Toyoshi Endo

Effects of vinpocetine on cyclic nucleotide metabolism in vascular smooth muscle

A novel vasodilating agent, vinpocetine (14-ethoxycarbonyl-(3 alpha,16 alpha-ethyl)-14,15-eburnamenine) inhibits Ca2+-dependent phosphodiesterase, selectively, among the three forms of cyclic nucleotide phosphodiesterase identified in the rabbit aorta. The concentration of vinpocetine producing 50% inhibition of Ca2+-dependent phosphodiesterase activity was approximately 21 microM, both in the presence and absence of Ca2+-calmodulin (CaM). Increasing the concentration of CaM in the presence of Ca2+ did not prevent vinpocetine-induced inhibition of Ca2+-dependent phosphodiesterase, thereby indicating that vinpocetine inhibited the enzyme by interacting with the enzyme and not with CaM. To determine the influence of vinpocetine-induced inhibition of Ca2+-dependent phosphodiesterase on cyclic nucleotide metabolism in vascular smooth muscle, cyclic nucleotide levels in isolated rabbit aortic strips were also investigated. Addition of vinpocetine produced dose-dependent increases in only the cyclic GMP levels and there was no significant effects on the cyclic AMP levels. These results provide pharmacological evidence that Ca2+-dependent phosphodiesterase mainly hydrolyzes cyclic GMP in vascular smooth muscle. Vinpocetine may induce vascular relaxation by increasing cyclic GMP contents in vascular smooth muscle through selective inhibition of Ca2+-dependent phosphodiesterase.

Distribution of S-100b protein in normal salivary glands and salivary gland tumors

Immunohistochemical studies were performed for the presence of S-100b protein in non-neoplastic and neoplastic salivary gland tissues by the peroxidase anti-peroxidase (PAP) method. Some cases of pleomorphic adenoma were investigated by immuno-electron microscopy. S-100b protein could not be detected in epithelial cells of intercalated ducts, acini, striated ducts and excretory ducts of non-neoplastic salivary gland. However, myoepithelial cells surrounding the acini and intercalated ducts were specifically stained by S-100b protein. In pleomorphic adenomas, S-100b protein-positive cells could be mostly observed in the myxoid and chondroid areas, and the basal layer cells of the double-layered ductal cells were also positive. In clear cell adenoma, the clear cells were also S-100b protein positive. In adenoid cystic carcinomas, S-100b protein-positive cells could be found in trabecular areas, but not in tumor cells showing cribriform-pattern. In other tumors (Warthins tumor, oxyphilic adenoma, basal cell adenoma, mucoepidermoid tumor and acinar cell carcinoma), S-100b protein positive cells were seldom observed. Immuno-electron microscopically, S-100b protein was diffusely distributed in the cytoplasm of myoepithelial cells as well as of tumor cells of pleomorphic adenoma, being distributed especially on the membrane of endoplasmic reticulum and the outer nuclear membrane.

Ca2+-phospholipid dependent phosphorylation of smooth muscle myosin

Abstract Isolated myosin light chain from chicken gizzard has been shown to serve as a substrate for Ca 2+ -activated phospholipid-dependent protein kinase. Autoradiography showed that Ca 2+ -activated phospholipid-dependent protein kinase phosphorylated mainly the 20,000-dalton light chain of chicken gizzard myosin. Exogenously added calmodulin had no effect on myosin light chain phosphorylation catalyzed by the enzyme. The 20,000-dalton myosin light chain, both in the isolated form and in the whole myosin form, served as the substrate for this enzyme. In contrast to the isolated myosin light chain, the light chain of whole myosin was phosphorylated to a lesser extent by the Ca 2+ -activated phospholipid dependent kinase. Our results suggest the involvement of phospholipid in regulating Ca 2+ -dependent phosphorylation of the 20,000-dalton light chain of smooth muscle myosin.

