VGCCs and PKC regulate SVEP1-integrin α4/9 inhibition of smooth muscle contraction
Increases in intracellular Ca2+ occur through Ca2+ release from the sarcoplasmic reticulum and via an influx of extracellular Ca2+ through VGCCs29. Aortas from C57BL/6J were either pre-incubated with BOP (Fig. 6A) or integrin α4 and α9 blocking antibodies (SF.10F) in the presence or absence of the VGCC inhibitor nifidepine (1 µM) prior to U46619 stimulation. VGCC inhibition significantly lowered both normal U46619-mediated vessel contraction (NS vs. NF, 0.75 (25 µg/ml) to 1.43 (100 µg/ml) mN/mm2,P <0.0001, concentration dependent, Fig. 6A), and the enhanced contraction caused by integrin α4/9 inhibition using BOP (BOP vs. BOP+NF, 1.05 (1 µg/ml) to 4.33 (100 µg/ml) mN/mm2,P <0.0001, concentration dependent, Fig. 6A). In Svep1+/-mice, inhibition of VGCCs also significantly reduced U46619-mediated contraction (Fig. 6B, 2.52 (25 µg/ml) to 6.76 (100 µg/ml) mN/mm2, P <0.0001, concentration dependent). VGCCs activity is regulated by protein kinase C (PKC)30. Inhibition of PKC using bisindolylmaleimide I (BIM) (I), 10 µM) significantly reduced normal U46619-mediated contraction (NS vs. BIM (I), 1.96 (25 µg/ml) to 2.97 (100 µg/ml) mN/mm2, P <0.0001, concentration dependent, Fig. 6C), the enhanced contraction caused by integrin α4/9 inhibition (BOP vs. BOP+BIM (I), 4.57 (25 µg/ml) to 5.18 (100 µg/ml) mN/mm2, P <0.0001, concentration dependent, Fig. 6C). BIM (I) inhibition of PKC also significantly reduced U46619-mediated contraction in Svep1+/-mice (Fig. 6D, 2.43 (25 µg/ml) to 5.98 (100 µg/ml) mN/mm2, P <0.0001, concentration dependent). These results show that SVEP1 regulation of GPCR-mediated contraction occurs through regulating PKC-mediated VGCC Ca2+ influx into the vessel.