Intrinsic defence capacity and therapeutic potential of natriuretic peptides in pulmonary hypertension associated with lung fibrosis.

BACKGROUND AND PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a progressive fibro-proliferative disorder refractory to current therapy commonly complicated by the development of pulmonary hypertension (PH); the associated morbidity and mortality are substantial. Natriuretic peptides possess vasodilator and anti-fibrotic actions, and pharmacological augmentation of their bioactivity ameliorates renal and myocardial fibrosis. Here, we investigated whether natriuretic peptides possess an intrinsic cytoprotective function preventing the development of pulmonary fibrosis and associated PH, and whether therapeutics targeting natriuretic peptide signalling demonstrate efficacy in this life-threatening disorder. EXPERIMENTAL APPROACH: Pulmonary haemodynamics, right ventricular function and markers of lung fibrosis were determined in wild-type (WT) and natriuretic peptide receptor (NPR)-A knockout (KO) mice exposed to bleomycin (1 mg·kg(-1) ). Human myofibroblast differentiation was studied in vitro. KEY RESULTS: Exacerbated cardiac, vascular and fibrotic pathology was observed in NPR-A KO animals, compared with WT mice, exposed to bleomycin. Treatment with a drug combination that raised circulating natriuretic peptide levels (ecadotril) and potentiated natriuretic peptide-dependent signalling (sildenafil) reduced indices of disease progression, whether administered prophylactically or to animals with established lung disease. This positive pharmacodynamic effect was diminished in NPR-A KO mice. Atrial natriuretic peptide and sildenafil synergistically reduced TGFß-induced human myofibroblast differentiation, a key driver of remodelling in IPF patients. CONCLUSIONS AND IMPLICATIONS: These data highlight an endogenous host-defence capacity of natriuretic peptides in lung fibrosis and PH. A combination of ecadotril and sildenafil reversed the pulmonary haemodynamic aberrations and remodelling that characterize the disease, advocating therapeutic manipulation of natriuretic peptide bioactivity in patients with IPF.

Comparative clinical- and cost-effectiveness of candesartan and losartan in the management of hypertension and heart failure: a systematic review, meta- and cost-utility analysis.

The UK National Health Service (NHS) currently spends in excess of £250 million per annum on angiotensin II receptor blockers (ARBs) for the treatment of hypertension and heart failure; with candesartan currently dominating the market. With the recent introduction of generic losartan, we set out to directly compare the branded market leader to its now cheaper alternative. The primary objectives were to compare the blood pressure (BP) lowering efficacy and cardiovascular outcomes of candesartan and losartan in the treatment of essential hypertension and chronic heart failure, respectively. The secondary objective was to model their comparative incremental cost-effectiveness in a UK NHS setting. The Cochrane Central Register of Controlled Trials (Cochrane Library 2009, issue 2), which contains the Hypertension and Heart Group's specialist register, Medline (1950-February 2010), and Embase (1980-February 2010) were included in the search strategy. Selection criteria were randomised studies of candesartan versus losartan in adults (> 18 years). The main outcome measures were as follows: Hypertension: mean change from baseline in trough (24 h postdose) systolic and diastolic BP. Heart failure: composite of cardiovascular death and hospital admission for management of heart failure. Two reviewers applied inclusion criteria, assessed trial quality, and extracted data. Eight (three of which met inclusion criteria) and zero trials compared candesartan directly with losartan in the treatment of hypertension and heart failure, respectively. A between-treatment difference of -1.96 mmHg [95% confidence interval (CI) -2.40 to -1.51] for trough diastolic BP and -3.00 mmHg (95% CI -3.79 to -2.22) for trough systolic BP in favour of candesartan was observed. Based on this differential, a 10-year Markov model estimates the cost per quality-adjusted life-year gained to exceed £40,000 for using candesartan in place of generic losartan. Candesartan reduces BP to a slightly greater extent when compared with losartan, however, such difference is unlikely to be cost-effective based on current acquisition costs, perceived NHS affordability thresholds and use of combination regimens. We could find no robust evidence supporting the superiority of candesartan over losartan in the treatment of heart failure. We therefore recommend using generic losartan as the ARB of choice which could save the UK NHS approximately £200 million per annum in drug costs.

Reciprocal regulation of human soluble and particulate guanylate cyclases in vivo.

