Nitric oxide generation mediated by β-adrenoceptors is impaired in platelets from patients with Type 2 diabetes mellitus

Aims/hypothesis Type 2 diabetic patients have been shown to have reduced basal platelet nitric oxide synthase activity, which is a possible contributor to the vascular complications seen in the disease. We investigated platelet nitric oxide generation stimulated by -adrenoceptors and adenylyl cyclase in Type 2 diabetic patients and control subjects.Methods Platelets isolated from blood taken from nine Type 2 diabetic patients and nine healthy control subjects of similar age were treated with isoproterenol 1 µmol/l, forskolin 1 µmol/l or vehicle. Platelet nitric oxide synthase activity was measured by L-[³H]-arginine to L-[³H]-citrulline conversion, cyclic GMP content by radioimmunoassay, and nitric oxide synthase type 3 expression by western blotting.Results Basal platelet nitric oxide synthase activity was lower in diabetic patients than in control subjects (0.01±0.02 pmol L-citrulline/10⁸ platelets, compared with 0.12±0.05; p<0.05), although no corresponding difference was seen in basal platelet cyclic GMP (0.61±0.39 and 0.13±0.22 pmol cyclic GMP/10⁸ platelets respectively; p=0.37). In control subjects isoproterenol 1 µmol/l and forskolin 1 µmol/l increased platelet nitric oxide synthase activity (to 0.27±0.08 and 0.27±0.07 pmol L-citrulline/10⁸ platelets respectively; p<0.05 for each in comparison with basal) and cyclic GMP (to 1.84±0.41 and 1.86±0.48; p<0.05 for each in comparison with basal). This effect was not achieved in diabetic patients. Isoproterenol- and forskolin-stimulated cyclic GMP correlated inversely with plasma glucose and HbA_1c. Platelet nitric oxide synthase type 3 expression was not different in control and diabetic subjects and was not changed by acute exposure of platelets to isoproterenol.Conclusions/interpretation Nitric oxide generation stimulated by -adrenoceptors and adenylyl cyclase is impaired in platelets of people with Type 2 diabetes mellitus, with no corresponding change in nitric oxide synthase type 3 expression. It is possible that this impairment contributes to the thrombotic and atherosclerotic complications of Type 2 diabetes.Abbreviations AR -adrenoceptors - GFP gel-filtered platelets - L-NAME N^G-nitro-L-arginine methyl ester - L-NMMA N^G-monomethyl-L-arginine - NO nitric oxide - NOS2 nitric oxide synthase type 2 - NOS3 nitric oxide synthase type 3     

Role of nitric oxide,tetrahydrobiopterin and peroxynitrite in glucose toxicity-associated contractile dysfunction in ventricular myocytes

Aims/hypothesis Local overproduction of nitric oxide is seen in early stages of diabetes, which can react with superoxide (O₂^–) to form peroxynitrite (ONOO^–). The aim of this study was to examine the effect of scavengers for nitric oxide, O₂^–, ONOO^– and NOS cofactor tetrahydrobiopterin (BH₄) on high glucose-induced cardiac contractile dysfunction.Methods Ventricular myocytes were cultured for 24 h with either normal (N, 5.5 mmol/l) or high (25.5 mmol/l) glucose, with or without the nitric oxide scavengers haemoglobin (100 nmol/l), PTIO (100 µmol/l), the NOS inhibitor L-NMMA (100 µmol/l), superoxide dismutase (SOD, 500 U/ml), the ONOO^– scavengers urate (100 µmol/l), MnTABP (100 µmol/l), BH₄ (10 µmol/l) and its inactive analogue NH₄ (10 µmol/l), and the GTP cyclohydrolase I inhibitor DAHP (1 mmol/l). Myocyte mechanics, NOS protein expression and activity were evaluated.Results High glucose myocytes showed reduced peak shortening, decreased maximal velocity of shortening/relengthening (± dL/dt), prolonged relengthening (TR₉₀) and normal shortening duration (TPS) associated with reduced cytosolic Ca²⁺ rise compared to normal myocytes. The high glucose-induced abnormalities were abrogated or attenuated by urate, MnTBAP, L-NMMA, BH₄, and SOD, whereas unaffected by haemoglobin, PTIO and NH₄. L-NMMA reduced peak shortening while PTIO and DAHP depressed ± dL/dt and prolonged TPS or TR₉₀ in normal myocytes. High glucose increased NOS activity, protein expression of eNOS but not iNOS, which were attenuated by L-NMMA and BH₄, respectively.Conclusion/interpretation These results suggested that NOS cofactor, NO and ONOO^– play a role in glucose-induced cardiomyocyte contractile dysfunction and in the pathogenesis of diabetic cardiomyopathy.Abbreviations PTIO 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide - DAHP 2,4-diamino-6-hydroxy-pyrimidine - E-C excitation-contraction - MnTABP manganese (III) tetrakis (4-benzoic acid) porphyrin - ± dL/dt maximal velocity of shortening and relengthening - L-NAME N-nitro-L-arginine methyl ester - L-NMMA L-N^G-monomethyl-arginine - NOS nitric oxide synthase - PS peak shortening - ONOO^– peroxynitrite - O₂^– superoxide anion - SOD superoxide dismutase - BH₄ tetrahydrobiopterin - NH₄ tetrahydroneopterin - TPS time-to-peak shortening - TR₉₀ time-to-90% relengthening     

