Antiplatelet activity of tetramethylpyrazine.

Tetramethylpyrazine (TMPZ) is an active ingredient of a Chinese herbal medicine. We have previously shown that TMPZ possesses antithrombotic activity in rat thrombotic models. Inhibition of platelet aggregation and promotion of disaggregation contributed to the antithrombotic activity of TMPZ. In this study, we evaluated the antiplatelet actions of TMPZ using human platelets and found that antiplatelet activity of TMPZ included the inhibition of intracellular Ca++ mobilization (mainly the release from internal stores), the enhancement of intracellular cAMP by inhibition of phosphodiesterase activity, and the reduction of GP IIb/IIIa exposure on the surface of activated platelets.

Antiplatelet structure-activity relationship of tetramethylpyrazine.

Tetramethylpyrazine (TMPZ) is an active component of certain plants previously found to have inhibitory effects on platelet function using both in vivo and in vitro assays(1). In this study, we examined the antiplatelet activity of structural analogues of TMPZ in order to assess structural requirements for activity. Functional requirements included nitrogen substitutions on the aromatic ring; substitutions could be either heterocyclic or ring substituted in the meta position. Enhanced antiplatelet activity was associated with increasing the number of alkyl groups on the pyrazine ring as well as increasing the length of unbranched alkyl side chains. Increased inhibition of platelet aggregation correlated with increased lipophilicity of the compounds tested. In addition, several compounds were identified which deserve further pharmacological investigation.

Electrospray ionization mass spectrometric characterization of acrylamide adducts to hemoglobin.

The most common procedure to identify hemoglobin adducts has been to cleave the adducts from the protein and characterize the adducting species, by, for example, derivatization and gas chromatography/mass spectrometry. To extend these approaches we used electrospray ionization mass spectrometry (ESI-MS) to characterize adducted hemoglobin. For this we incubated [14C]acrylamide with the purified human hemoglobin (type A0) under conditions that yielded high adduct levels. When the hemoglobin was separated by reversed-phase high-performance liquid chromatography (HPLC), 65% of the radioactivity copurified with the beta-subunit. Three adducted species were prominent in the ESI mass spectrum of the intact beta-subunit, indicating acrylamide adduction (i.e., mass increase of 71 Da) and two additional unidentified moieties with mass increments of 102 and 135 Da. Endoproteinase Glu-C digestion of the adducted beta-subunit resulted in a peptide mixture that, upon reversed-phase HPLC separation, provided several radiolabeled peptides. Using ESI-MS we identified these as the V91-101 and V102-122 peptides that represent the cysteine-containing peptides of the beta-subunit. These results provide definitive information on acrylamide-modified human hemoglobin and demonstrate that ESI-MS provides valuable structural information on chemically adducted proteins.

The inhibitory effect of 9-amino-1,2,3,4-tetrahydroacridine (THA) on platelet function.

Tetrahydroacridine (THA), or Cognex, is currently awaiting FDA approval for the treatment of Alzheimer's disease. In addition to reports indicating that THA improves the symptoms of patients with Alzheimer's disease, we have found that THA possesses potent antiplatelet activity. THA produced dose-dependent inhibition of human platelet aggregation induced by collagen, ADP, A23187, and phorbol ester. THA, when added to activated platelets, dispersed the platelet aggregates. We have also examined the effects of THA on intracellular Ca++ mobilization, ATP release, and production of cyclic AMP.

Interspecies comparison of cellular localization of the cyanide metabolizing enzyme rhodanese within olfactory mucosa.

The observation of high levels of xenobiotic metabolizing enzyme activity in the olfactory mucosa has produced speculation on the functional significance of these enzymes in the nose. Hypothesized roles include protection of the nasal epithelium, lung, and other downstream tissues, and termination or modification of olfactory responses. The enzyme rhodanese metabolizes cyanide, which is a commonly inhaled toxicant and an odorant and therefore of interest to both toxicologists and olfactory neurobiologists. The cellular localization of this enzyme within the olfactory mucosa will have important consequences for its ability to protect specific cells, as well as its ability to alter the concentration of inhaled cyanide at receptors, and therefore could provide clues as to its function in this tissue. We have compared the distribution of this enzyme in two species, the rat and the cow, using immunohistochemical localization techniques employing species-specific polyclonal antisera raised in our laboratory. In the rat, rhodanese-like immunoreactivity was greatest within the apical portion of the sustentacular cells, the basal cells, and the duct cells of Bowman's glands. Very little to no reaction was observed in the acinar cells of Bowman's glands. In the cow, however, the acinar cells and duct cells of Bowman's glands showed intense immunoreactivity with little to no reaction observed in the sustentacular or basal cells. The differences in localization of rhodanese in these two species may have important implications for cell types at risk during inhalation of cyanide or organonitrile compounds metabolized to cyanide within the nasal mucosa.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombotic/antiplatelet activity of tetramethylpyrazine.

