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J R Falck

Publications and source records attributed to J R Falck.

At least 145 records · Page 8Linked to original sources
PubMed1997

Cytochrome P450-derived renal HETEs: storage and release.

We have established an assay based on gas chromatography-mass spectrometry to profile and quantitate endogenous cytochrome P450 monooxygenase (P450)-hydroxyeicosatetraenoic acids (HETEs) exiting the isolated perfused rabbit kidney in response to hormonal stimulation. In response to angiotensin II (Ang II) P450-derived HETEs (16-, 17-, 18-, 19- and 20-) are released from the isolated Kreb's perfused rabbit kidney. Ang II produced a several-fold increase in the levels of P450-HETEs above basal levels in both urinary (such as for 20-HETE from 0.93 +/- 0.7 to 2.31 +/- 0.9 ng/min) and venous (from 0.1 +/- 0.05 to 0.3 +/- 0.05 ng/min) effluents. However, inhibition of P450, which reduced basal release, did not prevent Ang II-induced release of P450-AA products from the rabbit kidney; for example, urinary 20-HETE in the presence of 17-ODYA (1 microM) was undetectable and increased to 0.93 +/- 0.4 ng/min with Ang II and venous 20-HETE increased from 0.06 +/- 0.03 to 0.24 +/- 0.07 ng/min. Similar results were obtained with clotrimazole (1 microM). As 16-, 18-, 19- and 20-HETEs are vasodilators in the rabbit kidney and 16- and 17-HETEs inhibit proximal tubular ATPase activity, we investigated their possible sites of esterification. Cortical and medullary lipids were extracted, separated by HPLC and P450-HETEs quantitated following alkaline hydrolysis. The P450-HETEs were incorporated into both neutral lipids (NL) and phospholipids [phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and phosphatidylcholine (PC)]. However, the assignment of a HETE to a specific phospholipid pool must be regarded as tentative as the appropriate standards containing P450-HETEs in the Sn-2 position (such as 20-HETE-PF., 20-HETE-PC, etc.) were not available. Esterified HETEs were found in larger quantities in the cortex as compared to the medulla (34.40 +/- 1.12 versus 22.76 +/- 0.53 ng/g). The PI fraction in the cortex yielded the largest quantity of HETEs and the PC fraction the lowest. In the medulla, the largest quantities of esterified HETEs were found in neutral lipids and only slightly lesser amounts in PE and PI. Esterified 18-HETE was localized only to the NI fraction. This fraction also contained the other HETEs, 19- and 20-HETE being the most abundant. Notably only 16- and 17-HETE were present in PF, whereas, 19- and 20-HETE were also present in PI, PS and PC. Thus, P450-HETEs, like EETs are stored in the kidney and are, presumably, subject to release by peptide activation of acylhydrolases.

Animals↗
PubMed1997

Inhibition of 20-HETE production contributes to the vascular responses to nitric oxide.

Nitric oxide (NO) inhibits a variety of heme-containing enzymes, including NO synthase and cytochrome P4501A1 and 2B1. The present study examined whether NO inhibits the production of 20-hydroxyeicosatetraenoic acid (20-HETE) by cytochrome P4504A enzymes and whether blockade of the production of this substance contributes to the vascular effects of NO. Sodium nitroprusside (SNP; 10(-5), 10(-4), and 10(-3) mol/L) reduced the production of 20-HETE by renal microsomes incubated with arachidonic acid to 71 +/- 5%, 29 +/- 4%, and 4 +/- 2% of control, respectively (n = 5). Similar results were obtained with the use of 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) (n = 3). To determine whether inhibition of 20-HETE contributes to the vasodilatory effects of NO, the effects of dibromo-dodecenyl-methylsulfimide (DDMS), a selective inhibitor of the formation of 20-HETE, on the response to SNP (10(-7) to 10(-3) mol/L) were examined in rat renal arterioles preconstricted with phenylephrine (n = 5). SNP increased vascular diameter in a concentration-dependent manner to 82 +/- 4% of control. After DDMS (25 mumol/L), SNP (10(-3) mol/L) increased vascular diameter by only 17 +/- 3%. The effects of DDMS on the mean arterial pressure (MAP) and renal blood flow (RBF) responses to infusion of an NO donor and a synthase inhibitor were also examined in thiobutabarbital-anesthetized, Sprague-Dawley rats. Infusion of MAHMA NONOate at 1, 3, 5, and 10 nmol/min reduced MAP by 16 +/- 2, 30 +/- 3, 40 +/- 5, and 48 +/- 5 mm Hg and lowered renal vascular resistance (RVR) by 15 +/- 3%, 26 +/- 2%, 30 +/- 3%, and 34 +/- 4% of control. After DDMS (10 mg/kg, n = 7 rats), the MAP and RVR responses to 1-hexamine, 6-(2-hydroxy-1-methyl-2-nitrohydrazino)N-methyl (MAHMA NONOate) averaged only 20% of those seen during control. In other experiments, MAP increased by 32 +/- 4% and RBF fell to 56 +/- 5% of control after administration of N-nitro-L-arginine (L-NArg) (10 mg/kg IV). After DDMS (10 mg/kg, n = 7 rats), MAP increased by only 19 +/- 4% and RBF fell by only 7 +/- 4% after L-NArg. These results indicate that NO inhibits cytochrome P4504A enzymes and that inhibition of the production of 20-HETE contributes to the vasodilatory effects of NO.

