Effect of procainhydrochloride on phospholipase A2 catalytic activity in sodium taurocholate-induced acute experimental pancreatitis in rats.

BACKGROUND: In human acute pancreatitis (AP) the local anaesthetic procainhydrochloride (procain-HCl) is given intravenously for pain treatment. Procain has been shown to inhibit catalytic activity of pancreatic (group I) phospholipase A2 (PLA2) and non-pancreatic (group II) PLA2. Both enzymes are important mediators for the local and systemic inflammatory process in AP. To determine the effect of procain, we examined serum and tissue levels of both types of PLA2 activity in the experimental rodent taurocholate model of AP. METHODS: In 60 rats, severe pancreatitis was induced by taurocholate. Forty rats were treated with procain-HCl intravenously at a dosage of 2 mg/kg body weight/h either at or 1 h after induction of pancreatitis. Twenty rats served as controls. We measured catalytic activities of group I and group II PLA2 in serum and tissue samples of lung and pancreas. RESULTS: Serum group II PLA2 catalytic activity was significantly reduced 3 and 6 h after AP induction in rats treated with procain-HCl (p < 0.001) in both treatment groups. In pancreatic and lung tissue, group II PLA2 catalytic activity was significantly reduced compared with normal values (p < 0.001). CONCLUSION: Procain-HCl given intravenously either at or 1 h after induction of necrotizing pancreatitis significantly inhibits group II PLA2 catalytic activity in serum and tissues.

Expression of human group II PLA2 in transgenic mice results in epidermal hyperplasia in the absence of inflammatory infiltrate.

Group II PLA2 has been implicated in inflammatory processes in both man and other animals and has been shown to be involved in inflammatory conditions, such as arthritis and sepsis. Transgenic mice expressing the human group II PLA2 gene have been generated using a 6.2-kb genomic fragment. These mice express the group II PLA2 gene abundantly in liver, lung, kidney, and skin, and have serum PLA2 activity levels approximately eightfold higher than nontransgenic littermates. The group II PLA2 transgenic mice reported here exhibit epidermal and adnexal hyperplasia, hyperkeratosis, and almost total alopecia. The chronic epidermal hyperplasia and hyperkeratosis seen in these mice is similar to that seen in a variety of dermatopathies, including psoriasis. However, unlike what is seen with these dermatopathies, no significant inflammatory-cell influx was observed in the skin of these animals, or in any other tissue examined. These mice provide an important tool for examining group II PLA2 expression, and for determining the role of group II PLA2 in normal and disease physiology. They serve as an in vivo model for identifying inhibitors of group II PLA2 activity and gene expression.

CB2 receptor activation causes an ERK1/2-dependent inflammatory response in human RPE cells.

A chronic low-level inflammation contributes to the pathogenesis of age-related macular degeneration (AMD), the most common cause of blindness in the elderly in Western countries. The loss of central vision results from attenuated maintenance of photoreceptors due to the degeneration of retinal pigment epithelium (RPE) cells beneath the photoreceptor layer. It has been proposed that pathologic inflammation initiated in RPE cells could be regulated by the activation of type 2 cannabinoid receptors (CB2). Here, we have analysed the effect of CB2 activation on cellular survival and inflammation in human RPE cells. RPE cells were treated with the selective CB2 agonist JWH-133 in the presence or absence of the oxidative stressor 4-hydroxynonenal. Thereafter, cellular viability as well as the release of pro-inflammatory cytokines and potential underlying signalling pathways were analysed. Our results show that JWH-133 led to increased intracellular Ca2+ levels, suggesting that RPE cells are capable of responding to a CB2 agonist. JWH-133 could not prevent oxidative stress-induced cell death. Instead, 10 µM JWH-133 increased cell death and the release of proinflammatory cytokines in an ERK1/2-dependent manner. In contrast to previous findings, CB2 activation increased, rather than reduced inflammation in RPE cells.

Roles of secretory phospholipases A(2) in inflammatory diseases and trauma.

