Circulation research Editors' yearly report: 1999-2000
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Journal Article↗
Biomedical subjects
R Busse
Publications and source records attributed to R Busse.
An endothelium-derived hyperpolarizing factor distinct from NO and prostacyclin is a major endothelium-dependent vasodilator in resistance vessels of wild-type and endothelial NO synthase knockout mice.
In addition to nitric oxide (NO) and prostacyclin (PGI(2)), the endothelium generates the endothelium-derived hyperpolarizing factor (EDHF). We set out to determine whether an EDHF-like response can be detected in wild-type (WT) and endothelial NO synthase knockout mice (eNOS -/-) mice. Vasodilator responses to endothelium-dependent agonists were determined in vivo and in vitro. In vivo, bradykinin induced a pronounced, dose-dependent decrease in mean arterial pressure (MAP) which did not differ between WT and eNOS -/- mice and was unaffected by treatment with N(omega)-nitro-l-arginine methyl ester and diclofenac. In the saline-perfused hindlimb of WT and eNOS -/- mice, marked N(omega)-nitro-l-arginine (l-NA, 300 micromol/liter)- and diclofenac-insensitive vasodilations in response to both bradykinin and acetylcholine (ACh) were observed, which were more pronounced than the agonist-induced vasodilation in the hindlimb of WT in the absence of l-NA. This endothelium-dependent, NO/PGI(2)-independent vasodilatation was sensitive to KCl (40 mM) and to the combination of apamin and charybdotoxin. Gap junction inhibitors (18alpha-glycyrrhetinic acid, octanol, heptanol) and CB-1 cannabinoid-receptor agonists (Delta(9)-tetrahydrocannabinol, HU210) impaired EDHF-mediated vasodilation, whereas inhibition of cytochrome P450 enzymes, soluble guanylyl cyclase, or adenosine receptors had no effect on EDHF-mediated responses. These results demonstrate that in murine resistance vessels the predominant agonist-induced endothelium-dependent vasodilation in vivo and in vitro is not mediated by NO, PGI(2), or a cytochrome P450 metabolite, but by an EDHF-like principle that requires functional gap junctions.
Acetylcholine↗
A gp91phox containing NADPH oxidase selectively expressed in endothelial cells is a major source of oxygen radical generation in the arterial wall.
Reactive oxygen species (ROS) play an important role in regulating vascular tone and intracellular signaling; the enzymes producing ROS in the vascular wall are, however, poorly characterized. We investigated whether a functionally active NADPH oxidase similar to the leukocyte enzyme, ie, containing the subunits p22phox and gp91phox, is expressed in endothelial cells (ECs) and smooth muscle cells (SMCs). Phorbol 12-myristate 13-acetate (PMA), a stimulus for leukocyte NADPH oxidase, increased ROS generation in cultured ECs and endothelium-intact rat aortic segments, but not in SMCs or endothelium-denuded arteries. NADPH enhanced chemiluminescence in all preparations. p22phox mRNA and protein was detected in ECs and SMCs, whereas the expression of gp91phox was confined to ECs. Endothelial gp91phox was identical to the leukocyte form as determined by sequence analysis. In contrast, mitogenic oxidase-1 (mox1) was expressed in SMCs, but not in ECs. To determine the functional relevance of gp91phox expression, experiments were performed in aortic segments from wild-type, gp91phox(-/-), and endothelial NO synthase (eNOS)(-/-) mice. PMA-induced ROS generation was comparable in aortae from wild-type and eNOS(-/-) mice, but was attenuated in segments from gp91phox(-/-) mice. Endothelium-dependent relaxation was greater in aortae from gp91phox(-/-) than from wild-type mice. The ROS scavenger tiron increased endothelium-dependent relaxation in segments from wild-type, but not from gp91phox(-/-) mice. These data demonstrate that ECs, in contrast to SMCs, express a gp91phox-containing leukocyte-type NADPH oxidase. This enzyme is a major source for arterial ROS generation and affects the bioavailability of endothelium-derived NO.
Acetylcholine↗
Under new management: A six-month progress report on Circulation Research.
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Cardiovascular Diseases↗
Ultrastructure and molecular histology of rabbit hind-limb collateral artery growth (arteriogenesis).
