Platelet-activating factor increases mucosal permeability in rat intestine via tyrosine phosphorylation of E-cadherin. (57/947)

1. Platelet-activating factor (PAF), an inflammatory mediator, plays an important role in mediating intestinal injury. However, it remains unclear whether PAF has a function in the intestine. The production of PAF by normal intestine and by unstimulated intestinal epithelial cell lines suggests that PAF may have a regulatory function in the normal bowel. 2. In this study we investigated the role of PAF in modulating intestinal mucosal permeability in rats. Lumen-to-blood transit of FD-4 (dextran 4400), (an index of intestinal permeability), was assessed in sham-operated rats and rats injected with PAF (1.25 microg kg(-1), i.v., a dose insufficient to induce intestinal injury). 3. PAF-induced villus cytoskeletal changes were examined by staining the intestine for F-actin. The effect of PAF on tyrosine phosphorylation of the junctional protein E-cadherin was examined by immunoprecipitation. Some rats were pretreated with AG1288 (a tyrosine kinase inhibitor) before PAF injection, and mucosal permeability change was assessed. 4. To investigate the role of endogenous PAF upon mucosal permeability, we studied the effect of PAF antagonists on (intraluminal) glucose-induced increase in mucosal permeability. 5. We found that low dose PAF: (a) alters the cytoskeletal structure of intestinal epithelium, (b) causes the influx of FD4 from intestinal lumen to systemic circulation, (c) induces tyrosine phosphorylation of E-cadherin and cadherin-associated proteins. Glucose-induced mucosal permeability increase is abolished by using two structurally different PAF antagonists. 6. These results suggest that endogenous PAF modulates macromolecular movement across the intestinal mucosal barrier, probably via tyrosine phosphorylation of E-cadherin and cytoskeletal alteration of enterocytes.  (+info)

The expression and localization of plasma platelet-activating factor acetylhydrolase in endotoxemic rats. (58/947)

The production of platelet-activating factor (PAF) and PAF-like phospholipids that also bind the PAF receptor are implicated in numerous pathological situations including bacterial endotoxemia and injury-induced oxidative damage. PAF and PAF-like phospholipids are hydrolyzed and inactivated by the enzyme PAF acetylhydrolase. In the intact rat, infusion of lipopolysaccharide (LPS) into a mesenteric vein served as an acute, liver-focused model of endotoxemia. We determined that the liver responds to LPS exposure with the production of plasma-type PAF acetylhydrolase mRNA and protein expression specifically in the resident macrophages of the liver. Liver macrophages, defined immunohistochemically using antibodies against ED1, present in livers from saline-treated animals contained no detectable PAF acetylhydrolase. Twenty-four hours following in vivo LPS administration, immunohistochemistry detected a slight increase in the number of ED1 staining cells and the ED1-positive cells now contained an abundance of PAF acetylhydrolase. The systemic administration of LPS resulted in increased expression of PAF acetylhydrolase in several tissues. Of the tissues examined, the greatest increase in PAF acetylhydrolase expression was observed in lung followed by increases in spleen, liver, kidney, and thymus. Additionally, the expression of PAF acetylhydrolase mRNA increased in circulating leukocytes and in peritoneal macrophages in response to systemic exposure to LPS. We examined the regulation of PAF acetylhydrolase expression and demonstrated the administration of the PAF receptor antagonists, BN 50739 and WEB 2170, inhibited by 50% the increase in PAF acetylhydrolase expression in response to LPS. The up-regulation of the plasma-type PAF acetylhydrolase expression constitutes an important mechanism for elevating the local and systemic ability to inactivate PAF and oxidized phospholipids in order to minimize PAF-mediated pathophysiology consequent from exposure to endotoxin. The abundance of PAF acetylhydrolase production in the liver lobule likely limits endotoxin-mediated tissue damage due to PAF synthesis.  (+info)

Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants. (59/947)

Over 150 mutations within the coding sequence of the V2 vasopressin receptor (V2R) gene are known to cause nephrogenic diabetes insipidus (NDI). A large number of these mutant receptors fail to fold properly and therefore are not routed to the cell surface. Here we show that selective, nonpeptidic V2R antagonists dramatically increase cell-surface expression and rescue the function of 8 mutant NDI-V2Rs by promoting their proper folding and maturation. A cell-impermeant V2R antagonist could not mimic these effects and was unable to block the rescue mediated by a permeant agent, indicating that the nonpeptidic antagonists act intracellularly, presumably by binding to and stabilizing partially folded mutants. In addition to opening new therapeutic avenues for NDI patients, these data demonstrate that by binding to newly synthesized mutant receptors, small ligands can act as pharmacological chaperones, promoting the proper folding and maturation of receptors and their targeting to the cell surface.  (+info)

