Raised vascular calcium in an animal model: effects on aortic function.

STUDY OBJECTIVE - The aim of the study was to develop an animal model to study the relationships between raised tissue calcium and vascular function. DESIGN - Ectopic calcification was developed in the animal model using chronic vitamin D2 intoxication, after which functional studies were performed in isolated superfused aortic rings. Results were compared with control preparations. EXPERIMENTAL ANIMALS - 160 female Sprague-Dawley rats weighing 200-225 g were randomly divided into experimental (vitamin D2 1 mg.d-1) and control (vehicle only) groups. MEASUREMENTS and RESULTS - Aortas from vitamin D treated animals had a higher calcium content than control aortas, without concomitant increases in cardiac calcium. Aortas with high calcium content were found to develop greater tension than control aortas when exposed to noradrenaline in the absence of extracellular calcium, but the tension maxima achieved in response to noradrenaline in calcium containing media or to high potassium depolarising solution were the same. The rate of development of contraction in response to noradrenaline was greater in aortas from the vitamin D treated animals than in controls. Isoprenaline and sodium nitroprusside produced less relaxation in the animal model aortas than in the controls. CONCLUSIONS - The results suggest that increased aortic calcium affects the response of the tissue to vasoactive agents. It appears that the additional vascular calcium may be stored in an agonist releasable pool, probably within the sarcoplasmic reticulum. The enlarged or newly developed pools appear to be refillable from the extracellular medium but not by intracellular reuptake of calcium, suggesting a bicompartmental model of intracellular calcium release and reuptake.

Antagonism of the lethal effects of cyanide with resealed erythrocytes containing rhodanese and thiosulfate.

A new concept has been presented for the antagonism of cyanide and possibly other chemical toxicants. Until now, only a half dozen truly specific "antidotes" were known. There are many other "antidotes" which merely prevent the absorption or enhance the elimination of a toxic compound rather than specifically destroying the substance to prevent its toxic effect. This new approach has considerable conceptual significance in toxicology, as it suggests the encapsulating other enzymes to degrade various other chemical toxicants. There are many chemical toxicants for which there are no specific antidotes, and the conceptual approach of employing erythrocyte-encapsulated enzyme provides an innovative, specific approach to antagonize the toxic and lethal effects of these chemicals.

Rhodanese and sodium thiosulfate encapsulated in mouse carrier erythrocytes. II. In vivo survivability and alterations in physiologic and morphologic characteristics.

Biodegradable drug carrier mechanisms were employed in drug antagonism studies. Prior studies indicated that erythrocytes containing encapsulated rhodanese and sodium thiosulfate metabolized cyanide to thiocyanate in vitro. Studies were conducted to investigate the properties of these sulfurtransferase-loaded red blood cells in vivo by administering the carrier red blood cells intravenously. Approximately 40 to 50% of the cells were eliminated within the first few hours while the remaining loaded erythrocytes persisted in the circulation. The present studies were initiated to investigate the characteristics of the disposition of the loaded erythrocytes and to examine differences in the properties between carrier and noncarrier erythrocytes. Also, the disposition and viability of the erythrocytes in vivo were studied with relation to various biochemical, physiological, and morphological properties. These studies indicated that the carrier erythrocytes had a smaller cell volume and were more susceptible to hemolysis than normal erythrocytes. Morphologic studies by electron microscopy indicated that extensive morphologic changes occurred during the procedures after hypotonic dialysis, isotonicity adjustment, and resealing were completed. Differences were noted between those cells that were only resealed and those cells that were also subjected to annealing. The morphologic characteristics of most of the cells were restored to the "normal" morphologic appearance only after annealing. Annealed erythrocytes' in vivo survivability was correlated with the physical properties of these cells.

In vivo studies on rhodanese encapsulation in mouse carrier erythrocytes.

Resealed erythrocytes containing sodium thiosulfate and rhodanese (CRBC) are being employed as a new approach in the antagonism of cyanide intoxication. In earlier in vitro studies, the behavior of red blood cells containing rhodanese and sodium thiosulfate was investigated with regard to their properties and their capability of metabolizing cyanide to thiocyanate. The present studies are concerned with the properties of these rhodanese-containing carrier erythrocytes in the intact animal. These carrier erythrocytes were administered intravenously and the survival of the encapsulated enzyme was compared with the administration (iv) of free exogenous enzyme. Also, the amount of leakage of the encapsulated rhodanese from the red blood cell was determined. The survival of the carrier red blood cell. prepared by hypotonic dialysis, was found to be characterized by a biphasic curve. There was an initial rapid loss of approximately 40 to 50% of the carrier cells with a t1/2 = 2.5 hr. Subsequently the remaining resealed annealed carrier erythrocytes persisted in the vascular system with a t1/2 = 8.5 days. When free exogenous rhodanese was administered directly into the vascular system, it was rapidly eliminated with a t1/2 = 53 min. Red blood cells containing sodium thiosulfate and rhodanese apparently are effective in vivo in the biotransformation of cyanide. In animals pretreated with encapsulated rhodanese and sodium thiosulfate, blood cyanide concentrations are appreciably decreased with a concomitant increase in thiocyanate ion, a metabolite of cyanide. When erythrocytes, which contained no rhodanese or sodium thiosulfate, were subjected to hypotonic dialysis, cyanide was not metabolized to any appreciable extent. Furthermore, carrier erythrocytes containing rhodanese and sodium thiosulfate were found to increase the protection against the lethal effects of cyanide by approximately twofold. The ability of these carrier erythrocytes alone to metabolize cyanide and to antagonize the lethal effects of cyanide reflects the potential of this new antidotal approach in the antagonism of chemical toxicants.

