Effect of adrenochrome on calcium accumulation by heart mitochondria

The effects of adrenochrome (1–100 μg/ml or 5.6 × 10^?6 to 5.6 × 10^?4 M) on calcium binding, calcium uptake, and ATPase activities of rat heart mitochondria were investigated. Depression, by adrenochrome, of mitochondrial calcium binding and uptake was observed under in vitro conditions, whereas mitochondrial ATPase activity was not altered appreciably. The inhibitory effect of adrenochrome on calcium uptake activity was independent of the pH of the incubation medium, but it was dependent upon the dose of drug and the time of incubation. High concentrations of calcium in the incubation medium antagonized the adrenochrome-induced depression. The inhibition of mitochondrial calcium uptake by adrenochrome was of a mixed type. Furthermore, the depressed calcium uptake activity was observed after washing adrenochrome-treated mitochondria with buffer. Significant decreases in calcium accumulating activities were also seen in mitochondria isolated from hearts perfused with various concentrations of adrenochrome (5–50 μg/ml) for 10 min, or with 50 μg/ml adrenochrome for various time periods. Contractile force of the perfused rat heart with various concentrations of adrenochrome (5–50, μg/ml) decreased in a dose-dependent manner. It is suggested that the cardiac contractile failure and myocardial cell necrosis induced by adrenochrome may partly be due to its inhibitory effect on the calcium accumulating ability of mitochondria.     

Influence of some divalent cations on heart sarcolemmal bound enzymes and calcium binding.

The actions of Ni²⁺, Co²⁺ and Mn²⁺ on the rabbit heart sarcolemmal ATPases, calcium binding, and adenylate cyclase activities were studied. The ability of sarcolemma to hydrolyze ATP was stimulated by 0.1–4 mM concentrations of Ca²⁺, Mg²⁺, Co²⁺, Ni²⁺ and Mn²⁺. The sarcolemmal Ca²⁺ ATPase (22.8 if $\text{μmoles P}{}_{\mathrm{i}}\text{mg}$ of $\text{protein}\text{hr}$) and Mg²⁺ ATPase (21.6 $\text{μmoles P}{}_{\mathrm{i}}\text{mg}$ of $\text{protein}\text{hr}$ were depressed by 0.25–4 mM-Co²⁺, Ni²⁺ and Mn²⁺, and the order of their potency was Ni²⁺ > Co²⁺ > Mn²⁺. The sarcolemmal Na⁺-K ⁺ ATPase activity (9.4 $\text{μmoles P}{}_{\mathrm{i}}\text{mg}$ of $\text{protein}\text{hr}$) was decreased by 0.10–4 mM concentrations of Co²⁺, Ni²⁺ and Mn²⁺. The sarcolemmal calcium binding in the presence of 0.1 mM Ca²⁺ (98 $\text{nmoles}\text{mg}$ of $\text{protein}\text{5 min}$) was depressed by 0.25 mM or higher concentrations of Co²⁺, Ni²⁺ and Mn²⁺, whereas that in the presence of 1.25 mM-Ca²⁺ (772 $\text{nmoles}\text{mg}$ of $\text{protein}\text{5 min}$) was decreased by 2–4 mM-Co²⁺, Ni²⁺ and Mn²⁺. The sarcolemmal adenylate cyclase activities in the absence (124 pmoles cyclic $\text{AMP}\text{mg}$ of $\text{protein}\text{min}$) and presence of 2 mM-NaFI517 pmoles cyclic $\text{AMP}\text{mg}$ of $\text{protein}\text{min}$) were decreased by 0.1–4 mM-Co²⁺ or Ni²⁺ and stimulated by 0.1–4 mM-Mn²⁺. The contractile force of the isolated rabbit heart was decreased by varying degrees by 0.1–1 mM of divalent cations (Ni²⁺ > Co²⁺ > Mn²⁺). These results indicate sarcolemma is one of the sites involved in the cardiodepressant actions of Ni²⁺, Co²⁺ and Mn²⁺.     

