Pancreatic-type Phospholipase A2 Research Articles

Proliferative effect of phospholipase A2 on rat periacinar fibroblastoid cells of the pancreas.

We have shown previously that rat pancreatic periacinar fibroblastoid cells (PFCs) can be cultured from isolated pancreatic acini. In the present study, immunocytochemical examination of the PFC extracellular matrix was performed using antibodies against prolyl hydroxylase alpha and beta subunits, types I, III, and IV collagen, fibronectin, and laminin. The PFC content of alpha-smooth muscle actin and platelet-derived growth factor (PDGF) receptor were studied by immunoblotting. We demonstrated that PFCs synthesized extracellular matrix and expressed alpha-smooth muscle actin and PDGF receptors. These results suggested that PFCs resemble myofibroblasts and may play a critical role in pancreatic fibrosis. Conversely, pancreatic-type phospholipase A2 (P-PLA2), one of the pancreatic digestive enzymes, has been shown to induce DNA synthesis of Swiss 3T3 fibroblasts. To determine whether this enzyme is involved in pancreatic fibrosis, we studied P-PLA2's proliferative and chemotactic effects on PFCs as well as its digestive activity. The proliferative and chemotactic effects were investigated using 3H-thymidine incorporation and a chemotactic assay, respectively. P-PLA2 had both proliferative and chemotactic effects. P-PLA2 is considered a growth factor for PFCs and is implicated in pancreatic fibrosis.

Gene expression of pancreatic-type phospholipase-A2 in rat ovaries: stimulatory action on progesterone release.

We reported previously that pancreatic-type group I phospholipase-A2 (PLA2-I) stimulates DNA synthesis or prostaglandin E2 production in various cell types via its specific receptors on the cell surface. In the present study we examined the effect of PLA2-I on corpus luteum, which possesses specific PLA2-I receptors, and demonstrated gene expression of PLA2-I in rat ovaries. PLA2-I stimulated progesterone release from incubated corpora lutea at concentrations above 10 nM in a dose-dependent manner. Northern blot analysis revealed the presence of PLA2-I messenger RNA (mRNA) in rat ovaries. The level of PLA2-I mRNA was elevated during diestrus to proestrus, and decreased to almost nothing on the day of estrus. To further evaluate the timing of PLA2-I expression, we analyzed RNAs extracted from small follicles, large preovulatory follicles, and corpora lutea obtained from various phases of hCG-treated immature rats. During follicular development, mRNA levels remained negligible. The level of PLA2-I mRNA began to rise after luteinization of follicles and increased during the maturation of the corpora lutea. Levels of PLA2-I mRNA in ovaries from day 5 and day 10 pregnant rats were 5- to 10-fold higher than those in diestrous rats, whereas on day 21, the mRNA level was decreased. Immunocytochemical studies were performed to clarify the synthesis and localization of PLA2-I in the ovary using polyclonal anti-PLA2-I antibodies. Intense immunoreactivity was detected only in luteal cells of newly formed corpora lutea. However, the number and intensity of immunoreactive cells decreased in old corpora lutea. No immunoreactivity was detected in follicular cells of small- or middle-sized follicles. These results demonstrate the correlation among PLA2-I gene expression, estrous cycle, and progesterone secretion in rat ovaries and suggest a new function of PLA2-I as an intragonadal regulatory factor for steroidogenesis.

Pancreatic-type phospholipase A2 induces group II phospholipase A2 expression and prostaglandin biosynthesis in rat mesangial cells.

The effect of pancreatic group I phospholipase A2 (PLA2-I) on receptor-mediated expression of arthritic group II phospholipase A2 (PLA2-II) and its correlation with prostaglandin E2 (PGE2) synthesis were examined in cultured rat mesangial cells. Scatchard analysis using 125I-PLA2-I revealed the existence of a single class of specific binding sites for PLA2-I in rat mesangial cells with an equilibrium dissociation constant (Kd) of 1.6 nM and a maximum binding capacity of 10.1 fmol/10(6) cells. The mammalian mature type of PLA2-I specifically recognized this binding site, whereas its inactive zymogen and mammalian PLA2-II showed much lower affinities. PLA2-I markedly increased PLA2-II mRNA levels as well as PLA2-II secretion from the cells in a time- and dose-dependent manner that was closely correlated with PGE2 production. Both PLA2-II expression and PGE2 synthesis were completely suppressed by pretreatment of the cells with actinomycin D, cycloheximide, or dexamethasone. These results strongly suggest that there may be crosstalk between PLA2-I and PLA2-II via the specific PLA2-I receptor that elicits PGE2 synthesis.

