Nitric oxide formation in the vascular wall: regulation and functional implications.
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Amino Acid Oxidoreductases↗
Biomedical subjects
R Busse
Publications and source records attributed to R Busse.
Volatile and intravenous anesthetics selectively attenuate the release of endothelium-derived hyperpolarizing factor elicited by bradykinin in the coronary microcirculation.
In addition to nitric oxide (NO) and prostacyclin (PGI2) another endothelium-derived factor, which hyperpolarizes vascular smooth muscle cell via activation of K+ channels, contributes to the vasorelaxant effect of bradykinin in different vascular beds. Preliminary findings suggest that this endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation is attenuated by both volatile and intravenous anesthetics. Since EDHF may play an important role in the coronary microcirculation, we investigated the effects of isoflurane (2 vol.% equivalent to approximately 250 microM), etomidate (30 and 100 microM), phenobarbital (100 microM) and thiopental (30 and 100 microM) on the EDHF-mediated dilator response to bradykinin and on the endothelium-independent dilatation evoked by sodium nitroprusside (SNP) in the isolated saline-perfused rat heart (Langendorff preparation). None of the anesthetics tested affected the dilator response to bradykinin or SNP under basal conditions. However, following inhibition of NO and PGI2 formation with NG-nitro-L-arginine (100 microM) and diclofenac (1 microM) respectively, isoflurane, etomidate and thiopental, but not phenobarbital, significantly attenuated the NO/PGI2-independent, i.e. EDHF-mediated dilator response to bradykinin, while the vasorelaxant effect of SNP remained unaffected. Isoflurane, etomidate and thiopental, but not phenobarbital, display cytochrome P450-inhibiting properties, suggesting that these anesthetics impair the cytochrome P450-dependent synthesis of EDHF in the coronary microcirculation.
Analysis of Variance↗
Characterization of furoxans as a new class of tolerance-resistant nitrovasodilators.
The vasodilator effects of C92-4609 (4-hydroxymethyl-furoxan-3-carboxamide, CAS 1609), C92-4678(4-phenyl-furoxan-3-carboxylic acid (pyridyl-3-yl-methyl)-amide), C92-4679 (3-phenyl-furoxan-4-carboxylic acid (pyridyl-3-yl-methyl)-amide) and C93-4759 (3-hydroxymethyl-furoxan-4-carboxamide) were studied in the isolated rabbit femoral artery and jugular vein. All furoxans were potent vasodilators in the femoral artery (EC50 0.1-50 microM), while they were less potent in the jugular vein by at least one order of magnitude. Apart from C92-4679, the vasodilatory potency of the furoxans correlated well with their nitric oxide (NO)-releasing capacity which was estimated both by stimulation of purified soluble guanylyl cyclase activity and electron spin resonance spectroscopy with a trapping agent for NO. The hypothesis that furoxans stimulate soluble guanylyl cyclase in the smooth muscle by spontaneously releasing NO was supported by the marked attenuation of their vasodilator effect in the presence of oxyhaemoglobin (10 microM) or following treatment with methylene blue (30 microM). In contrast to earlier findings, NO release from these furoxans was not thiol-dependent, as demonstrated for C92-4609, the relaxant effect of which in the femoral artery was not altered in the presence of N-acetyl-L-cysteine (1 mM). Moreover the KCa+ channel inhibitor, tetrabutylammonium (3 mM), but not the KATP+ channel inhibitor, glibenclamide (3 microM), significantly attenuated the dilator response to C92-4679 in the femoral artery. Pretreatment of these segments with the cytochrome P450 inhibitor, SKF525a (30 microM), also reduced the C92-4679-induced relaxation in this vascular bed.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Control and consequences of endothelial nitric oxide formation.
The intention of this chapter is to give a brief overview of the continuously expanding field of endothelium-derived NO. Over the past few years it has become apparent that the mechanisms controlling the activation of NOS are more complex than was previously thought, with factors such as pHi, [Ca2+]i, shear stress, and gender all contributing to the control of "basal" NO production as well as the regulation of NOS levels in endothelial cells. The list of the functional consequences of endothelial NO formation has also grown, with antiproliferative, antihypertensive, and antiatherogenic effects all being described. Recent advances at the molecular biology level have facilitated the pioneering of a whole new field of research, and a number of groups have shown that NO can modulate the expression of several genes, such as that encoding MCP-1, an effect that is probably due to an interaction between NO and transcription factors. Further elucidation of the signals that influence the production and actions of NO will, without doubt, further the understanding of numerous physiological and pathophysiological processes.
