Furosemide accumulation by renal tissue.

Furosemide accumulation by rabbit kidney cortical slices increased with incubation time, reaching a plateau by 60–90 min. Furosemide uptake by kidney slices from rabbits, dogs, mice and rats was inhibited by dinitrophenol, ouabain and probenecid, and was decreased by incubation at 0°, anoxia or the use of sodium-free medium. Furosemide slice/medium ratios decreased with increasing concentration of furosemide in the incubation medium. Addition of furosemide to medium containing rat kidney slices and either p-aminohippurate (PAH) or N-methylnicotinamide (NMN) produced a dose-related inhibition of PAH, but not NMN, accumulation. It is concluded that furosemide is actively transported by the renal organic anion transport system.     

Effects of monovalent cations and insulin on glucose metabolism of the isolated rat diaphragm

The effects of monovalent cations on glucose utilization, glycogen synthesis and lactate formation of rat diaphragm incubated in vitro were studied in the presence and absence of insulin. It was found that the monovalent ions markedly influenced glucose metabolism and the action of insulin in this tissue. Sodium and particularly lithium ions promoted glucose uptake and glycogen synthesis, while in the presence of a high concentration of potassium, glucose utilization and glycogen synthesis were low and lactate formation increased. The maximal effect of insulin was obtained in media high in sodium or lithium ions. The sulfhydryl reagent, 2,2'-dithiodipyridine (DTP), inhibited glucose uptake and glycogen synthesis to the same extent. Lactate production was unchanged by DTP when the tissues were incubated in a sodium medium and was increased by DTP in the presence of lithium. The monovalent cations strongly influence the metabolic action of insulin in the diaphragm preparation and appear to act by affecting an early rapid reaction catalyzed by the hormone.     

Effects of epinephrine and the cyclic AMP phosphodiesterase inhibitor SQ 20009 on glucose and glycogen metabolism in skeletal muscle.

In rat diaphragm incubated in vitro, epinephrine and isoproterenol inhibited glucose uptake and the incorporation of radioactive glucose into glycogen. The metabolic effects of epinephrine were inhibited by the beta-adrenergic blocking agents, propranolol and sotalol, but not by the alpha-adrenergic blocking drug, phentolamine. The phosphodiesterase inhibitor, 1-ethyl-4-(isopropylidine-hydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, hydrochloride (SQ 20009), also decreased glucose uptake and glycogen synthesis. In contrast to the action of epinephrine, exogenous calcium ions were required for the effects of SQ 20009 on phosphorylase activation, increased tissue glucose- 6-phosphate levels and lactate production. It was concluded that the inhibition of glucose uptake that occurred when tissue cyclic AMP levels were increased by beta-adrenergic drugs or phosphodiesterase inhibitors was not due directly to accumulation of glucose-6-phosphate from glycogenolysis but was a result of a cyclic AMP-mediated inhibition of glycogen synthesis.     

Effects of metformin on glucose uptake by isolated diaphragm from normal and diabetic rats

The effects of the antidiabetic drug metformin (Nl, Nl-dimethylbiguanide) on glucose uptake by isolated rat diaphragm muscle have been studied. A therapeutic concentration of metformin (10 μg/ml) had no effect on glucose uptake by diaphragm muscle from normal rats incubated in the absence or presence of insulin (100 and 1000 μU/ml), but increased uptake by diaphragm muscle from alloxan-diabetic rats incubated in the presence of insulin (P < 0.05). Diaphragm muscle from normal rats incubated in a medium containing sodium butyrate (0.25 mg/ml) showed a reduction in glucose uptake similar to that seen in muscle from diabetic animals. Metformin (10 μg/ml) also increased glucose uptake by this preparation in the presence of insulin (P < 0.01). A higher concentration of metformin (100 μg/ml) caused a depression of glucose uptake by diaphragms from normal rats, and the necessity for studying therapeutic concentrations of the biguanide drugs is stressed. The relation of these findings to the antidiabetic effect of the drug in man is discussed. The mechanisms involved are discussed in terms of changes in glycogen metabolism.     

Stereospecific influence of oxazepam hemisuccinate on cyclic AMP accumulation elicited by adenosine in cerebral cortical slices.