Animals↗
PubMed1996

Acylation of glucosaminyl phosphatidylinositol revisited. Palmitoyl-CoA dependent palmitoylation of the inositol residue of a synthetic dioctanoyl glucosaminyl phosphatidylinositol by hamster membranes permits efficient mannosylation of the glucosamine residue.

Two critical steps in the assembly of yeast and mammalian glycosylphosphatidylinositol (GPI) anchor precursors are palmitoylation of the inositol residue and mannosylation of the glucosamine residue of the glucosaminyl phosphatidylinositol (GlcNalpha-PI) intermediate. Palmitoylation has been reported to be acyl-CoA dependent in yeast membranes (Costello, L. C., and Orlean, P. (1992) J. Biol. Chem. 267, 8599-8603) but strictly acyl-CoA independent in rodent membranes (Stevens, V. L., and Zhang, H. (1994) J. Biol. Chem. 269, 31397-31403), and thus poorly conserved. In addition, it was suggested that acylation must precede mannosylation in both yeast (Costello, L. C., and Orlean, P. (1992) J. Biol. Chem. 276, 8599-8603) and rodent (Urakaze, M., Kamitani, T., DeGasperi, R., Sugiyama, E., Chang, H.-M., Warren, C. D., and Yeh, E. T. H. (1992) J. Biol. Chem. 267, 6459-6462) cells because GlcNalpha-acyl-PI accumulates in vivo when mannosylation is blocked. However, GlcNalpha-acyl-PI accumulation would also be expected if mannosylation and acylation were independent of each other. These issues were addressed by the use of a synthetic dioctanoyl GlcNalpha-PI analogue (GlcNalpha-PI(C8)) as an in vitro substrate for GPI-synthesizing enzymes in Chinese hamster ovary cell membranes. GlcNalpha-PI(C8) was acylated in an manner requiring acyl-CoA. Thus, the process involving acyl-CoA reported for yeast has been conserved in mammals. Furthermore, both GlcNalpha-PI(C8) and GlcNalpha-acyl-PI(C8) could be mannosylated in vitro, but mannosylation of the latter was significantly more efficient. This provides direct support for the earlier suggestion that acylation precedes mannosylation in rodents cells. A similar result was also observed with the Saccharomyces cerevisiae mannosyltransferase. In contrast, it has been reported that mannosylation of endogenous GlcNalpha-PI by Trypansoma brucei membranes occurs without prior acylation. The same result was obtained with GlcNalpha-PI(C8), confirming that the mannosyltransferase of trypanosomes is divergent from those in yeasts and rodents.

Acyl Coenzyme A↗
PubMed1996

An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha.