Effects of free fatty acids, ethanol and development on gamma-aminobutyric acid and glutamate fluxes in rat nerve endings

The effects of type A (cis-unsaturated) and type B (trans-unsaturated and saturated) fatty acids, 1% and 3% ethanol (v/v), and development (7 days) on the thermodynamics of glutamate and gamma-aminobutyric acid (GABA) transport into cortical rat brain nerve endings were examined. The effects of the various manipulations, which are known to affect membrane fluidity, may be summarized. Three percent ethanol and oleic acid increased delta S degrees and delta S+ for glutamate transport and decreased delta H degrees and delta H+. Type B fatty acids had the opposite effects. In comparison to glutamate transport, GABA transport was less affected by the various manipulations and showed less specificity in terms of the fatty acid effects. Similarly, the effects of development on the thermodynamic parameters for glutamate and GABA transport were not consistent. Glutamate transport into 7-day nerve endings showed thermodynamic behavior similar to that seen when type A fatty acids were incorporated into adult nerve endings. In contrast, GABA transport into 7-day nerve endings had the character of adult nerve endings into which type B fatty acids were incorporated.     

Involvement of 5-hydroxytryptamine in the central control of respiration, blood pressure and heart rate in the anaesthetized rat.

5-Hydroxytryptamine (5-HT, 1 ng-25 μg) injected into the cerebral ventricles of urethananaesthetized rats produced a rise in blood pressure, a biphasic change in heart rate and a decrease in ventilation. Responses were largest after injections into the 3rd ventricle. Lateral ventricle injections produced smaller responses while 4th ventricle injections produced the least response. The cardiovascular responses were prevented or reduced by prior intraventricular injection of bromolysergic acid diethylamide. The pressor response was not blocked by hexamethonium (10 mg/kg i.v.) and was only partly reduced by pretreatment with either 6-hydroxydopamine (3 × 100 mg/kg i.v.) or atropine methonitrate (10 mg/kg i.v.). The pressor response was prevented by spinal transection at C1 or C3 but only slightly reduced by transection at C5 or C7. In animals curarized and artificially respired with air, the blood pressure response was reduced. It is conjectured that the rise in blood pressure may involve a direct effect of hypoxia or hypercapnia resulting from a decrease in respiratory activity which is in turn mediated by a direct action of 5-HT on structures lining the 3rd ventricles. There appears to be little or no involvement of the autonomic nervous system in the response. The fall in heart rate is mediated sympathetically.     

Isopropanol enhancement of cytochrome P-450-dependent monooxygenase activities and its effects on carbon tetrachloride intoxication

Acute or chronic treatment of rats with isopropanol caused a significant increase in hepatic cytochrome P-450 content and a two- to threefold increase in aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities, but no significant change in ethylmorphine N-demethylase or benzo(a)pyrene hydroxylase activity. In rats treated with isopropanol and challenged with CCl4, liver toxicity of CCl4 was characteristically potentiated, as assessed by elevation of serum glutamic-pyruvic transaminase (SGPT) levels. Isopropanol pretreatment also potentiated CCl4-induced damage to the hepatic monooxygenase system. In addition to a decrease in cytochrome P-450, rats treated with isopropanol and challenged with CCl4 showed a nonspecific decrease not only in aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities, but also in ethylmorphine N-demethylase, benzo(a)pyrene hydroxylase, and NADPH-cytochrome c reductase activities. These results were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized microsomes. The electrophoretic results showed that isopropanol pretreatment markedly potentiated the CCl4-caused destruction of cytochrome P-450 hemeproteins. The data strongly suggest that isopropanol increases one or more forms of cytochrome P-450 which selectively enhance the metabolism of CCl4 to an active metabolite. This active metabolite then causes a nonselective damage to the microsomal mixed-function oxidase system.     

