Human platelets as the object of investigation of the molecular mechanisms underlying nongenomic effects of glucocorticoid hormones

The effect of hydrocortisone (100 nM–10 μM) on the major biochemical parameters of platelet activity (intracellular free calcium concentration, thromboxane B₂ content, and baseline and stimulated levels of cAMP and cGMP) is examined. The results obtained indicate that the inhibitory effect of glucocorticoids on platelet aggregation is mediated by activation of the adenylate cyclase system and suppression of the calcium response. Presumably, neither guanylate cyclase nor phospholipase A₂-dependent systems are the targets of nongenomic actions of glucocorticoid hormones. Platelets can serve as a convenient tool for the investigation of nongenomic effects of glucocorticoid hormones. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 9, pp. 285–287, September, 1996     

A rapid phospholipase A2 bioassay using14C-oleate-labelledE. coli bacterias

Summary Two methods of phospholipase A₂ determination using¹⁴C-labelledE. coli bacterias as substrate were compared. One method works with a filter membrane for separation of cleaved¹⁴C-oleate from remaining phospholipids, the other uses the well-known thin-layer chromatography for lipid analysis. Some features of human serum phospholipase A₂ regarding pH and Ca²⁺ dependency were investigated. Possible sources of errors were discussed. It was shown that either method can differentiate between normal and pathologically elevated phospholipase A₂ levels, but that the filter method is superior in terms of sensitivity and workload.Abbreviations PLA₂ phospholipase A₂ - BSA bovine serum albumin - (F)FA (free) fatty acid - TLC thin-layer chromatography     

Phospholipase A2 activity and lipid peroxidation during endotoxemia under conditions of experimental peritonitis

The severity of endotoxemia in peritonitis depends on morphofunctional state of the intestine determined by the intensity of free-radical lipid peroxidation and phospholipase A₂ activity, which are the highest on day 1 postoperation. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 1, pp. 31–33, January, 2000     

Contribution of the reproductive hormones to the regulation of prostaglandin F2α production by immunocompetent cells

The ability of estradiol, progesterone, and chorionic gonadotropin to influence prostaglandin F_2α production by intact splenocytes of CBA mice was studied. Estradiol and progesterone similarly activated the processes of prostaglandin F_2α production. No relationship was revealed between the effect and the concentration of the hormones. Chorionic gonadotropin activated prostaglandin production by immunocompetent cells but only when used in a concentration reflecting the peak of its physiological secretion. Combining gonadotropin with estradiol or progesterone did not lead to any appreciable differences in the prostaglandin-stimulating action of each hormone alone. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 8, pp. 178–180, August, 1995 Presented by K. P. Kashkin, Member of the Russian Academy of Medical Sciences     

Effects of chlorpromazine on the enzymatic and toxic properties of the most basic phospholipase A2 fromVipera russelli snake venom

The effect of chlorpromazine on various biological activities of phospholipase A₂ VRV-PL-VIII_a fromVipera russelli snake venom was investigated. The drug inhibited the in vitro phospholipase A₂ activity of the enzyme by 55%. The ID₅₀ was found to be 1.15 µM. Increasing substrate concentration relieved the inhibition of phospholipase A₂ activity by the drug indicating probable interaction with the substrate. The drug totally quenched the fluorescence intensity of the enzyme. Chlorpromazine increased the LD₅₀ of the enzyme by 1.6 fold. The drug also inhibited hemolytic and anticoagulant potencies but failed to inhibit edema inducing activity and myotoxicity of the enzyme.     