Enhanced myocardial contractility but not tachycardia persists in isolated working hyperthyroid rat hearts

Summary It is generally believed that the increased contractility and tachycardia of the hyperthyroid heart are a result of thyroid hormone-induced alterations of the mechanical and electrical properties of the heart, respectively. We compared the contractility (dP/dt_max) and the spontaneous beating rate of hyperthyroid and euthyroid hearts perfused in vitro in either a non-working or a working mode. The dP/dt_max (4196±74 mm Hg s^–1) and beating rate (322±8 beats/min) of the non-working hyperthyroid hearts were significantly higher (p<0.001) than those of the euthyroid hearts (3267±115 mm Hg s^–1 and 260±6 beats/min at an external Ca²⁺ of 2.5 mM). At 2.5 mM Ca²⁺, the working hyperthyroid hearts again displayed enhanced contractility (5636±179 mm Hg s^–1 vs 4508±172 mm Hg s^–1; p<0.001) but the spontaneous beating rate (275±7 beats/min) was not significantly different from euthyroid (261±8 beats/min). When hearts were subjected to periods of alternate non-working and working perfusion, the beating rate of the hyperthyroid hearts was significantly higher than euthyroid during non-working (p<0.02) but not during working perfusion. Increasing the afterload on the non-working preparations in a stepwise fashion from 75 cm H₂O to 120 cm H₂O caused significant changes in left ventricular pressure and dP/dt_max in both heart types but the tachycardia in the hyperthyroid hearts persisted (at 120 cm H₂O; hyperthyroid, 294±9 beats/min; euthyroid, 224±10 beats/min; p<0.001). Alteration of the preload (10 to 25 cm H₂O) and afterload (75 to 105 cm H₂O) on working hyperthyroid and euthyroid hearts caused changes in both left ventricular pressure and dP/dt_max but the beating rates of both heart types were never significantly different. We conclude from our results that (i) the increased contractility of the hyperthyroid rat heart is due to thyroid hormone-induced alteration of the mechanical properties of the heart; (ii) the tachycardia of hyperthyroidism is not due to thyroid hormone-induced changes in the electrical properties of the heart, but probably involves some as yet unidentified chronotropic agent.     

Alterations in ventricular myocyte contraction caused by C-type natriuretic peptide and nitric oxide in eNOS-/- mice

Lack of endothelial nitric oxide synthase (eNOS) may affect the sensitivity of cyclic GMP signaling through soluble guanylyl cyclase (sGC). We hypothesized that in eNOS knockout (eNOS-/-) mice, stimulation of guanylyl cyclase would have enhanced effects inhibiting cardiac contraction. We measured cell shortening and calcium transients in isolated ventricular myocytes from adult eNOS-/- and wild-type (WT) mice after stimulating particulate guanylyl cyclase (pGC) with C-type natriuretic peptide (CNP, 10(-8) and 10(-7) M) or sGC with S-nitroso-N-acetyl-penicillamine (SNAP, NO donor, 10(-6) and 10(-5) M). Although sGC activity was increased by +71% in eNOS-/-, SNAP had similar effects in the two groups (%shortening -39% control vs. -37% eNOS-/-), suggesting that the cyclic GMP pathway was desensitized in eNOS-/- myocytes. CNP had significantly smaller effects on cell contraction (%shortening -34% control vs. -14% eNOS-/-) and pGC activity was not changed in eNOS-/- myocytes. Similar effects were also produced by guanylin and carbon monoxide, stimulators of pGC and sGC. CNP's effects on Ca(2+) transients were also attenuated in eNOS-/- myocytes. SNAP did not alter Ca(2+) transients in eNOS-/- or control cells. In the eNOS-/- mice, cyclic GMP-dependent protein kinase and cyclic AMP phosphodiesterase activity were reduced. This study demonstrated that the downstream cyclic GMP pathway was attenuated in eNOS-/- mice and this was partially compensated for by increased sGC, but not pGC activity in ventricular myocytes.     

