cDNA microarray analysis of vascular gene expression after nitric oxide donor infusions in rats: implications for nitrate tolerance mechanisms

Vascular nitrate tolerance is often accompanied by changes in the activity and/or expression of a number of proteins. However, it is not known whether these changes are associated with the vasodilatory properties of nitrates, or with their tolerance mechanisms. We examined the hemodynamic effects and vascular gene expressions of 2 nitric oxide (NO) donors: nitroglycerin (NTG) and S-nitroso-N-acetylpenicillamine (SNAP). Rats received 10 microg/min NTG, SNAP, or vehicle infusion for 8 hours. Hemodynamic tolerance was monitored by the maximal mean arterial pressure (MAP) response to a 30-microg NTG or SNAP bolus challenge dose (CD) at various times during infusion. Gene expression in rat aorta after NTG or SNAP treatment was determined using cDNA microarrays, and the relative differences in expression after drug treatment were evaluated using several statistical techniques. MAP response of the NTG CD was attenuated from the first hour of NTG infusion (P <.001, analysis of variance [ANOVA]), but not after SNAP (P >.05, ANOVA) or control infusion (P >.05, ANOVA). Student t-statistics revealed that 447 rat genes in the aorta were significantly altered by NTG treatment (P <.05). An adjusted t-statistic approach using resampling techniques identified a subset of 290 genes that remained significantly different between NTG treatment vs control. In contrast, SNAP treatment resulted in the up-regulation of only 7 genes and the down-regulation of 34 genes. These results indicate that continuous NTG infusion induced widespread changes in vascular gene expression, many of which are consistent with the multifactorial and complex mechanisms reported for nitrate tolerance.     

Lack of critical involvement of endothelial nitric oxide synthase in vascular nitrate tolerance in mice

We examined the direct involvement of endothelial nitric oxide (eNOS) in nitrate tolerance using eNOS knockout (eNOS (-/-)) and wild-type (eNOS (+/+)) mice. Animals were treated with either nitroglycerin (NTG, 20 mg kg(-1)s.c. 3 x daily for 3 days) or vehicle (5% dextrose, D5W), and nitrate tolerance was assessed ex vivo in isolated aorta by vascular relaxation studies and cyclic GMP accumulation. Western blot was performed to determine NOS expression after NTG treatment. In both the eNOS (-/-) and (+/+) mice, the EC(50) from NTG concentration-response curve was increased by approximately 3 fold, and vascular cyclic GMP accumulation was similarly decreased after NTG pretreatment. Vascular tolerance did not lead to changes in eNOS protein expression in eNOS (+/+) mice. These results indicate that vascular nitrate tolerance was similarly induced in eNOS (-/-) and (+/+) mice, suggesting that eNOS may not be critically involved in nitrate tolerance development in mice.     

Microarray analysis of differential gene expression in lead-exposed astrocytes

The toxic metal lead is a widespread environmental health hazard that can adversely affect human health. In an effort to better understand the cellular and molecular consequences of lead exposure, we have employed cDNA microarrays to analyze the effects of acute lead exposure on large-scale gene expression patterns in immortalized rat astrocytes. Our studies identified many genes previously reported to be differentially regulated by lead exposure. Additionally, we have identified novel putative targets of lead-mediated toxicity, including members of the family of calcium/phospholipid binding annexins, the angiogenesis-inducing thrombospondins, collagens, and tRNA synthetases. We demonstrate the ability to distinguish lead-exposed samples from control or sodium samples solely on the basis of large-scale gene expression patterns using two complementary clustering methods. We have confirmed the altered expression of candidate genes and their encoded proteins by RT-PCR and Western blotting, respectively. Finally, we show that the calcium-dependent phospholipid binding protein annexin A5, initially identified as a differentially regulated gene by our microarray analysis, is directly bound and activated by nanomolar concentrations of lead. We conclude that microarray technology is an effective tool for the identification of lead-induced patterns of gene expression and molecular targets of lead.     

Time-dependent expression pattern of nitric oxide and superoxide after myocardial infarction in rats.