Six distinct secretory small molecular weight phospholipases A(2) (PLA(2)) have been cloned and characterized from human tissues. Two of them, pancreatic group IB PLA(2) (PLA(2)-IB) and synovial-type group IIA PLA(2) (PLA(2)-IIA) have been studied as to their association to various inflammatory diseases. PLA(2)-IB is a digestive enzyme synthesized by pancreatic acinar cells. In acute pancreatitis, which is characterized by destruction of pancreatic tissue, PLA(2)-IB is released into the circulation, but its role in pancreatic and other tissue damage is still hypothetical. The concentration of PLA(2)-IIA increases in blood plasma in generalized inflammatory response resulting from infections, chronic inflammatory diseases, acute pancreatitis, trauma and surgical operations. PLA(2)-IIA is synthesized in a number of gland cells and is present in cellular secretions on mucosal surfaces including Paneth cells of intestinal mucosa, prostatic gland cells and seminal plasma, and lacrimal glands and tears. PLA(2)-IIA is expressed in hepatoma-derived cells in vitro and hepatocytes in vivo. PLA(2)-IIA is regarded as an acute phase protein and seems to function as an antibacterial agent especially effective against Gram-positive bacteria. Other putative functions in the inflammatory reaction include hydrolysis of cell membrane phospholipids and release of arachidonic acid for prostanoid synthesis.

Anaerobic glycolysis and the development of ischaemic contracture in isolated rat heart.

The relationship between myocardial ATP content and the increase in left ventricular resting tension during ischaemia (ischaemic contracture) was studied in isolated rat hearts perfused for 15 min either with aerobic buffer (pO2 greater than 500 mmHg) containing non-glycolytic substrate, acetate (5 mmol X litre-1), or with hypoxic buffer (pO2 less than 10 mmHg) with glucose (10 mmol X litre-1) before making them totally ischaemic for 10 min. ATP was determined spectrophotometrically from extracts of frozen whole hearts. Left ventricular tension was recorded by intraventricular balloon catheter. Myocardial ATP content was 15.4 +/- 1.0 mumol X g-1 dry weight (mumol X g-1) after 10 min stabilising period, 14.1 +/- 0.9 mumol X g-1 after 15 min aerobic perfusion (plus acetate) and 9.0 +/- 1.3 mumol X g-1 after 15 min hypoxic perfusion (plus glucose). During 10 min of ischaemia ATP decreased in aerobic hearts to 5.4 +/- 1.1 mumol X g-1 and to 7.9 +/- 1.0 mumol X g-1 in hypoxic hearts. The left ventricular resting tension increased during ischaemia in hypoxic hearts to 9 +/- 5% of control systolic pressure (0 = diastolic pressure, 100 = systolic pressure during stabilising period), whereas in aerobic hearts the tension began to increase immediately and was 84 +/- 22% of systolic pressure at the end of the ischaemic period. In parallel control experiments, hearts were also perfused either with glucose-containing aerobic buffer or acetate-containing hypoxic buffer. ATP was well preserved during aerobic perfusion (plus glucose) but decreased markedly during hypoxic perfusion (plus acetate). There was no increase in resting tension in the aerobic hearts (plus glucose) whereas the resting tension increased considerably during hypoxic perfusion (plus acetate). The results indicate that the initiation of ischaemic contracture occurs at much higher myocardial ATP level when ATP comes from mitochondrial sources than when ATP is generated by anaerobic glycolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased serum phospholipase A2 activity in schizophrenia: a replication study.

Phospholipase A2 (PLA2) is a key enzyme in the metabolism of phospholipids. Because a disordered phospholipid metabolism has frequently been reported in schizophrenia, we investigated the PLA2 activity in serum from 14 drug-free paranoid schizophrenic patients, 20 healthy controls, and 8 nonschizophrenic psychiatric patients. Schizophrenics showed significantly higher PLA2 activity than healthy controls and nonschizophrenic patients. The increment in schizophrenics was not due to increased concentration of pancreatic secretory PLA2, as concerning pancreatic PLA2 no differences were found among the 3 proband groups. The present findings confirm the results of our previous study and suggest that increased serum PLA2 activity might reflect an increment in the intracellular enzyme activity in schizophrenia. In the brain the activation of intracellular PLA2 results in changes in neuronal activity due to alterations in receptor sensitivity and in neurotransmitter metabolism. The possibility that such PLA2-induced mechanisms are involved in the pathogenesis of schizophrenia should be investigated in further experiments.

