The effect of reserpine treatment on the extraneuronal uptake of [3H]-isoprenaline into rat atria.

Treatment of rats with reserpine (1 mg kg-1 day-1) for up to 7 days resulted in a marked decrease in a corticosterone-sensitive component of the extraneuronal accumulation of [3H]-isoprenaline into their atria. The change in extraneuronal uptake did not appear to be due to a direct action of reserpine on the uptake mechanism, since it was several days before the treatment had a significant effect on the accumulation of [3H]-isoprenaline. Further, reserpine in vitro did not inhibit extraneuronal uptake. The reserpine-induced change in the accumulation of [3H]-isoprenaline was not an artifact due to changes in water balance, ion distribution, extracellular space or tissue atrophy. Nor was the change due to an increase in the efflux of [3H]-isoprenaline from the tissue. These experiments support the suggestion that the extraneuronal uptake is dependent upon a functional adrenergic innervation.     

The extent of neuronal re-uptake of 3H-noradrenaline in isolated vasa deferentia and atria of the rat

Summary After pretreatment of rats with reserpine and pargyline (to inhibit vesicular uptake and monoamine oxidase, respectively) and after inhibition of catechol-O-methyl transferase (by U-0521) and in calcium-free solution, the adrenergic neurones of isolated vasa deferentia and atria were loaded with ³H-noradrenaline. The spontaneous efflux of ³H-noradrenaline and 3H-dihydroxyphenylglycol was determined, as well as the steady-state effect of two concentrations of desipramine.On the basis of a mathematical model of the adrenergic nerve ending, fractional rates (FR = rate of flux divided by tissue tritium content) were calculated for unidirectional outward diffusion, for outward transport and for neuronal re-uptake (all for 3H-noradrenaline). Although the density of adrenergic innervation is lower in atria than in vasa deferentia, neuronal re-uptake amounted to about 90% of the spontaneous efflux of ³H-noradrenaline in both tissues.While the FR for unidirectional outward diffusion was virtually the same in both tissues, the FR for outward transport of ³H-noradrenaline was more than three times higher in atria than in vasa deferentia. There is, as yet, no explanation for this pronounced difference.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - FR fractional rate (= rate of flux divided by simultaneously determined tritium content of tissue) - FRL fractional rate of loss (= rate of net efflux divided by simultaneously determined tritium content of tissue) - MAO monoamine oxidase     

Comparison of the uptake of [3H]-gabapentin with the uptake of L-[3H]-leucine into rat brain synaptosomes.

1. Gabapentin is a novel anticonvulsant with an unknown mechanism of action. Homogenate binding studies described elsewhere have suggested that [3H]-gabapentin binds to a site in brain similar to the large neutral amino acid (LNAA) uptake site, termed system-L. 2. This study describes an investigation into the uptake of [3H]-gabapentin into a crude synaptosomal preparation from cerebral cortex of rat brain. Characterization studies showed that [3H]-gabapentin is taken up into synaptosomes by a system that is similar to that responsible for the uptake of L-[3H]-leucine. This system is sodium-independent, temperature-sensitive and requires ATP for function. 3. Kinetic studies of [3H]-gabapentin uptake produced a Michaelis constant (KM = 160 microM) similar to that observed for L-[3H]-leucine (KM = 110.3 microM). Vmax values were 837.1 pmol mg-1 protein min-1 and 2.192 nmol mg-1 protein min-1 respectively. 4. Gabapentin and L-leucine mutually inhibit their uptake. Lineweaver-Burke plots of these data demonstrate that inhibition occurs by a competitive mechanism. Further to this the Dixon transformation of the data illustrates that these two substrates share a common uptake site by the similarity between their calculated Ki and KM values (gabapentin inhibition of L-[3H]-leucine uptake: Ki = 160 microM; L-leucine inhibition of [3H]-gabapentin uptake: Ki = 262 microM). 5. Studies into the effect of gabapentin, the system-L-specific ligand 2-(-)-endoamino-bicycloheptane-2-carboxylic acid (BCH), and the system-A-specific ligand alpha-(methyl-amino)-isobutyric acid (MeAIB), on the initial rate of uptake of [3H]-glycine, L-[3H]-glutamate, L-[3H]-glutamine, and L-[3H]-leucine were performed. At 100 microM, gabapentin significantly inhibited initial rate of uptake of [3H]-glycine (29%), L-[3H]-glutamate (22%) and L-[3H]-leucine (40%). 6. Gabapentin is taken up into synaptosomes by a system similar to system-L, responsible for the uptake of large neutral amino acids. Gabapentin will also inhibit the uptake of certain excitatory amino acids in this synaptosomal preparation. The implications of these findings for the mechanism of action for gabapentin are unclear. The data presented here may suggest an intracellular site for mechanism of action for this compound. Similarly changes in levels of amino acid pools may be involved in the mechanism of gabapentin's anticonvulsant action.     

