Biomedical subjects

H L Fung

Publications and source records attributed to H L Fung.

At least 19 recordsLinked to original sources

PubMed1992

Biochemical mechanism of organic nitrate action.

Increasing evidence suggests that organic nitrate action derives from their metabolic conversion to nitric oxide (NO) in the vascular smooth muscle cell. The primary catalytic activity of this process appears to reside at the cellular plasma membrane. There is no concrete evidence to indicate that NO formation is preceded by the production of inorganic nitrite ion or that the NO produced needs to form S-nitrosothiols before it can activate guanylate cyclase to produce cyclic guanosine 3',5'-monophosphate (cGMP). Although sulfhydryl donors can partially reverse nitroglycerin-induced tolerance in patients, this phenomenon (by itself) is not sufficient to implicate intracellular sulfhydryl depletion as an operating mechanism of clinical nitrate tolerance. This is because sulfhydryl donors can react with nitroglycerin extracellularly to form S-nitrosothiols, and nonsulfhydryl compounds, such as enalapril and hydralazine, can prevent the development of in vivo nitrate tolerance. In addition to the cellular biochemical reactions, organic nitrates also produce systemic biochemical effects through altering neurohormonal status. These systemic effects may contribute significantly to the development of nitrate tolerance in therapeutic situations.

Animals↗

PubMed1992

A common enzyme may be responsible for the conversion of organic nitrates to nitric oxide in vascular microsomes.

We compared the nitric oxide (NO)-generating behavior of nitroglycerin (NTG), pentaerythritol trinitrate (PEtriN) and isosorbide dinitrate (ISDN), in the microsomal preparation of bovine coronary artery smooth muscle cells. The comparative NO generating activities among these nitrates were consistent with their relative reported vasodilating activities. Consistent with our previous observations with NTG, 400 microM bromosulfophthalein did not affect NO generation from PEtriN and ISDN in vascular microsomes while 400 microM 1-chloro-2,4-dinitrobenzene completely inhibited NO generation from these nitrates. Gel filtration chromatography with solubilized microsomes of bovine aortic smooth muscle cells showed the primary activity of NO generation from all three nitrates to be eluted at about 200 kD, consistent with that found with solubilized microsomes from the bovine coronary artery microsomes. These results suggest that organic nitrates may be converted to NO by one common enzyme in vascular microsomes.

Amino Acid Oxidoreductases↗

PubMed1992

Pharmacodynamic modeling of the in vitro vasodilating effects of organic mononitrates.

The in vitro dose-relaxation curves of four isomeric organic mononitrates: L-isoidide mononitrate (L-IIMN), isosorbide-2-mononitrate (IS-2-MN), isomannide mononitrate (IMMN), and isosorbide-5-mononitrate (IS-5-MN), were determined with rat aorta rings. These mononitrates relaxed vascular tissue in a concentration-dependent manner. Based upon the EC50 obtained from the Hill equation, the relative potency of L-IIMN: IS-2-MN: IMMN: IS-5-MN was 43.2:12.2:2.3:1. The Hill exponential coefficients were identical (value of about 1.5) in these four isomeric mononitrates, suggesting that they are likely to have a common mechanism of action. For all four isomers, relaxation was fairly immediate after addition of the tested compound into the tissue bath, with a 2- to 3-min-delay to reach steady-state effect. The rates of relaxation were then used to construct a pharmacodynamic model that described the time course of relaxation for these compounds. This theoretical analysis suggested that in vitro nitrate action is mediated by a catenary process, consistent with published biochemical evidence that suggests a series of reactions involving metabolic activation to nitric oxide, production of cyclic GMP, and myosin light-chain phosphorylation to produce relaxation. Via this pharmacodynamic model, the half-lives of nitric oxide and cGMP in the smooth muscle cells were estimated to be 15.2 and 23.1 sec, respectively, consistent with literature reports. Results from the present study indicated the potential use of in vitro pharmacodynamic modeling in confirming mechanism(s) of drug action obtained through biochemical or other methods.

Animals↗

PubMed1992

Structure activity relationship of organic nitrates: an exploratory hypothesis via molecular models.

