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H L Fung

Publications and source records attributed to H L Fung.

At least 91 records · Page 5Linked to original sources
PubMed1990

Identification of the subcellular site for nitroglycerin metabolism to nitric oxide in bovine coronary smooth muscle cells.

The vasodilating action of nitroglycerin (NTG) is thought to be mediated by its metabolic activation to nitric oxide (NO) in the vascular smooth muscle cell, but the site at which this process occurs has not been defined. To determine which cellular component is primarily responsible for this metabolic activation, subcellular fractions of bovine vascular smooth muscle cells were prepared and incubated with NTG along with cofactors. Time-dependent headspace NO concentrations generated in these preparations were determined directly by chemiluminescence detection. A mathematical model was developed to relate headspace NO with the NO-generating activity in each incubation, correcting for the concurrent processes of NO partitioning between the headspace and the incubation medium, and NO degradation in these two phases. The estimated NO-generating activities from different subcellular fractions were well correlated with the activities of two enzyme markers of the plasma membrane (K(+)-activated ouabain sensitive p-nitrophenyl phosphatase and 5'-nucleotidase), but not with those of the mitochondria, endoplasmic reticulum or the cytosol. These results indicate that the enzyme(s) responsible for the metabolic activation of NTG, and possibly other organic nitrate vasodilators, are associated with the plasma membrane in bovine coronary smooth muscle cells.

Analysis of Variance↗
PubMed1990

Spontaneous liberation of nitric oxide cannot account for in vitro vascular relaxation by S-nitrosothiols.

S-nitrosothiols are potent vasodilators in vivo and in vitro, and have recently been proposed as possible endogenous precursors of endothelium-derived nitric oxide (NO). NO release from S-nitrosothiols has generally been assumed to be spontaneous, but this has not been proven. This hypothesis was examined by altering the NO release profiles of two S-nitrosothiols, those of S-nitroso-N-acetylpenicillamine (SNAP) and S-nitroso-glutathione (GSNO), and observing their relaxation potency on isolated endothelium-denuded rat aortic rings. Spontaneous degradation of SNAP and GSNO in tissue bathing medium (monitored by high-performance liquid chromatography) and the associated NO release (assessed by chemiluminescence detection of headspace NO) were enhanced in the presence of 100 microM N-acetylpenicillamine and inhibited in the presence of 100 U/ml superoxide dismutase. However, the relaxant effects of SNAP and GSNO were enhanced in the presence of superoxide dismutase, and diminished in the presence of N-acetylpenicillamine. In addition, the relaxation potencies of SNAP, GSNO, S-nitrosocystein, S-nitroso-N-acetylcysteine and S-nitroso-coenzyme A were not correlated with spontaneous NO generation. These findings therefore argue against spontaneous liberation of NO as a predominant mechanism of S-nitrosothiol action. The highly polar SNAP, GSNO, S-nitrosocysteine and S-nitroso-N-acetylcysteine (octanol, pH 7.4 buffer partition coefficient from less than .025-.052) and the bulky and polar S-nitroso-coenzyme A (MW 797) have similar relaxation potencies, indicating that intracellular penetration of intact S-nitrosothiols may not be required for activity. NO generation from SNAP was examined in subcellular fractions of bovine coronary arterial smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Endothelial nitric oxide generating enzyme(s) in the bovine aorta: subcellular location and metabolic characterization.

The metabolic production of nitric oxide (NO) from bovine endothelial homogenates and subcellular fractions was examined. NO was quantified using a sensitive and specific ozone redox-chemiluminescence detector. Endogenously produced NO was detected in the headspace gas of bovine vascular endothelial homogenates supplemented with superoxide dismutase and an NADPH regenerating system, which had been incubated at 0 degrees C for 3 hr. An identical system incubated at 37 degrees C for 3 hr did not produce NO. Both superoxide dismutase and an NADPH regenerating system were required for observing endogenous NO production from endothelial homogenates. Among the various endothelial subcellular fractions tested, only the 1000 x g supernatant manifested significant generation of endogenous NO. Addition of 1.4 mM L-arginine (but not D-arginine, L-citrulline or L-lysine) resulted in significant enhancement of NO production from the 15,500 and 210,000 x g pellet fractions. L-arginine did not stimulate NO production from either homogenates or subcellular fractions of coronary vascular smooth muscle cells. Exogenously added calcium or magnesium was not required for NO generation in the 210,000 x g pellet stimulated with L-arginine, whereas L-canavanine (1.4 mM) and SKF 525-A (0.1 mM) significantly inhibited NO production with this preparation. Analysis of the enzyme marker data from various subcellular fractions suggests that the endothelial NO-generating enzyme system is membrane-bound and might be associated with the plasma membrane.

