The 650-kDa 12(S)-hydroxyeicosatetraenoic acid binding complex: occurrence in human platelets, identification of hsp90 as a constituent, and binding properties of its 50-kDa subunit.

A cytosolic 650-kDa complex which binds 12(S)-hydroxy-5,8,10, 14-eicosatetraenoic acid (12(S)-HETE) with high affinity and specificity has been found in various cell lines but not until now in platelet cytosol. After incubation of human platelets with 12(S)-[3H]HETE, a labeled cytosolic 650-kDa complex was isolated. As previously shown for the binding complex in Lewis lung carcinoma (LLC) cells, ATP treatment transformed the platelet complex into a 50-kDa ligand-binding subunit. These results are of interest for two reasons: (a) 12(S)-HETE is a major arachidonic acid metabolite in platelets, and (b) platelets contain large amounts of the cell adhesion molecule GpIIb/IIIa, the activation of which is regulated by 12(S)-HETE. Hsp90 was found to be a component of the 12(S)-HETE binding complex in Lewis lung carcinoma cells, and the 50-kDa ligand-binding subunit itself bound 12(S)-HETE with high affinity. Competition experiments showed that 12(R)-HETE, 15-deoxy-Delta12, 14-prostaglandin J2, and 5(S)-HETE had lower affinity for the 50-kDa subunit than 12(S)-HETE. The 12(S)-HETE binding protein appears to be distinct from known members of the steroid hormone receptor superfamily of nuclear receptors.     

Measurement of icosanoids. Report of the Group for Standardization of Methods in Icosanoid Research

This statement from laboratories highly qualified in icosanoid analysis identifies the urgent need for the availability of the following compounds in labeled (deuterium and tritium) and unlabeled form: PGE2 PGF2 alpha PGD2 6-keto-PGF1 alpha Thromboxane B2 9 alpha,20-dihydroxy-11,15-dioxo-2,3- dinorprost -5-enoic acid 9 alpha-hydroxy-11,15-dioxo-2,3,18,19- tetranorprost -5-ene-1,20-dioic acid 15-keto-13,14-dihydro-PGE2 15-keto-13,14-dihydro-PGF2 alpha 5 alpha-7 alpha-dihydroxy-11- ketotetranorprosta -1,16-dioic acid 7 alpha-hydroxy-5,11-diketo- tetranorprosta -1,16-dioic acid 2,3 dinor-thromboxane B2 2,3 dinor-6-keto-PGF1 alpha 2,3 dinor-6,15-diketo 13,14 dihydro-20-carboxyl-PGF1 alpha 2,3 dinor-13,14-dihydro-6,15-diketo-PGF1 alpha LTB4 LTC4 LTD4 LTE4 LTF4 20-OH-LTB4 20-COOH-LTB4 5-HETE 12-HETE 15-HETE omega-OH-12-HETE 5S, 12S-di HETE 5S, 15S-di HETE HHT other hydroxylated polyunsaturated fatty acids and their epoxides.     

Fatty acid modulation of tumor cell adhesion to microvessel endothelium and experimental metastasis.

Tumor cell interaction with the endothelium of the vessel wall is a rate limiting step in metastasis. The fatty acid modulation of this interaction was investigated in low (LM) and high (HM) metastatic B16 amelanotic melanoma (B16a) cells. 12(S)-HETE increased the adhesion of LM cells to endothelium derived from pulmonary microvessels. All other monohydroxy and dihydroxy fatty acids were ineffective. LTB4 induced a modest stimulation but LTC4, LTD4, LTE4 as well as LXA4 and LXB4 were ineffective. The 12(S)-HETE enhanced adhesion of B16a cells was inhibited by pretreatment with 13(S)-HODE but not by 13(R)-, 9(S)-HODE or 13-OXO-ODE. 13(S)-HODE decreased adhesion of HM B16a cells to endothelium. 12(S)-HETE enhanced surface expression of integrin alpha IIb beta 3 and monoclonal antibodies against this integrin but not against alpha 5 beta 1, blocked enhanced but not basal adhesion to endothelium. Intravenous injection of 12(S)-HETE treated LM cells resulted in increased lung colonization (experimental metastasis). This effect was specific for 12(S)-HETE and was inhibited by 13(S)-HODE but not by other HODE's. 12(S)-HETE also enhanced lung colonization by HM cells and 13(S)-HODE decreased lung colonization by HM cells. Our results suggest a highly specific bidirectional modulation of metastatic phenotype and lung colonization by 12(S)-HETE and 13(S)-HODE.     