Negative metabolic effects of cyclic GMP are altered in renal hypertension induced cardiac hypertrophy

We tested the hypothesis that increasing myocardial cyclic GMP levels would reduce myocardial O₂ consumption and that renal hypertension (One Kidney-One Clip, 1K1C)-induced cardiac hypertrophy would change this relationship. Four groups of anesthetized open-chest New Zealand white rabbits (N=26) were utilized. Either vehicle or 3-morpholinosydnonimine (SIN-1) (10^–4M, a guanylate cyclase activator) was topically applied to the left ventricular surface of control or 1K1C rabbits. Coronary blood flow (radioactive microspheres) and O₂ extraction (microspectrophotometry) were used to determine O₂ consumption. Myocardial cyclic GMP levels were determined by radioimmunoassay. Guanylate cyclase activity was measured by conversion of GTP to cyclic GMP. 1K1C rabbits had a greater heart weight-to-body weight ratio (3.29±0.15) than controls (2.63±0.19). Systolic blood pressure was higher in 1K1C rabbits than in controls. In control rabbits, cyclic GMP levels (pmoles/g) were higher in SIN-1-treated (EPI: 7.5±1.6; ENDO: 8.1±1.5) than in vehicle-treated animals (EPI: 5.4±0.4; ENDO: 5.6±0.6). This effect was enhanced in 1K1C rabbits, with cyclic GMP levels in the SIN-1-treated group (EPI: 11.9 ±1.3; ENDO: 13.0±1.5) almost double those observed in the vehicletreated group (EPI: 6.3±0.8; ENDO: 7.7±1.1). There were no significant differences in basal or maximally stimulated guanylate cyclase activity between controls and 1K1C rabbits. Myocardial O₂ consumption (ml O₂/min/100 g) was significantly less in the EPI region of control animals treated with SIN-1 (7.2±1.2) than in the same region of controls treated with vehicle (9.1±2.0). Myocardial O₂ consumption was also significantly less in SIN-1-than vehicle-treated 1K1C animals (SIN-1-treated: EPI: 6.9±0.8; ENDO: 6.2±0.7; vehicle-treated: EPI: 10.0±0.8; ENDO: 12.5±3.0). There was no significant difference in O₂ consumption between control and 1K1C animals after treatment with SIN-1. Thus, there was a greater elevation in cyclic GMP in 1K1C rabbits, but this did not result in a corresponding greater depression in O₂ consumption. This suggests that cyclic GMP plays a role in the control of myocardial metabolism, and that the sensitivity of myocardial O₂ consumption to changes in cyclic GMP is reduced by renal hypertension-induced cardiac hypertrophy.     

Role of force – frequency relation during AV–block,sinus node block and betaadrenoceptor block in conscious animals

Objective Myocardial contractility is regulated by adrenergic stimulation, the strength–length relationship and the force–frequency relationship or Bowditch effect. The latter mechanism was clearly demonstrated in muscle strips, in the isolated heart as well as in in–vivo experiments. The aim of this study was to further investigate the role of the force–frequency effect on the contractile response to exercise or isoproterenol infusion in conditions of restricted increases in heart rate i.e., AV–block, sinus node block and beta–adrenoceptor block. Methods Nineteen dogs were instrumented with a left ventricular miniature pressure gauge, catheters in the aorta, pulmonary artery and left atrium and pacing leads on the left atrium and left ventricle. In order to control the chronotropic response during sympathetic stimulation, permanent AV–block was induced in nine dogs, sinus node block using UL–FS 49 and beta–adrenoceptor block using propranolol was studied in ten dogs. Results Adrenergic stimulation (isoproterenol 0.4 µg/kg or exercise) after total AV–block failed to increase LVdP/dt. However, increasing LV pacing rate (from 50 up to 200 bpm) prior to adrenergic stimulation elicited a significant increase in LVdP/dt (4762 ± 166 mmHg/s vs. 6485 ± 381 mmHg/s, p < 0.05). In dogs in sinus rhythm, heart rate and LVdP/dt response to isoproterenol and exercise following pre–treatment with UL-FS 49 is significantly reduced, with heart rate increasing from 103 ± 7 up to 154 ± 5 bpm and LV dP/dt_max from 2925 ± 171 mmHg/s to 6249 ± 400 mmHg/s compared to the response in control conditions (HR 220 ± 3 bpm and LV dP/dt_max 7473 ± 616 mmHg/s) (p < 0.05). When heart rate is matched using atrial pacing, the LVdP/dt_max response reached comparable values as observed in control conditions (7310 ± 550 mmHg/s). Similar responses were obtained during exercise. Beta–adrenoceptor blockade attenuates considerably the heart rate and LVdP/dt response to sympathetic stimulation. Adjusting heart rate with atrial pacing restores only partially LVdP/dt_max. Conclusion During sympathetic stimulation, the chronotropic response plays a major role for the concomitant full expression of the inotropic response. In conditions where increases in heart rate are absent or severely restricted such as in permanent AV–block, sinus node block and beta–adrenoceptor block, the inotropic response will also be impaired.     

