotypes, significant heterogeneity in the rate of chemotaxis is also evident among proliferating VSMCs (i.e., only 5-10% chemotax at 4 hrs). Preliminary experiments in a novel microscope-adapted Boyden chamber, enabling simultaneous measurement of VSMC migration and Cai in individual cells, suggests that heterogeneous Cai activation patterns (i.e., in the magnitude/rate of Cai change) could underlie some cellular differences in chemotaxis among proliferating VSMCs. However, that """"""""equalization"""""""" of Cai- responses with ionomycin in these proliferating VSMCs fails to recruit significant additional chemotaxis (at least at the 4 hr assay time-point) suggests that an additional factor, such as the initial inability to produc and/or utilize activated CaMK II by a large subpopulation (at least 90% of cells), may also be a critical factor in the apparent heterogeneity of chemotaxis. Studies are in progress to measure the coordination of Cai and CaMK II activations during chemotaxis in individual cells to resolve these fundamental issues.
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