Antagonism of cyanide intoxication with murine carrier erythrocytes containing bovine rhodanese and sodium thiosulfate.

Murine carrier erythrocytes containing bovine rhodanese and sodium thiosulfate are being explored as a new approach to antagonize the lethal effects of potassium cyanide in mice. Prior studies indicated that these carrier erythrocytes persist in the vascular system for the same length of time as normal erythrocytes and can enhance metabolism of cyanide to thiocyanate. The present studies demonstrate the ability of these carrier red blood cells containing rhodanese and thiosulfate to antagonize the lethal effects of cyanide either alone or in various combinations with sodium nitrite and/or sodium thiosulfate. Potency ratios are compared in groups of mice treated with sodium nitrite, sodium thiosulfate, and carrier erythrocytes containing rhodanese and sodium thiosulfate either alone or in various combinations prior to the administration of potassium cyanide. These results indicate that the administration of carrier erythrocytes containing rhodanese and thiosulfate alone can provide significant protection against the lethal effects of cyanide. These carrier erythrocytes potentiate the antidotal effect of sodium thiosulfate alone or the combination of sodium nitrite and sodium thiosulfate. The mechanisms of cyanide antagonism by these carrier erythrocytes and their broader conceptual significance to the antagonism of other chemical toxicants are discussed.

Encapsulation of rhodanese and organic thiosulfonates by mouse erythrocytes.

A series of organic thiosulfonates were synthesized and studied as sulfur donor substrates for rhodanese encapsulated within murine carrier erythrocytes. Previous studies have indicated that resealed erythrocytes containing rhodanese (CRBC) and sodium thiosulfate can rapidly metabolize cyanide to the less toxic thiocyanate. This thiosulfate-rhodanese system was very efficacious as a new conceptual approach to antagonize cyanide intoxication both in vitro and in vivo. However, its potential is restricted because of the limited availability of thiosulfate due to its poor permeability through RBC membrane. Present studies suggest that there are advantages in using alternative sulfur donors, i.e., organic thiosulfonates in this rhodanese-containing resealed erythrocyte system, since these compounds have higher lipid solubility than inorganic thiosulfates and can readily penetrate the red blood cell membrane. Therefore, this system could provide a virtually unlimited amount of sulfur donor to the encapsulated rhodanese even if the substrates are in solution outside the cells. Moreover, the rhodanese reaction rate of any of these organic thiosulfonates is much faster than the rate observed with the classic cyanide antidote, sodium thiosulfate. This CRBC system will continue to detoxify cyanide even when these encapsulated sulfur donors are depleted, as the lipid soluble organic thiosulfonate outside the cells will diffuse past the membrane into the cell to replenish the sulfur donor. The encapsulation efficiency for rhodanese is about 30%, and the velocity of the rhodanese reaction increases linearly with the volume of enzyme-laden erythrocytes. Similarly, reaction velocity increases linearly with substrate concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Antagonism of the lethal effects of cyanide by a synthetic water-soluble cobalt(III) porphyrin compound.

Efficacy of hydroxocobalamin (vitamin B12) as a cyanide antidote is limited by its high molecular weight (1355 g/mol) and by the competitive binding of the cobalamin dimethylbenzimidazole. The present study describes experiments with a lower molecular weight cobalt porphyrin that has a high affinity for cyanide, Co(III)-5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrin (CoTPPS), which was prepared by the method of Herrmann et al. (1978). CoTPPS was synthesized and its efficacy as an antidote to the lethal effects of cyanide either alone or in various combinations with NaNO2 and/or Na2S2O3 was determined. The LD50 value for CoTPPS was found to be 334 mg/kg. These studies were conducted using the CoTPPS LD01, 200 mg/kg. The cyanide antagonists NaNO2 (0.1 g/kg, sc), Na2S2O3 (1.0 g/kg, ip), and CoTPPS (0.2 g/kg, ip) were administered at 45, 15, and 10 min respectively prior to graded doses of KCN (sc). The LD50 values for KCN in male Swiss-Webster mice were calculated by probit analysis at the 95% confidence level and the various treatments were compared by potency ratios. These results indicated that the administration of CoTPPS alone protects against the lethal effects of cyanide. Moreover, CoTPPS adds to the protection provided by Na2S2O3 and/or NaNO2. Efficacy of this antidote is probably related to the binding equilibrium between CoTPPS and cyanide.

