Calcium Signalling and Autacoid Production in Endothelial Cells Are Modulated by Changes in Tyrosine Kinase and Phosphatase Activity

Abstract

The vascular endothelium is the source of a number of vasodilator and vasoconstrictor autacoids and is thus a key regulator of vascular homeostasis. We studied the effects of altering the balance between protein tyrosine kinase and phosphatase activity on Ca²⁺ signalling and phosphotyrosine levels in cultured human endothelial cells, as well as on autacoid production in native endothelial cells. In isolated segments of rabbit aorta and carotid artery, as well as in bovine coronary arteries, the tyrosine phosphatase inhibitors phenylarsine oxide (PAO) and sodium orthovanadate initiated endothelium-dependent relaxations which could be attributed to the release of nitric oxide and the endothelium-derived hyperpolarizing factor. In cultured endothelial cells incubation with PAO resulted in a time-dependent accumulation in 6-keto prostaglandin F_1α, the stable metabolite of prostacyclin, as well as in an increase in the intracellular concentration of free Ca²⁺ ([Ca²⁺]i). Inhibition of tyrosine kinases attenuated both the PAO-induced relaxation and the increase in endothelial [Ca²⁺]i. Western blot analysis of endothelial cells treated with the tyrosine phosphatase inhibitors revealed a time-dependent increase in the tyrosine phosphorylation of a series of bands in both the Triton X-100-soluble and Triton X-100-insoluble (cytoskeletal) fractions. These observations suggest that alterations in cellular levels of phosphotyrosine may have profound effects on vascular homeostasis by modulating Ca²⁺ signalling and autacoid production in endothelial cells.

Ca 2+ -Independent Activation of the Endothelial Nitric Oxide Synthase in Response to Tyrosine Phosphatase Inhibitors and Fluid Shear Stress

“…For example, erbstatin A, which abolished the phenylarsine oxide-induced vasodilatation as well as the shear stress-induced production of NO, 9 only slightly attenuated the vasodilator response to acetylcholine in carotid arteries. 27 Although it was initially presumed that the concomitant effects of phenylarsine oxide on [Ca 2ϩ ] i and endothelial NO production were related, 27 the results of the present study demonstrate that phenylarsine oxide also stimulates NOS III via a Ca 2ϩ -independent mechanism involving tyrosine kinase activation. The endothelial response elicited by phenylarsine oxide therefore exhibits characteristics identical to those thought to be exclusive to the shear stress-induced production of NO.…”

“…In native endothelial cells, however, low concentrations of sodium orthovanadate are able to elicit the immediate release of NO and completely relax maximally constricted arterial segments. 27 Thus, it is conceivable that a slowly developing tyrosine hyperphosphorylation, such as that reported using multipassaged endothelial cells, inhibits rather than stimulates NOS III. Despite efforts to determine the effect of shear stress on the tyrosine dephosphorylation of NOS III, no quantifiable data could be obtained because of the weak NOS III/phosphotyrosine signal in cultured endothelial cells.…”

“…For example, tyrosine kinase inhibitors have been shown to selectively attenuate Ca 2ϩ influx into agonist-stimulated endothelial cells, 26,27 whereas the tyrosine phosphatase inhibitors phenylarsine oxide and vanadate are reported to activate transmembranous Ca 2ϩ influx via an inositol 1,4,5-trisphosphateindependent mechanism. 25 However, there do appear to be marked differences in the role played by phosphotyrosine in mediating the endothelial response to receptor-dependent and receptor-independent stimulation.…”

Simultaneous in situ monitoring of intracellular Ca2+and NO in endothelium of coronary arteries

“…However, this Ca 2ϩ -independent eNOS activation conflicts with published results that eNOS did not have Ca 2ϩ -independent activity in broken cell preparations or as a purified enzyme (14,23). In this regard, PAO-induced NOS activation was even reported also to depend on Ca 2ϩ increase in cultured endothelial cells (8). It appears that intracellular Ca 2ϩ may also participate in the regulation of eNOS activity induced by PAO, even though it may not directly mediate eNOS activation.…”

Nitric oxide generation mediated by ?-adrenoceptors is impaired in platelets from patients with Type 2 diabetes mellitus

“…Phosphorylation of Thr 495 is generally associated with a decrease in enzyme activity [41,42]. While endothelial NO production can be modulated by tyrosine kinase inhibitors and tyrosine phosphatase inhibitors [43,44], little is known about which tyrosine residues are phosphorylated [45]. Thus changes in threonine or tyrosine phosphorylation of NOS3 could also explain our findings.…”