Vasopressin-induced disruption of actin cytoskeletal organization and canalicular function in isolated rat hepatocyte couplets: Possible involvement of protein kinase C

Abstract

The effect of vasopressin (VP) on canalicular function and hepatocellular morphology, with particular regard to actin cytoskeletal organization and the concomitant plasma membrane bleb formation, was studied in isolated rat hepatocyte couplets. VP induced the concentration-dependent formation of multiple plasma membrane blebs as well as simultaneous impairment in both canalicular vacuolar accumulation (cVA) and retention (cVR) of the fluorescent bile acid, cholyl-lysyl-fluorescein (CLF), which evaluate couplet secretory function and tight-junction integrity, respectively. These effects were mimicked by the protein kinase C (PKC) activator, phorbol dibutyrate (PDB), but not by the protein kinase A (PKA) activator, dibutyryl-cAMP. VP-induced bleb formation and canalicular dysfunction were fully prevented by the protein kinase inhibitor, H-7, but not by the PKA inhibitor, KT5720, further suggesting a specific role of PKC. VP-induced alterations were also prevented by pretreatment with the Ca 2؉ -buffering agent, BAPTA/AM, but not with the calmodulin-dependent protein kinase II antagonist, calmidazolium. Neither the Ca 2؉ -activated neutral protease inhibitor, leupeptin, nor the antioxidants, ␣-tocopherol or deferoxamine, were able to prevent either VP-induced plasma membrane blebbing or canalicular dysfunction. The Ca 2؉ -ionophore, A23187, mimicked the VP-induced alterations, but its harmful effects were completely prevented by H-7. Bleb formation induced by VP and PDB was accompanied by an extensive redistribution of filamentous actin from the pericanalicular area to the cell body, and this effect was fully prevented by H-7. These results suggest that VP-induced canalicular and cytoskeletal dysfunction is mediated by PKC and that classical (Ca 2؉ -dependent) PKC appear to be involved because intracellular Ca 2؉ is required for VP to induce its harmful effects. (HEPATOLOGY 1998;28:1031-1041.)Vasopressin (VP), like other hormonal mediators including angiotensin II, epinephrine, and norepinephrine, exerts its biological actions by inducing liberation of inositol 1,4,5-trisphosphate and diacylglycerol. 1 Inositol 1,4,5-trisphosphate mediates increase in cytosolic Ca 2ϩ ([Ca 2ϩ ] cyt ), whereas diacylglycerol mediates activation of protein kinase C (PKC). VP induces bile flow impairment either by PKC activation or Ca 2ϩ mobilization. 2-4 A variety of mechanisms were proposed to account for the cholestatic effect of VP. This hormone decreases primary secretion in isolated rat hepatocyte couplets via both [Ca 2ϩ ] cyt elevation and PKC activation, 5 indicating that the effect is, at least in part, hepatocellular in origin.

The Hippo pathway: regulators and regulations

“…It is well known that some GPCR ligands, such as LPA, S1P, and thrombin, also induce contractive actin bundles in cells (Miller et al 2012;Mo et al 2012;Yu et al 2012b). On the other hand, some ligands for Ga s -coupled receptors, such as vasopressin, dopamine, and parathyroid hormone, have been shown to counteract the formation of actin bundles (Ding et al 1991;Egan et al 1991b;Roma et al 1998;Nguyen et al 1999;Zhang et al 2006). Mechanical cues such as cell spreading, cell geometry, and matrix stiffness all result in rearrangement of the actin cytoskeleton (Dupont et al 2011;Wada et al 2011;Zhao et al 2012).…”

Receptor-Mediated Biphasic Alteration of Hepatocellular Transport from Hepatocyte to Bile Canaliculi as Measured by Near-Infrared Spectroscopy: A Novel Test with Glucagon for Biliary Excretion

“…Therefore, an increase in cytosolic calcium by norepinephrine induces contraction of bile canaliculi in the doublet hepatocytes; the contraction is inhibited by wortmannin [7]. It has also been reported that vasopressin induced the disruption of actin cytoskeletal organization and canalicular function either via protein kinase C activation or calcium mobilization [26]. It has been reported that bile canalicular peristalsis is propagated by pericentral to periportal calcium waves via gap junctions, through which second messengers communicate between hepatocytes [8, 27, 28].…”

Regulation of organic cation transport

“…In the liver, insulin stimulates phosphatidylinositol 3-kinase (PI3K) activity in rat [67] and inhibits secretin-induced ductal secretion by activation of PKCa and inhibition of PKA activity [68]. In hepatocyte couplets vasopressin induces canalicular and cytoskeletal dysfunction that is probably mediated by PKC and intracellular Ca 2+ [69]. Stimulation of ATP receptors induces Ca 2+ signalling in bileduct epithelia [70] and angiotensin II activates a signalling pathway leading to phosphorylation of MAPK in hepatocytes; this event is mediated by a CaM-dependent protein kinase, a PKC-dependent pathway and a PI3K-dependent pathway [71].…”