Previous experiments by our group have demonstrated a subacute increased contribution of nitric oxide (NO) to vasoreactivity after myocardial infarction in rats. However, the activation pattern of NO may be phasic after infarction and has been described to be strongly associated with superoxide production. Therefore, the present study evaluated the morphological distribution and time-dependent induction of NO and superoxide at the protein (via immunohistochemistry, chemiluminescence and spectrophotometry) and mRNA (via real-time RT-PCR) level after experimental induction of a myocardial infarction in rats. Myocardial infarction led to a modest but lasting upregulation of endothelial NO synthase (eNOS) in blood vessels and cardiomyocytes. In contrast, inducible NOS (iNOS) showed dramatic de novo formation 1 week after infarction, predominantly in the infarcted area and cardiomyocytes. In addition, a gradually increased myocardial production of superoxide was detected during remodelling, probably related by an upregulation of NADH oxidase in the peri-infarcted and remote myocardium. Furthermore, peroxynitrite formation was increased after myocardial infarction, indicating strong interaction between NO and superoxide. In conclusion, during the early remodelling phase after myocardial infarction mRNA concentrations and protein levels of eNOS and iNOS show an augmentation in a biphasic manner. Moreover, upregulation of NADH oxidase leads to increased presence of superoxide and peroxynitrite and thereby to a reduction of the bioavailability and compensatory effects of NO, as such influencing post-infarction remodelling.     

Inhibition of inducible nitric oxide synthase gene expression and nitric oxide synthesis in vascular smooth muscle cells by granulocyte-colony stimulating factor in vitro.

Clinical and experimental evidence suggests that granulocyte-colony stimulating factor (G-CSF) acts as an anti-inflammatory modulator with beneficial effects in severe inflammatory diseases, e.g., sepsis and septic shock. Excessive production of nitric oxide (NO) is regarded as a potent mediator of the vascular changes leading to systemic hypotension that occurs during sepsis. Therefore, the aim of the present study was to investigate the influence of G-CSF on inducible nitric oxide synthase (iNOS) gene expression and NO synthesis in vascular smooth muscle cells (VSMC). Qualitative and quantitative analyses of iNOS cDNA revealed that G-CSF significantly reduced interferon-gamma/lipopolysaccharide (IFN-gamma/LPS) dependent iNOS gene expression (P < 0.05) following 6, 18, 24, and 48 h incubation periods. In addition, the co-application of G-CSF resulted in a decreased IFN-gamma/LPS mediated iNOS protein generation as detected by immunoblotting methods after 24 and 48 h. Measurement of the stable NO metabolites showed a significant reduction of nitrite/nitrate concentrations following co-incubation of VSMC with G-CSF + IFN-gamma/LPS (242.57 +/- 10.73 nmol NO2-/NO3-/mg cell protein, n = 8) as compared to IFN-gamma/LPS treatment (306.20 +/- 19.26 nmol NO2-/NO3-/mg cell protein, n = 8, P < 0.05) following a 24-h incubation protocol. This inhibitory effect of G-CSF was still present after a 48 h incubation period (G-CSF + IFN-gamma/LPS: 319.56 +/- 6.26 nmol NO2-/NO3-/mg cell protein; IFN-gamma/LPS: 489.20 +/- 27.15 nmol NO2-/NO3-/mg cell protein (P < 0.05), n = 8, respectively). The present findings suggest that inhibition of iNOS gene expression and NO generation in VSMC might be one of the protective anti-inflammatory effects of G-CSF during sepsis.     

贫铀对大鼠肺诱导型一氧化氮合酶基因表达的影响

目的通过研究贫铀(depleted uranium,DU)颗粒气管灌注大鼠肺中的诱导型一氧化氮合酶(iNOS)基因表达的变化,揭示DU对肺组织的毒性作用机制。方法Wistar大鼠20只随机分为4组,1个对照组,3个染铀组,剂量分别为1、3、5 mg/ml气管灌注不同剂量DU颗粒3个月后,将大鼠肺组织iNOS mRNA进行RT-PCR并通过凝胶成像分析系统扫描RT-PCR产物,用内参半定量法分析iNOS mRNA的变化。结果对照组无iNOS mRNA表达,各染铀组扩增产物电泳条带吸光度值(A)明显高于对照组(P<0.05);其中13、mg组产物电泳条带A值逐渐增高,3 mg组到达高峰,5 mg组产物电泳条带A值明显低于13、mg组(P<0.05)。结论DU颗粒气管灌注能使大鼠肺组织iNOS mRNA表达水平升高,并与DU剂量呈正相关。DU剂量增高到一定程度则使iNOS mRNA表达水平降低,这种变化可能与DU化学毒性和辐射损伤的复合作用有关。     

