Down-regulation of the expression of endothelial NO synthase is likely to contribute to glucocorticoid-mediated hypertension

Hypertension is a side effect of systemically administered glucocorticoids, but the underlying molecular mechanism remains poorly understood. Ingestion of dexamethasone by rats telemetrically instrumented increased blood pressure progressively over 7 days. Plasma concentrations of Na+ and K+ and urinary Na+ and K+ excretion remained constant, excluding a mineralocorticoid-mediated mechanism. Plasma NO2−/NO3− (the oxidation products of NO) decreased to 40%, and the expression of endothelial NO synthase (NOS III) was found down-regulated in the aorta and several other tissues of glucocorticoid-treated rats. The vasodilator response of resistance arterioles was tested by intravital microscopy in the mouse dorsal skinfold chamber model. Dexamethasone treatment significantly attenuated the relaxation to the endothelium-dependent vasodilator acetylcholine, but not to the endothelium-independent vasodilator S-nitroso-N-acetyl-d,l-penicillamine. Incubation of human umbilical vein endothelial cells, EA.hy 926 cells, or bovine aortic endothelial cells with several glucocorticoids reduced NOS III mRNA and protein expression to 60–70% of control, an effect that was prevented by the glucocorticoid receptor antagonist mifepristone. Glucocorticoids decreased NOS III mRNA stability and reduced the activity of the human NOS III promoter (3.5 kilobases) to ≈70% by decreasing the binding activity of the essential transcription factor GATA. The expressional down-regulation of endothelial NOS III may contribute to the hypertension caused by glucocorticoids.

Adenosine activates ATP-sensitive potassium channels in arterial myocytes via A2 receptors and cAMP-dependent protein kinase.

The mechanism by which the endogenous vasodilator adenosine causes ATP-sensitive potassium (KATP) channels in arterial smooth muscle to open was investigated by the whole-cell patch-clamp technique. Adenosine induced voltage-independent, potassium-selective currents, which were inhibited by glibenclamide, a blocker of KATP currents. Glibenclamide-sensitive currents were also activated by the selective adenosine A2-receptor agonist 2-p-(2-carboxethyl)-phenethylamino-5'-N- ethylcarboxamidoadenosine hydrochloride (CGS-21680), whereas 2-chloro-N6-cyclopentyladenosine (CCPA), a selective adenosine A1-receptor agonist, failed to induce potassium currents. Glibenclamide-sensitive currents induced by adenosine and CGS-21680 were largely reduced by blockers of the cAMP-dependent protein kinase (Rp-cAMP[S], H-89, protein kinase A inhibitor peptide). Therefore, we conclude that adenosine can activate KATP currents in arterial smooth muscle through the following pathway: (i) Adenosine stimulates A2 receptors, which activates adenylyl cyclase; (ii) the resulting increase intracellular cAMP stimulates protein kinase A, which, probably through a phosphorylation step, opens KATP channels.

Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and microfilament protein VASP (vasodilator-stimulated phosphoprotein).

VASP (vasodilator-stimulated phosphoprotein), an established substrate of cAMP- and cGMP-dependent protein kinases in vitro and in living cells, is associated with focal adhesions, microfilaments, and membrane regions of high dynamic activity. Here, the identification of an 83-kDa protein (p83) that specifically binds VASP in blot overlays of different cell homogenates is reported. With VASP overlays as a detection tool, p83 was purified from porcine platelets and used to generate monospecific polyclonal antibodies. VASP binding to purified p83 in solid-phase binding assays and the closely matching subcellular localization in double-label immunofluorescence analyses demonstrated that both proteins also directly interact as native proteins in vitro and possibly in living cells. The subcellular distribution, the biochemical properties, as well as microsequencing data revealed that porcine platelet p83 is related to chicken gizzard zyxin and most likely represents the mammalian equivalent of the chicken protein. The VASP-p83 interaction may contribute to the targeting of VASP to focal adhesions, microfilaments, and dynamic membrane regions. Together with our recent identification of VASP as a natural ligand of the profilin poly-(L-proline) binding site, our present results suggest that, by linking profilin to zyxin/p83, VASP may participate in spatially confined profilin-regulated F-actin formation.