Increased synthesis of thromboxane A(2) and expression of procoagulant activity by monocytes in response to arachidonic acid in diabetes mellitus.

Thromboxane A(2) (TXA(2)) synthesis and expression of procoagulant activity (PCA) were investigated in mononuclear cells and monocytes prepared from a control and a Type 2 diabetic group. Monocytes from the diabetic group produced 2.10+/-0.81 ng of TXB(2)/5 x 10(5) monocytes compared to 1.26+/-0.43 ng/5 x 10(5) monocytes by the control group (P<0.01, n=11) when incubated in autologous plasma containing arachidonic acid (200 microg/ml). When monocytes were incubated in buffer containing arachidonic acid (20 microg/ml), cells from the diabetic group produced 1.65+/-0.68 ng of TXB(2)/5 x 10(5) monocytes compared to 1.07+/-0.31 ng/5 x 10(5) monocytes by the control group (P<0.02, n=12). Expression of PCA was examined in mononuclear cell preparations. Basal and maximally stimulated PCA with lipopolysaccharide (4.2 microg/ml) were not different between control and diabetic groups. However, arachidonic acid induced a four-fold (P<0.001) increase in PCA in the diabetic group. This activity was characterized as tissue factor. Increased synthesis of TXA(2) and expression of PCA may potentiate thrombosis and increase fibrin deposition, events that play primary roles in the development of vascular disease.

Apoptosis and JNK activation are differentially regulated by Fas expression level in renal tubular epithelial cells.

BACKGROUND: In chronic renal disease, renal tubular epithelial cell (RTC) Fas expression is up-regulated, leading to apoptotic RTC deletion and tubular atrophy. In vitro, cytokine- or hypoxia-induced up-regulation of Fas expression is associated with RTC apoptosis. In contrast, constitutively expressed, low level RTC Fas does not mediate apoptosis, suggesting that Fas may be coupled to expression level-dependent RTC signaling pathways. Fas is known to signal through JNK in many systems, but the requirement of JNK activation for apoptosis remains controversial. METHODS: To determine if RTC Fas regulates JNK activity and apoptosis, human RTC were transfected with graded concentrations of a eukaryotic expression vector for murine Fas. Apoptosis was measured by annexin V, TUNEL and PARP cleavage assays. JNK activity was determined by immune complex kinase assay and/or immunoblots with phospho-specific JNK antibodies, in the presence or absence of co-expressed dominant negative JNK constructs. RESULTS: Fas antibody stimulation of RTC with high Fas expression levels (to model RTC phenotype in renal disease) caused a tenfold increase in apoptosis, while RTC with low level Fas expression (to model normal RTC phenotype) were apoptosis-resistant. Fas ligation activated JNK in RTC expressing low levels of Fas, but not in apoptosis-sensitive RTC with increased Fas expression. Dominant negative JNK co-expression failed to inhibit apoptosis in RTC expressing high levels of Fas, suggesting that JNK is neither necessary, nor sufficient, for Fas-dependent apoptosis. CONCLUSIONS: At high levels of expression, RTC Fas promotes apoptosis in a JNK-independent manner. At low basal expression, Fas induces JNK activation, but not apoptosis, consistent with novel roles for RTC Fas as a mediator of cell stress or chronic inflammation.

The IL-1 receptor and Rho directly associate to drive cell activation in inflammation.