Animals↗
Inhalation anesthetics inhibit the release of endothelium-derived hyperpolarizing factor in the rabbit carotid artery.
BACKGROUND: Inhalation anesthetics may interfere with the synthesis or action of endothelium-derived vasoactive factors. We investigated the effects of desflurane, enflurane, halothane, isoflurane, and sevoflurane on the release of nitric oxide and endothelium-derived hyperpolarizing factor (EDHF) in the isolated endothelium-intact carotid artery of the rabbit. METHODS: Isolated segments of the carotid artery were suspended in Krebs-Henseleit solution (37 degrees C) and preconstricted with phenylephrine (1 microM). Relaxations caused by acetylcholine (ACh) (0.03-10 microM) or sodium nitroprusside (0.01-10 microM) were compared in the presence or absence of the nitric oxide synthase inhibitor NG-nitro-L-arginine (0.1 mM) in segments exposed to desflurane (8%), enflurane (2-4%), halothane (2-3.5%), isoflurane (2-4%), or sevoflurane (2%) as well as in NG-nitro-L- arginine-treated segments exposed to enflurane (2%) in combination with the KCa(+)-channel blocker tetrabutylammonium (0.3 mM) or the cytochrome P450 inhibitor clotrimazole (3 microM). RESULTS: Desflurane, enflurane, and sevoflurane selectively inhibited the ACh-induced release of EDHF. Halothane and isoflurane also weakly affected the nitric oxide-mediated relaxant response to ACh. The inhibitory effect of these two anesthetics on EDHF release was concentration-dependent. Relaxations induced by sodium nitroprusside were not inhibited by any of the anesthetics tested. Three structurally unrelated cytochrome P450 inhibitors clotrimazole (0.1 mM), metyrapone (1 mM), and SKF525a (proadifen, 0.1 mM) abolished the EDHF-mediated relaxation elicited by ACh. The pharmacologic profile of the inhibitory effect of enflurane on the release of EDHF closely resembled that of clotrimazole but not that of tetrabutylammonium. Moreover, all anesthetics inhibited the cytochrome P450-catalyzed O-dealkylation of 7-ethoxycoumarin by rabbit liver microsomes in a concentration-dependent manner. CONCLUSIONS: Inhalation anesthetics significantly attenuate the EDHF-mediated relaxant response to ACh in the rabbit carotid artery. This effect appears to be attributable to inhibition of the cytochrome P450-dependent synthesis of EDHF by the endothelium.
Acetylcholine↗
Selective inhibition by barbiturates of the synthesis of endothelium-derived hyperpolarizing factor in the rabbit carotid artery.
1. Several lines of evidence suggest that both volatile and intravenous anaesthetics may interfere with the synthesis and release of endothelium-derived vasoactive factors. We have investigated the effects of three different barbiturates on the release of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in phenylephrine (1 microM)-preconstricted, endothelium-intact ring segments of the rabbit carotid artery. The segments were pretreated with the cyclo-oxygenase inhibitor, diclofenac (1 microM), to prevent the formation of vasoactive prostanoids, such as prostacyclin (PGI2). 2. Acetylcholine (ACh) elicited a concentration-dependent relaxation (EC50 0.15 microM) in control segments which was not significantly different from the relaxant responses of segments pretreated with methohexitone (0.03-0.3 mM), phenobarbitone (0.1-0.3 mM) or thiopentone (0.1-0.3 mM). 3. Inhibition of NO synthesis with NG-nitro-L-arginine (0.1 mM) significantly reduced the maximum relaxant response to ACh from 96 to 40%. This NO/PGI2-independent relaxation appeared to be mediated by the release of EDHF, since it was strongly diminished in the presence of the K+Ca inhibitors, tetrabutylammonium (1-3 mM) and charybdotoxin (10 nM), following preconstriction with potassium calcium (40 mM) or removal of the endothelium. Thiopentone or methohexitone markedly attenuated the EDHF-mediated relaxant response to ACh, while phenobarbitone had no effect. The endothelium-independent relaxation elicited by sodium nitroprusside (0.01-10 microM), on the other hand, was only marginally affected by these anaesthetics. 4. The cytochrome P450 inhibitor, clotrimazole (3-100 microM), mimicked the inhibitory effect of thiopentone and methohexitone on the NO/PGI2-independent relaxant response to ACh. Moreover the cytochrome P450-catalyzed O-dealkylation of 7-ethoxycoumarin by rabbit liver microsomes was inhibited in the presence of thiopentone or methohexitone (0.3-1 mM), while phenobarbitone was without effect.5. These findings suggest that thiopentone and methohexitone selectively attenuate the EDHF-mediated relaxant response to ACh in the rabbit carotid artery, presumably by interfering with its synthesis from arachidonic acid via the cytochrome P450 epoxygenase pathway.
