Convenient syntheses of [20,20,20-H(3)]-arachidonic acid and [20,20-H(2)]-20-hydroxyeicosatetraenoic acid.

Deuterated arachidonic acid and 20-HETE were prepared in good overall yields and high stereoselectivities. Key transformations include a trans-specific vinyl dibromide reduction and Suzuki cross-couplings to a lithium borate or a 9-BBN borane. These standards are three and two mass units higher, respectively, than their naturally occurring counterparts and are useful in mass spectrometry analysis.

Drofenine: A 2-APB Analogue with Greater Selectivity for Human TRPV3.

Transient receptor potential vanilloid-3 (TRPV3) is a member of the TRPV subfamily of TRP ion channels. The physiological functions of TRPV3 are not fully understood, in part due to a lack of selective agonists and antagonists that could both facilitate the elucidation of roles for TRPV3 in mammalian physiology, as well as potentially serve as therapeutic agents to modulate conditions for which altered TRPV3 function has been implicated. In this study, the Microsource Spectrum Collection was screened for TRPV3 agonists and antagonists using alterations in calcium flux in TRPV3 over-expressing HEK-293 cells. The antispasmodic agent drofenine was identified as a new TRPV3 agonist. Drofenine exhibited similar potency to the known TRPV3 agonists 2-aminoethoxydiphenylboronate (2-APB) and carvacrol in HEK-293 cells, but greater selectivity for TRPV3 based on a lack of activation of TRPA1, V1, V2, V4, or M8. Multiple inhibitors were also identified, but all of the compounds were either inactive or not specific. Drofenine activated TRPV3 via interactions with the residue, H426, which is required for TRPV3 activation by 2-APB. Drofenine was a more potent agonist of TRPV3 and more cytotoxic than either carvacrol or 2-APB in human keratinocytes and its effect on TRPV3 in HaCaT cells was further demonstrated using the antagonist icilin. Due to the lack of specificity of existing TRPV3 modulators and the expression of multiple TRP channels in cells/tissue, drofenine may be a valuable probe for elucidating TRPV3 functions in complex biological systems. Identification of TRPV3 as a target for drofenine may also suggest a mechanism by which drofenine acts as a therapeutic agent.

Characterization of 14,15-epoxyeicosatrienoyl-sulfonamides as 14,15-epoxyeicosatrienoic acid agonists: use for studies of metabolism and ligand binding.

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid. EETs mediate numerous biological functions. In coronary arteries, they regulate vascular tone by the activation of smooth muscle large-conductance, calcium-activated potassium (BK(Ca)) channels to cause hyperpolarization and relaxation. We developed a series of 14,15-EET agonists, 14,15-EET-phenyliodosulfonamide (14,15-EET-PISA), 14,15-EET-biotinsulfonamide (14,15-EET-BSA), and 14,15-EET-benzoyldihydrocinnamide-sulfonamide (14,15-EET-BZDC-SA) as tools to characterize 14,15-EET metabolism and binding. Agonist activities of these analogs were characterized in precontraced bovine coronary arterial rings. All three analogs induced concentration-dependent relaxation and were equipotent with 14,15-EET. Relaxations to these analogs were inhibited by the BK(Ca) channel blocker iberiotoxin (100 nM), the 14,15-EET antagonist 14,15-epoxyeicosa-5(Z)-enoylmethylsulfonamide (10 muM), and abolished by 20 mM extracellular K(+). 14,15-EET-PISA is metabolized to 14,15-dihydroxyeicosatrienoyl-PISA by soluble epoxide hydrolase in bovine coronary arteries and U937 cells but not U937 cell membrane fractions. 14,15-EET-P(125)ISA binding to human U937 cell membranes was time-dependent, concentration-dependent, and saturable. The specific binding reached equilibrium by 15 min at 4 degrees C and remained unchanged up to 30 min. The estimated K(d) and B(max) were 148.3 +/- 36.4 nM and 3.3 +/- 0.5 pmol/mg protein, respectively. These data suggest that 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA are full 14,15-EET agonists. 14,15-EET-P(125)ISA is a new radiolabeled tool to study EET metabolism and binding. Our results also provide preliminary evidence that EETs exert their biological effect through a membrane binding site/receptor.