Microarray analysis of changes in bone cell gene expression early after cadmium gavage in mice

We developed an in vivo model for cadmium-induced bone loss in which mice excrete bone mineral in feces beginning 8 h after cadmium gavage. Female mice of three strains [CF1, MTN (metallothionein-wild-type), and MT1,2KO (MT1,2-deficient)] were placed on a low-calcium diet for 2 weeks. Each mouse was gavaged with 200 microg Cd or vehicle only. Fecal calcium was monitored daily for 9 days, beginning 4 days before cadmium gavage, to document the bone response. For CF1 mice, bones were taken from four groups: +/- Cd, 2 h after Cd and +/- Cd, 4 h after Cd. MTN and MT1,2KO strains had two groups each: +/-Cd, 4 h after Cd. PolyA+ RNA preparations from marrow-free shafts of femura and tibiae of each +/- Cd pair were submitted to Incyte Genomics for microarray analysis. Fecal Ca results showed that bone calcium excreted after cadmium differed for the three mouse strains: CF1, 0.24 +/- 0.08 mg; MTN, 0.92 +/- 0.22 mg; and MT1,2KO, 1.7 +/- 0.4 mg. Gene array results showed that nearly all arrayed genes were unaffected by cadmium. However, MT1 and MT2 had Cd+/Cd- expression ratios >1 in all four groups, while all ratios for MT3 were essentially 1, showing specificity. Both probes for MAPK 14 (p38 MAPK) had expression ratios >1, while no other MAPK responded to cadmium. Vacuolar proton pump ATPase and integrin alpha v (osteoclast genes), transferrin receptor, and src-like adaptor protein genes were stimulated by Cd; other src-related genes were unaffected. Genes for bone formation, stress response, growth factors, and signaling molecules showed little or no response to cadmium. Results support the hypothesis that Cd stimulates bone demineralization via a p38 MAPK pathway involving osteoclast activation.     

Altered L-arginine/nitric oxide synthase/nitric oxide pathway in the vascular adventitia of rats with sepsis

1. In recent studies, the vascular adventitia has been established as an important source of inducible nitric oxide synthase (iNOS) and subsequent nitric oxide (NO) production, even more powerful than the media in response to certain inflammatory factors, such as lipopolysaccharide (LPS). The adventitia has an independent L-arginine (L-Arg)/NOS/NO pathway and is involved in the regulation of vascular function. In the present study, we explored the changes in and the pathophysiological significance of the L-Arg/NOS/NO pathway in the adventitia of rats with sepsis. 2. Sepsis was induced by caecal ligation and puncture in order to observe changes in L-Arg transport, NOS gene expression and activity and NO generation in the vascular adventitia to determine the mechanism of activation of the L-Arg/NOS/NO pathway. 3. Severe sepsis resulted in severe disturbance of haemodynamic features, with decreased mean arterial blood pressure, brachycardia and inhibited cardiac function (decreased left ventricular +/-dP/dt(max)). Left ventricular end-diastolic pressure was elevated threefold (P < 0.01) under anaesthesia. Rats with sepsis showed severe glucopenia and lacticaemia. Plasma levels of the inflammatory factors macrophage chemoattractant protein-1 and interleukin-8 were increased five- and 29-fold, respectively (P < 0.01). 4. In the adventitia of the thoracic and abdominal aortas, the L-Arg/NO pathway was similarly characterized: the uptake of [(3)H]-L-Arg was Na(+) independent, with the peak occurring at approximately 40 min incubation. Total NOS activity was largely calcium independent (> 90%). The V(max) of L-Arg transport in the sepsis group was increased by 83.5% (P < 0.01), but the K(m) value was not significantly different compared with controls. 5. The mRNA levels of cationic amino acid transporter (CAT)-1 and CAT-2B in the sepsis group were increased by 86 and 62%, respectively (both P < 0.01). Inducible NOS activity was increased 2.8-fold compared with controls (P < 0.01) and iNOS mRNA levels were elevated approximately sixfold (P < 0.01). The NO levels in the plasma and incubation media (incubation for 40 min) in the sepsis group were increased by 144 and 273%, respectively (both P < 0.01). 6. The Arg/NOS/NO pathway was activated in the vascular adventitia of rats with sepsis shock. The L-Arg/NOS/NO pathway in the aortic adventitia may play an important role in the pathogenesis of sepsis and septic shock.     

