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Epoxyeicosatrienoic acids (EETs) are endothelium-derived eicosanoids that activate potassium channels, hyperpolarize the membrane, and cause relaxation. We tested 19 analogs of 14,15-EET on vascular tone to determine the structural features required for activity. 14,15-EET relaxed bovine coronary arterial rings in a concentration-related manner (E[D.sub.50] = [10.sup.-6] M). Changing the carboxyl to an alcohol eliminated dilator activity, whereas 14,15-EET-methyl ester and 14,15-EET-methylsulfonimide retained full activity. Shortening the distance between the carboxyl and epoxy groups reduced the agonist potency and activity. Removal of all three double bonds decreased potency. An analog with a [DELTA]8 double bond had full activity and potency. However, the analogs with only a [DELTA]5 or [DELTA]11 double bond had reduced potency. Conversion of the epoxy oxygen to a sulfur or nitrogen resulted in loss of activity. 14(S),15(R)-EET was more potent than 14(R),15(S)-EET, and 14,15-(cis)-EET was more potent than 14,15-(trans)-EET. These studies indicate that the structural features of 14,15-EET required for relaxation of the bovine coronary artery include a carbon-1 acidic group, a [DELTA]8 double bond, and a 14(S),15(R)-(cis)-epoxy group. endothelium-derived hyperpolarizing factor; cytochrome P-450; arachidonic acid; epoxyeicosatrienoic acid

Epoxyeicosatrienoic acids (EETs) are synthesized in the endothelial cells of vascular tissues. They are released from the endothelial cells and produce relaxation of the smooth muscle cells by hyperpolarization. The present findings demonstrate that EETs also regulate aromatase activity in vascular smooth muscle cells. Exposure of cultured rat aortic smooth muscle cells to either 1 [micro]M 14,15-EET or 1 [micro]M 11,12-EET inhibits dibutyryl cAMP-induced aromatase activity by 80-100%. 11,12-Dihydroxyeicosatrienoic acid, the hydration product of 11,12-EET, has no effect on dibutyryl cAMP-induced vascular smooth muscle aromatase activity. In contrast to 14,15-EET, the N-methylsulfanilamide derivative of 14,15-EET (14,15-EET-SA) was neither metabolized nor incorporated into cell lipids, but it retained the ability to inhibit cAMP-induced aromatase activity. Furthermore, the 14,15-EET-SA inhibition of cAMP-induced aromatase activity persisted when the sulfanilamide derivative of 14,15-EET was covalently tethered to silica beads (average diameter, 0.5 [micro]m), which restricted 14,15-EET-SA from entering the cell. These data are consistent with the presence of a receptor for EETs in the plasma membrane and support the hypothesis that the inhibition of aromatase by EETs is initiated by the interaction of EET with the putative plasma membrane receptor. epoxyeicosatrienoic acid; receptor; adenosine 3',5'-cylic monophosphate; estrogen

We have recently reported that direct interaction between phosphatidylinositol bisphosphate (PI[P.sub.2]) and the COOH-terminal cytoplasmic domain of ROMK1 is important for opening of the channel. We identified arginine-188 of ROMK1 as a critical residue for this interaction. Here, we further report that substitution of a neutral amino acid for lysine-181, arginine-217, or lysine-218 decreases single-channel open probability for the full-conductance state and increases the frequency of opening at a subconductance state. Compared with wild-type ROMK1 channels, these substitution mutants also display an increased sensitivity to the block by anti-PI[P.sub.2] antibodies and to inhibition by intracellular protons. These results indicate that, like arginine-188, lysine-181, arginine-217, and lysine-218 are also involved in interactions with PI[P.sub.2] and are critical for ROMK1 to open at full conductance. Using synthetic phosphoinositides containing phosphates at different positions in the head group, we also examined the specificities of phosphoinositides in the regulation of ROMK1 channels. We found that phosphoinositides containing phosphate at both positions 4 and 5 of the inositol head group have the highest efficacy in activating ROMK1 channels. These results suggest that phosphatidylinositol 4,5-bisphosphate is likely the important phosphoinositide in the regulation of ROMK1 channels in a physiological membrane milieu. phosphatidylinositol bisphosphate; pleckstrin homology domain; inwardly rectifying potassium channels; giant patch-clamp recording; kidney