Previous studies in the canine heart had shown that the growth of collateral arteries occurs via proliferative enlargement of pre-existing arteriolar connections (arteriogenesis). In the present study, we investigated the ultrastructure and molecular histology of growing and remodeling collateral arteries that develop after femoral artery occlusion in rabbits as a function of time from 2 h to 240 days after occlusion. Pre-existent arteriolar collaterals had a diameter of about 50 microm. They consisted of one to two layers of smooth muscle cells (SMCs) and were morphologically indistinguishable from normal arterioles. The stages of arteriogenesis consisted of arteriolar thinning, followed by transformation of SMCs from the contractile- into the proliferative- and synthetic phenotype. Endothelial cells (ECs) and SMCs proliferated, and SMCs migrated and formed a neo-intima. Intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) showed early upregulation in ECs, which was accompanied by accumulation of blood-derived macrophages. Mitosis of ECs and SMCs started about 24 h after occlusion, whereas adhesion molecule expression and monocyte adhesion occurred as early as 12 h after occlusion, suggesting a role of monocytes in vascular cell proliferation. Treatment of rabbits with the pro-inflammatory cytokine MCP-1 increased monocyte adhesion and accelerated vascular remodeling. In vitro shear-stress experiments in cultured ECs revealed an increased phosphorylation of the focal contacts after 30 min and induction of ICAM-1 and VCAM-1 expression between 2 h and 6 h after shear onset, suggesting that shear stress may be the initiating event. We conclude that the process of arteriogenesis, which leads to the positive remodeling of an arteriole into an artery up to 12 times its original size, can be modified by modulators of inflammation.
Animals↗
EDHF: a cytochrome P450 metabolite in coronary arteries.
Endothelium-dependent relaxation cannot be fully attributed to the release of nitric oxide or prostacyclin (PGI2). In resistance-sized vessels and coronary arteries a high proportion of endothelium-dependent relaxation, in response to agonist-induced or mechanical stimulation of endothelial cells, can be attributed to the release of 1 or more endothelium-derived hyperpolarizing factor (EDHF). In coronary arteries EDHF has been pharmacologically characterized as a cytochrome P450 (CYP)-derived metabolite of arachidonic acid. We show here that a CYP 2C arachidonic acid epoxygenase, homologous to CYP 2C8/9, is expressed in cultured human endothelial cells and native porcine coronary artery endothelial cells. Down-regulation of CYP 2C protein by transfection of porcine coronary arteries with anti-sense oligonucleotides decreased EDHF-mediated vascular responses while EDHF-mediated hyperpolarisation and relaxation were potentiated by the CYP-inducing compound beta-naphthoflavone. Thus, CYP 2C appears to play a crucial role in the generation of EDHF-mediated responses in porcine coronary arteries.
Animals↗
Health technology assessment in Germany. Status, challenges, and development.
The objectives of this paper are to describe the status and development of health technology regulation with regard to coverage decisions and utilization, and to analyze the current situation of health technology assessment (HTA) in Germany. The relevant literature for controlling health technologies was identified by searching the literature and databases and through personal contacts. The literature was analyzed with regard to the different sectors in the healthcare system. For the analysis of the current state of HTA in Germany, a national survey was carried out. In addition, the names of topics under assessment were collected. The results show that coverage decisions in the ambulatory sector appear to be much more regulated than those in the inpatient sector. The same is true for diffusion and usage of technologies. The strict separation of the hospital and the ambulatory care sector in Germany constitutes a barrier to regulation and to making HTA an effective instrument in Germany. Until recently, HTA in Germany focused on biotechnology, such as gene technology. Recently the German Scientific Working Group of Technology Assessment in Health Care has adopted a systematic methodology to undertake HTAs. It can be concluded that regulation of health technologies in Germany is characterized by inconsistencies ranging from strict regulation in the ambulatory sector to almost no regulation in the rehabilitation sector. Increasing interest in HTA, in conjunction with a high priority assigned to evaluation of health technologies by the newly elected government, is likely to improve this situation in the future.
Ambulatory Care↗
The progestin levonorgestrel induces endothelium-independent relaxation of rabbit jugular vein via inhibition of calcium entry and protein kinase C: role of cyclic AMP.
