Differences in characteristics of ethacrynic acid accumulation in kidney cortex, medulla and papilla.

Ethacrynic acid (EA) accumulation in vitro by slices of rat kidney cortex, medulla and papilla was studied. Slice/medium (S/M) concentration ratio increased during incubation, approaching saturation after 150 min. S/M in slices from cortex reached 12 while in medullary and papillary slices it did not exceed 2. Increasing EA concentration in the medium from 10^?7 to 5 × 10^?3 M decreased the S/M in cortical slices from 12 to 2 while no significant effect was observed in slices of medulla or papilla. EA accumulation by cortical slices was drastically reduced in the absence of sodium in the medium, while no change in EA accumulation occurred in slices of medulla or papilla. In potassium-free media, EA accumulation was slightly but significantly reduced in cortical slices but no effect was observed on EA concentration in medullary or cortical slices. Probenecid reduced S/M of EA in cortical but not in medullary or papillary slices. N-ethylmaleimide reduced EA accumulation in slices of kidney cortex but not of medulla or papilla. These data suggest that EA accumulation in kidney cortex is through the organic acid transport process and differs from the mechanism of accumulation in medulla or papilla. Anoxia and elevation of pH to 8.5 reduced EA accumulation in slices from all three regions of the kidney.     

Differences in Na and K transport in kidney cortex and medulla indicated by ouabain, ethacrynic acid and other inhibitors.

The effect of various inhibitors, ouabain, ethacrynic acid (EA), thiomerin and N-ethyl maleimide (NEM) was studied on (Na + K)-ATPase in the rat kidney cortex and medulla and on sodium extrusion and potassium accumulation in slices from cortex and medulla. With each inhibitor the sensitivity of (Na + K)-ATPase in medulla was similar to that in the cortex. Mg-ATPase was more resistant than (Na + K)-ATPase to Thiomerin and NEM. Medullary Mg-ATPase was more sensitive than medullary (Na + K)-ATPase to EA. Na-extrusion and K-accumulation were completely inhibited in cortical and medullary slices by NEM. Thiomerin had no significant effect on Na extrusion or K accumulation in cortical slices: Thiomerin inhibited Na extrusion in the medulla but enhanced K-accumulation. Ouabain inhibited Na-extrusion and K-accumulation in the medulla significantly more than in the cortex. In the absence of K^? in the medium Na extrusion persisted in the cortex at 56 per cent of maximal extrusion rate while in the medulla only 30 per cent of the maximal extrusion rate persisted. EA inhibited Na-extrusion and K-accumulation significantly more in slices of cortex than in slices of kidney medulla. It is suggested that transport of sodium in kidney cortex and medulla involve quantitatively and qualitatively different mechanisms.     

Differential effect of ethacrynic acid on microsomal Mg2+-ATPase.

The effect of ethacrynic acid (EA) was studied on microsomal ATPase in various tissues of the rat and in kidneys of different species. Inhibition of Na,K-ATPase in microsomes of kidney medulla and cortex was observed only at 5 × 10^?3 M EA. At concentrations of 10^?5?10^?4 M, EA caused some enhancement of Na,K-ATPase activity. Microsomal Mg-ATPase of kidney medulla and cortex was inhibited by EA at concentrations of 10^?5?10^?3 M in various rodents (rat, mouse, guinea-pig and rabbit) as well as in human and cat kidney. EA showed the same differential effect on kidney microsomal Mg-ATPase when microsomes were prepared without treatment with desoxycholate, when chloride was replaced by sulfate in the medium. EA inhibited preferentially Mg-ATPase vs Na, K-ATPase in micro-somes of rat brain and rat submaxillary gland.     

Divalent cation transport in kidney slices. I. Properties of calcium transport in slices of rat kidney cortex and the effects of diuretics

Correlative studies on transports of Ca2+ and Na+ and the properties of Ca2+ transport were carried out in rat kidney cortex slices. Ouabain had no effect on the Ca2+ transport but did inhibit the Na+ transport extensively. With addition of 2,4-dinitrophenol to the incubation medium, or under the anaerobic conditions, the effluxes of Ca2+ and Na+ were inhibited while the Ca2+ influx was enhanced significantly. Sulfhydryl inhibitors such as ethacrynic acid, mersalyl and p-chloromercuribenzoic acid suppressed Ca2+ efflux and stimulated Ca2+ influx. When the slices of kidney cortex were treated with these inhibitors, there was a reduction in the content of cellular ATP. The present results suggest that Ca2+ transport may be partly independent of Na+ transport, and that Ca2+ efflux may require energy-yielding processes.     

