Biomedical subjects

H Nakazawa

Publications and source records attributed to H Nakazawa.

At least 55 records · Page 3Linked to original sources

PubMed2001

A defect in the cytochrome b large subunit in complex II causes both superoxide anion overproduction and abnormal energy metabolism in Caenorhabditis elegans.

A mev-1(kn1) mutant of the nematode Caenorhabditis elegans is defective in the cytochrome b large subunit (Cyt-1/ceSDHC) in complex II of the mitochondrial electron transport chain. We have previously shown that a mutation in mev-1 causes shortened life span and rapid accumulation of aging markers such as fluorescent materials and protein carbonyls in an oxygen-dependent fashion. However, it remains unclear as to whether this hypersensitivity is caused by direct toxicity of the exogenous oxygen or by the damage of endogenous reactive oxygen species derived from mitochondria. Here we report important biochemical changes in mev-1 animals that serve to explain their abnormalities under normoxic conditions: (i) an overproduction of superoxide anion from mitochondria; and (ii) a reciprocal reduction in glutathione content even under atmospheric oxygen. In addition, unlike wild type, the levels of superoxide anion production from mev-1 mitochondria were significantly elevated under hyperoxia. Under normal circumstances, it is well known that superoxide anion is produced at complexes I and III in the electron transport system. Our data suggest that the mev-1(kn1) mutation increases superoxide anion production at complex II itself rather than at complexes I and III. The mev-1 mutant also had a lactate level 2-fold higher than wild type, indicative of lactic acidosis, a hallmark of human mitochondrial diseases. These data indicate that Cyt-1/ceSDHC plays an important role not only in energy metabolism but also in superoxide anion production that is critically involved in sensitivity to atmospheric oxygen.

Adenosine Triphosphate↗

PubMed2001

Regulation of inducible nitric oxide synthase by self-generated NO.

A ferric heme-nitric oxide (NO) complex can build up in mouse inducible nitric oxide synthase (iNOS) during NO synthesis from L-arginine. We investigated its formation kinetics, effect on catalytic activity, dependence on solution NO concentration, and effect on enzyme oxygen response (apparent KmO2). Heme-NO complex formation was biphasic and was linked kinetically to an inhibition of electron flux and catalysis in iNOS. Experiments that utilized a superoxide generating system to scavenge NO showed that the magnitude of heme-NO complex formation directly depended on the NO concentration achieved in the reaction solution. However, a minor portion of heme-NO complex (20%) still formed during NO synthesis even when solution NO was completely scavenged. Formation of the intrinsic heme-NO complex, and the heme-NO complex related to buildup of solution NO, increased the apparent KmO2 of iNOS by 10- and 4-fold, respectively. Together, the data show heme-NO complex buildup in iNOS is due to both intrinsic NO binding and to equilibrium binding of solution NO, with the latter predominating when NO reaches high nanomolar to low micromolar concentrations. This behavior distinguishes iNOS from the other NOS isoforms and indicates a more complex regulation is possible for its activity and oxygen response in biologic settings.

Animals↗

PubMed2001

Analysis of the metabolites of the sodium salt of 6-hydroxy-5-(phenylazo)-2-naphthalenesulfonic acid in Sprague-Dawley rat urine.

The sodium salt of 6-hydroxy-5-(phenylazo)-2-naphthalenesulfonic acid (SS-AN), which is a subsidiary color present in Food Yellow No. 5 [Sunset Yellow FCF, disodium salt of 6-hydroxy-5-(4-sulfophenylazo)-2-naphthalenesulfonic acid], was orally administered to Sprague-Dawley rats. Metabolite A, metabolite B, and unaltered SS-AN were detected as colored metabolites in the rat urine. Analysis of the chemical structures showed that metabolite A (major peak) was 6-hydroxy-5-(4-sulfooxyphenylazo)-2-naphthalenesulfonic acid, the sulfuric acid conjugate of SS-AN, and metabolite B (minor peak) was 6-hydroxy-5-(4-hydroxyphenylazo)-2-naphthalenesulfonic acid (SS-PAP), which is a derivative of metabolite A without the sulfuric acid. The colorless metabolites p-aminophenol, o-aminophenol, and aniline present in the urine were analyzed by liquid chromatography-mass spectrometry. The orally administered SS-AN had been metabolized to the colorless metabolites (p-aminophenol 45.3%, o-aminophenol 9.4%, aniline 0.4%) in the 24-h urine samples. Analysis of the colored metabolites by high-performance liquid chromatography with detection at 482 nm indicated the presence of metabolite A (0.29%), SS-PAP (0.01%), and SS-AN (0.02%) were detected in the 24-h urine samples. Approximately 56% of SS-AN was excreted into the urine and the rest is probably excreted into feces.