Metabolism of [3H]-(+/-)-isoprenaline by isolated atria and coronary arteries of the kitten

1 Isolated coronary arteries of the kitten accumulated more unchanged isoprenaline and metabolized more amine than atria following incubation for 1 to 20 min with [3H]-(+/-)-isoprenaline (25 ng/ml or 5 micrograms/ml). 2 Cortisol (10 or 80 microM), U-0521 (120 microM) and oxytetracycline (100 microM) all reduced metabolite formation. 3 Cortisol inhibited 'Iso InfluxMin' (cellular isoprenaline accumulation plus total metabolite production). In contrast, it increased, decreased or did not alter accumulation of unmetabolized isoprenaline, depending upon the experimental conditions. 4 Isoprenaline accumulation was increased in atria and reduced in coronary arteries by U-0521, while oxytetracycline reduced accumulation in coronary arteries at the high amine concentration. 5 It is concluded that in atria, cortisol inhibits metabolism and has differential effects on a number of extraneuronal compartments which accumulate isoprenaline. Both cortisol and U-0521 appear to be extraneuronal uptake inhibitors and inhibitors of catechol-O-methyltransferase in coronary arteries. Oxytetracycline may have effects additional to inhibition of isoprenaline binding to connective tissue fibres.     

The effects of hydrocortisone on responses to and extraneuronal uptake of (—)-isoprenaline in cat and guinea-pig atria

1. (—)-Isoprenaline-induced positive inotropic effects were assessed in driven left atria and positive chronotropic effects in spontaneously beating right atria from guinea-pigs and cats. 2. Hydrocortisone produced dose dependent shifts to the left in cumulative concentration-effect curves to (—)-isoprenaline in left and right atrial preparations from the cat. 3. Maximal potentiations were produced by 160 μM and 40 μM hydrocortisone in cat left and right atria respectively. 4. No such effects were seen in similar preparations from the guinea-pig even in the presence of concentrations of hydrocortisone as high as 400 μM. 5. Hydrocortisone inhibited the extraneuronal uptake of [7—³H](—)-isoprenaline in a concentration dependent manner in both left and right atria of the cat, but not the guinea-pig. 6. These results are consistent with the idea that hydrocortisone potentiates responses to (—)-isoprenaline in cat atria by inhibition of the extraneuronal uptake process.     

Bay K 8644 enhances the outflow of [3H]-noradrenaline and [3H]-DOPEG from isolated rat atria

Summary The effect of Bay K 8644 (a dihydropyridine Ca²⁺-channel activator), was examined on spontaneous and stimulus-evoked release of tritium from isolated rat atria prelabelled with [³H]-noradrenaline. Bay K8644 (3mol/l) significantly increased atrial rate from 206±7 to 259±9 beats·min^–1 (P<0.05) and also tritium outflow (expressed as fractional rate of loss in min^– × 103) from 6.49±0.35 to 8.61±0.74 (P<0.05). Neither the maximal rate nor the overflow of tritium induced by stimulation of sympathetic nerve terminals was changed by the compound. The increase in basal tritium outflow produced by Bay K 8644 was calcium-dependent. However, it could not be antagonized by nitrendipine. The overflow of tritium induced by Bay K 8644 consisted mainly of 3,4-dihydroxyphenylglycol ([³H]-DOPEG), indicating that the compound produces a leakage from the storage vesicles of sympathetic nerve terminals of the isolated rat atria.Members of Consejo Nacional de Investigaciones Científicas - Técnicas (CONICET), Argentina Send offprint requests to M. C. Camilión de Hurtado at the above address     

Modification by prostaglandin E2 (PGE2) of the response of guinea-pig isolated vasa deferentia and atria to adrenergic stimuli

1. Prostaglandin E(2) (PGE(2)) exerted positive cardiostimulant effects on isolated guinea-pig atria. The response was not altered by treatment of the animal with reserpine or by addition of propranolol to the organ bath. These results suggest that the cardiostimulatory actions of PGE(2) are not mediated through the release of catecholamines or stimulation of adrenoceptors.2. On the electrically driven atria, PGE(2) consistently exerted a cardiostimulant action which was not appreciably altered by changes in calcium ion in the bathing medium. PGE(2) showed no effect on the transport of calcium by the fragments of heart sarcoplasmic reticulum.3. PGE(2) reduced the responses to both noradrenaline and tyramine in the isolated atria. The shifted dose-response curve was not parallel to the original.4. PGE(2) increased the contractor response of the isolated vas deferens to nerve stimulation or to direct electrical stimulation.5. PGE(2) antagonized the increase caused by noradrenaline in contractor response of isolated vas deferens to direct electrical stimulation, whereas it affected the potentiation by noradrenaline differently when the vas deferens was contracting in response to nerve stimulation. In low concentration it inhibited and in large concentrations, it slightly enhanced the potentiation by catecholamine.6. It is concluded that PGE(2) has actions on multiple sites. It has post-junctional as well as pre-junctional effects on adrenergic neurones.     

