05-611 Sigma-AldrichAnti-phospho-VASP (Ser239) Antibody, clone 16C2

In cultured human vascular smooth muscle cells, insulin increases cyclic GMP production by inducing nitric oxide (NO) synthesis. The aim of the present study was to determine whether in these cells the insulin-stimulated NO/cyclic GMP pathway plays a role in the regulation of glucose uptake.Glucose transport in human vascular smooth muscle cells was measured as uptake of 2-deoxy-d-[3H]glucose, cyclic GMP synthesis was checked by radioimmunoassay, and GLUT4 recruitment into the plasma membrane was determined by immunofluorescence. Insulin-stimulated glucose transport and GLUT4 recruitment were blocked by an inhibitor of NO synthesis and mimicked by NO-releasing drugs. Insulin- and NO-elicited glucose uptake were blocked by inhibitors of soluble guanylate cyclase and cyclic GMP-dependent protein kinase; furthermore, glucose transport was stimulated by an analog of cyclic GMP.Our results suggest that insulin-elicited glucose transport (and the corresponding GLUT4 recruitment into the plasma membrane) in human vascular smooth muscle cells is mediated by an increased synthesis of NO, which stimulates the production of cyclic GMP and the subsequent activation of a cyclic GMP-dependent protein kinase.

In experimental animal models, long-term in vivo treatment with nitroglycerin (NTG) induces both endothelial dysfunction and tolerance to nitrates. However, it is still controversial whether nitrate tolerance in humans is associated with both endothelial dysfunction and impaired vascular response to nitrovasodilator-derived NO.

Chronic in vivo treatment with nitroglycerin (NTG) induces tolerance to nitrates and cross-tolerance to nitrovasodilators and endothelium-derived nitric oxide (NO). We previously identified increased vascular superoxide formation and reduced NO bioavailability as one causal mechanism. It is still controversial whether intracellular downstream signaling to nitrovasodilator-derived NO is affected as well.

The development and functional analysis of a monoclonal antibody (16C2) are reported; the antibody recognizes vasodilator-stimulated phosphoprotein (VASP; an established substrate of both cAMP- and cGMP-dependent protein kinase) only when serine 239 is phosphorylated. VASP serine 239 represents one of the best characterized cGMP-dependent protein kinase phosphorylation sites in vitro and in intact cells. Experiments with purified, recombinant human VASP and various VASP constructs with mutated phosphorylation sites (S157A, S239A, T278A) and experiments with intact cells (human/rat platelets and other cells) treated with cyclic nucleotide-elevating agents demonstrated the specificity of the monoclonal antibody 16C2. Quantitative analysis of the VASP shift from 46 to 50 kDa (indicating VASP serine 157 phosphorylation) and the appearance of VASP detected by the 16C2 monoclonal antibody (VASP serine 239 phosphorylation) in human platelets stimulated by selective protein kinase activators confirmed that serine 239 is the VASP phosphorylation site preferred by cGMP-dependent protein kinase in intact cells. Immunofluorescence experiments with human platelets treated with cGMP analogs showed that the 16C2 monoclonal antibody also detects VASP serine 239 phosphorylation in situ at established intracellular localization sites. Analysis of VASP serine 239 phosphorylation by the 16C2 antibody appears to be the best method presently available to measure cGMP-dependent protein kinase activation in intact cells. Also, the 16C2 antibody promises to be an excellent tool for the evaluation of VASP function in intact cells.