Cholesterol and its oxysterol congeners are important constituents of cell membranes and function as intermediates in several crucial biosynthetic pathways. These compounds autoregulate their metabolic fate by end-product repression and activation of downstream catabolism. Although end-product repression by oxysterols is relatively well understood, the mechanism by which these compounds act as positive transcription signalling molecules is unknown. Here we identify a specific group of endogenous oxysterols that activate transcription through the nuclear receptor LXR alpha. Transactivation of LXR alpha by oxysterols occurs at concentrations at which these compounds exist in vivo. The most potent activators also serve as intermediary substrates in the rate-limiting steps of three important metabolic pathways: steroid hormone biosynthesis, bile acid synthesis, and conversion of lanosterol to cholesterol. Our results demonstrate the existence of a nuclear receptor signalling pathway for oxysterols and suggest that LXR alpha may be important as a sensor of cholesterol metabolites.

Animals↗
PubMed1996

The highly stereoselective oxidation of polyunsaturated fatty acids by cytochrome P450BM-3.

Cytochrome P450BM-3 catalyzes NADPH-dependent metabolism of arachidonic acid to nearly enantiomerically pure 18(R)-hydroxyeicosatetraenoic acid and 14(S), 15(R)-epoxyeicosatrienoic acid (80 and 20% of total products, respectively). P450BM-3 oxidizes arachidonic acid with a rate of 3.2 +/- 0.4 micromol/min/nmol at 30 degrees C, the fastest ever reported for an NADPH-dependent, P450-catalyzed reaction. Fatty acid, oxygen, and NADPH are utilized in an approximately 1:1:1 molar ratio, demonstrating efficient coupling of electron transport to monooxygenation. Eicosapentaenoic and eicosatrienoic acids, two arachidonic acid analogs that differ in the properties of the C-15-C-18 carbons, are also actively metabolized by P450BM-3 (1.4 +/- 0.2 and 2.9 +/- 0.1 micromol/min/nmol at 30 degrees C, respectively). While the 17,18-olefinic bond of eicosapentaenoic acid is epoxidized with nearly absolute regio- and stereochemical selectivity to 17(S),18(R)-epoxyeicosatetraenoic acid (>/=99% of total products, 97% optical purity), P450BM-3 is only moderately regioselective during hydroxylation of the eicosatrienoic acid omega-1, omega-2, and omega-3 sp3 carbons, with 17-, 18-, and 19-hydroxyeicosatrienoic acid formed in a ratio of 2.4:2.2:1, respectively. Based on the above and on a model of arachidonic acid-bound P450BM-3, we propose: 1) the formation by P450BM-3 of a single oxidant species capable of olefinic bond epoxidation and sp3 carbon hydroxylation and 2) that product chemistry and, thus, catalytic outcome are critically dependent on active site spatial coordinates responsible for substrate binding and productive orientation between heme-bound active oxygen and acceptor carbon bond(s).

8,11,14-Eicosatrienoic Acid↗
PubMed1996

A functional phosphatidylinositol 3,4,5-trisphosphate/phosphoinositide binding domain in the clathrin adaptor AP-2 alpha subunit. Implications for the endocytic pathway.

Clathrin-coated pits are sites of concentration of ligand-bound signaling receptors. Several such receptors are known to recruit, bind, and activate the heterodimeric phosphatidylinositol-3-kinase, resulting in the generation of phosphatidylinositol 3,4, 5-trisphosphate. We report here that dioctanoyl-phosphatidylinositol-3,4,5-P3 binds specifically and saturably to soluble AP-2 and with greater affinity to AP-2 within assembled coat structures. Soluble -myo-inositol hexakisphosphate shows converse behavior. Binding to bovine brain clathrin-coated vesicles is evident only after detergent extraction. These observations and evidence for recognition of the diacylglyceryl backbone as well as the inositol phosphate headgroup are consistent with AP-2 interaction with membrane phosphoinositides in coated vesicles and with soluble inositol phosphates in cytoplasm. A discrete binding domain is identified near the N terminus of the AP-2 alpha subunit, and an expressed fusion protein containing this sequence exhibits specific, high affinity binding that is virtually identical to the parent protein. This region of the AP-2 alpha sequence also shows the greatest conservation between a Caenorhabditis elegans homolog and mammalian alpha, consistent with a function in recognition of an evolutionarily unchanging low molecular weight ligand. Binding of phosphatidylinositol 3,4, 5-trisphosphate to AP-2 inhibits the protein's clathrin binding and assembly activities. These findings are discussed in the context of the potential roles of phosphoinositides and AP-2 in the internalization and trafficking of cell surface receptors.