Electrophoretic signal comparison applied to mRNA differential display analysis.

Gene expression analysis by electrophoretic methods is currently limited by the labor-intensive visual evaluation of the electrophoretic signal profiles. For this purpose, we present a flexible approach to computer-assisted comparison of quantitative electrophoretic patterns between multiple expression signals. Gaussian curves are first fitted to the complex peak mixtures, and the resulting approximate signals are then aligned and compared on a peak-by-peak basis with respect to specific patterns defined by the investigator. The rationale of the method is to produce a compressed list of exceptional expression patterns quantified by a set of associated numeric features. A score value is attached to each pattern in such a way that large values identify the most potential findings to be focused on in visual analysis instead of the vast amount of original electrophoretic results. The validity of the method is demonstrated by analyzing a large set of electrophoretic data from mRNA differential display experiments monitoring changes in gene expression patterns in human colonic carcinoma. The automated identification of variously defined gene expression patterns agrees well with the visual evaluation of the same electropherograms. The general comparison approach may also be found useful with other gene expression profiling instruments.

Expression of human group II phospholipase A2 in transgenic mice.

Group II phospholipase A2 (PLA2) has been proposed to play an important role in inflammation and defense against bacterial infection. We investigated tissues of transgenic mice expressing the human group II PLA2 gene by immunohistochemistry using rabbit anti-human group II PLA2 antibodies, and by in situ hybridization by probing with human group II PLA2 mRNA anti-sense (test) and sense (control) riboprobes. By immunohistochemistry, human group II PLA2 was found in various mouse tissues and cell types including hepatocytes, proximal tubule cells of the kidney, epithelial cells of the renal pelvis, urinary bladder and ureter, granulosa cells of Graafian follicles, aortic intima and media, cartilage, epiphyseal bone, bronchial epithelial cells, and connective tissue cells in the dermis. By in situ hybridization, group II PLA2 mRNA was localized in hepatocytes, epidermal cells, dermal cells, connective tissue fibroblasts, epithelial and smooth muscle cells of the urinary bladder, and cells of Bowman's capsule. These results show that human group II PLA2 is expressed in large amounts in hepatocytes and many extrahepatic tissues of the transgenic mice. These animals provide a useful new tool for studies on the metabolism, in vivo effects, and physiological and pathological roles of phospholipase A2.

Distribution of group II phospholipase A2 protein and mRNA in rat tissues.

Group II phospholipase A2 (PLA2) is an acute-phase protein and an important component of the host defense against bacteria. In this study we investigated the distribution of PLA2 protein by immunohistochemistry and the distribution of mRNA of PLA2 by Northern blotting and in situ hybridization in rat tissues. PLA2 protein was localized in the Paneth cells of the intestinal mucosa, chondrocytes and the matrix of cartilage, and megakaryocytes in the spleen. By Northern blotting, mRNA of PLA2 was found in the gastrointestinal tract, lung, heart, and spleen. By in situ hybridization, PLA2 mRNA was localized in the Paneth cells of the small intestinal mucosa but in no other cell types. Our results show specific distribution of PLA2 in a limited number of cell types in rat tissues. The reagents developed in this study (the anti-rat PLA2 antibody and probes for Northern blotting and in situ hybridization of mRNA of rat PLA2) will provide useful tools for future studies concerning the role of PLA2 in various experimental disease models.

Secretion of group 2 phospholipase A2 by lacrimal glands.