IL-1-stimulated mesenchymal cells model molecular mechanisms of inflammation. Binding of IL-1 to the type I IL-1 receptor (IL-1R) clusters a multi-subunit signaling complex at focal adhesion complexes. Since Rho family GTPases coordinately organize actin cytoskeleton and signaling to regulate cell phenotype, we hypothesized that the IL-1R signaling complex contained these G proteins. IL-1 stimulated actin stress fiber formation in serum-starved HeLa cells in a Rho-dependent manner and rapidly activated nucleotide exchange on RhoA. Glutathione S-transferase (GST) fusion proteins, containing either the full-length IL-1R cytosolic domain (GST-IL-1Rcd) or the terminal 68 amino acids of IL-1R required for IL-1-dependent signal transduction, specifically coprecipitated both RhoA and Rac-1, but not p21(ras), from Triton-soluble HeLa cell extracts. In whole cells, a small-molecular-weight G protein coimmunoprecipitated by anti-IL-1R antibody was a substrate for C3 transferase, which specifically ADP-ribosylates Rho GTPases. Constitutively activated RhoA, loaded with [gamma-32P]GTP, directly interacted with GST-IL-1Rcd in a filter-binding assay. The IL-1Rcd-RhoA interaction was functionally important, since a dominant inhibitory mutant of RhoA prevented IL-1Rcd-directed transcriptional activation of the IL-6 gene. Consistent with our previous data demonstrating that IL-1R-associated myelin basic protein (MBP) kinases are necessary for IL-1-directed gene expression, cellular incorporation of C3 transferase inhibited IL-1R-associated MBP kinase activity both in solution and in gel kinase assays. In summary, IL-1 activated RhoA, which was physically associated with IL-1Rcd and necessary for activation of cytosolic nuclear signaling pathways. These findings suggest that IL-1-stimulated, Rho-dependent cytoskeletal reorganization may cluster signaling molecules in specific architectures that are necessary for persistent cell activation in chronic inflammatory disease.

Interleukin-1 stimulates Jun N-terminal/stress-activated protein kinase by an arachidonate-dependent mechanism in mesangial cells.

BACKGROUND: We have studied interleukin-1 (IL-1)-stimulated signals and gene expression in mesangial cells (MCs) to identify molecular mechanisms of MC activation, a process characteristic of glomerular inflammation. The JNK1 pathway has been implicated in cell fate decisions, and IL-1 stimulates the Jun N-terminal/stress-activated protein kinases (JNK1/SAPK). However, early postreceptor mechanisms by which IL-1 activates these enzymes remain unclear. Free arachidonic acid (AA) activates several protein kinases, and because IL-1 rapidly stimulates phospholipase A2 (PLA2) activity release AA, IL-1-induced activation of JNK1/SAPK may be mediated by AA release. METHODS: MCs were grown from collagenase-treated glomeruli, and JNK/SAPK activity in MC lysates was determined using an immunocomplex kinase assay. RESULT: Treatment of MCs with IL-1 alpha induced a time-dependent increase in JNK1/SAPK kinase activity, assessed by phosphorylation of the activating transcription factor-2 (ATF-2). Using similar incubation conditions, IL-1 also increased [3H]AA release from MCs. Pretreatment of MCs with aristolochic acid, a PLA2 inhibitor, concordantly reduced IL-1-regulated [3H]AA release and JNK1/SAPK activity, suggesting that cytosolic AA in part mediates IL-1-induced JNK1/SAPK activation. Addition of AA stimulated JNK1/SAPK activity in a time- and concentration-dependent manner. This effect was AA specific, as only AA and its precursor linoleic acid stimulated JNK1/SAPK activity. Other fatty acids failed to activate JNK1/SAPK. Pretreatment of MCs with specific inhibitors of AA oxidation by cyclooxygenase, lipoxygenase, and cytochrome P-450 epoxygenase had no effect on either IL-1- or AA-induced JNK1/SAPK activation. Furthermore, stimulation of MCs with the exogenous cyclooxygenase-, lipoxygenase-, phosphodiesterase-, and epoxygenase-derived arachidonate metabolites, in contrast to AA itself, did not activate JNK1/SAPK. CONCLUSION: We conclude that IL-1-stimulated AA release, in part, mediates stimulation of JNK1/SAPK activity and that AA activates JNK1/SAPK by a mechanism that does not require enzymatic oxygenation. JNK1 signaling pathway components may provide molecular switches that mediate structural rearrangements and biochemical processes characteristic of MC activation and could provide a novel target(s) for therapeutic intervention.