Inhibition of porcine pancreas phospholipase A2 activation by gabexate mesilate

Summary We investigated the effect of gabexate mesilate on the catalytic activity of phospholipase A₂ in homogenized porcine pancreatic tissue. Gabexate mesilate is a potent inhibitor of serine proteases. There is no direct inhibition of phospholipase A₂ catalytic activity in concentrations up to 6 mmol/l. Preincubation of homogenized pancreatic tissue with gabexate mesilate leads to a reduction of phospholipase A₂ activity even in concentrations as low as 6 µmol/l. The activation of purified porcine prophospholipase A₂ added to pancreatic tissue can be completely inhibited. Thus, gabexate mesilate might influence the activation of phospholipase A₂ administered in therapeutic concentrations in inflamed pancreatic tissue.Abbreviations PLA₂ Phospholipase A₂ - ProPLA₂ Prophospholipase A₂     

Role of Phospholipase A2 in Activation of Isolated Cardiomyocyte Respiration in Postinfarction Cardiosclerosis

The rate of oxygen consumption by isolated cardiomyocytes was studied in rats with experimental postinfarction cardiosclerosis. The increase in oxygen consumption under these condition was comparable to that in melittin- and arachidonic acid-induced activation of phospholipase A₂ in cardiomyocytes of intact animals. Bromophenacyl bromide inhibition of phospholipase A₂ in cardiomyocytes of rats with postinfarction cardiosclerosis led to reduction of oxygen consumption rate to values characteristic of intact animal cardiomyocytes. The results confirm the hypothesis according to which high oxygen consumption in postinfarction cardiosclerosis is related to increased activity of phospholipase A₂. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 12, pp. 631–633, December, 2008     

Surface roughness mediates its effects on osteoblasts via protein kinase A and phospholipase A2

Earlier studies have shown that implant surface roughness influences osteoblast proliferation, differentiation, matrix synthesis and local factor production. Moreover, the responsiveness of osteoblasts to systemic hormones, such as 1,25-(OH)₂D₃, at the implant surface is also influenced by surface roughness and this effect is mediated by changes in prostaglandins. At present, it is not known which signaling pathways are involved in mediating cell response to surface roughness and how 1,25-(OH)₂D₃ treatment alters the activation of these pathways. This paper reviews a series of studies that have addressed this question. MG63 osteoblast-like cells were cultured on commercially pure titanium (cpTi) surfaces of two different roughnesses (R_a 0.54 and 4.92 μm) in the presence of control media or media containing 1,25-(OH)₂D₃ or 1,25-(OH)₂D₃ plus H8 (a protein kinase A inhibitor) or quinacrine (a phospholipase A₂ inhibitor). At harvest, the effect of these treatments on cell number and alkaline phosphatase specific activity was measured. Compared to cultures grown on the smooth surface, cell number was reduced on the rough surface. 1,25-(OH)₂D₃ inhibited cell number on both surfaces and inhibition of protein kinase A in the presence of 1,25-(OH)₂D₃ restored cell number to that seen in the control cultures. Inhibition of phospholipase A₂ in the presence of 1,25-(OH)₂D₃ caused a further reduction in cell number on the smooth surface, and partially reversed the inhibitory effects of 1,25-(OH)₂D₃ on the rough surface. Alkaline phosphatase specific activity was increased in cultures grown on the rough surface compared with those grown on the smooth surface; 1,25-(OH)₂D₃ treatment increased enzyme specific activity on both surfaces. Cultures treated with H8 and 1,25-(OH)₂D₃ displayed enzyme specific activity that approximated that seen in control cultures. Inhibition of phospholipase A₂ also inhibited the 1,25-(OH)₂D₃-dependent effect on the smooth surface, but on the rough surface there was an inhibition of the 1,25-(OH)₂D₃ effect as well as a partial inhibition of the surface roughness-dependent effect. The results indicate that surface roughness and 1,25-(OH)₂D₃ mediate their effects through phospholipase A₂, which catalyzes one of the rate-limiting steps in prostaglandin E₂ production. Further downstream, prostaglandin E₂ activates protein kinase A.     