Adaptor Proteins, Vesicular Transport↗
PubMed1996

Metabolism of arachidonic acid by canine polymorphonuclear leukocytes synthesis of lipoxygenase and omega-oxidized metabolites.

Both polymorphonuclear (PMN) leukocytes and metabolites of arachidonic acid, especially lipoxygenase products, have been reported to contribute to myocardial damage after coronary artery occlusion and reperfusion. While canine models of myocardial ischemia were used in many of these studies, very little is known about arachidonic acid metabolism by canine PMNs. Moreover, it is unclear whether arachidonic acid metabolites released by canine PMNs affect vascular tone. Therefore, we characterized arachidonic acid metabolism by canine PMNs and determined the effect of these metabolites on vascular tone of isolated canine coronary arteries. Suspensions of canine PMNs were incubated with [14C]arachidonic acid and the calcium ionophore A23187. The incubation media was extracted, and the metabolites resolved by HPLC. 20-Hydroxy-leukotriene B4 (LTB4), 12,20-dihydroxyeicosatetraenoic acid (diHETE), LTB4, 12-hydroxyheptadeclatrienoic acid (HHT), and 12-(S)-hydroxyeicosatetraenoic acid (HETE) were isolated, and their structures confirmed by gas chromatography/mass spectrometry. There was also evidence for the formation of 20-HETE, thromboxane B2 (TXB2), 5-HETE, and several isomers of LTB4. None of the arachidonic acid metabolites that were isolated from incubates of canine PMNs augmented vascular tone, but material migrating with 12,20-diHETE relaxed canine coronary arteries. Authentic 12(S),20-diHETE also produced a concentration-related relaxation of canine coronary artery. 12(R), 20-diHETE was inactive. 20-HETE inhibited A23187-induced PMN aggregation. Thus, arachidonic acid is metabolized in canine PMNs through the cyclooxygenase, lipoxygenases and cytochrome P-450 pathways. Whether these metabolites contribute to myocardial injury remains to be determined.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1996

Molecular cloning and expression of CYP2J2, a human cytochrome P450 arachidonic acid epoxygenase highly expressed in heart.

A cDNA encoding a human cytochrome P450 arachidonic acid epoxygenase was isolated from a human liver cDNA library. Sequence analysis revealed that this 1,876-base pair cDNA contained an open reading frame and encoded a new 502-amino acid protein designated CYP2J2. Blot hybridization analysis of RNA prepared from human tissues revealed that CYP2J2 was highly expressed in the heart. Recombinant CYP2J2 protein was prepared using the baculovirus expression system and purified to near electrophoretic homogeneity. The enzyme metabolized arachidonic acid predominantly via olefin epoxidation to all four regioisomeric cis-epoxyeicosatrienoic acids (catalytic turnover 65 pmol of product formed/nmol of cytochrome P450/min at 30 degrees C). Epoxidation of arachidonic acid by CYP2J2 at the 14,15-olefin was highly enantioselective for (14R, 15S)-epoxyeicosatrienoic acid (76% optical purity). Immunoblotting of microsomal fractions prepared from human tissues using a polyclonal antibody raised against the recombinant hemoprotein confirmed primary expression of CYP2J2 protein in human heart. The in vivo significance of CYP2J2 was suggested by documenting the presence of epoxyeicosatrienoic acids in the human heart using gas chromatography/mass spectroscopy. Importantly, the chirality of CYP2J2 products matched that of the epoxyeicosatrienoic acid enantiomers present, in vivo, in human heart. We propose that CYP2J2 is one of the enzymes responsible for epoxidation of endogenous arachidonic acid pools in human heart and that epoxyeicosatrienoic acids may, therefore, play important functional roles in cardiac physiology.

Amino Acid Sequence↗
PubMed1996

Effect of angiotensin II on the apical K+ channel in the thick ascending limb of the rat kidney.