Effects of trimethyltin on homecage behavior of rats

Diurnal patterns of feeding, drinking, locomotor activity, and rearing in male Fischer-344 rats were examined for 2 weeks after a single oral dose of trimethyltin chloride (TMT) at 0, 3, 5, or 7 mg/kg. Body weights and feeding and drinking efficiency ratios (ratios of amount of food or water consumed per unit effort) were also determined daily. TMT caused a dose- and time-related drop in body weight; two of five rats in the 7 mg/kg group were killed moribund on 15 days after dosing. Feed consumption fell to 25% of control within 5 days after 7 mg/kg TMT, and to 50% of control for Days 2 and 3 after 5 mg/kg TMT. Water consumption doubled within 2 days after 7 mg/kg TMT and remained elevated for 2 weeks. Feeding efficiency dropped to 40% of control after 7 mg/kg, but drinking efficiency was unchanged. The diurnal patterns of drinking and of rearing were disrupted at all doses of TMT; a normal peak in rearing activity, occurring immediately prior to light onset, was markedly attenuated after all doses on Day 3, and at 5 and 7 mg/kg on Days 5 and 7 post-TMT. These results suggest (1) that the regulation of feed and water intake is severely compromised after a high dose of TMT, and (2) that the rat's cyclical patterns of homecage behavior are sensitive to TMT doses as low as 3 mg/kg.     

Cytotoxicity of isoproterenol to cultured heart cells: effects of antioxidants on modifying membrane damage

Primary cultures of rat myocytes were used to study the cardiac damage induced by toxic doses of L-isoproterenol (ISO). Cultures were exposed to varying concentrations of ISO (2.4 X 10(-5), 1 X 10(-4), and 5 X 10(-4) M) for 0.5, 1.5, 4, and 12 hr. Mitochondrial membrane fragility, myocyte potassium content (as an index of sarcolemmal damage), and cellular glutathione content were used to evaluate cellular injury. A significant increase in mitochondrial fragility was observed 0.5 hr after treatment with 5 X 10(-4) M ISO. Lower doses caused an increase in mitochondrial fragility 1.5 hr after exposure. Longer durations of ISO exposure (4 and 12 hr) were necessary to decrease cellular potassium content. Glutathione levels were minimally affected by ISO. L-Ascorbic acid (5 X 10(-3) M) or sodium bisulfite (9.6 X 10(-4) M) were added to the cultures to determine if antioxidants prevent the toxicity caused by ISO. The presence of L-ascorbic acid or sodium bisulfite in ISO-treated myocyte cultures prevented the toxic changes in mitochondrial fragility and myocyte potassium content. The data indicate that antioxidants may be useful in reducing injury induced by toxic doses of ISO. Furthermore, mitochondrial injury may be involved significantly with the development of ISO-induced cardiotoxicity.     

Effect of phenobarbital on cephaloridine toxicity and accumulation in rabbit and rat kidneys

Cephaloridine causes necrosis of renal proximal tubules in humans and laboratory animals. This antibiotic nephrotoxicity in rats has been shown to be reduced by mixed-function oxidase (MFO) inhibitors such as piperonyl butoxide and cobaltous chloride. The purpose of this study was to determine the effect of phenobarbital, a MFO inducer, on cephaloridine nephrotoxicity in rats and rabbits. Phenobarbital induced rabbit renal MFO activities and also potentiated cephaloridine toxicity in rabbit kidneys. In contrast, a similar treatment with phenobarbital produced little effect on rat renal MFO activities and did not alter cephaloridine nephrotoxicity in rats. These results suggested that cephaloridine may have to be bioactivated within the kidney prior to producing toxicity. However, a higher renal cortical concentration of cephaloridine was detected in phenobarbital-treated rabbits. This higher concentration appeared to be due to a greater ability of renal cortical cells to accumulate cephaloridine. Therefore, rather than as a result of enzyme induction, the potentiating effect of phenobarbital on cephaloridine nephrotoxicity might be due to the increased renal cortical accumulation of the parent drug, cephaloridine.     

Inhibition of dog brain synaptosomal Na+ -K+ ATPase and K+-stimulated phosphatase activities by long chain n-alkyl-amine and -piperidine, and N'-alkylnicotinamide derivatives