Acetylcholine↗
Specificity of different organic nitrates to elicit NO formation in rabbit vascular tissues and organs in vivo.
1. In the present study we assessed the formation of nitric oxide (NO) from classical and thiol-containing organic nitrates in vascular tissues and organs of anaesthetized rabbits, and established a relationship between the relaxant response elicited by nitroglycerin (NTG) and NO formation in the rabbit isolated aorta. Furthermore, the effect of isolated cytochrome P450 on NO formation from organic nitrates was investigated. 2. Rabbits received diethyldithiocarbamate (DETC; 200 mg kg-1 initial bolus i.p. and 200 mg kg-1 during 20 min, i.v.) and either saline, or one of the following organic nitrates: nitroglycerin (NTG, 0.5 mg kg-1), isosorbide dinitrate (ISDN), N-(3-nitratopivaloyl)-L-cysteine ethylester (SPM 3672), S-carboxyethyl-N-(3-nitratopivaloyl)-L-cysteine ethylester (SPM 5185), at 10 mg kg-1 each. After 20 min the animals were killed, blood vessels and organs were removed, and subsequently analyzed for spin-trapped NO by cryogenic electron spin resonance (e.s.r.) spectroscopy. 3. In the saline-treated control group, NO remained below the detection limit in all vessels and organs. In contrast, all of the nitrates tested elicited measurable NO formation, which was higher in organs (liver, kidney, heart, lung, spleen) (up to 4.8 nmol g-1 20 min-1) than in blood vessels (vena cava, mesenteric bed, femoral artery, aorta) (up to 0.7 nmol g-1 20 min-1). Classical organic nitrates (NTG, ISDN) formed NO preferentially in the mesenteric bed and the vena cava, while the SPM compounds elicited comparable NO formation in veins and arteries. 4. Using a similar spin trapping technique, NO formation was assessed in vitro in phenylephrine-precontracted rabbit aortic rings. The maximal relaxation elicited by a first exposure (10 min) to NTG (0.3 to 10 microM) was positively correlated (r = 0.8) with the net increase (NTG minus basal) of NO spin-trapped during a second exposure to the same concentration of NTG in the presence of DETC. 5. Cytochrome P450 purified from rabbit liver enhanced NO formation in a NADPH-dependent fashion from NTG, but not from the other nitrates, as assessed by activation of purified soluble guanylyl cyclase. 6. We conclude that the vessel selective action of different organic nitrates in vivo reflects differences in vascular NO formation. Thus, efficient preload reduction by classical organic nitrates can be accounted for by higher NO formation in venous capacitance as compared to arterial conductance and resistance vessels. In contrast, NO is released from cysteine-containing nitrates (SPMs) to a similar extent in arteries and veins, presumably independently of an organic nitrate-specific biotransformation. Limited tissue bioavailability of NTG and ISDN might account for low NO formation in the aorta, while true differences in biotransformation seem to account for differences in NO formation in the other vascular tissues.
Animals↗
Calcium signaling in endothelial cells involves activation of tyrosine kinases and leads to activation of mitogen-activated protein kinases.