Regional asynchrony of segmental contraction may explain the “oxygen consumption paradox” in stunned myocardium

Summary Despite apparently depressed function, stunned myocardium maintains oxygen consumption and has the capacity to increase contractility with inotropic stimulation. We hypothesized that during stunning, O₂ demand is maintained because regional segment work is performed, but is asynchronous with global left ventricular contraction, and that inotropic stimulation would restore regional work and synchrony. Thirteen open-chest anesthetized dogs were subjected to three left anterior descending (LAD) coronary artery occlusions (10 min) and reperfusions (15 min) to produce regional myocardial stunning. Segment shortening and force development were measured independently and simultaneously in the LAD (experimental) region and circumflex (control) regions. Regional myocardial work was calculated as the integrated product of instantaneous force and shortening, during two periods: 1) over the entire cardiac cycle (Positive Work), and 2) limited to the systolic portion of the cardiac cycle (Systolic Work). Regional myocardial O₂ consumption (MVO₂) was calculated from regional blood flow (radiolabeled microspheres) and O₂ saturation data (microspectrophotometry). Occlusion of the LAD produced a delay in onset of segment shortening in the ischemic region, but not in regional force development. A time delay of 67–81 ms persisted through the three stages of occlusions and reperfusion. Systolic regional work was depressed to a greater extent (924±182 to 149±118 g*mm*min^–1) than total positive regional work (1437±337 to 857±174 g*mm*min^–1). Regional subepicardial MVO₂ in the stunned region was not different than in the control region (7.3±1.5 vs. 6.9±1.4 ml O₂*min^–1*100 g^–1). Local infusion of isoproterenol reversed the delay in regional shortening from 73±7 to 21±8 ms, thereby augmenting systolic work (298%) more than positive work (60%), without a significant increase in MVO₂ (7.3±1.5 to 10.5±3.2 ml O₂*min^–1*100 g^–1). It is concluded that myocardial stunning decreases regional systolic work due to regional mechanical asynchrony, while MVO₂ is used supported total positive work which was not significantly reduced. Isoproterenol restores regional work by restoring synchrony, without greatly affecting regional MVO₂.This study was supported in part by a research grant from the Schulz Foundation and USPHS grant HL40320.     

A New Enzymatic Assay for Guanosine 3′:5′-Cyclic Monophosphate and Its Application to the Ductus Deferens of the Rat

A sensitive enzymatic procedure has been developed for the determination of guanosine 3′:5′-cyclic monophosphate (cyclic GMP). It is based on the conversion of cyclic GMP to GMP by cyclic nucleotide phosphodiesterase and on the transfer of ³²P from [γ-³²P]ATP to GMP by the action of a specific ATP:GMP phosphotransferase (EC 2.7.4.8). The [³²P]GDP is separated from the remaining [³²P]ATP by enzymatic degradation of ATP by myosin and by precipitation of the ³²P_i formed. The reaction blank, which is mostly caused by the nucleotide content of the enzymes, is doubled by about 0.1 pmol of cyclic GMP. The procedure has advantages in speed and/or accuracy over other methods in current use.     

The Nitric Oxide Donor SIN-1 Protects Endothelial Cells from Tumor Necrosis Factor-α-Mediated Cytotoxicity: Possible Role For Cyclic GMP and Heme Oxygenase

In cultured endothelial cells, incubation with TNF-α(50 ng/ml) for 72 h markedly reduced viability of endothelial cells. A 6-h pre-incubation with the nitric oxide (NO) donor linsidomine (SIN-1, 10–150μ ) protected endothelial cells in a concentration-dependent manner and increased viability by up to 59% of control. The unmetabolized parent compound molsidomine and the NO-free metabolite of SIN-1 3-morpholinoiminoacetonitrile (SIN-1C) were without cytoprotective effect. Cytoprotection by SIN-1 was completely abolished by the NO scavenger 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO, 30μ ). A cytoprotective effect comparable to SIN-1 was observed when preincubating the cells with dibutyryl cyclic GMP (10–100μ ). Moreover, no protection by SIN-1 occurred in the presence of cycloheximide (1μ ) or 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 0.1μ ), a selective inhibitor of soluble guanylyl cyclase. Tin protoporphyrin-IX (SnPP, 25μ ), an inhibitor of heme oxygenase, was found to attenuate SIN-1-induced cytoprotection. Our results demonstrate that SIN-1 produces a long-term endothelial protection against cellular injury by TNF-α, presumably via a cyclic GMP-dependent pathway leading to up-regulation of protective proteins such as heme oxygenase.     

