Retention of indoles in the pineal organ of the bird (Melopsittacus undulatus, Shaw) during its preparation for radioautographic study

Since high-resolution radioautography (dipping technique) might be very useful for the study of indole metabolism in the pineal cells, the retention of [3H]-indoles has to be examined during the preparation of specimens for electron microscopy (EM). The pineal organ of the parakeet (Melopsittacus undulatus) was used in the present work. 1) Indole metabolism: following uptake of [3H]-5-hydroxytryptophan ([3H]-HW) in vivo and [3H]-5-hydroxytryptamine ([3H]-HT) in vitro in similar seasonal and nycthemeral conditions--all the known pineal indolic metabolites were recovered by thin layer chromatography. [3H]-5-methoxyindoles were also formed from [3H]-melatonin ([3H]-aMT). 2) The radioactivity of fluids used in the processing of pineal organs in EM was determined by liquid scintillation counting: (a) no exogenous [3H]-indoles could be revealed in EM solutions after [3H]-HW in vivo uptake. (b) 8.8 to 13.4% of [3H]-indoles were washed out by glutaraldehyde after [3H]-HT in vitro uptake. (c) most of the 5-methoxyindoles after in vitro uptake of [3H]-aMT were lost in glutaraldehyde. Our chromatography procedures did not permit the identification of [3H]-indoles extracted by the glutaraldehyde fixative. In previous experiments, [3H]-HW and [3H]-HT uptake showed the presence of selective radioautographic reactions in the cells of the receptor line; however, silver grains were scarce and diffusely distributed in the pineal parenchyma after [3H]-aMT uptake.     

Effect of Different Photoperiod Exposure on [3H]Imipramine Binding and Serotonin Uptake in the Rat Brain

Seasonal rhythmicity in the occurrence of acute depressive episodes and the therapeutic efficacy of light exposure suggest the possible involvement of the pineal gland or other biological oscillators in the pathophysiology of depressive illness. We have performed studies to clarify whether different light/dark (LD) cycle schedules may induce changes in the biochemical targets of antidepressants in the rat CNS. In particular, we have investigated the effect of short- (LD 8:16) or long-day (LD 14:10) photoperiods on different biochemical parameters of serotonergic neurons. A significant increase in the density of [3H]imipramine ([3H]IMI) binding and in the Vmax of 5-[3H]hydroxytryptamine (5-[3H]HT) uptake was found in the hypothalamus of LD 8:16-with respect to LD 14:10-exposed rats, whereas no difference was found in the kinetic properties of postsynaptic 5-HT receptors and in 5-HT metabolism in the hypothalami and cerebral cortices of rats exposed to the two different photoperiods. A seasonal rhythm of [3H]IMI binding sites and 5-HT uptake seems to exist only in certain brain areas, such as the hypothalamus, because no differences were found in the cerebral cortex of LD 14:10- and LD 8:16-accustomed rats. [3H]IMI binding and 5-HT uptake were significantly increased in the hypothalamus of rats accustomed to a light/dark-inverted cycle (DL 10:14) and killed 6 h after the stopping of lighting in comparison to rats exposed to normal LD 14:10 cycles and killed 6 h after the beginning of lighting. Therefore, a circadian modification of the serotonergic presynaptic sites seems to be present and related to light/dark exposure. Because the existence of endogenous compounds able to modulate [3H]IMI binding and 5-HT uptake, other than 5-HT, has been postulated in the mammalian brain, the involvement of these substances in the periodic changes observed could be suggested.     

Effects of 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine and Related Compounds on the Uptake of [3H]3,4-Dihydroxyphenylethylamine and [3H]5-Hydroxytryptamine in Neostriatal Synaptosomal Preparations

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) is known to cause a destruction of the dopaminergic nigrostriatal pathway in certain animal species including mice. MPTP and some structurally related analogs were tested in vitro for their capacity to inhibit the uptake of [3H]3,4-dihydroxyphenylethylamine-([3H]DA), [3H]5-hydroxytryptamine ([3H]5-HT), and [3H]gamma-aminobutyric acid [( 3H]GABA) in mouse neostriatal synaptosomal preparations. MPTP was a very potent inhibitor of [3H]5-HT uptake (IC50 value 0.14 microM), a moderate inhibitor of [3H]DA uptake (IC50 value 2.6 microM), and a very weak inhibitor of [3H]GABA uptake (no significant inhibition observed at 10 microM MPTP). In other experiments, MPTP caused some release of previously accumulated [3H]DA and [3H]5-HT, but in each case MPTP was considerably better as an uptake inhibitor than as a releasing agent. The 4-electron oxidation product of MPTP, i.e., 1-methyl-4-phenyl-pyridinium iodide (MPP+), was a very potent inhibitor of [3H]DA uptake (IC50 value 0.45 microM) and of [3H]5-HT uptake (IC50 value 0.78 microM) but MPP+ was a very weak inhibitor of [3H]GABA uptake. These data may have relevance to the neurotoxic actions of MPTP.     

