Glucocorticoid deficiency increases phospholipase A2 activity in rats.

An important mechanism for the antiinflammatory effect of pharmacological doses of glucocorticoids is the inhibition of arachidonic acid release from phospholipids by phospholipase A2 (PLA2). As a corollary, one might predict that low endogenous concentrations of glucocorticoids favor inflammatory disease states. Indeed, clinical and experimental observations revealed an association between glucocorticoid deficiency and disease states caused by immunological and/or inflammatory mechanisms. The purpose of the present investigation was to study the regulation of PLA2 mRNA, protein, and enzyme activity in adrenalectomized (ADX) rats where glucocorticoid concentrations were below physiological levels. The mRNA of group I and II PLA2 were measured by PCR. Group II PLA2 mRNA was increased by 126 +/- 9% in lung tissue of ADX rats, whereas group I PLA2 was increased only by 27 +/- 1.5%. The increase in group II mRNA in ADX rats was reflected by a corresponding increase of group II PLA2 protein (70-100%) in lung, spleen, liver, and kidney. This increase was reversed by the administration of exogenous corticosterone. After ADX, the percentage increase in total PLA2 activity was higher than that of mRNA or PLA2 protein, suggesting that the activity of the enzyme was modulated by inhibitors or activators. The concentration of lipocortin-I, an inhibitor of PLA2 enzyme was strongly correlated with the activity of PLA2 in the tissues (lung, spleen, liver, and kidney). In all these tissues, the concentrations of lipocortin-I declined after ADX. Thus upregulation of PLA2 enzyme and downregulation of lipocortin-I might account for the enhanced inflammatory response in hypoglucocorticoid states.     

Subcellular characteristics of phospholipase A2 activity in the rat kidney. Enhanced cytosolic, mitochondrial, and microsomal phospholipase A2 enzymatic activity after renal ischemia and reperfusion.

Phospholipase A2 (PLA2) activities in cytosolic, mitochondrial, and microsomal fractions of rat kidneys were characterized under control conditions, after ischemia, and subsequent to ischemia and reperfusion. Two forms of PLA2 activity were present in the cytosolic fraction: a high molecular weight form, active against phosphatidylcholine (PC), and phosphatidylethanolamine (PE), which upon purification has a molecular mass of 110 kD; and smaller form (Mr approximately 14 kD), active against PE. In mitochondrial and microsomal fractions a single form (Mr approximately 14 kD), active against both PC and PE, was dominant. Activities in each fraction were optimal at pH 8.5-9.5. Cytosolic PLA2 activity was enhanced when Ca2+ concentration [( Ca2+]) was increased over the range of 10(-7) to 10(-6) M. Mitochondrial PLA2 activity required higher [Ca2+] for activation (greater than 10(-6) M). After 45 min of ischemia cytosolic PLA2 activity was decreased, whereas mitochondrial and microsomal activities were increased. When ischemia was followed by 1 h of reperfusion, cytosolic, mitochondrial, and microsomal activities were enhanced. Ischemia alone did not change the gel filtration chromatography patterns of PLA2 activity, but ischemia and reperfusion resulted in the appearance of a new peak of activity in cytosolic and mitochondrial fractions (Mr approximately 2-3 kD). Thus, the rat kidney has multiple forms of PLA2 activity, likely representing distinct enzymes, with Ca2+ dependencies suggesting regulation by Ca2+ in vivo. Ischemia and reperfusion result in stable increases of PLA2 activity in each subcellular fraction, perhaps related to covalent modifications of PLA2's, which likely account for membrane phospholipid degradation, and increased tissue levels of unsaturated free fatty acids.     

Serum phospholipase A2 activity in chronic pancreatic diseases.

This study was performed to investigate the phospholipase A2 (PLA2) serum activity in patients with chronic pancreatic disease. PLA2, elastase-1, total, and pancreatic isoamylase were evaluated in 40 control subjects, 28 patients with pancreatic cancer, 51 with chronic pancreatitis, and 36 with extrapancreatic diseases, mainly of gastrointestinal origin. Elastase-1, PLA2, and pancreatic isoamylase were increased in 56%, 25%, and 15% of patients with pancreatic cancer, and in 40%, 31%, and 41% of subjects with chronic pancreatitis. All four enzymes gave pathological values in a number of patients with extrapancreatic diseases. We conclude that the diagnostic efficacy of phospholipase A2 in chronic pancreatic disease is similar to that of other well known pancreatic enzymes, with an unsatisfactory sensitivity and specificity.     

Membrane structure, toxins and phospholipase A2 activity.

