Koji Yashiro

Alteration of membrane phospholipids in hypertrophied rat salivary glands induced by chronic administration of isoproterenol

Hypertrophy of parotid and submandibular glands, and elevated phospholipid content per gland in both were induced. However, phospholipid content per tissue weight increased only in the parotid gland. Sublingual gland phospholipids were unaffected with regard to the gland weight and phospholipid content; phospholipid class compositions were altered in each of the three glands, typically an increase of phosphatidylcholine and decreases of sphingomyelin and phosphatidic acid. Concerning fatty-acyl groups in phospholipid, prominent increase of octadecadienoic acid was observed in total and individual phospholipids of both parotid and submandibular glands. However, eicosatetraenoic acid decreased in the total phospholipids of these two glands. In the sublingual gland, phospholipids, fatty-acyl compositions were not changed by isoproterenol. The results suggest that isoproterenol-induced changes of membrane phospholipids are regulated by at least two different effects, one for fatty-acyl groups and another for base moieties of phospholipids.

Phospholipid metabolism in rat submandibular gland. Positional distribution of fatty acids in phosphatidylcholine and microsomal lysophospholipid acyltransferase systems concerning proliferation

Rat submandibular gland phosphatidylcholine mainly consisted of the 1-saturated acyl-2-unsaturated acyl type. The high occupancy of unsaturated fatty acid at the C-2 position is in part explained by the preference of microsomal acyl-CoA:1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC) acyltransferase for unsaturated fatty acyl-CoAs. This enzyme activity was partially inhibited by divalent cations. Ca2+ may be important for regulation of a deacylation-reacylation cycle, suggested because Ca2+ is also known to activate the deacylation enzyme, phospholipase A2. Although the presence of 1-acyl-GPC acyltransferase activity is also observed in plasma membrane of the submandibular gland, the microsomal enzyme showed properties different from the enzyme in plasma membrane in terms of its susceptibility to neural salts and detergents. Cell proliferation caused by chronic administration of isoproterenol resulted in an increase of linoleic acid at the C-2 position of phosphatidylcholine. However, this alteration did not correlate with the changes of activity and substrate specificity of 1-acyl-GPC acyltransferase and the other C-2 acylation enzyme, 1-acyl-sn-glycero-3-phosphate acyltransferase, which suggests that the alteration of fatty acid by isoproterenol treatment is due to a change of supply of substrates or specific acyl breakdown of phosphatidylcholine.

Comparison of membrane phospholipid and its fatty acid compositions in developing rat salivary glands

1. Membrane phospholipid and its fatty acid compositions have been analyzed in 3- and 9-week-old rat salivary glands. 2. When compared between the three major glands (parotid, submandibular, sublingual) in adult rats, phospholipid compositions were similar, but for their fatty acid, characteristic properties from each phospholipid were shown. 3. Alterations in their compositions were also observed during development of the salivary glands.

Ca2+-independent phospholipase A2 activity in apical plasma membranes from the rat parotid gland

An apical-enriched plasma membrane fraction (A-PM) was prepared from rat parotid gland by Mn2+ precipitation. In this fraction, phosphatidylcholine (PC) labelled at the sn-2 position was mainly decomposed into two labelled compounds (free fatty acid and 1,2-diacylglycerol) under Ca2+-free conditions. Studies using double-labelled PC and 2,3-diphosphoglycerate (as a phospholipase D inhibitor) showed that they were produced through different pathways: free fatty acid was released by phospholipase A2 (PLA2) while 1,2-diacylglycerol may be produced by sequential action of phospholipase D and phosphatidate phosphatase. The PLA2 in A-PM did not require Ca2+ for its activity and was highly activated by Triton X-100 and ATP. The inhibitor of the well-documented Ca2+-independent PLA2, bromoenol lactone, did not inhibit the PLA2 activity in A-PM. Although PLA2 activity was detected in other subcellular fractions, the highest specific activity was in A-PM. Its distribution among various fractions was roughly similar to that of the marker enzyme of apical plasma membranes. These findings suggested that Ca2+-independent PLA2 activity is present in apical plasma membranes from rat parotid gland. In addition, to clarify the involvement of the PLA2 in exocytosis, the fusion of exogenous PLA2-treated membranes with secretory granules was examined by fluorescence dequenching assay. This study clearly demonstrated the facilitation of fusion by PLA2 treatment, which suggests some involvement of apical PLA2 in saliva secretion.

SUBSTRATE SPECIFICITY OF MICROSOMAL 1-ACYL-SN-GLYCERO-3-PHOSPHOINOSITOL ACYLTRANSFERASE IN RAT SUBMANDIBULAR GLAND FOR POLYUNSATURATED LONG-CHAIN ACYL -COAS