Mechanical Restitution of Contractility in Stunned Myocardium of Open-Chest Dogs

The aim of the present study was to determine the interval–contractility relationship in the same myocardial area before and after ischemia, thereby testing the hypothesis that calcium handling is impaired in stunned myocardium. Myocardial stunning was created by 15-min occlusion of the left anterior descending branch of coronary artery (LAD) followed by reperfusion in 10 open-chest dogs. Complete atrioventricular (A-V) block was achieved by injecting formaldehyde into the A-V node and the heart was paced by right ventricular electrodes. Extrasystoles at various intervals were then produced by electrical stimulation after cessation of regular pacing. Miniature force gauges were used to assess contractility. Mechanical restitution curves (MRCs) were constructed in the LAD area before and after stunning, by plotting normalized contractility against extrasystolic intervals (ESIs). The MRC was fitted to the monoexponential function FR_n = FR_max {1 – Exp[(ESI-ESI₀)/T _c]}, where FR_n is normalized contractile force, FR_max is the maximal value of a contractile response, T _c is the time constant for restitution, and ESI₀ is the smallest ESI that produced a mechanical response. After occlusion–reperfusion, FR_max decreased from (136.5 ± 8.2)% to (111.2 ± 5.4)%, whereas T _c and ESI₀ remained unchanged. These results suggested preserved sarcoplasmic reticulum function in stunned myocardium, but that the responsiveness of the contractile machinery is depressed.     

Leucocyte Na+/H+ antiport activity in Type 1 (insulin-dependent) diabetic patients with nephropathy

Summary The development of proteinuria in Type 1 (insulin-dependent) diabetic patients may depend on predisposition to essential hypertension in addition to poor glycaemic control. Previous work has shown increased leucocyte Na^–/H⁺ antiport activity in essential hypertension and increased erythrocyte Li⁺/Na⁺ exchange in Type 1 diabetic patients with proteinuria. To test whether susceptibility to nephropathy in Type 1 diabetes was linked to abnormalities of leucocyte Na⁺/H⁺ antiport activity, we measured the intracellular pH and kinetics of the Na⁺/H⁺ antiport in 19 Type 1 diabetic subjects with, and 15 diabetic subjects without albuminuria and compared them to 25 matched normal control subjects. Intracellular pH (mean ±SD 7.59 ± 0.14) and maximal transport capacity of the antiport (V_max 87.7 ±24.9 mmol· 1^–1·min^–1) were higher in diabetic subjects with albuminuria compared to normotensive control subjects (pH 7.44±0.09; V_max 55.6±10.3 mmol·1^–1min^–1; p <0.001 for both), similar to the defect described in essential hypertension. These differences were not seen in diabetic subjects with normal urinary albumin/creatinine ratios (pH 7.46 ±0.09; V_max 61.0 ±13.6mmol·1^–1min^–1). Buffering characteristics of the leucocytes at different pH in the Type 1 diabetic subjects with albuminuria differed from normal control subjects and diabetic subjects with normal urinary albumin/creatinine ratios. We conclude that increased leucocyte Na⁺/H⁺ antiport activity, a known marker of essential hypertension, is usually associated with nephropathy in Type 1 diabetes.     

Insulin action and insulin binding following pancreas transplantation

Insulin action and insulin specific binding to erythrocytes were examined in ten recipients of a pancreatic segment and renal graft (Group 1), in nine non-diabetic kidney recipients (Group 2) and in ten age- and weight-matched healthy control subjects (Group 3). All transplant recipients were normoglycaemic without need of insulin, received the same immunosuppression and had good renal graft function at 11–18 months post-transplantation, when the investigation was performed. Using the insulin clamp technique, insulin action was expressed as the metabolic clearance rate of glucose at insulin infusion rates of 1.0 (MCR_submax) and 10.0 (MCR_max) mU·kg^–1·min^–1. In comparison with the healthy control subjects, fasting free insulin and C-peptide levels were significantly higher in Groups 1 and 2, but no differences between Groups 1 and 2 were found (p>0.05). Mean values±SEM of MCR_submax in Groups 1, 2 and 3 were 6.30±0.55, 6.09 ±0.69 and 10.52±1.10 ml·kg^–1·min^–1 respectively, and of MCR_max 12.65±0.78, 13.14±0.92 and 19.28±1.42 ml·kg^–1·min^–1 respectively. Insulin action was significantly decreased in Groups 1 and 2 at the low as well as the high insulin infusion rates but there was no difference between the two groups of recipients (p>0.05). No differences in binding data (specific binding, number of binding sites per cell) were found. It is concluded that insulin resistance is common to all immunosuppressed organ recipients and is not related to the pancreas graft. The decreased maximal response to insulin and normal insulin binding to erythrocytes tend to suggest a post-receptor defect in insulin action.     