The encapsulation of squid diisopropylphosphorofluoridate-hydrolyzing enzyme within mouse erythrocytes.

This study describes the entrapment of squid-type diisopropylphosphorofluoridate-hydrolyzing enzyme (DFPase) within mouse red blood cells. These erythrocytes thereby gain the ability to rapidly hydrolyze alkylphosphate cholinesterase (ChE) inhibitors such as diisopropyl fluorophosphate (DFP). DFPase rapidly hydrolyzes DFP to diisopropyl phosphate. Resealed erythrocytes provide a stable carrier system that can preserve the activity of encapsulated enzymes against otherwise rapid in vivo degradation; thus, ChE inhibitors can be degraded to relatively nontoxic metabolites by these erythrocyte carriers. Squid DFPase was purified from the hepatopancreas of Atlantic squid and DFPase activity was determined by measuring changes in fluoride ion concentration using a fluoride ion selective electrode. Mouse erythrocytes in suspension with excess squid DFPase were dialyzed against hypotonic buffer to allow the encapsulation of the enzyme to occur. Cells were then resealed by returning the suspension to isosmotic with saline. Rate of DFP hydrolysis observed with these cells was much greater than the rate of nonenzymatic hydrolysis and was directly proportional to the amount of the erythrocyte suspension added to the assay solution. The rate of hydrolysis was first order in substrate. Erythrocyte controls showed no endogenous DFPase activity. These results suggest that enzyme entrapment may be developed as a method to prevent and antagonize organophosphate poisoning.

Cyanide antagonism with carrier erythrocytes and organic thiosulfonates.

Previous studies reported that resealed erythrocytes containing rhodanese (CRBC) and NA2S2O3 rapidly metabolize cyanide to the less toxic thiocyanate both in vitro and in vivo. This provided a new conceptual approach to prevent and treat cyanide intoxication. Although the rhodanese-containing carrier cells with thiosulfate as the sulfur donor were efficacious, this approach has potential disadvantages, as thiosulfate has limited penetration of cell membrane and product inhibition of rhodanese can occur due to inorganic sulfite accumulation. In order to circumvent substrate limitation and product inhibition by sodium thiosulfate, organic thiosulfonates were explored. These thiosulfonates have higher lipid solubility than thiosulfate and therefore can replenish the depleted sulfur donor, as they can readily penetrate cell membranes. Also, product inhibition of rhodanese is less apt to occur. This change in sulfur donors should greatly enhance cyanide detoxication, replenish the sulfur donor, and minimize product inhibition of rhodanese. Present studies demonstrate the enhanced efficacy of exogenous organic thiosulfonates over sodium thiosulfate in the CRBC antidotal system to detoxify the lethal effects of cyanide either alone or in combinations with exogenously administered NaNO2. Murine carrier erythrocytes containing purified bovine liver rhodanese were administered intravenously into male Balb/C mice. Subsequently, butanethiosulfonate (BTS) or Na2S2O3 (ip), and NaNO2 (sc) were co-administered prior to KCN (sc). Potency ratios, derived from the LD50 values, were compared in groups of mice treated with CRBC-Na2S2O3 or CRBC-BTS either alone or in combination with NaNO2.(ABSTRACT TRUNCATED AT 250 WORDS)

GT160-246, a toxin binding polymer for treatment of Clostridium difficile colitis.

GT160-246, a high-molecular-weight soluble anionic polymer, was tested in vitro and in vivo for neutralization of Clostridium difficile toxin A and B activities. Five milligrams of GT160-246 per ml neutralized toxin-mediated inhibition of protein synthesis in Vero cells induced by 5 ng of toxin A per ml or 1.25 ng of toxin B per ml. In ligated rat ileal loops, 1 mg of GT160-246 neutralized fluid accumulation caused by 5 microg of toxin A. At doses as high as 80 mg/loop, cholestyramine provided incomplete neutralization of fluid accumulation caused by 5 microg of toxin A. GT160-246 protected 80% of the hamsters from mortality caused by infection with C. difficile, whereas cholestyramine protected only 10% of animals. Treatment of C. difficile-infected hamsters with metronidazole initially protected 100% of the hamsters from mortality, but upon removal of treatment, 80% of the hamsters had relapses and died. In contrast, removal of GT160-246 treatment did not result in disease relapse in the hamsters. GT160-246 showed no antimicrobial activity in tests with a panel of 16 aerobic bacteria and yeast and 22 anaerobic bacteria and did not interfere with the in vitro activities of most antibiotics. GT160-246 offers a novel, nonantimicrobial treatment of C. difficile disease in humans.