Expression and regulation of endothelial nitric oxide synthase.

Endothelium-derived nitric oxide (NO) is a key determinant of blood pressure homeostasis and platelet aggregation and is synthesized by the endothelial isoform of nitric oxide synthase (eNOS). In the vascular wall, eNOS is activated by diverse cell-surface receptors and by increases in blood flow, and the consequent generation of NO leads to vascular smooth-muscle relaxation. Endothelium-dependent vasorelaxation is deranged in a variety of disease states, including hypertension, diabetes, and atherosclerosis, but the roles of eNOS in endothelial dysfunction remain to be clearly defined. The past several years have witnessed important advances in understanding the molecular and cellular biology of eNOS regulation. In endothelial cells, eNOS undergoes a complex series of covalent modifications, including myristoylation, palmitoylation, and phosphorylation. Palmitoylation of eNOS dynamically targets the enzyme to distinct domains of the endothelial plasma membrane termed caveolae; caveolae may serve as sites for the sequestration of signal-transducing proteins and are themselves subject to dynamic regulation by ligands and lipids. Originally thought to be expressed only in endothelial cells, eNOS is now known to be expressed in a variety of tissues, including blood platelets, cardiac myocytes, and brain hippocampus. Paradigms established in endothelial cells for the molecular regulation and subcellular targeting of eNOS are being extended to the investigation of eNOS expressed in nonendothelial tissues. This review summarizes recent advances in understanding the molecular regulation of eNOS and the other NOS isoforms and identifies important parallels between eNOS and other cell-signaling molecules. © 1997, Elsevier Science Inc. (Trends Cardiovasc Med 1997;7:28-37).

Preferential inhibition of inducible nitric oxide synthase by ebselen.

Ebselen, 2-phenyl-1,2-benzisoselenazole-3(2H)-one can preferentially inhibit the activity of inducible nitric oxide (NO) synthase with little inhibition of endothelial constitutive NO synthase within a certain concentration range. This suggests that ebselen deserves further in vivo studies to examine its possible application to the therapy of septic shock where inducible NO synthase is responsible for vasodilation.

Expression of constitutive endothelial nitric oxide synthase in human blood platelets.

Nitric oxide (NO) activates the soluble isoform of guanylate cyclase in platelets and inhibits platelet function. Several studies suggest the existence of a pathway for NO synthesis in platelets as a form of feedback inhibition, but the identity of the NO synthase (NOS) isoform present within platelets is unknown. We isolated human platelets, and synthesized cDNA from platelet RNA for analysis by PCR. Primers for human neuronal or inducible NOS failed to yield a PCR signal. However, primers specific for endothelial NOS (ecNOS) amplified a DNA band of the expected size. Analysis of nucleotide sequence revealed that the amplified DNA is ecNOS. NOS enzyme activity was detected in the platelet particulate subcellular fraction, as previously demonstrated for ecNOS in other cells. Thus, ecNOS is present in human platelets, and may play a role in the regulation of platelet function by an endogenous NO pathway.

Structure and function of nitric oxide synthases.

Nitric oxide (NO), which accounts for the biological activity of endothelium-derived relaxing factor, is now thought to play a variety of roles in the nervous system and in immunologic reactions. NO is synthesized from L-arginine by nitric oxide synthase (NOS). There are three isoforms of NOS; type I (neuronal), type II (inducible), and type III (endothelial). The fundamental structure of the three isoforms, which contain calmodulin-, FMN-, FAD-, and NADPH-binding domains, is the same. Calmodulin is already bound to inducible NOS without requiring Ca2+, while the others are Ca2+/calmodulin-dependent. Endothelial NOS is bound to membranes by N-myristoylation, while the other isoforms are soluble. The human endothelial NOS gene has been cloned. It has several highly repetitive regions which could provide potential sites for DNA polymorphism. It might be of interest to examine the relationship between such polymorphism and cardiovascular disorders.

Mass trial of hypoallergenic rice (HRS-1) produced by enzymatic digestion in atopic dermatitis with suspected rice allergy. HRS-1 Research Group.