Animals↗

PubMed2001

Anti-human vWF monoclonal antibody, AJvW-2 Fab, inhibits repetitive coronary artery thrombosis without bleeding time prolongation in dogs.

The antithrombotic and antihaemostatic effects of the monoclonal antibody against human vWF (AJvW-2 Fab) were investigated in comparison with those of the monoclonal antibody against platelet GPIIb/IIIa (abciximab) in dogs. The ex vivo platelet aggregation and template bleeding time were measured before, 5, 90, 210 min and 24 h after injection of either AJvW-2 Fab or abciximab in anesthetized beagle dogs. Plasma concentration, vWF occupancy and plasma vWF antigen level were also measured by ELISA. In addition, the antithrombotic effect was evaluated in a canine model of repetitive coronary thrombosis (Folts model). AJvW-2 Fab significantly inhibited the ex vivo botrocetin-induced platelet aggregation at 0.18 mg/kg (53% plasma vWF occupancy) and also inhibited cyclic flow reductions (CFRs) at 0.06 mg/kg (31% occupancy). A significant prolongation of the bleeding time was observed at 1.8 mg/kg (95% occupancy), which was 30 times as high as the antithrombotic effective dose. Whereas, abciximab significantly inhibited both the ex vivo ADP-induced platelet aggregation and CFRs at 0.8 mg/kg, which was the minimally effective dose, also resulting in a significant prolongation of the bleeding time. These results suggest that blockade of the GPIb-vWF axis with AJvW-2 Fab leads to the inhibition of thrombus formation in the stenosed coronary arteries without less bleeding time prolongation than the GPIIb/IIIa blockade with abciximab.

Animals↗

PubMed2001

Inducible nitric oxide synthase deficiency does not affect the susceptibility of mice to atherosclerosis but increases collagen content in lesions.

BACKGROUND: Although endothelial nitric oxide synthase (NOS) is antiatherogenic, the role of inducible NOS (iNOS) in the development of atherosclerosis is not established. METHODS AND RESULTS: We compared the susceptibility of iNOS knockout (iNOS(-/-)) and wild-type (iNOS(+/+)) mice to the development of atherosclerosis induced by feeding an atherogenic diet for 15 weeks. Plasma lipid level, atherosclerotic lesion size, and cellular density in the lesions were all similar in the 2 strains (lesion size: iNOS(+/+) 285+/-73x10(3) microm(2), iNOS(-/-) 293+/-82x10(3) microm(2), n=10). iNOS mRNA was detected in the lesions of iNOS(+/+) but not iNOS(-/-) mice through RT-PCR. Immunohistochemically, iNOS(+/+) mice showed iNOS staining in macrophages and medial smooth muscle cells in the lesions. Nitrotyrosine staining showed a similar distribution, whereas it was absent in iNOS(-/-) mice. There was no apparent difference in the intensity or distribution of vascular cell adhesion molecule-1 staining in the lesions of the 2 strains. However, the lesions of iNOS(+/+) mice showed a markedly decreased extracellular collagen content compared with those of iNOS(-/-) mice CONCLUSIONS: iNOS induction does not affect the development of atherosclerosis in mice fed an atherogenic diet, but the resulting lesions show decreased levels of extracellular collagen and may be more fragile.

Animals↗

PubMed2001

N-methyl-D-aspartate receptor antagonist reduces nitrotyrosine formation in caudate-putamen in rat focal cerebral ischemia-reperfusion.

The aim of this study is to determine experimentally whether N-methyl-D-aspartate (NMDA) receptor is involved in nitrotyrosine formation in rat brain subjected to focal ischemia-reperfusion, by using the NMDA receptor antagonist MK-801. Halothane-anesthetized and artificially ventilated rats were given MK-801 (5 mg/kg, i.p.) or vehicle prior to 2 h of focal cerebral ischemia followed by 0.5 h of reperfusion. The brain was then removed and divided into four sections, cortical ischemic core, peri-ischemic cortex, lateral caudate-putamen and non-ischemic cortex. Tissue nitrotyrosine was measured by means of hydrolysis/HPLC. MK-801 significantly attenuated nitrotyrosine formation in the lateral caudate-putamen. We conclude that nitrotyrosine formation required activation of NMDA receptors, at least in part.