BACKGROUND & PURPOSE: We demonstrated previously that reciprocal regulation of soluble (sGC) and particulate (pGC) guanylate cyclases by NO and natriuretic peptides coordinates cyclic cGMP-mediated vasodilatation in vitro. Herein, we investigated whether such an interaction contributes to vascular homeostasis in mice and humans in vivo. EXPERIMENTAL APPROACH: Mean arterial blood pressure (MABP) changes in anaesthetized mice were monitored in response to i.v. administration of cGMP- and cAMP-dependent vasodilators in wild-type (WT), endothelial NO synthase (eNOS) and natriuretic peptide receptor (NPR)-A knockout mice. Forearm blood flow (FBF) in response to intra-brachial infusion of ANP (25, 50, 100, 200 pmol min(-1)) in the absence and presence of the NOS inhibitor NG-methyl-L-arginine (L-NMA; 4 micromol min(-1)) and the control constrictor noradrenaline (240 pmol min(-1)) was assessed in healthy volunteers. KEY RESULTS: Sodium nitroprusside (SNP; NO-donor) and atrial natriuretic peptide (ANP) produced dose-dependent reductions in MABP in WT animals that were significantly enhanced in eNOS KO mice. In NPR-A K mice, SNP produced a dose-dependent reduction in MABP that was significantly greater than that in WT mice. Responsiveness to the cAMP-dependent vasodilator epoprostenol was similar in WT, eNOS KO and NPR-A KO animals. ANP caused vasodilatation of the forearm resistance vasculature that was significantly greater in individuals lacking endothelium-derived NO (i.e. L-NMA treated). CONCLUSIONS & IMPLICATIONS: These data demonstrate that crosstalk occurs between the NO-sGC and ANP-pGC pathways to regulate cGMP-dependent vasodilatation in vivo in both mice and humans. These findings have implications for understanding the link between natriuretic peptide activity and cardiovascular risk.

KATP channel activation induces ischemic preconditioning of the endothelium in humans in vivo.

BACKGROUND: Endothelial dysfunction contributes to ischemia-reperfusion injury (IRI) and is reduced by ischemic preconditioning (IPC). IPC may involve activation of ATP-sensitive potassium channels (K(ATP)). We determined whether modulation of K(ATP) channels occurs in endothelial IPC in humans. METHODS AND RESULTS: IRI of the forearm was induced by inflating a blood pressure cuff to 200 mm Hg for 20 minutes in healthy volunteers. K(ATP) activation was modulated by intra-arterial glibenclamide (blocker) and diazoxide (opener). Endothelial function (response to intra-arterial acetylcholine) was assessed with forearm plethysmography before and after (1) 15-minute reperfusion, (2) IRI preceded by IPC (3 five-minute periods of ischemia), (3) IRI preceded by IPC with glibenclamide, (4) IPC followed by glibenclamide before IRI, (5) IRI preceded by diazoxide, and (6) IRI preceded by coinfusion of glibenclamide with diazoxide. IRI caused endothelial dysfunction (P=0.002), which IPC prevented (P=0.40). Glibenclamide abolished IPC when given contemporaneously with (P=0.003) or during IRI (P=0.0005). Diazoxide prevented endothelial dysfunction after IRI (P=0.68) but not when coinfused with glibenclamide. CONCLUSIONS: Glibenclamide abolishes and diazoxide mimics endothelial IPC in humans. The time course of the effect of glibenclamide suggests involvement of K(ATP) channels as effectors of endothelial IPC in vivo. These data may have implications for understanding the therapeutic role of agents that modulate K(ATP) channel function.

Protection against lipopolysaccharide-induced endothelial dysfunction in resistance and conduit vasculature of iNOS knockout mice.

