K. Kishta Reddy

High affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of RAC/protein kinase B and their influence on kinase activity

The influence of inositol phosphates and phosphoinositides on the α isoform of the RAC-protein kinase B (RAC/PKB) was studied using purified wild type and mutant kinase preparations and a recombinant pleckstrin homology (PH) domain. Binding of inositol phosphates and phosphoinositides to the PH domain was measured as the quenching of intrinsic tryptophan fluorescence. Inositol phosphates and D3-phosphorylated phosphoinositides bound with affinities of 1-10 μM and 0.5 μM, respectively. Similar values were obtained using RAC/PKB expressed and purified from baculovirus-infected Sf9 cells in the fluorescence assay. The influence of synthetic dioctanoyl derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate on the activity of RAC/PKB purified from transfected COS-1 cells was studied. Phosphatidylinositol 3,4,5-trisphosphate was found to inhibit the RAC/PKB kinase activity with half-maximal inhibition at 2.5 μM. In contrast, phosphatidylinositol 3,4-bisphosphate stimulated kinase activity (half-maximal stimulation at 2.5 μM). A mutant RAC/PKB protein lacking the PH domain was not affected by D3-phosphorylated phosphoinositides. These results demonstrate that the PH domain of RAC/PKB binds inositol phosphates and phosphoinositides with high affinity, and suggest that the products of the phosphatidylinositide 3-kinase can act as both a membrane anchor and modulator of RAC/PKB activity. The data also provide further evidence for a link between phosphatidylinositide 3-kinase and RAC/PKB regulation.

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.

Regulation of AP-3 Function by Inositides IDENTIFICATION OF PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE AS A POTENT LIGAND

As part of the growing effort to understand the role inositol phosphates and inositol lipids play in the regulation of vesicle traffic within nerve terminals, we determined whether or not the synapse-specific clathrin assembly protein AP-3 can interact with inositol lipids. We found that soluble dioctanoyl-phosphatidylinositol 3,4,5-trisphosphate (DiC8PtdIns(3,4,5)P3) was only 7.5-fold weaker a ligand than D-myo-inositol hexakisphosphate in assays that measured the displacement of D-myo-[3H]inositol hexakisphosphate. In functional assays we found that both of these ligands inhibited clathrin assembly, but DiC8-PtdIns(3,4,5)P3 was more potent and exhibited a larger maximal effect. We also examined the structural features of DiC8-PtdIns(3,4,5)P3 that establish specificity. Dioctanoyl-phosphatidylinositol 3,4-bisphosphate, which does not have a 5-phosphate, and 4,5-O-bisphosphoryl-D-myo-inosityl 1-O-(1,2-O-diundecyl)-sn-3-glycerylphosphate, which does not have a 3-phosphate, were, respectively, 2-fold and 4-fold less potent than DiC8-PtdIns(3,4,5)P3 as inhibitors of clathrin assembly. Deacylation of DiC8-PtdIns(3,4,5)P3 reduced its affinity for AP-3 almost 20-fold, and also dramatically lowered its ability to inhibit clathrin assembly. The deacylated products of the soluble derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 4,5-bisphosphate were both not significant inhibitors of clathrin assembly. It therefore appears that the interactions of inositides with AP-3 should not be considered simply in terms of electrostatic effects of the highly charged phosphate groups. Ligand specificity appears also to be mediated by hydrophobic interactions with the fatty-acyl chains of the inositol lipids.

Diphospho‐myo‐inositol phosphates in Dictyostelium and Polysphondylium: identification of a new bisdiphospho‐myo‐inositol tetrakisphosphate

The two major diphospho inositol phosphates from the axenic strain Dictyostelium discoideum AX2 were previously investigated and identified as 6‐PP‐InsP5 and 5,6‐bis‐PP‐InsP4. In order to examine whether these findings are representative of Dictyostelids in general, five non‐axenic wild‐type species of Dictyostelium and two of Polysphondylium were studied. It was found that all of the Dictyostelium species exhibit similar patterns of diphospho inositol phosphates. By contrast, both of the Polysphondylium species contain 5‐PP‐InsP5 as the predominant isomer. Besides 5,6‐bis‐PP‐InsP4, a new bis‐PP‐InsP4 was detected in Polysphondylium. This compound is either 1,5‐bis‐PP‐InsP4 or its corresponding enantiomer 3,5‐bis‐PP‐InsP5. The structures were elucidated by two‐dimensional 1H‐1H and 1H‐31P NMR analysis. Additionally, they were confirmed using a specific 6‐PP‐InsP5‐5‐kinase from D. discoideum AX2 as an enantio‐specific tool and enantiomerically pure reference standards.

Insulin second messengers : synthesis of 6-O-(2-amino-2-deoxy-α-D-glucopyranosyl)-D-chiro-inositol-1-phosphate

Abstract An efficient enantiospecific route to protected chiro-inositol 7 from (−)-quinic acid was exploited for the synthesis of the title disaccharide which displayed insulin mimetic activity.

Synthesis of 2- and 5-diphospho-myo-inositol pentakisphosphate (2- and 5-PP-InsP5), intracellular mediators

Abstract The title pyrophosphates were prepared in good overall yield from a readily available bis-disiloxanylidene derivative of myo -inositol.

Wortmannin and LY294002 inhibit the insulin-induced down-regulation of IRS-1 in 3T3-L1 adipocytes.

The insulin receptor substrate-1 (IRS-1) is expressed in 3T3-L1 adipocytes and is involved in at least some insulin responses, notably mitogenesis. Chronic exposure to insulin down regulates IRS-1 in these cells by stimulating its degradation (Rice, K.M., Turnbow, M.A. and Garner, C.W. (1993) Biochem. Biophys. Res. Commun. 190, 961-967). This insulin response was completely inhibited by wortmannin and LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), two inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase). Neither wortmannin nor LY294002 had any effect on the calcium-dependent degradation of IRS-1 in vitro nor did they inhibit the phosphorylation of IRS-1 in vitro. In addition, neomycin, a cationic aminoglycoside antibiotic that binds to phosphoinositides, inhibited the insulin-induced down-regulation of IRS-1 in 3T3-L1 adipocytes and, also, the C8-PIP3-stimulated degradation of IRS-1 in vitro. These results suggest that PI 3-kinase and its 3-phosphoinositide products mediate the insulin-induced down-regulation of IRS-1 in 3T3-L1 adipocytes.

Eicosanoid biosynthesis: Differential inhibition of cytochrome P450 epoxygenase and ω-hydroxylase

Abstract Biphenyl 4 and vinyldibromide 8 were prepared on a multigram scale and shown to be comparatively specific arachidonic acid epoxygenase and ω-hydroxylase inhibitors, respectively, in rat kidney microsomal fractions.

Synthesis and structure revision of the myo-inositol monophosphatase inhibitor l-671,776

Concomitant deprotection/spiro-heteroannulation of 6 utilizing (EtO)3SiI was exploited for the asymmetric total synthesis of the title tetracyclic terpenoid whose structure was revised to 13.

Concise synthesis of L-α-phosphatidyl-D-myo-inositol 3,4-bisphosphate, an intracellular second messenger

A highly efficient, asymmetric total synthesis of the title phospholipid as well as short chain diester and cross-linkable diether analogs was achieved in six steps from the readily available cyclitol 1.