We have used the patch-clamp technique to study the effect of angiotensin II (AII) on the activity of the apical 70 pS K+ channel and used Na(+)-sensitive fluorescent dye (SBFI) to investigate the effect of AII on intracellular Na+ concentration (Na+i) in the thick ascending limb (TAL) of the rat kidney. Addition of 50 pM AII reversibly reduced NPo, a product of channel open probability (Po) and channel number (N), to 40% of the control value and reduced the Na+i by 26%. The AII (50 pM)-induced decrease in channel activity defined by NPo was partially reversed by addition of 5 microM 17-octadecynoic acid (17-ODYA), an agent which blocks the cytochrome P450 monooxygenase. The notion that P450 metabolites of arachidonic acid (AA) may mediate the inhibitory effect of AII was further suggested by experiments in which addition of 10 nM of 20-hydroxyeicosatetraenoic acid (20-HETE) blocked the channel activity in cell-attached patches in the presence of 17-ODYA. We have used gas chromatography mass spectrometry (GC/MS) to measure the production of 20-HETE, a major AA metabolite of the P450-dependent pathway in the TAL of the rat. Addition of 50 pM AII increased the production of 20-HETE to 260% of the control value, indicating that 20-HETE may be involved in mediating the effect of AII (50 pM). In contrast to the inhibitory effect of 50 pM AII, addition of 50-100 nM AII increased the channel activity to 270% of the control value and elevated the Na+i by 45%. The effect of AII on the activity of the 70 pS K+ channel was also observed in the presence of 5 microM 17-ODYA and 5 microM calphostin C, an inhibitor of protein kinase C. However, addition of 100 microM NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, abolished completely the AII (50-100 nM)-induced increase in channel activity and addition of an exogenous nitric oxide (NO) donor, S-nitroso-N-acetyl-penicillamine (SNAP), increased channel activity in the presence of L-NAME. These data suggest that the stimulatory effect of AII is mediated by NO. We conclude that AII has dual effects on the activity of the apical 70 pS K+ channel. The inhibitory effect of AII is mediated by P450-dependent metabolites whereas the stimulatory effect may be mediated via NO.

Angiotensin II↗
PubMed1996

Cytochrome P450, the arachidonic acid cascade, and hypertension: new vistas for an old enzyme system.

As a participant of the endogenous arachidonic acid metabolic cascade, microsomal cytochrome P450 metabolizes the fatty acid to biologically active hydroxyeicosatetraenoic and epoxyeicosatrienoic acids. Studies from several laboratories have documented the powerful vasoactive properties of these P450-derived eicosanoids. Associated changes in cell membrane ion permeability and fluxes may provide the molecular basis underlining their vasoactivity. Furthermore, a role for the P450 arachidonic acid monooxygenase in renal physiology and pathophysiology has been suggested by: 1) an association between the activities of the arachidonic acid omega/omega-1 oxygenase and the development of hypertension in spontaneously hypertensive rats, and 2) a relationship between acquired or inherited abnormalities in the renal epoxygenase activities and/or regulation and salt-sensitive hypertension in Dahl rats. These studies provide significant evidence to indicate that microsomal P450, in addition to its recognized traditional toxicological and pharmacological roles, may also play an important physiological role in the control of tissue and body homeostasis.

Animals↗
PubMed1996

Formation and actions of 20-hydroxyeicosatetraenoic acid in rat renal arterioles.

The present study examined whether preglomerular arterioles of the rat produce 20-hydroxyeicosatetraenoic acid (20-HETE) and whether 20-HETE is vasoactive on these vessels. Raf preglomerular arterioles produced 20-HETE (4.8 +/- 1.0 pmol.min-1.mg-1, n = 7) and, to a lesser extent, 14-, 15-, 11-, and 12-dihydroxyeicosatetraenoic acid, 6-ketoprostaglandin F/alpha and prostaglandin E2 when incubated with [14C]larachidonic acid. The results of immunoblotting and reverse-transcription polymerase chain reaction experiments indicate that these vessels express mRNA and protein for a P-450 4A2 enzyme. With the use of a rat juxtamedullary nephron microvascular preparation perfused in vitro with a cell-free media, addition of 20-HETE (1 nM-1 microM) to the bath reduced the diameter of proximal and distal portions of the efferent arterioles. At a concentration of 1 microM, the diameter of the proximal and distal portions of the afferent arteriole fell by 14 +/- 1 and 16 +/- 3% after 20-HETE. The response to 20-HETE (1 microM) was not altered by blockade of cyclooxygenase, lipoxygenase, and p-450 pathways. Blockade of the large-conductance Ca(2+)-activated K+ channel with tetraethylammonium (1 mM) reduced the diameter of afferent arterioles by 10% and blocked the vasoconstrictor response to 20-HETE (1 microM). These results indicate that 20-HETE is an endogenous constrictor of preglomerular arterioles and suggest a role for the P-450 4A2 enzyme in the regulation of renal vascular tone.