Factors affecting morphine uptake into kidney slices.

The uptake of [n-methyl¹⁴C]morphine by mouse kidney slices was saturable, reversible, temperature- and pH-dependent, and was inhibited by strong metabolic poisons and by structural analogs, thereby satisfying criteria for mediation by active transport processes. There were species differences: rat kidney cortex slices took morphine up at rates similar to those of mouse kidney, but guinea pig kidney cortex had lower uptake. Thin-layer chromatography (t.l.c.) of slice extracts after incubation with [¹⁴C]morphine did not indicate significant metabolism of morphine. Morphine efflux after equilibrium uptake from 30 nM or 10 μM initial medium levels required 20 min for 50 per cent of the [¹⁴C]morphine to exit. The uptake at 5 min was proportional to the equilibrium level at 30 min from 5 nM to 10 μM. No evidence for counter-transport was observed. Tissue/medium levels of 10–12 at 39–90 nM morphine (after 30 min at 37°) were reduced 50 per cent at pH 6.5 or at 20°, or by mitochondrial enzyme inhibitors, e.g. rotenone. Narcotic antagonists and analogs (methadone, nalorphine and levorphanol) and quinine also reduced the uptake of morphine from medium levels of 0.01 to 0.1 μM. However, at morphine concentrations above 10 μM, narcotic analogs or antagonists up to 50 μM did not inhibit uptake. Transport system inhibitors, quarternary bases, reducing agents and SH-oxidants also inhibited morphine uptake from 30 nM to 0.5 mM. Phloretin, phloridzin and atractyloside did not block uptake, while glucose, ouabain and NaF were very weak inhibitors. The results suggest that uptake of morphine in kidney slices involves SH groups and mitochondrial activity rather than glycolytic or ion-pump mechanisms. With a few exceptions, the characteristics and inhibitor sensitivity of morphine uptake by kidney slices and of amino acid uptake by brain slices appear similar.     

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.     

Regional cyclic GMP content in incubated tissue slices of rat brain

Incubated tissue slices from different regions of the rat brain contained cyclic guanosine 3′,5′-monophosphate (cyclic GMP) in the following descending order of content: cerebellum hypothalamus striatum thalamus-midbrain brain stem hippocampus cerebral cortex. Cholinomimetic agents at 10^?5 M (carbamylcholine, pilocarpine, acetylcholine and eserine) and papaverine at 10^?4 M significantly elevated cyclic GMP accumulation in the cerebral cortex. Three min incubation with carbamylcholine (10^?5 M) did not increase cyclic nucleotide accumulation in the remaining brain regions.     

The stimulating and inhibitory effect of carbamoylcholine on decamethonium uptake by slices of mouse kidney

The present study was performed to decide whether a common mechanism is involved in the accumulation by mouse kidney slices of the quaternary ammonium compounds decamethonium and carbamoylcholine. Slices were incubated (1 hr) in Krebs-Ringer bicarbonate medium (37°, pH 7.4) containing [¹⁴C]decamethonium (2 × 10⁻⁶M) in presence or absence of carbamoylcholine. The decamethonium uptake was significantly increased by 10⁻⁴M-3 × 10⁻³M carbamoylcholine, whereas 2 × 10⁻²M and 3 × 10⁻²M carbamoylcholine significantly inhibited the uptake. Initial decamethonium uptake (3 min incubation) was significantly stimulated when slices were preincubated (1 hr) with 3 × 10⁻³M carbamoylcholine before transfer to a carbamoylcholine-free medium containing decamethonium. This suggests a relationship between the stimulating effect and the presence of carbamoylcholine in the slices. No efflux of decamethonium (incubation period 15 min) occurred from slices preincubated (1 hr) with decamethonium (2 × 10⁻⁶M), which means that stimulation cannot be attributed to inhibition of decamethonium efflux by carbamoylcholine. The above results support the assumption that decamethonium and carbamoylcholine share a common transport mechanism. The stimulation phenomenon can be interpreted as an example of accelerative exchange diffusion, which should mean that carbamoylcholine efflux accelerates decamethonium influx.     

