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The endothelial nitric oxide synthase (eNOS), the expression of which is regulated by a range of transcriptional and posttranscriptional mechanisms, generates nitric oxide (NO) in response to a number of stimuli. The physiologically most important determinants for the continuous generation of NO and thus the regulation of local blood flow are fluid shear stress and pulsatile stretch. Although eNOS activity is coupled to changes in endothelial cell [Ca.sup.2+] levels, an increase in [Ca.sup.2+] alone is not sufficient to affect enzyme activity because the binding of calmodulin (CaM) and the flow of electrons from the reductase to the oxygenase domain of the enzyme is dependent on protein phosphorylation and dephosphorylation. Two amino acids seem to be particularly important in regulating eNOS activity and these are a serine residue in the reductase domain ([Ser.sup.1177]) and a threonine residue ([Thr.sup.495]) located within the CaM-binding domain. Simultaneous alterations in the phosphorylation of [Ser.sup.1177] and [Thr.sup.495] in response to a variety of stimuli are regulated by a number of kinases and phosphatases that continuously associate with and dissociate from the eNOS signaling complex, eNOS associated proteins, such as caveolin, heat shock protein 90, eNOS interacting protein, and possibly also motor proteins provide the scaffold for the formation of the protein complex as well as its intracellular localization. calmodulin; caveolin; mRNA stability; phosphatase; serine phosphorylation; threonine phosphorylation

Angiogenesis is one of the most recent physiological functions attributed to products of cytochrome P-450 (CYP450) enymes. To test this at a molecular level in human cells, we used a cloned cDNA for the human endothelial enzyme CYP450 2C9 (CYP2C9) to study growth as well as differentiation of human microvascular endothelial cells from the lung (HMVEC-L). Using adenoviral vectors overexpressing mRNA for CYP2C9, we show that the presence of CYP2C9 doubles thymidine incorporation and stimulates proliferation of primary cultures of endothelial cells compared with Ad5-GFP (control) in 24 h. In addition, there is a significant increase of tube formation in Matrigel after infection of HMVEC-L with Ad5-2C9 than with Ad5-GFP. More interestingly, Ad5-2C9 expressing the antisense product of CYP2C9 (2C9AS) inhibited tube formation compared with both Ad5-GFP as well as the Ad5-2C9 constructs. Finally, we tested the most abundant arachidonic acid metabolite of CYP2C9, 14,15-epoxyeicosatrienoic acid, which induced angiogenesis in vivo when embedded in Matrigel plugs and implanted in adult rats. These data support an important role for CYP2C9 in promoting angiogenesis. cytochrome P-450 2C9; recombinant adenovirus

Endothelial cells, which are situated at the interface between blood and the vessel wall, have a crucial role in controlling vascular tone and homeostasis, particularly in determining the expression of pro-atherosclerotic and anti-atherosclerotic genes. Many of these effects are mediated by changes in the generation and release of endothelium-derived autacoids [from the Greek autos (self) and akos (remedy)], which are generally short-lived and locally acting. In vivo, endothelial cells are constantly subjected to mechanical stimulation, which in turn determines the acute production of autacoids and the levels of autacoid-producing enzymes.

Endothelial cells synthesize and release vasoactive mediators in response to various neurohumoural substances (e.g. bradykinin or acetylcholine) and physical stimuli (e.g. cyclic stretch or fluid shear stress). The best-characterized endothelium-derived relaxing factors are nitric oxide and prostacyclin. However, an additional relaxant pathway associated with smooth muscle hyperpolarization also exists. This hyperpolarization was originally attributed to the release of an endothelium-derived hyperpolarizing factor (EDHF) that diffuses to and activates smooth muscle [K.sup.+] channels. More recent evidence suggests that endothelial cell receptor activation by these neurohumoural substances opens endothelial cell [K.sup.+] channels. Several mechanisms have been proposed to link this pivotal step to the subsequent smooth muscle hyperpolarization. The main concepts are considered in detail in this review.

It has been established that endothelial 11,12-epoxyeicosatrienoic acid production and endothelium-derived hyperpolarizing factor (EDHF)-mediated hyperpolarization and relaxation in porcine coronary arteries can be selectively modulated by changing the expression and activity of cytochrome P450 (CYP) 2C8/34. Transfection of coronary arteries with CYP 2C8/34 antisense oligonucleotides leads to lower levels of CYP 2C and attenuates EDHF-mediated vascular responses. It is clear that a CYP-epoxygenase product is a vital element of EDHF-mediated relaxation in the porcine coronary artery.

The production of nitric oxide (NO) by the endothelial NO synthase (eNOS) is fundamental in determining cardiovascular homeostasis. Viscous drag is an important stimulus for continued formation of NO. It is shown that the serine/threonine protein kinase Akt/PKB controls the activation of eNOS, resulting in increased NO production. This represents a novel Ca(super2+)-independent regulatory mechanism for eNOS activation.

The role of isometric contraction in initiating a signaling pathway similar to that elicited by fluid shear stress was studied. Whether the process had a role in enhanced endothelial NO production was also taken into account. The results showed that the application of a NOS inhibitor to endothelium-intact rabbit aortic rings elicited a major additional increase in tension dependent on the level of vascular smooth muscle precontraction. Isometric contraction was found to be a determinant for the synthesis of NO in arteries.

A study was conducted to assess the process by which nitric oxide (NO) and prostacyclin (PGI2) are let out from rabbit femoral arteries containing endothelium, in resting conditions and after incitement by shear stress or acetylcholine. The increased release of NO and PGI2 from the endothelium of perfused rabbit femoral arteries is due to an enhanced fluid shear pressure, increased flow at constant diameter or by vasoconstriction at constant flow.

The effects of the selective K+ -channel blocker glibenclamide on prostacyclin-induced vasodilation in rabbit hearts was investigated. The purpose was to test the hypothesis that prostacyclin and its stable analogue iloprost induce vasodilation in the coronary circulation by activating ATP-dependent K+ channels (K-ATP). The results supported the hypothesis that prostacyclin and iloprost-induced vasodilation in the coronary circulation is due to activation of K-ATP in coronary vascular smooth muscle.