Involvement of RhoA and its interaction with protein kinase C and Src in CCK-stimulated pancreatic acini

We evaluated intracellular pathways responsible for the activation of the s mall GTP-binding protein Rho p21 in rat pancreatic acini. Intact acini were incubated with or without CCK and carbachol, and Triton X-100-soluble and crude microsomes were used for Western immunoblotting. When a RhoA-specific antibody was used, a single band at the location of 21 kDa was detected. C CK (10 mu M-10 nM) and carbachol (0.1-100 mu M) dose dependently increased the amount of immunodetectable RhoA with a peak increase occurring at 3 min . High-affinity CCK-A-receptor agonists JMV-180 and CCK-OPE (1-1,000 nM) di d not increase the intensities of the RhoA band, suggesting that stimulatio n of RhoA is mediated by the low-affinity CCK-A receptor. Although an incre ase in RhoA did not require the presence of extracellular Ca2+, the intrace llular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic ac id-AM abolished the appearance of the RhoA band in response to CCK and carb achol. The G(q) protein inhibitor G protein antagonist-2A (10 mu M) and the phospholipase C (PLC) inhibitor U-73122 (10 mu M) markedly reduced RhoA ba nds in response to CCK. The protein kinase C (PKC) activator phorbol ester (10-1,000 nM) dose dependently increased the intensities of the RhoA band, which were inhibited by the PKC inhibitor K-252a (1 mu M). The pp60(c-src) inhibitor herbimycin A (6 mu M) inhibited the RhoA band in response to CCK, whereas the calmodulin inhibitor W-7 (100 mu M) and the phosphoinositide 3 -kinase inhibitor wortmannin (6 mu M) had no effect. RhoA was immunoprecipi tated with Src, suggesting association of RhoA with Src. Increases in mass of this complex were observed with CCK stimulation. In permeabilized acini, the Rho inhibitor Clostridium botulinum C3 exoenzyme dose dependently inhi bited amylase secretion evoked by a Ca2+ concentration with an IC50 of C3 e xoenzyme at 1 ng/ml. We concluded that the small GTP-binding protein RhoA p 21 exists in pancreatic acini and appears to be involved in the mediation o f pancreatic enzyme secretion evoked by CCK and carbachol. RhoA pathways ar e involved in the activation of PKC and Src cascades via G(q) protein and P LC.