Nitric oxide activates PKCalpha and inhibits Na+-K+-ATPase in opossum kidney cells.
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Nitric oxide (NO) reduces the molecular activity of Na+-K+-ATPase in opossum kidney (OK) cells, a proximal tubule cell line. In the present study, we investigated the cellular mechanisms for the inhibitory effect of NO on Na+-K+-ATPase. Sodium nitroprusside (SNP), a NO donor, inhibited Na+-K+-ATPase in OK cells, but not in LLC-PK1 cells, another proximal tubule cell line. Similarly, phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, inhibited Na+-K+-ATPase in OK, but not in LLC-PK1, cells. PKC inhibitors staurosporine or calphostin C, but not the protein kinase G inhibitor KT-5823, abolished the inhibitory effect of NO on Na+-K+-ATPase in OK cells. Immunoblotting demonstrated that treatment with NO donors caused significant translocation of PKCalpha from cytosolic to particulate fractions in OK, but not in LLC-PK1, cells. Furthermore, the translocation of PKCalpha in OK cells was attenuated by either the phospholipase C inhibitor U-73122 or the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. U-73122 also blunted the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. The phospholipase A2 inhibitor AACOCF3 did not blunt the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. AACOCF3 alone, however, also decreased Na+-K+-ATPase activity in OK cells. In conclusion, our results demonstrate that NO activates PKCalpha in OK, but not in LLC-PK1, cells. The activation of PKCalpha in OK cells by NO is associated with inhibition of Na+-K+-ATPase. (+info)
Involvement of the cAMP/protein kinase A pathway and of mitogen-activated protein kinase in the anti-proliferative effects of anandamide in human breast cancer cells.
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Anandamide (ANA) inhibits prolactin- and nerve growth factor (NGF)-induced proliferation of human breast cancer cells by decreasing the levels of the 100 kDa prolactin receptor (PRLr) and the high affinity trk NGF receptor, respectively, and by acting via CB(1)-like cannabinoid receptors. However, the intracellular signals that mediate these effects are not known. Here, we show that, in MCF-7 cells: (i) forskolin and the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 prevent, and the protein kinase A inhibitor RpcAMPs mimics, the inhibitory effects of ANA on cell proliferation and PRLr/trk expression and (ii) ANA inhibits forskolin-induced cAMP formation and stimulates Raf-1 translocation and MAPK activity, in a fashion sensitive to the selective CB(1) antagonist SR141716A. ANA stimulation of MAPK was enhanced by inhibitors of ANA hydrolysis. Forskolin inhibited MAPK and ANA-induced Raf-1 translocation. These findings indicate that, in MCF-7 cells, ANA inhibits adenylyl cyclase and activates MAPK, thereby exerting a down-regulation on PRLr and trk levels and a suppression of cell proliferation. (+info)
Increased concentrations of nonesterified arachidonic acid, 12L-hydroxy-5,8,10,14-eicosatetraenoic acid, prostaglandin E2, and prostaglandin F2alpha in epidermis of psoriasis.
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Lesional epidermis of psoriasis has a probable reduction in the cyclic AMP/cyclic GMP ratio. This altered ratio may in part be responsible for the characteristic glycogen storage, rapid cell proliferation, and reduced differentiation in lesional epidermis. The concentrations of prostaglandins E2 and F2alpha, free arachidonic acid, and 12L-hydroxy-5,8,10,14-eicosatetrawnoic acid in specimens of uninvolved and involved epidermis of psoriasis were measured with deuterium-labeled carriers and multiple ion analysis. Snap frozen specimens contained: 1.4 +/- 0.4 mug/g (wet weight) of arachidonic acid in uninvolved in contrast to 36.3 +/- 16.7 mug/g in involved epidermis (P = 0.015); less than 0.05 +/- 0.01 mug/g of hydroxyeicosatetraenoic acid in uninvolved in contrast to 4.1 +/- 1.9 mug/g in involved epidermis (P = 0.015); 23.6 +/- 5.0 ng/g of prostaglandin E2 in uninvolved in contrast to 33.1 +/- 5.7 ng/g in involved epidermis (P less than 0.01); and 21.0 +/- 4.4 ng/g of prostaglandin F2alpha in uninvolved in contrast to 39.0 +/- 5.9 ng/g in involved epidermis (P less than 0.01). The arachidonic acid and hydroxyeicosatetraenoic acid levels in involved epidermis were strongly correlated (r = 0.97). The increased levels of arachidonic acid and 12L-hydroxy-5,8,10,14-eicosatetraenoic acid in involved epidermis may have diagnostic and pathophysiological importance. (+info)
Evidence that 2-arachidonoylglycerol but not N-palmitoylethanolamine or anandamide is the physiological ligand for the cannabinoid CB2 receptor. Comparison of the agonistic activities of various cannabinoid receptor ligands in HL-60 cells.
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We examined the effect of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, on the intracellular free Ca(2+) concentrations in HL-60 cells that express the cannabinoid CB2 receptor. We found that 2-arachidonoylglycerol induces a rapid transient increase in intracellular free Ca(2+) concentrations in HL-60 cells. The response was affected by neither cyclooxygenase inhibitors nor lipoxygenase inhibitors, suggesting that arachidonic acid metabolites are not involved. Consistent with this notion, free arachidonic acid was devoid of any agonistic activity. Importantly, the Ca(2+) transient induced by 2-arachidonoylglycerol was blocked by pretreatment of the cells with SR144528, a CB2 receptor-specific antagonist, but not with SR141716A, a CB1 receptor-specific antagonist, indicating the involvement of the CB2 receptor but not the CB1 receptor in this cellular response. G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the cells abolished the response. We further examined the structure-activity relationship. We found that 2-arachidonoylglycerol is the most potent compound among a number of naturally occurring cannabimimetic molecules. Interestingly, anandamide and N-palmitoylethanolamine, other putative endogenous ligands, were found to be a weak partial agonist and an inactive ligand, respectively. These results strongly suggest that the CB2 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic natural ligand for the CB2 receptor that is abundant in the immune system. (+info)
Cheek cell phospholipids in human infants: a marker of docosahexaenoic and arachidonic acids in the diet, plasma, and red blood cells.