The activation of endothelial cells following exposure to a variety of receptor-dependent and -independent stimuli is associated with the release of Ca2+ from intracellular stores as well as the influx of Ca2+ from the extracellular space. In the present study, we investigated the interaction between Ca2+ signaling in cultured human umbilical vein endothelial cells and tyrosine phosphorylation. Stimulation of endothelial cells with either bradykinin (100 nmol/L), histamine (1 mumol/L), or the Ca(2+)-ATPase inhibitor thapsigargin (30 nmol/L) resulted in a slightly delayed but prolonged tyrosine phosphorylation of two low molecular weight proteins (approximately 42 and approximately 44 kD). These proteins were identified by immunoprecipitation as the 42- and 44-kD isoforms of mitogen-activated protein kinase (MAP kinase). The agonist-induced tyrosine phosphorylation of the 42-/44-kD doublet was sensitive to the tyrosine kinase inhibitors genistein (100 mumol/L) and piceatannol (10 mumol/L) and was inhibited by the removal of Ca2+ from the extracellular medium. In fura 2-loaded endothelial cells, inhibition of tyrosine kinases attenuated Ca2+ signaling after stimulation with either bradykinin (30 nmol/L) or thapsigargin (30 nmol/L). Since inhibition of tyrosine kinases specifically attenuates the plateau phase of the Ca2+ response after stimulation, the effect of tyrosine kinase inhibition appeared to be mostly associated with the influx of Ca2+ from the extracellular space.(ABSTRACT TRUNCATED AT 250 WORDS)
Bradykinin↗
Nitric oxide modulates the expression of monocyte chemoattractant protein 1 in cultured human endothelial cells.
The recruitment of monocytes into the arterial wall is one of the earliest events in the pathogenesis of atherosclerosis. Since monocyte chemoattractant protein 1 (MCP-1) plays a key role in the subendothelial recruitment of monocytes, we tested whether nitric oxide (NO) modulates the expression of MCP-1 in cultured human endothelial cells. Inhibition of basal NO production by NG-nitro-L-arginine (L-NAG) upregulates endothelial MCP-1 mRNA expression (250 +/- 20%) and protein secretion. Exogenous addition of NO dose-dependently decreased MCP-1 mRNA expression and secretion. Changes in MCP-1 mRNA expression and protein secretion were paralleled by corresponding changes in chemotactic activity of cell-conditioned media for monocytes. An MCP-1 antibody reduced monocyte chemotactic activity by 85% and completely abolished the increased monocyte chemotactic activity induced by the inhibition of NO production. Elevation of endothelial cGMP levels had no significant effect on MCP-1 mRNA expression. Inhibition of basal endothelial NO production by L-NAG increased binding activity of a nuclear factor kappa B (NF-kappa B)-like transcriptional regulatory factor, whereas exogenous addition of NO decreased NF-kappa B-like binding activity during stimulation with tumor necrosis factor-alpha. Thus, NO modulates MCP-1 expression and monocyte chemotactic activity secreted by human umbilical vein endothelial cells (HUVECs) in culture. The activation of NF-kappa B-like transcriptional regulatory proteins by inhibition of NO suggests a molecular link between an oxidant-sensitive transcriptional regulatory mechanism and NO synthesis in HUVECs.
Arteriosclerosis↗
Regulation and functional consequences of endothelial nitric oxide formation.
Since its discovery, endothelium-derived nitric oxide (NO) has become one of the most intensely investigated molecules in the field of cardiovascular physiology. Although initial investigations centred on the role of NO in mediating vasodilation and inhibition of platelet activation it has since become clear that this small, atypical signal molecule is also involved in regulating cell growth and proliferation as well as affecting the transcription of certain genes, the products of which have been implicated in the pathogenesis of such states as atherosclerosis and hypertension. Our understanding of the intracellular regulation of the NO synthases has also progressed and the constitutive endothelial enzyme is now known to be controlled by both intracellular Ca2+ and pH. In addition it would appear that this enzyme can also be upregulated in response to stimuli such as fluid shear stress and oestrogen. This review is intended to give the reader a glimpse of the multifaceted actions of endothelium-derived NO.
Chemokine CCL2↗
[Radiology, health care structural reform and health system research--status, developments and challenges].
Through the health care reform act of 1993 and the next reform step currently under discussion, the financing mechanisms of both the ambulatory and the hospital sector of the German health care system will be changed drastically which will effect radiology earlier and more than other specialties. In the ambulatory care sector, fee-for-service has led to the tendency of more services, especially through self-referral to diagnostic imaging. Currently, managed care structures are under discussion which will lead to fewer services. In the hospital sector, reimbursement of all costs on a per-diem basis also had the effect of much diagnostic imaging. Beginning in 1996, diagnostic-related groups with pre-determined fixed reimbursements will supplement per-diem charges. Internal financial billing will lead to fewer demands for imaging services. Health services research in radiology is necessary to demonstrate the efficacy of imaging procedures at all levels from structure (technical efficacy) through process (clinical efficacy) to outcome (outcome efficacy).
Ambulatory Care↗
Thrombin prevents the expression of inducible nitric oxide synthase in vascular smooth muscle cells by a proteolytically-activated thrombin receptor.
