The endothelial nitric-oxide synthase (eNOS) is a key determinant of vascular homeostasis. Like all known nitric-oxide synthases, eNOS enzyme activity is dependent on Ca 2؉ -calmodulin. eNOS is dynamically targeted to specialized cell surface signal-transducing domains termed plasmalemmal caveolae and interacts with caveolin, an integral membrane protein that comprises a key structural component of caveolae. We have previously reported that the association between eNOS and caveolin is quantitative and tissue-specific (Feron, O., Belhassen, L., Kobzick, L., Smith, T. W., Kelly, R. A., and Michel, T. (1996) J. Biol. Chem. 271, 22810 -22814). We now report that in endothelial cells the interaction between eNOS and caveolin is importantly regulated by Ca 2؉ -calmodulin. Addition of calmodulin disrupts the heteromeric complex formed between eNOS and caveolin in a Ca 2؉ -dependent fashion. In addition, overexpression of caveolin markedly attenuates eNOS enzyme activity, but this inhibition is reversed by purified calmodulin. Caveolin overexpression does not affect the activity of the other NOS isoforms, suggesting eNOSspecific inhibition of NO synthase by caveolin. We propose a model of reciprocal regulation of eNOS in endothelial cells wherein the inhibitory eNOS-caveolin complex is disrupted by binding of Ca 2؉

Nitric oxide production in the vascular endothelium is promoted by diverse agonists that transiently increase intracellular Ca 2؉ concentration and activate the endothelial nitric-oxide synthase (eNOS), a Ca 2؉ /calmodulindependent enzyme. eNOS is acylated by the fatty acids myristate and palmitate and is targeted thereby to plasmalemmal signal-transducing domains termed caveolae. eNOS enzyme activity is markedly attenuated by its interactions with caveolin, the structural scaffolding protein of caveolae. We have discovered that in living cells, the eNOS-caveolin heteromeric complex undergoes cycles of dissociation and re-association modulated by Ca 2؉ -mobilizing agonists. Calcium ionophore A23187 and the muscarinic cholinergic agonist carbachol both promote the dissociation of eNOS from caveolin in cultured cells, associated with translocation of eNOS from caveolae. As [Ca 2؉ ] i returns to basal levels, eNOS reassociates with caveolin, and the inhibited enzyme complex is then restored to caveolae, a process accelerated by palmitoylation of the enzyme. These data establish an eNOS-caveolin regulatory cycle, wherein enzyme activation is modulated by reversible protein-protein interactions controlled by Ca The endothelial isoform of nitric-oxide synthase (eNOS) 1 is robustly expressed in the vascular endothelium and in cardiac myocytes, and the cellular regulation of eNOS may represent an important determinant of cardiovascular homeostasis (reviewed in Ref. 1). In endothelial cells and in cardiac myocytes, eNOS is targeted to specialized invaginations of the plasmalemma termed caveolae (2). Plasmalemmal caveolae serve as sites for the sequestration of signaling proteins and are further characterized by the presence of caveolin, an intrinsic membrane protein that forms a structural "scaffold," organizing both proteins and lipids within this key membrane organelle (3, 4). Caveolin directly interacts with several structurally distinct signaling proteins in caveolae, including G proteins and cellular oncogenes (4) as well as eNOS (2, 5-9). The activity of purified eNOS, a Ca 2ϩ /calmodulin-dependent enzyme (10, 11), is markedly attenuated by its interaction with caveolin (5-9). We have also shown that purified Ca 2ϩ /calmodulin can overcome the inhibitory interaction between eNOS and caveolin in vitro (5, 7, 9), but the relevance of these observations to the dynamic regulation of eNOS in endothelial cells is less well understood. In vascular endothelial cells and in cardiac myocytes, the cycle of eNOS activation and deactivation is intimately coupled to the changes in intracellular Ca 2ϩ that are promoted by stimulation of diverse G protein-coupled receptors (12,13). In this report, we describe a series of experiments that have explored the relationships between intracellular Ca 2ϩ regulation and the dynamics of eNOS-caveolin interactions in living cells. We also document the role of eNOS palmitoylation in the reversible caveolar targeting of the eNOS-caveolin complex following muscarinic cholinergic stimulat...

We have shown previously that a GC-rich element (GGGGCGGGGTGGGGGG) conferring epidermal growth factor (EGF) responsiveness to the human gastrin promoter binds Sp1 and additional undefined complexes. A rat GH4 cell line expression library was screened by using a multimer of the gastrin EGF response element, and three overlapping cDNA clones were identified. The full-length rat cDNA encoded an 89-kDa zinc finger protein (ZBP-89) that was 89% identical to a 49-kDa human factor, ht(beta), that binds a GTGGG/CACCC element in T-cell receptor promoters. The conservation of amino acids between the zinc fingers indicates that ZBP-89 is a member of the C2H2 zinc finger family subclass typified by the Drosophila Krüppel protein. ZBP-89 is ubiquitously expressed in normal adult tissues. It binds specifically to the gastrin EGF response element and inhibits EGF induction of the gastrin promoter. Collectively, these results demonstrate that ZBP-89 functions as a repressor of basal and inducible expression of the gastrin gene.