The effects of the d-, dl- and l-isomers of the water-soluble benzodiazepine, oxazepam sodium hemisuccinate, on cyclic AMP levels in superfused slices of guinea-pig and rat cerebral cortex were investigated. At 100 μM these drugs decreased the accumulation of cyclic AMP elicited by adenosine in a manner closely correlated to their stereostructure and relative anticonvulsant and anxiolytic potencies in vivo. The drugs inhibited uptake of low concentrations of radioactive adenosine into the slices in a similar manner. Addition of theophylline or adenosine deaminase to the superfusion medium sharply decreased both cyclic AMP basal levels and levels elicited by the d-isomer, suggesting mediation by adenosine of the effects of benzodiazepines on cyclic AMP and hence their psychotropic action.     

The effect of human plasma on the glucose uptake of the rat diaphragm before and after administration of carbutamide

The glucose uptake of the rat diaphragm has been determined in the presence of the plasma of young volunteers before and after administration of carbutamide without and with added insulin. The increase in the glucose uptake of the rat diaphragm due to the added plasma above that in the medium alone has been termed plasma effect. The increase in the glucose uptake of the rat diaphragm with plasma and added insulin above that with only insulin in the medium has been termed plasma+insulin effect. There was a significant increase in the plasma effect and the plasma+insulin effect after carbutamide administration. The increase in the plasma+insulin effect was significantly greater than the increase in the plasma effect. From these observations it has been suggested that carbutamide potentiates the action of insulin peripherally. Observed facts about carbutamide do not contradict this mechanism of action.     

Uptake and efflux of 14C-paraquat by rat lung slices: the effect of imipramine and other drugs.

Paraquat accumulation by rat lung slices incubated at 10 or 100 μm concentration was linear with time and the accumulated paraquat was “noneffluxable.” Imipramine (100 or 500 μm) inhibited paraquat (10 μm) uptake by 38 and 85%, respectively, and 500 μm imipramine enhanced paraquat efflux by 40%. The combination of impaired uptake and enhanced efflux suggested the possibility that imipramine might reduce the toxicity of paraquat in intact animals. However, at the doses used, imipramine did not alter paraquat toxicity in vivo. Eleven other drugs were shown to inhibit uptake, but only five enhanced paraquat efflux.     

The effect of potassium dichromate on renal tubular transport processes

Several studies have been conducted to examine the effects of potassium dichromate on the morphology of renal tissue, but relatively few studies have been done on the functional aspects of the renal damage. This study was designed to examine with in vitro techniques the effects of dichromate on a number of renal tubular transport processes. The accumulation of paraaminohippurate (PAH) and of tetraethylammonium chloride (TEA) by both rat and rabbit slices was inhibited by 10^?4m potassium dichromate added to the bathing solution. On the other hand, the accumulation of the nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB), was only modestly reduced. In addition, the uptake of TEA was stimulated significantly by low concentrations (10^?6m) of potassium dichromate. Although, in general, similar results were obtained using renal cortical slices taken from pretreated animals significant differences occurred. For example, at none of the doses tested (5–20 mg/kg) was the accumulation of PAH, TEA, or AIB stimulated. This was true whether the measurements were made as soon as 0.5 hr after administration of potassium dichromate or as long as 5 days after its administration. At an intermediate dose of potassium dichromate (10 mg/kg) the lactate-stimulated accumulation of PAH was depressed, but the control or nonsubstrate-stimulated accumulation was not affected. In addition to the effects on uptake of organic compounds, potassium dichromate, whether administered to the intact animal or added to the extra-cellular fluid compartment, caused a decrease in the intracellular concentration of potassium and an increase in the intracellular concentration of sodium. From a quantitative point of view, however, the effect of the nephrotoxin administered to the intact animal was always greater on tissue electrolytes than when the nephrotoxin was added in vitro.     