Platelet-activating factor receptors in lamb lungs are downregulated immediately after birth. (60/947)

Platelet-activating factor (PAF) is a phospholipid with diverse biological functions mediated by a G protein-coupled receptor. We determined PAF receptor binding in lung membranes of four groups of perinatal lambs. Membrane protein (100 microg/ml) was incubated for 60 min at 30 degrees C with 0.5-24 nM of acetyl-[(3)H]PAF in 30 mM Tris buffer, pH 7.2, containing 0.25% BSA, 10 mM MgCl(2), and 125 mM choline chloride. PAF bound to membrane was isolated and quantified by scintillation spectrometry, followed with Scatchard analysis for receptor density (B(max)). The B(max) (means +/- SE, fmol/mg protein) were 445.8 +/- 12.3, 244.2 +/- 3.3, 250.6 +/- 3.6, and 419. 9 +/- 8.6 for the fetal, 90-min-old, <1-day-old, and 6- to 12-day-old lambs, respectively. The B(max) for the 90-min-old and <1-day-old lambs were not different but were significantly lower than those of either the term fetal or 6- to 12-day-old lambs. These data show a significant decrease in PAF binding to its receptor and in PAF B(max) in lung membranes of immediate newborn lambs. The dissociation constants (K(D), nM) were 7.7 +/- 0.52, 11.5 +/- 0.34, 6.9 +/- 0.48, and 5.0 +/- 0.53 for fetal, 90-min-old, <1-day-old, and 6- to 12-day-old newborn lamb lungs, respectively. The K(D) of the 90-min-old lamb was the highest of all. PAF receptor gene measured by RT-PCR showed a significant downregulation of PAF receptor gene mRNA in lungs of lambs <1 day old, suggesting a transcriptional regulation of PAF receptor gene expression in the immediate newborn period. We speculate that decreased PAF receptor binding immediately after birth will facilitate the fall in pulmonary vascular resistance in the immediate newborn period.  (+info)

Scorpion venom-induced neutrophilia is inhibited by a PAF receptor antagonist in the rat. (61/947)

A dramatic blood neutrophilia is an important feature of the severe envenoming caused by the Brazilian scorpion Tityus serrulatus and may contribute to the development of lung injury in children. We examined the effects of an intravenous injection of T. serrulatus scorpion venom (TsV) on the total number of leukocytes and neutrophils in the blood of anesthetized rats. Injection of TsV (250 microg/kg) induces a significant leukocytosis 2 and 3 h after its injection, explained by an increase in the number of neutrophils. The release of catecholamines and action on adrenoceptors is responsible for most of the systemic manifestations of TsV. However, pretreatment with the beta-adrenoceptor antagonists metoprolol and propranolol or the alpha1-adrenoceptor antagonist prazosin (0.25 mg/kg) did not prevent TsV-induced neutrophilia. Blood neutrophilia induced by TsV occurred simultaneously with a significant reduction of mature neutrophils in bone marrow. Pretreatment with the platelet-activating factor (PAF) receptor antagonists UK-74505 or WEB-2086 prevented TsV-induced increase in blood neutrophils and reduction in the number of neutrophils in the bone marrow. It is concluded that scorpion venom induces blood neutrophilia in rats, explained by a PAF receptor-dependent mobilization of neutrophils from the bone marrow.  (+info)

Sphingomyelinase and ceramide analogs induce contraction and rises in [Ca(2+)](i) in canine cerebral vascular muscle. (62/947)

Studies were designed to investigate effects of neutral sphingomyelinase (N-SMase) and ceramide analogs as well as phosphorylcholine on vascular tone and Ca(2+) mobilization in isolated canine cerebral arterial smooth muscle. N-SMase (0.001-0.1 U/ml) provoked a gradual but sustained vasoconstriction of arterial rings in a concentration-related manner that was endothelium independent. Incubation of denuded arterial rings in Ca(2+)-free medium or pretreatment with verapamil in extracellular Ca(2+) resulted in a reduction of the N-SMase-evoked constriction. Exposure of arterial rings to 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid (BAPTA)-AM did not, however, result in a reduction of N-SMase-induced constriction. Both staurosporine and bisindolymaleimide I attenuated N-SMase-induced contractions to 66% and 72% of control, respectively. N-SMase caused gradual and sustained rises in intracellular Ca(2+) concentration ([Ca(2+)](i)) in primary cultured cerebral vascular smooth muscle cells. Pretreatment of these cultured cells with nimodipine and verapamil caused a steady decline in N-SMase-induced rises in [Ca(2+)](i). Exposure of the cells to Ca(2+)-free solution reversed the [Ca(2+)](i)-induced rise triggered by N-SMase to the resting baseline. Both C(8) and C(16) ceramide (10(-9)-10(-6) M), but not phosphorylcholine, constricted denuded canine arterial rings in a concentration-related manner and elevated [Ca(2+)](i). Our results suggest that the sphingomyelin-signaling pathway, via a probable release of ceramide molecules, may play an important role in regulation of cerebral arterial wall tone.  (+info)