Low-dose hydrocortisone infusion attenuates the systemic inflammatory response syndrome

There is increasing evidence that the hypercortisolemia in inflammatory diseases suppresses the elaboration of proinflammatory cytokines, thus protecting the host from its own defence reactions. In severe sepsis and septic shock cortisol levels are usually elevated, but some patients may have relative adrenal insufficiency. This may contribute to the overwhelming systemic inflammatory response syndrome. We evaluated the impact of low-dose hydrocortisone infusion (10 mg/h) on the course of the systemic inflammatory response syndrome. This dose corresponds to a maximum secretory rate of cortisol achieved in corticotropin-stimulated healthy humans. In a prospective observational study 57 surgical patients with severe sepsis or septic shock were studied, of which in addition to the conventional treatment 12 patients were infused with low-dose hydrocortisone, and 45 were treated without any corticosteroid. In the longitudinal analysis the systemic inflammatory response — as judged by body temperature, cardiovascular response, and kinetics of inflammatory mediators such as phospholipase A₂, C-reactive protein, and neutrophil elastase — started to differ in favor of the hydrocortisone-treated patients after 2 days of treatment (P < 0.05, Mann-Whitney U test). The difference disappeared after withdrawal of exogenous cortisol. Shock reversal was achieved in all patients treated with low-dose hydrocortisone. The data provide evidence that low-dose hydrocortisone infusion attenuates the systemic inflammatory response in human septic shock. From an immunological point of view a relative cortisol deficiency may contribute to the amplified immune response in systemic inflammatory diseases. A randomized clinical trial must clarify the impact of low-dose hydrocortisone infusion on the clinical course and outcome of septic shock patients.Abbreviations TNF Tumor necrosis factor - PLA₂ phospholipase A₂ - CRP C-reactive protein - E-PI neutrophil elastase-₁-proteinase inhibitor complex - IL interleukin Correspondence to: J. Briegel     

Serum phospholipase A2 in intensive care patients with peritonitis,multiple injury,and necrotizing pancreatitis

Summary To study the source and role of circulating phospholipase A₂ (PLA₂) catalytic activity we monitored the serum from patients with necrotizing pancreatitis (n=8), diffuse peritonitis (n=6), and multiple injuries (n=11). Immunoreactive PLA₂ serum protein concentration was analysed using a fluoroimmunoassay based on an antibody against human pancreatic PLA₂. Serum PLA₂ catalytic activity was analysed using a radiochemical method based on a substrate with tritiated palmitic acid in beta position. In necrotizing pancreatitis immunoreactive PLA₂ and PLA₂ catalytic activity both increased. Obviously, in necrotizing pancreatitis the major part of serum catalytic activity stems from the pancreas. In patients with diffuse peritonitis and multiple injuries, as a rule, immunoreactive phospholipase A₂ serum concentration appears to be within the normal range. In contrast, in these patients we demonstrated high serum catalytic PLA₂ activity comparable to that in necrotizing pancreatitis. The source of catalytic PLA₂ activity in peritonitis and multiple injuries seems not to be the pancreas. There was a correlation between pulmonary insufficiency and serum PLA₂ catalytic activity in patients with necrotizing pancreatitis, peritonitis, and multiple injuries.     

In vitro inhibition of phospholipase A2 by gabexate mesilate,camostate, and aprotinine

Summary Gabexate mesilate (FOY, ethyl-p-(6-guanidino-hexanoyl-oxy)-benzoate-methansulfonate), camostate (N,N-dimethyl-carb-amoylmethyl-4-(guanidinobenzoyloxy)-phenyl-acetate) methansulfonate and aprotinine (Trasylol) were tested for possible inhibition of phospholipase A₂. Gabexate mesilate at a concentration of 5×10^–4 mol/l and camostate at a concentration of 10^–3 mol/l caused a 50% reduction in enzyme activity. There was almost no inhibition by aprotinine at clinical doses; 40 million KIU/l were necessary to reduce phospholipase A₂ activity by 20%. From the therapeutic dose (4,000 mg/day per i.v. infusion) and the half-life of gabexate mesilate in blood circulation (1 min) it can be calculated that the in vitro concentration of gabexate mesilate is only 10^–6 to 10^–7 mol/l. Under these conditions gabexate mesilate cannot diminish the in vivo enzyme activity of phospholipase A₂.     