Microsomal 1-acyl-sn-glycero-3-phosphoinositol (1-acyl-GPI) acyltransferase in the rat submandibular gland showed the highest specific activities for eicosanoid-related polyunsaturated acyl-CoAs, such as arachidonoyl-, bishomo-gamma-linolenoyl- and 5,8,11,14,17-eicosapentaenoyl-CoAs, with low Km values. High activities were also obtained with acyl-CoAs having long (more than 14 carbon atoms) and n - 6 unsaturated (more than 3 double bonds) acyl chains. This enzyme also utilized acyl-CoAs having trans-unsaturated or branched chains, but not short-chains, as substrates, although the activity levels for trans-unsaturated acyl-CoAs were lower than those for cis-unsaturated acyl-CoAs. Chronic administration of isoproterenol induced decreases of this enzyme activity and the content of arachidonic, bishomo-gamma-linolenic and 5,8,11,14,17-eicosapentaenoic acids at the sn-2 position of phosphatidylinositol. These results suggest that enrichment of arachidonic acid in the sn-2 position of phosphatidylinositol is established by the high specificity and affinity of 1-acyl-GPI acyltransferase for arachidonoyl-CoA. On the other hand, the low level of bishomo-gamma-linolenic and 5,8,11,14,17-eicosapentaenoic acids in the sn-2 position of phosphatidylinositol may be explained by their limited availability.

Properties of membrane phospholipids and their fatty acyl compositions of secretory granules from rat parotid gland

A secretory granular fraction isolated from rat parotid glands was remarkably different from a microsomal fraction in its phospholipid composition. It had higher levels of lysophospholipids (8%) and phosphatidylethanolamine (31%), while there were lower levels of phosphatidylcholine (40%) and phosphatidylserine (2.1%) than the microsomal fraction. However, fatty acid compositions of individual phospholipid classes from the two subfractions were found to be nearly similar to each other. ESR analysis demonstrated that extracted phospholipids from the secretory granular fraction were more fluid than those from microsomes. The relevance of these observations to physiological function of secretory granules is discussed.

Comparison of phospholipid synthesis in rat salivary glands: Properties of 1-acylglycerophosphorylcholine and 1-acylglycerophosphate acyltransferase systems in microsomes

1. 1-Acyl-sn-glycerol-3-phosphorylcholine and 1-acyl-sn-glycerol-3-phosphate acyltransferase activities were characterized in rat salivary gland microsomes. 2. The acyl-CoA selectivities between these two kinds of lysophospholipid acyltransferase activities were very different. 3. When the three major glands were compared (parotid, submandibular, sublingual), they showed their own particular acyltransferase activity, but they had very similar in acyl-CoA selectivity. 4. Those observations were also compared in rat liver microsomes.

Properties of plasma membrane-induced amylase release from rat parotid secretory granules: effects of Ca2+ and Mg-ATP

A secretory granular fraction (SG) and a plasma membrane rich fraction (PM) have been isolated from rat parotid gland by differential and Percoll gradient centrifugation. With these two fractions, a cell-free interaction system has been reconstituted to clarify the exocytotic interaction between the secretory granules and plasma membranes, and the conditions of amylase release from SG have been characterized in vitro. The addition of PM into this assay system induced a rapid and transient release of amylase from SG. Some other membranes such as erythrocyte ghosts also mimicked the effect of PM. This release was increased by Ca2+, but was not completely blocked by EGTA. Simultaneous addition of 1 mM ATP with 1 mM MgCl2 (Mg-ATP) in the presence of Ca2+ reduced this release. However, in spite of the existence of Mg-ATP, the stimulation of PM-induced amylase release was caused by Ca2+ in a concentration-dependent manner (10(-7)-10(-3) M). These results suggest that Ca2+ and Mg-ATP should participate as important regulators in the exocytotic interaction between secretory granules and plasma membranes in this system. Furthermore, the differences between our system and intact cells are also discussed.

Comparison of phospholipid N-methylation activity in rat submandibular salivary gland and liver

Successive phospholipid N-methylation from phosphatidylcholine to phosphatidylethanolamine in submandibular gland and liver microsomes proceeded without the addition of exogenous phospholipid substrate. Methylation activity in the submandibular microsomes showed different susceptibilities to various detergents than the liver enzyme and also partially required Mg2+. However, the three methylation steps could not be distinguished by their Mg2+ requirements. Ca2+ had no effect on the activity. The methylation activity in submandibular gland was much lower than in liver. Chronic administration of isoproterenol, which causes an increase of phosphatidylcholine in membrane phospholipids of salivary glands, decreased methylation activity in the submandibular gland. Thus the increase in phosphatidylcholine in isoproterenol-treated rat salivary glands may not be derived from the phospholipid methylation pathway, but may be due to stimulation of other routes of phosphatidylcholine metabolism.

Glutathione-dependent Peroxidase Activities in Rat Submandibular Gland

The activities of Se-dependent glutathione peroxidases and non-Se-dependent glutathione peroxidase in the submandibular gland were observed using specific substrates. The activities for H2O2, cumene hydroperoxide, tert-butyl hydroperoxide, and phosphatidylcholine hydroperoxide were strongly inhibited by iodoacetate. After correction for the activity toward cumene hydroperoxide, it was shown that cumene hydroperoxide is mainly reduced by cytosolic glutathione peroxidase. Although the specific activity was lower than that of cytosolic glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase showed activity toward not only phosphatidylcholine hydroperoxide but also phosphatidylethanolamine hydroperoxide. These results suggest that cytosolic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase share a role in the reduction of hydroperoxide, but non-Se-dependent glutathione peroxidase (glutathione S-transferase) plays a lesser role.