Cytosolic domain of the type I interleukin-1 receptor spontaneously recruits signaling molecules to activate a proinflammatory gene.

Immediate postreceptor events activated by IL-1-IL-1R interaction remain undefined. We have initiated studies to identify candidate signal transducers that associate with the cytosolic domain (cd) of the IL-1R. Immunocomplex kinase assays demonstrated an IL-1-activated myelin basic protein kinase activity that coprecipitated with the IL-1R from rat mesangial, mouse EL-4, and HeLa cells. Using glutathione-S-transferase (GST) fusion proteins, HeLa cell lysates next were assayed for kinases that associated with IL-1R cytoplasmic sequences. A GST-IL-1R fusion protein containing the entire cd (amino acids 369-569; GST-IL-1Rcd) recruited a kinase activity in the absence and presence of IL-1 stimulation. In contrast, a GST-IL-1R membrane-proximal region mutant (amino acids 369-501; GST-IL-1RcdDelta), which lacks COOH-terminal amino acid residues required for nuclear factor-kappaB activation, poorly phosphorylated MBP. In gel, kinase assays demonstrated 63-, 83-, and 100-kD kinases that specifically coprecipitated with the HeLa IL-1R and the GST-IL-1Rcd, but not GST-IL-1RcdDelta. 35S-labeled proteins, with Mrs identical to the kinase activities, stably associated with GST-IL-1Rcd. Transient transfection assays of 293 cells were used to evaluate the functional significance of these findings. Simply increasing IL-1cd expression in 293 cells stimulated 5'-IL-6 flanking region-regulated CAT activity threefold above control, an effect blocked by the kinase inhibitors staurosporine and calphostin C. In summary, we have identified two previously unrecognized 63- and 83-kD kinases as well as a protein with an Mr similar to the recently cloned IL-1R-associated kinase, all of which associate spontaneously with the IL-1Rcd. Ectopic IL-1Rcd expression was sufficient to trigger cellular activation, suggesting that the extracellular domain of the intact receptor represses signal transduction until IL-1 is bound. Given that the IL-1Rcd signaling domain has been conserved in a functionally diverse group of transmembrane receptors, further characterization of this signaling process may define novel molecular mechanisms controlling cellular function and differentiation.

Angiotensin II activates the beta 1 isoform of phospholipase C in vascular smooth muscle cells.

Vascular smooth muscle cells (VSMC) contribute to the pathophysiology of hypertension through cell growth and contraction, and phospholipase C (PLC) is a critical effector enzyme in growth factor and vasoconstrictor signaling. There is indirect evidence that angiotensin II (ANG II) receptors are linked to the PLC-beta isoform signaling pathways. However, recent studies suggest that PLC-beta isoforms may not be expressed in VSMC. Our data demonstrate that in human aortic VSMC, PLC-beta 1 and PLC-gamma 1 proteins were detected by immunoblot analysis, and PLC-beta 1 mRNA was identified by reverse transcriptase-polymerase chain reaction in rat aortic VSMC. Incubation of permeabilized VSMC with anti-PLC-beta 1 or anti-Gq alpha antibodies inhibited ANG II-dependent inositol polyphosphate (IP) formation, while anti-PLC-gamma 1 antibodies did not inhibit ANG II-regulated IP formation. Conversely, anti-PLC-gamma 1 antibodies completely abolished platelet-derived growth factor (PDGF)-dependent IP generation, whereas anti-PLC-beta 1 antibodies had no effect on PDGF-induced PLC activation. Inhibition of tyrosine phosphorylation with genistein or herbimycin A did not diminish ANG II-stimulated IP formation or cytosolic free Ca2+ concentration transients, thereby confirming that ANG II signals via a PLC-gamma 1-independent mechanism. In summary, PLC-beta 1 and PLC-gamma 1 are expressed in human aortic VSMC, and PLC-beta 1 is the isoform that is critical for ANG II-regulated PLC signaling in these cells.