Animals↗

PubMed2001

Inducible nitric oxide production is an adaptation to cardiopulmonary bypass-induced inflammatory response.

BACKGROUND: Cardiopulmonary bypass (CPB) increases nitric oxide (NO) production by the activation of NO synthases (NOS). However, the role of NO from inducible NOS (iNOS) in CPB-induced inflammatory response remains unclear. We examined the effect of a selective iNOS inhibitor, aminoguanidine, on CPB-induced inflammatory response in a rat-CPB model. METHODS: Adult Sprague-Dawley rats underwent 60 minutes of CPB (100 mL x kg(-1) x min(-1), 34 degrees C). Group A (n = 10) received 100 mg/kg of aminoguanidine intraperitoneally 30 minutes before the initiation of CPB, and group B (n = 10) served as controls. RESULTS: There were significant time-dependent changes in plasma interleukin (IL)-6, IL-8, nitrate + nitrite, the percentage ratio of nitrotyrosine to tyrosine (%NO2-Tyr, an indicator of peroxynitrite formation), and respiratory index (RI). Three hours after CPB termination, IL-6, IL-8, and RI were significantly higher in group A (IL-6, 397.5+/-80.6 pg/mL; IL-8, 26.99+/-6.57 ng/mL; RI, 1.87+/-0.31) than in group B (IL-6, 316.5+/-73.9 pg/mL, p <0.05; IL-8, 17.21+/-3.12 ng/mL, p < 0.01; RI, 1.57+/-0.24, p < 0.05) although nitrate + nitrite (31.8+/-4.1 micromol/L) and %NO2-Tyr (1.15%+/-0.20%) were significantly lower in group A than in group B (nitrate + nitrite, 50.2+/-5.0 micromol/L, p < 0.01; %NO2-Tyr, 1.46%+/-0.21%, p < 0.01). Western immunoblot analysis from lung tissue of group A identified marked iNOS inhibition without inhibiting endothelial-constitutive NOS, and neutrophil accumulation in the lung specimens was significantly greater in group A (6.5+/-0.7/alveoli) than in group B (4.4+/-0.4/alveoli, p < 0.01). CONCLUSIONS: These results suggest that NO production from iNOS may be an adaptive response for attenuating the CPB-induced inflammatory response.

Animals↗

PubMed2001

Peroxynitrite formation from human myocardium after ischemia-reperfusion during open heart operation.

BACKGROUND: Current experimental studies have demonstrated that peroxynitrite (ONOO-) has both cytotoxic and cytoprotective effects on myocardial ischemia-reperfusion injury. However, even myocardial ONOO- formation has not yet been investigated in humans undergoing open heart operation. We measured plasma nitrotyrosine as an indicator of ONOO- formation during open heart operation and examined its association with myocardial ischemia-reperfusion injury. METHODS: Twenty adult patients undergoing mitral valve replacement under cardiopulmonary bypass between 1997 and 1998 were enrolled in this study (6 men and 14 women). Arterial blood (Ao) and coronary sinus effluent (CS) were obtained: (1) before the initiation of cardiopulmonary bypass, (2) just after aortic unclamping, (3) at 5 minutes, (4) at 10 minutes, (5) at 15 minutes, and (6) at 20 minutes after aortic unclamping. RESULTS: At every sampling point after reperfusion, plasma nitrate and nitrite was significantly lower in CS than in Ao, and the percentage ratio of nitrotyrosine to tyrosine (%NO2-Tyr; an index of ONOO- formation) was significantly higher in CS than in Ao. The CS-Ao difference in %NO2-Tyr, myocardium-derived ONOO-, reached its peak at 5 minutes after reperfusion (2.17+/-0.74%), which was significantly correlated with the peak CS-Ao difference in plasma malondialdehyde, and with postoperative maximum creatine kinase-MB. CONCLUSIONS: These results first demonstrate that ONOO- is produced from human myocardium after ischemia-reperfusion during open heart operation, and myocardium-derived ONOO- can be determined by the CS-Ao difference in %NO2-Tyr.

Adult↗

PubMed2001

GC/MS analysis of polybrominated diphenyl ethers in fish collected from the Inland Sea of Seto, Japan.