The progestin and oestrogen component of oral contraceptives have been involved in the development of venous thromboembolic events in women. In the present study we determined the vasoactive effects of sex steroids used in oral contraceptives in isolated preconstricted rabbit jugular veins in the presence of diclofenac and examined the underlying mechanisms. The natural hormone progesterone, the synthetic progestins levonorgestrel, 3-keto-desogestrel, gestodene and chlormadinone acetate, and the synthetic estrogen 17 alpha-ethinyloestradiol induced concentration-dependent relaxations of endothelium-intact veins constricted with U46619. Levonorgestrel also inhibited constrictions evoked by either a high potassium (K(+)) solution or phorbol myristate acetate (PMA) in the absence and presence of extracellular calcium (Ca(2+)). In addition, levonorgestrel depressed contractions evoked by Ca(2+) and reduced (45)Ca(2+) influx in depolarized veins. Relaxations to levonorgestrel in U46619-constricted veins were neither affected by the presence of the endothelium nor by the inhibitor of soluble guanylyl cyclase, NS2028, but were significantly improved either by the selective cyclic AMP phosphodiesterase inhibitor rolipram or in the absence of diclofenac, and decreased by the protein kinase A inhibitor, Rp-8-CPT-cAMPS. Rolipram also potentiated relaxations to levonorgestrel in PMA-constricted veins in the presence, but not in the absence of extracellular Ca(2+). Levonorgestrel increased levels of cyclic AMP and inhibited PMA-induced activation of protein kinase C in veins. These findings indicate that levonorgestrel caused endothelium-independent relaxations of jugular veins via inhibition of Ca(2+) entry and of protein kinase C activation. In addition, the cyclic AMP effector pathway contributes to the levonorgestrel-induced relaxation possibly by depressing Ca(2+) entry.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
Phosphorylation and activation of the endothelial nitric oxide synthase by fluid shear stress.
Fluid shear stress activates the endothelial nitric oxide (NO) synthase (eNOS) by a mechanism which does not require an increase in the intracellular concentration of free Ca2+ ([Ca2+]i), and is sensitive to several kinase inhibitors. Although phosphorylation of eNOS has been suggested to regulate enzyme activity, the mechanism of eNOS activation is still unclear. Here we demonstrate that fluid shear stress elicits the phosphorylation of eNOS on tyrosine and serine residues. Inhibition of phosphatidylinositol 3-kinase (PI3K), using wortmannin or a dominant negative mutant of its downstream target, Akt (protein kinase B), prevented the maintained serine phosphorylation and activation of eNOS. Enhancing eNOS phosphorylation by inhibiting serine/threonine phosphatases, increased eNOS activity by approximately twofold, as assessed by the accumulation of intracellular cyclic GMP, without increasing the intracellular concentration of free Ca2+. These data suggest that shear stress activates a pathway involving PI3K and the serine/threonine kinase Akt, which phosphorylates eNOS. This phosphorylation directly increases eNOS activity at resting [Ca2+]i, thus rendering the shear stress-induced activation of eNOS apparently Ca2+-independent.
Amino Acid Sequence↗
Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca(2+) changes and dilation in isolated resistance arteries.
Using a novel vessel culture technique in combination with antisense oligonucleotide transfection, we tested whether the endothelium-derived hyperpolarizing factor (EDHF) is a cytochrome P450 (CYP)-related compound. Isolated resistance arteries from hamster gracilis muscle (n=19) were perfused and exposed to antisense (As), sense (S), or scrambled (Scr) oligonucleotides against the coding region of CYP2C8/9, an isoform expressed in endothelial cells. Thereafter, NO- and prostaglandin-independent, EDHF-mediated vascular responses associated with hyperpolarization [i.e., decrease in smooth muscle calcium (Fura 2) and vasodilation] were studied after the application of acetylcholine (ACh). These EDHF-mediated responses were markedly attenuated (by 70%) by As- but not by S- or Scr-oligonucleotide treatment. However, the responses to norepinephrine (0.3 micromol/l), the NO donor sodium nitroprusside (1 micromol/l), and the K(Ca) channel activator NS1619 (100 micromol/l) were unaltered. As treatment, which specifically targeted the endothelial layer (as assessed by confocal microscopy), had no inhibitory effect on increases in endothelial calcium to ACh. It is concluded that a CYP2C8/9-related isoform functions as an EDHF synthase in hamster resistance arteries and that a product of this enzyme is an EDHF, or at least an integral part of the signaling cascade leading to EDHF-mediated responses.-Bolz, S.-S., Fisslthaler, B., Pieperhoff, S., de Wit, C., Fleming, I., Busse, R., Pohl, U. Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca(2+) changes and dilation in isolated resistance arteries.
Acetylcholine↗
Increased nitrovasodilator sensitivity in endothelial nitric oxide synthase knockout mice: role of soluble guanylyl cyclase.
