Alterations in heart failure of cyclic AMP-dependent inotropic and lusitropic properties of cardiac and skeletal muscle.

A central working hypothesis in our laboratory is that deficient cellular cyclic AMP concentrations may be responsible, at least in part, for striated muscle dysfunction, both cardiac and skeletal, in heart failure. These results suggest that therapy aimed at restoring cyclic AMP to normal levels may be effective with regard to improving systolic and diastolic function in the heart and may decrease the development of fatigue in skeletal muscle of patients with failure. The use of cyclic AMP-dependent drugs in clinical practice has been limited by side effects associated with raising total cellular content of this cyclic nucleotide. However, evidence suggesting that separate pools of cyclic AMP may exist within the cell raises the possibility that those pools associated with excitation/contraction coupling could serve as more specific therapeutic targets.

Negative inotropic effect of methylecgonidine, a major product of cocaine base pyrolysis, on ferret and human myocardium.

This study examined the physiological effects and potential mechanisms of action of methylecgonidine (MEG), the major pyrolysis product from smoking "crack cocaine," on cardiac function. Ferret right ventricular papillary muscles and human ventricular trabeculae were isolated and placed in a physiological solution at 30 degrees C containing 2.5 mM Ca2+ and stimulated at 0.33 Hz. MEG decreased peak tension and peak intracellular Ca2+ transients in a concentration-dependent manner (10 microM-1 mM). The negative inotropic effect (NIE) of MEG was reversible by atropine (1 microM). Atropine shifted the concentration-response curve of MEG rightward (pA2 = 9.17) similar to that of carbachol (pA2 = 8.70). With prior addition of histamine (1 microM) and Ca2+ (4.5 mM) in equiinotropic concentrations, MEG and carbachol decreased contractility to a greater extent in the histamine-stimulated muscles. To clarify whether the treatments altered responsiveness of the contractile elements to Ca2+, the effect of 2,3-butanedione monoxime (BDM), an agent that interferes with the interaction of actin and myosin, was tested after prior addition of histamine or increased Ca2+. No differential effect occurred. Moreover, the nitric oxide synthase inhibitor NG-nitro-L-arginine methylester (L-NAME; 0.1 mM), lessened the NIE of MEG compared with prior (pre-L-NAME) values. Furthermore, in human ventricular trabeculae (n = 7), MEG exhibited an NIE that was also reversible by atropine. We concluded that the NIE of MEG is caused by decreased calcium availability; the effect is not the result of a local anesthetic action but is mediated by stimulation of cholinergic receptors. This effect is potentiated by the nitric oxide pathway.

Effect of cocaine and methylecgonidine on intracellular Ca2+ and myocardial contraction in cardiac myocytes.

We evaluated the cardiac effects of the principle pyrolysis product of crack cocaine smoking, methylecgonidine (MEG), in comparison with cocaine. Peak cell shortening and intracellular Ca2+, as detected by the Ca2+ indicator indo 1, were recorded in enzymatically isolated ferret myocytes. Both cocaine and MEG decreased peak cell shortening and peak intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner (10(-8)-10(-4) M). MEG shifted the peak [Ca2+]i-to-peak shortening relationship downward and was more potent than cocaine. Atropine (10(-6) M) upwardly shifted the dose-response curves of MEG, cocaine, and carbachol but not of procaine. The negative inotropic effects of MEG were inhibited by methoctramine, a selective M2 receptor blocker but not by M1 (pirenzepine) or M3 (4-diphenylacetoxy-N-methylpiperidine methiodide) blocking agents. In contrast to cocaine, the effects of large doses of MEG were irreversible over the time course of our experiments, raising the possibility of structural damage. We conclude that MEG acts primarily on M2 cholinergic receptors in the heart to produce acute cardiac intoxication and, in contrast to cocaine, may decrease the myofilament Ca2+ responseness and cause structural damage to myocytes by a direct toxic effect.

A robotics-based liquid chromatographic assay for the measurement of atovaquone in plasma.