Effects of 2-substituted-4-phenylquinolines on uptake of serotonin and norepinephrine by isolated brain synaptosomes

In this present communication, the in vitro inhibition of the uptake of [3H]-L-norepinephrine ([3H] NE) and [3H]-Serotonin ([3H] 5-HT) by eleven synthesized 2-substituted-4-phenyl quinolines were studied using rat brain synaptosomal preparations. Compounds with an open side chain were relatively weak inhibitors of the synaptosomal uptake of [3H] NE and [3H] 5HT. Compounds having a distance of three atoms between the terminal basic nitrogen of the side chain and the quinoline ring were better inhibitors of serotonin uptake than those compounds having a four-atom distance. The replacement of the side chain with a piperazine ring produced compounds which were more potent and selective inhibitors of the uptake of either [3H] 5-HT or [3H] NE. Further structure-activity relationships are also discussed.     

Regional Differences in 5-Hydroxytryptamine and Catecholamine Uptake in Primary Astrocyte Cultures

The uptake of 3H-labelled 5-hydroxytryptamine (5-HT, serotonin) norepinephrine ([3H]NE), and 3,4-dihydroxyphenylethylamine ([ 3H]dopamine, [3H]DA) was studied in primary astrocyte cultures prepared from the cerebral cortex, corpus striatum, and hippocampal regions of neonatal rat brain. Na+-dependent uptake showed marked regional differences. For [3H]5-HT the magnitude of uptake was corpus striatum greater than or equal to cerebral cortex greater than hippocampus, whereas for [3H]NE the order was hippocampus greater than corpus striatum greater than cerebral cortex. For [3H]DA, only the hippocampal cultures showed significant Na+-dependent uptake. [3H]5-HT uptake was specifically inhibited by 10(-7) M fluoxetine whereas [3H]NE uptake was preferentially inhibited by 10(-7) M desipramine. These results may reflect regional brain specialization and/or different developmental patterns of high affinity uptake of serotonin and catecholamines by astrocytes in situ.     

H-Pro-[3H]Leu-Gly-NH2: Uptake and Metabolism in Rat Brain

The uptake and metabolism of H-Pro-[3H]Leu-Gly-NH2 ([3H]PLG) in rat brain was investigated by reverse-phase paired-ion high pressure liquid chromatography. Following in vitro incubation of [3H]PLG with rat brain subcellular preparations, the microsomal-cytosol fraction was about twice as active in degrading PLG as the crude mitochondrial-synaptosomal fraction. For both enzyme preparations the pH optimum was found at pH 7-7.5. The major labeled metabolite was [3H]leucine, whereas 3H]labeled Leu-Gly-NH2 as the only labeled peptide intermediate was found in trace amounts. After intravenous injection of [3H]PLG the uptake of unmetabolized peptide in the brain appeared to be very low: 0.008% and 0.001% of the administered dose/g tissue at 2 and 5 min after injection respectively, while at longer survival times intact peptide was below the detection limit. Compared with the intravenous route of administration, intracerebroventricular injection of [3H]PLG yielded much higher brain concentrations of unmetabolized PLG. Following both routes of administration, the metabolite profile was in agreement with that obtained after in vitro incubation. However, the in vivo experiments also showed considerable incorporation of [3H]leucine liberated from [3H]PLG into proteins. Both the in vitro and in vivo results indicate that the initial cleavage of PLG in rat brain occurs at the NH2-terminus and that the dipeptide intermediate H-Leu-Gly-NH2 is subsequently hydrolyzed to its constituent amino acids very rapidly.     

Characterization of carrier-mediated efflux in human embryonic kidney 293 cells stably expressing the rat serotonin transporter: a superfusion study