Endothelial dysfunction is a characteristic of, and may be pathogenic in, inflammatory cardiovascular diseases, including sepsis. The mechanism underlying inflammation-induced endothelial dysfunction may be related to the expression and activity of inducible nitric oxide synthase (iNOS). This possibility was investigated in isolated resistance (mesenteric) and conduit (aorta) arteries taken from lipopolysaccharide (LPS)-treated (12.5 mg/kg i.v.) or saline-treated iNOS knockout (KO) and wild-type (WT) mice. LPS pretreatment (for 15 h, but not 4 h) profoundly suppressed responses to acetylcholine (ACh) and significantly reduced sensitivity to the NO donor spermine-NONOate (SPER-NO) in aorta and mesenteric arteries of WT mice. This effect was temporally associated with iNOS protein expression in both conduit and resistance arteries and with a 10-fold increase in plasma NOx levels. In contrast, no elevation of plasma NOx was observed in LPS-treated iNOS KO animals, and arteries dissected from these animals did not express iNOS or display hyporeactivity to ACh or SPER-NO. The mechanism underlying this phenomenon may be suppression of eNOS expression, as observed in arteries of WT animals, that was absent in arteries of iNOS KO animals. These results clearly demonstrate that iNOS induction plays an integral role in mediation of the endothelial dysfunction associated with sepsis in both resistance and conduit arteries.

Acute administration of L-arginine does not improve arterial endothelial function in chronic renal failure.

BACKGROUND: Reduced activity of the nitric oxide (NO) pathway has been implicated in the endothelial dysfunction that occurs in patients with renal failure. NO is generated from L-arginine by NO synthase, and certain uremic toxins including asymmetrical dimethyl-L-arginine (ADMA), inhibit NO synthase and might contribute to endothelial dysfunction. We hypothesized that exogenous L-arginine might improve endothelial function in patients with renal failure by overcoming the effects of uremic toxins. METHODS: Endothelial function of the forearm resistance vasculature was assessed using plethysmography to measure the dilator response to intra-arterial acetylcholine (25 to 100 nmol/min). Endothelial function of radial and brachial arteries was assessed using vascular ultrasound to measure the dilator response to flow during reactive hyperemia (flow-mediated dilation; FMD). Studies were performed before and after administration of L-arginine by intra-arterial infusion (50 micromol/min) in 8 pre-dialysis patients or by intravenous infusion (10 g) in 18 hemodialysis patients. RESULTS: Local L-arginine did not improve the dilator response of forearm resistance vessels (AUC 23.1 +/- 6.4 pre, 23.1 +/- 5.1 post; P = 0.9) or FMD of the radial artery (6.5 +/- 1.2% pre, 6.3 +/- 0.8% post; P = 0.8). Systemic L-arginine did not improve FMD of the brachial artery (4.1 +/- 1.1% pre, 3.0 +/- 1.1% post; P = 0.07). These data demonstrate that acute local or systemic administration of L-arginine did not improve endothelial function in resistance or conduit arteries of patients with chronic renal failure. CONCLUSION: The results suggest that competitive inhibition of nitric oxide synthase (NOS) by circulating inhibitors is not the principal explanation for impaired endothelial dilator function in chronic renal failure.

Dialysis improves endothelial function in humans.

BACKGROUND: Circulating inhibitors of endothelial function have been implicated in the pathogenesis of vascular disease in chronic renal failure. The aim of this study was to determine if lowering the plasma concentration of these and other dialysable toxins improves endothelial function. To do this we compared the acute effects on endothelial function of single episodes of haemodialysis with automated peritoneal dialysis. We hypothesized that endothelial function would improve after dialysis, with a greater effect seen after haemodialysis due to more substantial clearance of endothelial toxins per-treatment. METHODS: Subjects with end-stage renal failure undergoing haemodialysis (n=16) or automated peritoneal dialysis (n=14) were investigated. Endothelial function was determined using vascular ultrasound to measure flow-mediated dilatation of the brachial artery and was compared with the dilatation caused by sublingual glyceryl trinitrate. Endothelial function was assessed before and after a single dialysis treatment. Plasma concentrations of the inhibitors of endothelial function, asymmetric dimethyl-l-arginine and homocysteine were measured. Flow-mediated dilatation was expressed as percentage change from basal diameter and analysed using Student's t test. RESULTS: The plasma concentration of circulating inhibitors of endothelial function was reduced after haemodialysis but not peritoneal dialysis. Haemodialysis increased flow-mediated dilatation from 4.0+/-1.0% to 5.8+/-1.2% (P<0.002). These changes persisted for 5 h but returned to baseline by 24 h. Automated peritoneal dialysis had no acute effect on flow-mediated dilatation (5.9+/-1.1% vs 5.4+/-0.8% after, P>0.5). There were no effects of either dialysis modality on dilatation to glyceryl trinitrate. CONCLUSIONS: Short-term reduction of circulating inhibitors of endothelial function by haemodialysis is associated with increased flow-mediated dilatation. These data suggest that dialysable endothelial toxins have deleterious effects on endothelial function that are rapidly reversible.