Development of an analytical method for polybrominated diphenyl ethers (PBDEs) in fish and their concentration in Japanese marine fish were investigated. Fish homogenate was extracted with diethyl ether/hexane (1 + 3). The extract was cleaned up by automated gel permeation chromatography (GPC) and then by mini-column chromatography, which consisted of three layers of silica gel and sulfuric acid-impregnated silica gel. The PBDE fraction was concentrated and injected into a GC/MS with negative chemical ionization (NCl). Recoveries of the 15 individual PBDEs (BDE-15, 28, 37, 47, 66, 71, 75, 77, 85, 99, 100, 119, 153, 154, and 209) each at a fortification level of 4 ng/g lipid were in the range of 88-128% and the relative standard deviations (RSD) were 0.43-7.6% (n = 4). Seven species of marine fish (conger eel, flounder, gray mullet, horse mackerel, red sea bream, sea bass, and yellowtail) were collected from the Inland Sea of Seto, and were analyzed with the developed method. Seven PBDEs (BDE-28, 47, 66, 99, 100, 153, and 154) were detected in all the samples. The most abundant PBDE congener was BDE-47 found in all the samples. Relatively high levels of PBDEs were found in the gray mullets and yellowtails.

Animals↗

PubMed2001

Increased plasma tetrahydrobiopterin in septic shock is a possible therapeutic target.

Hemoperfusion with a column of polymyxin B immobilized on fibers (PMX) has been used to adsorb endotoxin in-patients with septic shock. PMX hemoperfusion (PMX-H) increases blood pressure (BP) too rapidly for the effect to be attributable to endotoxin removal. Since inducible NO synthase (iNOS) is known to be involved in the profound hypotension, we hypothesized that a decrease of tetrahydrobiopterin (BH(4)), an essential cofactor of iNOS, might account for the rapid effect of PMX-H on BP, if plasma BH(4) is increased concomitantly with NO in septic shock patients and if PMX can decrease BH(4). BH(4) was evaluated by measuring total biopterin, which can include derivatives of BH(4) by using high-performance liquid chromatography (HPLC). We confirmed that PMX fabric time dependently decreased total biopterin concentration in vitro. The plasma level of total biopterin in shock patients was indeed markedly elevated compared with that in volunteers (131.1+/-33.4 vs. 10.4+/-1.1 nM, n=5, P<0.01). Level of NO metabolites (NOx) were 173.9+/-104.7 versus 28.7+/-11.6 µM (P<0.01). In beagles, plasma total biopterin was 44.7+/-6.9 nM at baseline, reached 118+/-28.6 nM after lipopolysaccharide (LPS) injection, and fell to 70.2+/-15.8 nM after PMX-H. Plasma NOx concentration was increased from 15.2+/-4.2 to 41.0+/-7.5 µM by LPS treatment. BP was 130+/-11.3 mmHg at baseline, 82.2+/-8.3 mmHg at 14 h after LPS, and 115.2+/-16.0 mmHg after PMX-H. In rats, plasma total biopterin was 88.8+/-1.5 nM at baseline, 383.7+/-144.2 nM after LPS and 177.0+/-14.2 nM after PMX-H. Plasma NOx was also increased after LPS (from 34.6+/-4.4 to 1445.6+/-376.0 nM). The marked increase in total biopterin concomitantly with NOx in septic shock patients and its reduction by PMX-H in animal models of septic shock are consistent with our hypothesis, and appear to justify further research on BH(4) removal as a potential therapeutic target.

Journal Article↗

PubMed2001

No evidence of NO-induced damage in potential donor organs after brain death.

Brain death induces multiple-organ dysfunction, with undesirable consequences for organ transplantation. However, the mechanisms are not completely clear. In the hearts, lungs, livers, and kidneys of rats, we investigated whether brain death leads to changes in nitric oxide (NO) production or to the formation of nitrotyrosine (the footprint of peroxynitrite, formed from NO and superoxide) or to lipid peroxidation products. To produce a rat model of brain death, we inflated a subdurally placed balloon catheter. We used the Griess reaction to assay plasma nitrite and nitrate. Proteolytic digestion followed by high-performance liquid chromatography (HPLC) with electrochemical detection determined nitrotyrosine formation in the tissues. Tissues were also examined immunohistochemically with anti-nitrotyrosine antibody. We used a thiobarbituric acid method to assay lipid peroxidation. An intense, transient hemodynamic activation occurred at the onset of brain death (heart rate, 496 beats/min; mean arterial pressure (AP), 181 mm Hg; dP/dt(max), 11,500 mm Hg/sec). A constant hypotensive phase (mean AP, 50 mm Hg; dP/dt(max), 2,674 mm Hg/sec) followed. Plasma concentration of nitrite plus nitrate remained unchanged 2 hours after brain death (32.8 +/- 1.5 vs 31.3 +/- 2.2 micromol/liter at zero time). Neither HPLC nor immunohistochemistry detected significant nitrotyrosine formation in the tissues. We detected no increase in lipid peroxidation products.Our results indicate that changes in the generation of reactive nitrogen and active oxygen species do not play an important role in post-brain-death organ dysfunction, at least not at the early stage.

