Johann Bauersachs

Characterization of endothelium-derived hyperpolarizing factor as a cytochrome P450-derived arachidonic acid metabolite in mammals.

1. In addition to nitric oxide (NO) and prostacyclin (PGI2) an as yet unidentified endothelium‐derived hyperpolarizing factor (EDHF) contributes to the dilator effect of bradykinin in different vascular beds. We have investigated the nature and mechanism of action of this factor in freshly isolated bovine and porcine coronary artery segments which were preconstricted with the thromboxane mimetic U46619 (9,11‐dideoxy‐11 alpha, 9 alpha‐epoxymethano‐prostaglandin F2 alpha, 10‐30 nM). 2. The concentration‐response curve of bradykinin was significantly shifted to the right after inhibition of NO synthesis with NG‐nitro‐L‐arginine (L‐NNA, 30 microM), whereas cyclo‐oxygenase blockade with diclofenac (1 microM) had no effect. Preconstriction of the segments with potassium chloride (40‐60 mM) completely abrogated the NO/PGI2‐independent dilator response to bradykinin. In sandwich bioassay experiments, both the luminal and abluminal release of NO, but not that of EDHF, was readily detectable. 3. Inhibitors of Ca(2+)‐activated K+ channels (K+Ca), such as apamin (1 microM) and tetrabutylammonium (TBA, 3 mM), strongly attenuated the EDHF‐mediated bradykinin‐induced relaxation, while glibenclamide (3 microM), an inhibitor of K+ATP channels, had no effect. 4. These relaxations were also significantly inhibited by the phospholipase A2 inhibitor, quinacrine (30 microM), and the cytochrome P450 inhibitors, SKF525a (30‐100 microM) and clotrimazole (100 microM). Moreover, incubation of endothelium‐denuded coronary artery rings with a cytochrome P450‐derived arachidonic acid metabolite, 11,12‐epoxyeicosatetraenoic acid, elicited a concentration‐dependent (1‐10 microM) dilatation which was abolished both in the presence of TBA (3 mM) and following preconstriction of the segments with potassium chloride instead of U46619.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelial Dysfunction in Chronic Myocardial Infarction Despite Increased Vascular Endothelial Nitric Oxide Synthase and Soluble Guanylate Cyclase Expression Role of Enhanced Vascular Superoxide Production

BACKGROUND Endothelial dysfunction of the peripheral vasculature is a well-known phenomenon in congestive heart failure that contributes to the elevated peripheral resistance; however, the underlying mechanisms have not yet been clarified. METHODS AND RESULTS Dilator responses, the expression of protein and mRNA of the endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and soluble guanylate cyclase (sGC), and superoxide anion (O(2)(-)) and peroxynitrite production were determined in aortic rings from Wistar rats 8 weeks after myocardial infarction and compared with those in sham-operated animals. In rats with heart failure, the concentration-response curve of the endothelium-dependent vasodilator acetylcholine (after preconstriction with phenylephrine) was significantly shifted to the right, and the maximum relaxation was attenuated. Determination of expression levels of the 2 key enzymes for NO-mediated dilations, eNOS and sGC, revealed a marked upregulation of both enzymes in aortas from rats with heart failure, whereas iNOS expression was not changed. Pretreatment with exogenous superoxide dismutase partially restored the acetylcholine-induced relaxation in aortas from rats with heart failure. Aortic basal and NADH-stimulated O(2)(-) production assessed by use of lucigenin-enhanced chemiluminescence was significantly elevated in rats with chronic myocardial infarction. Peroxynitrite-mediated nitration of protein tyrosine residues was not different between the 2 groups of rats. CONCLUSIONS These results demonstrate that endothelial dysfunction in ischemic heart failure occurs despite an enhanced vascular eNOS and sGC expression and can be attributed to an increase in vascular O(2)(-) production by an NADH-dependent oxidase. By inactivation of NO, O(2)(-) production appears to be an essential mechanism for the endothelial dysfunction observed in heart failure.