Endogenously produced nitric oxide (NO) modulates nitrovasodilator-induced relaxation. We investigated the underlying mechanism in wild-type (WT) mice and endothelial NO synthase knockout (eNOS(-/-)) mice to determine whether a chronic lack of endothelial NO alters the soluble guanylyl cyclase (sGC) pathway. In aortic segments from eNOS(-/-) mice, the vasodilator sensitivity to sodium nitroprusside (SNP) was significantly greater than that in WT mice. There was no difference in sensitivity to the G-kinase I activator 8-para-chlorophenylthio-cGMP or to cromakalim. N(omega)-Nitro-L-arginine had no effect on the SNP-induced relaxation in eNOS(-/-) but increased the sensitivity in WT mice so it was no longer different than that of eNOS(-/-). Basal cGMP levels in aortic rings were significantly lower in eNOS(-/-) mice than in WT mice. SNP (300 nmol/L) induced a significantly greater cGMP accumulation in eNOS(-/-) mice than in WT mice. The maximal SNP-induced (10 micromol/L) increase in cGMP was similar in both strains. SNP-stimulated sGC activity was significantly greater in eNOS(-/-) mice than in WT mice. Incubation of aortic segments from WT mice with N(omega)-nitro-L-arginine increased sGC activity, an effect prevented by coincubation with SNP (10 micromol/L). The aortic expressions of the sGC alpha1 and beta1 subunits in WT and eNOS(-/-) mice were identical as determined with Western blot analysis. These data suggest that chronic exposure to endothelium-derived NO, as well as acute exposure to nitrovasodilator-derived NO, desensitizes sGC to activation by NO but does not alter sGC expression. Both the acute cessation of endothelial NO formation in WT mice and the chronic deficiency of NO in eNOS(-/-) mice restore the NO sensitivity of sGC and enhance vascular smooth muscle relaxation in response to nitrovasodilator agents.
Animals↗
Nifedipine increases cytochrome P4502C expression and endothelium-derived hyperpolarizing factor-mediated responses in coronary arteries.
In addition to NO and prostacyclin, endothelial cells release a factor that elicits vasodilatation by hyperpolarizing the underlying vascular smooth muscle cells. In some vascular beds, this so-called endothelium-derived hyperpolarizing factor (EDHF) displays the characteristics of a cytochrome P450 (CYP)-derived arachidonic acid metabolite, such as an epoxyeicosatrienoic acid. Native porcine and cultured human coronary artery endothelial cells were screened for CYP epoxygenases, and CYP2B, CYP2C, and CYP2J were detected with reverse transcription-polymerase chain reaction. The CYP inducer beta-naphthoflavone and the Ca(2+) antagonist nifedipine significantly increased CYP2C mRNA but did not change the expression of CYP2J or CYP2B. To determine the relationship between CYP2C expression and EDHF production in native endothelial cells, we incubated porcine coronary arteries with nifedipine. Nifedipine enhanced endothelial CYP2C protein expression, as well as the generation of 11,12-epoxyeicosatrienoic acid. In organ bath experiments, pretreatment with nifedipine enhanced bradykinin-induced, EDHF-mediated relaxations as well as the concomitant hyperpolarization of smooth muscle cells. The specific CYP2C9 inhibitor sulfaphenazole, on the other hand, significantly attenuated EDHF-mediated hyperpolarization and relaxation. These results demonstrate that in porcine coronary arteries, the elevated expression of a CYP epoxygenase, homologous to CYP2C8/9, is associated with enhanced EDHF-mediated hyperpolarization in response to bradykinin. Therefore, we propose that an isozyme of CYP2C is the most likely candidate for the CYP-dependent EDHF synthase in porcine coronary arteries.
8,11,14-Eicosatrienoic Acid↗
Drug-drug interactions in medical patients: effects of in-hospital treatment and relation to multiple drug use.