The effects of piperidine, primary amine, and nicotinamide aliphatic derivatives on dog brain snyaptosomal Na⁺-K⁺ ATPase were investigated. These derivatives inhibited the enzyme activity in a manner dependent on alkyl chain length. Kinetic studies revealed that inhibition of Na⁺-K⁺ ATPase activity by long chain alkyl derivatives (C₁₂-C₁₈) was biphasic and non-competitive with respect to the inhibitor and substrate (ATP) concentrations respectively. These long alkyl derivatives caused changes in the Hill coefficient that suggest the occurrence of a possible conformational change in the enzyme molecule. Dual-inhibitor experiments showed that both saturated and unsaturated pentadecylpiperidine (C₁₅-pip) derivatives inhibited Na⁺-K⁺ ATPase by the same mechanism. Oleylamine (C_18:1-NH₂), and N-dodecylnicotinamide (C₁₂-NA⁺Cl^?) derivatives apparently inhibited the enzyme activity by a mechanism different from that of piperidine derivatives. Low concentrations of C_15:1-pip, C_18:1-NH₂, and C₁₂-NA⁺Cl^? inhibited dog brain Na⁺-K⁺ ATPase activity only, but at higher inhibitor concentrations K⁺-stimulated phosphatase activity was inhibited as much as Na⁺-K⁺ ATPase. It is concluded that long chain n-alkyl derivatives of piperidines, amines, and nicotinamide have a cationic detergent-like action on Na⁺-K⁺ ATPase, possibly by the disruption of protein-phospholipid interactions. The mechanism by which these compounds inhibit the overall Na⁺-K⁺ ATPase may involve two different inhibitory sites: one, a high affinity inhibitory site, binding to which inhibits the Na⁺-stimulated phosphorylation reaction, and the other, a low affinity inhibitory site, binding to which inhibits the K⁺-stimulated dephosphorylation reaction. These possible mechanisms may provide an explanation for the observed biphasic inhibition kinetics.     

Effects of diuretics on calcium uptake and release in renal microsomes

A variety of diuretics were tested for their effects on Ca²⁺ uptake and release by microsomes isolated from rat kidneys. Mersalyl acid, ethacrynic acid, furosemide and bumetanide inhibited Ca²⁺ uptake by microsomes. Cysteine abolished the inhibitory effects of mersalyl acid and ethacrynic acid on Ca²⁺ uptake. No appreciable differences in the inhibition were seen between microsomes from kidney cortex and those from medulla. Acetazolamide had no significant effect on Ca²⁺ uptake; however. hydrochlorothiazide stimulated Ca²⁺ uptake activity. The effects of the diuretics on Ca²⁺ uptake by microsomes were entirely on the process of Ca²⁺ accumulation in microsomes, since release of Ca²⁺ that had been taken up previously was not influenced by any of the diuretics tested. These results provide information concerning the biochemical mechanisms of action of diuretics on Ca²⁺ reabsorption in the kidney.     

Sulphinpyrazone prevents in vivo the inhibitory effect of aspirin on rat platelet cyclo-oxygenase activity

Sulphinpyrazone reportedly inhibits in vitro platelet cyclo-oxygenase activity. This study shows that Sulphinpyrazone administration (200 mg/kg) to rats was followed by long lasting inhibition of platelet cyclo-oxygenase, as measured by malondialdehyde generation by sodium arachidonate. The inhibition was apparently competitive and could in fact be ascribed to supposed metabolites of the drug. When given 30min–6 hours before aspirin (5–25mg/kg), Sulphinpyrazone and to a considerable extent its metabolites significantly prevented permanent inhibition of platelet cyclo-oxygenase activity normally produced in vivo by aspirin. Sulphinpyrazone at 100 mg/kg was unable by itself to modify platelet malondialdehyde or thromboxane B₂ generation, yet if effectively interfered with aspirin activity. This suggests that Sulphinpyrazone and its metabolites may interact with a binding site on cyclo-oxygenase not directly involved with the enzyme activity. Interaction of aspirin with this binding site would be a prerequisite for its inhibitory effect on the enzyme active site.The clinical implications of this study include a reappraisal of the pharmacological basis for the association of Sulphinpyrazone and aspirin in thrombosis prevention trials.     