Nature of Na+-independent stimulation of renal transport of p-aminohippurate by exogenous metabolites

The relationship between p-aminohippurate (PAH) and Na⁺ transport in rabbit kidney cortical slices was examined under optimal metabolic conditions. Addition of lactate, pyruvate and acetate to the incubation medium stimulated PAH transport and accumulation, but had no effect on active Na⁺ efflux from the slices. Conversely, small concentration of F^?, in the presence of acetate, decreased PAH transport and accumulation, but had no effect on Na⁺ efflux. These observations constitute evidence that, in addition to Na⁺ cotransport [M. I. Sheikh and J. V. Møller Biochem. J.208, 243 (1982)], PAH uptake is metabolically stimulated by a Na⁺-independent pathway. This could occur either by a direct metabolic effect on the PAH carrier, or by exchange with intracellular anions that are generated in the presence of exogenous substrate. In support of the latter possibility the PAH carrier is demonstrated to function as an anion exchanger of PAH and fumurate after preloading of the slices with fumurate under anaerobic conditions.     

In vitro analysis of the renal handling of paraquat.

Accumulation of paraquat by mouse renal cortical slices was related to the concentration of paraquat in the medium and the duration of incubation. Paraquat accumulation was depressed by incubation of slices under nitrogen or by addition of metabolic inhibitors. Accumulation of a second organic base, N-methylnicotinamide (NMN), was depressed by a concentration of paraquat which failed to influence accumulation of the organic acid, p-aminohippurate (PAH). The uptake component of NMN accumulation was inhibited by paraquat. The data suggest that paraquat is accumulated by an energy-requiring process and that this accumulation occurs via the organic base transport system. In addition, an apparently toxic effect of paraquat on cortical slice function was observed when the incubation temperature was raised from 25 to 37°C. At this temperature, 10^?3m paraquat depressed not only NMN accumulation but PAH accumulation and slice oxygen consumption as well. Thus, paraquat can be toxic to the function of kidney slices and this effect appears to be temperature-dependent.     

The stimulating and inhibitory effect of monoquaternary ammonium compounds on decamethonium uptake by rat kidney cortex slices

Decamethonium is concentrated as the unchanged compound in rat kidney cortex slices incubated (oxygen-carbon dioxide atmosphere 95:5 v/v %) in Krebs-Ringer bicarbonate medium (37°, pH 7.4) containing [¹⁴C]-decamethonium. Decamethonium uptake was a saturable and metabolically dependent process. When added to medium at relatively low concentrations carbamoylcholine, choline, neostigmine and tetraethylammonium stimulated 1 hr uptake of decamethonium(2 × 10⁻⁶ M), whereas these monoquaternary ammonium compounds at higher concentrations inhibited uptake. Initial decamethonium uptake (5 min) was increased when slices were preincubated (90 min) with a monoquaternary ammonium compound (without decamethonium) before transfer to a medium containing only decamethonium (2 × 10⁻⁶ M). This suggests that stimulation is related to the presence of monoquaternary ammonium compounds in the slices. No decamethonium efflux (30 min) occurred from slices preincubated (1 hr) with decamethonium (2 × 10⁻⁶ M), which means that stimulation cannot be attributed to inhibition of decamethonium efflux by monoquaternary ammonium compounds. The stimulation phenomenon can be interpreted as an example of substrate-facilitated carrier transport, which implies that efflux of monoquaternary ammonium compounds accelerates decamethonium influx. This interpretation is consistent with the conclusion from the experiments on mutual inhibition that the polymethylene-bisquaternary ammonium compound decamethonium and the above monoquaternary ammonium compounds share a common carrier mechanism.     

Paraquat accumulation: Tissue and species specificity

Rat tissues have been examined in vitro for their ability to accumulate paraquat or diquat to concentrations in excess of those present in the incubation medium. With a concentration of 10^?6 M. lung slices were able to accumulate paraquat to concentrations nearly ten times that of the medium. and brain slices to concentrations double that of the medium, over a period of two hours. Neither slices of lung nor brain accumulated diquat significantly from a concentration in the medium of 10^?6 M. The accumulation of paraquat by brain slices, like that of lung slices, has been shown to he energy-dependent. Other organs examined showed little, if any, ability to accumulate either paraquat or diquat. Lung slices from dog. monkey and rabbit have also been shown to possess the ability to accumulate paraquat in vitro. After oral dosing of paraquat to rats. the lung concentration increased with time to six times that of the plasma after 30 hr. Other organs, with the exception of the kidney, did not concentrate paraquat to the same extent. Kidney concentrations after oral dosing of both paraquat and diquat were high throughout the period of time studied. It is. therefore, suggested that the apparent selectivity exhibited by paraquat for the lung is associated with the accumulation process.     