Endothelial Dysfunction Coincides With an Enhanced Nitric Oxide Synthase Expression and Superoxide Anion Production

We investigated the effects of aortic banding-induced hypertension on the endothelium-dependent vasodilator responses in the aorta and coronary circulation of Sprague-Dawley rats. We studied the influence of hypertension on the endothelial nitric oxide synthase (NOS III) expression, assessed by Western blot and reverse transcription-polymerase chain reactions experiments, and on the superoxide anion (O2-) production. Two weeks after aortic banding, the endothelium-dependent relaxations were not altered. At this time, the expression of NOS III in the aorta and in confluent coronary microvascular endothelial cells (RCMECs) exhibited no marked changes, whereas O2- production was enhanced 1.9-fold in aortas from aortic-banded rats. Six weeks after aortic banding, the endothelium-dependent dilations were markedly impaired in the heart (50% decrease) and aorta (35% decrease). Analysis of NOS III protein and mRNA levels revealed marked increases in both aortas and confluent RCMECs (2.6- to 4-fold) from aortic-banded compared with sham-operated rats. There was no further increase in O2production in both the aorta and confluent RCMECs from aortic-banded rats. An enhanced nitrotyrosine protein level was also detected in the aorta from 6-week aortic-banded rats. These findings indicate that in hypertension induced by aortic banding, an enhanced O2- production alone is not sufficient to produce endothelial dysfunction. Endothelial vasodilator hyporesponsiveness was observed only when NOS III expression and O2- production were increased and was associated with the appearance of enhanced nitrotyrosine residues. This would suggest that the development of endothelial dysfunction is linked to an overproduction of not one, but two, endothelium-derived radicals that might lead to the formation of peroxynitrite.

Ca2+-Independent Activation of the Endothelial Nitric Oxide Synthase in Response to Tyrosine Phosphatase Inhibitors and Fluid Shear Stress

Fluid shear stress enhances NO formation via a Ca2+-independent tyrosine kinase inhibitor-sensitive pathway. In the present study, we investigated the effects of the protein tyrosine phosphatase inhibitor phenylarsine oxide and of fluid shear stress on endothelial NO production as well as on the membrane association and phosphorylation of the NO synthase (NOS) III. Phenylarsine oxide (10 micromol/L) induced an immediate and maintained NO-mediated relaxation of isolated rabbit carotid arteries, which was insensitive to the removal of extracellular Ca2+ and the calmodulin antagonist calmidazolium. This phenylarsine oxide-induced vasodilatation was unaffected by genistein but abrogated by the tyrosine kinase inhibitor erbstatin A. Incubation of native or cultured endothelial cells with phenylarsine oxide resulted in a time-dependent tyrosine phosphorylation of mainly Triton X-100-insoluble (cytoskeletal) proteins, along with a parallel change in the detergent solubility of NOS III, such that the enzyme was recovered in the cytoskeletal fraction. A similar, though slightly delayed, phenomenon was also observed after the application of fluid shear stress but not in response to any receptor-dependent agonist. Although Ca2+-independent NO formation was sensitive to erbstatin A, phenylarsine oxide treatment was associated with the tyrosine dephosphorylation of NOS III rather than its hyperphosphorylation. Proteins that also underwent redistribution in response to the tyrosine phosphatase inhibitor included paxillin, phospholipase C-gamma1, mitogen-activated protein kinase, and the tyrosine kinases Src and Fyn. We envisage that fluid shear stress and tyrosine phosphatase inhibitors may alter the conformation and/or protein coupling of NOS III, facilitating its interaction with specific phospholipids, proteins, and/or protein kinases that enhance/maintain its Ca2+-independent activation.

Effect of YC-1, an NO-independent, superoxide-sensitive stimulator of soluble guanylyl cyclase, on smooth muscle responsiveness to nitrovasodilators