Tetramethylpyrazine (TMPZ) is an active component that can be extracted from certain plants. In China, this herbal extract is used to treat ischemic vascular diseases. We have assessed the antithrombotic activity of TMPZ using arterial and venous thrombosis models in rats. Antithrombotic activity of TMPZ is at least partly due to its potent antiplatelet effect. We have shown that TMPZ inhibits platelet aggregation induced by ADP, collagen, thrombin, and A23187 but not by diacylglycerol (DAG). Platelets that were induced to produce maximal aggregation could be dispersed by the addition of TMPZ. Platelets could then be reaggregated by the addition of DAG. Studies monitoring intracellular ionized calcium suggest that TMPZ inhibits calcium mobilization from both the extracellular medium and from intracellular stores.

Tyrosine- and phenylalanine-restricted formula diet augments immunocompetence in healthy humans.

Previous studies indicate that limiting tyrosine and phenylalanine intake in the diet decreases tumor growth and metastasis. General health status, immune status, and platelet function were studied in nine healthy human subjects consuming low-protein foods supplemented with formula diets free of tyrosine and phenylalanine to maintain total daily intake of tyrosine at 2.4 mg/kg body wt and phenylalanine at 3.5 mg/kg body wt. This regimen decreased plasma tyrosine (p less than 0.05) but not phenylalanine. Blood indicators of protein status were not changed. Platelet aggregation decreased in response to adenosine diphosphate and platelet activating factor in seven of nine subjects. Natural killer, T-helper, and T-cytotoxic/suppressor lymphocyte numbers proportionally increased relative to neutrophils (p less than 0.05). Natural killer cell activity increased in six of nine subjects. Increased natural killer cell activity and decreased platelet aggregation are two indices associated with decreased tumor growth and metastasis.

Augmentation of antimetastatic activity of interferon and tumor necrosis factor by heparin.

Interferon (IFN) and tumor necrosis factor (TNF) suppress the development of experimental metastasis and when used together, TNF and IFN show synergistic activity. However, the use of TNF is limited by its ability to initiate inappropriate hemostasis. Hemostatic effects are exaggerated by the procoagulant activity of certain tumor cell lines. Therapy with anticoagulants are indicated to block the effects of tumor cell products as well as chemotherapeutic side effects. Heparin is a glycosaminoglycan with diverse biological activity, including the ability to rapidly accelerate the inactivation of active clotting factors. The present studies have explored the therapeutic effects of combining heparin with TNF or interferon on experimental metastasis in mice using a melanoma cell line (B16BL6). Our data indicate that continued heparinization augments the antitumor activity of both interferon and TNF. Alterations of the hemostatic and immune systems play a role in the producing the observed effect.

Stereospecific effect of naloxone hydrochloride on cyanide intoxication.

Cyanide intoxication in mice can be antagonized by the opiate antagonist, (-)naloxone HCl, alone or in combination with sodium thiosulfate and/or sodium nitrite. Potency ratios, derived from LD50 values, were compared in groups of mice pretreated with sodium nitrite (sc, 100 mg/kg), sodium thiosulfate (ip, 1 g/kg), and (-)naloxone HCl (sc, 10 mg/kg) either alone or in various combinations. These results indicate that naloxone HCl provides a significant protection against the lethal effects of potassium cyanide. The protective effect of sodium thiosulfate, but not sodium nitrite, was enhanced with (-)naloxone HCl. The combined administration of sodium nitrite and sodium thiosulfate was further enhanced with (-)naloxone HCl. This protective effect of naloxone HCl against the lethal effect of cyanide appears to be restricted to the (-)stereoisomer, as the (+)stereoisomer, the inactive opiate antagonist, is also inactive in protecting against the lethal effects of cyanide. The mechanism of antagonism is discussed.

Encapsulation of thiosulfate: cyanide sulfurtransferase by mouse erythrocytes.