The influence of drugs on the uptake of acetylcholine by slices of rat cerebral cortex

1. The acetylcholine content of cortical slices from rat brain, was determined after incubation for 30 min in a medium containing acetylcholine (4 mug/ml.). The cholinesterase activity of the slices had been inhibited by pretreatment with 3,3-dimethyl-n-butyl 2-methylphosphonofluoridate (soman).2. Acetylcholine accumulated in the tissue slices, up to a concentration of about six times that in the medium.3. The uptake of acetylcholine was partly inhibited by potassium in high concentrations.4. Hemicholinium-3, O-ethyl S-diethylaminoethyl ethylphosphonothiolate, physostigmine, atropine and choline, in that order of potency, inhibited the accumulation of acetylcholine in the cortical slices, but soman and ethyl N,N-dimethyl phosphonoamidocyanate (tabun) had no effect on the uptake of acetylcholine.5. Substances interfering with energy metabolism, such as 2,4-dinitrophenol, oligomycin, sodium azide, amylobarbitone sodium and p-chloromercuribenzoate inhibited the uptake of acetylcholine. Ouabain had little inhibitory effect.6. In anaerobic conditions the accumulation of acetylcholine in the tissue slices was nearly blocked.7. The uptake of acetylcholine in the tissue slices was dependent on temperature. The Q(10) was about 2.8. Autoradiography of sections from slices in which (3)H-acetylcholine had accumulated showed a diffuse distribution of radioactivity in the cytoplasm of all cells. There was no visible preference for certain cells or cell structures.     

Actin cytoskeleton, tubular sodium and the renal synthesis of dopamine.

The present study has examined the effect of colchicine and cytochalasin B, two cytoskeleton disrupter compounds, on the formation of dopamine in slices of rat renal cortex loaded with exogenous L-3,4-dihydroxyphenylalanine (L-DOPA); the deamination of newly formed dopamine into 3,4-dihydroxyphenylacetic acid (DOPAC) was also examined. The accumulation of newly formed dopamine and DOPAC in kidney slices loaded with L-DOPA (10-100 microM) was found to be dependent on the concentration of L-DOPA, being similar in control conditions and in preparations treated with increasing concentrations of colchicine (5, 10 and 50 microM). By contrast, cytochalasin B (5, 10 and 50 microM) was found to produce a concentration-dependent reduction in the formation of dopamine and of its deaminated metabolite DOPAC in kidney slices loaded with L-DOPA (10-100 microM). The inhibitory effect of cytochalasin B on the formation of dopamine was found to be completely abolished in kidney slices pretreated with ouabain (500 microM) or when sodium concentration in the incubation was reduced from 120 to 20 mM. On its own, ouabain (500 microM) was found to reduce the formation of dopamine by 55%; the effect of reducing sodium concentration in the incubation medium to 20 mM was also a significant reduction (53% decrease) in the formation of dopamine. The accumulation of DOPAC did always parallel that of its parent amine. It is concluded that the renal formation of dopamine is dependent on the concentration of sodium in the medium and the integrity of the tubular transport of sodium, namely on the association between actin cytoskeleton and Na+,K(+)-ATPase, appears to be determinant.     