1 We studied the effects of 3‐(5′‐hydroxymethyl‐2′furyl)‐1‐benzyl indazole (YC‐1) on the activity of purified soluble guanylyl cyclase (sGC), the formation of guanosine‐3′: 5′cyclic monophosphate (cyclic GMP) in vascular smooth muscle cells (VSMC), and on the tone of rabbit isolated aortic rings preconstricted by phenylephrine (PE). In addition, we assessed the combined effect of YC‐1, and either NO donors, or superoxide anions on these parameters. 2 YC‐1 elicited a direct concentration‐dependent activation of sGC (EC50 18.6 ± 2.0 μm), which was rapid in onset and quickly reversible upon dilution. YC‐1 altered the enzyme kinetics with respect to GTP by decreasing Km and increasing Vmax. Activation of sGC by a combination of sodium nitroprusside (SNP) and YC‐1 was superadditive at low and less than additive at high concentrations, indicating a synergistic activation of the enzyme by both agents. A specific inhibitor of sGC, 1H‐(1,2,4)‐oxdiazolo‐(4,3‐a)‐6‐bromo‐quinoxazin‐l‐one (NS 2028), abolished activation of the enzyme by either compound. 3 YC‐1 induced a concentration‐dependent increase in intracellular cyclic GMP levels in rat cultured aortic VSMC, which was completely inhibited by NS 2028. YC‐1 applied at the same concentration as SNP elicited 2.5 fold higher cyclic GMP formation. Cyclic GMP‐increases in response to SNP and YC‐1 were additive. 4 YC‐1 relaxed preconstricted endothelium‐denuded rabbit aortic rings in a concentration‐dependent manner (50% at 20 μM) and markedly increased cyclic GMP levels. Relaxations were inhibited by NS 2028. A concentration of YC‐1 (3 μm), which elicited only minor effects on relaxation and cyclic GMP, increased the vasodilator potency of SNP and nitroglycerin (NTG) by 10 fold and markedly enhanced SNP‐and NTG‐induced cyclic GMP formation. 5 Basal and YC‐1‐stimulated sGC activity was sensitive to inhibition by superoxide (O2−) generated by xanthine/xanthine oxidase, and was protected from this inhibition by superoxide dismutase (SOD). YC‐1‐stimulated sGC was also sensitive to inhibition by endogenously generated (O2− in rat preconstricted endothelium‐denuded aortic rings. Relaxation to YC‐1 was significantly attenuated in aortae from spontaneously hypertensive rats (SHR), which generated O2− at a higher rate than aortae from normotensive Wistar Kyoto rats (WKY). SOD restored the vasodilator responsiveness of SHR rings to YC‐1. 6 In conclusion, these results indicate that YC‐1 is an NO‐independent, O2−‐sensitive, direct activator of sGC in VSMC and exerts vasorelaxation by increasing intracellular cyclic GMP levels. The additive or even synergistic responses to NO‐donors and YC‐1 in cultured VSMC and isolated aortic rings apparently reflect the direct synergistic action of YC‐1 and NO on the sGC. The synergism revealed in this in vitro study suggests that low doses of YC‐1 may be of therapeutic value by permitting the reduction of nitrovasodilator dosage.

Display of the characteristics of endothelium-derived hyperpolarizing factor by a cytochrome P450-derived arachidonic acid metabolite in the coronary microcirculation.

1 In addition to nitric oxide (NO) and prostacyclin (PGI2) an endothelium‐derived factor, which hyperpolarizes vascular smooth muscle cells via activation of K+ channels, contributes to the dilator effect of bradykinin in different vascular beds. Since this so‐called endothelium‐derived hyperpolarizing factor (EDHF) also seems to play an important role in the coronary circulation, we investigated its nature and mechanism of action in the rat isolated perfused heart (Langendorff preparation). 2 Bolus injections of bradykinin (1, 10, and 100 pmol) elicited a transient dose‐dependent dilator response (e.g., 12 ± 2% decrease in coronary perfusion pressure (CPP) at 10 pmol bradykinin, n = 41). Administration of the cyclo‐oxygenase inhibitor, diclofenac (1 μm), augmented the bradykinin‐induced dilation approximately twofold (n = 9 P < 0.01). Combined treatment with the NO synthase inhibitor, NG‐nitro‐L‐arginine (30 μm) and diclofenac (1 μm) significantly reduced the duration, but increased the amplitude of the dilator response to bradykinin (27 ± 2% decrease in CPP, n = 24, P < 0.01). 3 The abolition of this NG‐nitro‐L‐arginine/diclofenac‐insensitive dilator response to bradykinin by tetrabutylammonium (0.3 μm), an inhibitor of Ca2+‐dependent K+ channels (4 ± 1% decrease in CPP, n = 6, P < 0.01), supports the view that the dilator compound released in the coronary microcirculation is EDHF. 4 This EDHF‐type dilation was reversibly inhibited by the phospholipase A2 inhibitor, quinacrine (3 μm, 9 ± 3% decrease in CPP, n = 6, P < 0.01) and by the cytochrome P450 inhibitor SKF525a (3 μm, 6 ± 1% decrease in CPP, n = 6, P < 0.01). 5 Tetrabutylammonium, quinacrine or SKF 525a did not affect the endothelium‐independent dilator response to sodium nitroprusside (1 nmol), indicating that these compounds did not affect smooth muscle relaxation in a non‐specific manner. 6 These findings suggest that in the coronary microcirculation bradykinin stimulates the release of a cytochrome P450‐derived arachidonic acid metabolite, which exhibits the characteristic features of EDHF.