Characterization of a high affinity binding site for pancreatic-type phospholipase A2 in the rat. Its cellular and tissue distribution.

We showed in an earlier study (Arita, H., Hanasaki, K., Nakano, T., Oka, S., Teraoka, H., and Matsumoto, K. (1991) J. Biol. Chem. 266, 19139-19141) that there is a high affinity and specific binding site for mammalian group I phospholipase A2 (PLA2-I) in Swiss 3T3 fibroblast cells. Analysis of the cellular distribution in rat using 125I-PLA2-I as a radioligand indicated the presence of this site in various cells, including vascular smooth muscle cells (VSMC), vascular endothelial cells, synovial cells, chondrocytes, and gastric mucosal cells. Scatchard analysis of rat VSMC revealed the existence of a single class of binding site with a Kd value of 1.60 nM and a Bmax value of 51.0 fmol/10(6) cells. The mammalian mature type of PLA2s-I derived from several animal species specifically recognized the same site in cells and stimulated DNA synthesis, whereas its inactive zymogen, mammalian group II PLA2s, and snake and bee venom PLA2s showed much lesser activities. 125I-PLA2-I bound to VSMC was rapidly internalized and subsequently released from the cells as trichloroacetic acid-soluble radioactivity. Down-regulation of the PLA2-I site was observed in the treatment of VSMC with cAMP-elevating agents, as well as glucocorticoids. Affinity labeling experiments indicated that PLA2-I binds to a single polypeptide with a mass of approximately 200,000 daltons. These results suggest a novel PLA2-I action on the cellular function via its specific binding site.

Novel proliferative effect of phospholipase A2 in Swiss 3T3 cells via specific binding site.

Phospholipase A2 (PLA2), EC 3.1.1.4, which catalyzes the release of free fatty acids from the sn-2 position of glycerophospholipids, has been extensively studied from the viewpoint of eicosanoid production (Arita, H., Nakano, T., and Hanasaki, K. (1989) Prog. Lipid Res. 28, 273-301). Several lines of evidence suggest that extracellular PLA2 is pathophysiologically related to some disorders, including inflammation and hypersensitivity. Despite this, little is known of the precise mechanism of the pathological processes as well as their intrinsic correlation with dysfunction. Here, we report a novel PLA2 action on the proliferation of Swiss 3T3 fibroblasts via specific binding sites of approximately Mr 200,000. Pancreatic type PLA2 in the active form specifically recognized the sites and stimulated thymidine incorporation in DNA. Its inactive zymogen and other PLA2s from platelets, snake, and bee venoms showed much lesser activities. Although the physiological significance remains to be identified, our finding is the first to offer a new viewpoint on the effect of mammalian extracellular PLA2 on cellular function.

Immunocytochemical studies on the localization of pancreatic-type phospholipase A2 in rat stomach and pancreas, with special reference to the stomach cells

Using a specific polyclonal antibody raised against rat pancreatic phospholipase A2 (PLA2), we investigated the localization of the enzyme in the rat pancreas and stomach by light and electron microscopy. In the pancreas, the enzyme was localized in the acinar cells, whereas the pancreatic islets showed no immunoreaction. In the stomach, the PLA2 reactive with the anti-pancreatic PLA2 antibody was distributed exclusively in the gastric glands, but not in the gastric pits or the pyloric glands. On the section of the stomach subjected to immuno- and PAS-staining, immunopositive cells were not the PAS-positive cells located in the gastric pit and the neck region of the gastric gland. Immunopositive cells were present from the neck to the bottom of the gastric gland. Immunoelectron microscopic observation revealed that the immunogold-labeled cell had a highly-developed rough endoplasmic reticulum in the basal cytoplasm and characteristic zymogen granules in the apical cytoplasm. Taking into account the cell position in the gastric gland, the immunopositive cell could therefore be identified as a chief cell. Since no double stainability with PLA2 and PAS was observed in the same cell, it is suggested that PLA2 could be used cytochemically as a marker enzyme of the chief cell in the gastric gland at the light-microscopic level. From the immunoelectron microscopic findings, we believe that the PLA2 in the stomach is released into the lumen of the stomach by exocytosis and could function as a digestive enzyme in the alimentary tract, like the PLA2 secreted from the pancreas. Other possible roles of the PLA2 in the stomach are discussed.