The identification of hydroxy fatty acids in psoriatic skin

Psoriasis is a common chronic inflammatory and proliferative skin disease characterised by epidermal neutrophil infiltration which may be induced by chemotactic substances in the involved epidermis. Superficial psoriatic scale was shown to contain biologically active amounts of leukotriene B₄ and monohydroxy-eicosatetraenoic acid (HETE)- like material as determined by assay for chemokinetic activity in high performance liquid chromatography (HPLC) fractions of scale extracts. Extracts of scale and chamber fluid from abraded lesional and uninvolved psoriatic skin were purified by HPLC and appropriate fractions were analysed by gas chromatography - mass spectrometry (GC-MS). The following monohydroxy metabolites of arachidonic, linoleic and 11,14-eicosadienoic acids were identified : 15-HETE, 12-HETE, 11-HETE, 9-HETE, 8-HETE, 5-HETE, 13-hydroxy-octadecadienoic acid (13-HODD), 9-HODD and 15-hydroxy-eicosadienoic acid (15-HEDE). The results suggested that 12-HETE, 13-HODD and 9-HODD are the most abundant monohydroxy fatty acids in the psoriatic skin extracts described above. Assays of 13-HODD, 9-HODD and 15-HEDE for chemokinetic activity were negative with concentrations up to 10^?4M. The biological significance of these three compounds in not known, but some of the hydroxylated metabolites of arachidonic acid may, by virtue of their chemotactic properties, be relevant to the pathogenesis of the psoriatic neutrophil infiltrate.     

The priming effect of 12(S)-hydroxyeicosatetraenoic acid on lymphocyte phospholipase D involves specific binding sites.

We have previously shown that 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE)-enrichment primed human peripheral blood mononuclear cells for phospholipase D activation by mitogens. Given that 12(S)-HETE-enriched cells stimulated with concanavalin A released free 12(S)-HETE in the extracellular medium, and that the priming effect of 12(S)-HETE on phospholipase D was suppressed by the non-permeant drug, suramin, we hypothesized an extracellular mechanism for 12(S)-HETE-induced PLD activation. Using [3H]12(S)-HETE as a ligand and a rapid filtration technique, we have pointed out the presence of specific low-affinity 12(S)-HETE binding sites on intact human mononuclear cells and lymphocytes. [3H]12(S)-HETE binding was efficiently displaced by other monohydroxylated and n-3 fatty acids but not by oleate and arachidonate, and was also significantly inhibited by suramin and pertussis toxin. Furthermore, 12(S)-HETE-induced PLD activation was strongly inhibited by pertussis toxin and genistein, but was not PKC-dependent. In addition, 12(S)-HETE also potentiated the ConA-induced tyrosine phosphorylation of a 46-50 kDa protein, which was inhibited by genistein. Collectively, these results suggest that 12(S)-HETE binding sites on human lymphocytes may be coupled to phospholipase D through pertussis toxin sensitive G-proteins and tyrosine kinases.     

