Cleland's reagents block lethal and hypnotic effects of pentobarbital.

We studied the effect of the reducing agents dithiothreitol and dithioerythritol (Cleland's reagents) on the hypnotic and toxic effects of pentobarbital in rodents. In pentobarbital anesthetized rats (50 mg/kg), the i.v. infusion of dithioerythritol (84 mumol/kg/min for 10 min) accelerated the return of eyeblink and righting reflexes (to 4 +/- 1 min vs. 22 +/- 2 min,k and 15 +/- 1 vs. 65 +/- 8 min respectively (P < 0.001). In mice receiving lethal doses of pentobarbital, the 4 h LD50 was 128 +/- 2 mg/kg; when dithioerythritol was given simultaneously, LD50 was > 165 mg/kg. Dithiothreitol also had a protective effect in rats. Oxidized dithiothreitol or dithioerythritol had no effect on pentobarbital sleeping time or mortality. The protective effect of dithioerythritol was dose-dependent; a high dose (294 mg/kg) gave complete protection in the short term, but killed the mice subsequently. The study shows that Cleland's reagents or their derivatives can act as pentobarbital antagonists in rodents, although side effects limit that usefulness. It also suggests caution in the use of dithiothreitol and related compounds during pentobarbital anesthesia.

Protection against doxorubicin toxicity in mice by chlorpromazine hypothermia.

Chlorpromazine increases the sensitivity of cells to doxorubicin; in vivo it also produces hypothermia, which has the contrary effect. To determine the net effect of these factors, we tested the toxicity of doxorubicin in mice which had developed chlorpromazine-induced hypothermia. Groups of 10 female Swiss albino mice received chlorpromazine (5 mg/kg sc). One hour later, when rectal temperature was 30 degrees C, doxorubicin. HCl (20 mg/kg ip) was injected. Median survival time was 10 +/- 2 days for controls and 48 +/- 5 days for chlorpromazine treated mice (4 experiments each). A high chlorpromazine dose (100 mg/kg) did not protect. Chlorpromazine (5 mg/kg) also reversed the slowing of weight gain by 5 mg/kg doxorubicin. If the drop in rectal temperature was prevented by keeping the mice at 32-35 degrees C, the protection by chlorpromazine was abolished. The results show that chlorpromazine protects against the toxicity of ip doxorubicin; the hypothermia produced by chlorpromazine seems to be essential for the protection.

Temperature-dependent ethanol inhibition of [14C]leucine incorporation in mouse heart slices.

The incorporation of [C14]leucine into mouse heart slice proteins was temperature dependent, being maximal at 37 degrees C. Ethanol (200mM) produced an irreversible inactivation of the incorporation which was strongly temperature dependent, being only 8 percent at 34 degrees C, but 66 percent at 42.8 degrees C. The inactivation was proportional to the concentration. At 41.8 degrees C, even 40 mM ethanol produced a significant inhibition of incorporation. Ethanol affected the oxygen consumption of the slices much less than the [C14] leucine incorporation. Both acetone and dimethylsulfoxide produced similar, although less pronounced, effects. It is concluded that ethanol potentiates the heat inactivation of [C14] leucine incorporation into mouse heart slices due to its solvent effect.

Chlorpromazine and dithioerythritol protection against acute ethanol toxicity.

Our previous work has shown that an increase in body temperature increases the acute toxicity of ethanol in mice. To determine whether a decrease in body temperature would have the opposite effect, we studied the effect of two substances that decrease body temperature (chlorpromazine (CPZ) and dithioerythritol (DE)) on ethanol toxicity. Matched groups of 10 mice were injected sc with CPZ (5 mg/kg), DTE (80 mg/kg), or saline (controls). CPZ and DTE significantly depressed the rectal temperature to 32.8 and 34.5 degrees C, respectively. One hour later, all three groups received a specified dose of ethanol (6.5-10.7 g/kg, 24% w/v, intraperitoneal). The experiment was repeated 17 times at different ethanol doses. The pretreatments increased the 1 hr LD50 from 7.8 +/- 0.1 g/kg for the controls to 8.6 +/- 0.2 g/kg (DTE) and 10.0 +/- 0.3 g/kg (CPZ) (p less than 0.001). The protective effect of CPZ was maximal around 5 mg/kg, and less at both lower and higher CPZ doses. When the temperature drop was prevented by directly heating the mice, the protective effect of DTE could be eliminated, but the effect of CPZ was only partially prevented. Placing the CPZ-treated mice in a warmer environment only produced a major reversal of CPZ protection when rectal temperature reached 38 degrees C. In conclusion, CPZ and DTE both decrease body temperature and protect against acute ethanol toxicity. The protection seems to be due at least partially to the decrease in body temperature prior to ethanol injection.

