Calcium glycerophosphate preserves transepithelial integrity in the Caco-2 model of intestinal transport.

AIM: To assess the direct effects of ischemia on intestinal epithelial integrity. Furthermore, clinical efforts at mitigating the effect of hypoperfusion on gut permeability have focused on restoring gut vascular function. METHODS: We report that, in the Caco-2 cell model of transepithelial transport, calcium glycerophosphate (CGP), an inhibitor of intestinal alkaline phosphatase F3, has a significant effect to preserve transepithelial electrical resistance (TEER) and to attenuate increases in mannitol flux rates during hypoxia or cytokine stimulation. RESULTS: The effect was observable even at concentrations as low as 1 µmol/L. As celiac disease is also marked by a loss of gut epithelial integrity, the effect of CGP to attenuate the effect of the α-gliadin peptide 31-55 was also examined. In this instance, CGP exerted little effect of preservation of TEER, but significantly attenuated peptide induced increase in mannitol flux. CONCLUSION: It appears that CGP treatment might synergize with other therapies to preserve gut epithelial integrity.

Inhibition of long chain fatty acyl-CoA synthetase (ACSL) and ischemia reperfusion injury.

Various triacsin C analogs, containing different alkenyl chains and carboxylic acid bioisoteres including 4-aminobenzoic acid, isothiazolidine dioxide, hydroxylamine, hydroxytriazene, and oxadiazolidine dione, were synthesized and their inhibitions of long chain fatty acyl-CoA synthetase (ACSL) were examined. Two methods, a cell-based assay of ACSL activity and an in situ [(14)C]-palmitate incorporation into extractable lipids were used to study the inhibition. Using an in vivo leukocyte recruitment inhibition protocol, the translocation of one or more cell adhesion molecules from the cytoplasm to the plasma membrane on either the endothelium or leukocyte or both was inhibited by inhibitors 1, 9, and triacsin C. The results suggest that inhibition of ACSL may attenuate the vascular inflammatory component associated with ischemia reperfusion injury and lead to a decrease of infarct expansion.

Rat brain docosahexaenoic acid metabolism is not altered by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide.

In a rat model of neuroinflammation, produced by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide (LPS), we reported that the brain concentrations of non-esterified brain arachidonic acid (AA, 20:4 n-6) and its eicosanoid products PGE(2) and PGD(2) were increased, as were AA turnover rates in certain brain phospholipids and the activity of AA-selective cytosolic phospholipase A(2) (cPLA(2)). The activity of Ca(2+)-independent iPLA(2), which is thought to be selective for the release of docosahexaenoic acid (DHA, 22:6 n-3) from membrane phospholipid, was unchanged. In the present study, we measured parameters of brain DHA metabolism in comparable artificial cerebrospinal fluid (control) and LPS-infused rats. In contrast to the reported changes in markers of AA metabolism, the brain non-esterified DHA concentration and DHA turnover rates in individual phospholipids were not significantly altered by LPS infusion. The formation rates of AA-CoA and DHA-CoA in a microsomal brain fraction were also unaltered by the LPS infusion. These observations indicate that LPS-treatment upregulates markers of brain AA but not DHA metabolism. All of which are consistent with other evidence that suggest different sets of enzymes regulate AA and DHA recycling within brain phospholipids and that only selective increases in brain AA metabolism occur following a 6-day LPS infusion.

Valproic acid selectively inhibits conversion of arachidonic acid to arachidonoyl-CoA by brain microsomal long-chain fatty acyl-CoA synthetases: relevance to bipolar disorder.

