Tokuro Ichida

Phorbol ester stimulates amylase secretion from rat parotid cells

Phorbol myristate acetate (PMA), a potent activator of Ca2+‐ and phospholipid‐dependent protein kinase (protein kinase C), evoked amylase release from rat parotid cells. In dose‐response studies, PMA stimulated amylase release independently of db‐cAMP, but potentiated the effect of carbachol. PMA and A23187, a Ca2+ ionophore, synergistically increased amylase release. The maximum effect of carbachol was further enhanced by PMA but not by A23187, suggesting that protein kinase C is not fully activated by the muscarinic‐cholinergic agonist under the condition where calcium is fully utilized for amylase secretion.

Does cyclic AMP mobilize Ca2+ for amylase secretion from rat parotid cells?

Both dibutyryl cAMP and carbachol stimulated amylase released from rat parotid cells incubated in Ca2+-free medium containing 1 mM EGTA. Cells preincubated with 10 microM carbachol in Ca2+-free, 1 mM EGTA medium for 15 min lost responsiveness to carbachol, but maintained responsiveness to dibutyryl cAMP. Dibutyryl cAMP still evoked amylase release from cells preincubated with 1 microM ionophore A23187 and 1 mM EGTA for 20 min. Although carbachol stimulated net efflux of 45Ca from cells preequilibrated with 45Ca for 30 min, dibutyryl cAMP did not elicit any apparent changes in the cellular 45Ca level. Inositol trisphosphate, but not cAMP, evoked 45Ca release from saponin-permeabilized cells. These results suggest that cAMP does not mobilize calcium for amylase release from rat parotid cells.

Evidence for the involvement of protein phosphorylation in cyclic AMP-mediated amylase exocytosis from 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, 867‐871]. 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 (μM since 33 μM 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 μM. 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.

OKADAIC ACID INHIBITS AMYLASE EXOCYTOSIS FROM PAROTID ACINI STIMULATED BY CYCLIC AMP

To evaluate the role of protein phosphorylation in amylase exocytosis, we studied the effects of okadaic acid, a potent inhibitor of protein phosphatase types I 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.

Protein phosphatase inhibitor calyculin A induces hyperphosphorylation of cytokeratins and inhibits amylase exocytosis in the rat parotid acini

Calyculin A, a protein phosphatase inhibitor with a chemical structure completely different from that of okadaic acid, reproduced the inhibitory effect of okadaic acid on cyclic AMP‐mediated amylase release from rat parotid acinar cells. Calyculin A markedly enhanced phosphorylation of cytokeratins in the cytoskeletal fraction of the cells, whereas cAMP had apparently no effect on the phosphorylation. Microscopic observations showed that parotid acini incubated with 100 nM calyculin A for 15 min had large vacuoles in the cytoplasm and conspicuous blebs on the basal plasma membrane. K252a, a nonselective protein kinase inhibitor, clearly reduced calyclin A‐induced phosphorylation of cytokeratins, and it markedly blocked the inhibition of amylase release and morphological changes evoked by calyculin A. These results suggest that hyperphosphorylation of cytokeratins profoundly affects the morphology and secretory activity of parotid acinar cells.

Evidence for the putative docking/fusion complex of exocytosis in parotid acinar cells

© 1997 Federation of European Biochemical Societies.

Effect of genistein on amylase release and protein tyrosine phosphorylation in parotid acinar cells

We evaluated the role of protein tyrosine phosphorylation in amylase exocytosis from parotid acinar cells by using genistein, a tyrosine kinase inhibitor. Amylase release stimulated by isoproterenol was dose‐dependently inhibited by genistein. Genistein also inhibited the exocytosis evoked by dibutyryl‐ or 8‐chlorophenylthio‐cAMP. Daidzein, a negative control agent of genistein, elicited no inhibitory effect. Isoproterenol had dual effects on protein tyrosine phosphorylation; it increased the phosphorylation of 190‐ and 210‐kDa proteins and decreased that of a 90‐kDa one. The phosphorylation was dose‐dependently inhibited by genistein but not by daidzein. These results suggest that protein tyrosine phosphorylation plays a role in the process of amylase exocytosis from parotid acinar cells.

Regulation of CREB phosphorylation by cAMP and Ca2 in parotid acinar cells

Since various secretory stimuli regulate not only secretion but also protein, RNA, and DNA syntheses in salivary glands, we evaluated the effect of secretory stimuli on the phosphorylation state of CREB (cAMP response element‐binding protein). Isoproterenol, forskolin, and CPS‐cAMP markedly stimulated the phosphorylation of CREB in parotid acinar cells, and PKA inhibitors H‐8 and H‐89 dose‐dependently inhibited it. In contrast, carbachol (CCH) and A23187 decreased CREB phosphorylation, but CCH did not decrease it in the absence of extracellular Ca2+. Although protein phosphatase inhibitor calyculin A alone markedly increased the phosphorylation, it could not prevent CCH‐induced dephosphorylation of CREB. CaM kinase IV, a putative protein kinase for CREB in response to Ca2+ elevation, was undetectable in parotid acinar cells.

Regulation of functional mRNA levels for trypsin-like esteroproteases by 5α-dihydrotestosterone and triiodothyronine in mouse submandibular salivary gland

The effects of 5 alpha-dihydrotestosterone and triiodothyronine on the levels of mRNA for trypsin-like esteroproteases in the submandibular glands of young female mice were examined using a translation system in a reticulocyte lysate. Immunoprecipitation of the trypsin-like esteroproteases synthesized in vitro gave almost the same isozyme profile as that of enzymes synthesized in vivo on isoelectric focusing in acrylamide gel containing 8 M urea. Although 5 alpha-dihydrotestosterone and triiodothyronine induced the enzyme activity 3-5 times, these hormones increased their functional mRNA levels only 45-70 per cent. These results suggest that there is little if any post-translational processing of esteroproteases and that hormones increase both the amount of mRNA and the efficacy of its translation.

Basic isozymes of chymotrypsin-like esteroprotease from the mouse submandibular gland

Basic isozymes of chymotrypsin-like esteroprotease from mouse submandibular glands were purified 60-80-fold by a rather simple procedure consisting of CM-Sepharose CL6B chromatography and gel filtration on Sephadex G-100. The purified sample contained three major isozymes (A, B, C) and some minor ones. Their isoelectric points were between pH 10 and 11. The molecular weights of the main isozymes were estimated at 28000 by SDS-polyacrylamide gel electrophoresis. The acidic isozyme (A) separated into two polypeptide chains whose molecular weights were 21500 and 6500. Specific activities of these isozymes using Bz-Tyr-OEt as substrate were comparable to that of bovine pancreatic alpha-chymotrypsin, but they hydrolyzed casein 10 times slower than did alpha-chymotrypsin. The hydrolytic activities of these isozymes on Bz-Tyr-OEt were inhibited by diisopropylfluorophosphate, tosyl-L-phenylalanine chloromethyl ketone and chymostatin, but they were 400 times less sensitive to chymostatin than was alpha-chymotrypsin.