The phospholipid-hydrolyzing enzyme phospholipase A2 (PLA2) (EC 3.1.1.4) exists in several forms which can be located in the cytosol or on cellular membranes. We review briefly cellular regulatory mechanisms involving covalent modification by protein kinase C and the action of Ca2+, cytokines, G proteins and other cellular proteins. The major focus is the role of phospholipid structure on PLA2 activity, including (1) the mechanism of PLA2 action on synthetic phospholipid bilayers, (2) perturbation of synthetic and cellular membranes with lipophilic agents and membrane-interactive peptides and (3) the ability of these agents to activate endogenous PLA2 activity, with emphasis on the venom and plant toxins melittin, cardiotoxin and Pyrularia thionein.     

Some antiphospholipid antibodies inhibit phospholipase A2 activity.

Certain antiphospholipid antibodies, particularly those associated with arterial thrombosis, reduce vascular prostacyclin production. Studies were conducted to determine whether antibody-mediated inhibition of phospholipase A2 accounts for this effect. In this report we present evidence that purified antiphospholipid antibodies reduce phospholipase A2 activity toward phospholipid substrates, both in vitro and in a defined system. Purified immunoglobulin, obtained from patients at risk for thrombosis who had plasma antiphospholipid antibodies, impaired prostacyclin generation after endothelial stimulation with thrombin or the calcium ionophore A23187. The release of arachidonate in response to A23187 was reduced in endothelial cells pretreated with antibody; the metabolism of exogenous arachidonate to prostacyclin was normal. Thrombin-induced synthesis of platelet-activating factor, which follows phospholipase A2-mediated generation of lysophosphatidylcholine, was also inhibited in parallel with the inhibition of prostacyclin generation. Phospholipase A2 activity was determined in a defined test system with two phospholipases A2. The hydrolysis of fatty acid was less in the presence of patient immunoglobulin than in buffer alone or with normal immunoglobulin. Inhibition by antibody was present at a range of phospholipase concentrations. Antiphospholipid antibodies, purified from patient serum by adsorption to and subsequent elution from immobilized cardiolipin or phosphatidylserine, also inhibited phospholipase A2 activity. The data support our conclusions that purified antiphospholipid antibodies inhibit endothelial phospholipase A2 activity in response to thrombin or ionophore and that phosphatidylcholine in a common metabolic precursor of both prostacyclin and platelet-activating factor. In a defined enzyme assay, inhibition by antiphospholipid antibody of phospholipase A2 activity does not require additional cofactors.     

磷脂酶A2激活在鼠急性缺血性脑损伤中的作用机制

目的 探讨急性脑缺血后脑组织内磷脂酶A2（PLA2）激活及细胞内[Ca^2 ]i与脑损伤的关系，为预防和治疗急性缺血性脑损伤提供理论基础和新的思路。方法 将局灶性脑缺血模型大鼠分5组（假手术组、缺血30、60、90、120min组），测定脑组织PLA2活力、脑细胞[Ca^2 ]i、脑含水量及缺血120min组脑组织PLA2表达量的改变。结果 脑缺血120min脑组织PLA2活性、[Ca^2 ]i、脑含水量较假手术组明显升高，并与时间呈正相关，缺血120min后脑组织中出现sPLA2-ⅡAmRNA表达，且cPLA2-ⅣmRNA表达水平较假手术组明显增强。结论 磷脂酶A2激活参与了脑缺血后神经细胞内钙超载及脑损伤的整人病理过程。     

Arteriovenous shunting in experimental liver cirrhosis in rats

The extent of systemic arteriovenous shunting (arteriovenous anastomotic blood flow) was assessed in rats with experimental liver cirrhosis and control rats by injecting 15 micron microspheres into the left ventricle and measuring the percentage of injected spheres trapped in the pulmonary circulation. Cirrhotic rats with body temperature maintained at 38 degrees C were found to have increased trapping of microspheres in the pulmonary bed when compared with control rats studied under similar conditions (8.2% +/- 2.2% vs. 3.6% +/- 0.8%, P less than 0.05). The extent of arteriovenous shunting was also measured in control rats maintained at various body temperatures (34 degrees to 40 degrees C) with and without prior alpha-adrenergic blockade with phenoxybenzamine (0.1 mg/kg). Both body warming and alpha-adrenergic blockade independently increased the extent of arteriovenous shunting. In cirrhotic rats, body warming also increased shunting. However, in contrast to findings in control rats, alpha-adrenergic blockade did not further increase shunting in cirrhotic rats warmed to 40 degrees C. Moreover, the degree of shunting after combined body warming and alpha-adrenergic blockade was similar in cirrhotic and control rats. Our results indicate that increased peripheral arteriovenous shunting occurs in this model of experimental cirrhosis, and that the increased shunting may be related to altered physiologic regulation of arteriovenous anastomotic blood flow.     