An exploratory attempt to explain the structure/activity relationship of organic nitrates was initiated. In addition to the partition coefficient, a new empirical parameter, F(5.8A), was used in this correlation. This new parameter denotes the number of times that two oxygen atoms in the molecule can be arranged at a distance of 5.8A. Vasodilating potencies (EC50s) and partition coefficients of 11 organic nitrates were obtained from a literature report, while their corresponding F(5.8A) values were obtained from manual determination using two molecular models, viz: CPK and Drieding. Via linear regression comparisons, we showed that F(5.8A) was a better physicochemical parameter than the partition coefficient in explaining the differences in the observed pharmacologic activity. When the two parameters were combined, greater than 90% of the variability of the potency was accounted for. These results suggest that the pertinent protein (most likely the activating enzyme) for organic nitrate activity may contain at least two 'oxygen-philic' attachment sites which are separated by a distance of 5.8A.

Animals↗

PubMed1992

Do nitrates differ?

1. The organic nitrates all share a common biochemical and physiological mechanism of action. 2. The organic nitrates differ substantially in their pharmacologic potency and pharmacokinetics. In vitro potency differences appear larger than the corresponding in vivo activities. 3. The duration of action of organic nitrates, after a single immediate-release dose, is governed by the pharmacokinetics of the drug. However, the duration of action of available sustained-release preparations, whatever the nitrate or formulation, is limited to about 12 h, due to the development of pharmacologic tolerance. 4. Nitrates do not appear to differ in their production of undesirable effects.

Animals↗

PubMed1992

Metabolic activation of sodium nitroprusside to nitric oxide in vascular smooth muscle.

Sodium nitroprusside (SNP) is thought to exert its vasodilating activity, at least in part, by vascular activation to nitric oxide (NO), but the activation mechanism has not been delineated. This study has examined the potential for vascular metabolism of SNP to NO in bovine coronary arterial smooth muscle subcellular fractions using a sensitive and specific redox-chemiluminescence assay for NO. SNP was readily metabolized to NO in subcellular fractions, and the dominant site of metabolism appeared to be located in the membrane fractions. NO-generating activity was significantly enhanced by, but did not absolutely require, the addition of a NADPH-regenerating system, NADPH per se, NADH or cysteine. A correlation analysis of NO-generating activity (in the presence of a NADPH-regenerating system) with marker enzyme activities indicated that the SNP-directed NO-generating activity was primarily membrane-associated. Radiation inactivation target-size analysis revealed that the microsomal SNP-directed NO-generating activity was relatively insensitive to inactivation by radiation exposure, suggesting that the functioning catalytic unit might be quite small. A molecular weight of 5 to 11 kDa was estimated. NO-generating activity could be solubilized from the crude microsomes with 3-[(3-cholamidopropyl)- dimethylammonio]-1-propane sulfonate, and the solubilized extract was subjected to gel filtration chromatography. NO-generating activity was eluted in two peaks: one peak corresponding to an approximate molecular weight of 4 kDa, thus confirming the existence of a small molecular weight NO-generating activity, and a second activity peak corresponding to a molecular weight of 112 to 169 kDa, the functional significance of which is unclear at present.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Pharmacokinetic/pharmacodynamic relationship of the duration of vasodilating action of organic mononitrates in rats.

Conflicting information existed regarding the relevance of pharmacokinetics in relation to the duration of action of organic nitrates. We examined this question using three geometric isomers of organic mononitrates, L-isoidide mononitrate, isosorbide-2-mononitrate and isosorbide-5-mononitrate, which produce no active metabolites but possess diverse biological half-lives (T1/2). These compounds were given i.v. to rats at doses which were predetermined to produce equal peak effects on pulse pressure. The relationship between percentage change in pulse pressure and plasma mononitrate level could be described by the classical Hill equation, with similar slope but different EC50 values. A duration of action, defined as the time taken for the pulse pressure to change from -30% to -20% from base line, was measured experimentally. Theoretical analysis suggests that a linear relationship should exist between this duration of action and T1/2/slope, and this was indeed observed (r = 0.99, P less than .01, n = 12), affirming the dependency of pulse pressure pharmacodynamics on the pharmacokinetics of organic mononitrates. Pharmacokinetic modeling of the in vivo time-dependent hemodynamic effects further indicated that the blood compartment data were more consistent with a mechanism of mononitrate action that involves metabolic activation to form nitric oxide and cyclic GMP, rather than direct receptor activation. Using this pharmacodynamic model, the in vivo half-lives of nitric oxide and cGMP were estimated to be 1.6 and 2.0 min, respectively.