Pituitary adenylate cyclase activating peptide induces cGMP-mediated relaxation in guinea-pig airways

The mechanism of relaxation of the guinea-pig trachea induced by pituitary adenylate cyclase activating peptide (PACAP)-27 was investigated. We examined whether modulators of nitric oxide (NO) and carbon monoxide (CO) affect PACAP-induced response of tracheal strips in vitro. Pretreatment with N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) and L-arginine (L-arg) had no effect, while 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), haemoglobin and zinc protoporphyrin IX (ZnPP-9) partially abolished the PACAP-induced relaxation. PACAP-27 elevated cyclic GMP level in airway smooth muscle tissue. These results indicate that PACAP-27 not only induces cyclic AMP-mediated responses, but also cyclic GMP-mediated responses in the airway. In addition, CO is related to the PACAP-induced elevation of cGMP level in the tracheal tissue.     

Involvement of cyclo-oxygenase-generated vasodilating eicosanoid(s) in addition to nitric oxide in endothelin-1-induced endothelium-dependent vasorelaxation in guinea pig aorta

Summary This study investigates the vasodilatory effects of endothelin-1 (ET-1) in isolated guinea pig aortic rings in vitro. Cumulative dose-response curves to ET-1 were constructed and ET-1 actions on prostaglandin F₂ (PGF₂)-precontraction were studied in both endothelium-intact and endothelium-denuded preparations, in the presence or absence of a cyclooxygenase inhibitor (indomethacin) and/or nitric oxide inhibitors (N^G-nitro-L-arginine methyl ester and hemoglobin). In endothelium-intact preparations, pretreatment with indomethacin (10^–5M, 30 min), alone or in combination with N^G-nitro-L-arginine methyl ester (L-NAME, 10^–4M), significantly augmented the constrictive responses to ET-1, whereas indomethacin, L-NAME, and hemoglobin (10^–5M) had no significant effects in the endothelium-denuded preparations. Furthermore, in PGF₂-precontracted, endothelium-intact preparations, ET-1, at a dose of 10^–9M, induced initial relaxation followed by subsequent contraction, while it only contracted the endothelium-denuded preparations. The initial relaxation was abolished by indomethacin, but not by L-NAME or hemoglobin. In addition, this relaxation was not inhibited by a specific ET_A receptor antagonist, BQ-123 (6 × 10^–6M). In addition to the involvement of nitric oxide, these results show the involvement of cyclo-oxygenase-generated vasodilating eicosanoid(s) derived from endothelium in ET-1-induced vasorelaxation in guinea pig aorta in vitro. The results also indicate that this vasorelaxation is mediated by ET_B receptor activation.This study was financially supported by Toyobo Biotechnology Foundation, and by Grants-in-Aid for scientific research from the Ministry of Education, Science and Culture of Japan (project numbers: 3770533 and 04670571) and the Ministry of Health and Welfare of Japan.     

Kinetic studies on the formation of nitrosamines I

Summary The kinetics of nitrosation of dimethylamine (DMA) in aqueous perchloric acid solution haves been studied using a differential spectrophotometric technique. The rate law is Initial rate=e[DMA]₀ [nitrite] ₀ ² [H⁺]/(f+[H⁺])² where [DMA]₀ and [nitrite]₀ represent initial stoichiometric concentrations. At 310.0 K and =2.0 M, e=(2.2±0.2)×10^–5 M^–1 s^–1 and f=(1.28±0.02) ×10^–3M. The associated activation energy is 56±3kJ mol^–1. A clear inhibition of the nitrosation rate by ionic strength has been observed in which only the kinetic parameter (f) has an effective change. It is concluded that under the experimental conditions of this work only the dinitrogen trioxid is the effective carrier for the nitrosation.     