PURPOSE: Tears are known to have antimicrobial properties. The authors investigated the presence of the antibacterial enzyme phospholipase A2 (PLA2) in tears and lacrimal glands. METHODS: The catalytic activity of PLA2 and the amount of pancreatic group 1 PLA2 and nonpancreatic group 2 PLA2 were measured in homogenates of eight human lacrimal glands from autopsied subjects and in tears from four healthy volunteers. The localization of PLA2s in lacrimal gland sections was studied by immunohistochemistry. Skeletal muscle was used as a control. RESULTS: The catalytic activity of PLA2 was significantly higher in lacrimal glands than in skeletal muscle. Immunochemical analysis showed significantly higher amounts of group 2 PLA2 in lacrimal gland than in skeletal muscle homogenates. Group 1 PLA2 was present in trace amounts only. The concentration of group 2 PLA2 in tears was high (1451.3 micrograms/l) compared to that in the serum of healthy individuals (3.7 micrograms/l). By immunohistochemistry, a granular reaction of group 2 PLA2 was localized in the glandular cells of lacrimal glands. The apical cytoplasm of many duct cells also was labeled. CONCLUSIONS: The lacrimal glands secreted nonpancreatic group 2 PLA2, which most likely acts as an antiinfectious factor in tears.

Synthesis of group II phospholipase A2 and lysozyme in lacrimal glands.

PURPOSE: The aim of this study was to demonstrate the synthesis and cellular distribution of group II phospholipase A2 and lysozyme in the main and accessory lacrimal glands. METHODS: The authors studied samples of normal main lacrimal glands of seven autopsied subjects and accessory lacrimal glands of eight patients who underwent ptosis surgery. The specimens were immunostained with a rabbit antiserum against group II phospholipase A2 and a monoclonal antibody against lysozyme. Expression of group II phospholipase A2 gene was shown using Northern hybridization and in situ hybridization. RESULTS: Lysozyme was present in the secretory granules of most acini, whereas group II phospholipase A2 was seen in a minority of acinar cells, primarily in the central parts of lobules in the main and accessory lacrimal glands. Synthesis of group II phospholipase A2 in the glandular cells was confirmed by Northern hybridization and by in situ hybridization. CONCLUSIONS: There are two specialized cell types in the main and accessory lacrimal glands, one synthesizing group II phospholipase A2 and the other synthesizing lysozyme. These enzymes are important nonspecific antibacterial factors in tears.

Group II PLA(2) content of tears in normal subjects.

PURPOSE: To determine the concentration of group II phospholipase (PL) A(2), an antimicrobial molecule, in tears of normal subjects in different age and sex groups. METHODS: PLA(2) content of tears was measured in 122 healthy volunteers with ages ranging from 20 to 89 years (mean, 49.5 years) by a time-resolved fluoroimmunoassay using a polyclonal rabbit antibody to recombinant human PLA(2). RESULTS: The mean concentration of PLA(2) in tears was 54.5 +/- 33.9 microg/ml. It was highest in the age group 20 to 29 years (81.6 +/- 32.0 microg/ml), and a decrease of concentration occurred with an increase of age. PLA(2) values were statistically significantly lower in the age group 60 to 69 years (P = 0.0013) and 70 years or more (P = 0.0001) than in the age group 20 to 29 years. There were no statistically significant differences in PLA(2) content of tears between the genders in any age group (P = 0.798). CONCLUSIONS: The results indicate that tears contain a high concentration of PLA(2) and that PLA(2) levels decrease with an increase of age and/or reflex tear component of the sample analyzed.

Immunohistochemical characterization of an amphicrine mucinous islet-cell carcinoma of the pancreas. Case report.

Immunohistochemical characteristics of a mucinous islet-cell carcinoma of the pancreas are described. The tumour presented with jaundice in a 59-year-old male. It consisted of polygonal atypical cells forming a reticular pattern, and invaded the common bile duct. In DNA flow cytometry, the tumour cells showed a clear-cut aneuploid peak. Intercellular mucin was abundant. A panel of antisera and monoclonal markers was applied in the immunohistochemical analysis. In addition to general epithelial and endocrine markers, the tumour cells showed a focal positive immunoreaction with anti-glucagon, anti-insulin, anti-vasoactive intestinal polypeptide, anti-pancreatic secretory trypsin inhibitor and anti-phospholipase A2 antigen. At the ultrastructural level, mucous and neuroendocrine granules were demonstrated in the same tumour cells.