Animals↗

PubMed1992

Conversion of nitroglycerin to nitric oxide in microsomes of the bovine coronary artery smooth muscle is not primarily mediated by glutathione-S-transferases.

The pharmacological action of organic nitrate vasodilators [e.g., nitroglycerin (NTG)] is thought to be mediated through metabolic conversion to nitric oxide (NO); conversion leads to vasodilatation, whereas diminished conversion in chronic therapy may lead to pharmacological tolerance. The biochemical nature of this process, however, is poorly understood. Glutathione-S-transferases (GST) have been shown to metabolize organic nitrates in the liver, but it is not known whether these enzymes are involved in this pharmacologically relevant process. We, therefore, compared the activities of conversion of NTG to NO vs. those of GST in microsomal suspensions of bovine coronary artery smooth muscle tissue. A classical GST substrate, 1-chloro-2,4-dinitrobenzene, inhibited NO production in microsomes, suggesting possible involvement of GST in organic nitrate activation. However, GST activity derived from microsomes exhibited a different heat lability profile compared to that of NO generation. Known inhibitors of GST (viz., indomethacin and bromosulfophthalein) did not alter the NO-generating activity in microsomes. Glutathione was a critical cofactor for GST, but not for NO generation from NTG, and thiols other than glutathione (e.g., N-acetyl-L-cysteine and thiosalicylic acid) also could facilitate NO production. Moreover, comparison to a commercially available purified liver GST preparation showed that, at the same GST activity toward 1-chloro-2,4-dinitrobenzene, the microsomal incubation produced about 8 times more NO than the purified liver GST. Radiation inactivation analysis of the functional molecular sizes of GST and the NO-producing enzyme(s) suggested that the enzymes were of different molecular weights (54 kD and 160 kD, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Gas chromatographic analysis of isomeric organic mononitrates in plasma.

A specific, sensitive and precise capillary gas chromatographic method using electron-capture detection was developed for the determination of four isomeric vasodilating organic mononitrates, viz. L-isoidide mononitrate (L-IIMN), isosorbide-2-mononitrate (IS-2-MN), isomannide mononitrate (IMMN) and isosorbide-5-mononitrate (IS-5-MN), in rat plasma. With a sample size of 100 microliters of rat plasma, the detection limits were found to be between 0.5 and 2 ng/ml for these mononitrates, and the absolute recovery was found to range from 83 to 90%. The within-day coefficients of variation for the assay of the four isomers were less than 5%, while the between-day coefficients of variation were less than 10%. Because of the short retention times of these isomers in this assay, routine analyses of about sixty plasma samples per day can be carried out. The possibility of in vivo interconversion among these four isomers in rats was investigated after individual administration of each isomer. No interconversion was found based on examination of plasma samples. The gas chromatographic method was applied to the pharmacokinetic studies of these four isomers in rats; at an intravenous dose of 2 mg/kg, the biological half-lives of L-IIMN, IMMN, IS-2-MN and IS-5-MN were found to be 13.2, 25.2, 54.6 and 112 min, respectively.

Animals↗

PubMed1991

Biochemical and pharmacological interactions between nitroglycerin and thiols. Effects of thiol structure on nitric oxide generation and tolerance reversal.