Animals↗
PubMed1989

Kinetic mechanisms for the concentration dependency of in vitro degradation of nitroglycerin and glyceryl dinitrates in human blood: metabolite inhibition or cosubstrate depletion?

The in vitro degradation of nitroglycerin (NTG) and its dinitrate metabolites in human blood and red blood cells (RBC) has been shown to exhibit apparent first-order kinetics. The decay rates of NTG and its dinitrate metabolites, however, were dependent on the initial concentration. We showed that this unusual kinetic behavior can be described mathematically by models of Michaelis-Menten kinetics combined with either competitive product inhibition or cosubstrate depletion. Experimental studies were conducted to determine the relative contribution of these two mechanisms to the observed kinetics. The effect of added thiols (the likely cosubstrates) on [14C]NTG degradation was studied separately in whole blood, reconstituted RBC, lysed RBC, and plasma. N-Acetylcysteine, L-cysteine, and D-cysteine accelerated NTG degradation in whole blood, while a similar concentration of glutathione had no effect. However, all four thiols exerted no effect on NTG kinetics in reconstituted and lysed RBC. In contrast, these thiols, as well as dithiothreitol, produced a marked increase (3-14 fold) in NTG degradation rate in plasma compared with buffer controls. Since thiol replenishment in reconstituted and lysed RBC did not abolish the concentration dependency, cosubstrate depletion due to thiols appeared unimportant as a contributor to the kinetic phenomenon. In human blood, metabolite inhibition of NTG degradation occurred along with the existence of concentration dependency. Both phenomena, however, were absent when NTG degradation was examined in rat blood. Concentration-dependent degradation in human blood was not observed for glyceryl-1-mononitrate, a compound that does not produce a nitrated metabolite.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Effect of intermittent exposure and drug-free intervals on the in vitro vascular tolerance to nitroglycerin.

Recent reports suggest that intermittent nitroglycerin (NTG) treatment, incorporating a daily nitrate-free interval, can avoid the tolerance associated with continuous NTG therapy. This study has investigated whether an in vitro model of NTG tolerance could be used to examine the mechanisms of tolerance avoidance (by intermittent NTG exposure) and tolerance reversal. Isolated rat abdominal aortic rings were exposed to 55 microM NTG at varying intervals over a 60 min period, and the concentration-relaxation curves to NTG were subsequently determined. Intermittent NTG exposure (either 12 x 0.5 min or 3 x 2 min) significantly reduced NTG tolerance compared to continuous exposure over the same period of time (60 min). The diminished tolerance was apparently due to the reduced total exposure time, since the NTG responsiveness of aortic rings exposed to NTG intermittently or continuously for 6 min of the incubation period was not significantly different. Under the present conditions, in vitro NTG tolerance could be reversed if sufficient washout time was allowed. Thus, aortic rings exposed to NTG for the initial, but not the final, 6 min of incubation were not tolerant to NTG. In addition, rings exposed to NTG for 3 min exhibited near-maximal tolerance after 5 min washout, but no tolerance after 60 or 120 min washout. It appears, therefore, that the isolated vessel retains the "repair" mechanism responsible for tolerance reversal under the present conditions. This study suggests that the in vitro model of NTG tolerance may be useful for investigating the characteristics and mechanisms of tolerance avoidance and reversal, as well as tolerance induction.

Animals↗
PubMed1989

An in vitro model to evaluate muscle damage following intramuscular injections.

An isolated rat muscle preparation was developed to screen for muscle damage (myotoxic potential) following intramuscular injections. Myotoxicity is evaluated by the total cumulative efflux of the enzyme creatine kinase from the extensor digitorum longus muscle into the incubation medium over a 2-hr period or by the slope of the cumulative creatine kinase efflux curve. The system allows for rapid screening of compounds and/or formulations regarding their myotoxic potential and is not sensitive to fluctuations of in vivo creatine kinase levels caused by animal handling or patient conditions. A good rank-order correlation was obtained between this in vitro technique and the in vivo myotoxicity of a number of pharmaceutical formulations, as indicated by circulating creatine kinase levels and histological observations.

Animals↗
PubMed1989

Use of an in vitro model for the assessment of muscle damage from intramuscular injections: in vitro-in vivo correlation and predictability with mixed solvent systems.