Specific incorporation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid into phosphatidylcholine in human endothelial cells

The incorporation of hydroxyeicosatetraenoic acids (HETEs) into cellular lipids was studied in cultures of human umbilical vein endothelial cells. 5-[3H]HETE was incorporated into the phospholipids (8%) and neutral lipids (15.5%). The uptake was at half maximum after 15 min and reached a plateau after 1 h. The incorporation occurred mainly into phosphatidylcholine (6.3%) with minimal uptake into phosphatidylserine and phosphatidylinositol (0.6%) or phosphatidylethanolamine (1.2%). There was no uptake of 12-[3H]HETE, 15-[3H]HETE or [3H]leukotriene B4 into phospholipids. Treatment of the phosphatidylcholine fraction with phospholipase A2 released 64% of the 5-[3H]HETE with 26% remaining in the lysophosphatidylcholine fraction. This indicates that the majority of the 5-HETE was in the sn-2 position. Unlabeled 5-HETE and arachidonic acid inhibited the uptake of 5-[3H]HETE into phosphatidylcholine with an ID50 of 2.5 and 1.25 microM, respectively. Stearic acid and 15-HETE were not effective inhibitors. Histamine, which activates phospholipases, increased the uptake of 5-[3H]HETE into phosphatidylcholine by 3-fold. Both 5-[3H]HETE and 12-[3H]HETE were incorporated into the neutral lipids of the cells. Analysis of the neutral lipid fraction revealed that 5-[3H]HETE was incorporated into mono-, di- and triacylglycerols but not cholesterol esters. Incorporation of 5-HETE into cellular lipids reduced histamine- and arachidonic acid-stimulated synthesis of 6-ketoprostaglandin F1 alpha and prostaglandin E2 in a concentration-related manner. Angiotensin I converting enzyme activity was not changed. Thus, 5-HETE is incorporated specifically into phosphatidylcholine and glycerol esters of human endothelial cells and this incorporation inhibits prostaglandin synthesis in these cells.     

Investigation of non-CB1, non-CB2 WIN55212-2-sensitive G-protein-coupled receptors in the brains of mammals,birds, and amphibians

Abstract

Purpose: Previous studies have found non-CB₁ non-CB₂ G-protein-coupled receptors in rodents that are activated by the aminoalkylindole cannabinoid agonist WIN55212-2. This work obtained evidence for the presence or absence of similar receptors in the brains of other mammals, birds and amphibians.

Materials and methods: Antagonism of the stimulation of [³⁵S]GTPγS binding by WIN55212-2 and CP55940 was assessed in multiple CNS regions of rat and canine, and in whole brain membranes from shrew, pigeon, frog and newt. A bioinformatics approach searched for orthologs of GRP3, GPR6, and GPR12 (closely related to cannabinoid receptors) in the genomes of these or related species. Orthologs were examined for amino acid motifs known to impart functionality to receptors.

Results: In mammals and pigeon, but not amphibians, a significant fraction of the stimulation of [³⁵S]GTPγS binding by WIN55212-2 was not blocked by the CB₁ antagonist SR141716A. BLAST searches found that GPR3 was restricted to mammals. GPR12 orthologs existed in all species, and they shared identical amino acid motifs. GPR6 orthologs existed all species, but with significant departures in the identity of some critical amino acids in bird, more so in amphibian.

Conclusions: The portion of WIN55212-2-stimulated [³⁵S]GTPγS binding that was antagonized by SR141716A was consistent with stimulation via CB₁ receptors, indicating that antagonist-insensitive activity was via a different G-protein coupled receptor. Pharmacological evidence of this receptor was found in the brains of mammals and pigeon, but not frog or newt. Bioinfomatics results implicate GPR6 as a possible candidate for the additional WIN55212-2-sensitive receptor.     