Endothelin-1 stimulates cytosolic phospholipase A2 activity and gene expression in rat glomerular mesangial cells.

Endothelin-1 (ET-1) stimulates vascular smooth muscle and mesangial cells to release prostaglandin E2 (PGE2), which can attenuate the vasoconstrictor and mitogenic effects of this peptide. Phospholipase A2 (PLA2)-mediated release of arachidonic acid from the sn-2 position of membrane phospholipids is thought to be one of the rate-limiting steps in prostaglandin (PG) synthesis. We evaluated the role of ET-1 to regulate gene expression, protein synthesis and enzymatic activity of cytosolic PLA2 (cPLA2), an intracellular form of the PLA2 enzyme family, in cultured rat mesangial cells using both acute and chronic incubation protocols. Acute ET-1-induced stimulation of cPLA2 activity was maximal after 10 minutes (181.1 +/- 6.84% of control), persisted for 40 minutes and did not require new protein synthesis. Heparin, a potent inhibitor of intracellular Ca2+ increase as well as mitogen-activated protein (MAP) kinase activation and cell proliferation, did not affect the rapid cPLA2 stimulation by ET-1. Chronic incubation of glomerular mesangial cells with ET-1 (1 to 24 hr) led to time- and dose-dependent increases in cPLA2 mRNA expression which was maximal after six hours, persisted up to 24 hours and which was accompanied by both cPLA2 protein formation, as assessed by Western analysis, as well as by stimulation of enzymatic activity. Inhibition of protein synthesis by cycloheximide increased basal cPLA2 mRNA accumulation in quiescent mesangial cells, and the combination of ET-1 and cycloheximide resulted in a greater induction of cPLA2 gene expression when compared to ET-1 alone. Actinomycin D treatment blocked the effect of ET-1 on cPLA2 mRNA accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoid uncoupling of antiogensin II-dependent phospholipase C activation in rat vascular smooth muscle cells.

Vascular tone is maintained by both angiotensin II (Ang II) and glucocorticoids, but the effect of glucocorticoids on Ang II function in vascular smooth muscle cells (VSMC) is unclear. To determine the direct influence of glucocorticoids on VSMC Ang II receptor function, the effects of dexamethasone on Ang II receptor binding, Ang II-induced phospholipase C (PLC) activation, and Ang II-dependent cell growth were studied in cultured rat VSMC. Dexamethasone caused concentration- and time-dependent increases in Ang II binding which were prevented by glucocorticoid receptor inhibition with RU 38486. Dexamethasone-induced enhancement of Ang II binding resulted from increased AT1 receptors, as indicated by Northern blot analysis and competitive binding assays. Despite causing increased Ang II receptor number, dexamethasone preincubation prevented Ang II-induced PLC activation, as indicated by phosphatidylinositol 4,5-bisphosphate degradation and inositol trisphosphate formation. When PLC activity was directly measured in VSMC soluble and membrane fractions, Ang II receptor activation caused decreased soluble and increased membrane PLC activity, consistent with the interpretation that Ang II caused cytosol-to-membrane PLC translocation. The effect of Ang II on PLC translocation was prevented by dexamethasone preincubation. Finally, prolonged incubation with dexamethasone and Ang II had additive effects on VSMC hypertrophy. In conclusion, glucocorticoids directly altered Ang II function in VSMC by causing increased Ang II receptor number, Ang II receptor/PLC uncoupling, and enhanced Ang II-dependent hypertrophy.

Interleukin 1 alpha causes rapid activation of cytosolic phospholipase A2 by phosphorylation in rat mesangial cells.