Animals↗

PubMed2001

The prevalence of renal cell carcinoma: a nation-wide survey in Japan in 1997.

BACKGROUND: The present study was conducted to investigate the incidence of renal cell carcinoma by sex, age group and different regions in Japan. METHODS: The survey was conducted from the beginning of January 1997 to the end of December 1997. A total of 1306 Institutions in all 47 prefectures throughout Japan were requested to register cases. RESULTS: There were 6358 persons with renal cell carcinoma, consisting of 4372 men and 1986 women. The age-specific incidence rates showed a peak in the age group of 65-70 years in both men and women. The crude incidence rates per 100 000 population for men and women were 7.1 and 3.1, respectively, and age-standardized incidence rates per 100 000 population for men and women were 4.9 and 1.8, respectively. The incidence rates in the Hokkaido region were significantly higher than in other regions (P < 0.05), among which there was no significant difference in incidence rates. CONCLUSIONS: The present study showed that the incidence rates of renal cell carcinoma in Japan were approximately the same as among Japanese in Los Angeles. The rates were, however, lower than North American and European countries, but higher than China, Central or South American countries and African countries. The reasons for the high incidence of renal cancer in the Hokkaido region are not entirely clear. Further epidemiologic research is required.

Adult↗

PubMed2001

Migration of 4-nonylphenol from polyvinyl chloride food packaging films into food simulants and foods.

Migration of 4-nonylphenol (NP) from polyvinyl chloride (PVC) films for food packaging into food simulants and foods has been studied in domestic applications such as wrapping of food and reheating in a microwave oven. The migration of NP from the PVC films was determined by high-performance liquid chromatography with electrochemical coulometric-array detection (LC/ED). Twelve PVC films intended for commercial use and ten for domestic applications (total: 22 samples) were analysed. Some of the PVC films (two home-use and ten retail-use) contained NP at concentrations of between 500 and 3300 microg/g. Migration of NP from the films was influenced by the test conditions (n-heptane at 25 degrees C for 60 min, distilled water at 60 degrees C for 30 min and 4% acetic acid at 60 degrees C for 30 min). The amount of NP migrating from the PVC films into n-heptane (0.33-1.6 microg/cm2) was higher than the amount migrating into distilled water or 4% acetic acid (up to 9.7 ng/cm2) for the 11 films in which NP was detected. Up to 0.23% of the NP migrated into distilled water and 4% acetic acid and up to 62.5% into n-heptane. In addition, we investigated NP migration into cooked rice samples wrapped in PVC film. Using spiked samples the method gave an average recovery of 83.7% (n = 5) with a standard deviation of 2.5%. Migration of NP ranged from not detectable (< 1.0 ng/g) to 410.0 ng/g by reheating samples in a microwave oven for 1 min and from not detectable to 76.5 ng/g by keeping samples at room temperature for 30 min.

Chromatography, High Pressure Liquid↗

PubMed2001

Analysis of plasticizers in cap-sealing resins for bottled foods.

A survey of plasticizers in cap-sealing resins for bottled foods has been undertaken. During 1997-1999 di-(2-ethylhexyl)phthalate (DEHP) was found in seven out of 21 samples on the Japanese domestic market and in 10 out of 61 imported samples as well as a further two samples which contained di-(2-ethylhexyl)adipate (DEHA). In the period 1993-1999, of the other plasticizers diacetyl lauroyl glycerol (DALG) was only detected in domestic samples whereas diisodecyl phthalate (DIDP) and diisononyl phthalate (DINP) were only in imported samples. It was observed overall that DEHP and DEHA were restricted to use in cap-sealing resins for bottled foods. Whilst phthalates, DEHA or DALG were detected in samples in 1993 and 1995, the investigation in 1997-1999 showed fewer samples in which these plasticizers were found.

Chromatography, Gas↗

PubMed2001

Determination of bisphenol A in canned vegetables and fruit by high performance liquid chromatography.