Human embryonic kidney 293 cells stably transfected with the rat plasmalemmal serotonin transporter (rSERT) were incubated with 5-[3H]hydroxytryptamine ([3H]5-HT) and superfused. Substrates of the rSERT, such as p-chloroamphetamine (PCA) or methylenedioxymethamphetamine, concentration-dependently increased basal efflux of [3H]5-HT. 5-HT reuptake blockers (e.g., imipramine, citalopram) also caused an enhancement of [3H]5-HT efflux, reaching about half the maximal effect of the rSERT substrates. In uptake experiments, both groups of substances concentration-dependently inhibited 5-HT uptake. EC50 values obtained in superfusion experiments significantly correlated with IC50 values from uptake studies (r2 = 0.92). Addition of the Na+,K(+)-ATPase inhibitor ouabain (100 microM) to or the omission of K+ from the superfusion buffer accelerated basal efflux. The effect of PCA (10 microM) was markedly enhanced by both measures, whereas the effect of uptake inhibitors remained unchanged. When [3H]MPP+, a substrate with low affinity for the rSERT, was used instead of [3H]5-HT for labeling the cells, uptake inhibitors failed to augment efflux. By contrast, PCA accelerated [3H]MPP+ efflux, and its effect was strongly enhanced in the presence of ouabain. The results suggest that the [3H]5-HT efflux caused by substrates of rSERT is carrier-mediated, whereas efflux induced by uptake inhibitors is a consequence of interrupted high-affinity reuptake that is ongoing even under superfusion conditions.     

Sodium Dependence of [3H]]Paroxetine Binding and 5- [3H]]Hydroxytryptamine Uptake in Rat Diencephalon

The sodium dependence of binding of [3H]-paroxetine, a selective serotonin uptake inhibitor, to the serotonin transporter in rat diencephalon was studied in both brain membranes and tissue sections and compared with that of 5-[3H]hydroxytryptamine ([3H]5-HT) uptake by synaptosomes from the same region. Binding of [3H]-paroxetine in both the membranes and sections displayed clear sodium dependence until a plateau occurring at 60 nM NaCl, the EC50 for sodium being 8 and 25 mM, respectively. The affinity (1/KD) of [3H]paroxetine binding was a simple hyperbolic function of sodium concentration. In contrast, the density of [3H]paroxetine sites was not affected by external Na+ concentration. The uptake of [3H]5-HT showed a similar pattern of sodium dependence with an EC50 for Na+ of 25 mM. Both the affinity (1/Km) and the rate (Vmax) of [3H]5-HT uptake were dependent on external [Na+] with sodium-dependence curves fitting a rectangular hyperbola. The kinetic analysis of results indicates that one sodium ion is required for the binding of [3H]paroxetine as well as for the binding and translocation of each [3H]5-HT molecule. The results concur with a single-site model of the sodium-dependent serotonin transporter with common or overlapping domains for 5-HT and 5-HT uptake inhibitors.     

Selective effect of microembolization on pulmonary removal of biogenic amines

Pulmonary amine extraction (E) was measured by triple-indicator dilution techniques from bolus injections of trace amounts of 5-hydroxy[14C]tryptamine ([14C]HT)m [3H]norepinephrine ([3H]NE), indocyanine green dye before and after glass-bead embolization in 23 anesthetized dogs. Control E(5-[14C]-HT) was 89.7 +/- 1.7%; 10 min after embolization (which approximately doubled pulmonary artery pressure and pulmonary vascular resistance), E(5-[14C]HT) was significantly reduced to 65.9 +/- 3.0% (kappa +/- SE; n = 10) (P less than 0.01). Control E([3H]NE) (40.1 +/- 4.5%) was unaffected by embolization. Imipramine (8 mg/kg) depressed control E(5-[14C]HT) to 38.7 +/- 1.5% and control E([3H]NE)d to 35.0 +/- 3.9% (P less than 0.05; n = 4). In these animals, pulmonary hemodynamic changes secondary to embolization were comparable to those in non-drug-treated dogs, but E(5-[14C]HT) and E([3H]NE) were not further depressed. Progressive pulmonary lobar artery ligation (n = 5) did not affect amine extraction until perfusion was limited to one lobe. The selectivity of the effect of embolization on E(5-[14C]HT), the lack of an effect on imipramine-insensitive E(5-[14C]HT) extraction, and the much smaller changes after progressive lobar ligation indicate that, although derecruitment of vascular surface area secondary to mechanical obstruction may contribute to postembolization depression of E(5-[14C]HT), additional mechanisms such as local saturation of 5-HT uptake or selective damage to endothelial cell transport of 5-HT may underlie these observations.     

Evidence that [3H]Dopamine Is Taken Up and Released from Nondopaminergic Nerve Terminals in the Rat Substantia Nigra In Vitro