We have shown previously that interleukin 1 (IL-1) stimulates eicosanoid production in glomerular mesangial cells (MC) by de novo synthesis of a 14-kD, group II phospholipase A2 (PLA2). IL-1-stimulated prostaglandin E2 synthesis precedes expression of this enzyme, suggesting that another PLA2 isoform must be more rapidly activated. In the presence but not absence of calcium inophore, [3H]arachidonate release is increased significantly as early as 5 min after addition of IL-1, and IL-1 concurrently stimulates a Ca(2+)-dependent phospholipase activity, which was characterized as the cytosolic form of PLA2 (cPLA2). IL-1 does not alter either cPLA2 mRNA expression or mass in serum-stimulated MC, suggesting that cPLA2 activity is increased by a posttranslational modification. IL-1 treatment for 30 min doubles 32P incorporation into immunoprecipitable cPLA2 protein, concordant with the increase in enzyme activity. Immunoblot analysis of extracts derived from IL-1-treated (30 min) cells demonstrates a decreased mobility of cPLA2, and treatment of MC lysates with acid phosphatase significantly reduces cytokine-activated cPLA2 activity, further indicating that IL-1 stimulates phosphorylation of the enzyme. IL-1 treatment (24 h) of serum-deprived MC doubled cPLA2 mRNA, protein, and activity. In summary, IL-1 increases cPLA2 activity in a biphasic, time-dependent manner both by posttranslational modification and de novo synthesis. We consider cPLA2 activation a key step in IL-1-stimulated synthesis of pro-inflammatory, lipid mediators, and an integral event in the phenotypic responses induced in target cells by this cytokine.

Endothelin-1 stimulates cytosolic phospholipase A2 in Chinese hamster ovary cells stably expressing the human ETA or ETB receptor subtype.

The ability of endothelin-1 (ET-1) to activate cytosolic phospholipase A2 (cPLA2) has been studied in Chinese Hamster ovary (CHO) cells stably expressing either the human ETA (CHO/ETA) or the human ETB (CHO/ETB) receptor subtype. ET-1 dose-dependently increased a dithiothreitol-insensitive cPLA2 activity in both cell types. In CHO/ETA cells, BQ-123, an ETA-selective antagonist, completely blocked ET-1-induced cPLA2 stimulation. In CHO/ETB cells, the ET-1 response was mimicked by 4AlaET-1 which could be blocked partially by PD 145065, a nonselective antagonist of ETA and ETB. As expected, ET-1 stimulated PGE2 synthesis in CHO cells transfected with ET receptors. We conclude that ET-1 can stimulate cPLA2 via both the human ETA and ETB receptor subtype.

Cell-specific regulation of type II phospholipase A2 expression in rat mesangial cells.

IL-1 stimulates mesangial cells to synthesize specific proteins, including a non-pancreatic (Type II) phospholipase A2 (PLA2). We have studied the regulation of PLA2 by proinflammatory mediators, implicated in the pathogenesis of glomerulonephritis, and have assessed whether the activation of second messenger systems modulates or mimics PLA2 gene expression by cytokines. IL-1 alpha and beta, TNF alpha, and LPS, but not serum, IL-2, or PDGF, potently induce PLA2 mRNA, and enzyme expression. IL-1-stimulated mesangial cells express a 1.0 kB PLA2 mRNA transcript that is induced in a dose- and time-dependent manner. IL-1-stimulated increases in steady-state PLA2 mRNA abundance result from a moderate increase in PLA2 transcription rate that is amplified by the prolonged persistence of the transcript. Forskolin and dibutyryl cAMP potentiate IL-1-induced PLA2 mRNA and enzyme expression, but have no effect in the absence of cytokine. 12-tetradecanoyl phorbol 13-acetate, sn-1, 2-dioctanoyl glycerol or 1-oleoyl-2-acetyl-sn-glycerol fail to induce PLA2 expression or to alter the effect of IL-1 when coincubated with the cytokine. In contrast, serum deprivation for 24 h specifically enhances IL-1-stimulated PLA2. Genistein potentiates PLA2 mRNA expression in cells exposed to both IL-1 and serum. The inhibitory effect of serum on IL-1-induced PLA2 mRNA abundance is reproduced by PDGF but not dexamethasone. These data demonstrate that the signaling pathways directly engaged by IL-1 to induce PLA2 expression in mesangial cells interact with several second messenger systems in a cell-specific manner. We speculate that IL-1 induces specialized changes in mesangial cell structure and function through direct activation of a transcription factor(s), that result in induction of a specific gene set.