Differential Metabolism of Hydroxyeicosatetraenoic Acid Isomers by Mouse Cerebromicrovascular Endothelium

Hydroxyeicosatetraenoic acid (HETE) derivatives of arachidonic acid are produced in the brain and have been implicated as pathologic mediators in various types of brain injury. To understand better their fate in the brain, particularly in cerebral microvessels, several HETEs were incubated with cultured mouse cerebromicrovascular endothelium for 1, 2, and 4 h, followed by HPLC analysis of medium and cellular lipids. 5(S)-, 8(RS)-, and 9(RS)-HETE were not metabolized by the cells, but were extensively incorporated, unmodified, into cell lipids. On the other hand, 11(RS)-, 12(S)-, and 15(S)-HETE were extensively metabolized and only minimally incorporated into cell lipids. Previously, the major 12-HETE metabolite was identified as 8-hydroxyhexadecatrienoic acid. In the present study, we identified the major 11-HETE metabolite as 7-hydroxyhexadecatrienoic acid and the major 15-HETE metabolite as 11-hydroxyhexadecatrienoic acid. omega-3 compounds, 15(S)- and 12(S)-hydroxyeicosapentaenoic acids (HEPE), were also metabolized to more polar compounds, but to a lesser extent than their tetraenoic acid, omega-6 counterparts. Comparison of 5-, 12-, and 15-HETE enantiomers revealed no differences in metabolism or incorporation between the R and S stereoisomers. These data suggest that many isomers of HETE and HEPE can be incorporated into cell lipids or metabolized by pathways that do not distinguish between enantiomers. These pathways, however, are sensitive to the position or number of double bonds and are selective based on the position of the hydroxyl group.     

Sphingosylphosphorylcholine and lysophosphatidylcholine are ligands for the G protein-coupled receptor GPR4

Sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC) are bioactive lipid molecules involved in numerous biological processes. We have recently identified ovarian cancer G protein-coupled receptor 1 (OGR1) as a specific and high affinity receptor for SPC, and G2A as a receptor with high affinity for LPC, but low affinity for SPC. Among G protein-coupled receptors, GPR4 shares highest sequence homology with OGR1 (51%). In this work, we have identified GPR4 as not only another high affinity receptor for SPC, but also a receptor for LPC, albeit of lower affinity. Both SPC and LPC induce increases in intracellular calcium concentration in GPR4-, but not vector-transfected MCF10A cells. These effects are insensitive to treatment with BN52021, WEB-2170, and WEB-2086 (specific platelet activating factor (PAF) receptor antagonists), suggesting that they are not mediated through an endogenous PAF receptor. SPC and LPC bind to GPR4 in GPR4-transfected CHO cells with K(d)/SPC = 36 nm, and K(d)/LPC = 159 nm, respectively. Competitive binding is elicited only by SPC and LPC. Both SPC and LPC activate GPR4-dependent activation of serum response element reporter and receptor internalization. Swiss 3T3 cells expressing GPR4 respond to both SPC and LPC, but not sphingosine 1-phosphate (S1P), PAF, psychosine (Psy), glucosyl-beta1'1-sphingosine (Glu-Sph), galactosyl-beta1'1-ceramide (Gal-Cer), or lactosyl-beta1'1-ceramide (Lac-Cer) to activate extracellular signal-regulated kinase mitogen-activated protein kinase in a concentration- and time-dependent manner. SPC and LPC stimulate DNA synthesis in GPR4-expressing Swiss 3T3 cells. Both extracellular signal-regulated kinase activation and DNA synthesis stimulated by SPC and LPC are pertussis toxin-sensitive, suggesting the involvement of a G(i)-heterotrimeric G protein. In addition, GPR4 expression confers chemotactic responses to both SPC and LPC in Swiss 3T3 cells. Taken together, our data indicate that GPR4 is a receptor with high affinity to SPC and low affinity to LPC, and that multiple cellular functions can be transduced via this receptor.     

