Yutaka Yokota

Ca2+-independent phospholipase A2 activity associated with secretory granular membranes in rat parotid gland

Phospholipase A2 activity was detected in a secretory granular fraction (SG) purified by Percoll gradient centrifugation from rat parotid gland using [3H]phosphatidylcholine (PC) as a substrate. High activity of this enzyme was observed at neutral pH. The enzyme was activated by Triton X-100 and did not require Ca2+ for its activity. In the absence of Ca2+, its apparent Km for exogenous PC was 28 μM while it was slightly increased by adding 5 mM CaCl2 (73 μM). Furthermore, the enzyme was located essentially in a granular membrane fraction separated from granular lysate. The deacylation activities were also detected in other subcellular fractions, which showed a different detergent-susceptibility of pH-dependency from that in SG. These results suggest that secretory granules have membrane-bound phospholipase A2 which has properties different from that found in other organelles.

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.

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.

Localization of a low Mr GTP-binding protein, rap1 protein, in plasma membranes and secretory granule membranes of rat parotid gland

Subcellular fractions were prepared from rat parotid gland by sequential centrifugation, Percoll gradient centrifugation and divalent-cation precipitation, and the localization of a low Mr GTP-binding protein, rap1 protein (rap1p) was analyzed by immunoblotting using a specific antibody. rap1p was found to be located in apical and basolateral plasma membranes, and secretory granule membranes in rat parotid gland. On the other hand, the beta gamma subunits of heterotrimeric Gt protein was localized in plasma membranes but not in granule membranes.

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.

Biochemical and physical analysis of subcellular membranes in rat parotid gland enlarged by chronic isoproterenol administration

1. Chronic administration of isoproterenol caused similar alterations of membrane lipid profile in at least two rat parotid subcellular fractions, secretory granular and microsomal. 2. Typical changes in phospholipid classes and fatty-acyl chain groups were an increase of phosphatidylcholine and a decrease of sphingomyelin, and an increase of octadecadienoyl chain and a decrease of eicosatetraenoyl chain, respectively. 3. Electron spin resonance study showed that the isoproterenol-treatment also affected a membrane physical property, which may be through these compositional changes in membrane constituents.