Reduction of infarct size by short-term pretreatment with atorvastatin

Previous studies have suggested that the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors attenuate ischemia-reperfusion injury by increasing the activity of endothelial nitric oxide synthase (eNOS). We assessed whether short-term treatment with atorvastatin reduces myocardial infarct size in the rat. Rats (male Sprague-Dawley) received atorvastatin 2 mg/kg per day (n = 9), 10 mg/kg per day (n = 8), or 75 mg/kg per day (n = 11), or placebo (n = 11) by gastric gavage for 3 days. Two additional groups received atorvastatin 10 mg/kg (n = 7) or placebo (n = 7) for 3 days and the nonselective nitric oxide synthase inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME), 15 mg/kg intravenously 15 min before surgery. All rats underwent 30 min of coronary artery occlusion followed by 180 min of reperfusion. Ischemic myocardium at risk was assessed with blue dye and infarct size by triphenyltetrazolium chloride. Ischemic myocardium at risk was comparable among groups. Infarct size, expressed as a percentage of the myocardium at risk, was significantly smaller in the atorvastatin 75 mg/kg group (22.6 ± 2.8%; p = 0.035 vs. placebo) and atorvastatin 10 mg/kg (20.3 ± 3.8%; p = 0.022 vs. placebo) compared with placebo (37.5 ± 4.3%). The effect of atorvastatin 2 mg/kg was of smaller magnitude and did not reach statistical significance (infarct size 30.6 ± 4.2% of the myocardium at risk). L-NAME abolished the protective effect of atorvastatin 10 mg/kg per day. Infarct size was 43.0 ± 4.1% in the atorvastatin group and 39.4 ± 3.3% in the placebo group (p = 0.503).In conclusion, short-term (3 days) atorvastatin (10–75 mg/kg/d) significantly reduced myocardial infarct size. The protective effect was completely abolished by L-NAME, strongly suggesting that this protective effect is mediated via the nitric oxide synthase pathway.     

Insulin increases cyclic nucleotide content in human vascular smooth muscle cells: a mechanism potentially involved in insulin-induced modulation of vascular tone

Summary It has been suggested that insulin exerts a vasodilating effect, but the mechanisms involved are not completely understood. Since cyclic nucleotides mediate the vasodilation induced by endogenous substances, such as prostacyclin and nitric oxide, we aimed to investigate the influence of insulin (concentration range 240–960 pmol/l) on both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) content in human vascular smooth muscle cells. Insulin dose-dependently increased both nucleotides (cAMP: from 0.7±0.1 to 2.6±0.4 pmol/10⁶ cells, p=0.0001; cGMP: from 1.3±0.2 to 3.4±0.7 pmol/10⁶ cells, p=0.033). This increase is receptor-mediated, since it was blunted when cells were preincubated with the tyrosine kinase inhibitor genistein. The effect of insulin remained significant (p=0.0001) when preincubation with the phosphodiesterase inhibitor theophylline prevented cyclic nucleotide catabolism. The increase of cGMP was blunted when the cells were preincubated with the guanylate cyclase inhibitor methylene blue, and with the nitric oxide-synthase inhibitor N^G-monomethyl-l-arginine. At all the concentrations tested, insulin potentiated the increase of cAMP induced by the stable prostacyclin analogue Iloprost (p=0.0001), whereas only at 1920 pmol/l did it potentiate the cGMP increase induced by glyceryltrinitrate (p=0.05). This study demonstrates that the vasodilating effects exerted by insulin may at least in part be attributable to an increase of both cGMP and cAMP via a receptor-mediated activation of adenylate and guanylate cyclases in human vascular smooth muscle cells and that the insulin effect on cGMP is mediated by nitric oxide.Abbreviations cAMP cyclic adenosine monophosphate - cGMP cyclic guanosine monophosphate - PDE phosphodiesterases - NO nitric oxide - hVSMC human vascular smooth muscle cells - l-NMMA N^G-monomethyl-l-arginine - GTN glyceryltrinitrate - BSA bovine serum albumin - NIDDM non-insulin-dependent diabetes mellitus - MEM minimal essential medium - RIA radioimmunoassay     

Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase

Cardiac myocytes have been shown to express constitutively endothelial nitric oxide synthase (eNOS) (nitric oxide synthase 3), the activation of which has been implicated in the regulation of myocyte L-type voltage-sensitive calcium channel current (I_Ca-L) and myocyte contractile responsiveness to parasympathetic nervous system signaling, although this implication remains controversial. Therefore, we examined the effect of the muscarinic cholinergic agonist carbachol (CCh) on I_Ca-L and contractile amplitude in isoproterenol (ISO)-prestimulated ventricular myocytes isolated from adult mice, designated eNOS^null mice, with targeted disruption of the eNOS gene. Although both eNOS^null and wild-type (WT) ventricular myocytes exhibited similar increases in I_Ca-L in response to ISO, there was no measurable suppression of I_Ca-L by CCh in cells from eNOS^null mice, in contrast to cells from WT mice. These results were reflected in the absence of an effect of CCh on the positive inotropic effect of ISO in eNOS^null myocytes. Also, unlike myocytes from WT animals, eNOS^null myocytes failed to exhibit an increase in cGMP content in response to CCh. Nevertheless, the pharmacologic nitric oxide donors 3-morpholino-sydnonimine and S-nitroso-acetyl-cystein increased cGMP generation and suppressed ISO-augmented I_Ca-L in eNOS^null cells, suggesting that the signal transduction pathway(s) downstream of eNOS remained intact. Of importance, activation of the acetylcholine-activated K⁺ channel by CCh was unaffected in atrial and ventricular eNOS^null myocytes. These results confirm the obligatory role of eNOS in coupling muscarinic receptor activation to cGMP-dependent control of I_Ca-L in cardiac myocytes.     

The effects of cyclooxygenase and nitric oxide synthase inhibition on cardiodynamic parameters and coronary flow in isolated rat hearts

BACKGROUND:

Despite the widespread clinical use of cyclooxygenase (COX) inhibitors, dilemmas regarding the potential impact of these drugs on the cardiovascular system persist.

OBJECTIVE:

To estimate the effects of different COX inhibitors (meloxicam, acetylsalicylic acid [ASA] and SC-560) on cardiac function and coronary flow in isolated rat hearts, with special focus on the L-arginine/nitric oxide system.

METHODS:

The hearts of eight-week-old male Wistar albino rats (n=72; 12 rats per group; body mass 180 g to 200 g) were retrogradely perfused according to the Langendorff technique at gradually increased perfusion pressure (40 cmH₂O to 120 cmH₂O). After control experiments, the hearts were perfused with the following drugs: 100 μM ASA, alone or in combination with 30 μM N(ω)-nitro-L-arginine monomethyl ester (L-NAME), 0.3 μM meloxicam with or without 30 μM L-NAME, 3 μM meloxicam with or without 30 μM L-NAME, 30 μM L-NAME and 0.25 μM SC-560. In the control and experimental groups, the following parameters of heart function were continuously recorded: maximum rate of left ventricular pressure development, minimum rate of left ventricular pressure development, systolic left ventricular pressure, diastolic left ventricular pressure, heart rate and mean blood pressure. Coronary flow was measured flowmetrically. The amount of released NO₂⁻ was determined spectrophotometrically in coronary venous effluent.

RESULTS:

While meloxicam and SC-560 were found to have an adverse influence on cardiac function and coronary perfusion, ASA did not negatively affect the intact model of the heart.

CONCLUSION:

It appeared that interaction between COX and the L-arginine/nitric oxide system truly exists in coronary circulation and may explain the causes of the observed effects.     

Distribution of Nitric Oxide Synthase and Secretory Role of Exogenous Nitric Oxide in the Isolated Rat Pancreas

Summary Background. Pancreatic production and in vivo effects of nitric oxide (NO) have been shown by several studies. In order to examine the direct actions of the NO donor sodium nitroprusside (SNP), this study used in vitro specimens of the rat pancreas where the distribution of neuronal nitric oxide synthase (NOS) and the secretory effects of SNP and the cyclic GMP (cGMP) analog 8-bromo cyclic GMP (8-Br cGMP) were investigated. Methods. NO containing pancreatic nerves were visualized by NOS immunohistochemistry. Basal and stimulated amylase output from rat pancreatic segments was measured by an on-line fluorimetric method Stimulation was achieved by either acetylcholine (ACh) or electrical field stimulation (EFS). Intracellular free calcium concentration ([Ca²⁺]_i) was measured in dispersed pancreatic acinar cells. Results. NOS containing nerves were demonstrated in the vicinity of pancreatic acini and blood vessels. SNP and 8-Br cGMP inhibited both basal and EFS evoked amylase output but failed to inhibit ACh induced amylase output. Basal [Ca²⁺]_i was decreased by both SNP and 8-Br cGMP but neither SNP nor 8-Br cGMP influenced the ACh evoked increase in [Ca²⁺]_i. Conclusion. NO is well distributed in the rat exocrine pancreas. Exogenous nitric oxide may have a dual action in the isolated rat pancreas: Inhibition of basal amylase secretion in acinar cells and inhibition of ACh release from intrinsic nere terminals. Both effects seem to be calcium dependent and possibly mediated by cGMP.