Expression of group II phospholipase A2 in Paneth cells of an adenoma of the rectum. A case report.

A case of tubulovillous adenoma in the rectum of a 51-year-old man is presented. The tumour contained numerous Paneth cells which formed well-developed glands in the basal areas. Group II phospholipase A2 and lysozyme were found in the tumour cells by immunohistochemistry. mRNA of group II phospholipase A2 was localized in the tumour cells by in situ hybridization. It was concluded that a considerable part of this rare type of tumour consisted of Paneth cells which were capable of synthesizing group II phospholipase A2.

Acinar cell carcinoma of the pancreas. Report of three cases.

Pancreatic acinar cell carcinoma is a rare neoplasm (comprising about 1% of pancreatic tumours). We studied three cases (61-year-old female; 42-year-old male; 57-year-old male), whose survival after diagnosis ranged from 1 year 2 months to 6 years 8 months. There were widespread metastases in each case. The tumours had acinar, trabecular and solid growth patterns. By immunohistochemistry, pancreatic acinar cell markers including carboxyl ester lipase, pancreatic secretory trypsin inhibitor and pancreatic phospholipase A2 (group I PLA2) gave a strong positive reaction in all three cases. By electron microscopy, zymogen granules were seen in the cytoplasm of the tumour cells. Immunostaining for prostate-specific antigen was positive in all three cases. Above-normal concentrations of pancreatic PLA2 were measured in the serum of one patient and the values decreased during chemotherapy concomitantly with the reduction in the size of the tumour mass. In conclusion, immunohistochemical demonstration of the secretory products of acinar cells including the new marker pancreatic PLA2 is useful in the differential diagnosis of pancreatic acinar cell carcinoma. Determination of the concentration of pancreatic group I PLA2 in serum may be helpful in the evaluation of therapy.

Absence of group II phospholipase A2, a Paneth cell marker, from the epididymis.

Paneth cell-like metaplasia has been reported in the epithelium of the epididymis and prostatic adenocarcinomas. We studied the expression of group II phospholipase A2 (PLA2), a marker of Paneth cell differentiation, in six orchiectomy specimens with Paneth cell-like metaplasia. Both immunohistochemistry for group II PLA2 protein and in situ hybridization for the mRNA of group II PLA2 gave negative results in all six cases but positive reaction for lysozyme. The results show that the cells of the Paneth cell-like metaplasia are not true Paneth cells.

DNA ploidy in pancreatic neuroendocrine tumors.

The nuclear DNA content of 17 pancreatic neuroendocrine tumors was measured from paraffin-embedded tissue with flow cytometry. The tumors were classified by immunostaining with antisera for synaptophysin, insulin, gastrin, glucagon, pancreatic polypeptide, somatostatin, and vasoactive intestinal polypeptide. Eight (47%) of the 17 tumors were aneuploid, and two (12%) were multiploid (had two aneuploid stemlines of cells). Seven of the eight insulinomas, one of the four gastrinomas, and two of the four nonspecified neuroendocrine tumors had an abnormal nuclear DNA content. The DNA indices of the aneuploid and multiploid cases ranged from 1.13 to 1.93, and three cases had a DNA index greater than 1.50. During the follow-up for up to 16 years (mean, 7 years), one patient with diploid nonspecified tumor died of the disease, another patient with a multiploid gastrinoma had metastatic disease develop, and a third patient with a multiploid nonspecified tumor was alive with the disease. The authors conclude that many neuroendocrine tumors of the pancreas have an abnormal nuclear DNA content as measured by DNA flow cytometry. DNA multiploid pancreatic neuroendocrine tumors may be associated with a less favorable clinical course, but this needs to be confirmed in additional studies.