On the role of the adrenergic receptors for extraneuronal amine uptake and retention in rat salivary glands in vitro

Summary The effect of an -receptor blocking agent, phenoxybenzamine, and a series of -receptor blocking agents on extraneuronal uptake and retention of radioactivity after incubation of rat salivary gland slices with ³H-noradrenaline or ³H-isoprenaline has been investigated. In some experiments with ³H-noradrenaline as substrate, neuronal uptake was prevented by adding protriptyline to the incubation medium. Phenoxybenzamine reduced extraneuronal accumulation of radioactivity after both ³H-noradrenaline and ³H-isoprenaline in a concentration-dependent manner, whereas after propranolol the levels of extraneuronally retained radioactive material were markedly increased, the efflux from the slices not being diminished. Some other nonselective and selective -receptor blocking agents with and without intrinsic activity were found to produce the same effect as propranolol upon the retention of radioactivity after incubation with ³H-noradrenaline and ³H-isoprenaline. When both phenoxybenzamine and propranolol were present in the incubation medium the effect of extraneuronally retained radioactivity after ³H-noradrenaline as well as after ³H-isoprenaline was the same as when only phenoxybenzamine was used. The metabolic pattern of the radioactivity in the slices revealed that the extraneuronally retained radioactive compounds consisted mainly of tritiated catabolites, especially 3-O-methylated ones. The relationship between the extraneuronal accumulation and adrenergic receptor mechanisms is discussed.     

Concomitant development of [3H]-dopamine and [3H]-5-hydroxytryptamine uptake systems in rat brain regions.

1 Synaptosomal uptake mechanisms of 5-hydroxytryptamine and dopamine were examined in cerebral cortex, corpus striatum and midbrain plus brainstem of developing rats. 2 In all regions, there was generally a parallel biphasic development of both uptake systems; the most rapid increases occurred in the first two weeks postpartum, followed by a slower rate of increase. 3 Kinetic studies with dopamine indicated that the maturation involved increases in maximal uptake without a change in the substrate Km, suggesting that there is a change in the number or terminals but not in the uptake system per se.     

Tryptophan inhibits the [3H]glutamate uptake into Xenopus oocytes injected with rat brain mRNA.

We characterized the glutamate (Glu) uptake in Xenopus oocytes injected with rat brain mRNA. The Glu uptake into oocytes was higher in mRNA-injected oocytes than in vehicle-injected ones. Na+ omission or addition of tryptophan inhibited the uptake in mRNA-injected oocytes, although it did not affect that in vehicle-injected oocytes. These results suggest that Glu transporters with a tryptophan sensitivity different from that of Glu transporters in native oocytes are expressed after injection of rat brain mRNA.     

Trimetazidine increases [3H]glucose uptake in rat brain

Trimetazidine, a clinically effective antianginal agent with no negative inotropic or vascular properties, acts by optimizing cardiac energy metabolism through inhibition of free fatty acid oxidation, shifting substrate utilization from fatty acids to glucose. Up to now there has been no study associating trimetazidine's effect on metabolic processes with glucose utilization in the mammalian brain. The objective of the present study was to determine if trimetazidine altered [(3)H]glucose uptake in rat brain. Adult male Wistar rats were administered trimetazidine (Metazydyna, Polfa) either as a single dose (10.0 mg/kg po) or for 14 consecutive days (5.0 mg/kg po per day) or vehicle saline (2.0 ml/kg po). Sixty minutes after the single dose or 14th dose of trimetazidine, and 15 min before experiment termination and brain dissection, 6-[(3)H]D-glucose (500 Ci/kg ip; Amersham) was administered. Using liquid scintillation counting, trimetazidine, either in a single or multiple dose regimen, was found to increase [(3)H]glucose uptake (DPM/100 mg of wet tissue) in all dissected regions of the brain (i.e., striatum, hippocampus, frontal cortex, thalamus with hypothalamus, pons with medulla oblongata, and cerebellum). Therefore, central effects need to be taken into consideration as possibly adding to known beneficial cardiac effects of trimetazidine.     