A precise and specific robotics-based liquid chromatographic (LC) method for measuring atovaquone concentrations in plasma was developed and validated, and the method was compared with an existing manual LC method. The compound was isolated from plasma by liquid-liquid extraction, separated by reversed-phase LC, and quantitated against an internal standard with UV detection. Least-squares linear regression with 1/concentration2 weighting was used as the calibration model. The range of the calibration curve for the assay under routine conditions was 0.25-50 micrograms ml-1. No endogenous interferences with the compound or the internal standard were noted in either untreated human plasma or in plasma from patients enrolled in Phase III clinical trials of atovaquone. The accuracy of the assay (determined as the percent bias) ranged from -4.8% to -9.4% in the validation runs. The intra- and interassay precisions (determined as the relative standard deviation) were less than 6.8% and 6.4%, respectively. The contribution of an internal standard on assay accuracy and precision also was examined. Interassay variability was marginally improved by the incorporation of an internal standard to the assay; accuracy and intra-assay precision were essentially unchanged. A paired t-test between estimates of atovaquone concentrations in healthy volunteer and HIV + patient human plasma samples assayed by the automated and manual methods demonstrated no significant difference (p = 0.31) between the values determined by each method.

Differences in the metabolism of the aromatic amino acid hydroxylase cofactor, tetrahydrobiopterin, in mutant mice with neurological and immunological defects.

Tetrahydrobiopterin (BH4) levels and GTP cyclohydrolase activity (GTP-CH) were measured in tissues from mutants and controls of 24 different mouse strains to identify mutants that might be suitable models for diseases which are characterized by a deficiency of the biopterin cofactor, such as parkinsonism and atypical phenylketonuria. BH4 levels and GTP-CH activity were determined in brain, liver, and spleen obtained from 24 mutants with neurological or immunological defects. BH4 levels in brain were slightly but significantly decreased in only two mutants, spastic (spa) and jittery (ji), while GTP-CH activity in brain was not significantly lower than controls in any of the strains examined. GTP-CH levels in liver were significantly decreased in four mutant strains (jittery, ji; leaner, tgla; reeler, rl; and anorexia, anx); however, BH4 levels were significantly lower only in the mutant anorexia (anx). The most significant and widespread changes in both BH4 levels and GTP-CH activity were observed in spleen. In those mutants which were most affected, BH4 levels and GTP-CH activity were decreased 85-90%.

Regulation of tetrahydrobiopterin biosynthesis in cultured adrenal cortical tumor cells by adrenocorticotropin and adenosine 3',5'-cyclic monophosphate.

Y-1 adrenal cortical tumor cells in culture, which contain substantial amounts of tetrahydrobiopterin [6R-(L-erythro-1',2'-dihydroxypropyl)5,6,7,8-tetrahydropterin] (BH4) and GTP cyclohydrolase (GTP-CH), were used to study the regulation of BH4 biosynthesis by ACTH and cAMP. ACTH produced a dose-dependent increase in steroidogenesis, BH4 levels and GTP-CH activity. Maximal stimulation of BH4 biosynthesis occurred at the same concentration of ACTH that caused maximal stimulation of steroidogenesis. ACTH-(1-24) was more potent than ACTH-(1-39). The stimulation of BH4 biosynthesis by ACTH was dependent on cell density, being greater at lower cell densities, but was independent of time in culture. The lack of stimulation by ACTH at higher cell densities was due to an increase in the specific activity of GTP-CH in the control cells as density increased. This increase may be due in part to the increased release of steroids, since exogenous steroids added to low density cultures also resulted in an increase in the specific activity of the enzyme. Addition of steroids had no effect on ACTH-dependent stimulation of BH4 biosynthesis at low cell densities. (Bu)2cAMP, 8-bromo-cAMP, and forskolin all produced time- and dose-dependent increases in BH4 levels, GTP-CH activity, and steroidogenesis. Maximum increases in GTP-CH and BH4 occurred at concentrations similar to those required for maximal stimulation of steroidogenesis. In the Kin-8 mutant of Y-1 cells, which has a type 1 cAMP-dependent protein kinase with an altered regulatory subunit, ACTH was unable to increase BH4 levels or GTP-CH activity at a concentration that produced maximal stimulation of BH4 and steroid biosynthesis in the parent Y-1 line. These studies indicate that Y-1 cells in culture are useful for studying the regulation of BH4 biosynthesis in the adrenal cortex.

Urinary biopterin and neopterin excretion and pituitary-adrenal activity in psychiatric patients.