We have previously demonstrated that the protein level of c-Src, a nonreceptor type of protein tyrosine kinase (PTK), was higher in the renal medulla from rats on a K-deficient (KD) diet than that in rats on a high-K (HK) diet (Wang WH, Lerea KM, Chan M, and Giebisch G. Am J Physiol Renal Physiol 278: F165-F171, 2000). We have now used the patch-clamp technique to investigate the role of PTK in regulating the apical K channels in the medullary thick ascending limb (mTAL) of the rat kidney. Inhibition of PTK with herbimycin A increased [NP.sub.o], a product of channel number (N) and open probability ([P.sub.o]), of the 70-pS K channel from 0.12 to 0.42 in the mTAL only from rats on a KD diet but had no significant effect in tubules from animals on a HK diet. In contrast, herbimycin A did not affect the activity of the 30-pS K channel in the mTAL from rats on a KD diet. Moreover, addition of N-methylsulfonyl-12,12-dibromododec-11-enamide, an agent that inhibits the cytochrome P-450-dependent production of 20-hydroxyeicosatetraenoic acid, further increased [NP.sub.o] of the 70-pS K channel in the presence of herbimycin A. Furthermore, Western blot detected the presence of PTP-1D, a membrane-associated protein tyrosine phosphatase (PTP), in the renal outer medulla. Inhibition of PTP with phenylarsine oxide (PAO) decreased [NP.sub.o] of the 70-pS K channel in the mTAL from rats on a HK diet. However, PAO did not inhibit the activity of the 30-pS K channel in the mTAL. The effect of PAO on the 70-pS K channel was due to indirectly stimulating PTK because pretreatment of the mTAL with herbimycin A abolished the inhibitory effect of PAO. Finally, addition of exogenous c-Src reversibly blocked the activity of the 70-pS K channel in inside-out patches. We conclude that PTK and PTP have no effect on the low-conductance K channels in the mTAL and that PTK-induced tyrosine phosphorylation inhibits, whereas PTP-induced tyrosine dephosphorylation stimulates, the apical 70-pS K channel in the mTAL. hypokalemia; hyperkalemia; c-Src; ROMK channel; herbimycin A; phenylarsine oxide

Frisbee, Jefferson C., Richard J. Roman, U. Murali Krishna, John R. Falck, and Julian H. Lombard. 20-HETE modulates myogenic response of skeletal muscle resistance arteries from hypertensive Dahl-SS rats. Am J Physiol Heart Circ Physiol 280: H1066-H1074, 2001.--The present study determined the role of 20-hydroxyeicosatetraenoic acid [20-HETE; produced by [Omega]-hydroxyation of arachidonic acid via cytochrome P-450 (CP450) 4A enzymes] in regulating myogenic activation of skeletal muscle resistance arteries from normotensive (NT) and hypertensive (HT) Dahl salt-sensitive (SS) rats. Gracilis arteries (GA) were isolated from each rat and viewed via television microscopy, and changes in vessel diameter with altered transmural pressure were measured with a video micrometer. Under control conditions, GA from both groups exhibited strong, endothelium-independent myogenic activation. Treatment of GA with 17-octadecynoic acid (17-ODYA; inhibitor of CP450 4A enzymes) did not alter myogenic activation in NT rats, but impaired this response in HT animals. Treatment of GA from HT rats with dibromo-dodecynyl-methylsulfimide (DDMS; inhibitor of 20-HETE production) impaired myogenic activation, as did application of 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, an antagonist for 20-HETE receptors. Application of iberiotoxin, a [Ca.sup.2+]-activated potassium ([K.sub.Ca]) channel inhibitor, restored myogenic activation from HT rats treated with DDMS. These results suggest that myogenic activation of skeletal muscle resistance arteries from NT Dahl-SS rats does not depend on CP450, whereas myogenic activation of these vessels in HT Dahl-SS rats is partly a function of 20-HETE production, inhibiting [K.sub.Ca] channels through a receptor-mediated process. cytochrome P-450 4A enzymes; cytochrome P-450 [Omega]-hydroxylase; potassium channels; vascular smooth muscle; dibromododecynyl-methylsulfimide; 17-octadecynoic acid

Examines the structural determinants and specificities for ROMK1-phosphoinositide interaction. Importance of arginine-188 in the interaction; Inhibition of intracellular protons; Regulation of ROMK channels by multiple signaling pathways.