Changes in endothelium-dependent vasodilatation and alpha-adrenergic responses in resistance vessels during the menstrual cycle in healthy women.

During the menstrual cycle, changes in endothelium-dependent vasodilatation have been demonstrated in conduit vessels in vivo, but responses in resistance vessels have not been studied. The aim of this study was to examine endothelium-dependent vasodilatation, the effects of local nitric oxide synthesis, and alpha-adrenergic constriction in resistance vessels during the menstrual cycle in 15 healthy female volunteers (mean age, 28.07 +/- 2.1 yr). Forearm blood flow in response to intrabrachial infusion of bradykinin (10, 30, and 100 pmol/min; endothelium-dependent vasodilator), glyceryl trinitrate (4, 8, and 16 nmol/min; endothelium-independent vasodilator), noradrenaline (60, 120, and 240 pmol/min; alpha-adrenergic receptor agonist), and N(G)-monomethyl-L-arginine (1, 2, and 4 micromol/min; nitric oxide synthase inhibitor) was assessed by venous occlusion plethysmography. All subjects were studied in early menstrual phase (days 1--4) and midcycle (days 10-13). Vasodilator response to bradykinin, expressed as the within-subject mean difference in the area under the dose-response curve between phases, was significantly increased at midcycle compared with that in the early menstrual phase (486.5 +/- 165.0; P = 0.01), whereas there was no significant difference in response to glyceryl trinitrate (185.8 +/- 239.0; P = 0.45). The vasoconstrictor response to noradrenaline was significantly greater at midcycle (97.1 +/- 39.4; P = 0.027), but the response to N(G)-monomethyl-L-arginine was not significantly different (17.5 +/- 35.2; P = 0.63). Serum estradiol was approximately 3-fold higher at midcycle, with a mean difference of 252.3 +/- 56.0 pmol/L (P = 0.0005). Progesterone concentrations were not significantly different (-0.11 +/- 0.1 nmol/L; P = 0.28). Differences in endogenous estrogen levels between menstrual phases may underlie changes in bradykinin and noradrenaline responses. If exogenous estrogens have similar effects, the balance of these two opposing actions may determine whether estrogen replacement in postmenopausal women has beneficial or harmful effects on the vasculature.

Reciprocal regulation of cGMP-mediated vasorelaxation by soluble and particulate guanylate cyclases.

Nitric oxide (NO) and atrial natriuretic peptides (ANP) activate soluble (sGC) and particulate guanylate cyclase (pGC), respectively, and play important roles in the maintenance of cardiovascular homeostasis. However, little is known about potential interactions between these two cGMP-generating pathways. Here we demonstrate that sGC and pGC cooperatively regulate cGMP-mediated relaxation in human and murine vascular tissue. In human vessels, the potency of spermine-NONOate (SPER-NO) and ANP was increased after inhibition of endogenous NO synthesis and decreased by prior exposure to glyceryl trinitrate (GTN). Aortas from endothelial NO synthase (eNOS) knockout (KO) mice were more sensitive to ANP than tissues from wild-type (WT) animals. However, in aortas from WT mice, the potency of ANP was increased after pretreatment with NOS or sGC inhibitor. Vessels from eNOS KO animals were less sensitive to ANP after GTN pretreatment, an effect that was reversed in the presence of an sGC inhibitor. cGMP production in response to SPER-NO and ANP was significantly greater in vessels from eNOS KO animals compared with WT animals. This cooperative interaction between NO and ANP may have important implications for human pathophysiologies involving deficiency in either mediator and the clinical use of nitrovasodilators.

Heterogenous nature of flow-mediated dilatation in human conduit arteries in vivo: relevance to endothelial dysfunction in hypercholesterolemia.