The role of nitric oxide in diabetes-induced changes of morphine tolerance in rats

Several neuroendocrine complications including diabetes change the morphine antinociception and the development of tolerance to the drug. Morphine antinociception was reduced significantly in morphine tolerant diabetic rats compared to the non-diabetic animals. The exact mechanism of this effect is not known. This study was performed to determine the role of nitric oxide (NO) on morphine tolerance in diabetic state. Nociceptive responses in alloxan-induced diabetic morphine tolerated rats were measured by the hot-plate test. The urinary nitric oxide level was measured spectrophotometrically with Griess reagent. For the conversion of nitrate to nitrite, vanadium chloride was used. The results showed that experimental diabetes increased morphine analgesia. Conversely, degree of tolerance to morphine was diminished in diabetic state. The urinary nitrite content in diabetic morphine tolerated rats was higher than non-diabetic groups. L-arginine significantly increased the NO production in diabetic morphine tolerated animals, whereas aminoguanidine decreased it. Appropriately, L-arginine increased the latency time of reaction to noxious stimuli in diabetic compared to non-diabetic rats. L-arginine-treated animals also showed more tolerance to morphine analgesia. As expected, aminoguanidine deducted the level of morphine tolerance in diabetic animals. It is suggested that NO has a modulatory role in the effects of diabetes on morphine analgesia and tolerance.     

Microarray analysis of glomerular gene expression in murine lupus nephritis

To elucidate the molecular mechanism of glomerular events in lupus nephritis, we performed genome-wide mRNA expression analysis of glomeruli microdissected from lupus mice. MRL/lpr mice (12-week-old) were orally given vehicle or prednisolone (10 mg/kg per day) for 4 weeks. Renal histology of MRL/lpr mice revealed mesangial proliferative glomerulonephritis with cellular infiltration of macrophages, T cells, and neutrophils. We identified 567 up-regulated genes in MRL/lpr glomeruli compared to control congenic mice. Those included complement components, adhesion molecules, chemokines and their receptors, and molecules related to antigen presentation. Over 130 genes were considered preferentially or exclusively expressed in hematopoietic cell lineages possibly reflecting leukocytes accumulation. Of note is the finding that chemokines and chemokine receptors (CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11, CXCL16, CCR5, CXCR3, and CXCR6) that are related to T helper 1 (Th1) cells accumulation were up-regulated concomitantly with increased expression of Ebi3, a subunit of IL-27 that plays a role in Th1 predominance. These changes were accompanied by increased mRNA expression of many genes that were inducible by Th1 cytokine interferon-gamma. Prednisolone markedly attenuated glomerular lesion and leukocyte influx parallel with the reduction of enhanced gene expression. The present study shows additional evidence supporting glomerular Th1 cells accumulation and their role. Our data also provide an important resource in seeking new therapeutic targets to lupus nephritis. Supplemental table: available only at http://dx.doi.org/10.1254/jphs.FP0071337.     

Increased renal vascular resistance in zinc-deficient rats: role of nitric oxide and superoxide

1. Zinc deficiency (ZD) induces many kinds of pathological states. However, the effects of ZD on haemodynamics remain unclear. In the present study, we measured mean blood pressure (BP) and renal blood flow (RBF) under anaesthesia and calculated renal vascular resistance (RVR) from these parameters in rats maintained on a ZD diet (0.5 p.p.m. zinc) for 4 weeks. 2. Zinc deficiency did not change mean BP, but significantly reduced RBF and increased RVR (each P < 0.01). In addition, these effects of ZD were reversible. 3. Because Cu/Zn superoxide dismutase (SOD) is a zinc-containing enzyme and superoxide is a potent scavenger of nitric oxide (NO), a vasodilator, we hypothesized that one of the mechanisms by which ZD increases RVR is by decreasing NO bioavailability by the enhanced formation of superoxide due to low Cu/Zn SOD activity. To test this hypothesis, we observed the roles of NO and superoxide in the mechanism, after having confirmed the low activity of Cu/Zn SOD in the kidneys of ZD rats. 4. Administration of the SOD mimetic tempol (5 mg/kg per min) decreased RVR to a significantly greater extent in ZD rats compared with control, suggesting that superoxide was responsible for the mechanism. Low doses of the NO donor sodium nitroprusside (SNP; 2.0 micro g/kg per min, continuous) decreased RVR to a significantly smaller extent in ZD rats compared with control, whereas a high dose of SNP (0.75 mg/kg, bolus) decreased RVR to a significantly greater extent in ZD rats compared with control, suggesting that the mechanism includes an inhibition of NO activity in ZD, which is most likely to be a scavenging of NO by the activated superoxide. 5. In summary, ZD may increase RVR. The mechanism probably includes changes in NO and superoxide activities.     