Ambiguous effect of chlorpromazine on doxorubicin activity against P388D1 tumours in mice.

Chlorpromazine (CPZ) can decrease the toxicity of doxorubicin (DOX). We wanted to determine whether this CPZ pretreatment could affect the response of tumours to therapeutic doses of DOX. Six groups of eight female CDF1 mice received 1 million leukaemia P388D1 cells i.p. For 5 days, they received DOX i.p. (total dose 0, 6 or 12 mg/kg), preceded by saline or 5 mg/kg CPZ s.c. CPZ in the absence of DOX had no effect on survival [median survival time (MST) of 19 days vs. 20]. In mice receiving DOX only, MST was greater than 60 days. Mice receiving DOX + CPZ were similar to DOX till day 25, but subsequently died earlier (MST of 27 and 34 days, for DOX 6 and 12 mg/kg). At death or day 60, 31% (5/16) of DOX mice and 88% (14/16) of DOX + CPZ had macroscopic tumours (P less than 0.005, both DOX dose groups combined). However, only 19% (3/16) of DOX and 6% (1/16) of DOX + CPZ had tumours in the abdominal cavity, the others being in the abdominal wall close to the site of injection. The results suggests that while CPZ did not affect the killing of cancer cells in the abdominal cavity, it did block the effect of DOX on cells in the abdominal wall and skin. This block may be caused by decreased local delivery of DOX, due to the hypothermia produced by CPZ.

Similarity of acetaldehyde and sulfhydryl reagent actions on the perfused guinea pig heart.

The effects of acetaldehyde (1 mM) and two known sulfhydryl reagents, diamide (0.25 mM) and cystamine (0.5 mM), on the perfused guinea pig heart were compared and were found to be markedly similar. All compounds increased heart rate and coronary flow. Dichloroisoproterenol blocked the increase in heart rate but not the increase in coronary flow. It is concluded that acetaldehyde may produce its effects by reaction with cellular thiols.

The effect of sulfhydryl reagents on the heart rate and coronary flow of the isolated perfused guinea-pig heart.

The action of a number of compounds able to react with thiols was tested on guinea-pig hearts perfused at constant pressure. The SH reagents used were NaNO2, oxidized glutathione, cystamine, diamide, 1,5-difluoro-2,4-dinitrobenzene, nitroglycerol, sodium nitroferricyanide and HgCl2. 6,6'-Dithiodinicotinic acid, an SH reagent that does not penetrate the cell, produced no effect. All the other SH reagents produced an increase in coronary flow. All except oxidized glutathione and nitroglycerol increased the heart rate. The increase in heart rate and oxygen consumption could be completely blocked by dichloroisoproterenol; the increase in coronary flow was not affected. Difluorodinitrobenzene, diamide, cystamine and NaNO2 significantly decreased the acid-soluble thiol content of the heart. For these compounds, there was a significant correlation between the decrease in coronary flow and the decrease in thiols. We conclude that in the isolated heart, most SH reagents, if used at the appropriate concentration, will increase the heart rate, probably by relaasing catecholamines. They will also decrease the coronary resistance, probably by a direct effect on the coronary vessels.

Assessment of myocardial free fatty acid metabolism in humans during heparin infusion.

Free fatty acids (FFA) have been shown to be a major myocardial substrates during the postabsorptive state in humans. In order to document the utilization of these substrates by the heart while heparin was infused, the following study was designed. During heparin infusion, total and individual FFA were measured in arterial and coronary sinus plasma during the postabsorptive state in 13 patients with coronary artery disease and in 4 patients with no evidence of cardiac disease. Oleic-1-14C acid bound to albumin was infused at a constant rate for at least 7 min into the left coronary artery. Left coronary blood flow was determined in all patients. It was assumed the oleic-1-14C acid behaves like endogenous FFA. Arterial FFA concentration was high (mean and SEM: 1 +/- 0.21 mumoles/ml) and varied by less than 10% during the course of the study in 14 of the 17 patients. Oleic acid constituted 44% of all arterial FFA. The relative concentrations of the various individual FFA were similar in arterial and coronary sinus plasma. The average FFA extraction was 19.9 +/- 2.1%. In contrast, the oleic-1-14C acid extraction was relatively more steady during the infusion, and the mean extraction of this tracer was 36.0 +/- 2.1%. Myocardial uptake for the entire group was 52.3 +/- 6.3 mumoles/min. Although a definite plateau for 14CO2 release from oleic-1-14C acid oxidation was not obtained, at least 31.8 +/- 4.3% of the removed FFA was oxidized, representing 49.3 +/- 6.4% of myocardial oxygen consumption. The failure of oleic-1-14C acid oxidation to reach a steady state was not due to delay in the myocardial CO2 pool, since 14CO2 release from infused NaH 14CO3 and lactate-1-14C in 11 other subjects revealed that in equilibrium was obtained within 5 min. These findings indicate that the generation of 14CO2 from oleic-1-14C acid oxidation is relatively slow, and the uptake of labeled FFA is partly matched by release of unlabeled FFA, suggesting that the human heart has important lipid pools.