Animals↗
PubMed1996

20-HETE is an endogenous inhibitor of the large-conductance Ca(2+)-activated K+ channel in renal arterioles.

The present study examined the effects of 20-hydroxyeicosatetraenoic acid (20-HETE) and 17-octadecynoic acid (17-ODYA), an inhibitor of the metabolism of arachidonic acid by P-450, on K(+)-channel activity in vascular smooth muscle cells (VSM) isolated from renal arterioles of the rat. Two types of K+ channels were characterized using inside-out excised membrane patches. One channel exhibited a large conductance (250.3 +/- 5 pS), was activated by membrane depolarization and elevations in cytoplasmic Ca2+ concentration, and was blocked by low concentrations (< 1 mM) of tetraethylammonium (TEA). The other K+ channel exhibited an intermediate conductance (46.3 +/- pS), was activated by membrane depolarization but not by changes in intracellular Ca2+ concentration, and was blocked by 4-aminopyridine (5 mM). Addition of 20-HETE to the bath (1-100 nM), reduced the frequency of opening of the large-conductance Ca(2+)-activated K+ channel recorded using cell-attached patches on VSM. It had no effect on the intermediate-conductance K+ channel: 17-ODYA (1 microM) increased the activity of the large-conductance Ca(2+)-activated K+ channel, and this effect was reversed by 20-HETE (10 nM). 20-HETE (1-1000 nM) reduced the diameter of isolated perfused small renal arteries of the rat by approximately 15% TEA (1 mM) blocked the vasoconstrictor response to 20-HETE (100 nM). These studies suggest that 20-HETE is an endogenously formed vasoconstrictor that acts in part by inhibiting the opening of the large-conductance Ca(2+)-activated K+ channel in renal arteriolar VSM.

Animals↗
PubMed1996

Cytochrome P-450-dependent HETEs: profile of biological activity and stimulation by vasoactive peptides.

The cytochrome P-450 pathway is capable of metabolizing arachidonic acid to omega- and subterminal hydroxylase metabolites, 16-, 17-, 18-, 19-, and 20-hydroxyeicosatetraenoic acids (P-450 HETEs). We have quantitated, by gas chromatography-mass spectrometry (GC/MS), endogenous HETEs exiting the rabbit isolated perfused kidney elicited by hormonal stimulation. Kidneys were perfused with Krebs-Henseleit solution containing indomethacin (2.8 microM) to prevent further metabolism of HETEs by cyclooxygenase. Phenylephrine (2-3 microM) was added to the perfusate to raise perfusion pressure to approximately 80 mmHg. Angiotensin II (ANG II), arginine vasopressin (AVP), and bradykinin (BK) were injected into the renal artery and perfusates collected throughout the vasoactive response. After addition of an internal standard, deuterated 19-HETE, perfusates were extracted and purified and P-450 HETEs were derivatized for GC/MS analysis. Under basal conditions, 16-, 18-, 19-, and 20-HETEs were released (range: 50-270 pg/ml), 19-HETE being the highest and fivefold greater than 16-HETE, the lowest. Injection of 50 ng ANG II increased by two- to sixfold P-450 HETE release associated with an increase of 40 +/- 11 mmHg in perfusion pressure. An equipressor dose of AVP (50 ng) did not release P-450 HETEs nor did a 5-micrograms dose of the vasodilator peptide BK, which decreased perfusion pressure by 22 +/- 6 mmHg. Authentic 19- and 20-HETE isomers resulted in dose-dependent dilation, as did 18(R)- and 16(R)-HETEs, whereas their enantiomers and 17-HETE isomers were without effect on perfusion pressure. The vasodilator effects of 18(R)- and 16(R)-HETEs, like 20- and 19-HETEs, were inhibited by indomethacin. Furthermore, P-450 HETEs exhibited both regio- and stereoselective inhibition of proximal tubule adenosine triphosphatase (ATPase) activity. The (S) enantiomers of 16- and 17-HETE potently inhibited activity, whereas their (R) isomers and other P-450 HETEs had negligible effects on ATPase activity. The quantity of HETEs released from the kidney, either under basal conditions or when stimulated by ANG II, and their biological profile suggest that subterminal HETEs may participate in renal mechanisms affecting vasomotion and tubular transport.