P2X receptor-stimulated calcium responses in preglomerular vascular smooth muscle cells involves 20-hydroxyeicosatetraenoic acid.

The current study tested the hypothesis that endogenous 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to the increase in intracellular calcium ([Ca2+]i) elicited by P2X receptor activation in renal microvascular smooth muscle cells. Vascular smooth muscle cells obtained from rats were loaded with fura-2 and studied using standard single cell fluorescence microscopy. Basal renal myocyte [Ca2+]i averaged 96 +/- 5 nM. ATP (10 and 100 microM) increased vascular smooth muscle cell [Ca2+]i by 340 +/- 88 and 555 +/- 80 nM, respectively. The cytochrome P450 hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), or the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE), significantly attenuated the peak myocyte [Ca2+]i responses to 10 and 100 microM ATP. ATP (100 microM) increased vascular smooth muscle cell [Ca2+]i by 372 +/- 93 and 163 +/- 55 nM in the presence of DDMS or 20-HEDE, respectively. The P2X receptor agonist, alpha,beta-methylene-ATP (10 microM), increased myocyte [Ca2+]i by 78 +/- 12 nM, and this response was significantly attenuated by DDMS (40 +/- 15 nM). In contrast, the vascular smooth muscle cell [Ca2+]i evoked by the P2Y agonist, UTP (100 microM), was not altered by DDMS or 20-HEDE. The effect of 20-HETE on [Ca2+]i was also assessed, and the peak increases in [Ca2+]i averaged 62 +/- 12 and 146 +/- 70 nM at 20-HETE concentrations of 1 and 10 microM, respectively. These results demonstrate that 20-HETE plays a significant role in the renal microvascular smooth muscle cell [Ca2+]i response to P2X receptor activation.

Transfection of CYP4A1 cDNA decreases diameter and increases responsiveness of gracilis muscle arterioles to constrictor stimuli.

Cytochrome P-450-4A1 (CYP4A1) is an omega-hydroxylase that catalyzes the metabolism of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). The goal of this study was to determine the vasomotor consequences of vascular overexpression of CYP4A1. Isolated rat gracilis muscle arterioles transfected ex vivo with an expression plasmid containing CYP4A1 cDNA expressed more CYP4A protein than vessels transfected with the control plasmid. In arterioles pressurized to 80 mmHg, the internal diameter of vessels transfected with CYP4A1 cDNA (55 +/- 3 microm) was surpassed (P < 0.05) by that of vessels transfected with control plasmid (97 +/- 4 microm). Treatment with a CYP4A inhibitor (N-methylsulfonyl-12,12-dibromododec-11-enamide; DDMS) or with an antagonist of 20-HETE actions [20-hydroxyeicosa-6(Z),15(Z)-dienoic acid; 20-HEDE] elicited robust dilation of arterioles transfected with CYP4A1 cDNA, whereas the treatment had little or no effect in vessels transfected with control plasmid. Examination of the intraluminal pressure-internal diameter relationship revealed that pressure increments over the range of 40-100 mmHg elicited a more intense (P < 0.05) myogenic constrictor response in arterioles transfected with CYP4A1 cDNA than in those with control plasmid. Arterioles transfected with CYP4A1 cDNA also displayed enhanced sensitivity to the constrictor action of phenylephrine. Treatment with DDMS or 20-HEDE greatly attenuated the constrictor responsiveness to both constrictor stimuli in vessels overexpressing CYP4A1, whereas the treatment had much less effect in control vessels. These data suggest that CYP4A1 overexpression promotes constriction of gracilis muscle arterioles by intensifying the responsiveness of vascular smooth muscle to constrictor stimuli. This effect of CYP4A1 overexpression appears to be mediated by a CYP4A1 product.

Transfection of CYP4A1 cDNA increases vascular reactivity in renal interlobar arteries.