GPR75 receptor mediates 20-HETE-signaling and metastatic features of androgen-insensitive prostate cancer cells

PurposeRecent studies have shown that 20-hydroxyeicosatetraenoic acid (20-HETE) is a key molecule in sustaining androgen-mediated prostate cancer cell survival. Thus, the aim of this study was to determine whether 20-HETE can affect the metastatic potential of androgen-insensitive prostate cancer cells, and the implication of the newly described 20-HETE receptor, GPR75, in mediating these effects.MethodsThe expression of GPR75, protein phosphorylation, actin polymerization and protein distribution were assessed by western blot and/or fluorescence microscopy. Additionally, in vitro assays including epithelial-mesenchymal transition (EMT), metalloproteinase-2 (MMP-2) activity, scratch wound healing, transwell invasion and soft agar colony formation were used to evaluate the effects of 20-HETE agonists/antagonists or GPR75 gene silencing on the aggressive features of PC-3 cells.Results20-HETE (0.1 nM) promoted the acquisition of a mesenchymal phenotype by increasing EMT, the release of MMP-2, cell migration and invasion, actin stress fiber formation and anchorage-independent growth. Also, 20-HETE augmented the expression of HIC-5, the phosphorylation of EGFR, NF-κB, AKT and p-38 and the intracellular redistribution of p-AKT and PKCα. These effects were impaired by GPR75 antagonism and/or silencing. Accordingly, the inhibition of 20-HETE formation with N-hydroxy-N′-(4-n-butyl-2-methylphenyl) formamidine (HET0016) elicited the opposite effects.ConclusionsThe present results show for the first time the involvement of the 20-HETE-GPR75 receptor in the activation of intracellular signaling known to be stimulated in cell malignant transformations leading to the differentiation of PC-3 cells towards a more aggressive phenotype. Targeting the 20-HETE/GPR75 pathway is a promising and novel approach to interfere with prostate tumor cell malignant progression.     

Temporal changes in 12-HETE formation in two models of canine myocardial infarction

Arachidonic acid (AA) metabolism by infarcted canine myocardium was studied and correlated with matched histologic analyses following permanently occluded or reperfused infarction. Histologic analysis of tissues from reperfused infarcts showed a marked acceleration of inflammatory cell invasion and of granulation tissue formation when compared to the occlusive infarct. In the reperfused infarct, polymorphonuclear leukocytes (PMNs) were very prominent at one day after infarction while in the occlusive infarcts the neutrophilic invasion was less intense but more sustained. At one day following reperfused infarction the major arachidonate product, which co-migrated by thin layer chromatography with the mono-hydroxyeicosatetraenoic acids (HETEs), was significantly elevated (254 +/- 49 pmoles/gm wet weight, n = 3) when compared to normal tissue (48 +/- 6 pmoles/gm n = 19). This occurred at a time when the number of PMNs was maximal in the infarcted tissue. Addition of the calcium ionophore A23187 caused a further marked stimulation in HETE production in the one day reperfused infarct but not at the other time points studied. The production of HETE was not significantly different in the infarcted tissue than in the normal tissue at three and seven days following reperfused infarction or at one, three, or seven days after occlusive infarction. The identity of this HETE product was investigated using reverse phase high performance liquid chromatography (RP-HPLC) and gas chromatography-mass spectrometry (GC-MS) and found to be predominantly 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) with a small amount of 15-HETE. Thus the production of 12-HETE parallels the number of neutrophils invading the infarcted area of the heart.     

Novel membrane target proteins for lipoxygenase-derived mono(S)hydroxy fatty acids.

Hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic acids (HODEs) are major bioactive lipids formed via the lipoxygenase oxygenation of arachidonic and linoleic acid, respectively. These metabolites appear to be involved in various cellular actions including cell proliferation, migration and regulation of enzyme activities such as phospholipases and kinases. In view of the diversity of biological effects of these hydroxy fatty acids, it seems likely that multiple mechanisms are involved. Previous reports showed that 15(S)-HETE inhibited the 5-lipoxygenase in rat basophilic leukemia (RBL-1) cell homogenates and established the presence of specific cellular HETE binding sites in these and other cells. The present study used 15(S)-HETE biotin hydrazide and 15(S)-HETE biotin pentyl amide as probes to identify membrane target proteins present in RBL-1 cells that specifically interact with HETEs and HODEs. Two membrane-associated proteins, with apparent molecular weights of 43 and 58 kDa, were identified that specifically interact with these probes and competition experiments indicated that 13(S)-HODE and 15(S)-HETE were the most effective competitors for the hydrazide probe, followed in decreasing effectiveness by 5(S)-HETE, arachidonic acid, 15(R)-HETE, stearic acid and 12(S)-HHT, a cyclooxygenase product. The two proteins were isolated and microsequencing analysis established their identities as actin and the alpha-subunit of mitochondrial ATP synthase, respectively. In vitro binding studies confirmed that purified actin is a potential 15-HETE binding protein. Subcellular cytosolic fractions exhibited fewer protein-probe complexes than membrane fractions. The association of HETEs and HODEs with these cytoskeletal and mitochondrial proteins, respectively, represents a new development in the potential actions of these hydroxy fatty acids.     

