Cofilin is an actin-depolymerizing protein, whose depolymerizing activity is supposed to be regulated in part by phosphorylation and dephosphorylation. Thus, we studied the phosphorylation states of cofilin in rat parotid acinar cells during stimulation for amylase exocytosis. Isoproterenol and carbachol induced rapid and extensive dephosphorylation of cofilin; 60-70% dephosphorylation was clearly detectable within 1 min. Membrane-permeable cyclic AMP (CPS-cAMP), phorbol ester (PMA), and Ca ionophore A23187 mimicked the effect of isoproterenol and carbachol. Protein phosphatase inhibitors (calyculin A or FK506 plus cyclosporin A) did not block the dephosphorylation in response to isoproterenol or carbachol. Furthermore, calyculin A alone strongly dephosphorylated cofilin. Although no exogenous protein phosphatases tested dephosphorylated cofilin in the homogenate, the cofilin that was isolated by immunoprecipitation was clearly dephosphorylated by protein phosphatases 1, 2A, and 2C.

(1) Rabbits and rats were fed a high-fat, high-cholesterol diet to produce lipid deposition in the wall of the aorta. The intima and inner media were examined for their content of lipids, protein-bound carbohydrates, and individual glycosaminoglycans. In addition, the rate of uptake of 35S from sulfate into the sulfated glycosaminoglycans was measured in some animals.(2) In the rat, lipid accumulation was minimal; there were increases in the protein-bound hexosamine and sialic acid and decreases in all the glycosaminoglycans (hyaluronic acid, heparan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate).(3) In the rabbit, lipid accumulation was marked in the normal appearing tissue and still greater in the "fatty streaks". There were associated increases in protein-bound hexosamine and hexose and, in the fatty streaks, of sialic acid also. The concentrations of all sulfated glycosaminoglycans were higher in the fatty streaks than in the surrounding tissue.(4) In the rat, the rate of synthesis of chondroitin sulfates, as reflected in sulfate-35S incorporation, was not increased, but the turnover of chondroitin sulfates as reflected in the fractional rate of 35S incorporation was increased; this is taken as evidence that the fall in concentration was due to an increase in the rate of removal. In the rabbit, both synthesis and turnover of chondroitin sulfates were greatly increased in the fatty streaks; the change in synthesis was greater and led to the increase in the concentrations of these substances.(5) The concentration of hyaluronic acid was increased in the aortae of alloxan diabetic animals. Sulfate incorporation was reduced, indicating decreased synthesis of sulfated glycosaminoglycans. Despite this, the aortae of diabetic animals had virtually the same composition (except for hyaluronic acid) as those of non-diabetics given the same diet.

We evaluated the role of cytosolic phospholipase A2 (PLA2) in the exocytosis of amylase from parotid acinar cells. The exocytosis stimulated by isoproterenol was dose-dependently inhibited by bromoenol lactone (BEL), a potent suicide inhibitor of Ca2+-independent cytosolic PLA2. The IC50 value of BEL was approximately 7 microM. AACOCF3, a selective inhibitor of Ca2+-dependent cytosolic PLA2, did not inhibit the exocytosis at least up to 30 microM. BEL also inhibited amylase release evoked by forskolin and membrane-permeable cAMP, but it did not inhibit cAMP-dependent protein kinase activity. PLA2 activity in parotid acinar cells was found to be predominantly Ca2+-independent, and was strongly inhibited by BEL, whose IC50 value was approximately 2 microM when it was applied to intact acini. Although isoproterenol scarcely enhanced [3H]arachidonic acid release from intact acinar cells, BEL dose-dependently decreased the basal arachidonic acid release to approximately one half of the control value. These results suggest that the cytosolic Ca2+-independent PLA2 activity plays a role in the membrane fusion process of exocytosis in parotid acinar cells.

We evaluated the role of protein phosphorylation in CAMP-mediated amylase exocytosis from parotid acinar cells by using H89, a new protein kinase A (PKA) inhibitor, which is more lipophilic and 25 times more potent than H8. In our previous studies, H8 markedly inhibited protein phosphorylation without decreasing amylase release [Takuma, T. (1988) Biochem. J. 256, 86778711. These findings were completely reproduced even in the small acini that were prepared by trypsin treatment before collagenase digestion. In the present study, however, H89 strongly inhibited both amylase release and protein phosphorylation in a dose-dependent manner. The inhibibory effect was specific for PKA at least up to 33 ,uM, since 33 ,uM H89 did not block amylase release stimulated by PMA. H85, a closely related compound of H89 without inhibitory effect on PKA, did not prevent amylase release or protein phosphorylation at least up to 33 PM. These results suggest that protein phosphorylation by PKA is involved in CAMP-mediated amylase exocytosis. The inhibition of protein phosphorylation by H8 might be insufficient or inadequate for blocking of amylase release.

To evaluate the role of protein phosphorylation in amylase exocytosis. we studied the effects of okadaic acid, a potent inhibitor of protein phosphatase types 1 and 2A, on amylase release and protein phosphorylation in rat parotid acini. Although okadaic acid by itself weakly stimulated amylase release, it did not potentiate amylase release stimulated by half-maximum doses of isoproterenol or CAMP, and markedly inhibited their maximum effects. Okadaic acid dose-dependently increased CAMP-independent phosphorylation of some proteins and enhanced CAMP-dependent phosphorylation of 21. and 26-kDa proteins. These results indicate that increase in protein phosphorylation does not necessarily enhance the exocytosis of amylase from parotid acini.