The endothelial nitric-oxide synthase (eNOS) is activated by transient increases in intracellular Ca2؉ elicited by stimulation of diverse receptors, including bradykinin B 2 receptors on endothelial cells. eNOS and B 2 receptors are targeted to specialized signal-transducing domains in the plasma membrane termed plasmalemmal caveolae. Targeting to caveolae facilitates eNOS activation following receptor stimulation, but in resting cells, eNOS is tonically inhibited by its interactions with caveolin, the scaffolding protein in caveolae. We used a quantitative approach exploiting immunofluorescence microscopy to investigate regulation of the subcellular distribution of eNOS in endothelial cells by bradykinin and Ca 2؉. In resting cells, most of the eNOS is localized at the cell membrane. However, within 5 min following addition of bradykinin, nearly all the eNOS translocates to structures in the cell cytosol; following more protracted incubations with bradykinin, most of the cytosolic enzyme subsequently translocates back to the cell membrane. The bradykinin-induced internalization of eNOS is completely abrogated by the intracellular Ca 2؉ chelator BAPTA; conversely, Ca 2؉ -mobilizing drugs and agonists promote eNOS translocation. These results establish that eNOS targeting to the membrane is labile and is subject to receptor-regulated Ca 2؉ -dependent reversible translocation, providing another point for regulation of NO-dependent signaling in the vascular endothelium.

Although estrogen is known to stimulate nitric oxide synthesis in vascular endothelium, the molecular mechanisms responsible for this effect remain to be elucidated. Using quantitative immunof luorescence imaging approaches, we have investigated the effect of estradiol on the subcellular targeting of endothelial nitric oxide synthase (eNOS) in bovine aortic endothelial cells. In unstimulated endothelial cells, eNOS is predominantly localized at the cell membrane. Within 5 min after the addition of estradiol, most of the eNOS translocates from the membrane to intracellular sites close to the nucleus. On more prolonged exposure to estradiol, most of the eNOS returns to the membrane. This effect of estradiol is evident at a concentration of 1 pM, and a maximal estradiol effect is seen at a concentration of 1 nM. Neither progesterone nor testosterone has any effect on eNOS distribution. After estradiol addition, a transient rise in intracellular Ca 2؉ concentration precedes eNOS translocation. Both the Ca 2؉ -mobilizing and eNOS-translocating effects of estradiol are completely blocked by the estrogen receptor antagonist ICI 182,780, and the intracellular Ca Estrogen was identified as a vasodilator nearly 60 years ago. In 1940, Reynolds and Foster reported marked dilation of the ear microvasculature within minutes after injection of estrogen into ovariectomized rabbits (1). The molecular mechanism underlying the estrogen-induced vasodilation is not defined. Several studies suggest that a key mediator of this vasodilator response could be the endothelium-derived relaxing factor, nitric oxide (NO), and that estrogen stimulates NO synthesis in vascular endothelium. Kawano et al. (2) found changes in endothelium͞NO-dependent vasomotion in parallel with the cyclical hormonal changes in premenopausal women, with the greatest potentiation at peak plasma levels of estrogen. Several groups (3-5) reported that estrogen potentiates or restores endothelium-dependent coronary vasodilation in postmenopausal women; Guetta et al. (6) showed that these effects are mediated by NO. Van Buren et al. (7) and Rosenfeld et al. (8) identified NO as the principal mediator of estrogen-induced dilation of ovine uterine vasculature. More recently, LantinHermoso et al. (9) and found that estrogen activates the endothelial isoform of NO synthase (eNOS) in cultured endothelial cells. Together, these studies suggest that estrogen acts as a vasodilator by stimulation of endothelial NO synthesis, although the molecular mechanism underlying this effect remains to be elucidated.eNOS is a Ca 2ϩ ͞calmodulin-dependent enzyme and is subject to a complex pattern of intracellular regulation, including co-and post-translational modifications and diverse interactions with other proteins and ligands (reviewed in refs. 11 and 12). In endothelial cells and cardiac myocytes eNOS is localized in specialized plasmalemmal signal-transducing domains termed caveolae; acylation of the enzyme by the fatty acids myristate and palmitate is required for targ...

Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases. The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal-transducing membrane domains termed plasmalemmal caveolae. Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca 2؉ -calmodulin (Michel, J. B., Feron, O., Sacks, D., and Michel, T. (1997) J. Biol. Chem. 272, 15583-15586). Caveolin also interacts with other structurally distinct signaling proteins via a specific region identified within the caveolin sequence (amino acids 82-101) that appears to subserve the role of a "scaffolding domain." We now report that the co-immunoprecipitation of eNOS with caveolin is completely and specifically blocked by an oligopeptide corresponding to the caveolin scaffolding domain. Peptides corresponding to this domain markedly inhibit nitric oxide synthase activity in endothelial membranes and interact directly with the enzyme to inhibit activity of purified recombinant eNOS expressed in Escherichia coli. The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reversed by Ca 2؉ -calmodulin. These studies establish that caveolin, via its scaffolding domain, directly forms an inhibitory complex with eNOS and suggest that caveolin inhibits eNOS by abrogating the enzyme's activation by calmodulin.The mammalian nitric oxide (NO) 1 synthases comprise a family of three related proteins and modulate diverse biological processes ranging from neurotransmission to vascular homeostasis to immunological surveillance (1, 2). These homodimeric proteins share similar overall catalytic schemes to produce NO by the NADPH-, heme-, and O 2 -dependent oxidation of L-arginine in a complex reaction involving numerous redox cofactors, and are absolutely dependent on the ability of the enzymes to be allosterically activated by Ca 2ϩ -calmodulin. The roles of calmodulin in NOS catalysis have been extensively studied, and the binding of calmodulin appears to facilitate interdomain electron transfer for all three enzyme isoforms. However, the NOS isoforms differ in important aspects of their Ca 2ϩ -dependence for enzyme activation by calmodulin. For the endothelial and neuronal nitric oxide synthase isoforms (termed eNOS and nNOS, respectively), transient changes in intracellular Ca 2ϩ promote calmodulin binding and enzyme activation; eNOS and nNOS are characteristically activated as a short-term response to receptor-dependent calcium transients. By contrast, the inflammation-related NOS (iNOS) binds calmodulin avidly and appears to be fully active even at low ambient intracellular calcium levels in immunostimulated cells.The different NOS isoforms may also be distinguished by their subcellular distribution. eNOS is unique among the NOS isoforms in being targeted to the signal-transducing membrane microdomains termed plasmalemmal caveolae (3). Plasmalemmal caveola...