Medium-chain fatty acids as ligands for orphan G protein-coupled receptor GPR84

Free fatty acids (FFAs) play important physiological roles in many tissues as an energy source and as signaling molecules in various cellular processes. Elevated levels of circulating FFAs are associated with obesity, dyslipidemia, and diabetes. Here we show that GPR84, a previously orphan G protein-coupled receptor, functions as a receptor for medium-chain FFAs with carbon chain lengths of 9-14. Medium-chain FFAs elicit calcium mobilization, inhibit 3',5'-cyclic AMP production, and stimulate [35S]guanosine 5'-O-(3-thiotriphosphate) binding in a GPR84-dependent manner. The activation of GPR84 by medium-chain FFAs couples primarily to a pertussis toxin-sensitive G(i/o) pathway. In addition, we show that GPR84 is selectively expressed in leukocytes and markedly induced in monocytes/macrophages upon activation by lipopolysaccharide. Furthermore, we demonstrate that medium-chain FFAs amplify lipopolysaccharide-stimulated production of the proinflammatory cytokine interleukin-12 p40 through GPR84. Our results indicate a role for GPR84 in directly linking fatty acid metabolism to immunological regulation.     

Selective orthosteric free fatty acid receptor 2 (FFA2) agonists: identification of the structural and chemical requirements for selective activation of FFA2 versus FFA3

Free fatty acid receptor 2 (FFA2; GPR43) is a G protein-coupled seven-transmembrane receptor for short-chain fatty acids (SCFAs) that is implicated in inflammatory and metabolic disorders. The SCFA propionate has close to optimal ligand efficiency for FFA2 and can hence be considered as highly potent given its size. Propionate, however, does not discriminate between FFA2 and the closely related receptor FFA3 (GPR41). To identify FFA2-selective ligands and understand the molecular basis for FFA2 selectivity, a targeted library of small carboxylic acids was examined using holistic, label-free dynamic mass redistribution technology for primary screening and the receptor-proximal G protein [(35)S]guanosine 5'-(3-O-thio)triphosphate activation, inositol phosphate, and cAMP accumulation assays for hit confirmation. Structure-activity relationship analysis allowed formulation of a general rule to predict selectivity for small carboxylic acids at the orthosteric binding site where ligands with substituted sp(3)-hybridized α-carbons preferentially activate FFA3, whereas ligands with sp(2)- or sp-hybridized α-carbons prefer FFA2. The orthosteric binding mode was verified by site-directed mutagenesis: replacement of orthosteric site arginine residues by alanine in FFA2 prevented ligand binding, and molecular modeling predicted the detailed mode of binding. Based on this, selective mutation of three residues to their non-conserved counterparts in FFA3 was sufficient to transfer FFA3 selectivity to FFA2. Thus, selective activation of FFA2 via the orthosteric site is achievable with rather small ligands, a finding with significant implications for the rational design of therapeutic compounds selectively targeting the SCFA receptors.     

