6A, GM6001 had no effect on KK activity in a cell-free biological activity assay, confirming that the inhibition of KK-induced AR shedding did not result from direct inhibition of plasma KK. As shown in Fig. 6B, we observed a rapid 3-fold increase in EGF receptor autophosphorylation (ErbB1 pTyr1068) in R-VSMC upon KK treatment that was sensitive to GM6001, consistent with ADAM-dependent EGF receptor ligand shedding in KK-treated cells. FIGURE 5. Plasma kallikrein stimulates ADAM-dependent amphiregulin and TNF-�� release from vascular smooth muscle cells. A, serum-deprived H-VSMC in 10-cm dishes were incubated in the presence or absence of 50 nm human plasma KK for 1�C3 h, after … FIGURE 6. Plasma kallikrein stimulates ADAM-dependent EGF receptor transactivation in vascular smooth muscle cells.

A, 20 nm human plasma KK was combined with the chromogenic substrate for plasma KK, S2302 (0.6 ��m), in the presence or absence of GM6001 … Kallikrein-stimulated ERK1/2 and JNK Activity in Primary Aortic Vascular Smooth Muscle Is Partially ADAM- and EGF Receptor-dependent EGF receptor transactivation contributes to ERK1/2 activation by diverse stimuli, including many G protein-coupled receptors (26, 27). To test whether transactivation was involved in KK-induced activation of the ERK1/2 and JNK cascades in primary VSMC, we determined whether the activation of these pathways by PK or KK in R-VSMC was sensitive to inhibition by GM6001 or the EGF receptor kinase inhibitor, AG1478. As shown in Fig. 7A, 15 min of exposure to either PK or KK produced ERK1/2 activation that was significantly inhibited in the presence of GM6001.

Fig. 7B illustrates similar effects on PK- and KK-induced JNK1/2 phosphorylation, implicating MMP-dependent ectodomain shedding in both signals. As shown in Fig. 7C, KK-induced ERK1/2 activation was substantially, but incompletely, inhibited by AG1478 at a concentration sufficient to completely block ERK1/2 activation by exogenously supplied EGF. These results indicate that MMP-dependent EGF receptor transactivation plays a significant role in the VSMC response to KK exposure. The incomplete effect of GM6001 and AG1478 indicates that other pathways, possibly also mediated via KK-activated PAR1/2, contribute to ERK1/2 activation, as has been shown for other G protein-coupled receptors Anacetrapib (50). FIGURE 7. Activation of ERK1/2 by plasma kallikrein in vascular smooth muscle cells is partially ADAM- and EGF receptor-dependent. A, serum-deprived R-VSMCs in 6-well plates were preincubated in the presence or absence of GM6001 (10 ��m) for 15 min prior … DISCUSSION The RAS and KKS constitute an interlocking signaling network involved in the regulation of vascular function (7,�C10). Coordinate regulation is achieved through shared pathway components.