Interleukin-1 alpha stimulates prostaglandin biosynthesis in serum-activated mesangial cells by induction of a non-pancreatic (type II) phospholipase A2.

Enhanced prostaglandin (PG) biosynthesis is a hallmark of inflammation, and interleukin-1 (IL), a proinflammatory cytokine, is a potent stimulus of PG production. We investigated the mechanisms of IL-1 alpha-enhanced PG synthesis in serum-stimulated mesangial cells. The rIL-1-stimulated increase in PGE2 synthesis was dose- and time-dependent and inhibited by both cycloheximide and actinomycin D. Phospholipase (PL) activity was increased 5- to 10-fold in acid extracts of rIL-1-treated cells as measured by arachidonate release from exogenous [14C]arachidonyl-phosphatidyl-ethanolamine. This induced phospholipase activity was Ca(2+)-dependent and inhibited by the PLA2 inhibitors, aristocholic acid, 7,7-dimethyl-5,8-eicosadienoic acid, and p-bromophenacylbromide, but not by the 1,2-diacylglycerol lipase inhibitor RHC 80267. The rIL-1-stimulated PLA2 had an alkaline pH optimum, and phosphatidylethanolamine was preferred over phosphatidylcholine as substrate. The PLA2 activity increased by rIL-1 was inhibited in cells coincubated with cycloheximide and was measurable after 6 h. A sensitive and specific solution hybridization assay demonstrated a coordinate time-dependent induction of non-pancreatic PLA2 mRNA expression which was increased at least 6-fold by 24 h. In whole cells, IL-1 had no effect on basal [3H]arachidonic acid release but vasopressin (1 microM)-stimulated release was potentiated 2- to 3-fold, suggesting that IL-1 may prime cells for increased PG synthesis via increased PLA2 activity. Thus IL-1 directly stimulates, as well as primes cells for, enhanced PG synthesis, in part, by increasing PLA2 activity through new synthesis of a non-pancreatic (Type II) PLA2.

Cytokine- and LPS-induced synthesis of interleukin-8 from human mesangial cells.

The cytokine neutrophil-activating peptide-1/interleukin-8 (NAP/IL-8) activates neutrophils (PMN) and elicits selective diapedesis of PMN into the extracellular space. The glomerular mesangial cell (MC) is a specialized pericyte that controls glomerular filtration and synthesizes and responds to a variety of cytokines. Because of its location within the glomerulus, the MC is in a pivotal position to orchestrate events underlying immune injury. Since immune-injured glomeruli have been shown to produce NAP/IL-8 activity in vitro, we assessed whether lipopolysaccharide (LPS)- or cytokine-activated MC might be a source of this activity. Pure human MC, devoid of monocyte/macrophage and fibroblast contamination, were grown by explant from collagenase-treated glomeruli. Human recombinant interleukin-1 alpha (IL-1 alpha, 20 ng/ml), IL-1 beta (50 ng/ml), tumor necrosis factor alpha (TNF, 100 ng/ml) and lipopolysaccharide (LPS, 10 micrograms/ml) stimulated release of a neutrophil chemotactic factor from cultured MC. Both concentrated (fivefold) and unconcentrated MC supernatants stimulated directed neutrophil migration under agarose at a level similar to that of the bacterial chemotactic factor, FMLP. In contrast, unstimulated MC-conditioned media and IL-1 alpha, IL-1 beta. TNF and LPS in medium alone did not directly induce PMN migration. Molecular sizing studies using sequential membrane ultrafiltration identified significant TNF-stimulated, MC-derived chemotactic activity in the 3000 to 10000 kD fraction. An anti-NAP/IL-8 monoclonal antibody, 46E5, significantly inhibited PMN chemotaxis stimulated by TNF-stimulated, MC-conditioned media in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of prostaglandin endoperoxide synthase gene expression in cultured rat mesangial cells: induction by serum via a protein kinase-C-dependent mechanism.