Presence of a phospholipase A2 inhibitor in porcine serum.

Porcine pancreatic phospholipase A2 (PLA2) was immobilized to Sepharose 4B and porcine serum was passed through this affinity column. Bound substances were eluted by an EDTA-containing buffer and fractionated in a Sepharose 6B column. A single protein peak of the eluate from the latter column was found to inhibit PLA2 activity in a dose-dependent manner in an assay system using radioactive lecithin as a substrate and porcine pancreatic PLA2 as the enzyme source. The serum fraction containing the PLA2 inhibitory protein(s) (PIP) appeared inhomogeneous on SDS-polyacrylamide gel electrophoresis with two major bands close to each other, corresponding to a molecular weight of approximately 60,000. It was concluded that PIP might act as a protective principle against autodigestion in acute pancreatitis and other inflammatory diseases as well as playing a regulatory role in prostaglandin metabolism.

Hyperamylasemia after cardiopulmonary bypass: pancreatic cellular injury or impaired renal excretion of amylase?

BACKGROUND: Postoperative hyperamylasemia and even acute pancreatitis are associated with coronary artery bypass grafting (CABG). The mechanism of hyperamylasemia and pancreatic acinar cell damage was studied in 20 patients undergoing CABG. METHODS: Serial blood and urine samples at eight time points before, during, and 24 hours after the CABG were collected. Salivary and pancreatic isoamylases, the fractional clearance of isoamylases (i.e., relative to creatinine clearance), pancreatic phospholipase A2 (a specific serum marker of pancreatic acinar cell injury), and cystatin C (a sensitive marker of glomerular filtration rate) were measured. RESULTS: Mild serum hyperamylasemia (300 to 1000 units/L) was found in 11 of 20 (55%) and severe (> 1000 units/L) in 6 of 20 (30%) patients with no signs of clinical acute pancreatitis. Hyperamylasemia occurred from 6 to 24 hours after the CABG and was mainly caused by pancreatic isoamylase. Serum pancreatic phospholipase A2 concentration remained unchanged, which excludes acinar cell damage. Although renal glomerular filtration was normal during CABG as measured by serum cystatin C and creatinine clearance, the fractional clearance of isoamylases decreased. CONCLUSIONS: The decreased rate of excretion into urine, rather than pancreatic cellular damage, is the major source of hyperamylasemia after CABG.

Pancreatic phospholipase A2 in proximal tubules of rat kidney in experimental acute pancreatitis and after intravenous injection of the enzyme.

Kinetics and distribution of i.v. human pancreatic phospholipase A2 (h-PLA2) were determined in intact and nephrectomized rats, and tissue localization of rat pancreatic PLA2 (r-PLA2) was studied by immunohistochemistry in experimental acute pancreatitis. The concentration of h-PLA2 and the catalytic activity of phospholipase A2 in plasma decreased exponentially in intact and nephrectomized animals after the injection. The initial 15-min half-life was considerably longer in nephrectomized animals, and higher h-PLA2 concentrations and PLA2 catalytic activities were found in plasma. h-PLA2 was localized in endocytotic vesicles and apical cytoplasmic vacuoles in proximal tubule cells of the kidney. The intensity of the immunoreaction decreased considerably between 15 and 50 min in these cells. No signs of tubular damage were seen by light microscopy. Neither immunoreactive h-PLA2 nor PLA2 catalytic activity was found in urine. r-PLA2 was observed in proximal tubule cells 15 min after an injection of sodium taurocholate (necrotizing pancreatitis group) or saline (edematous pancreatitis group) into the pancreatic duct. Signs of tubular damage were present in necrotizing pancreatitis, but tubular morphology was normal in the animals with edematous pancreatitis. We conclude that the proximal tubule cells of the kidney participate in the metabolism of circulating pancreatic PLA2, and considerably higher PLA2 levels persist in plasma in nephrectomized animals. Endogenous pancreatic PLA2 is detected in kidneys in acute pancreatitis.