Heparin-enhanced plasma phospholipase A2 activity and prostacyclin synthesis in patients undergoing cardiac surgery.

Although eicosanoid production contributes to physiological and pathophysiological consequences of cardiopulmonary bypass (CPB), the mechanisms accounting for the enhanced eicosanoid production have not been defined. Plasma phospholipase A2 (PLA2) activity, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and thromboxane B2 (TXB2) levels were measured at various times during cardiac surgery. Plasma PLA2 activity increased after systemic heparinization, before CPB. This was highly correlated with concurrent increases in plasma 6-keto-PGF1 alpha, TXB2 concentrations did not increase with heparin administration but did increase significantly after initiation of CPB. High plasma PLA2 activity, 6-keto-PGF1 alpha, and TXB2 concentrations were measured throughout the CPB period. Protamine, administered to neutralize the heparin, caused an acute reduction of both plasma PLA2 activity and plasma 6-keto-PGF1 alpha, but no change in plasma TXB2 concentrations. Thus the ratio of TXB2 to 6-keto-PGF1 alpha increased significantly after protamine administration. Enhanced plasma PLA2 activity was also measured in patients with lower doses of heparin used clinically for nonsurgical applications. Human plasma PLA2 was identified as group II PLA2 by its sensitivity to deoxycholate and dithiothreitol, its substrate specificity, and its elution characteristics on heparin affinity chromatography. Heparin addition to PMNs in vitro resulted in dose-dependent increases in cellular PLA2 activity and release of PLA2. The PLA2 released from the PMN had characteristics similar to those of post-heparin plasma PLA2. In conclusion, plasma PLA2 activity and 6-keto-PGF1 alpha concentrations are markedly enhanced with systemic heparinization. Part of the anticoagulant and vasodilating effects of heparin may be due to increased plasma prostacyclin (PGI2) levels. In addition the pulmonary vasoconstriction sometimes associated with protamine infusion during cardiac surgery might be due to decreased plasma PLA2 activity, with an associated increased TXB2/6-keto-PGF1 alpha ratio.     

Evaluation of renal tubular damage in liver cirrhosis by urinary enzymes and beta-2-microglobulin excretions

To assess the renal tubular damage in liver cirrhosis the fractional clearances of beta-2-microglobulin (B2m-fr.cl) and malate-dehydrogenase (MDH-fr.cl) were measured respectively in sixty-four and in forty-six out of seventy-nine patients with liver cirrhosis of different aetiology; furthermore the fractional excretions of gammaglutamyl-transpeptidase (fr-GGT) and of alpha-glucosidase (fr-AGL) were determined in fifty-three and in forty of them respectively. In all patients glomerular filtration rate (GFR) and renal plasma flow (RPF) were also measured. Twenty-five subjects were studied as a control group for the enzyme excretions, sixteen for B2m-fr.cl. B2m-fr.cl and MDH-fr.cl--indexes of tubular functions--on the average were normal and only slightly increased respectively in cirrhotics compared to controls. Nevertheless fr-GGT and fr-AGL--indexes of cytolysis of tubular cells--on the average were massively increased in cirrhosis compared to controls, particularly in those with reduced RPF and/or GFR. No clear relationship between the indexes of tubular damage studied and the indexes of liver function was found. Our results show that (1) A renal tubular anatomical damage was found by means of an increase in the release of enzyme from tubular cells in patients with liver cirrhosis, particularly in those with a significant reduction of RPF and/or GFR; even so renal reabsorption of low molecular weight proteins is generally maintained. (2) The tubular damage does not seem to be related to the degree of liver impairment.     

Glucagon-independent renal hyperaemia and hyperfiltration after an oral protein load in Child A liver cirrhosis

The work was designed to study the effects of a meat meal on glomerular filtration rate (GFR), renal plasma flow (RPF), and plasma concentrations of glucagon, insulin, growth hormone, renin, aldosterone, total amino acids, and NH3 in healthy humans (H) as well as in patients with Child A liver cirrhosis (LC). The meat meal produced renal hyperaemia and hyperfiltration without changes in the filtration fraction. Fractional Na excretion in urine increased significantly after the meat meal only in LC. Hyperinsulinaemia and hyperglucagonaemia were seen at baseline in LC and were not affected by the meat meal, whereas in H glucagon concentration increased significantly over baseline within 30 min from the meat meal and insulin within 60 min. Growth hormone concentration was normal at baseline in LC and increased significantly 120-180 min after the meal, whereas it was not affected in H. Renin and aldosterone were stable in both H and LC. Plasma amino acid concentration began to increase 60 min after the meat meal, when hyperfiltration was present. The data indicate that in human Child A cirrhosis of the liver renal haemodynamic response to a meat meal is independent of changes in glucagon.     