Recent evidence suggests that the steady state level of prostaglandin (PG) endoperoxide synthase (PES) might regulate long term increases in PG synthesis, but the mechanism(s) regulating PES gene expression remains unclear. The present experiments investigated the regulation of PES expression in quiescent mesangial cells treated with serum. Serum induced time-dependent increases in PES steady state mRNA, protein level, and enzyme activity. Both the kinetics and cycloheximide sensitivity of PES mRNA induction suggest that PES was induced as a member of the delayed-early gene set. Beta-adrenergic-mediated cAMP accumulation was similar in Go and cycling cells, suggesting that the increase in PES expression did not reflect global up-regulation of signaling cascades in serum-treated cells. Three lines of evidence suggest that protein kinase-C (PKC) mediates serum-stimulated PES gene expression: 1) 12-tetradecanoyl phorbol 13-acetate mimicked the serum response; 2) PES gene expression by serum was attenuated in cells depleted of PKC; and 3) a PKC inhibitor, sangivamycin, blocked serum-stimulated PES gene induction. In addition, the ability of dexamethasone to block serum-induced PES enzyme activity without affecting the increase in PES mRNA points to posttranscriptional mechanisms of regulation. In summary, we conclude that in glomerular mesangial cells the PES gene is inducible, not constitutive, and that expression of this gene in Go cells is induced by serum and results in chronic elevations of PGE2. These findings are consistent with the hypothesis that differential regulation of PES gene expression might play a role in diverse cellular responses, such as mitogenesis and inflammation.

Suppression of foam cell lesions in hypercholesterolemic rabbits by inhibition of thromboxane A2 synthesis.

Hypercholesterolemia induces adhesion of blood-borne monocytes to vascular endothelium and their subsequent migration into the intima, where foam cell lesions subsequently develop. The regulating mechanisms for the adhesion and migration are unclear. In this study, a specific thromboxane A2 (TXA2) synthetase inhibitor, UK-38485, was used to treat rabbits fed an atherogenic diet to determine whether inhibition of TXA2, the major metabolite of arachidonic acid in monocytes, affects lesion development. Rabbits were fed a diet supplemented with 2% cholesterol and 8% peanut oil for 12 weeks with or without UK-38485 at a dosage that maintained 80% to 90% inhibition of TXA2 formation in serum. The treatment with UK-38485 had no effect on total serum cholesterol. Both the treated and untreated groups developed subpopulations of high and low responders with respect to the extent of lesion coverage, forming a bimodal distribution. The treatment with UK-38485 significantly (p less than 0.001) reduced the percentage of the thoracic aorta covered by lesions when treated low responders (5.3 +/- 1.0%, n = 12) were compared to untreated low responders (23.6 +/- 2.9%, n = 12). However, UK-38485 had no effect when treated high responders (76.3%, n = 3) were compared to untreated high responders (72.0%, n = 12). Lesion coverage was not correlated with serum cholesterol levels. Stimulation of isolated rabbit monocytes in autologous plasma with 0.66 mM arachidonic acid in the presence of increasing concentrations of UK-38485 caused a dose-dependent inhibition of TXA2 and a concurrent increase in prostaglandin E2 (PGE2).(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium transport in rat renal papillary collecting tubule cells in culture.