On the present and future role of Lp-PLA2 in atherosclerosis-related cardiovascular risk prediction and management

Circulating concentration and activity of secretory phospholipase A₂ (sPLA₂) and lipoprotein-associated phospholipase A₂ (Lp-PLA₂) have been proven as biomarkers of increased risk of atherosclerosis-related cardiovascular disease (ASCVD). Lp-PLA₂ might be part of the atherosclerotic process and may contribute to plaque destabilisation through inflammatory activity within atherosclerotic lesions. However, all attempts to translate the inhibition of phospholipase into clinically beneficial ASCVD risk reduction, including in randomised studies, by either non-specific inhibition of sPLA₂ (by varespladib) or specific Lp-PLA₂ inhibition by darapladib, unexpectedly failed. This gives us a strong imperative to continue research aimed at a better understanding of how Lp-PLA₂ and sPLA₂ regulate vascular inflammation and atherosclerotic plaque development. From the clinical viewpoint there is a need to establish and validate the existing and emerging novel anti-inflammatory therapeutic strategies to fight against ASCVD development, by using potentially better animal models and differently designed clinical trials in humans.     

A radiochemical test for phospholipase A2 catalytic activity

Summary A position-specifically labelled phosphatidylcholine is the substrate for the selective determination of Phospholipase A₂ in serum, ascites and tissue samples. Optimal reaction conditions and simplifications of handling are discussed.A control group of human serum samples ranged up to 2.1 U/l. The maximum serum activity in samples of patients with acute pancreatitis was 126 U/l. In human ascites activities up to 380 U/l were measured. The method described here turned out to be a practicable instrument for the determination of phospholipase A₂ activity using only commercially available reagents.Abbreviation PlA₂ Phospholipase A₂     

Characterization of specific binding sites for high-density lipoproteins on rat hepatocytes: Effects of estradiol and testosterone

Two types of binding sites for high-density lipoproteins (HDL): P₁ and P₂ K_dl=20 and K_d2=2.5μg/ml, N₁=130 and N₂=35 ng/mg cell protein) are identified on the surface of rat hepatocytes. Conditions for predominant determination of P₂ are created by employing radiolabeled lipoproteins (¹²⁵I-HDL) with a high specific activity (1000 dpm) and the differences in the kinetics of the P₁-and P₂-¹²⁵I-HDL complex formation. P₂ predominate on hepatocytes from females. The addition of estradiol to a culture of hepatocytes from males increases the content of P₂, while the addition of testosterone to hepatocytes from females decreases the content of P₂ to the levels determined in males. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 12, pp. 629–634, December, 1996     

Acid phospholipase A1 in liver — A brief survey

Summary Acid phospholipase A₁ activity in liver (rat, human) is predominantly localized in lysosomes. A minor proportion (less than 3% of the total activity) is also present in the Golgi apparatus and the endoplasmic reticulum, presumably due to enzymatically active precursors of the corresponding lysosomal enzyme. Lysosomal phospholipase A₁ is the most important enzyme initiating the intralysosomal catabolism of diacylphosphoglycerides. It has been purified 50,600-fold, with a yield of about 26%. The enzyme prefers phosphatidylethanolamine as a substrate, which at 200 µM and pH 4.5, is hydrolysed at a rate of approximately 8.2 U/mg. Lysosomal phospholipase A₁ is a glycoprotein of about 29 kDa with an isoelectric point of pH 5.3. Unspecific extralysosomal endogenous inhibitors of this enzyme are pH range, inorganic cations, and various proteins. Divalent cations are more potent inhibitors than monovalent ones. Most endogenous intra- and extracellular proteins inhibit the enzyme, the cationic species exhibiting high inhibitory potencies, glycoproteins only little. Inhibitory proteins act through their binding to the substrate. Lysosomal phospholipase A₁ seems to be an important target in drug-induced lipidosis. This lipid storage disease is caused by various cationic amphiphilic drugs that are trapped intralysosomally by protonation. In lysosomes such compounds raise the pH, interact with the polar lipids to be degraded and the lysosomal lipolytic enzymes, such as phospholipase A₁. These mechanisms result in impaired intralysosomal phospholipid degradation and hence in intralysosomal phospholipid accumulation.Dedicated to Professor Walther Vogt on the occasion of his 70th birthday     