Flow-mediated dilatation (FMD) of conduit arteries is dependent on an intact endothelium, although the mechanisms are not fully understood. Using high-resolution ultrasound, we examined the role of endothelial mediators in radial artery dilatation in response to transient (short period of reactive hyperemia) and sustained (prolonged period of reactive hyperemia, hand warming, or an incremental infusion of acetylcholine into the distal radial artery) hyperemia. After short episodes of reactive hyperemia, FMD was abolished by local infusion of the nitric oxide synthesis inhibitor N:(G)monomethyl-L-arginine (5.3+/-1.2% versus 0.7+/-0.7%, P:<0.001). In contrast, basal vessel diameter and dilatation after prolonged episodes of reactive hyperemia, hand warming, and distal infusion of acetylcholine were not attenuated by nitric oxide synthesis inhibition. Inhibition of cyclooxygenase or local autonomic nervous system blockade also had no effect on FMD. Patients with hypercholesterolemia exhibited reduced FMD in response to transient hyperemia, but the response to sustained hyperemia was normal. These data suggest heterogeneity of endothelial responses to blood flow that are dependent on the characteristics of the flow stimulus. Dilatation after brief episodes of hyperemia is mediated by release of nitric oxide, whereas dilatation during sustained hyperemia is unaffected by NO synthesis inhibition. Hypercholesterolemia seems to differentially affect these pathways with impairment of the nitric oxide-dependent pathway and preservation of non nitric oxide-mediated dilatation to sustained flow stimuli.

Acute systemic inflammation impairs endothelium-dependent dilatation in humans.

BACKGROUND: We tested the hypothesis that endothelial dysfunction underlies the association between an acute inflammatory episode and the transiently increased risk of a cardiovascular event by examining the effects of an experimental inflammatory stimulus on endothelium-dependent vasodilation. METHODS AND RESULTS: Salmonella typhi vaccine was used to generate a systemic inflammatory response in healthy volunteers. In 12 subjects, dilatation of the brachial artery to flow and to sublingual nitroglycerin (NTG) was recorded (conduit vessel response), and in 6 subjects, venous occlusion plethysmography was used to measure forearm blood flow during intrabrachial infusion of the endothelium-dependent dilators acetylcholine (ACh) and bradykinin (BK) and the endothelium-independent dilators NTG and verapamil (resistance vessel response). Responses were assessed 16 hours before and 8 and 32 hours after vaccination. Vaccination resulted in elevations in white cell count and serum levels of interleukin-6 and interleukin-1 receptor antagonist. Eight hours after vaccination, resistance vessel responses to BK (P:=0.0099) and ACh (P:=0.0414) were markedly attenuated, and brachial artery flow-mediated dilatation was depressed. Resistance vessel responses to verapamil and NTG were unchanged, as was the conduit vessel response to NTG. Thirty-two hours after vaccination, resistance vessel responses to BK and ACh had returned to normal. CONCLUSIONS: S typhi vaccine generates a mild inflammatory reaction associated with temporary but profound dysfunction of the arterial endothelium in both resistance and conduit vessels to both physical and pharmacological dilator stimuli. This finding might explain the association between infection and inflammation and the enhanced risk of an acute cardiovascular event.

Effects of sodium intake on the pressor and renal responses to nitric oxide synthesis inhibition in normotensive individuals with different sodium sensitivity.

OBJECTIVE: The present study evaluated the role of nitric oxide (NO) in the systemic vascular and renal adaptation to changes in dietary sodium intake. DESIGN AND METHODS: Seven healthy normotensive male subjects were randomized to high or low sodium diets in a double blind crossover design (7 days on each diet). The NO synthesis inhibitor, NGmonomethyl-L-arginine (L-NMMA) was infused systemically (1.8 mg/kg over 30 min) at the end of each dietary period and its effects on blood pressure, renal plasma flow, glomerular filtration rate, urinary flow rate and sodium excretion were measured. RESULTS: Blood pressure increased in response to L-NMMA on a high sodium diet only (area under time curve percentage change in mean blood pressure, low sodium = -94.5 +/- 164.3; high sodium = 391.1 +/- 228.6; P < 0.05 low versus high). The increase in blood pressure was directly and significantly associated with the individual salt sensitivity, defined by the difference in systemic mean blood pressure between high and low sodium diets (r = 0.756; P < 0.05). L-NMMA also reduced renal plasma flow and urinary flow rate in subjects on high sodium diet. CONCLUSIONS: The data support a significant influence of endogenous NO in the systemic and renal vascular adaptation to a high sodium diet in normotensive men. In addition, the direct association between the individual sodium-sensitivity and the pressor response to L-NMMA suggests that there is increased dependence of vascular tone on NO in normotensive subjects whose blood pressure is more sodium sensitive.