Murine carrier erythrocytes, prepared by hypotonic dialysis, were employed in the encapsulation of several compounds including [14C]sucrose, [3H]inulin, and bovine thiosulfate:cyanide sulfurtransferase (rhodanese), a mitochondrial enzyme which converts cyanide to thiocyanate. Approximately 30% of the added [14C]sucrose, [3H]inulin, and rhodanese was encapsulated by predialyzed erythrocytes, and a decrease in the mean corpuscular volume and mean corpuscular hemoglobin was observed. In the encapsulation of rhodanese a recovery of 95% of the erythrocytes was achieved and an 85% equilibrium was established. The addition of potassium cyanide (50 mM) to intact, rhodanese-loaded erythrocytes containing sodium thiosulfate resulted in its metabolism to thiocyanate. These results establish the potential use of erythrocytes as biodegradable drug carrier in drug antagonism.

Effects of thiosulfate on cyanide pharmacokinetics in dogs.

One method to treat cyanide poisoning involves the administration of a combination of sodium thiosulfate and sodium nitrite. Sodium thiosulfate is believed to exert its antidotal effect by serving as a sulfur donor, thereby increasing the rate of rhodanese catalyzed biotransformation of cyanide to thiocyanate. To gain insight into the mechanism of action of thiosulfate on cyanide toxicity, a pharmacokinetic analysis of cyanide distribution and metabolism with and without sodium thiosulfate was conducted in mongrel dogs. A compartmental model for thiocyanate, the major metabolite of cyanide, was developed from plasma concentrations determined at various times after iv administration of thiocyanate; sodium thiosulfate did not alter thiocyanate-model parameters. The model for thiocyanate was coupled to a model for cyanide, and model based equations were fitted to the blood levels of both cyanide and thiocyanate that were measured after iv administration of cyanide. This kinetic analysis showed that thiosulfate increased the rate of conversion of cyanide to thiocyanate over 30-fold. The mechanism of thiosulfate protection appeared to be due to extremely rapid formation of thiocyanate in the central compartment, which thereby limited the amount of cyanide distribution to sites of toxicity.

Amino acid changes in a genetic strain of epileptic beagle dogs.

A neurochemical evaluation of beagle dogs with naturally occurring spontaneous generalized convulsive seizures was performed. Amino acid profiles of serum, cerebrospinal fluid (CSF), and biopsied cerebral cortex from epileptic dogs were compared with those from seizure-free siblings. No differences in absolute levels were noted. However, when levels were normalized as a percent of total free amino acids, seizures was performed. Amino acid profiles of serum, cerebrospinal fluid (CEF), and biopsied cerebral cortex from epileptic dogs were compared with those seizure-free siblings. No differences also the two groups differed in certain respects. Ten significant correlations between amino acid pairs appeared in epileptic dogs, but only one was seen in seizure-free animals. Seven of these ten correlations involved glutamate or taurine. It was noted that the highly correlated amino acids (taurine, glutamate, glycine, glutamine, alanine) all utilize sodium-dependent membrane transport processes. The sum of glutamate, aspartate, and glycine levels (competing sodium-dependent high-affinity systems) was significantly lower in epileptic beagles. Since this difference was noted in serum but not CSF or brain, it may indicate a diminished capacity of sodium-dependent high-affinity renal transport for acidic and certain small neutral amino acids.

[Gamma-aminobutyric acid system and bioelectric activity of the brain of rats during alcoholic intoxication]. / Sistema gamma-aminomaslianoi kisloty i bioélektricheskaia aktivnost' golovnogo mozga krys pri alkogol'noi intoksikaktsii.

The changes of the GABA metabolizing enzyme activities in rat brain are connected with the phases of the functional condition of the central nervous system and doses of ethanol. Increase in the dose of ethanol caused the distinct decrease of locomotion degree and vertical component of motor activity. Chronic ethanol consumption caused an increase of the enzyme activities of GABA metabolism without any change of the brain GABA level. The brain glutamine concentration and the GAbA-T activity were increased but there was no change in the brain GABA level of rats after 24-hr ethanol withdrawal. Significant increases in mean energy content of EEG occurred throughout the withdrawal period in both dietary groups of rats receiving ethanol. In 24 hr after withdrawal, prodromal and ictal activity were apparent in the spontaneous EEG records of "ethanol" group.