The renin-anglotensin system (RAS) is the most important regulatory system of electrolyte homeostasis and blood pressure. We report here the development of transgenic rats carrying the human angiotensinogen TGR-(hAOGEN) and human renin TGR(hREN) genes. The plasma levels and tissue distribution of the transcription and translation products from both genes are described. A unique species specificity of the enzyme kinetics was observed. The human RAS components in the transgenic rats did not interact with the endogenous rat RAS in vivo. Insead, infusions of exogenous human RAS components specifically interacted with human transgene translation products. Thus, iion of human renin in TGR(hAOGEN) led to an increase of angioensin H and an elevation of blood pressure, which could not be antagonized by the human-specific renin enzyme inhibitor Ro 42-5892. Rat renin also elevated blood pressure and angiotensin H in TGR(hAOGEN); however, this effect was not antagonized by the human renin inhibitor. Compared to mice, rats offer the advantage of chronic instrumentation and repetitive, sophisticated, hemodynamic, and endocrinological investigations. Thus, transgenic rat models with human-specific enzyme kinetics permit primate-specific analyses in non-primate in vivo and in vitro experimental systems.The successful incorporation of renin-angiotensin (RAS) genes into transgenic mice has been reported (1, 2). Rats, on the other hand, present specific technical problems with respect to the generation of transgenic animals. The transgenic rats TGR(mREN2)27, which harbor a mouse renin gene and exhibit fulminant hypertension, provided evidence for a monogenetic form of hypertension (3).Transgenic mice harboring the genes of mouse renin and angiotensinogen (2), rat renin and angiotensinogen (4), as well as human renin (1) have been described. Thus far, most ofthe RAS gene constructs used for the generation of transgenic animals interacted directly with the host RAS (2, 4).Since the genetic background for the transgenes appears to be of primary importance for the development of hypertension (5), we developed transgenic rats harboring the complete human renin TGR(hREN) and human angiotensinogen TGR(hAOGEN) genes. These rats allow studies into the regulation of human genes in non-primate animal models (6). In vitro, the species specificity of human renin and angiotensinogen is well known. Transgenic animals permit the study of specific interactions with the human transgene products in vivo, without interference from the host RAS.EXPERIMENTAL PROCEDURES Transgenic Techniques. Linear DNA fiagments consisting of either the entire human renin gene (1, 7) or the entire human angiotensinogen gene (8), as described elsewhere (1, 3), were injected into the male pronuclei offertilized, outbred Sprague-Dawley (SD) rat eggs.Immnnalyses of Renin and en. Human renin was immunologically measured in transgenic rat plasma. The direct and specific measurement of human renin used two pairs of monoclonal antibodies in an immunoradiometric assay. ...

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...

N-Terminal myristoylation and thiopalmitoylation of the endothelial isoform of nitric oxide synthase (eNOS) are required for targeting the enzyme to specialized signal-transducing microdomains of plasma membrane termed caveolae. We have previously documented that the subcellular localization of eNOS is dynamically regulated by agonists such as bradykinin, which promotes enzyme depalmitoylation and translocation from caveolae. More recently, we have shown that association of eNOS with caveolin, the principal structural protein in caveolae, leads to enzyme inhibition, in a reversible process modulated by Ca2+-calmodulin (CaM). We now report studies of the respective roles of acylation and caveolin interaction for regulating eNOS activity. Using eNOS truncation and deletion mutants expressed in COS-7 cells, we have identified an obligatory role for the N-terminal half of eNOS in stabilizing its association with caveolin. By exploring the differential effects of detergents (CHAPS vs octyl glucoside), we have shown that this direct interaction between both proteins is facilitated by, but does not require, eNOS acylation, and, importantly, that treatment of intact aortic endothelial cells with the calcium ionophore A23187 leads to the rapid disruption of the eNOS-caveolin complexes. Finally, using transiently transfected COS-7 cells, we have observed that the myristoylation-deficient cytosol-restricted eNOS mutant (myr-) as well as the cytosolic fraction of the palmitoylation-deficient eNOS mutant (palm-) may both interact with caveolin; this association also leads to a marked inhibition of enzyme activity, which is completely reversed by addition of calmodulin. We conclude that the regulatory eNOS-caveolin association is independent of the state of eNOS acylation, indicating that agonist-evoked Ca2+/CaM-dependent disruption of the caveolin-eNOS complex, rather than agonist-promoted depalmitoylation of eNOS, relieves caveolin's tonic inhibition of enzyme activity. We therefore propose that caveolin may serve as an eNOS chaperone regulating NO production independently of the enzyme's residence within caveolae or its state of acylation.

A synthetic oligonucleotide probe complementary to messenger ribonucleic acid (mRNA) encoding for the C-terminal portion of atrial natriuretic factor (ANF) has been used to study the expression of the ANF gene in rat myocardium. Four experimental models were studied: binephrectomy (for 48 h); ligature of both ureters (for 48 h); deoxycorticosterone acetate-salt (for 3 wk); and aortocaval fistula (for 2 wk). Analysis of atrial RNA by gel-blot hybridization detected a single band, corresponding in length to that of mRNA coding for ANF. Such an mRNA was also detected in ventricular RNA but was 1/50th as abundant. In the four experimental groups ANF mRNA was increased significantly as compared with controls. In all rats there was no significant difference in the ANF mRNA content between the left and the right atrium. Each experimental condition was accompanied by a highly significant increase in ANF gene expression in the left ventricle, where all of the ventricular tissue could be recruited and with a negative gradient from the base to the apex of the left ventricle. These data were confirmed by in situ hybridization. Thus all of the atrial and ventricular myocardium can express the ANF gene. Recruitment increases in response to passive stretch of the cardiac chambers.

Estradiol increases rat aorta EDRF activity in the absence of changes in endothelial NO synthase gene expression. The decreased endothelium-derived generation of O2-. in response to estrogens could account for enhanced EDRF-NO bioactivity and decreased peroxynitrite release. All of these effects could contribute to the vascular protective properties of estrogens.