Autoregulation of nitric oxide-soluble guanylate cyclase-cyclic GMP signalling in mouse thoracic aorta.

1. The sensitivity of the soluble guanylate cyclase (sGC)-cyclic guanosine-3',5'-monophosphate (cyclic GMP) system to nitric oxide (NO) was investigated in mouse aorta from wild type (WT) and NO synthase (NOS) knockout (KO) animals. 2. The NO donor, spermine-NONOate (SPER-NO) was more potent in aortas from eNOS KO mice compared to WT (pEC50 7.30+/-0.06 and 6.56+/-0.04, respectively; n=6; P<0.05). In contrast, the non-NO based sGC activator, YC-1 was equipotent in vessels from eNOS WT and KO mice. The sensitivity of aortas from nNOS and iNOS KO animals to SPER-NO was unchanged. Forskolin (an adenylate cyclase activator), was equipotent in vessels from eNOS WT and KO animals. 3. The cyclic GMP analogue, 8-Br-cGMP was equipotent in eNOS WT and KO mice (pEC50 4. 38+/-0.04 and 4.40+/-0.05, respectively; n=5; P>0.05). Zaprinast (10-5 M) a phosphodiesterase type V (PDE V) inhibitor, had no effect on the response to SPER-NO in vessels from eNOS WT or KO mice. 4. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 3x10-4 M) increased the potency of SPER-NO in aortas from WT mice (pEC50 6. 64+/-0.02 and 7.37+/-0.02 in the absence and presence of L-NAME, respectively; n=4; P<0.05). 5. In summary, there is increased sensitivity of vessels from eNOS KO animals to NO. Cyclic AMP-mediated dilatation is unchanged, consistent with a specific up-regulation of sGC - cyclic GMP signalling. The functional activity of cyclic GMP-dependent protein kinase (G-kinase) and PDE V was also unchanged, suggesting that sGC is the site of up-regulation. These alterations in the sensitivity of the sGC - cyclic GMP pathway might represent a mechanism for the dynamic regulation of NO bioactivity.

Biochemical characterization of S-nitrosohemoglobin. Mechanisms underlying synthesis, no release, and biological activity.

S-Nitrosohemoglobin (SNO-Hb) has been suggested to act as an endogenous NO donor and physiological regulator of blood pressure. However, the mechanisms responsible for the formation of SNO-Hb and those underlying the release of NO and subsequent biological activity have yet to be elucidated. In the present study, a number of nitrosated oxyhemoglobin (HbO(2)) derivatives have been synthesized and characterized. HbO(2) can be nitrosated at up to three distinct residues, one in the alpha-globin chain and two in the beta-chain. A beta-chain mononitrosated species (designated "SNO-Hb"), generated by the reaction of HbO(2) and S-nitrosoglutathione, released NO via a thiol-dependent mechanism involving nucleophilic attack at the nitrosated thiol functionality of SNO-Hb; in the case of glutathione, this process was associated with the formation of a mixed disulfide. In contrast, multinitrosated hemoglobin species released NO and relaxed vascular smooth muscle by a thiol-independent mechanism. HbO(2) scavenged potently NO released from SNO-Hb and inhibited its vasorelaxant properties. These data show that the predominant vasoactive species released from SNO-Hb is NO, with HNO a putative intermediate; the presence of a low molecular weight thiol is a prerequisite for this process. Such observations have important implications for the generation, metabolic fate, and biological activity of S-nitrosothiols.

Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial arginine deiminases.

Methylarginines inhibit nitric oxide synthases (NOS). Cellular concentrations of methylarginines are determined in part by the activity of dimethylarginine dimethylaminohydrolase (DDAH; EC 3.5.3. 18). We have cloned human DDAH and identified and expressed a second novel DDAH isoform (DDAH I and II respectively). DDAH I predominates in tissues that express neuronal NOS. DDAH II predominates in tissues expressing endothelial NOS. These results strengthen the hypothesis that methylarginine concentration is actively regulated and identify molecular targets for the tissue and cell-specific regulation of methylarginine concentration.

Determinants of the response of human blood vessels to nitric oxide donors in vivo.