Reversible elimination of myofibrillar Ca2+ sensitivity by diamide and other sulfhydryl reagents: comparison with reversible contracture produced in intact cells.

Cardiac contractile activity is usually controlled by intracellular Ca2+, but it can also be modified by oxidizing agents. Incubation of guinea pig heart myofibrils with diamide (3 mM, 1 h) increased basal (no Ca2+) ATPase activity by 580% and abolished Ca2+ dependence. The effect was proportional to diamide concentration (0.01-1 mM) and duration of preincubation (up to 2 h). Dithiothreitol (5 mM, 1 h) reversed most of the basal ATPase activation and restored Ca2+ sensitivity. Other sulfhydryl reagents produced a similar effect but also produced inhibition of total ATPase. In intact cell preparations, diamide produced a slow tonic contraction, consistent with myofibril activation. In the perfused rat heart, 1 mM diamide slowly increased diastolic ventricular pressure; this increase was partially reversed by dithioerythritol. In isolated rat heart myocytes, 1 mM diamide produced a slow tonic contraction, increased contractility in response to stimulation. Cardiocytes superfused for 1 h with buffer containing EGTA to deplete Ca2+ did not contract in response to stimulation but showed a slow tonic contraction with diamide. This contraction could be slowly and only partially reversed by dithioerythritol. Response to stimulation was restored by addition of Ca2+. The results show that diamide can produce contraction in viable cells. This contraction does not require extracellular Ca2+ and is unlikely to involve intracellular Ca2+. The direct activation of myofibrillar ATPase may contribute to the increased myocardial stiffness seen in ischemia and to ischemic contracture.

A comparison of fatty acid patterns of arterial plasma, pericardial fluid and cardiac lymph in dog.

The fatty acid composition of cardiac lymph and pericardial fluid in anesthetized open-chest dogs, was analysed and compared with that of arterial plasma. The fatty acid content of triglyceride (TG) and total fatty acid fractions of pericardial fluid and cardiac lymph was significantly lower than that of plasma. On the other hand, free fatty acid (FFA) concentrations of pericardial fluid, cardiac lymph and plasma were not significantly different. The FFA fraction of cardiac lymph contained relatively more stearic acid and less oleic acid whereas, in plasma, the FFA fraction contains less myristic acid. The data are consistent with the hypothesis that endothelial permeability is a limiting factor for the transvascular exchanges of large molecules such as TG and that the FFA distribution in intersititial spaces may have been modified by the contribution of fatty acids from endogenous cardiac reserves or from the hydrolysis of plasma TG.

Effects of chronic administration of delta 9-transtetrahydrocannabinol (delta9-THC) in guinea-pigs.

The guinea-pigs were divided into three groups: (1) absolute control, (2) solvent control (tween 4%), and (3) delta9-THC group (3 mg/kg). The selected dose of delta9-THC corresponds to the minim amount producing physiological effects in acute administration and was given for six months at the rate of five injections/week. The results showed that THC produced no changes on these parameters: serum glucose, urea nitrogen, total proteins, Mg, Ca, Na, and K. However, the fatty acids and alpha1 globulin were significantly decreased. There was a significant increase in gamma globulin. The body weight gain of delta9-THC treated animals was lower than of the two controls. Delta9-THC decreased the relative weight of liver and spleen; however, it did not significantly affect the relative weight of heart, adrenals, and kidneys. Similarly, the morphological examinations showed no alteration in these tissues, except in the liver tissue, where a perturbation of the autodigestion of glycogen was noted. These observations suggest that the toxic effect of the drug is caused by its accumulation in the liver, which provokes an inhibition of certain liver enzymatic systems.

Ethanol delays and reverses lysophosphatidylcholine-induced calcium overload in neonatal rat heart cells.