Proteolytically active forms of thrombin ( alpha- and gamma-thrombin) and thrombin receptor peptides inhibited the release of nitrite, a stable endproduct of nitric oxide, evoked by interleukin-1 beta (IL-1 beta ) in cultured vascular smooth muscle cells while proteolytically inactive forms [D-Phe-Pro-Arg chloromethyl ketone-alpha-thrombin (PPACK-alpha-thrombin) and diisopropylphosphoryl-alpha-thrombin (DIP-alpha-thrombin)] had either no or only minimal inhibitory effects. Under bioassay conditions, perfusates from columns containing IL-1 beta-activated vascular smooth muscle cells or cells treated with IL-1 beta plus PPACK-alpha-thrombin relaxed detector blood vessels. These relaxations were abolished by the inhibitor of nitric oxide synthesis, NG-nitro-L-arginine. No relaxations were obtained with untreated cells or IL-1 beta-treated cells in the presence of alpha-thrombin. The expression of inducible nitric oxide synthase mRNA and protein in vascular smooth muscle cells by IL-1 beta was impaired by alpha-thrombin. These results demonstrate that thrombin regulates the expression of the inducible nitric oxide synthase at a transcriptional level via the proteolytic activation of the thrombin receptor in vascular smooth muscle cells.
Amino Acid Sequence↗
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)
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
Nitric oxide promotes seizure activity in kainate-treated rats.
L-Arginine-derived nitrogen monoxide (NO) formation was determined in different regions of the rat brain during kainate-induced seizures. NO was trapped in vivo as a paramagnetic mononitrosyl-iron diethyldithiocarbamate complex, the concentration of which was determined ex vivo by cryogenic electron spin resonance spectroscopy. Basal NO formation (0.3-0.8 nmol g-1 tissue 30 min-1) was detected in the brain of control rats. In kainate-injected rats NO formation was increased six-fold within 30-60 min in the amygdala/temporal cortex region, and up to 12-fold, though more slowly, in the remaining cortex. The kainate-elicited convulsions and NO formation were attenuated in animals pretreated with either 7-nitroindazole, a specific inhibitor of neuronal NO synthase, or diazepam. These findings identify NO as a proconvulsant mediator in kainate-evoked seizures.
Amino Acid Oxidoreductases↗
Pyrrolidine dithiocarbamate selectively prevents the expression of the inducible nitric oxide synthase in the rat aorta.
Exposure of rat aortic rings without endothelium to interleukin-1 beta for 5 h significantly attenuated the contractions due to phenylephrine and increased the tissue content of guanosine 3',5'-cyclic monophosphate (cyclic GMP) due to the induction of nitric oxide synthase. The presence of pyrrolidine dithiocarbamate, a specific inhibitor of nuclear transcription factor kappa B activation, during the exposure of the rings to interleukin-1 beta prevented these responses to interleukin-1 beta. Rat aortic rings which had been incubated for 5 h with interleukin-1 beta in the absence and presence of pyrrolidine dithiocarbamate prior to the organ chamber experiment had a similar concentration-dependent relaxation curve for acetylcholine in rings with endothelium, and for 3-morpholino-sydnonimine (SIN-1) in rings without. Pyrrolidine dithiocarbamate applied acutely did not alter the tone elicited by phenylephrine in rings with or without endothelium and had no effect on the subsequent relaxation induced by acetylcholine in rings with endothelium or by SIN-1 in rings without endothelium. These observations suggest that pyrrolidine dithiocarbamate prevents the interleukin-1 beta-mediated expression of the inducible nitric oxide synthase without affecting the activity of the constitutive enzyme in the rat aorta.
Acetylcholine↗
Subcellular localization and characterization of nitric oxide synthase(s) in endothelial cells: physiological implications.