Adenosine Triphosphatases↗
PubMed1996

Stereospecific effects of epoxyeicosatrienoic acids on renal vascular tone and K(+)-channel activity.

The present study examined the effects of 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) on the diameter of small renal arteries of the rat and assessed their action on K(+)-channel activity in vascular smooth muscle (VSM) cells isolated from these vessels. The R,S-isomer of 11,12-EET (1, 10, and 100 nM) increased the diameter of small renal arteries preconstricted with phenylephrine; however, the S,R-isomer was inactive. Both the R,S- and S,R-isomers of 14,15-EET had little effect on the diameter of these vessels even at a high concentration (100 nM). The vasodilator effect of 11(R),12(S)-EET was attenuated by tetraethylammonium (TEA, 1 mM) and iberiotoxin (100 nM), selective inhibitors of the large-conductance Ca(2+)-activated K+ (KCa) channel. In contrast, apamin (100 nM) and 4-aminopyridine (2 mM), which are inhibitors of other types of K+ channels, had no effect on the vasodilatory effect of 11,12-EET. In patch-clamp experiments, 100 nM racemic 11,12-EET increased outward K+ currents in VSM cells. Addition of the R,S-isomer or racemic 11,12-EET (1-100 nM), but not the S,R-isomer, increased the activity of KCa channel recorded from renal VSM cells with cell-attached patches. However, racemic EET had no effect on this channel when added to the internal (inside-out) or external (outside-out) face of excised membrane patches. These results suggest that 11,12-EET is a potent dilator of small renal arteries and that the R,S-isomer is the active enantiomer. The vasodilator effect of 11,12-EET appears to involve activation of KCa channel.

8,11,14-Eicosatrienoic Acid↗
PubMed1996

Actions of epoxygenase metabolites on the preglomerular vasculature.

Epoxygenase metabolites of arachidonic acid are produced by the kidney and have been implicated in the control of renal blood flow. This study examined the preglomerular actions of various epoxyeicosatrienoic acids (EET). By use of the in vitro blood-perfused juxtamedullary nephron preparation, interlobular and afferent arteriolar diameter responses to 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET were determined. Diameters of interlobular and afferent arterioles preconstricted with 0.5 microM norepinephrine averaged 24 +/- 1 microns (N = 27) and 17 +/- 1 microns (N = 32), respectively, at a renal perfusion pressure of 100 mm Hg. Superfusion with 0.01 to 100 nM 11,12-EET caused graded increases in diameters of the interlobular and afferent arterioles. At a dose of 100 nM, 11,12-EET increased the diameters of the interlobular and afferent arterioles by 18 +/- 2% (N = 10) and 20 +/- 3% (N = 9), respectively. The vasodilatory response to 11,12-EET was stereoselective because 11,12(R,S)-EET but not 11,12(S,R)-EET increased the diameters of the interlobular and afferent arterioles. 14,15-EET had a much smaller effect and increased the diameters of the these vessels by 10%; 8,9-EET did not significantly affect vascular diameters. In contrast, 5,6-EET constricted the interlobular and afferent arterioles by 16 +/- 3% (N = 6) and 21 +/- 3% (N = 7), respectively. The corresponding diols, 5,6-DIHETE and 11,12-DIHETE, had no effect on diameters of the interlobular and afferent arterioles at concentrations up to 1 microM. The vasodilatory response to 11,12-EET was not affected by removal of the endothelium or by inhibition of cyclooxygenase with indomethacin. In contrast, the vasoconstrictor response to 5,6-EET was abolished by both removal of the endothelium or cyclooxygenase inhibition. The thromboxane/ enderoperoxide receptor inhibitor, SQ 29,548, resulted in a 60% attenuation of the afferent arteriolar vasconstriction to 5,6-EET. These results indicate that the preglomerular vasoconstriction to 5,6-EET is cyclooxygenase dependent and requires an intact endothelium, whereas the vasodilation to 11,12-EET is stereoselective and is the result of direct action of the epoxide on the preglomerular vascular smooth muscle.