Co-administration of N-acetylcysteine (NAC) with nitroglycerin (NTG) has been shown to partially reverse nitrate tolerance and to potentiate the hypotensive effect of NTG in humans. However, a high clinical dose of NAC was required for this pharmacologic interaction resulting in the production of unwanted side-effects. Therefore, sulfhydryl compounds more active than NAC need to be identified if this interaction is to be exploited clinically. We previously suggested that the effect of sulfhydryl compounds on NTG may be mediated by the formation of S-nitrosothiol or nitric oxide (NO) extracellularly to the vascular smooth muscle cell (e.g. in plasma) (Fung et al., J. Pharmacol Exp Ther 245: 524-530, 1988). In an attempt to understand the structural features which govern this thiol-catalyzed NO generation from NTG, nineteen different aliphatic and ten aromatic sulfhydryl compounds were examined with respect to their catalytic activity to generate NO from NTG in plasma. Significantly enhanced production of NO was observed with most sulfhydryl compounds examined when compared to buffer control. Among the aliphatic thiols, only mercaptosuccinic acid was more potent than NAC (2x), whereas among the aromatic thiols, both thiosalicylic acid (TSA, 10x) and TSA-methyl ester (3x) were more potent than NAC. Comparative in vitro relaxation studies were carried out using isolated (and nitrate-tolerant) rat aortic rings with NTG/TSA and NTG/NAC, in the presence of 0.5% (v/v) plasma. Under these conditions, partial reversal of NTG tolerance could be achieved with TSA, but not with NAC. These data are consistent with the view that extracellular production of NO or S-nitrosothiol serves as a tolerance-reversing mechanism of thiols on NTG. TSA appears to be a more potent sulfhydryl compound than NAC in this biochemical and pharmacologic interaction.

Acetylcysteine↗

PubMed1991

Chemical stabilization of a vasoactive S-nitrosothiol with cyclodextrins without loss of pharmacologic activity.

S-Nitrosothiols have been proposed as the endogenous chemical representing the vasoactive endothelium-derived relaxing factor, as well as the active cellular intermediates responsible for the therapeutic action of organic nitrates. The relatively stable analogue S-nitroso N-acetyl penicillamine (SNAP) is a potent vasodilator producing less pharmacologic tolerance than nitroglycerin upon prolonged administration. The therapeutic potential of this new class of vasodilators, however, may be limited by their chemical instability in solution (t1/2 of SNAP is 26 hr in 5% dextrose). We examined the usefulness of several cyclodextrins (CD) to stabilize this polar compound in solution. At cyclodextrin concentrations of 12 mM, hydroxypropyl-beta CD was most effective at stabilizing SNAP (t1/2 = 77 hr) when compared to alpha CD (41 hr), beta CD (69 hr), gamma CD (36 hr), and beta CD-tetradecasulfate (38 hr). Stability constants for the complexation of SNAP with the various cyclodextrins were determined by the classical solubility technique and were found to range from 26 to 435 M-1. Increased complexation brought about better SNAP stability. Complexation of SNAP with cyclodextrins, however, did not decrease the relaxation potency of SNAP as determined in an in vitro blood vessel preparation. Cyclodextrin complexation may be a useful approach to stabilize labile and polar compounds, such as S-nitrosothiols, without loss of pharmacologic activity.

Animals↗

PubMed1991

Regulation of K+ and Ca2+ channels in experimental cardiac failure.

To examine the status of ATP-sensitive K+ (K+ATP) channels and 1,4-dihydropyridine-sensitive Ca2+ (Ca2+DHP) channels during experimental cardiac failure, we have measured the radioligand binding properties of [3H]glyburide and [3H]PN 200 110, respectively, in tissue homogenates from the rat cardiac left ventricle, right ventricle, and brain 4 wk after myocardial infarction induced by left coronary artery ligation. The maximal values (Bmax) for [3H]glyburide and [3H]PN 200 110 binding were reduced by 39 and 40%, respectively, in the left ventricle, and these reductions showed a good correlation with the right ventricle-to-body weight ratio in heart-failure rats. The ligand binding affinities were not altered. In the hypertrophied right ventricle, Bmax values for both the ligands were not significantly different when data were normalized to DNA content or right ventricle weights but showed an apparent reduction when normalized to unit protein or tissue weight. Moderate reductions in channel densities were observed also in whole brain homogenates from heart failure rats. Assessment of muscarinic receptors, beta-adrenoceptors and alpha 1-adrenoceptors by [3H]quinuclidinyl benzilate, [3H]dihydroalprenolol, and [3H]prazosin showed reductions in left ventricular muscarinic and beta-adrenoceptor densities but not in alpha 1-adrenoceptor densities, consistent with earlier observations. It is suggested that these changes may in part contribute to the pathology of cardiac failure.