The potential of binary mixtures of propylene glycol-water, ethanol-water, and polyethylene glycol 400-water to cause skeletal muscle damage (myotoxicity) following intramuscular injection was examined with an in vitro model using the isolated rat muscle. At moderate concentrations (20-40%, v/v) of the organic cosolvent, the order of myotoxicity was propylene glycol greater than ethanol much greater than polyethylene glycol 400. The in vitro results were then compared with in vivo toxicity in rabbits after injection of normal saline, 40% (v/v) polyethylene glycol 400, 40% (v/v) propylene glycol, indocyanine green in normal saline, and indocyanine green in 40% (v/v) propylene glycol. Employing the area under the creatine kinase activity curve from 0 to 72 hr as the index of skeletal muscle damage, an excellent in vitro-in vivo correlation was observed. The basic myotoxicity relationships obtained from the binary cosolvent systems were then used to examine the myotoxicity of ternary organic cosolvent mixtures. Several mixed solvent systems with the same theoretical molar solubilization power for a model compound, diazepam, were selected to determine (1) if myotoxicity can be reduced by changing the composition of the ternary mixtures and (2) if myotoxicity of the individual components is additive. For the solvent systems containing propylene glycol, ethanol, and water, the total myotoxicity equaled the sum of the individual myotoxicity of each component. In contrast, for the solvent systems containing polyethylene glycol 400, the total myotoxicity was only half of the sum of individual toxicities. These results suggest that polyethylene glycol 400 in mixed cosolvent systems might have a protective effect on the myotoxicity generated by intramuscular injections.

Animals↗
PubMed1989

Mechanisms of nitrate action and vascular tolerance.

The various potential mechanisms contributing to nitrate tolerance are discussed. Pharmacokinetic alterations of the organic nitrate in the systemic circulation do not readily reflect pharmacologic tolerance. Neurohormonal changes do accompany continuous nitrate therapy, but the causative factor of tolerance, if it exists, has not been identified. Vascular metabolism of organic nitrates is impaired during in vitro nitrate tolerance, but it is still uncertain whether reduction in intracellular sulfhydryl availability is the operative mechanism. Vascular cyclic GMP production may be reduced during tolerance, but this change may not parallel that observed in vascular relaxation.

Blood Vessels↗
PubMed1989

Cellular mechanisms of nitrate action.

It is now generally accepted that organic nitrates generate their vasodilator action via production of nitric oxide. However, the cellular location of the metabolic enzyme(s) responsible for such conversion has not been defined. We examined the production of nitric oxide, via chemiluminescence detection, by various cellular fractions of the bovine coronary artery. We were able to show that the highest activity resides in the plasma membrane. Future isolation and characterization of such metabolic systems will greatly assist our understanding of nitrate action and tolerance. Several cellular mechanisms for nitrate tolerance have been proposed. Among the most popular theories is the "intracellular sulfhydryl depletion hypothesis" originally proposed by Needleman et al. The primary supportive data for this mechanism are that exogeneously added thiols (such as N-acetylcysteine) can potentiate the in vivo activity of nitroglycerin and can partially reverse nitrate tolerance. We showed that a cellular-impermeant thiol, viz: glutathione, can also potentiate the hemodynamic effect of nitroglycerin in rats. We subsequently showed that exogenously administered thiols can promote the formation of vasoactive S-nitrosothiols in blood. Thus, the beneficial effects of thiols on nitrate action might be mediated through an extracellular pathway. Another cellular mechanism for nitrate tolerance suggested that tolerance is caused by an alteration of the enzyme, guanylate cyclase. We showed, however, that blood vessels made tolerant to nitroglycerin remain fully responsive (in terms of in vitro relaxation) toward nitric oxide and S-nitrosothiols. These data showed that, as far as relaxation is concerned, nitrate tolerance did not cause a significant alteration of guanylate cyclase activity toward nitric oxide and S-nitrosothiols.

Animals↗
PubMed1989

Characterization of the pharmacokinetic interaction between nifedipine and ethanol in the rat.