8,11,14-Eicosatrienoic Acid↗
PubMed1996

Effects of metabolites of leukotriene B4 on human neutrophil migration and cytosolic calcium levels.

Leukotriene B4 (LTB4) is metabolized by beta-oxidation, omega-oxidation and the 12-hydroxyeicosanoid dehydrogenase/delta 10-reductase pathway. We have investigated the effects of metabolites formed by the latter pathway on calcium mobilization and migration in human neutrophils and have compared their potencies with those of other LTB4 derivatives. 12-Oxo-LTB4 and 10,11-dihydro-LTB4 were 60 to 100 times less potent than LTB4 in stimulating neutrophils, whereas 10,11-dihydro-12-oxo-LTB4 and 10,11-dihydro-12-epi-LTB4 exhibited still lower potencies. The 6-trans isomers of 12-oxo-LTB4 and 10,11-dihydro-12-oxo-LTB4 were much less potent than the 6-cis compounds. The EC50 values for biologically and chemically (6-cis) synthesized 12-oxo-LTB4 were similar, indicating that the 6,7-double bond is retained in the cis configuration in the biologically formed compound. Methylation of LTB4 markedly reduced its effect on cytosolic calcium levels, whereas addition of a 3-hydroxyl group had a much more modest effect. Modifications of the omega end of the molecule also resulted in lower potencies for calcium mobilization. Nearly all of the compounds tested desensitized neutrophils to LTB4-induced calcium mobilization, which suggests that their effects were mediated by receptors for the latter compound. However, modifications in the carboxyl end of the molecule had smaller effects on desensitization than on calcium mobilization, whereas the reverse was true for modifications in the omega end of the molecule. This suggests that the structural requirements for agonist-induced desensitization to LTB4 may differ to some extent from the requirements for calcium mobilization.

Calcium↗
PubMed1995

Phosphorylation of the platelet p47 phosphoprotein is mediated by the lipid products of phosphoinositide 3-kinase.

Platelet stimulation by thrombin or the thrombin receptor activating peptide (TRAP) results in the activation of phosphoinositide 3-kinase and the production of the novel polyphosphoinositides phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3). We have shown previously that these lipids activate calcium-independent protein kinase C (PKC) isoforms in vitro (Toker, A., Meyer, M., Reddy, K. K., Falck, J. R., Aneja, R., Aneja, S., Parra, A., Burns, D. J., Ballas, L. M. and Cantley, L. C. (1994) J. Biol. Chem. 269, 32358-32367). Activation of platelet PKC in response to TRAP is detected by the phosphorylation of the major PKC substrate in platelets, the p47 phosphoprotein, also known as pleckstrin. Here we provide evidence for two phases of pleckstrin phosphorylation in response to TRAP. A rapid phase of pleckstrin phosphorylation (< 1 min) precedes the peak of PtdIns-3,4-P2 production and is unaffected by concentrations of wortmannin (10-100 nM) that block production of this lipid. However prolonged phosphorylation of pleckstrin (> 2 min) is inhibited by wortmannin concentrations that block PtdIns-3,4-P2 production. Phorbol ester-mediated pleckstrin phosphorylation was not affected by wortmannin and wortmannin had no effect on purified platelet PKC activity. Phosphorylation of pleckstrin could be induced using permeabilized platelets supplied with exogenous gamma-32P[ATP] and synthetic dipalmitoyl PtdIns-3,4,5-P3 and dipalmitoyl PtdIns-3,4-P2 micelles, but not with dipalmitoyl phosphatidylinositol 3-phosphate or phosphatidylinositol 4,5-bisphosphate. These results suggest two modes of stimulating pleckstrin phosphorylation: a rapid activation of PKC (via diacylglycerol and calcium) followed by a slower activation of calcium-independent PKCs via PtdIns-3,4-P2.

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