Animals↗

PubMed1991

Concurrent hydralazine administration prevents nitroglycerin-induced hemodynamic tolerance in experimental heart failure.

BACKGROUND: Organic nitrates such as nitroglycerin and isosorbide dinitrate are useful in the treatment of congestive heart failure (CHF), but tolerance develops rapidly during continuous administration. Because combination therapy of nitrate and hydralazine has been shown to provide both short- and long-term benefit but nitrate alone produces hemodynamic tolerance, we questioned whether hydralazine can preserve the favorable preload effects of nitroglycerin. METHODS AND RESULTS: Using an in vivo model of nitroglycerin tolerance in the CHF rat, we examined the effects of hydralazine bolus dosing during continuous nitroglycerin infusion. Continuous infusion of nitroglycerin alone (10 micrograms/min) produced initial reductions in left ventricular end-diastolic pressure of 40-50%, which returned to baseline by 8 hours (tolerance development). Coadministration of hydralazine (2 x 0.1 mg) maintained the effects of nitroglycerin infusion on left ventricular end-diastolic pressure (45% reduction at 10 hours). This hydralazine dose alone reduced left ventricular peak systolic pressure by approximately 12 +/- 3% but had no effect on left ventricular end-diastolic pressure. Hydralazine dosing did not affect steady-state plasma concentrations of nitroglycerin or metabolites, and hydralazine was unable to prevent nitroglycerin tolerance induced in vitro. CONCLUSIONS: The beneficial interaction of hydralazine on the preload effects of nitroglycerin may explain the long-term clinical efficacy of hydralazine/nitrate combination in CHF. Our results also suggest that the mechanism of in vivo nitrate tolerance in CHF may be systemic rather than vascular in origin.

Animals↗

PubMed1991

Vascular nitric oxide-generating activities for organic nitrites and organic nitrates are distinct.

Like organic nitrates, the organic nitrites are assumed to undergo vascular metabolic activation to nitric oxide (NO) to produce vasodilating activity, but this assumption has never been examined. In this study, isobutyl nitrite and amyl nitrite were incubated with subcellular fractions of bovine coronary arterial smooth muscle at 37 degrees C, and NO generation was assessed by redox chemiluminescence assay of headspace NO. Bovine vascular subfractions exhibited substantial and significant catalytic activity for NO generation towards both organic nitrite substrates, and NO-generating activity could be inhibited by heating and irradiation. These observations suggest that organic nitrites are converted enzymatically to NO in vascular smooth muscle. Analysis of marker enzyme activities suggested that the primary NO-generating activity was associated with the cytosol, whereas a distinct, but relatively minor NO-generating activity was also identified in the microsomal fraction. The cytosolic and microsomal NO-generating activities exhibited different temperature dependencies for heat-induced inhibition of activity. The molecular weights of the NO-generating enzymes, determined by radiation inactivation target-size analysis, were 263 kDa (95% confidence interval: 236-298 kDa) for the cytosolic enzyme and 79 kDa (95% confidence interval: 54-148 kDa) for the microsomal enzyme. Previous studies in this laboratory have shown that the representative organic nitrate, nitroglycerin (NTG), is metabolized to NO by an enzyme associated with the plasma membrane of bovine vascular smooth muscle cells. Thus, the enzymes responsible for vascular bioconversion of organic nitrates and organic nitrites to NO are apparently different.(ABSTRACT TRUNCATED AT 250 WORDS)

Amyl Nitrite↗

PubMed1991

Nitrate therapy: is there an optimal substance and formulation?