The clinical usefulness of a hypoallergenic rice (HRS-1) which was produced by enzymatic decomposition of the constituent proteins of original rice was evaluated in a multicentre study in 44 subjects with recalcitrant atopic dermatitis (AD), who were suspected of having rice allergy. The subjects were fed for at least 4 weeks with HRS-1 instead of eliminating both regular rice and wheat from their daily diet. The extent of overall skin lesions was expressed by using the AD affected area and severity index (ADASI). A statistically significant decrease in ADASI was observed at the 2nd and the 4th week readings and at the end of the study. A provocation test with regular rice was carried out in 5 of 44 subjects following the HRS-1 therapy. An obvious increase in ADASI was found in all of these 5 cases just after this procedure. On final evaluation, 77% of the patients tested showed 'moderate' to 'remarkable' improvement, and 59% of the patients a 'moderate' to 'remarkable' reduction in use of the steroid ointment concomitantly used for the treatment. Finally, HRS-1 was evaluated as 'useful' or 'very useful' in 69% of the subjects.

Stabilization of inducible nitric oxide synthase by monoclonal antibodies.

We have produced 14 monoclonal antibodies to inducible nitric oxide synthase purified from rat peritoneal cytotoxic activated macrophages. None of the antibodies showed neutralizing activity, but some of them enhanced the enzyme activity through stabilization of the enzyme.

[Clinical effect of hypoallergenic rice (HRS-1) in atopic dermatitis. HRS-1 Research Group].

The usefulness of hypoallergenic rice (HRS-1) was clinically evaluated in 43 patients with severe atopic dermatitis (AD), who were suspected of having rice allergy, in collaboration with 13 hospitals. The patients were fed with HRS-1 instead of eliminating both regular rice and wheat from their daily diet. AD area and severity index (ADASI) was calculated as an indicator of the degree of cutaneous symptoms. Significant decrease of ADASI were observed in the 2nd and 4th week and at the end of the replacement therapy (5.6 weeks on average). On final evaluation, 74% of the patients tested showed "moderate" to "remarkable" improvement, and in 53% of the patients HRS-1 resulted in a "moderate" to "remarkable" reduction in the dosage and the grade of potency of the steroid ointment concomitantly used for the treatment. Finally, HRS-1 was evaluated as "useful" to "very useful" as the elimination diet in 70% of the patients.

Dynamic regulation of endothelial nitric oxide synthase: complementary roles of dual acylation and caveolin interactions.

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.

Primary percutaneous transluminal coronary angioplasty performed for acute myocardial infarction in a patient with idiopathic thrombocytopenic purpura.

A 72-year-old female with idiopathic thrombocytopenic purpura (ITP) complained of severe chest pain. Electrocardiography showed ST-segment depression and negative T wave in I, aVL and V4-6. Following a diagnosis of acute myocardial infarction (AMI), urgent coronary angiography revealed 99% organic stenosis with delayed flow in the proximal segment and 50% in the middle segment of the left anterior descending artery (LAD). Subsequently, percutaneous transluminal coronary angioplasty (PTCA) for the stenosis in the proximal LAD was performed. In the coronary care unit, her blood pressure dropped. Hematomas around the puncture sites were observed and the platelet count was 28,000/mm3. After transfusion, electrocardiography revealed ST-segment elevation in I, aVL and V1-6. Urgent recatheterization disclosed total occlusion in the middle segment of the LAD. Subsequently, PTCA was performed successfully. Then, intravenous immunoglobulin increased the platelet count and the bleeding tendency disappeared. A case of AMI with ITP is rare. The present case suggests that primary PTCA can be a useful therapeutic strategy, but careful attention must be paid to hemostasis and to managing the platelet count.

Production and epitope specificity of monoclonal antibody against mouse peptidylarginine deiminase type II.

Peptidylarginine deiminase catalyzes the conversion of arginyl residues in proteins to citrullyl residues in the presence of Ca2+. We described the preparation of monoclonal antibody (subclass type IgG1) specific to mouse peptidylarginine deiminase type II. The antibody had no effect on the enzyme activity and its specific epitope was localized in the eight-residue segment at the amino-terminal portion of the enzyme.

HA-1077 suppress both proliferation of vascular smooth muscle cells and c-fos mRNA induction.

