Chapter 6: MOLECULAR AND CELLULAR BIOLOGY OF BLOOD VESSELS ENDOTHELIAL CELL-VASCULAR SMOOTH MUSCLE INTERACTIONS Endothelial Control of Vascular Tone
The endothelium serves a dual function in the control of vascular tone Fig. 6-4). It secretes relaxing factors such as nitric oxide and adenosine, and constricting factors such as the endothelins. Vessel tone thus depends on the balance between these factors, as well as on the ability of the smooth muscle cell to respond to them. The most important regulatory molecules are discussed separately.
ENDOTHELIUM-DERIVED RELAXING FACTOR/NITRIC OXIDE
An EDRF was first described by Furchgott and Zawadzki,2 who observed that aortic rings dilated in response to acetylcholine only when the rings maintained an intact endothelium. The predominant form of EDRF, derived from L-arginine by the action of the enzyme nitric oxide synthase (NOS), is nitric oxide (NO), or a closely related nitroso compound.62
Many factors have been shown to regulate the release of EDRF/NO69 by increasing intracellular Ca2+. These include hormones such as acetylcholine, norepinephrine, bradykinin, thrombin, ATP, and vasopressin; the platelet-derived factors, serotonin and histamine; fatty acids; ionophores; and physical forces. NO easily crosses the smooth muscle cell membrane and binds to the heme moiety of the soluble guanylate cyclase, thereby enhancing the formation of cyclic GMP. Cyclic GMP, in turn, reduces intracellular Ca2+ concentrations leading todephosphorylation of the myosin light chain and relaxation.70 It should be noted that the drug nitroglycerin exerts its vasodilator effects by being converted to NO, thus substituting for a natural product. Deficiency in release of active NO is an important contributing factor leading to vasospasm.
NO is produced by the action of the enzyme NOS, which oxidizes the guanidino nitrogens of L-arginine to form citrulline and NO. This enzyme has been cloned from brain (nNOS, for neuronal NOS, type I),74 macrophages (iNOS, for inducible NOS, type II),72 and endothelial cells (eNOS, for endothelial NOS, type III).73 The three isoforms of NOS share important consensus sequences for NADPH, flavin adenine dinucleotide, and flavin mononucleotide cofactor-binding sites, as well as a Ca2+-calmodulin-binding site. During the past several years, a great deal has been learned about how these enzymes function.74 All NO synthases function as homodimers, and each subunit consists of a carboxy-terminal reductase domain and an amino-terminal oxygenase domain, connected by a calmodulin-binding region. The NADPH- and flavin-binding sites reside in the reductase domain, where electrons derived from NADPH are stored by the flavins. For both the neuronal and endothelial isoforms of the enzymes, increases in intracellular calcium lead to calmodulin binding to the calmodulin-binding site, which in turn enables electrons to flow from the reductase domain to the amino-terminal oxygenase domain. This region contains binding sites for heme, tetrahydrobiopterin, and L-arginine. Electrons transferred from the reductase domain are initially bound by the ferrous iron in the prosthetic heme group. The precise role of tetrahydrobiopterin remains unknown, although it appears critical in allowing electrons to be transferred from the heme to the guanidino nitrogens of L-arginine, resulting in the formation of NO. Interestingly, when tetrahydrobiopterin or L-arginine is absent, the electron flows to molecular oxygen, resulting in the formation of the superoxide anion.75 This phenomenon has been termed uncoupling of NOS, and there are substantial data that this may occur in a variety of disease states, perhaps because of oxidation of tetrahydrobiopterin.
Although increases in intracellular calcium clearly activate eNOS via stimulation of calmodulin, there are additional ways that the enzyme is activated that seem independent of calmodulin or calcium. For example, shear stress acutely stimulates the release of NO from the endothelium, and this depends only on calcium during the first few seconds of the response.76 The continued activation of eNOS in response to several minutes or hours of shear seems independent of calcium and calmodulin. Phosphorylation of eNOS is almost certainly important in this calcium-independent stimulation.77 Recently, specific sites of the enzyme have been identified that are phosphorylated in response to shear.78 Phosphorylation by the kinase Akt leads to a calcium-independent activity of the enzyme.79 Other phosphorylation sites have also been implicated.
Although expression of the endothelial enzyme (eNOS) was originally thought to be constitutive, it is now clear that its expression is highly regulated. Increases in shear stress rather markedly enhance expression of eNOS.80 Likewise, low shear is associated with a decrease in eNOS expression. Exercise training dramatically increases eNOS expression in endothelial cells, likely because of the increased shear stress caused by the high cardiac output that accompanies sustained exercise.84 In contrast, inflammatory cytokines such as TNF-'J decrease eNOS expression.^3 This is caused by destabilization of eNOS mRNA, rather than by decreasing the rate of eNOS transcription.82 Several other conditions and stimuli seem to alter eNOS expression by changing the half-life of mRNA. These include exposure to oxidized LDL,83 hypoxia,84 and changes in endothelial cell growth state.85 The mechanisms underlying regulation of eNOS mRNA stability are incompletely understood, but are the focus of intense investigation.
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