The action of hypoglycaemic sulphonylureas on carbohydrate metabolism in the fasted rat

Tolbutamide and carbutamide given orally to fasted rats cause a rise in the liver glycogen content 1½ to 3½ hr. after administration of the drugs. Glycogen accumulates preferentially in the right lobe. Subcutaneously injected tolbutamide has the same effect. Both sulphonylureas cause inhibition of glucose-6-phosphatase activity of rat liver homogenates in vitro, but at drug concentrations comparable with those found in plasma of treated patients the degree of inhibition is less than 10%. Livers from treated rats show normal glucose-6-phosphatase activity. The glucose uptake of the isolated rat diaphragm is unaffected by the sulphonylureas added in vitro. Diaphragms from treated rats show normal glucose uptake in the presence or absence of insulin. The inferences to be drawn from these results are discussed in the light of previous work. It is concluded that the sulphonylureas exert hypoglycaemic action by inhibiting glycogenolysis and it is suggested that they might do so by inhibiting release of glucagon from the pancreas.     

Effect of parathion and its metabolites on calcium uptake activity of rat skeletal muscle sarcoplasmic reticulum in vitro.

The insecticide parathion and its potent anticholinesterase metabolite paraoxon induce skeletal muscle necrosis when administered in vivo to rats. In the present study the effects in vitro of parathion and its metabolites on microsomal (sarcoplasmic reticular) calcium uptake activity in rat diaphragm skeletal muscle are examined. Parathion (0.05 mM) is a potent inhibitor of this calcium uptake. The inhibition is apparently competitive with calcium in the system. Parathion (0.05 mM) is also shown to inhibit calcium-dependent ATPase activity associated with the microsomal calcium uptake. Paraoxon, the active anticholineslerase metabolite of parathion, and p-nitrophenol, a hydrolytic metabolite of paraoxon, have no inhibitory effects at this level. At 1.5 mM levels they do inhibit the skeletal muscle microsomal calcium uptake. Eserine, a chemically unrelated anticholinesterase agent, also has inhibitory effects at 1.5 mM. When these same compounds are incubated with isolated rat hemidiaphragms they antagonize the muscle contraction elicited by direct stimulation of the muscle. The skeletal muscle necrosis caused by parathion and paraoxon appear to relate to the anticholinesterase activity in vivo. The relatively potent inhibition of calcium uptake activity of sarcoplasmic reticulum in vitro seen with parathion appears to be an independent action and not related to cholinesterase inhibition.     

Effects of opiod narcotic drugs on energy reserves of skeletal muscle—I: Glycogen

Morphine, at concentrations of 0·77 to 8 mM, did not significantly alter the rate of glucose uptake by diaphragm muscles from normal rats. At concentrations of 5 mM and higher, however, morphine reduced the glycogen content of diaphragm muscle. In isolated intact extensor digitorum longus and soleus (EDL + SOL)muscles of the rat, glucose uptake was likewise unaffected by morphine but the glycogen content was decreased. Of the narcotic drugs that were tested in vitro for glycogenolytic activity, methadone was the most potent, followed in order by meperidine, morphine and hydromorphone. After incubation in vitro, the glycogen content of diaphragm muscles from rats injected with a single dose of morphine was lower than that of muscles from corresponding control rats. Under identical experimental conditions, the glycogen content of diaphragm muscles from rats chronicall treated with increasing doses of morphine was essentially the same as that of muscles from corresponding control rats, suggesting that the sensitivity of the muscle glycogen system to morphine decreased during chronic morphine treatment. Tolerance was not observed in vitro where methadone had a greater glycogenolytic effect in the diaphragm muscles from rats chronically treated with morphine than in muscles from the control rats. Propranolol, in a concentration sufficient to block the glycogenolytic action of epinephrine in EDL + SOL muscles, did not block the glycogenolytic action of methadone. The activities of both phosphorylase and glycogen synthase were greatly depressed, while phosphoglucomutase activity was unchanged in muscles incubated with methadone. It is concluded that the glycogenolytic effect of methadone in skeletal muscle is not mediated through the release of catecholamines in the muscles, nor can it be explained by a differential action on the activities of the enzymes involved in the last stage of synthesis and the first stage of breakdown of muscle glycogen.     