The potency of the nitric oxide (NO) donors glyceryltrinitrate (GTN) and 3-morpholinosydnonimine was compared in human dorsal hand veins, the radial artery, and the forearm resistance vessels. NO donors were more potent in veins and the radial artery (vessels with minimal basal NO-mediated dilatation) than in the resistance vascular bed (where basal NO is a major determinant of vascular tone). In contrast, 8-bromoguanosine 3',5'-cyclic monophosphate (a cGMP mimetic) was approximately equipotent in resistance arteries and veins and was less potent in the radial artery. Inhibition of phosphodiesterase V with dipyridamole did not alter the arteriovenous profile of GTN. Increasing the local concentration of NO in veins (by infusing sodium nitroprusside) reduced their sensitivity to GTN but not to 8-bromoguanosine 3',5'-cyclic monophosphate. Conversely, reducing endogenous NO production in the resistance vasculature led to time-dependent increases in the response to GTN. These data suggest that soluble guanylate cyclase rather than cGMP-dependent protein kinase or phosphodiesterase V is the site in the second messenger pathway that determines the arteriovenous profile of NO donors. Moreover, the sensitivity of soluble guanylate cyclase to NO donors might be regulated by the ambient concentration of NO, with increased local NO down-regulating the dilator response to NO donors.

Endogenous inhibitors of nitric oxide synthesis: how important are they?

In animals and humans, inhibition of nitric oxide (NO) production has widespread effects. Reduced activity of the NO:cyclic GMP pathway has been documented in disease states, including hypertension, diabetes and certain types of renal disease. Inhibitors of NO synthesis occur endogenously, and have been implicated in the regulation of the NO pathway in health and disease. Here we review the possible biological roles of endogenous NO synthase inhibitors, with particular reference to renal disease.

Dose- and time-dependency of the dilator effects of the endothelin antagonist, BQ-123, in the human forearm.

AIMS: Endothelin-1 is a potent endogenous vasoconstrictor that acts on the endothelin A (ET[A]) receptor. The dose-response and time-course of the dilator effect of the ET(A) receptor antagonist, BQ-123, was investigated in the forearm of healthy volunteers. METHODS: Forearm blood flow was measured using mercury-in rubber strain gauge venous occlusion plethysmography. RESULTS: Following intra-arterial infusion of BQ-123 (50 nmol min-1) for 5 min, forearm blood flow increased by approximately 60% over the next 60 minutes; lower doses were without significant effect. The degree of dilatation was similar to that observed in previous studies using 20-fold larger doses. CONCLUSIONS: This study confirms that basal endothelin-1 has a role in the physiological regulation of vascular tone. It is possible that at low doses, BQ-123 might be a more sensitive pharmacological tool for the detection of abnormal endothelin-1 mediated constriction.

Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase.

1. Dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes the endogenous nitric oxide synthase inhibitors NG-monomethyl-arginine and NG,NG-dimethy-L-arginine to citrulline, was identified by Western blotting in rat and human tissue homogenates. 2. S-2-amino-4(3-methylguanidino)butanoic acid (4124W) inhibited the metabolism of [14C]-NG-monomethyl-L-arginine to [14C]-citrulline by rat liver homogenates (IC50 416 +/- 66 microM; n = 9), human cultured endothelial cells (IC50 250 +/- 34 microM; n = 9) and isolated purified dimethylarginine dimethylaminohydrolase. 3. Addition of 4124W to culture medium increased the accumulation of endogenously-generated NG,NG-dimethy-L-arginine in the supernatant of human cultured endothelial cells from 3.1 +/- 0.3 to 5 +/- 0.7 microM (n = 15; P < 0.005). 4. 4124W (1 microM - 1 mM) had no direct effect on endothelial nitric oxide synthase activity but caused endothelium-dependent contraction of rat aortic rings (1 mM 4124W increased tone by 81.5 +/- 9.6% of that caused by phenylephrine 100 nM). This effect was reversed by L-arginine (100 microM). 4124W reversed endothelium-dependent relaxation of human saphenous vein (19.2 +/- 6.7% reversal of bradykinin-induced relaxation at 1 mM 4124W). 5. These data suggest that inhibition of dimethylarginine dimethylaminohydrolase increases the intracellular contraction of NG,NG-dimethyl-L-arginine sufficiently to inhibit nitric oxide synthesis. Inhibiting the activity of DDAH may provide an alternative mechanism for inhibition of nitric oxide synthases and changes in the activity of DDAH could contribute to pathophysiological alterations in NO generation.