The urinary excretion of biopterin and neopterin, pterin compounds related to tetrahydrobiopterin, the cofactor for the initial steps in monoamine synthesis, was serially measured in a heterogeneous group of psychiatric patients and compared to excretion in control subjects, to state of illness, and to the results of the dexamethasone suppression test. Patients with major depression had increased excretion of biopterin compared to normal subjects. There was no relationship between biopterin or neopterin excretion and postdexamethasone cortisol levels. Pterin excretion did not significantly change with improvement in mood or with conversion from nonsuppressor to suppressor status. The meaning of increased urinary biopterin is presently unclear, although its relation to hormonal state and sympathoadrenal tone deserves further study.

Biopterin cofactor biosynthesis: GTP cyclohydrolase, neopterin and biopterin in tissues and body fluids of mammalian species.

Levels of GTP cyclohydrolase, neopterin and biopterin were determined in tissues and body fluids of humans, monkey, dog and mouse. Highest levels of GTP cyclohydrolase and biopterin were found in pineal, liver, spleen, bone marrow, whole adrenal gland and small intestine. High levels of biopterin were found in the urine of all species examined. High levels of neopterin were found only in the urine of humans and monkeys, very low levels could be detected in dog, while none could be detected in mouse, rat, guinea pig or hamster urine.

Urinary excretion of biopterin and neopterin in psychiatric disorders.

Levels of urinary neopterin and biopterin were determined in patients having a diagnosis of schizophrenia, unipolar depression, or bipolar depression. Both neopterin and biopterin levels were significantly higher in the urine of patients with unipolar depression than in the urine of the control group. Subclassification of patients into primary and secondary depression demonstrated a significant elevation of urinary biopterin in both groups, whereas urinary neopterin was elevated only in those patients with primary depression. In patients with bipolar depression, neopterin excretion was elevated, but biopterin excretion did not differ from controls. No significant differences were found in schizophrenic patients.

Endogenous effector of the benzodiazepine binding site: purification and characterization.

A protein has been isolated from the small intestine and bile duct which inhibits the binding of [3H]diazepam to specific benzodiazepine binding sites on synaptosomal membranes. When ion-exchange chromatography and gel filtration chromatography are used, this protein has been purified to apparent homogeneity. "Nepenthin" has been chosen as a name for this protein, which has an approximate molecular weight of 16 000, as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography. Purified nepenthin is a competitive inhibitor of [3H]diazepam binding with a Ki = 4.6 X 10(-8) M. It does not inhibit the binding of specific ligands to the enkephalin, beta-adrenergic, gamma-aminobutyrate, or dopamine binding sites in the CNS. Neither gamma-aminobutyric acid nor glycine alters the inhibition of [3H]diazepam binding by this protein. Nepenthin can be extensively treated with proteases (trypsin, chymotrypsin, and Pronase), and inhibition of diazepam binding remains stable, indicating that a lower molecular weight fragment retains activity. Antibodies raised against this purified effector have been used in in situ double antibody labeling studies with rat brain slices. These studies indicate that cells containing an immunologically similar material are present in the deep cortical region of the forebrain.

Studies of yeast alcohol dehydrogenase with 3-aminopyridine monucleotide.

3-Aminopyridine mononucleotide, a nicotinamide mononucleotide analog, was prepared by enzymatic cleavage of 3-aminopyridine adenine dinucleotide by a snake venom phosphodiesterase and isolated by means of ion exchange chromatography. The spectrophotometric and fluorometric properties of this analog were studied. Several anions were shown to quench the fluorescence intensity of this analog. pH was shown to have a pronounced effect on the fluorescence intensity. 3-Aminopyridine mononucleotide was shown to be a coenzyme-competitive inhibitor of yeast alcohol dehydrogenase. The 3-aminopyridine mononucleotide was diazotized with the use of nitrous acid. A time dependent irreversible inactivation of yeast alcohol dehydrogenase resulted from incubation with the diazotized 3-aminopyridine mononucleotide at pH 7.0. Incubation of the enzyme with NAD prior to the addition of the diazotized 3-aminopyridine mononucleotid protected the enzyme against inactivation.

Superoxide anion independent reduction of nitrobluetetrazolium by human renal tissue.

The rate of nitrobluetetrazolium reduction by extracts from rodent and human kidney tissue has been measured in the presence and absence of exogeneous superoxide dismutase and catalase. These oxygen metabolite scavengers had no effect on that reduction rate. Oxygen was inhibitory to nitrobluetetrazolium reduction whereas anaerobiosis enhanced the rate. These data suggest that the reduction rate in renal tissue is not superoxide anion radical mediated as had been previously suggested.