Effects of dicoumarol on cytotoxicity caused by tert-butylhydroquinone in isolated rat hepatocytes

The effects of dicoumarol, an inhibitor of DT-diaphorase, on the cytotoxicity of tert-butylhydroquinone (tBHQ) were studied in freshly isolated rat hepatocytes. Addition of tBHQ (0.5 mM) to hepatocytes resulted in a time-dependent cell death accompanied by depletion of intracellular ATP, glutathione (GSH), and protein thiols. Pretreatment of hepatocytes with dicoumarol (30 μM) did not affect cell viability or cellular levels of ATP, GSH, or protein thiols during the incubation period; however, dicoumarol did promote the appearance of cell blebs and the depletion of ATP and protein thiols induced by tBHQ and ultimately enhanced the cytotoxicity of tBHQ.     

Bromocriptine, an ergot alkaloid, inhibits excitatory amino acid release mediated by glutamate transporter reversal

Bromocriptine, a dopamine D₂ receptor agonist, has widely been used for patients with Parkinson's disease. The aim of the present study was to investigate the effect of bromocriptine on glutamate transporter. Since the astroglial glutamate transporter GLT-1 (EAAT2) is the predominant isoform in the forebrain, we generated EAAT2-expressing human embryonic kidney cells and immortalized mouse astrocytes. In the present studies, we observed a GLT-1-immunoreactive band and significant Na⁺-dependent d-[³H] aspartate uptake. Furthermore, the glutamate transporter inhibitors, dl-threo-β-benzyloxyaspartic acid (TBOA) and dihydrokainate (DHK), displayed a dose-dependent reduction of d-[³H] aspartate uptake in both types of cells. In contrast, cells exposed to either chemical anoxia or high KCl elicited a marked release of d-[³H] aspartate, and the release was inhibited by TBOA and DHK, implying the contribution of glutamate transporter reversal. Interestingly, we found that bromocriptine dose-dependently inhibits d-[³H] aspartate release elicited by chemical anoxia or high KCl, while no changes occurred in the uptake. The inhibitory action of bromocriptine was not affected by sulpiride, a dopamine D₂ receptor antagonist. On the other hand, bromocriptine had no effect on swelling-induced d-[³H] aspartate release, which is mediated by volume-regulated anion channels. In vivo studies revealed that bromocriptine suppresses the excessive elevation of glutamate levels in gerbils subjected to transient forebrain ischemia in a manner similar to DHK. Taken together, these results provide evidence that bromocriptine inhibits excitatory amino acid release via reversed operation of GLT-1 without altering forward transport.     

The local anaesthetic activity of a benzotriazinium salt.

1 The local anaesthetic properties of 2-n-propyl-4-p-tolylamino-1,2,3-benzotriazinium iodide (TnPBI) were compared with those of lignocaine hydrochloride on intact and desheathed sciatic nerves of the frog, on the phrenic nerve-hemidiaphragm preparation of the rat, and by the intradermal wheal test in the guinea-pig. 2 Both TnPBI and lignocaine were more potent on desheathed than on intact sciatic nerves. The potency of TnPBI was affected more than that of lignocaine by the presence of the sheath in intact nerves. 3 Both drugs inhibited conduction in the rat phrenic nerve, as shown by the reduction in twitch tension of the diaphragm elicited by nerve stimulation. TnPBI also caused an initial augmentation of the twitch tension of the diaphragm when applied directly to the muscle. 4 TnPBI was shown to be approximately twice as potent as lignocaine by the guinea-pig intradermal wheal test. 5 These results are discussed in view of the known effects of TnPBI on intracellular calcium storage.     

Lipoic acid improves glucose utilisation and prevents protein glycation and AGE formation

The present study investigates the antiglycating effect of alpha-lipoic acid (LA) in high fructose-fed rats in vivo and its potential to inhibit the process of glycation in vitro. In addition, the effect of LA on glucose utilisation in rat diaphragm was also studied. Rats fed a high fructose diet (60% total calories) were administered with 35 mg/kg b.w, lipoic acid (LA) intraperitoneally for 20 days. The effects of LA on plasma glucose, fructosamine, protein glycation and glycated haemoglobin in high fructose rats and on in vitro glycation were studied. In vitro utilization of glucose was carried out in normal rat diaphragm in the presence and absence of insulin in which LA was used as an additive. The contents of glucose, glycated protein, glycated haemoglobin and fructosamine were significantly lowered on LA administration to high fructose-fed rats. LA prevented in vitro glycation and the accumulation of advanced glycation end products. Further LA enhanced glucose utilization in the rat diaphragm. This effect was additive to that of insulin and did not interfere with the action of insulin. The findings provide evidence for the therapeutic utility of lipoic acid in diabetes and its complications.     