A transferable, beta‐naphthoflavone‐inducible, hyperpolarizing factor is synthesized by native and cultured porcine coronary endothelial cells.

1. The vascular endothelium releases a hyperpolarizing factor (endothelium‐derived hyperpolarizing factor, EDHF) tentatively identified as a cytochrome P450‐derived arachidonic acid metabolite. However, there is still controversy concerning its transferability and identity. We designed a bioassay system for assessing EDHF release in which the membrane potential was recorded in cultured vascular smooth muscle cells located downstream from donor endothelial cells. 2. Under combined nitric oxide (NO) synthase and cyclo‐oxygenase blockade with NG‐nitro‐L‐arginine (100 mumol l‐1) and diclofenac (10 mumol l‐1), the superfusate from bradykinin (30 mumol l‐1)‐stimulated, cultured porcine coronary endothelial cells induced a distinct hyperpolarization followed by a depolarization. Direct application of bradykinin to the smooth muscle cells resulted solely in membrane depolarization. Similar results were obtained using bradykinin‐stimulated porcine coronary arteries as donor. 3. Single‐channel current measurements suggest that this EDHF‐induced hyperpolarization was elicited by the activation of Ca(2+)‐dependent K+ channels. 4. Increasing the transmural pressure within the donor segment significantly enhanced the duration, but not the amplitude of the hyperpolarization induced by the effluate from bradykinin‐stimulated donor segments. 5. Inhibition of P450 oxygenase activity with clotrimazole (3 mumol l‐1) or 17‐octadecynoic acid (3 mumol l‐1) abolished EDHF release from the coronary endothelium, while the P450‐derived arachidonic acid metabolite, 5,6‐epoxyeicosatrienoic acid, induced a hyperpolarization of detector smooth muscle cells almost identical to that induced by EDHF. Moreover, induction of P450 activity by beta‐naphthoflavone (3 mumol l‐1, 48 h), significantly increased the bradykinin‐induced release of EDHF. 6. These findings suggest that the vascular endothelium releases a transferable hyperpolarizing factor, chemically distinct from NO and prostacyclin, in response to agonists and mechanical stimulation. This beta‐naphthoflavone‐inducible EDHF appears to be a cytochrome P450‐derived metabolite of arachidonic acid, which elicits hyperpolarization by activation of Ca(2+)‐dependent K+ channels.

Vasodilator dysfunction in aged spontaneously hypertensive rats: changes in NO synthase III and soluble guanylyl cyclase expression, and in superoxide anion production

OBJECTIVE/METHODS Genetic hypertension is associated with an apparent endothelial dysfunction and impaired endothelium-dependent vasodilatation in response to increased flow and receptor-dependent agonists. However, the link between impaired vasodilatation and nitric oxide (NO) synthase expression is still unclear. In the present study, dilator responses were determined in the aorta and coronary circulation of 16 month old spontaneously hypertensive (SHR) and Wistar Kyoto rats (WKY). Changes in vascular reactivity were compared with alterations in superoxide anion production as well as endothelial NO synthase (NOS III) and soluble guanylyl cyclase expression. RESULTS In the isolated perfused heart both the bradykinin- and sodium nitroprusside-induced vasodilator responses were attenuated in SHR compared to WKY. Western blot analysis revealed a parallel reduction in NOS III expression in coronary microvascular endothelial cells from SHR. Superoxide anion production in aortae from SHR was markedly elevated over that of aortae from WKY, and was almost completely abolished by pretreatment with superoxide dismutase. Superoxide dismutase induced similar relaxations in phenylephrine-preconstricted aortic rings from both SHR and WKY, but failed to restore the attenuated acetylcholine- and sodium nitroprusside-induced relaxations in SHR. No difference in NOS III expression was detected in the aortae from either strain whereas soluble guanylyl cyclase expression was markedly decreased in SHR. CONCLUSIONS These results demonstrate that NOS III expression in different tissues is differentially affected by hypertension. Moreover, although an elevated superoxide anion production is apparent in the aorta, a reduced soluble guanylyl cyclase expression appears to account for the observed vasodilator dysfunction in SHR.