OBJECTIVES: Adverse drug reactions (ADRs) are a major cause of hospital admissions, thereby leading to significant medical and economical problems. Drug interactions contribute to a major part of ADRs, especially in elderly patients and in patients under polymedication. As a peculiarity of the German health care system the general practitioner is less involved in patient care during hospital stay than in other countries. Consequently, changes in medication at the transition point from out-patient to in-patient care and back may contribute to drug-related problems. PATIENTS: In the present study we investigated potential interactions in 169 consecutive patients with the diagnosis CHD (coronary heart disease) or COLD (chronic obstructive lung disease) who were admitted to the University Hospitals of Hannover and Magdeburg. METHODS: For each patient, potential interactions between prescribed drugs at admission, at discharge, and 3 months after discharge were assessed by using drug interaction data bases. RESULTS: We found that the number of drugs taken per patient as well as the number of interactions per patient are higher during hospitalization than before admission (pre-admission), and fall back after the hospital stay (post-discharge), but not to the pre-admission level. The number of potential interactions was significantly correlated in a polynomial manner to the number of drugs taken by each patient. The number of patients without a potential interaction was 44.4% pre-admission, 39.6% at discharge, and 39.1% post-discharge. Patients with potential interactions had a mean of 2.8, 2.7, and 2.4 interactions at each of the time points. Drug classes mainly involved with potential interactions were kaliuretic diuretics (recorded by 43.3% at discharge), ACE inhibitors (30.3%), anticoagulants and aggregation inhibitors (20.4%) and digitalis glycosides (14.7%). Regarding the frequency of the interaction categories, 68-70% of the potential interactions demand clinical attention, while 1-2% are life-threatening. 17-19% may result in therapeutic benefit; 10-12% are without clinical relevance. CONCLUSIONS: The risk of drug interactions increases exponentially with the number of drugs given to a patient. Although we have no data on the fraction of interactions that became clinically manifest, our study indicates that prescribing fewer drugs can reduce the risk of suffering from sickness secondary to drug interactions. Taking this risk into account may help to improve the quality of drug treatment and to save costs.
Adult↗
Oxidative stress and expression of p22phox are involved in the up-regulation of tissue factor in vascular smooth muscle cells in response to activated platelets.
Vascular injury after balloon angioplasty results in the rapid activation of platelets leading to the release of growth factors and vasoactive substances. In addition, up-regulation of tissue factor (TF) and an increased production of reactive oxygen species (ROS) have been detected at sites of vascular injury. We investigated whether platelet-derived products (PDP) released from activated human platelets increase ROS production, resulting in the induction of TF expression in vascular smooth muscle cells (SMC). PDP induced a time- and concentration-dependent increase in ROS generation in cultured SMC that was mediated mainly by PDGF-AB and TGF-beta1 and impaired by the flavin inhibitor diphenylene iodonium. Increased ROS formation was associated with enhanced mRNA levels of the small NAD(P)H oxidase subunit p22phox or its smooth muscle isoform. Transient transfection with a p22phox antisense vector decreased PDP-induced ROS generation. PDP up-regulated TF mRNA expression, which was redox sensitive and reduced by transfection of the p22phox antisense vector. In addition, PDP-stimulated reporter gene activity of two TF promoter constructs was decreased by coexpression of the p22phox antisense vector. These results indicate that activated platelets up-regulate TF expression and that this response involves ROS generation and a p22phox-containing NAD(P)H oxidase in SMC.
Animals↗
[Cost comparison of hospital and ambulatory i.v. therapy in adult cystic fibrosis patients. Results of a controlled prospective study].
BACKGROUND: The regular i.v.-therapy of adults with cystic fibrosis (CF) on an in-patient basis is regarded as expensive. Home treatment is supposed to be cheaper. During a prospective controlled study to compare in-patient treatment (SIT) with home i.v.-treatment (HIT) in regard to clinical, psychosocial and economic parameters, delivered health services and costs in the German setting were evaluated in a comparable manner. PATIENTS AND METHODS: During the study period 4/95 to 9/96 45 patients with altogether 56 hospital and 40 home i.v.-courses of approximately 14 days were included in the study. Principal instruments to record the resource consumption were documentation sheets kept by the medical staff and the patients. In addition, pharmacy accounts in HIT and patient records and hospital controlling data in SIT were used. RESULTS: The average costs of a course were 14,038 DM for HIT and 18,702 DM for SIT. Striking are the large differences in medication costs. Two main reasons could be identified for the cost difference: 1, the use of a mobile infusion system in HIT and 2, the much higher prices of pharmaceuticals in the ambulatory care sector compared to the hospital sector, where extremely high profits of the home care service firms or the pharmacy can be supposed. The social costs of HIT (when antibiotics are valued to opportunity costs) are estimated at less than 10,500 DM. From a societal perspective HIT is preferable, from the perspective of the statutory health insurance funds hospital therapy is preferable. CONCLUSIONS: 1. The widely accepted hypothesis that ambulatory care is cheaper than hospital care is--at least in the German setting--not a-priori true. 2. In the treatment of CF patients incentive failures exist which induce unnecessary and avoidable hospital stays if the perspective of the statutory sickness funds is dominant. 3. Changes in system conditions as e.g. the permission of mail-order pharmacies might help to implement a more rational allocation of resources.