Contribution of nitric oxide produced by inducible nitric oxide synthase to vascular responses of mesenteric arterioles in streptozotocin-diabetic rats

1. The functional changes in mesenteric arterioles of streptozotocin-induced diabetes were investigated by intravital microscopy. The mesentery was exteriorized from anesthetized rats, spread in a chamber, and superfused with Tyrode solution. All drugs tested were applied to the superfusing Tyrode solution. 2. Compared with age-matched controls, the diabetic rats showed enhanced vascular sensitivity to phenylephrine, an alpha(1)-adrenoceptor agonist. The preincubation of the mesentery with N(G)-nitro-l-arginine (l-NNA), a nitric oxide synthase (NOS) inhibitor, shifted the phenylephrine-concentration-response curves to the left in both the diabetic and control rats. Even in the presence of l-NNA, the sensitivity to phenylephrine was higher in the diabetic rats than in the control. 3. Acetylcholine relaxed the mesenteric arterioles in both groups, but to a significantly greater extent in the control than in the diabetic rats. However, the l-NNA-induced constriction of arterioles did not differ significantly between the groups. In contrast, the amplitude of the constrictions of mesenteric arterioles induced by S-ethylisothiourea, an inducible NOS (iNOS) inhibitor, was significantly greater in the diabetic rats than in the control. 4. Immunostaining of the mesentery with a specific antibody for iNOS revealed iNOS in the microvessels of only the diabetic rats. 5. These results suggest that constrictor responses to alpha(1)-adrenoceptor stimulation are sensitized in the mesenteric arterioles of STZ-diabetic rats, and that iNOS expressed in the arteriolar smooth muscle plays a role in suppressing the basal tone and the reactivity of the arterioles in STZ-diabetic rats.     

Cardiac and vascular effects of nitric oxide synthase inhibition in lipopolysaccharide-treated rats

In the present study, intraperitoneal injection of lipopolysaccharide (10 mg/kg) to anaesthetized rats produced a gradual fall in mean arterial pressure in 6 h. Aortic rings from lipopolysaccharide-treated rats showed a significant reduction in the contractile response to vasoconstrictors. Pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine, two nitric oxide synthase (NOS) inhibitors, abolished this vascular hyporeactivity. In ventricular myocytes isolated from lipopolysaccharide-treated rats, both electrically induced Ca(2+) transients and the intracellular Ca(2+) response to beta-adrenergic stimulation were significantly depressed when compared with those recorded from myocytes from sham control rats. L-NAME and aminoguanidine alone had no effects on electrically stimulated Ca(2+) transients in ventricular myocytes either from control or lipopolysaccharide-treated rats. However, these two NOS inhibitors augmented the intracellular Ca(2+) response to beta-adrenergic stimulation in myocytes from lipopolysaccharide-treated rats, but not in control myocytes. In addition, 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ), an inhibitor of nitric oxide (NO)-sensitive guanylyl cyclase, also reversed the intracellular Ca(2+) hyporesponsiveness to beta-adrenergic stimulation in myocytes from lipopolysaccharide-treated rats. In cardiac myocytes from lipopolysaccharide-rats pretreated with aminoguanidine, the intracellular Ca(2+) hyporesponsiveness to beta-adrenergic stimulation was abolished. However, there still existed a depressed Ca(2+) response to electrical field stimulation. These data indicate that NO following lipopolysaccharide stimulation contributes to vascular hyporeactivity and the depressed intracellular Ca(2+) response to beta-adrenergic stimulation in lipopolysaccharide-treated rats, but is not responsible for the reduced Ca(2+) response to electrical stimulation in our experimental conditions.     