The treatment of hyponatraemia using vasopressin antagonists. (63/947)

Hyponatraemia is a frequent electrolyte disorder. It is primarily attributable to vasopressin excess plus sustained fluid intake. Hyponatraemia causes CNS symptoms, especially during the first 2-4 days; these symptoms are related to brain swelling. Hyponatraemia occurs in the setting of liver cirrhosis and congestive cardiac failure, in which it is related to stimulation by low arterial blood pressure acting through baroreceptors. Hyponatraemia also occurs in the syndrome of inappropriate antidiuretic hormone secretion, usually from neoplasms releasing vasopressin. The conventional treatment of hyponatraemia used to be fluid restriction and treatment of the underlying disorder. This kind of treatment has been unreliable, cumbersome and difficult to comply with for the patient. In the future, effective vasopressin V2 antagonists will become available for clinical use in the treatment of hyponatraemia, and are expected to improve the management of hyponatraemia. Pharmacological characteristics and observations of biological effects of three antagonists are reported in the present article.  (+info)

Activation of platelet-activating factor (PAF) receptor stimulates nitric oxide (NO) release via protein kinase C-alpha in HEC-1B human endometrial epithelial cell line. (64/947)

BACKGROUND: Impairment of the fertility in the platelet-activating factor (PAF) receptor transgenic female mice suggests changes in PAF functions can influence uterine receptivity. We hypothesized that vasodilatory actions of PAF in the uterus was exerted by PAF-mediated nitric oxide (NO) release via activation of isoenzyme-specific protein kinase C (PKC). MATERIALS AND METHODS: Inducible and endothelial NOS was shown by Reverse transcription polymerase chain reaction RT-PCR in cDNA synthesized from RNA extract of proliferative and secretory endometrium as well endometrial epithelial cell lines HEC-1B. The effect of WEB2170, N(G)-monomethyl-L-arginine (L-NMMA) and Ro31-8220 on PAF mediated NO release by HEC-1B cell was determined. PAF induced translocation of PKCalpha in HEC-1B cell and its antagonist effect by Ro 31-8220 was studied by Western immunoblot analysis. PKC isoenzyme regulated by PAF was determined in HEC-1B cell lysate by immunoprecipitation. RESULTS: PAF-evoked a rapid and concentration-dependent biphasic increase in total NO in human HEC-1B endometrial epithelial cell line [as measured by a Sievers NOA 280A NO Chemiluminescent Analyser.] This increase in NO release was attenuated by the PAF receptor antagonist, WEB2170. Inhibition of NO synthesis by N(G)-monomethyl-L-arginine produced marked dose-dependent attenuation of PAF-mediated NO release, indicating nitric oxide synthase (NOS) activation. PAF-mediated NO release was also inhibited by the PKC inhibitor Ro 31-8220 and by the removal of extracellular calcium, suggesting a dependency on PKC and calcium, respectively. RT-PCR analysis showed expression of inducible NOS and endothelial NOS in human endometrium, myometrium and HEC-1B cells. Western immunoblot analysis showed PKCalpha, betaII and iota were the principal isozymes present in the HEC-1B cell line and normal endometrium, suggesting that both HEC-1B cells and normal endometrium have similar PKC isozymes. PAF induced the translocation of both PKCalpha and PKCiota within the time frame of NO release. The translocation of PKCalpha, but not PKCiota, was susceptible to inhibition by Ro 31-8220 that also inhibited PAF-evoked NO release, suggesting that PKCalpha is the principal isozyme involved in this process and that eNOS may be a substrate for PKCalpha. Kinase assays performed using immunoprecipitated PKCalpha showed that PAF (1 nM) activated PKCalpha that was inhibited by co-incubation with Ro31-8220 and Ca(2+)-free medium. CONCLUSIONS: This study demonstrates that PAF-stimulated NO release via PKCalpha in epithelial cells might regulate endometrial functions such as implantation and menstruation.  (+info)