Adult↗
Cytochrome P450 2C is an EDHF synthase in coronary arteries.
In most arterial beds a significant endothelium-dependent dilation to various stimuli persists even after inhibition of nitric oxide synthase and cyclo-oxygenase. This dilator response is preceded by an endothelium-dependent hyperpolarization of vascular smooth muscle cells, which is sensitive to a combination of the calcium-dependent potassium-channel inhibitors charybdotoxin and apamin, and is assumed to be mediated by an unidentified endothelium-derived hyperpolarizing factor (EDHF). Here we show that the induction of cytochrome P450 (CYP) 2C8/34 in native porcine coronary artery endothelial cells by beta-naphthoflavone enhances the formation of 11,12-epoxyeicosatrienoic acid, as well as EDHF-mediated hyperpolarization and relaxation. Transfection of coronary arteries with CYP 2C8/34 antisense oligonucleotides results in decreased levels of CYP 2C and attenuates EDHF-mediated vascular responses. Thus, a CYP-epoxygenase product is an essential component of EDHF-mediated relaxation in the porcine coronary artery, and CYP 2C8/34 fulfils the criteria for the coronary EDHF synthase.
8,11,14-Eicosatrienoic Acid↗
Aggregating human platelets stimulate expression of vascular endothelial growth factor in cultured vascular smooth muscle cells through a synergistic effect of transforming growth factor-beta(1) and platelet-derived growth factor(AB).
BACKGROUND: Vascular endothelial growth factor (VEGF), an endothelial mitogen and chemoattractant, has been implicated in the recovery of the endothelium after balloon injury. The increased expression of VEGF in vascular smooth muscle cells (SMC) at sites of injury suggests that this cell type may be a major cellular source of VEGF. This study examined whether aggregating platelets stimulate VEGF expression in cultured SMC. METHODS AND RESULTS: ++VEGF expression in SMC was assessed by Northern blot analysis and by reverse transcription followed by polymerase chain reaction and the release of VEGF by Western blot analysis and immunoassay. Platelet-derived products (PDP) released by aggregating human platelets time-dependently and concentration-dependently enhanced VEGF mRNA levels, mainly that coding for the soluble splice variant VEGF(165/164), and stimulated the release of VEGF protein. These effects were potentiated by transient acidification of PDP, which release bioactive transforming growth factor (TGF)-beta(1), and mimicked by platelet-derived growth factor (PDGF)(AB) and TGF-beta(1) in a synergistic manner. Both a TGF-beta-neutralizing antibody and a PDGF-neutralizing antibody significantly attenuated the effect of acidified PDP on VEGF production. CONCLUSIONS: Aggregating human platelets induce VEGF mRNA expression in cultured SMC and the subsequent release of VEGF protein. This effect can be attributed to a supra-additive action of PDGF(AB) and TGF-beta(1) and may represent a novel mechanism by which platelets contribute to the recovery of the endothelial lining at sites of balloon-injured arteries.
Angioplasty, Balloon↗
Signal transduction of eNOS activation.
Consistent with its classification as a Ca2+/calmodulin-dependent enzyme the constitutive endothelial nitric oxide (NO) synthase (eNOS) can be activated by receptor-dependent and -independent agonists as a consequence of an increase in the intracellular concentration of free Ca2+ ([Ca2+]i) and the association of the Ca2+/calmodulin complex with eNOS. Additional post-translational mechanisms regulate the activity of eNOS, including the interaction of eNOS with caveolin-1, heat shock protein 90 (Hsp90), or membrane phospholipids, as well as enzyme translocation and phosphorylation. In response to fluid shear stress the maintained production of NO by native and cultured endothelial cells is associated with only a transient increase in [Ca2+]i. In the absence of extracellular Ca2+ and in the presence of calmodulin antagonists, shear stress stimulates a maintained production of NO which is insensitive to the removal of extracellular Ca2+, but sensitive to tyrosine kinase inhibitors, Hsp90-binding proteins and phosphatidylinositol 3-kinase inhibitors. A pharmacologically identical activation of eNOS can be induced by protein tyrosine phosphatase inhibitors suggesting that the phosphorylation of eNOS, and possibly that of an associated regulatory protein(s), is crucial for its Ca(2+)-independent activation.
Animals↗