Calcium-Dependent and Calcium-Independent Activation of the Endothelial NO Synthase

Largely assumed to be a Ca2(+)-/calmodulin-dependent enzyme, the endothelial constitutive nitric oxide (NO) synthase (NOS III) can be activated by agonists as a consequence of an increase in the intracellular concentration of free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is elicited by an increase in inositol 1,4,5-trisphosphate which is the consequence of tyrosine phosphorylation and activation of phospholipase C-gamma1 as well as protein tyrosine phosphatases. Following the mobilization of intracellular Ca2+, the depleted Ca2+ stores signal to cation channels in the plasma membrane by a pathway which appears to involve activation of both tyrosine and serine/threonine kinases since this portion of the Ca2+ response is attenuated by both tyrosine kinase inhibitors and serine phosphatase inhibitors. In response to fluid shear stress the continuous production of NO by native and cultured endothelial cells is associated with only a transient and minimal increase in [Ca2+]i. In the absence of extracellular Ca2+ and in the presence of the calmodulin antagonist, shear stress stimulates a continuous production of NO which is sensitive to the nonspecific kinase inhibitor staurosporine and the tyrosine kinase inhibitor erbstatin A. A pharmacologically identical activation of NOS III can be induced by protein phosphatase inhibitors suggesting that the tyrosine phosphorylation of NOS III or an associated regulatory protein is crucial for its Ca2(+)-independent activation. Thus in a departure from widely held beliefs, we propose that the endothelial cells are able to respond to mechanical and humoral stimuli activating NOS III by at least two separate pathways.

Inhibition of Inducible Nitric Oxide Synthase Restores Endothelium-Dependent Relaxations in Proinflammatory Mediator-Induced Blood Vessels

Endothelium-dependent relaxations mediated by nitric oxide (NO) are attenuated in arteries exposed to proinflammatory mediators. Because proinflammatory mediators stimulate the expression of the inducible NO synthase (iNOS) in vascular cells, the role of iNOS-derived NO in the impaired endothelium-dependent relaxation was examined in arterial ring preparations. Exposure of rabbit carotid arteries to interleukin-1 beta (IL-1 beta; 100 U/mL for 7 hours) and porcine coronary arteries to a combination of tumor necrosis factor-alpha (1000 U/mL), interferon-gamma (500 U/mL), and lipopolysaccharide (10 micrograms/mL) for 15 hours (conditions that are associated with iNOS expression) markedly attenuated relaxations to receptor-dependent agonists, whereas those to the calcium ionophore A23187 and sodium nitroprusside were virtually unchanged. The impaired relaxation was not associated with a reduced level of the constitutive endothelial NOS (cNOS) but was accompanied by a reduced formation of biologically active NO as assessed in a bioassay system. The attenuated relaxation of carotid arteries to acetylcholine was not affected by superoxide dismutase and was neither found in arteries exposed to IL-1 beta for only 15 minutes nor in IL-1 beta-treated arteries for 7 hours followed by a 17-hour incubation period without the cytokine. Furthermore, no impaired relaxation was found in rings exposed to IL-1 beta in combination with either cycloheximide or N-alpha-tosyl-L-lysine chloromethyl ketone or pyrrolidine dithiocarbamate, treatments that prevent iNOS expression. In addition, selective inhibition of iNOS with S-methylisothiourea (10 mumol/L) completely restored acetylcholine-induced relaxations. These findings indicate that the continuous generation of NO induced by proinflammatory mediators plays a major role in the inhibition of endothelium-dependent relaxation, most likely by impairing a step in the signal transduction cascade that links activation of endothelial receptors to the calcium-calmodulin-dependent activation of NOS.