Factors affecting the efflux of paraquat from rat lung slices

In an attempt to reduce the toxicity of paraquat several compounds were examined for their ability to increase the rate of efflux of paraquat from the lung. The compounds were selected because they were known, from in vitro studies, to reduce the accumulation of paraquat into the lung. Histamine (100 μM), promethazine (100 μM), putrescine (100 μM), bromthymol blue (300 μM) and the metabolic inhibitors iodoacetate (1 mM), rotenone (100 μM) and KCN (1 mM) have been shown to reduce the accumulation of paraquat into rat lung slices, as did the incubation of slices under nitrogen.The efflux of paraquat from lung slices prepared from rats dosed intravenously with paraquat was biphasic, having a very fast component and a slow component. The slow component was first order and was characterised by a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 17 h. This half life is similar to that seen in vivo (24h) following intravenous dosing. When lung slices prepared from rats dosed intravenously with paraquat were incubated in the presence of iodoacetate (1 mM) or under nitrogen, the half life of paraquat in the slices was reduced to approximately 3 h. In the presence of rotenone (100 μM) it was reduced to approximately9 h. Histamine (100 μM) and promethazine (100 μM) did not affect the efflux of paraquat from lung slices. Bromthymol blue, a dye which forms “ion pair” complexes with paraquat, also significantly increased the efflux of paraquat from lung slices. The effect of bromthymol blue, however, decreased with time and thus paraquat efflux in the presence of bromthymol blue did not obey first order kinetics.In order to measure cellular viability of lung slices, oxygen comsumption, glucose oxidation and the rate of the efflux of protein from the slices into the incubation medium were determined. Iodoacetate (1 mM) and rotenone (100 μM) almost abolished oxygen consumption and glucose oxidation whereas these activities were inhibited to a lesser extent by bromthymol blue (300 μM) (18% and 30%, respectively). During the first 30 min of incubation in the presence of KCN (1 mM) oxygen consumption was almost abolished but between 30 min and 4 h returned to control levels. The effect of KCN could therefore be divided into 2 phases. Over 4 h incubation glucose oxidation was inhibited by 36%. Iodoacetate (1 mM) and incubation under nitrogen caused the most pronounced increases in the rate of protein efflux from slices. KCN (1 mM) and rotenone (100 μM) also increased the rate of protein efflux but to a lesser extent. We have therefore suggested that the effect of KCN (1 mM) on cellular viability, while severe, may be less than that of iodoacetate (1 mM) or incubation under nitrogen.We have concluded from these studies that: (1) the reduction in the accumulation of paraquat or increase in its efflux produced by metabolic inhibitors may be a consequence of lung cell damage; (2) bromthymol blue and putrescine cause an increase in the efflux of paraquat from the lung in vitro without damaging the tissue suggesting that in principle this approach may be useful in vivo; and (3) histamine and promethazine reduce the uptake of paraquat into the lung by direct competition with paraquat since they neither increase the rate of efflux of paraquat from the lung nor reduce cellular viability.     

Evidence for carrier-mediated efflux of dopamine from corpus striatum

This study was designed to further evaluate the hypothesis that the efflux of cytoplasmic dopamine from nerve endings of the corpus striatum can occur by carrier-mediated facilitated diffusion. Since dopamine (DA) and sodium ions are thought to be cotransported by the neuronal uptake carrier, the change in DA efflux upon alterations induced in the sodium gradient was observed. Ouabain was used to inhibit Na⁺K⁺-ATPase and thus increase intracellular sodium concentration, while metabolic inhibitors resulted in the same effect by reducing the amount of ATP available for Na⁺K⁺-ATPase. Rats were pretreated with reserpine and incubated with [³H]DA in the presence of pargyline so that the efflux of cytoplasmic [³H]DA could be assessed. Under these conditions, ouabain produced a concentration-related increase in the efflux of [³H]DA from nerve endings of the corpus striatum. The ouabain-induced release of [³H]DA was reduced by 10^?M benztropine, a dopamine uptake inhibitor. Benztropine (10^?5M) also reduced the increase in release of pHjDA when the same tissue preparation was incubated with the metabolic inhibitors sodium cyanide, iodoacetic acid and dinitrophenol. Ouabain at a concentration which caused slight release of [³H]DA potentiated the efflux produced by 3 × 10^?6 M amphetamine, 3 × 10^?6M unlabeled DA and 10^?4M fenfluramine. These results suggest that release of [³H]DA by sympathomimetic amines may be a carrier-mediated process. The Q₁₀ for 10^?4 M ouabain-induced release of [³H]DA was much higher than the Q₁₀ for the above processes and, therefore, the rate-limiting step in ouabain-induced release may involve inactivation of an enzyme such as Na⁺K⁺-ATPase which is coupled to the transport of sodium. Thus, by changing the availability of sodium with ouabain or metabolic inhibitors and by changing the cytoplasmic accessibility of the membrane carrier with benztropine or phenethylamines, the efflux of cytoplasmic [³H]da can be altered markedly.     