Intracellular signaling in the regulation of renal Na-K-ATPase. I. Role of cyclic AMP and phospholipase A2.

We have reported that dopamine (DA) inhibits Na-K-ATPase activity in the cortical collecting duct (CCD) by stimulating the DA1 receptor, and the present study was designed to evaluate the mechanism of this effect. Short-term exposure (15-30 min) of microdissected rat CCD to DA, a DA1 agonist (fenoldopam), vasopressin (AVP), forskolin, or dibutyryl cAMP (dBcAMP), which increase cAMP content by different mechanisms, strongly (approximately 60%) inhibited Na-K-ATPase activity. 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, completely blocked Na-K-ATPase inhibition by DA or fenoldopam, and IP20, an inhibitor peptide of cAMP-dependent protein kinase A (PKA), abolished the Na:K pump effect of all the cAMP agonists listed above. To verify whether the mechanism of pump inhibition by agents that increase cell cAMP involves phospholipase A2 (PLA2), we used mepacrine, a PLA2 inhibitor, which also abolished Na-K-ATPase inhibition by DA or fenoldopam, as well as by AVP, forskolin, or dBcAMP. Arachidonic acid (10(-7) - 10(-4) M) inhibited Na-K-ATPase activity in dose-dependent fashion. Corticosterone, which induces lipomodulin, a PLA2 inhibitor protein inactivated by PKA, equally abolished the pump effects of DA, fenoldopam, forskolin, and dBcAMP, suggesting that lipomodulin might act between PKA and PLA2 in cAMP-dependent pump regulation. We conclude that dopamine inhibits Na-K-ATPase activity in the CCD through a DA1 receptor-mediated cAMP-PKA pathway that involves the stimulation of PLA2 and arachidonic acid release, possibly mediated by inactivation of lipomodulin. This pathway is shared by other agonists that increase cell cAMP and thus stimulate PKA activity.     

Importance of adenosine triphosphate in phospholipase A2-induced rabbit renal proximal tubule cell injury.

The pathogenesis of ischemic renal tubular cell injury involves a complex interaction of different processes, including membrane phospholipid alterations and depletion of high-energy phosphate stores. To assess the role of membrane phospholipid changes due to activation of phospholipases in renal tubule cell injury, suspensions enriched in rabbit renal proximal tubule segments were incubated with exogenous phospholipase A2 (PLA2). Exogenous PLA2 did not produce any significant change in various metabolic parameters reflective of cell injury in control nonhypoxic preparations despite a significant decrease in phosphatidylethanolamine (PE) and moderate increases in lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). In contrast, exogenous PLA2 treatment of hypoxic tubules resulted in a severe degree of cell injury, as demonstrated by marked declines in tubule K+ and ATP contents and significant decreases in tubule uncoupled respiratory rates, and was associated with significant phospholipid alterations, including marked declines in phosphatidylcholine (PC) and PE and significant rises in LPC, LPE, and free fatty acids (FFA). The injurious metabolic effects of exogenous PLA2 on hypoxic tubules were reversed by addition of ATP-MgCl2 to the tubules. The protective effect of ATP-MgCl2 was associated with increases in tubule PC and PE contents and declines in LPC, LPE, and FFA contents. These experiments thus indicate that an increase in exogenous PLA2 activity produces renal proximal tubule cell injury when cell ATP levels decline, at which point phospholipid resynthesis cannot keep pace with phospholipid degradation with resulting depletion of phospholipids and accumulation of lipid by-products. High-energy phosphate store depletion appears to be an important condition for exogenous PLA2 activity to induce renal tubule cell injury.     

血小板型磷脂酶A2抗菌活性的研究进展

磷脂酶A2(PLA2)是一类能水解磷脂Sn-2位脂键的酶,也是生物化学、基因工程和药理学研究的热点酶类.近几年来一个重要发现就是某些型别PLA2具有抗菌功能,研究得最早且最多的是血小板型PLA2,它主要对G+菌具有强的抗菌活性,在宿主防御细菌感染中发挥重要作用.现就血小板型PLA2抗菌活性的研究进展作一综述.     

Liver cirrhosis and coagulopathy

In patients with chronic liver disease profound coagulation changes and thrombocytopenia may lead to life-threatening bleeding that requires thorough diagnosis and immediate blood product replacement.