Effect of S-100 protein on microtubule assembly—disassembly

S‐100 protein was examined for its ability to affect microtubule assembly—disassembly in vitro. Addition of S‐100 protein inhibited microtubule assembly in a dose‐dependent manner in the presence of Ca2+, but this effect was not observed in the presence of 2 mM EGTA. The interaction of microtubule proteins with S‐100 protein was also demonstrated by Ca+‐dependent affinity chromatography on S‐100 protein—Sepharose. The proteins bound to the Sepharose column comigrated with tubulin as judged by sodium dodecyl sulfate—polyacrylamide gel electrophoresis. These results might suggest a possible physiological role of S‐100 protein in the microtubule assembly—disassembly system.

Vitamin A acid-induced activation of Ca2+-activated, phospholipid-dependent protein kinase from rabbit retina.

Ca2+-activated, phospholipid-dependent protein kinase from rabbit retina was partially purified. Vitamin A acid (retinoic acid) stimulated this protein kinase in the presence of Ca2+, while other metabolites of vitamin A such as retinol or retinal were less effective. The order of the extent of phosphorylation of the various substrate proteins by this protein kinase was identical in the presence of vitamin A acid or phosphatidylserine. The major spots of the 32P labeled peptide from histone H1 phosphorylated in the presence of vitamin A acid by this protein kinase did not differ from those obtained from histone H1 phosphorylated in the presence of phosphatidylserine. Retinol caused a further enhancement of the enzymatic activity, whereas the addition of retinal inhibited the activation by vitamin A acid. Thus, vitamin A and its metabolites may play an important role in the regulation of Ca2+-activated, phospholipid-dependent protein kinase activity in the retina.

S-100 antigen in human T lymphocytes

The contents of the S-100 antigen in human blood components were determined using a sensitive enzyme-immuno assay system specific to S-100. The S-100 antigen was mainly present in the white blood cells, especially in the T lymphocytes, in blood from healthy humans. A remarkable decrease in S-100 antigen content of lymphocytes from patients with acute T lymphocytic leukemia was observed. On the other hand, the calmodul in contents in the lymphocytes did not decrease. The possibility that S-100 plays a role not only in tissues of neuronal or neuroectodermal origin but also in non-neuroectodermal cells such as T lymphocytes warrants further investigation.

Ca2+-Calmodulin dependent phosphorylation of myelin isolated from rabbit brain

Abstract The phosphorylation of myelin (basic protein) purified from rabbit brain was markedly stimulated by exogenously added calmodulin in the presence of calcium and inhibited by W-7(N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), a calmodulin interacting agent, in a dose-dependent fashion. However, exogenously added myelin basic protein free from protein kinase activity could not serve as a substrate of this calmodulin dependent protein kinase, suggesting that this kinase catalyzes the phosphorylation of the enzyme-substrate complex. These results suggest that a calmodulin-dependent protein kinase complex with the substrate (basic protein) is located in the myelin membrane of the central nervous system.

Phospholipids and regulation of protein kinase reaction

Abstract Naturally occurring phospholipids such as phosphatidylinositol and phosphatidylserine inhibited cAMP-dependent protein kinase by interacting with the substrate protein (phosphate acceptor). This inhibition was observed both in the presence and absence of cAMP when histone H2B, protamine, and myelin basic protein were used, but was not detected when casein was used as the substrate. Other phospholipids such as phosphatidylethanolamine and phosphatidylcholine did not inhibit the kinase but did stimulate the kinase when protamine served as the substrate. Both cAMP-dependent and cGMP-dependent protein kinases were inhibited by phosphatidylserine when histone H2B was used as substrate. The substrate protein binding to phosphatidylinositol and phosphatidylserine was observed when these phospholipids were added to the incubation mixture, suggesting that direct interaction between the substrate protein and the phospholipids resulted in inhibition of cAMP and cGMP-dependent protein kinase. Thus the substrate protein for protein kinase probably plays an important role in regulating the kinase activity related to various phospholipids.