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.