Increased lysophosphatidylcholine (LPC) production by the ischemic heart is associated with tissue damage. In vitro, LPC produces an increase in cytosolic [Ca2+], usually followed by cell contracture and lysis. Since ethanol reportedly protect cells during ischemia-reperfusion, we wished to determine whether ethanol could protect heart cells against LPC-induced Ca2+ overload. Newborn rat heart cells in culture were loaded with Fura-2 and [Ca2+]i recorded in individual cells. The presence of 22 or 44 mM ethanol increased the time required for 10 microM x L-palmitoyl-LPC to produce maximal Ca2+ accumulation from 8.4+/-0.4 min (n=47) to 21.1+/-2.1 x min (n=32; P<0.01) and 23.8+/-1.8 min (n=10; P<0.01) respectively. The onset of the [Ca2+]i increase could be reversed partially by the addition of ethanol (44 or 88 mM). After the addition of 22 mM ethanol, the cells retained the Fura-2 three times longer than under control conditions. Ethanol (88 mM) decreased the critical micelle concentration of LPC, thus decreasing the LPC monomer concentration in this solution. La3+ also protected the cells against LPC but no further protection was afforded by the addition of ethanol. Our results suggest that ethanol concentrations commonly found in the blood of social drinkers protect heart cells against the deleterious effect of LPC.

Cardiovascular defects among the progeny of mouse phenylketonuria females.

In a genetic mouse model of human phenylketonuria we have examined the offspring of hyperphenylalaninemic mothers for the presence of cardiovascular defects, an important feature of the pathology of the human maternal phenylketonuria syndrome. Beginning at 14.5 d after conception (75% through gestation), a variety of cardiovascular defects became apparent among the progeny of the hyperphenylalaninemic females. These defects ranged from mild to serious and correlated with the maternal but not the fetal Pah genotype. Nearly all of the defects were vascular, however, whereas the most reported in humans so far have been cardiac. The predisposing biochemical condition in this mouse disease model seems to be the same as in the human disease; elevated maternal blood phenylalanine levels concentrated across the placental barrier to produce a teratogenic developmental environment. This model for congenital cardiovascular defects should enhance two related areas of research. 1) It should allow a more thorough investigation of the relationship between maternal diet and maternal phenylketonuria birth defects, and 2) it should provide an experimental tool to gain insight into the normal process of cardiovascular development.

Role of cardiac lymph and interstitial fluid in lipid metabolism of canine heart.

To determine whether cardiac interstitial spaces participate in cardiac fatty acid pool, the relationship between cardiac lymph and arterial plasma free palmitate and triglycerides was studied in anesthetized dogs [14C]Sucrose, infused at a constant rate in a femoral vein, appeared in the lymph at 90% of its arterial concentration within 60 min. On the other hand, when [1-14C]palmitate was infused at the same rate and at the same site, the ratio of lymph to arterial plasma 14C-labelled free fatty acids (FFA) was only 21% at 60 min and 25% at 120 min, even though the concentrations of endogenous FFA in lymph and arterial plasma were the same. The ratio reached 90% only 24 h after a bolus injection of [3H]palmitate. [1-14C]Palmitate in the lymph triglyceride fraction was only 8% of that in plasma. Although the lymph composition may be influenced by the metabolism of heart muscle, cardiac adipose tissue, and serum lipoproteins, these results indicate the presence of a pool of myocardial fatty acids which may be partly located in the interstitial spaces.

Free fatty acid content of myocardial interstitial spaces of dog.

Morphological and biochemical observations from our laboratory have shown the presence of lipids in the cardiac interstitial spaces of the dog. The present study was designed to assess the importance of free fatty acids (FFA) in these lipids using FFA or sucrose tracers in 14 anesthetized fasting open-chest mongrel dogs. Endogenous FFA and labeled tracers were measured in arterial and coronary sinus plasma; they were also determined in lymph collected from cardiac efferent lymphatic trunks. [14C] sucrose was infused at a constant rate in the femoral vein of 5 dogs. The concentration of the tracer in the lymph was 90% of the arterial concentration after 60 min of infusion. On the other hand, when [1-14C] palmitate was infused at the same rate in 7 dogs, the ratio of lymph to arterial tracer concentration was only 20% (60 min) and 25% (120 min), even though the myocardial extraction and oxidation of the tracer were stable. This ratio tended to reach a plateau (greater than or equal to 90%) only 24 hr after a bolus injection of the tracer. This tracer study shows the presence of a pool of myocardial fatty acids with a relatively slow turnover rate that may constitute an important reservoir of FFA.