SCFAs induce mouse neutrophil chemotaxis through the GPR43 receptor

Short chain fatty acids (SCFAs) have recently attracted attention as potential mediators of the effects of gut microbiota on intestinal inflammation. Some of these effects have been suggested to occur through the direct actions of SCFAs on the GPR43 receptor in neutrophils, though the precise role of this receptor in neutrophil activation is still unclear. We show that mouse bone marrow derived neutrophils (BMNs) can chemotax effectively through polycarbonate filters towards a source of acetate, propionate or butyrate. Moreover, we show that BMNs move with good speed and directionality towards a source of propionate in an EZ-Taxiscan chamber coated with fibrinogen. These effects of SCFAs were mimicked by low concentrations of the synthetic GPR43 agonist phenylacetamide-1 and were abolished in GPR43(-/-) BMNs. SCFAs and phenylacetamide-1 also elicited GPR43-dependent activation of PKB, p38 and ERK and these responses were sensitive to pertussis toxin, indicating a role for Gi proteins. Phenylacetamide-1 also elicited rapid and transient activation of Rac1/2 GTPases and phosphorylation of ribosomal protein S6. Genetic and pharmacological intervention identified important roles for PI3Kγ, Rac2, p38 and ERK, but not mTOR, in GPR43-dependent chemotaxis. These results identify GPR43 as a bona fide chemotactic receptor for neutrophils in vitro and start to define important elements in its signal transduction pathways.     

Sphingosine-1-phosphate is a high-affinity ligand for the G protein-coupled receptor GPR6 from mouse and induces intracellular Ca2+ release by activating the sphingosine-kinase pathway

We identified and cloned the mouse orthologue of human GPR6 as a new member of the lysophospholipid-receptor family. Sphingosine-1-phosphate (S1P) activated GPR6, transiently expressed in frog oocytes or in Chinese hamster ovary (CHO) cells, with high specificity and nanomolar affinity. The GPR6 gene was found to be located on chromosome 10B1 and a single exon coded for the entire open-reading frame. Signal transduction of S1P was inhibited by pertussis toxin, suggesting a coupling of GPR6 to an inhibitory G protein. In CHO cells transfected with GPR6, the sphingosine-kinase pathway mediated Ca(2+) mobilization from internal stores. Apoptotic cell death was induced by serum deprivation or H(2)O(2) treatment and was prevented by S1P in GPR6-, but not in vector-transfected CHO cells. The antiapoptotic effect of S1P required activation of sphingosine kinase and was accompanied by an increase in MAP-kinase phosphorylation.     

Temporal changes in 12-HETE formation in two models of canine myocardial infarction

Arachidonic acid (AA) metabolism by infarcted canine myocardium was studied and correlated with matched histologic analyses following permanently occluded or reperfused infarction. Histologic analysis of tissues from reperfused infarcts showed a marked acceleration of inflammatory cell invasion and of granulation tissue formation when compared to the occlusive infarct. In the reperfused infarct, polymorphonuclear leukocytes (PMNs) were very prominent at one day after infarction while in the occlusive infarcts the neutrophilic invasion was less intense but more sustained. At one day following reperfused infarction the major arachidonate product, which co-migrated by thin layer chromatography with the mono-hydroxyeicosatetraenoic acids (HETEs), was significantly elevated (254 ± 49 pmoles/gm wet weight, n=3) when compared to normal tissue (48 ± 6 pmoles/gm n=19). This occured at a time when the number of PMNs was maximal in the infarcted tissue. Addition of the calcium ionophore A₂₃₁₈₇ caused a further marked stimulation in HETE production in the one day reperfused infarct but not at the other time points studied. The production of HETE was not significantly different in the infarcted tissue than in the normal tissue at three and seven days following reperfused infarction or at one, three, or seven days after occlusive infarction. The identity of this HETE product was investigated using reverse phase high performance liquid chromatography (RP-HPLC) and gas chromatography-mass spectrometry (GC-MS) and found to be predominantly 12 - hydroxy - 5,8,10,14 - eicosatetraenoic acid (12-HETE) with a small amount of 15-HETE. Thus the production of 12-HETE parallels the number of neutrophils invading the infarcted area of the heart.