Effect of topical application of defined constituents of coal tar on skin and liver aryl hydrocarbon hydroxylase and 7-ethoxycoumarin deethylase activities

A single topical application of coal tar solution (USP) to neonatal rats resulted in the induction of aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase activities in skin, liver, lung, kidney, and intestine. The induction response of AHH in these tissues was in the order of skin > liver > intestine > lung > kidney. The induction response of 7-ethoxycoumarin O-deethylase was highest in the lung and followed the order of lung > kidney > skin > liver > intestine. Twenty-eight defined chemical constituents present in coal tar solution were individually analyzed for their induction effect on skin and liver AHH and 7-ethoxycoumarin O-deethylase activities. A single topical application of anthracene, 1,2-benzanthracene, 2,3-benzanthracene, benzo(a)pyrene, 2,3-benzofluorene, carbazole, chrysene, coronene, 1,2,3,4-dibenzanthracene, 1,2,5,6-dibenzanthracene, 7,12-dimethylbenzanthracene, fluorene, isoquinoline, 3-methylcholanthrene, or phenanthrene at a dose of 1 mg/10 g body weight to neonatal rats resulted in statistically significant induction of skin and liver AHH and 7-ethoxycoumarin O-deethylase activities. The induction response varied from chemical to chemical. Acridine was found to be an inducer of the skin enzymes only. Topical application of triphenylene, benzo(ghi)perylene, fluoranthene, 1-methylphenanthrene, and perylene caused induction of liver enzymes without producing effects on skin enzymes. These studies indicate that several hydrocarbons present in coal tar solution have induction effects on drug and carcinogen metabolism in skin and liver and that there are tissue-specific responses to some of these hydrocarbons.     

The effects of nitrogen mustard (HN2) on activities of the plasma membrane of PC6A mouse plasmacytoma cells

Nitrogen mustard, HN2 (10^?5 M), inhibited the transport of the potassium congener ⁸⁶rubidium into PC6A mouse plasmacytoma cells by 45% after a 4 hr incubation at 37° in vitro. HN2 (10^?3 M) had a rapid effect on the profile of ⁸⁶rubidium transport into PC6A cells when added simultaneously with the ⁸⁶rubidium whereas a monofunctional analogue of HN2((2-chloroethyl)dimethylamine) had no effect at 10^?3 M. The transport of the amino acid analogues α-aminoisobutyric acid and cycloleucine into PC6A cells was inhibited by 19% and 5% respectively after a 4 hr incubation with 10^?5 M HN2. The results suggest that the activity of plasma membrane Na⁺K⁺-ATPase may be affected by HN2. This enzyme may play a pivotal role in controlling cell growth and division. Crude cell membrane preparations from PC6A cells had variable Na⁺K⁺-ATPase activity which was possibly due to contamination with mitochondrial Mg²⁺-ATPase. Incubation of a crude cell membrane preparation in the presence of 40 nM dicyclohexylcarbodiimide gave constant Na⁺K⁺-ATPase activity which was inhibited by 44% on incubation with HN2 (10^?3 M) for 0.5 hr. The monofunctional analogue of HN2 inhibited this preparation by only 7% under the same conditions. It is suggested that inhibition of Na⁺K⁺-ATPase by HN2 may be an important facet of its cytotoxic activity.     

Sex difference in stimulatory actions of cofactors on prostaglandin synthetase in microsomes from rat kidney medulla

The stimulatory action of cofactors on PG synthetase in the microsomal fraction of rat kidney medulla has been examined in relation to the sex of the animals. Norepinephrine stimulated the activity of PG synthetase to the same degree in males and females. Reduced glutathione caused a much greater increase in the PG synthetase activity in females than in males. In females reduced glutathione was a stronger activator of PG synthetase than norepinephrine, whilst in males it was a poorer stimulator. These results suggest that there may be a sex difference in the PGE isomerase which converts PG endoperoxides to PGE, possibly due to sex hormones.     

Nature of the toxicity of cyclosporin A in the rat

The potent immunosuppressive agent Cyclosporin A (CyA) causes a spectrum of toxicological effects in rats, of which the most striking is weight loss. Pair-feeding experiments have shown that this is caused, in part, by a short period of anorexia. However, even when the food intake has become normal the rats receiving CyA fail to gain weight. That CyA at the doses used causes increased protein catabolism is also indicated by a fall in serum albumin and a marked rise in blood urea unaccompanied by a corresponding rise in creatinine. CyA is mildly and reversibly hepatotoxic and there is slight nephrotoxicity in the rat on the basis of histology and small elevations in creatinine.     

