Fig. 6. Inhibitors of CaM and CaMKII do not change intracellular Ca²⁺ levels but block NE and ionomycin (ION)-induced cPLA₂ phosphorylation. VSMCs were treated with inhibitors of CaM (10 µM W-7) and CaMKII (10 µM KN-93) or their vehicle (VEH) as described in Materials and Methods. (A) Effect of inhibitors of CaM (10 µM W-7) and CaMKII (10 µM KN-93) or vehicle (VEH) on cytosolic Ca²⁺ ([Ca]_i) was measured using fura-2 in the presence of NE (10 µM), ION (1 µM) or vehicle (V). The figure shows a representative of three experiments performed with each agonist and vehicle, and in the presence of different inhibitors on different batches of cells grown on coverslips. (B) Phosphorylation of cPLA₂ in response to NE (10 µM), ION (1 µM) or vehicle (V) in the presence (1.8 mM) or absence of extracellular Ca²⁺. (C,D) Phosphorylation of cPLA₂ in response to NE (10 µM) and ION (1 µM) in the presence or absence of inhibitors of CaM (10 µM W-7), CaMKII (10 µM KN-93). (E,F) Phosphorylation of cPLA₂ in response to NE (10 µM) and ION (1 µM) in the presence or absence of the corresponding structural analogues of W-7 and KN-93 (10 µM W-5; 10 µM KN-92). Phosphorylation of cPLA₂ was determined by incorporation of ³²P and detected by autoradiography as described in Materials and Methods. The figure shows a representative autoradiogram and the densitometric analysis from three experiments repeated with each agonist and the vehicle in the presence and absence of each inhibitor performed in different batches of cells grown in 100 mm tissue culture dishes. The density of cPLA₂ phosphorylation was quantified using NIH Image 1.62 (n=3). ^*Value significantly different from the corresponding value obtained in the absence of Ca²⁺ (B), in the presence of V of NE (C,E) or ION (D,F) (P<0.05).