20-HETE, a cytochrome P-450 4A (CYP4A1)-derived arachidonic acid metabolite, is a major eicosanoid formed in renal and extrarenal microcirculation. 20-HETE inhibits Ca(2+)-activated K(+) channels in vascular smooth muscle cells and thereby may modulate vascular reactivity. We transfected renal interlobar arteries with an expression plasmid containing the cDNA of CYP4A1, the low-K(m) arachidonic acid omega-hydroxylase, and examined the consequences of increasing 20-HETE synthesis on constrictor responses to phenylephrine. CYP4A1-transfected interlobar arteries demonstrated a twofold increase in CYP4A protein levels and 20-HETE production compared with arteries transfected with the empty plasmid; they also showed increased sensitivity to phenylephrine, as evidenced by a decrease in EC(50) from 0.37 +/- 0.04 microM in plasmid-transfected arteries to 0.07 +/- 0.01 microM in CYP4A1-transfected arteries. The increased sensitivity to phenylephrine was greatly attenuated by N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), a selective inhibitor of 20-HETE synthesis, and by 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, a specific 20-HETE antagonist. This effect of DDMS was reversed by addition of 20-HETE, further substantiating the notion that increased levels of 20-HETE contribute to the increased sensitivity to phenylephrine in vessels overexpressing CYP4A1. These data suggest that 20-HETE of vascular origin sensitizes renal vascular smooth muscle to phenylephrine.

20-hydroxyeicosatetraenoic acid (20-HETE): structural determinants for renal vasoconstriction.

The effects of natural and synthetic eicosanoids on the diameter of rat interlobular arteries studied in vitro were compared to that of the potent, endogenous vasoconstrictor 20-HETE. Vasoconstrictor activity was optimum for chain lengths of 20-22 carbons with at least one olefin or epoxide between located between C(13)-C(15) and an oxygen substituent at C(20)-C(22). The presence of delta (Zou et al. Am. J. Physiol. 1996, 270, R228; Gebremedhin, D. et al. Am. J. Physiol. 1998, 507, 771)-, delta (Carroll et al. Am. J. Physiol. 1996, 271, R863; Vazquez et al. Life Sci. 1995, 56, 1455)-, or delta (Imig et al. Hypertension 2000, 35, 307; Lopez et al. Amer. J. Physiol. 2001, 281, F420)-olefins had no influence on the vasoconstrictor response whereas the introduction of a C(7)-thiomethylene enhanced potency. A sulfonamide or alcohol, but not a lactone, could replace the C(1)-carboxylate. These data were used to construct a putative binding domain map of the 20-HETE receptor consisting of: (i) a comparatively open, hydrophilic binding site accommodating the C(1)-functionality; (ii) a hydrophobic trough spanning the olefins; (iii) a shallow pocket containing a critical pi-pi binding site in the vicinity of the pi (Ito et al. Am. J. Physiol. 1998, 274, F395; Quigley, R.; Baum, M.; Reddy, K. M.; Griener, J. C.; Falck, J. R. Am. J. Physiol. 2000, 278, F949)-olefin; and (iv) an oxyphilic binding site proximate to the omega-terminus.

20-hydroxyeicosatetraenoic acid (20-HETE) metabolism in coronary endothelial cells.

We have investigated the role of endothelial cells in the metabolism of 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoactive mediator synthesized from arachidonic acid by cytochrome P450 omega-oxidases. Porcine coronary artery endothelial cells (PCEC) incorporated 20-[(3)H]HETE primarily into the sn-2 position of phospholipids through a coenzyme A-dependent process. The incorporation was reduced by equimolar amounts of arachidonic, eicosapentaenoic or 8,9-epoxyeicosatrienoic acids, but some uptake persisted even when a 10-fold excess of arachidonic acid was available. The retention of 20-[(3)H]HETE increased substantially when methyl arachidonoyl fluorophosphonate, but not bromoenol lactone, was added, suggesting that a Ca(2+)-dependent cytosolic phospholipase A(2) released the 20-HETE contained in PCEC phospholipids. Addition of calcium ionophore A23187 produced a rapid release of 20-[(3)H]HETE from the PCEC, a finding that also is consistent with a Ca(2+)-dependent mobilization process. PCEC also converted 20-[(3)H]HETE to 20-carboxy-arachidonic acid (20-COOH-AA) and 18-, 16-, and 14-carbon beta-oxidation products. 20-COOH-AA produced vasodilation in porcine coronary arterioles, but 20-HETE was inactive. These results suggest that the incorporation of 20-HETE and its subsequent conversion to 20-COOH-AA in the endothelium may be important in modulating coronary vascular function.