RATIONALE: Several drugs used to treat bipolar disorder (lithium and carbamazepine), when administered chronically to rats, reduce the turnover of arachidonic acid, but not docosahexaenoic acid, in brain phospholipids by decreasing the activity of an arachidonic acid-selective phospholipase A(2). Although chronic valproic acid produces similar effects on brain arachidonic acid and docosahexaenoic acid turnover, it does not alter phospholipase A(2) activity, suggesting that it targets a different enzyme in the turnover pathway. MATERIALS AND METHODS/RESULTS: By isolating rat brain microsomal long-chain fatty acyl-CoA synthetases (Acsl), we show in vitro that valproic acid is a non-competitive inhibitor of Acsl, as it reduces the maximal velocity of the reaction without changing the affinity of the substrate for the enzyme. While valproic acid inhibited the synthesis of arachidonoyl-CoA, palmitoyl-CoA, and docosahexaenoyl-CoA, the K (i )for inhibition of arachidonoyl-CoA synthesis (14.1 mM) was approximately one fifth the K (i) for inhibiting palmitoyl-CoA (85.4 mM) and docosahexaenoyl-CoA (78.2 mM) synthesis. As chronic administration of valproic acid in bipolar disorder achieves whole-brain levels of 1.0 to 1.5 mM, inhibition of arachidonoyl-CoA formation can occur at brain concentrations that are therapeutically relevant to this disease. Furthermore, brain microsomal Acsl did not produce valproyl-CoA. CONCLUSIONS: This study shows that valproic acid acts as a non-competitive inhibitor of brain microsomal Acsl, and that inhibition is substrate-selective. The study supports the hypothesis that valproic acid acts in bipolar disorder by reducing the brain arachidonic acid cascade, by inhibiting arachidonoyl-CoA formation.

Gö 6983: a fast acting protein kinase C inhibitor that attenuates myocardial ischemia/reperfusion injury.

Reperfusion injury is characterized by a decrease in endothelial release of nitric oxide within 5 min after reperfusion, increased leukocyte-endothelium interaction, and transmigration of leukocytes into the myocardium, producing cardiac contractile dysfunction. Gö 6983 is a fast acting, lipid soluble, broad spectrum protein kinase C inhibitor. When administered at the beginning of reperfusion, it can restore cardiac function within 5 min and attenuate the deleterious effects associated with acute ischemia/reperfusion. Gö 6983 may offer greater cardioprotection than other broad-spectrum PKC inhibitors in postischemic reperfusion injury because it inhibits PKC(zeta) as well as four other isoforms. The cardioprotection is associated with decreased leukocyte superoxide release and increased endothelial derived nitric oxide from vascular tissue. In vitro studies of human tissue showed that Gö 6983 significantly inhibited antigen-induced superoxide release from leukocytes of patients previously sensitized to tree pollen. In human vascular tissue, Gö 6983 inhibited intracellular Ca(2+) accumulation, suggesting a mechanism for its vasodilator properties. These studies suggest that Gö 6983 would be an effective compound to use in a clinical ischemia/reperfusion setting of organ transplantation and/or cerebral ischemia where inhibiting superoxide release and vasoconstriction in postischemic tissues would allow for better restoration of organ function during reperfusion. However, given the broad-spectrum action of Gö 6983, careful titration of the dose regimen would be recommended to ensure a successful outcome in the setting of organ transplantation and/or cerebral ischemia.

Inhibiting long-chain fatty acyl CoA synthetase does not increase agonist-induced release of arachidonate metabolites from human endothelial cells.

BACKGROUND: Triacsin C, a fatty acid analog, inhibits endothelial nitric oxide synthetase (eNOS) palmitoylation, increases nitric oxide synthesis and enhances methacholine-induced relaxation of vascular rings. The experiments presented here tested the hypothesis that triacsin C increases the synthesis of PGI(2) and/or endothelial-derived hyperpolarizing factor. METHODS: Long-chain fatty acyl CoA synthetase activity (LCFACoAS), agonist-induced prostacyclin synthesis and agonist-induced release of radioactivity in endothelial cells labeled with [(3)H]arachidonic acid were measured in the presence and absence of triacsin C. RESULTS: Inhibition by triacsin C of palmitoyl CoA formation was significantly greater than inhibition of arachidonoyl CoA formation in solubilized endothelial cell preparations. While 24-hour triacsin C treatment significantly reduced basal 6-keto synthesis, it had no effect on agonist-stimulated synthesis. The release of arachidonic acid metabolites was examined in [(3)H]arachidonate-labeled cells. Triacsin C treatment had no effect on basal or vasopressin-, angiotensin-II-, bradykinin- or ionomycin-induced release of radioactivity, but significantly reduced release in response to isoproterenol or phenylephrine. Expression of neither immunoreactive eNOS nor immunoreactive inducible nitric oxide synthetase (iNOS) was changed by triacsin C treatment, but the fraction of immunoreactive eNOS in the cytoplasm of treated cells was significantly greater as compared to vehicle control cells. Phorbol myristoyl acetate or fenofibrate significantly increased in vitro LCFACoAS activity, and significantly decreased the nitrite/eNOS ratio. CONCLUSIONS: These data indicate that, while triacsin C can inhibit arachidonoyl CoA synthetase in endothelial cells, it does not increase the availability of endogenous substrate for basal or agonist-induced PGI(2) synthesis, nor does it enhance release of arachidonic acid or its metabolites.