HA1077 is a newly synthesized vasodilator with unique intracellular calcium antagonistic action. In this study, its effect on the growth of vascular smooth muscle cells (VSMC) stimulated by fetal calf serum was examined. Both the proliferation and [3H]thymidine incorporation into DNA of the growth-arrested VSMC was dose-dependently inhibited by HA1077. The expression of a proto-oncogene, c-fos, which reached the maximum 30 min after addition of serum, was similarly inhibited by this agent in a dose-dependent manner. Thus, HA1077 is expected to be a useful vasodilator agent capable of suppressing the growth of VSMC which is thought to be an important underlying mechanism of atherosclerosis or restenosis after angioplasty.

Caveolin versus calmodulin. Counterbalancing allosteric modulators of endothelial nitric oxide synthase.

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 Ca2+-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 Ca2+-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.

Expression of endothelial nitric oxide synthase in human and rabbit gastrointestinal smooth muscle cells.

The aim of this study was to identify the nitric oxide synthase (NOS) isoform expressed in freshly dispersed rabbit gastric smooth muscle cells and in cultured rabbit gastric, human intestinal, and guinea pig taenia coli smooth muscle cells. RT-PCR products of the predicted size (354 bp) were obtained with endothelial NOS (eNOS)-specific primers, but not neuronal NOS (nNOS)- or inducible NOS (iNOS)-specific primers, in all smooth muscle preparations except guinea pig taenia coli. Control RT-PCR studies showed absence of the endothelial markers, platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial growth factor receptor (VEGFR), and the interstitial cell marker, c-kit, from cultures of smooth muscle cells. Cloning and sequence analysis showed that the predicted amino acid sequence (117 residues) in rabbit and human smooth muscle cells differed by only one residue from that of human eNOS. Northern blot analysis, using the PCR-generated and cloned eNOS cDNA from rabbits and humans as probes, demonstrated the expression of eNOS mRNA (4.4 kb) in both species. eNOS, but not nNOS or iNOS, transcripts were localized by in situ RT-PCR in single, freshly dispersed rabbit gastric smooth muscle cells; expression was evident in the majority of cells in each preparation. We conclude that eNOS is selectively expressed in rabbit gastric and human intestinal smooth muscle cells. The results confirm functional evidence for the existence of a constitutive NOS in smooth muscle cells of the gut in different species, except for guinea pig taenia coli.

A stable L-arginine-dependent relaxing factor released from cytotoxic-activated macrophages.

It has been known that cytotoxic-activated macrophages release an unstable vasorelaxing substance, nitric oxide. We have found that a more stable relaxing factor is released from those cells. This factor seems to be synthesized from L-arginine. It acts through the activation of soluble guanylate cyclase without affecting membrane potential. It has little charge at nearly neutral pH, and its molecular weight is < 500.

Cloning and structural characterization of the human endothelial nitric-oxide-synthase gene.

Nitric oxide accounts for the activity of endothelium-derived relaxing factor, which seems to have an important role in vasodilation and inhibition of platelet aggregation. In endothelial cells, one isoform of nitric-oxide synthase is constitutively expressed. Analysis of the cDNA encoding the human endothelial nitric-oxide synthase revealed that the mRNA is 4.1 kb in size and that the translated protein consists of 1203 amino acids. We have cloned a genomic DNA encoding the human endothelial nitric-oxide synthase and analyzed the entire nucleotide sequence of the gene. The gene consists of 26 exons with a total size of 21 kb. The 5' flanking region of the gene lacks TATA boxes, but it contains putative Sp1-binding sites in (G+C)-rich regions. Of particular interest is the fact that a shear-stress-responsive element is located at position -985, which probably regulates the nitric-oxide-synthase gene in response to fluid mechanical forces at the transcriptional level in the vascular endothelium. Two minisatellite sequences are detectable in introns 2 and 8; a 32-bp consensus sequence repeats 38 times and a 57-bp consensus sequence repeats ten times. We found polymorphisms of the BamHI fragment containing the former minisatellite sequence in genomic DNA from pedigree family members. Furthermore, five tandem repeats of a 27-bp core consensus sequence and 35 repeats of a dinucleotide (CA) are located in introns 4 and 13, respectively. These repeat sequences will probably provide genetic markers for gene mapping and linkage analysis of inherited diseases including cardiovascular diseases.