15-hydroxy-11-alpha-9-alpha-(epoxymethano)prosta-5-13-dienoic-acid and rottlerin

Vascular endothelial cells (ECs), which regulate vascular tonus, serve as a barrier at the interface of vascular tissue. It is generally believed that alteration of this barrier is correlated with diabetic complications; however, a detailed mechanism has not been elucidated. This study examined alteration of bovine arterial EC functions stimulated by a thromboxane A2 analog (9,11-dideoxy-11 alpha,9 alpha-epoxymethano prostaglandin F(2 alpha); U46619) under normal and high-glucose (HG) conditions. U46619 treatment increased EC layer permeability in a time- and dose-dependent fashion. This response initially disrupted calcium-dependent EC-to-EC connections, namely, vascular endothelial cadherin (VE-CaD). Thereafter, EC force development in association with morphological changes was detected employing a reconstituted EC fiber technique, resulting in paracellular hole formation in the EC layer. Thus, we confirmed that U46619-induced enhancement of EC layer permeability involves these sequential steps. Similar trials were performed using a concentration twice that of normal glucose (22.2 mM glucose for 48 h). This treatment significantly enhanced U46619-induced EC layer permeability; furthermore, increases in both rate of VE-CaD disruption and EC fiber contraction were evident. Inhibition of calcium-independent protein kinase C and diacylglycerol kinase indicated that the glucose-dependent increase in VE-CaD disruption was mediated by a calcium-independent mechanism. Moreover, EC contraction was regulated by a typical calcium-independent pathway associated with rho kinase and actin stress fiber. Contraction was also enhanced under HG conditions. This investigation revealed that glucose-dependent enhancement of EC layer permeability is related to increases in VE-CaD disruption and EC contraction. Increases in both parameters were mediated by alteration of a calcium-independent pathway.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetophenones; Animals; Benzopyrans; Benzoquinones; Cadherins; Calcium; Capillary Permeability; Cattle; Cell Communication; Cells, Cultured; Egtazic Acid; Endothelial Cells; Enzyme Inhibitors; Extracellular Space; Glucose; Image Processing, Computer-Assisted; Immunohistochemistry; Isometric Contraction; Muscle Contraction; Muscle, Smooth, Vascular; Vasoconstrictor Agents

The effects of Gö-6976, a Ca(2+)-dependent protein kinase C (PKC) isozyme inhibitor, and rottlerin, a PKC-delta isozyme/calmodulin (CaM)-dependent kinase III inhibitor, on responses to vasopressor agents were investigated in the feline pulmonary vascular bed. Injections of angiotensin II, norepinephrine (NE), serotonin, BAY K 8644, and U-46619 into the lobar arterial constant blood flow perfusion circuit caused increases in pressure. Gö-6976 reduced responses to angiotensin II; however, it did not alter responses to serotonin, NE, or U-46619, whereas Gö-6976 enhanced BAY K 8644 responses. Rottlerin reduced responses to angiotensin II and NE, did not alter responses to serotonin or U-46619, and enhanced responses to BAY K 8644. Immunohistochemistry of feline pulmonary arterial smooth muscle cells demonstrated localization of PKC-alpha and -delta isozymes in response to phorbol 12-myristate 13-acetate and angiotensin II. Localization of PKC-alpha and -delta isozymes decreased with administration of Gö-6976 and rottlerin, respectively. These data suggest that activation of Ca(2+)-dependent PKC isozymes and Ca(2+)-independent PKC-delta isozyme/CaM-dependent kinase III mediate angiotensin II responses. These data further suggest that Ca(2+)-independent PKC-delta isozyme/CaM-dependent kinase III mediate responses to NE. A rottlerin- or Gö-6976-sensitive mechanism is not involved in mediating responses to serotonin and U-46619, but these PKC isozyme inhibitors enhanced BAY K 8644 responses in the feline pulmonary vascular bed.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Acetophenones; Angiotensin II; Animals; Benzopyrans; Calcium Channel Agonists; Carbazoles; Cats; Drug Interactions; Enzyme Inhibitors; Female; Free Radical Scavengers; Immunohistochemistry; Indoles; Isoenzymes; Male; Muscle, Smooth, Vascular; Norepinephrine; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-alpha; Pulmonary Circulation; Serotonin; Vasoconstriction; Vasoconstrictor Agents