Endothelial cells (EC) contain a constitutive Ca2+/calmodulin-dependent nitric oxide (NO) synthase (cNOS) which plays an important role in the local control of vascular tone. We compared the subcellular distribution of this enzyme in cultured and freshly isolated pig EC by determination of specific cNOS activity and immunoblot analysis. Similar studies were also performed with cultured and freshly isolated bovine and cultured human EC. Enzyme activity was predominantly (> 70%) associated with the particulate fraction of all EC types tested and was highest in freshly isolated porcine EC. Both specific cNOS activity and immunoreactivity were substantially higher (> 3-fold) in the microsomal as compared with the soluble fraction of all EC types tested. In freshly isolated pig EC, these two fractions also differed in terms of their Ca(2+)-dependency, pH optimum and inhibitor specificity. EC may thus contain either two different cNOS isoenzymes or a single enzyme, the conformation of which differs between the soluble and membrane-bound state. Moreover, detailed subcellular fractionation of freshly isolated pig EC revealed that the distribution of cNOS activity closely resembled that of the plasma membrane marker 5'-nucleotidase, suggesting that most, if not all, of the cNOS activity in these cells is associated with the plasma membrane. This localization might render the enzyme more susceptible to activation by physical stimuli, such as a shear stress-induced change in the fluidity of the plasma membrane. Moreover, the continuous exposure to shear stress in vivo may also upregulate cNOS expression in EC, since specific enzyme activity, immunoreactivity and basal NO release were significantly higher in freshly isolated EC as compared with cultured EC.
Amino Acid Oxidoreductases↗
Subcellular localization and characterization of neuronal nitric oxide synthase.
In contrast to the predominantly particulate, Ca2+/calmodulin-dependent nitric oxide (NO) synthase in endothelial cells, the corresponding neuronal isoenzyme is considered to be mainly soluble, presumably owing to the lack of a posttranslational myristoylation. However, preliminary findings from this and other laboratories suggest that a substantial portion of the neuronal NO synthase activity may in fact be membrane bound. We have therefore investigated the distribution of this enzyme among subcellular fractions of the rat and rabbit cerebellum in more detail. Up to 60% of the total NO synthase activity was found in the particulate fraction and, according to density gradient ultracentrifugation, associated mainly with the endoplasmic reticulum fraction. There was no apparent difference between the soluble and particulate enzymes with respect to their specific activity, Ca2+ and pH dependency, inhibitor sensitivity, or immunoreactivity, suggesting that both rat and rabbit cerebella contain a single Ca2+/calmodulin-dependent NO synthase. The inhibition by the cytochrome P450 inhibitor SKF-525A of the NO synthase activity in these subcellular fractions (IC50 = 90 microM) and the fact that mammalian cytochrome P450 enzymes are endoplasmic reticulum-bound proteins support the notion that the cerebellar NO synthase is a cytochrome P450-type hemoprotein. Moreover, the aforementioned findings suggest that posttranslational myristoylation may not be the only factor determining the intracellular localization of NO synthase.
Amino Acid Oxidoreductases↗
Attenuation of coronary autoregulation in the isolated rabbit heart by endothelium derived nitric oxide.
OBJECTIVE: The aim was to investigate the role of endothelium derived nitric oxide (EDNO/EDRF) in the control of coronary autoregulation. METHODS: In isolated saline perfused rabbit hearts coronary flow responses to stepwise increases in perfusion pressure were studied under control conditions, during maximum dilatation with sodium nitroprusside, and in the presence of the inhibitor of EDNO synthesis, NG-nitro-L-arginine (L-NNA), or the vasoconstrictors endothelin-1 and arginine vasopressin. RESULTS: At a constant perfusion pressure of 60 mm Hg, infusion of L-NNA (30 microM), but not D-NNA, reduced the coronary flow from 24.7(SEM 2) to 13.6(2.2) ml.min-1 and abolished flow increases induced by the EDRF stimulator acetylcholine. Under these conditions, pressure induced coronary flow increases were reduced (p < 0.05 compared to control) over the whole range of perfusion pressures studied (45 to 120 mm Hg). Arginine vasopressin [2(0.6) nM] and endothelin-1 [1.5(1) nM] induced similar reductions of coronary resting flow but the pressure induced flow increases were significantly greater than in the presence of L-NNA. Moreover, inhibition of EDRF synthesis reduced the peak reactive hyperaemia after a 30 s interruption of coronary flow from 47(2) to 32(2) ml.min-1. These changes occurred in spite of a decrease in the myocardial oxygen uptake from 5.1(0.6) to 3.4(0.5) ml.100 g-1.min-1 (p < 0.01) and a concomitant increase in the lactate release from 46(7) to 95(54) mumol.min.100 g-1 (p < 0.01), indicating myocardial ischaemia. CONCLUSIONS: EDNO attenuates coronary autoregulatory responses which, if unopposed, potentially impair a functionally adequate myocardial perfusion. It is suggested that the modulator role of EDNO is, at least in part, specific and most likely to be due to shear dependent alterations of EDNO release.
Acetylcholine↗