We characterized the pharmacokinetic interaction between nifedipine and ethanol as a function of route of administration with pharmacokinetic monitoring of both compounds. Nifedipine was infused (8.5 micrograms/min/kg) for 9 hr either i.a. or intraportally to assess the extent of hepatic first-pass metabolism of nifedipine in the rat. Nifedipine clearance and volume of distribution were similar for both routes of administration. Thus, hepatic first-pass metabolism of nifedipine was apparently negligible in our animal preparation. The pharmacokinetics of ethanol were also examined in the animal preparation in order to design a suitable ethanol dosing protocol for the interaction study. The apparent ethanol Vmax and volume of distribution were 1.85 +/- 0.25 mg/min and 723 +/- 91 ml/kg (mean +/- S.D.), respectively. The pharmacokinetic interaction between nifedipine and ethanol was assessed by the following protocol: nifedipine was infused (either i.a. or intraportally) for 9 hr. When nifedipine concentrations reached steady-state at 4 hr, a bolus dose of ethanol (2.0 g/kg) was administered, followed by ethanol infusion (1.85 mg/min). Constant concentrations of ethanol were obtained with this dosing protocol. Nifedipine clearance was decreased by 49% in the presence of blood ethanol concentrations of 2.5 to 2.7 mg/ml, after either route of administration. The volume of distribution of nifedipine was not changed. The ethanol concentrations observed with nifedipine coinfusion were similar to the ethanol concentrations predicted by the ethanol pharmacokinetic parameters. Thus, ethanol altered nifedipine pharmacokinetics in the rat, but not vice versa.

Animals↗
PubMed1989

Blood and liver biochemical indices of the dystrophic chicken model.

The dystrophic chicken line 413 (University of California, Davis) and its genetically related normal line 412 were investigated for possible differences in serum blood chemistry indices and hepatic cytochrome P-450 content at two and three different ages ex ovo, respectively. There were statistical differences in the serum levels of creatine kinase, SGOT, uric acid, and phosphorus between normal and dystrophic animals at 34 and 48 days ex ovo. In the dystrophic animals, the elevations in uric acid and phosphorus levels may be suggestive of renal dysfunction, while the elevations in SGOT levels may indicate the presence of hepatic abnormalities. The hepatic concentrations of cytochrome P-450 and cytochrome b5 were not different between normal and dystrophic animals at 34, 48, and 70 days ex ovo.

Animals↗
PubMed1988

Complexation of nifedipine with substituted phenolic ligands.

The bioavailability of nifedipine in man is highly variable. This may be partly due to its poor aqueous solubility (5-6 micrograms/ml over pH 2.2-10.0, as determined in this laboratory). We initiated this study to examine the enhancement of aqueous nifedipine solubility via complexation. A series of substituted aromatic ligands was studied to identify those structural features important for complexation with nifedipine. The studies were performed at 25 degrees C employing the solubility technique, using pH 2.2 or 7.0 buffers at an ionic strength of 0.25 M. The apparent equilibrium complexation constants for the 1:1 and/or 1:2 complexes were determined, where appropriate. A linear free-energy approach was used to relate K1:1 with Hammett's sigma (sigma) and fractional partition coefficient (pi) parameters. The following correlation was obtained: log (K1:1/K0) = 0.31 sigma + 0.10 pi + 0.36 (r2 = 0.86, P less than 0.003, N = 9), where K0 is the complexation constant for phenol. Statistical analyses showed that sigma was more important than pi in affecting nifedipine complexation. The exact location of this interaction on the nifedipine molecule is undefined at present.

Calorimetry, Differential Scanning↗
PubMed1988

Interpretation of nitrate plasma concentrations. Effect of cardiac output on nitroglycerin pharmacokinetics in experimental animals.

We tested the hypothesis that the pharmacokinetics of nitroglycerin might be governed by haemodynamics, viz: cardiac output. The steady state pharmacokinetics of nitroglycerin, both in arterial and in venous plasma, were investigated in 11 rats after sequential infusions either of nitroglycerin alone (10 micrograms kg-1 min-1) or of nitroglycerin plus vasopressin (the latter at 5.5 mU kg-1 min-1). Cardiac output was estimated twice in each animal using 85Sr and 141Ce microspheres. Nitroglycerin systemic clearance in arterial plasma was found to be correlated strongly with cardiac output (r = 0.784, N = 22, P less than 0.001). Using the distribution ratio of nitroglycerin between red blood cells and plasma, we determined the systemic clearance of nitroglycerin in arterial blood to be about 3/4 of cardiac output. Vasopressin co-infusion decreased both the cardiac output and the arterial nitroglycerin clearance, but it also increased the arteriovenous extraction of nitroglycerin. Thus, vasopressin had no net effect on the venous plasma clearance of nitroglycerin. In animals infused with nitroglycerin alone, cardiac output also significantly correlated with nitroglycerin venous plasma clearance (P less than 0.01) and arteriovenous extraction (P less than 0.05). These data indicate that haemodynamic alterations may have profound effects on the observed plasma concentrations of nitroglycerin. These parameters need to be standardized and controlled if meaningful plasma concentrations of organic nitrates are to be obtained and interpreted.