Influence of mercury on uptake of [3H]dopamine and [3H]norepinephrine by rat brain synaptosomes

Mercuric compounds have been shown to alter several membrane-bound enzymes and associated receptor activities. The present studies were initiated to investigate the in vitro effects of mercuric chloride (HgCl2) and methylmercury chloride (CH3HgCl) on the uptake of [3H]dopamine (3HDA), [3H]norepinephrine (3HNE), and Na+, K+-ATPase in rat brain synaptosomes. Brain synaptosomes were prepared by the ficoll-sucrose gradient method from normal, adult male Sprague-Dawley rats, weighing approx. 200 g. The effect of mercury on Na+, K+-ATPase was determined by using a coupled enzymatic method. Uptake of DA and NE by brain synaptosomes was determined by filtration in the presence and absence of 0-30 microM HgCl2 and 0-100 microM CH3HgCl. A parallel inhibition in the synaptosomal uptake of 3HDA and 3HNE, and the activity of the synaptosomal membrane Na+, K+-ATPase, was observed in both mercuric chloride and methylmercury treatments. The mercury compounds also significantly inhibited the mitochondrial ATPase (Mg2+-oligomycin-sensitive ATPase). The inhibitory influences of the toxins were concentration-dependent. The results suggest that the mercury compound mediated decrease in DA and NE uptake in brain synaptosomes may be related to the inhibition of Na+, K+-ATPase by the same toxins.     

The role of calcium in the regulation of [H]hemicholinium-3 binding sites in rat brain

The role of calcium in the regulation of sodium-dependent high-affinity uptake of choline was assessed in vitro in slices of the rat brain, by measuring the specific binding of [^3H]hemicholinium-3 ([^3H]HCh-3) and the uptake of [^3H]choline. Depolarization with potassium of slices of hippocampus, cortex, or striatum significantly increased the specific binding of [^3H]HCh-3 when compared to control slices. However, the observed potentiation of specific binding of [^3H]HCh-3 was markedly inhibited by the removal of calcium from the incubation medium in cortex or hippocampus, but not in slices of striatum. Alterations in the uptake of [^3H]choline directly paralleled the observed changes in the specific binding of [^3H]HCh-3 in striatum of the rat and were unaffected by the reduction of calcium in the incubation medium. The inorganic calcium channel antagonists, cadmium and cobalt, but not magnesium, zinc, manganese or lanthanum, significantly inhibited the 40 mM potassium chloride-induced stimulation of the binding of [^3H]HCh-3 in the striatum. Finally, the calcium ionophore A23187 significantly increased the binding of [^3H]HCh-3 in slices of striatum, either in the presence or absence of calcium in the bathing medium. This study demonstrates regional differences in the role of extracellular calcium in the regulation of the uptake of choline and suggests the involvement of intracellular release of calcium in the in vitro regulation of the sodium-dependent high-affinity uptake of choline in the striatum     

Effects of phencyclidine on [3H]catecholamine and [3H]serotonin uptake in synaptosomal preparations from rat brain

Phencyclidine inhibited uptake in vitro of [³H]norepinephrine (ic₅₀ 0.52 μM), [³H]dopamine (ic₅₀ 0.73 μM) and [³H]serotonin (ic₅₀ 0.80 μM) in crude synaptosomal preparations from rat brain through a competitive mechanism. Phencyclidine was fairly similar in potency to d-amphetamine and methylphenidate in inhibiting catecholamine uptake but was 8 times more potent than d-amphetamine and 34 times more potent than methylphenidate in inhibiting [³H]serotonin uptake.     

Effect of cocaine on tritium overflow evoked from vasa deferentia previously loaded with [3H]noradrenaline by stimulation using different types of electrode.

In mouse and guinea-pig vasa deferentia previously incubated with [3H]noradrenaline, electrical stimulation applied through parallel electrodes (transmurally) increased overflow of tritium 2- to 5-fold above the resting value. Electrical stimulation applied using methods involving more substantial conduction of nerve impulses in neuronal elements in the tissues evoked a tritium overflow which was smaller (70%) than that evoked by transmural stimulation. Cinchocaine (25 microM), tetrodotoxin (0.5 microM) or the absence of calcium effectively abolished evoked overflow in both tissues whichever method of stimulation was used. In mouse vas deferens, cocaine (10 microM) did not alter overflow evoked by either transmural or axonal stimulation while 100 microM produced a reduction. In guinea-pig vas deferens, cocaine (10 microM) produced a statistically significant increase in evoked overflow of about 50% or more with both transmural and axonal stimulation. As in mouse vas deferens, 100 microM cocaine produced a reduction. It is concluded that the action of cocaine is independent of these methods of stimulation and that some difference in the arrangement of the noradrenergic nerves in the two species may account for the differential effect of cocaine observed.