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BACKGROUND: Assessment of essential fatty acid status requires collection of blood or adipose tissue samples. However, these invasive techniques cannot always be used in studies involving infants, young children, or subjects from whom it is difficult to obtain blood. A body tissue that is easy to access is the buccal mucosa (cheek cells). OBJECTIVE: The objective was to investigate the degree to which fatty acids of cheek cells reflect the fatty acid content of plasma, red blood cells, and the diet. DESIGN: Thirty-one infants aged 12 mo were enrolled. Five infants were fed human milk and 26 infants received formulas that provided a wide range of arachidonic acid and docosahexaenoic acid (DHA) intakes. Cheek cells were collected on a small piece of gauze by gently swabbing the inside of the cheek 3 times. Lipids were extracted from the gauze and the phospholipid fatty acid content of the cheek cells was determined. RESULTS: Cheek cell DHA and arachidonic acid in phospholipids were significantly correlated with DHA and arachidonic acid in plasma [r = 0.61 (P < 0.001) and r = 0.37 (P <0.05), respectively], red blood cells [r = 0.58 (P < 0.001) and r = 0.37 (P < 0.05), respectively], and the diet [r = 0.65 (P < 0.001) and r = 0. 51 (P < 0.01), respectively]. CONCLUSIONS: Given these correlations and the ease and noninvasive nature of this technique, cheek cell fatty acids may serve as a marker of the essential fatty acid content, especially of DHA and arachidonic acid, in plasma, red blood cells, and the diet. (+info)
Activation of high levels of endogenous phospholipase A2 in cultured cells.
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Activatable cellular phospholipase A2 (PLase; phosphatide 2-acyl-hydrolase, EC 3.1.1.4) has been proposed to constitute the first and rate-limiting step in prostaglandin synthesis and to regulate membrane function by altering the levels in the membrane of the detergent lipids lysolecithin and free fatty acids. We have observed that a wide variety of cells in culture contain high levels of endogenous PLase that can be activated by polypeptide toxins, such as melittin purified from bee venom and direct lytic factor purified from the venom of African Ringhals cobra (Hemachatus hemachatus). Activation of PLase by sublytic concentrations of these agents results in the synthesis and release of prostaglandins. Melittin concentrations greater than or equal to 10 microgram/ml activate sufficient PLase in 3T3-4a mouse fibroblasts to hydrolyze 10% of the cellular lecithin in less than 5 min and virtually all of it within 30 min, demonstrating the existence of sufficient activatable PLase to provide the basis for the proposed mechanism of regulation of membrane function by alteration of membrane lipid composition. Lipases, phospholipases B and C, and sphingomyelinases are not activated by melittin. The PLase activated in 3T3-4a cells exhibits little, if any, specificity for individual phosphoglycerides. The PLase activated by direct lytic factor exhibits a Ca2+ dependence characteristic of lysosomal PLase, wherease the Ca2+ dependence of PLase activated by melittin is consistent with the activation of a cell-surface enzyme. The extent of cell death correlates with percent of maximal PLase activation. (+info)
Inhibition of arachidonic acid release from cells as the biochemical action of anti-inflammatory corticosteroids.
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Serum stimulates the production of prostaglandins by transformed mouse fibroblasts. Hydrocortisone (cortisol) inhibits this stimulation. The half-maximal inhibition occurs at 6x10-9 M. Studies with cells labeled with [3H]arachidonic acid in their lipids show that the stimulation by serum results in the release of arachidonic acid from the cellular lipids, mostly phospholipids. Hydrocrotisone inhibits this release but does not inhibit the production of prostaglandins from exogenously supplied arachidonic acid. This inhibition of arachidonic acid release from phospholipids may be the mechanism for the anti-inflammatory action of corticosteroids. (+info)
Phosphatidylinositide 3-kinase localizes to cytoplasmic lipid bodies in human polymorphonuclear leukocytes and other myeloid-derived cells.
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Phosphatidylinositide 3-kinase (PI3K) is a key enzyme implicated in intracellular signaling of diverse cellular responses including receptor-mediated responses and neutrophil activation. Several PI3K subunits have been cloned and shown to be localized to plasma membrane receptors, the cytosol, or intracellular vesicles or caveolae. We report the localization of PI3K to a distinct intracellular site, cytoplasmic lipid bodies, in leukocytes. In U937 monocyte cells, PI3K p85 regulatory and p110beta catalytic subunits were localized to lipid bodies by immunocytochemistry and/or immunoblotting and enzyme assays of subcellular fractions. In RAW murine macrophages, p55, p85alpha, and p85beta PI3K subunits were present at isolated lipid bodies. PI3K p85 was also shown to colocalize and, by co-immunoprecipitation, to be physically associated with phosphorylated Lyn kinase in lipid bodies induced to form in human polymorphonuclear leukocytes. These findings, therefore, indicate a novel site for PI3K compartmentalization and suggest that PI3K-mediated signaling is active within cytoplasmic lipid bodies in leukocytes. (+info)