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Biomedical subjects

U Murali Krishna

Publications and source records attributed to U Murali Krishna.

9 recordsLinked to original sources
PubMed2004

A short, concise route to diphosphatidylglycerol (cardiolipin) and its variants.

A new approach is described for the synthesis of the cardiolipin family of phospholipids that uses phosphonium salt methodology. The method involves the reaction of 2-O-protected glycerol with a trialkyl phosphite derived from 1,2-diacylsn-glycerol in the presence of pyridinium bromide perbromide and triethylamine to afford the phosphoric triesters. The synthesis involves three steps and allows the preparation of a wide range of cardiolipins with different substitution patterns and chain lengths, including unsaturated derivatives. The use of inexpensive protecting groups and the ease of purification facilitate this synthetic route and allow its scale-up in a higher overall yield (72%) than the literature methods.

Cardiolipins↗
PubMed2003

Regulation of diacylglycerol kinase alpha by phosphoinositide 3-kinase lipid products.

Diacylglycerol kinase alpha (DAGK alpha), like all type I DAGKs, has calcium regulatory motifs that act as negative regulators of enzyme activity and localization. Accordingly, DAGK alpha is activated by phospholipase C-coupled receptors in a calcium-dependent manner. One of the first functions attributed to DAGK alpha 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 calcium-independent DAGK alpha activation, and show that DAGK alpha 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 DAGK alpha activation, allowing DAGK alpha relocation to the intact lymphocyte plasma membrane. This activation is isoform-specific, 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 receptor-regulated calcium increase, DAGK alpha activation and membrane localization is a direct consequence of PI3K activation.

Animals↗
PubMed2002

Comparison of vasodilatory properties of 14,15-EET analogs: structural requirements for dilation.

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 (ED(50) = 10(-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 Delta8 double bond had full activity and potency. However, the analogs with only a Delta5 or Delta11 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 Delta8 double bond, and a 14(S),15(R)-(cis)-epoxy group.

8,11,14-Eicosatrienoic Acid↗
PubMed2002

14,15-Epoxyeicosa-5(Z)-enoic acid: a selective epoxyeicosatrienoic acid antagonist that inhibits endothelium-dependent hyperpolarization and relaxation in coronary arteries.

Endothelium-dependent hyperpolarization and relaxation of vascular smooth muscle are mediated by endothelium-derived hyperpolarizing factors (EDHFs). EDHF candidates include cytochrome P-450 metabolites of arachidonic acid, K(+), hydrogen peroxide, or electrical coupling through gap junctions. In bovine coronary arteries, epoxyeicosatrienoic acids (EETs) appear to function as EDHFs. A 14,15-EET analogue, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) was synthesized and identified as an EET-specific antagonist. In bovine coronary arterial rings preconstricted with U46619, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET induced concentration-related relaxations. Preincubation of the arterial rings with 14,15-EEZE (10 micromol/L) inhibited the relaxations to 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET but was most effective in inhibiting 14,15-EET-induced relaxations. 14,15-EEZE also inhibited indomethacin-resistant relaxations to methacholine and arachidonic acid and indomethacin-resistant and L-nitroarginine-resistant relaxations to bradykinin. It did not alter relaxation responses to sodium nitroprusside, iloprost, or the K(+) channel activators (NS1619 and bimakalim). Additionally, in small bovine coronary arteries pretreated with indomethacin and L-nitroarginine and preconstricted with U46619, 14,15-EEZE (3 micromol/L) inhibited bradykinin (10 nmol/L)-induced smooth muscle hyperpolarizations and relaxations. In rat renal microsomes, 14,15-EEZE (10 micromol/L) did not decrease EET synthesis and did not alter 20-hydroxyeicosatetraenoic acid synthesis. This analogue acts as an EET antagonist by inhibiting the following: (1) EET-induced relaxations, (2) the EDHF component of methacholine-induced, bradykinin-induced, and arachidonic acid-induced relaxations, and (3) the smooth muscle hyperpolarization response to bradykinin. Thus, a distinct molecular structure is required for EET activity, and alteration of this structure modifies agonist and antagonist activity. These findings support a role of EETs as EDHFs.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2002

Evidence for a membrane site of action for 14,15-EET on expression of aromatase in vascular smooth muscle.

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 microM 14,15-EET or 1 microM 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 microm), 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.

8,11,14-Eicosatrienoic Acid↗
PubMed2002

Structural determinants and specificities for ROMK1-phosphoinositide interaction.

We have recently reported that direct interaction between phosphatidylinositol bisphosphate (PIP(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-PIP(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 PIP(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.

Alanine↗
PubMed2001

Altered mechanisms underlying hypoxic dilation of skeletal muscle resistance arteries of hypertensive versus normotensive Dahl rats.

OBJECTIVE: To determine mechanisms underlying hypoxic dilation of skeletal muscle resistance arteries from normotensive (NT) and hypertensive (HT) Dahl salt-sensitive (SS) rats. METHODS: Isolated gracilis arteries (GA) from both rat groups were viewed via television microscopy and vascular responses to a reduction in PO2 from 145 mm Hg to 40 mm Hg were measured with a video micrometer. Responses were determined following endothelium removal and following inhibition of specific biochemical pathways regulating vascular tone. RESULTS: Hypoxic dilation was impaired in HT rats versus NT controls. Endothelium removal abolished hypoxic dilation in NT rats, although a significant dilation to hypoxia remained in vessels from HT animals. Inhibition of cytochrome P450 (CP450) 4A enzymes blunted hypoxic dilation in both groups, while inhibition of epoxyeicosatrienoic acid (EET) production impaired responses in NT rats only. Inhibition of 20-hydroxyeicosatetraenoic acid (20-HETE) production or blockade of membrane receptors for 20-HETE reduced hypoxic dilation in HT rats, with minimal effects in NT animals. Nitric oxide synthase inhibition had no effect on hypoxic dilation in either group, while cyclooxygenase inhibition significantly reduced this response in both groups. CONCLUSIONS: These results suggest that the mechanisms of hypoxic dilation in GA from NT Dahl-SS rats are altered with HT, impairing the response to reduced PO2. While hypoxia induces substantial prostanoid release in both groups, the role of CP450 4A enzymes is shifted from EET production in NT rats toward inhibition of 20-HETE production in HT rats.

Animals↗