Inhibiting long chain fatty Acyl CoA synthetase increases basal and agonist-stimulated NO synthesis in endothelium.

OBJECTIVES: Endothelial nitric oxide synthase (eNOS) activation/deactivation is associated with cyclic depalmitoylation/repalmitoylation of specific Cys residues. The mechanism of depalmitoylation has been identified recently, but repalmitoylation remains undefined. We hypothesized that long chain fatty acyl CoA synthetase (LCFACoAS) modulates endothelial nitric oxide synthase repalmitoylation by limiting palmitoyl CoA availability. METHODS: Human coronary endothelial cells were treated with triacsin-C, an inhibitor of long chain fatty acyl CoA synthetase, for 24 h. Media nitrite accumulation, eNOS activity, and eNOS palmitoylation were measured. Methacholine-induced NO synthesis or vascular relaxation were measured in endothelium-intact rat aortae in the presence and absence of triacsin-C. RESULTS: Triacsin-C significantly reduced incorporation of [3H] palmitate into immunoreactive endothelial nitric oxide synthase and over a concentration range of 0.1 to 10 microM, increased media nitrite accumulations 2- to 2.5-fold over baseline. Total in vitro catalytic activity of nitric oxide synthase in triacsin-C treated cells did not differ significantly from control. Triacsin-C significantly increased methacholine-induced NO synthesis in the isolated rat aorta, and significantly enhanced methacholine-induced relaxation of rat aortic rings. CONCLUSIONS: These data are consistent with the interpretation that inhibition of palmitoylation increases endothelial nitric oxide synthase activity without changing endothelial nitric oxide synthase expression, suggesting that inhibiting palmitoylation increases the catalytically active fraction of endothelial nitric oxide synthase.

Molecular characterization of a rabbit long-chain fatty acyl CoA synthetase that is highly expressed in the vascular endothelium.

The formation of coenzyme A thioesters from long-chain fatty acids represents a metabolic branch point. We have isolated, cloned and sequenced a long-chain fatty acyl CoA synthetase (LCFACoAS) that is localized to the endothelium of rabbit heart and aorta. Immunofluoresence and in situ hybridization studies show intense staining of the intimal layer of the aorta and coronary vessels. The microvessels, including the capillaries, of the coronary circulation also show intense immunofluoresence. The enzyme shares only about 30% to 70% homology with the primary amino acid sequence of the other known LCFACoAS. There is a region of 44 amino acids at the carboxy terminus, which is unique to the vascular enzyme. This domain contains the most hydrophobic region of the molecule, indicating that it may function as a membrane anchoring site. These results suggest that this LCFACoAS represents a novel isoform, whose functional significance remains to be determined.

Mg2+ efflux from the isolated perfused rabbit heart is mediated by two states of the beta1-adrenergic receptor.

The non-selective beta-adrenergic receptor agonist isoproterenol stimulates Mg(2+) efflux from the perfused heart. The beta-adrenergic receptor subtype governing Mg(2+) efflux was determined in rabbit hearts perfused by the method of Langendorff with Mg(2+)-free Krebs Henseleit buffer. Magnesium efflux was examined during infusion of isoproterenol (a non-selective beta-adrenergic agonist), dobutamine (beta(1)-selective), salbutamol (beta(2)-selective), BRL37344 in the presence of 200 nM propranolol (beta(3)-selective conditions) or CGP12177 (beta(3)/low affinity state beta(1)-selective). Isoproterenol increased Mg(2+) efflux in a dose-dependent manner, and was the most potent and efficacious agent used. Dobutamine and CGP12177 each significantly increased Mg(2+) efflux, but with markedly different time characteristics. Dobutamine induced significantly less Mg(2+) release than isoproterenol. Although the maximal effect of CGP12177 on Mg(2+) release was 30% less than that of isoproterenol, the difference was not statistically significant. Neither salbutamol nor BRL37344 had any effect on Mg(2+) efflux. These results suggest that isoproterenol-induced Mg(2+) efflux is mediated by both the high and low affinity states of the beta(1)AR, with the low affinity state making the larger contribution.