The intracellular mechanisms that mediate cytochalasin-induced increase in intestinal epithelial tight junction (TJ) permeability are unclear. In this study, we examined the involvement of myosin light chain kinase (MLCK) in this process, using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin B (Cyto B) (5 microg/ml) produced an increase in Caco-2 MLCK activity, which correlated with the increase in Caco-2 TJ permeability. The inhibition of Cyto B-induced MLCK activation prevented the increase in Caco-2 TJ permeability. Additionally, myosin-Mg(2+)-ATPase inhibitor and metabolic inhibitors (which inhibit MLCK induced actin-myosin contraction) also prevented the Cyto B-induced increase in Caco-2 TJ permeability. Cyto B caused a late-phase (15-30 min) aggregation of actin fragments into large actin clumps, which was also inhibited by MLCK inhibitors. Cyto B produced a morphological disturbance of the ZO-1 TJ proteins, visually correlating with the functional increase in Caco-2 TJ permeability. The MLCK and myosin-Mg(2+)-ATPase inhibitors prevented both the functional increase in TJ permeability and disruption of ZO-1 proteins. These findings suggested that Cyto B-induced increase in Caco-2 TJ permeability is regulated by MLCK activation.

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Branched-chain a-keto acid dehydrogenase (BCKDH) phosphatase was purified about 8000-fold from extracts of bovine kidney mitochondria. The highly purified phosphatase exhibited a molecular weight of -460,000, as estimated by gel-permeation chromatography. Another form of the phosphatase, with an apparent molecular weight of =230,000, was also detected under conditions of high dilution. In contrast to pyruvate dehydrogenase phosphatase, BCKDH phosphatase was active in the absence of divalent cations.BCKDH phosphatase was inactive toward 32P-labeled phosphorylase a, but exhibited -10% maximal activity with 32p-labeled pyruvate dehydrogenase complex. BCKDH phosphatase activity was inhibited by GTP, GDP, ATP, ADP, UTP, UDP, CTP, and CDP. Half-maximal inhibition occurred at about 60, 200, 200, 400, 100, 250, 250, and 400 jIM, respectively. These inhibitions were reversed completely by 2 mM Mg2e. GTP was replaceable by guanosine 5'-(i,y-imido)triphosphate. GMP, AMP, UMP, CMP, NAD, and NADH showed little effect, if any, on BCKDH phosphatase activity at concentrations up to 1 mM. Heparin showed half-maximal inhibition at 2 g/mIl. This inhibition was only partially (30%) reversed by 2 mM Mg2+. CoA and various acyl-CoA compounds exhibited half-maximal inhibition at 150-300 ,uM. These inhibitions were not reversed by 2 mM M&+. BCKDH phosphatase activity was stimulated 1.5-to 3-fold by protamine, poly(L-lysine), and poly(L-arginine) at 3.6 pg/mI.The mammalian branched-chain a-keto acid dehydrogenase (BCKDH) complex, like the analogous pyruvate dehydrogenase complex, is located within the mitochondrial inner membrane-matrix compartment and is subject to regulation by phosphorylation and dephosphorylation (1)(2)(3)(4)(5)(6). BCKDH is inactivated by phosphorylation of its a subunit (Mr, 46,000), catalyzed by a protein kinase that copurifies with the BCKDH complex (7-10). Although this protein kinase has not been isolated in a homogeneous state, its properties (9,11,12) indicate that it is different from pyruvate dehydrogenase kinase (13). The existence of a specific BCKDH phosphatase has been uncertain. Previous attempts to detect and purify a BCKDH phosphatase have been unsuccessful (8,14). Also contributing to this uncertainty are conflicting reports (2,4,8,14) concerning the activity of pyruvate dehydrogenase phosphatase toward the phosphorylated BCKDH complex. Dephosphorylation and reactivation of phosphorylated BCKDH complex by a broad-specificity cytosolic phosphatase have been reported (14). Although this mechanism is not considered to be physiologically relevant because of the mitochondrial localization of the BCKDH complex, cytosolic phosphatases could interfere in the assay for BCKDH phosphatase unless well-washed mitochondria are used. Activation of phosphorylated BCKDH complex by a protein factor without accompanying dephosphorylation has been observed (15). The molecular basis of this activation is not known. Low levels of a Mg2+-dependent phosphatase activity were detected in purified preparations of BCKD...

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