Nitrate therapy has been shown to be beneficial for the treatment of coronary artery diseases and a number of chemical entities and their respective pharmaceutical formulations are available for clinical use. In this report, the performance of these nitrate drug/formulation combinations is discussed in terms of the relative onset of action, duration of action, tolerance properties/regeneration of reactivity and patient acceptance. A pharmacodynamic action scheme is presented and this allows a systematic assessment of how changes in the nitrate and/or formulation may impact on therapeutic activity. Data suggest that several sustained-release preparations of various nitrates can provide protection against exercise-induced angina for about 12 h on repeated dosing, provided a 'nitrate-free' or 'nitrate-poor' interval is also instituted. The 'ideal' nitrate substance and formulation, which theoretically can provide around-the-clock protection, is not yet available.

Angina Pectoris↗

PubMed1991

Use of refractometers to detect controlled-substance tampering.

Hospital pharmacies presently lack a simple and cost-effective procedure to monitor the integrity of solutions of controlled substances that they distribute. Thus, the use of refractive-index values, measured by inexpensive hand-held refractometers, in monitoring such solutions was studied. Four refractometers were used to measure the refractive index or % Brix (an index of the percentage of solid in solution) of solutions of a number of controlled substances, including fentanyl citrate, morphine sulfate, hydromorphone hydrochloride, and meperidine hydrochloride. The hand-held refractometers provided precise readings with small variabilities. Although this method does not determine the actual drug concentration per se, subversion of the monitoring procedure for many solutions would require considerable forethought and scientific knowledge. A refractometric survey of 83 controlled-substance solutions returned to the hospital pharmacy showed the procedure to be capable of identifying a solution of unexpected concentration. The described refractometric procedure is rapid, simple, reproducible, inexpensive, and applicable to a wide array of drug solutions. Hospital pharmacies may consider using the procedure for routine monitoring of solutions of controlled substances.

Drug and Narcotic Control↗

PubMed1991

Differential hemodynamic effects and tolerance properties of nitroglycerin and an S-nitrosothiol in experimental heart failure.

S-nitrosothiols are potent in vitro vasodilators, but little is known about their in vivo action. In this study, we compared the effects of S-nitroso N-acetyl penicillamine (SNAP) and nitroglycerin (NTG) on left ventricular (LV) hemodynamics in congestive heart failure rats. By using a twoday crossover design, stepwise i.v. infusions of SNAP or NTG at 3, 5 and 8 micrograms/min were administered for 30 min each, followed by a dose of 10 micrograms/min over the next 10 h. LV end-diastolic and peak-systolic pressures (LVEDP and LVPSP, respectively) were measured at selected intervals. SNAP and NTG produced maximal LVEDP reductions of 46 and 44%, respectively, at the highest infusion rate. However, at the lower doses, greater reductions of LVEDP were seen with SNAP. NTG had a smaller effect on LVPSP (maximum 6% reduction) than SNAP (maximum reduction of 15%). During the 10-h infusion of NTG, LVEDP gradually returned to base-line values, indicating the development of tolerance, despite relatively constant plasma levels of NTG over the infusion period. Tolerance in LVEDP effects was not observed during the 10-h infusion of SNAP. In the presence of NTG tolerance, rats were still responsive to SNAP (mean reduction of LVEDP 24%), suggesting the absence of cross-tolerance between these two nitrovasodilators. These results suggest that SNAP is a more potent in vivo vasodilator than NTG, has more arterial action than NTG and is less prone to produce LV hemodynamic tolerance.

Animals↗

PubMed1990

Dissociation of nitrovasodilator-induced relaxation from cyclic GMP levels during in vitro nitrate tolerance.

The effects of nitroglycerin tolerance on relaxation and cyclic GMP accumulation by nitroglycerin, nitric oxide and S-nitroso-N-acetylpenicillamine were examined in isolated rate aortic rings. Cyclic GMP accumulation by nitroglycerin, S-nitroso-N-acetylpenicillamine and nitric oxide was diminished in nitroglycerin-tolerant aorta. In contrast, only relaxation by nitroglycerin, but not S-nitroso-N-acetylpenicillamine and nitric oxide, was attenuated. These data suggest that cyclic GMP levels might represent an inadequate index for mechanistic studies of nitroglycerin relaxation tolerance.

Animals↗