Animals↗
PubMed1988

Antianginal effects of intravenous nitroglycerin over 24 hours.

To determine the constancy of hemodynamic and antianginal effects of the constant infusion of intravenous nitroglycerin (NTG) and their relationship to infusion rate and plasma NTG concentration, we administered maximal tolerated doses of intravenous NTG (range 10 to 120 micrograms/min, mean = 52 +/- 33 micrograms/min) and placebo to 10 patients with chronic stable angina for 25 hr each in a randomized, double-blind fashion. Sublingual NTG (0.4 mg) was given at 24.5 hr of infusion as a positive control. Bicycle exercise time (NIH protocol), blood pressure, heart rate, exercise ST response, and venous plasma NTG were determined before and at 1, 4, 8, 24, and 24.5 hr. Plasma NTG was linearly related to infusion rate, reached a steady state within 15 min and was unchanged over 24 hr (mean = 5.5 +/- 1.2 ng/ml). Mean plasma NTG clearance was 9.3 liters/min. However, during dose titration, patients demonstrated different relationships between plasma NTG and hemodynamic effects, with widely varying slopes and intercepts. Intravenous NTG produced a sustained reduction in blood pressure and a rise in heart rate at rest, and a reduction in blood pressure during submaximal exercise at as late as 24 hr, associated with reduced submaximal ST segment abnormality. In contrast, exercise tolerance to onset of angina showed a marked initial increase on intravenous NTG but fell progressively and did not differ from that with placebo at 24 hr. Increased exercise tolerance was associated with an increase in maximal heart rate and double product (heart rate X blood pressure), suggesting that direct coronary vasodilation and/or reduced left ventricular volume were the principal determinants of increased exercise tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Sublingual↗
PubMed1988

Mechanisms for the pharmacologic interaction of organic nitrates with thiols. Existence of an extracellular pathway for the reversal of nitrate vascular tolerance by N-acetylcysteine.

Recent reports have shown that the coadministration of N-acetylcysteine (NAC) potentiated the hemodynamic actions of i.v. nitroglycerin (NTG) and reversed NTG tolerance in humans. This study has investigated the feasibility of various pharmacokinetic and biochemical mechanisms for the thiol-organic nitrate interaction, using the rat as an animal model. In order to establish that the potentiating interaction between NAC and NTG can be reproduced in the rat, NTG dose-blood pressure response curves were determined before and during concurrent thiol infusion. The hypotensive effect of NTG was enhanced significantly by NAC and glutathione, but not by N-acetylserine, showing clearly that the potentiating effect of NAC was due specifically to its thiol functional group. The systemic clearance of NTG was not affected significantly by NAC coinfusion. In addition, the intracellular metabolism of NTG in thoracic aorta segments from rats infused previously with NAC or N-acetylserine was similar, both with respect to total production of metabolites and their distribution. Thus, the enhancement of NTG action could not be attributed apparently to an effect of NAC on NTG systemic pharmacokinetics or vascular metabolism of NTG. Because glutathione, which does not enter cells readily, also potentiated the effects of NTG, the possibility of an extracellular pathway for the thiol-organic nitrate interaction was examined. In vitro degradation of NTG in plasma and blood was accelerated in the presence of NAC (or glutathione). NAC also promoted the formation of S-nitroso-N-acetylcysteine from NTG in rat and human plasma and human blood.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcysteine↗
PubMed1987

Tolerance to relaxation in rat aorta: comparison of an S-nitrosothiol with nitroglycerin.

Tolerance and cross-tolerance to the relaxant effects of S-nitroso-N-acetylpenicillamine (SNAP) and nitroglycerin were examined in rat aortic rings. Nitroglycerin-tolerant rings remained fully responsive to SNAP and sodium nitroprusside. Thus, reduced metabolic activation of nitroglycerin, rather than impaired guanylate cyclase activity, appeared to be the operating mechanism for nitrate tolerance in this preparation. Under similar conditions, SNAP incubation induced less tolerance than nitroglycerin. S-nitrosothiols, therefore, appear to be less tolerance-producing than nitroglycerin.

Animals↗