Subcellular distribution of deslanatoside C-3H in isolated guinea pig hearts. interaction of other drugs

Guinea-pig hearts were perfused with Krebs-Henseleit (K-H) medium 30 min for equilibration, then 4, 16, 32 or 64 min with Deslanatoside C-³H 10^?7 M in K-H medium and then again with K-H medium alone to wash extracellular spaces at a constant flow of 3 ml/min at 28 C. Deslanatoside C incorporated into the ventricles and atria increased from 4 to 64 min, the ventricles invariably showing a higher degree of incorporation. The concentration of Deslanatoside C also increased from 4 to 64 min in microsome. mitochondrial and nuclear fractions, the microsome traction invariably showing the highest specific concentration. Glycoside in the supernatant fraction was not tightly bound to any macromolecular structure. A β-adrencrgie blocking agent SD 1601 10^?7 M, and Creatinol O-phosphate 10^?5 M both increased myocardial uptake of Deslanatoside C-³H and its incorporation into the pellet fractions. Daunomycin 10^?5 M increased the myocardial uptake of the labelled glycoside and its concentration in both supernatant and pellet fractions. Neither phenobarbital sodium 10^?6 M nor Ouabain 10^?7 M affected uptake or the subcellular distribution of Deslanatoside C. Ouabain 10^?6 M decreased both uptake by the heart and the subcellular concentrations of Deslanatoside C.     

The effects of 1-methyl-4-phenylpyridinium ion (MPP+) on the efflux and metabolism of endogenous dopamine in rat striatal slices

1-Methyl-4-phenylpyridinium ion (MPP+) was shown to accumulate concentration-dependently in slices from rat striatum. At 10 microM, MPP+, the tissue concentration was found to be 118 +/- 9 microM following 75 min of incubation. The accumulation of MPP+ was reduced in the presence of 10 microM of the selective dopamine uptake inhibitor GBR 12909 (-50%) or by destruction of the dopaminergic terminals by complete hemisection of the forebrain 4 days before the experiments (-75%). Accumulation of MPP+ in the catecholamine-poor occipital cortex and cerebellum was only 25% of that obtained in striatum. Reserpine pretreatment of the rats in-vivo did not modify the accumulation of MPP+ in the striatal slices. MPP+ (1-10 microM) increased the net efflux of dopamine and reduced the efflux of the dopamine metabolite DOPAC from the striatal slices. The effect on dopamine was readily diminished if MPP+, after a 15 min incubation, was then omitted from the medium. In contrast, the DOPAC efflux was reduced for 75 min even though MPP+ was present in the incubation medium only for the first 15 min. In the presence of the monoamine oxidase inhibitor, pargyline (350 microM), MPP+ also produced an increase in dopamine efflux. In normal medium, the presence of the dopamine uptake inhibitor GBR 12909 (10 microM), or the absence of calcium, failed to modify the MPP+-induced increase in dopamine efflux. MPP+ also increased dopamine efflux from slices from reserpinized rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro experiments on the accumulation and release of 14C-histamine by snail (Helix pomatia) nervous tissue.

When isolated snail ganglia were incubated at 25° in a medium containing ¹⁴C-histamine, tissue:medium ratios of about 4 were obtained after only 5 min. Metabolism of the accumulated amine is rapid, for even within this short incubation period, 20 per cent of the substance was metabolised. The process responsible for this accumulation showed properties of an active transport system: it was temperature-sensitive, sodium dependent, and particularly inhibited by ouabain, phenoxybenzamine, chlorpromazine and desimipramine. The accumulation of ¹⁴C-histamine showed saturation kinetics typical of a carrier-mediated process and can be divided into sodium-sensitive and -insensitive components. Kinetic analysis of the data revealed similar K_m values for both sodium-sensitive and sodium-insensitive plus -sensitive components, i.e. 10^?5 M.A rapid efflux of radioactivity from tissue loaded with ¹⁴C-histamine was observed when exposed to 70 mM KCl. This release was inhibited when the calcium content in the medium was replaced by sucrose. Moreover, cobalt ions added to the incubation medium counteracted the effect of KCl.The discussion of the present results is based on the hypothesis that histamine has a transmitter function in the snail CNS.     

Effect of papaverine on Ca2+ uptake by a mitochondrial fraction isolated from rat uterine smooth muscle

A mitochondrial fraction was isolated from rat uterine smooth muscle. The effect of papaverine (3 × 10^?6, 10^?5, 3 × 10^?5, 10^?4 M) on Ca²⁺ accumulation by the fraction was studied. The effect of papaverine was changed by using different substrates for Ca²⁺ accumulation. Papaverine inhibited the Ca²⁺ accumulation supported by glutamate plus ATP concentration-dependently. The Ca²⁺ accumulation supported by ATP alone was also inhibited. On the other hand, the Ca²⁺ accumulation supported by succinate and ATP was increased by papaverine. The effect was not concentration-dependent; papaverine (3 × 10^?5 M) was the most effective concentration for the effect with a shorter incubation (2 min).