Effects of methotrexate on thymidine triphosphate levels in Chinese hamster cell cultures.

Chinese hamster ovary cells were incubated in medium containing hypoxanthine and glycine and supplied with 10^?5 M methotrexate to inhibit the endogenous synthesis of thymidine nucleotides. Within 1–5 min after addition of the inhibitor, incorporation of [6-³H]deoxyuridine into DNA was as low as 1 to 1.5 per cent of control values, indicating that endogenous synthesis of thymidine nucleotides was blocked rapidly and almost completely. The cellular dTTP content was determined under various culture conditions as a function of time after addition of methotrexate. If the medium used contained undialyzed fetal calf serum, dTTP levels decreased relatively slowly in asynchronous as well as in synchronous S-phase cell populations. Similar results were obtained with asynchronous cultures incubated in medium with dialyzed serum. In contrast, if synchronous cultures consisting predominantly of cells in S phase were incubated in medium containing dialyzed serum and supplied with methotrexate, dTTP levels decreased within 15 min from control values of 15–20 pmoles/10⁶ cells to levels of 1.5 to 3 pmoles/10⁶ cells or lower. The previously reported failure of methotrexate to cause a rapid depletion of cellular dTTP may reflect, therefore, maintenance of thymidine nucleotide pools by cells that are not in the S phase and/or uptake by cells of thymidine present in undialyzed serum used for preparation of culture media.     

Effects of the sea anemone Anemonia sulcata toxin II on skeletal muscle and on neuromuscular transmission

Effects of anemone toxin II (ATX II) have been analysed on the neuromuscular junction of the frog and different twitch muscles. Amplitudes of evoked endplate potentials and endplate currents are increased by ATX II, without effects on the amplitudes of miniature endplate potentials and endplate currents resulting from ionophoretically applied transmitter. The increase in evoked transmitter release is due to an increase in quantal content caused by an effect of the toxin on the presynaptic action potentials. ATX II is also effective on muscle fibers. The action potentials of frog twitch muscles are reversibly prolonged by ATX II. Their rate of rise and amplitudes are increased, while there is no effect on resting membrane potential. Similarly, action potentials of fast twitch muscle (extensor digitorum longus, EDL) of the mouse are reversibly prolonged by ATX II. In slow twitch muscle (soleus, SOL) of the mouse the toxin induces repetitive action potentials following the generation of a single action potential. Tetrodotoxin resistant action potentials of both denervated EDL and SOL are greatly and irreversibly prolonged by ATX II. The effects on muscle are due to a Na+ channel specific action of ATX II. Na+ current inactivation is slowed with the time constant tau h increasing towards positive membrane potentials. The steady state inactivation curve hoo was shifted to more positive potentials and its slope reduced.     

Effect of honeybee (Apis mellifera) venom on the course of adjuvant-induced arthritis and depression of drug metabolism in the rat

The ability of honeybee venom to suppress Mycobacterium butyricum-induced arthritis was studied in Lewis rats. Bee venom, 2 mg.kg-1.day-1 for 24 days, suppressed but did not abolish the primary and secondary inflammatory responses to the adjuvant as monitored by decreases in the swelling of the left and right hind paws and adjuvant-induced arthritis on heme metabolism were also examined. Bee venom or adjuvant had no effect on hepatic delta-aminolevulinic acid synthase, porphyrin content, or ferrochelatase activity. However, with both treatments cytochrome P-450 and the associated enzymic activities of ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase were depressed markedly. In contrast, both treatments caused several-fold enhancement of hepatic microsomal heme oxygenase activity. Adjuvant-treated rats receiving bee venom showed changes in heme metabolism which were of a magnitude similar to those observed when either agent was administered to the experimental animals. Although the bee venom appears to suppress adjuvant-induced arthritis to a greater extent in female than in male rats, the alterations in heme metabolism were similar in bee venom-treated male and female rats. The observed changes in heme metabolism elicited by the venom or by the adjuvant are strongly suggestive of perturbations of the immune system causing alterations in hepatic microsomal enzymes.