Steroid hormones modulate lipid spectrum in lysosomal membranes of skin fibroblasts

It is shown that estradiol and hydrocortisone modulate lipid composition and affect lipidprotein interaction in fibroblast lysosome membranes, which can promote enzyme release from lysosomes. These effects are particular mechanisms of hormone regulation of functional lysosome activity in the skin. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 2, pp. 165–167, February, 1998     

Phospholipases and acyltransferases in macrophages

Summary In contrast to many other cells, macrophages contain a phospholipase A₂, which preferentially liberates arachidonic acid from the main phospholipids. In unstimulated macrophages this acylchain-specific phsopholipase A₂ is localized in the lipid-free cytosol and thus without function. After activation of protein kinase C with diacylglycerols, the cytosolic phospholipase A₂ is translocated to cellular membranes. The same activator of protein kinase C causes an inhibition of the acyl-CoA: lysophosphatide acyltransferase. This enzyme regulates the availability of free arachidonic acid for eicosanoid synthesis by reacylation into phospholipids. Thus protein kinase C seems to regulate the level of free arachidonic acid by opposite effects on the two major enzymes, which are responsible for the control of free arachidonic acid.     

Effects of muscarine agonists and antagonists on vasopressin-stimulated water transport in the amphibian bladder

The effects of M₁ and M₂ cholinoceptors on stimulated water transport in the urinary bladder of the common frogRana temporaria L. are described. In the presence of pirenzepine, a selective M₁ cholinoceptor antagonist, carbachol stimulated water transport. Activation of M₂ cholinoceptors by oxotremorine in concentrations of 0.5–5.0 μM inhibited water transport, whereas their activation by this compound in higher concentrations (10–100 μM) stimulated it. The use of the phospholipase C inhibitor neomycin (0.5 mM) and the calmodulin inhibitor W-7 (1 mM) indicated that activation of M₂ cholinoceptors switches on phospholipid-Ca²⁺-calmodulin-dependent mechanisms. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 9, pp. 252–254, September, 1995 (Presented by P. V. Sergeev, Member of the Russian Academy of Medical Sciences)     

Balysum-2 inhibitis evoked activity of the pyramidal neurons in hippocampus

The effects of Balysum-2 on the responsiveness of field CA1 hippocampal pyramidal neurons was studied in experiments with cultured cerebral slices. Addition to the medium of 10⁻⁴M Balysum-2 or its active ingredient (comenic acid) led to a reversible decrease in the peak amplitude of focal response (pop-spike) recorded in the pyramidal layer CA1 during stimulation of the radial layer. Perfusion of hippocampal slices with a solution containing the noncompetitive GABA_A-antagonist picrotoxin prevented the effects of Balysum-2 and comenic acid. Inhibition of hippocampal pyramids by Balysum-2 and comenic acid is probably caused by an incease in the inhibitory effect mediated by GABA_A-receptors. Translated fromByulleten' Eksperimental'noi Biologii I Meditsiny, Vol. 125, No. 1, pp. 63–65, January, 1998     

Der Einfluß desP A CO 2 auf die Wirkung der Sinusnervenreizung bei intakten und ausgeschalteten Nn. vagi

Summary In rabbits rebreathing oxygen one carotid sinus nerve was stimulated repeatedly by electrical stimuli of constant intensity, both before and after inactivation of vagi by cold blocking or sectioning. In animals with intact vagi the reflex hyperpnea elicited by the nerve stimulation decreased only slightly with increasing hypercapnia. After inactivation of the vagi the stimulatory effect was, at normalP _{A CO 2}, mostly greater than in the intact condition. But with increasing hypercapnia the decrease of the reflex hyperpnea was generally steeper than in the intact preparation, sometimes reaching even lower values than before inactivation. The depressant effects of the stimulation on systemic blood pressure and heart rate were not distinctly changed by the increase ofP _{A CO 2}, neither with intact nor with inactivated vagi. It is concluded that the ventilatory effect of chemoreceptor afferents is modified both by centralP _{CO 2} and by vagal afferents.

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