Effects of protein tyrosine kinase and protein tyrosine phosphatase on apical K[sup +] channels in the TAL. Am J Physiol Cell Physiol 281: Cl188-Cl195, 2001.—We have previously demonstrated that the protein level of c-Src, a nonreceptor type of protein tyrosine kinase (PTK), was higher in the renal medulla from rats on a K-deficient (KI)) diet than that in rats on a high-K (HK) diet (Wang WH, Lerea KM, Chan M, and Giebisch G. Am J Physiol Renal Physiol 278: F165-F171, 2000). We have now used the patch-clamp technique to investigate the role of PTK in regulating the apical K channels in the medullary thick ascending limb (mTAL) of the rat kidney. Inhibition of PTK with herbimycin A increased NP[sub o], a product of channel number (N) and open probability (P[sub o]), of the 70-pS K channel from 0.12 to 0.42 in the mTAL only from rats on a KD diet but had no significant effect in tubules from animals on a HK diet. In contrast, herbimycin A did not affect the activity of the 30-pS K channel in the mTAL from rats on a KD diet. Moreover, addition of N-methylsulfonyl-12,12-dibromododec11-enamide, an agent that inhibits the cytochrome P-450dependent production of 20-hydroxyeicosatetraenoic acid, further increased NP[sub o] of the 70-pS K channel in the presence of herbimycin A. Furthermore, Western blot detected the presence of PTP-1D, a membrane-associated protein tyrosine phosphatase (PTP), in the renal outer medulla. Inhibition of PTP with phenylarsine oxide (PAO) decreased NP[sub o] of the 70-pS K channel in the toTAL from rats on a HK diet. However, PAO did not inhibit the activity of the 30-pS K channel in the mTAL. The effect of PAO on the 70-pS K channel was due to indirectly stimulating PTK because pretreatment of the toTAL with herbimycin A abolished the inhibitory effect of PAO. Finally, addition of exogenous c-Src reversibly blocked the activity of the 70-pS K channel in inside-out patches. We conclude that PTK and PTP have no effect on the... [ABSTRACT FROM AUTHOR]

A phosphoramidite approach was utilized for the first time to synthesize cardiolipin. Optically active 1,2-di-O-acyl-sn-glycerol was coupled with 2-O-protected glycerols utilizing mono- and bifunctional phosphitylating agents to yield, after final removal of protecting groups, the title compound. [Copyright &y& Elsevier]

Mild, room temperature CrCl2 reduction of 1,1,1-trichloroalkanes stereoselectively generates (Z)-1-chloro-2-substituted-1-alkenes in excellent yields. [Copyright &y& Elsevier]

A series of 11,12-EET analogues were synthesized and compared using a human endothelial cell based TNF-α-induced VCAM-1 expression assay. The resulting data were used to map a putative recognition/binding domain for 11,12-EET. [Copyright &y& Elsevier]

Diacylglycerol kinase α (DAGKα), like all type I DAGKs, has calcium regulatory motifs that act as negative regulators of enzyme activity and localization. Accordingly, DAGKα is activated by phospholipase C-coupled receptors in a calcium-dependent manner. One of the first functions attributed to DAGKα in lymphocytes was that of regulating interleukin 2-induced cell cycle entry. Interleukin-2 nonetheless exerts its action in the absence of cytosolic calcium increase. We have studied alternative receptor-derived signals to explain calciumindependent DAGKα activation, and show that DAGKα is stimulated by Src-like kinase-dependent phosphoinositide 3 kinase (PI3K) activation in lymphocytes. Our results demonstrate that, in vivo, the increase in cellular levels of PI3K products is sufficient to induce DAGKα activation, allowing DAGKα relocation to the intact lymphocyte plasma membrane. This activation is isoformspecific, because other type I DAGKs are not subject to this type of regulation. These studies are the first to describe a pathway in which, in the absence of receptorregulated calcium increase, DAGKα activation and membrane localization is a direct consequence of PI3K activation. [ABSTRACT FROM AUTHOR]