Abstract-Ca2ϩ sparks are localized intracellular Ca 2ϩ events released through ryanodine receptors (RyRs) that control excitation-contraction coupling in heart and smooth muscle. Ca 2ϩ spark triggering depends on precise delivery of Ca 2ϩ ions through dihydropyridine (DHP)-sensitive Ca 2ϩ channels to RyRs of the sarcoplasmic reticulum (SR), a process requiring a very precise alignment of surface and SR membranes containing Ca 2ϩ influx channels and RyRs. Because caveolae contain DHP-sensitive Ca 2ϩ channels and may colocalize with SR, we tested the hypothesis that caveolae are the structural element necessary for the generation of Ca 2ϩ sparks. Using methyl-␤-cyclodextrin (dextrin) to deplete caveolae, we found that dextrin dose-dependently decreased the frequency, amplitude, and spatial size of Ca 2ϩ sparks in arterial smooth muscle cells and neonatal cardiomyocytes. However, temporal characteristics of Ca 2ϩ sparks were not significantly affected. We ruled out the possibility that the decreases in Ca 2ϩ spark frequency and size are caused by changes in DHP-sensitive L-type channels, SR Ca 2ϩ load, or changes in membrane potential. Our results suggest a novel signaling model that explains the formation of Ca 2ϩ sparks in a caveolae microdomain. The transient elevation in [Ca 2ϩ ] i at the inner mouth of a single caveolemmal Ca 2ϩ channel induces simultaneous activation and thus opens several RyRs to generate a local Ca 2ϩ release event, a Ca 2ϩ spark. Alterations in the molecular assembly and ultrastructure of caveolae may lead to pathophysiological changes in Ca 2ϩ signaling. Thus, caveolae may be intimately involved in cardiovascular cell dysfunction and disease. Materials and MethodsSingle SMCs were isolated enzymatically from myogenic cerebral (100 to 800 m in diameter posterior and basilar) arteries from adult Sprague-Dawley rats (12 to 14 weeks; 200 to 280 g), as previously described. 14 Single cardiomyocytes were isolated enzymatically from newborn rats. 21 For Ca 2ϩ imaging, the cells were incubated with the Ca 2ϩ indicator fluo-3-AM (5 m) and pluronic acid (0.005% wt/vol) for 30 minutes at room temperature in Ca 2ϩ -free Hanks solution. 3,14 SMCs and cardiomyocytes were imaged using a BioRad laser scanning confocal microscope attached to a Nikon Diaphot microscope. Whole-cell membrane currents and potentials in freshly isolated cerebral artery myocytes were measured using the perforated patch configuration of the patch-clamp technique configuration with amphotericin B or nystatin. 22 Currents were recorded from holding potentials of Ϫ80 mV (Ϫ100 mV) during lineage voltage ramps at 0.67 V/s from Ϫ100 to ϩ100 mV or 300-ms step pulses to different potentials; pulse frequency 0.2 Hz. 22,23 An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org. ResultsWe used a laser scanning confocal microscope and the Ca Figure 1 online, available at http:// www.circresaha.org) by membrane depolarization (using 60 mmol/L external K ϩ ) or by th...

Urokinase (uPA)-and urokinase receptor (uPAR)-dependent cell adhesion to the extracellular matrix protein vitronectin (Vn) is an important event in wound healing, tissue remodeling, immune response, and cancer. We recently demonstrated that in human vascular smooth muscle cells (VSMC) uPA/uPAR are functionally associated with the ectoprotein kinase casein kinase-2 (CK2). We now asked whether CK2 regulates uPA-dependent cell adhesion to Vn, since the latter is a natural CK2 substrate. We found that Vn is indeed selectively phosphorylated by CK2 and that this phosphorylation is uPA-regulated in VSMC. Vn induces release of ecto-CK2 from the cell surface via a process termed as "shedding." CK2-mediated Vn phosphorylation was decisive for the uPA-dependent VSMC adhesion. Specific inhibition of CK2 completely abolished the uPA-induced cell adhesion to Vn. This effect was specific for cell adhesion to Vn and required participation of both uPAR and ␣ v ␤ 3 integrins as adhesion receptors. CK2 localization at the cell surface was highly dynamic; Vn induced formation of clusters where CK2 colocalized with uPAR and ␣ v ␤ 3 integrins. These results indicate that the uPA-dependent VSMC adhesion is a function of selective Vn phosphorylation by the ectoprotein kinase CK2 and suggest a regulatory role for Vn phosphorylation in the uPA-directed adhesive process.The urokinase (uPA) 1 /urokinase receptor (uPAR) system has multiple functions serving as a molecular link between pericellular proteolysis and signaling cascades, regulating cell adhesion, migration, and proliferation (1). Several reports suggest a possible role for uPA/uPAR in these processes independent of the uPA enzymatic activity. For instance, uPAR has a nonproteolytic action as a cellular adhesion receptor for vitronectin (Vn). Vn is a multifunctional glycoprotein present in blood, extracellular matrix, inflammatory sites, and in atherosclerotic plaques (2). UPAR contains a high affinity Vn-binding site, which is different from the uPA-binding site. This binding site makes uPAR uniquely poised to regulate extracellular proteolysis and the adhesion process (3). Vn has been shown to contain consensus sequences for phosphorylation by various protein kinases, such as cAMP-dependent protein kinase A (PKA), protein kinase C (PKC) and, and as recently described, the protein kinase casein kinase-2 (CK2) (4). Vn can be phosphorylated at specific sites that modulate its conformation and influences its subsequent functions (5-7). However, whether or not Vn phosphorylation plays any direct role in the uPA-directed cell adhesion has not been explored.We recently demonstrated that in human vascular smooth muscle cells (VSMC), the cell surface CK2 is associated with uPAR and might be activated by uPA. Activated CK2 mediates phosphorylation of the cell-surface shuttle protein nucleolin, which translocates then in its phosphorylated form into the cell and regulates mitogenic effects of uPA (8). However, ectoprotein kinases operating on numerous cell types can provide for the pho...