A photosensitive vascular smooth muscle store of nitric oxide in mouse aorta: no dependence on expression of endothelial nitric oxide synthase

(1) Photorelaxation is the reversible relaxation of vascular smooth muscle (VSM) when irradiated with ultraviolet (UV) light resulting from the release of nitric oxide (NO). In this study we characterize the involvement of endothelial nitric oxide synthase (eNOS) in the photorelaxation response of thoracic aorta from endothelial NOS deficient (-/-) and control (C57BL/6j) mice. (2) Cirazoline contracted aortae were repeatedly exposed to 30 s of UV light every 3-4 min. Equal levels of photorelaxation (45+/-2%; n=34) was observed in both strains. (3) 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), K(+), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), 4-aminopyridine (4-AP) and ethacrynic acid significantly reduced the photorelaxation response. In C57BL/6j mice diethyldithiocarbamate (DETCA) also reduced photorelaxation. (4) Control endothelium-intact and -denuded aorta and L-NAME (100 micro M) treated and untreated eNOS (-/-) aortae were repeatedly exposed to UV light for 5 min every 10 min until no photorelaxation response was observed. After 1 h of rest in the dark the vessels showed between 30-70% recovery of the photorelaxation response indicating regeneration of the store in the absence of the endothelium and eNOS. (5) The results of this study suggest that photorelaxation in mouse aorta VSM results from the release of NO from a stable store of RSNOs, which activates soluble guanylate cyclase (sGC), leading to cGMP-dependent relaxation that is partially mediated by an increase in K(V) channel activation and hyperpolarization. In addition, the eNOS isoform is not essential for the formation of the photorelaxation store and a non-NOS source of NO may be involved in the maintenance of this store.     

Reversal of iron-induced nephrotoxicity in rats by molsidomine, a nitric oxide donor.

Fe-NTA is a very potent nephrotoxic agent and causes oxidative renal injury as shown in various studies. Reactive oxygen species as well as nitric oxide (NO) play an important role in acute renal failure (ARF). Present study was designed to investigate the effect of NO donor, molsidomine (Mol) and inducible NO synthase inhibitor (iNOS), aminoguanidine (AG) in Fe-NTA-induced renal toxicity. Rats were pretreated with Mol (5, 7.5 and 10 mg/kg, p.o.), and AG (100 mg/kg, i.p.) before Fe-NTA challenge (8 mg iron/kg body weight, i.p.) to determine the urea and creatinine levels along with biochemical analysis of oxidative stress. Fe-NTA administration markedly increased the BUN and serum creatinine level which was coupled with a marked lipid peroxidation, reduced activity of glutathione and decreased total nitric oxide levels of rat kidneys coupled with significant morphological alterations. Fe-NTA also markedly increased the levels of tumor necrosis factor-alpha (TNF-alpha) in serum. Concomitant treatment with molsidomine significantly reduced the serum creatinine and BUN levels, decreased lipid peroxidation in a significant manner, restored the levels of reduced glutathione, increased total nitric oxide levels and restored the normal morphology. Molsidomine treatment also attenuated the serum levels of TNF-alpha. Prior administration of AG did not reverse the protective effects produced by molsidomine. Present findings strongly suggest that protection afforded by molsidomine may be due to its direct NO donor ability but not through nitric oxide synthase activity as pretreatment with aminoguanidine did not abolish the protective effects of molsidomine.     

Platelet inhibitory effects of the nitric oxide donor drug MAHMA NONOate in vivo in rats

The platelet inhibitory effects of the nitric oxide (NO) donor drug MAHMA NONOate ((Z-1-[N-methyl-N-[6-(N-methylammoniohexyl)amino]]diazen-1-ium-1,2-diolate) were examined in anaesthetised rats and compared with those of S-nitrosoglutathione (GSNO; an S-nitrosothiol). Bolus administration of the aggregating agent ADP dose-dependently reduced the number of circulating free platelets. Intravenous infusions of MAHMA NONOate (3-30 nmol/kg/min) dose-dependently inhibited the effect of 0.3 micromol/kg ADP. MAHMA NONOate was approximately 10-fold more potent than GSNO. MAHMA NONOate (0.3-10 nmol/kg/min) also reduced systemic artery pressure and was again 10-fold more potent than GSNO. Thus MAHMA NONOate has both platelet inhibitory and vasodepressor effects in vivo. The dose ranges for these two effects overlapped, although blood pressure was affected at slightly lower doses. The platelet inhibitory effects compared favourably with those of GSNO, even though NONO-ates generate free radical NO which, in theory, could have been scavenged by haemoglobin. Therefore platelet inhibition may be a useful therapeutic property of NONOates.