A high performance liquid chromatography (HPLC) method was developed for the determination of bisphenol A (BPA) that had migrated into canned fruit and vegetables. BPA was extracted with acetonitrile from the solid portion of canned food, and with an OASIS HLB cartridge from the aqueous portion, respectively. Both extracts were cleaned up on a Florisil cartridge. The HPLC separation was carried out on a Wakosil II 3C18 RS column (4.6 x 150 mm) with acetonitrile-water (40:60, v/v) as a mobile phase with a flow rate of 0.8 ml/min. BPA was detectable by UV detector at 228 nm and determined with the similarity of chromatographic peak spectrum by multiwavelength detector (similarity index was 0.99 or above). The quantification limits were 10 ng/g for the solid portion and 5 ng/ml for the aqueous portion, respectively. BPA was mainly detected in the solid portion of canned food and found at the maximum level of 11 micrograms per can. To verify migration into the solid portion of canned food, a partitioning experiment was carried out.

Benzhydryl Compounds↗

PubMed2001

Contribution of chloride channel activation to the elevated muscular tone of the pulmonary artery in monocrotaline-induced pulmonary hypertensive rats.

In monocrotaline-treated rat pulmonary artery from which endothelium was removed, greater spontaneous muscular tone was observed under resting conditions than in vehicle-treated artery. The aim of the present study was to show the possible contribution of Cl- channels in the mechanism of the elevated tone. Verapamil almost completely inhibited the elevated spontaneous muscular tone by decreasing [Ca2+]i. The elevated muscular tone was also inhibited by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS), a Cl- channel inhibitor. After the inhibition of muscular tone by DIDS, verapamil did not induce further relaxation. Quantitative RT-PCR analysis indicated that the mRNA levels of ClC3 and Ca2+-activated Cl- channels did not change in the pulmonary hypertensive pulmonary artery from those of vehicle-treated rats. These results suggest that the elevated muscular tone observed in the monocrotaline-induced hypertensive pulmonary artery is due to membrane depolarization of smooth muscle cells and that this phenomenon might be mediated by the activation of DIDS-sensitive Cl- channels.

Animals↗

PubMed2001

Simultaneous determination of phenolic xenoestrogens by solid-phase extraction and high-performance liquid chromatography with fluorescence detection.

A highly sensitive and selective method for simultaneous determination of some hydroxyl group-containing endocrine disruptors, including bisphenol A (BPA), bisphenol B (BPB), bisphenol E (BPE), bisphenol F (BPF) and 4-nonylphenol (4-NP), was developed. The method consists of precolumn derivatization of the analytes, solid-phase extraction (SPE) and subsequent chromatographic analysis by high-performance liquid chromatography (HPLC) with fluorescence detection. 4,4'-Cyclohexylidenebisphenol (BPZ) was used as an internal standard. Derivatization was carried out using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label. Parameters of the derivatization reaction (temperature, time, concentration of reagent, stability, etc.) and of the solid-phase extraction (recovery, solvent, etc.) were studied in detail. Detection limits of compounds studied in standard solutions ranged from 0.08-1.3 ppb (ng/ml). The proposed method was successfully applied to plastic samples; BPA was found in both polycarbonate and polyvinyl chloride plastics, while 4-NP was found in plastics made of polyvinyl chloride and another polymer.

Benzhydryl Compounds↗

PubMed2001

Physiological concentration of nitric oxide induces positive inotropic effects through cGMP pathway in isolated rat ventricular myocytes.

We used authentic NO or NO from NO donors to show that the physiological levels of NO (<1 microM) induce a positive inotropic effect and demonstrated that the effect is evoked through a cGMP-dependent pathway. In isolated rat ventricular myocytes, authentic NO at 588 nM increased both cell shortening and the intracellular Ca(2+) ([Ca(2+)]i) transient (133 and 117%, respectively; p < 0.05 vs. baseline), and 0.16-1.7 microM NO elicited reproducible dose-dependent increases in cell shortening. NOC18 (0.1 mM: actual NO concentration 673 nM) or SNAP (0.1 mM: actual NO concentration 285 nM) showed similar effects (shortening 215% and [Ca(2+)]i transient 160% increases, and shortening 148% and [Ca(2+)]i transient 117% increases, respectively). The NO-induced increases in cell shortening and the [Ca(2+)]i transient were inhibited by an inhibitor of soluble guanylate cyclase (ODQ, 30 microM) or by an inhibitor of cAMP-dependent protein kinase (KT5720, 0.1 microM). In the presence of an inhibitor of cGMP-inhibited cAMP-phosphodiesterase (milrinone, 10 microM), NO failed to increase both cell shortening and the [Ca(2+)]i transient. These results suggest that physiological levels of NO induce positive inotropy through a cGMP-dependent pathway.

3',5'-Cyclic-AMP Phosphodiesterases↗