Comparative effects of chlorpromazine and its free radical on membrane functions

Chlorpromazine, chlorpromazine free radical and a sulfhydryl inhibitor, p-hydroxymercuribenzoate, were compared for their effects on several biochemical phenomena associated with membrane function. Chlorpromazine free radical was slightly more potent than chlorpromazine in inhibiting calcium accumulation by rat brain microsomes, but less potent in protecting synaptosomes from osmotic shock, and in inhibiting synaptosomal uptake of dopamine and norepinephrine. p-Hydroxymercuribenzoate failed to protect synaptosomes from osmotic shock. It was less effective than chlorpromazine and chlorpromazine free radical in inhibiting synaptosomal uptake of catecholamines. The present data indicate that biochemical effects of chlorpromazine such as the protection of synaptosomal membrane from osmotic shock and the inhibition of synaptosomal uptake of dopamine and norepinephrine are caused by chlorpromazine itself but not by chlorpromazine free radical. Although chlorpromazine free radical was a more potent inhibitor of synaptosomal calcium uptake than chlorpromazine, the free radical was a more potent inhibitor of synaptosomal calcium uptake than chlorpromazine, the differences in potency were markedly smaller than those on microsomal Na⁺, K⁺-ATPase previously reported from this laboratory.     

Effects of dicoumarol, insulin and anoxia on rat diaphragm. I. Effect on respiration, extra-cellular space and water content

Dicoumarol (0'5 mM) inhibits the respiration of muscle tissue, but at a low concentration (0-05 mM) enhances respiration. The effect of insulin when given with dicoumarol, when acting as a stimulus, is one of enhancement.     

The complexity of the inhibitory action of dicoumarol on the mixed function oxidase system of rat liver microsomes

The inhibitory effect of dicoumarol on the mixed function oxidase system of liver microsomes from phenobarbital treated rats were studied. Lineweaver-Burk plots of p-nitroanisole O-demethylation were non-linear in the presence of dicoumarol but linear in its absence. At high concentrations of p-nitroanisole the inhibition was partially overcome indicating a competition between the substrate and dicoumarol for the enzyme. Furthermore, it was found that dicoumarol promotes a time and concentration dependent conversion of cytochrome P-450 to P-420 and that it inhibits cytochrome P-450 reduction. While these fenomena seem directly related to the inhibitory action of dicoumarol on the overall p-nitroanisole demethylase reaction, an inhibitory effect of dicoumarol on NADH : ferricyanide reductase, on the contrary, was shown not to be related to the inhibition of the demethylation process.     

Actions of lithium ions and insulin on glucose utilization, glycogen synthesis and glycogen synthase in the isolated rat diaphragm

The effects of lithium ions and insulin on carbohydrate metabolism of the isolated rat diaphragm were studied and compared. Like insulin, lithium ions caused a conversion of glycogen synthase D to the more active I form of the enzyme. Maximal activation of the enzyme was produced by about 5 mM LiCl. Lithium ions markedly increased glucose utilization and glycogen synthesis by the diaphragm, but the action of this ion appears to be exerted by a mechanism different from that of insulin, since the effects of maximal concentrations of the two agents are additive. In their action on glucose metabolism, lithium ions had a unique ability to direct the glucose taken up by the cell toward glycogen. Insulin and lithium ions had opposite effects on the tissue content of glucose 6-phosphate; insulin increased the tissue level of this metabolite, whereas lithium ions decreased it. ATP and creatine phosphate concentrations were not affected by insulin or lithium ions. The effects of lithium ions on carbohydrate metabolism are exerted at relatively low concentrations of Li, and our results indicate that significant alterations of carbohydrate metabolism may occur when therapeutic or toxic amounts of lithium salts are ingested by man.