Sodium transport in the papillary collecting duct (PCD) is poorly understood because of the inaccessibility of the distal nephron to micropuncture. Cultured rat renal papillary collecting tubule (RPCT) cells were investigated as a model for the PCD. RPCT cells have the morphologic appearance and hormonal responsiveness of the papillary collecting tubule. Sodium transport was studied using 22Na+ uptake measurements. Sodium uptake, measured at 23 degrees C in the absence of K+ and in the presence of 0.5 mM ouabain, was saturable at 100 mM extracellular NaCl, and half-maximal uptake occurred at 40 mM NaCl. The accumulation of 22Na+ appeared to be intracellular and was regulated by (Na+,K+)-ATPase activity, since activation of the Na+/K+ pump with K+ reduced 22Na+ accumulation by 90%. The time course for uptake was linear, showed only a single component, and followed first order kinetics with a t1/2 of 16 min. Amiloride and lithium inhibited 22Na+ influx, and a Dixon plot was linear, with a Ki of 16 microM amiloride. Chloride replacement of 1 mM furosemide, with or without K+, reduced uptake by only 20%. Sodium efflux from RPCT cells in the presence of ouabain showed a similar time course (t1/2, 15 min) and was also inhibited by amiloride (IC50 = 20 microM). Increased extracellular pH stimulated 22Na+ uptake and inhibited 22Na+ efflux. Addition of permeable organic acids, acetate, and bicarbonate, enhanced 22Na+ uptake. These results are consistent with Na+/H+ and Na+/Na+ exchange as mechanisms of 22Na+ uptake in the RPCT cell. This exchanger may be important in regulation of transepithelial sodium flux, maintenance of intracellular pH and cell volume, and hormonal stimulation of the papillary collecting duct.

Vasopressin-mediated protein phosphorylation in intact toad urinary bladder.

Endogenous protein phosphorylation was examined in intact 32P-labeled toad bladders in response to arginine vasopressin (AVP) and other agents under conditions where the bladders were undergoing the normal hydroosmotic response. AVP increased 32P incorporation into proteins with apparent MW of 17,000, 28,000 and 34,000 and decreased 32P incorporation into a protein with MW 15,500. The cyclic AMP analog 8-(p-chloro-phenylthio)-cyclic AMP mimicked the effects of AVP on 32P incorporation. AVP-dependent changes in protein phosphorylation were found to be specific for the epithelium of the bladder and were blocked by the antagonist d(CH2)5-D-TyrVAVP. AVP caused increased phosphorylation even in the absence of an osmotic gradient, but the AVP-mediated decrease in 32P content of the 15,500 MW band was observed only in the presence of an osmotic gradient. Isolated epithelial cells also displayed AVP-stimulated increases in 32P incorporation into the MW 17,000 and 34,000 phosphoproteins, but no decrease in 32P incorporation into the 15,500-dalton band. Phosphorylation of the MW 34,000 band was maximal within 3 min. These data are consistent with the hypothesis that physiological effects of AVP may, in part, be mediated by cyclic AMP-dependent phosphorylation of specific proteins.

Renal cyclo-oxygenase and lipoxygenase products in health and disease.

Renal glomeruli have cyclo-oxygenase and lipoxygenase enzymes which convert arachidonic acid to prostaglandins, thromboxane and 12-hydroxyeicosatetraenoic acid. Glomerular epithelial and mesangial cells, in culture, also synthesize these arachidonate products. Angiotensin and vasopressin contract mesangial cells and stimulate mesangial synthesis of PGE2. PGE2, in the glomerulus, antagonizes the actions of angiotensin on the mesangium and hence reduces angiotensin-mediated glomerular contraction. Glomerular immune injury (nephrotoxic serum nephritis) augments glomerular production of prostaglandins and thromboxane. Thromboxane reduces glomerular function and inhibition of thromboxane synthesis preserves glomerular filtration rate and renal plasma flow in this disease model. Spontaneously hypertensive rats also have enhanced glomerular prostaglandin and thromboxane synthesis. Although acute inhibition of thromboxane synthesis will vasodilate the hypertensive rat kidney, chronic inhibition does not reduce blood pressure or increase renal blood flow.