Potassium chloride (25 mM) and (+)-amphetamine (100 microM) both stimulated the release of radioactivity from slices of substantia nigra preincubated with [3H]3,4-dihydroxyphenylethylamine [( 3H]dopamine). Potassium chloride (25 mM) released radioactivity from slices of both zona compacta and zona reticulata. Prior 6-hydroxydopamine (6-OHDA) lesions of one nigrostriatal pathway did not reduce the spontaneous release of radioactivity, or the potassium chloride- or amphetamine-induced release of radioactivity from slices of nigra ipsilateral to the lesion after preincubation with [3H]dopamine. The accumulation of radioactivity following incubation of nigral slices from 6-OHDA-lesioned animals with [3H]dopamine was increased when compared to uptake into slices from intact tissue. In synaptosomal preparations of striatum, nomifensine but not desipramine or fluoxetine inhibited [3H]dopamine uptake. In contrast, nomifensine, desipramine, and fluoxetine all inhibited [3H]dopamine uptake in nigral synaptosomal preparations. Following 6-OHDA lesions of one nigrostriatal pathway the uptake of [3H]dopamine into nigral synaptosomal preparations was unchanged but uptake into striatal preparations was substantially decreased. In contrast, bilateral electrolesions of the dorsal and medial raphe nuclei reduced [3H]dopamine uptake into nigral preparations but not into striatal synaptosomes. The uptake of [3H]5-hydroxytryptamine ([3H]5-HT) into synaptosomal preparations of substantia nigra was abolished by fluoxetine and reduced by desipramine, but was unaffected by nomifensine. In contrast, fluoxetine, desipramine, and nomifensine all inhibited [3H]5-HT uptake into striatal synaptosomal preparations. Following 6-OHDA lesions of one nigro-striatal pathway the uptake of [3H]5-HT into nigral synaptosomal preparations was unchanged but uptake into striatal preparations was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

One-step purification of the serotonin transporter located at the human platelet plasma membrane.

A 68-kDa glycoprotein bearing the biological activity of the plasma membrane serotonin (5-hydroxytryptamine, 5-HT) transporter has been purified from human blood platelets, a classical cell model for the study of 5-HT uptake. After treatment of the whole platelet population or its plasma membrane fraction by sulfhydryl-dependent bacterial protein toxins or by digitonin, purification was reproducibly obtained by a one-step affinity chromatography using two different columns with 5-HT or 6-fluorotryptamine as ligands and elution by 5-HT or Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa and exhibited an apparent isoelectric point of 5.6-6.2. Two sialic acid residues were detected in the purified material. The purified glycoprotein bound the 5-HT uptake blocker [3H]paroxetine with a Kd (0.25 nM) similar to the one observed for intact human platelets. It also bound [3H] 5-HT but neither [3H]hydroxytetrabenazine nor [3H] ouabain, the respective markers of the granular monoamine transporter and of the Na+,K(+)-ATPase associated to the plasma membrane 5-HT transporter. 5-HT derivatives and 5-HT uptake inhibitors exhibited similar Ki values for 5-HT uptake and paroxetine binding in intact human platelets and in the purified glycoprotein. Under laser UV irradiation, 40% of this purified glycoprotein could be labeled by either [3H]paroxetine or [3H]cyanoimipramine. No labeling was detected with either [3H] gamma-aminobutyric acid or [3H]GBR 12783, the respective markers of gamma-aminobutyric acid and dopamine carriers. The purified 68-kDa protein is therefore likely to correspond at least to the binding domain of the 5-HT transporter located at the human platelet plasma membrane.     

5-HT uptake blockade prevents the increasing effect of K(ATP) channel blockers on electrically evoked [3H]-5-HT release in rat and mouse neocortical slices

To explore if prolonged--as opposed to acute--5-HT uptake blockade can lead to changes in the function of ATP-dependent potassium (K(ATP)) channels, we investigated in rat and mouse neocortical slices the effects of K(ATP) channel blockers on electrically evoked [3H]-serotonin ([3H]-5-HT) release after short- and long-term exposure to 5-HT uptake blockers. Glibenclamide (1 microM), a K(ATP) channel blocker, enhanced the electrically evoked [3H]-5-HT release by 66 and by 77%, respectively, in rat and in mouse neocortex slices. This effect was confirmed in the rat by tolbutamide (1 microM), another K(ATP) channel antagonist. After short-term blockade (45 min) of 5-HT uptake, glibenclamide still increased the release of [3H]-5-HT in the rat. Glibenclamide, however, failed to enhance [3H]-5-HT release after long-term uptake blockade (210 min). In the mouse, however, both short- and long-term inhibition of 5-HT reuptake by citalopram (1 microM) prevented the facilitatory effect of glibenclamide. The Na(+)/K(+)-ATPase inhibitor ouabain (3.2 microM) abolished the glibenclamide-induced increase in [3H]-5-HT release in both rat and mouse, suggesting that an operative Na(+)/K(+)-ATPase is a prerequisite for activation of K(ATP) channels. The terminal 5-HT(1B) autoreceptor-mediated feedback control was involved in the glibenclamide-induced increase in [(3)H]-5-HT release only in mouse neocortical tissue, as evident from the use of the 5-HT(1B) autoreceptor ligands metitepin (1 microM) and cyanopindolol (1 microM). These results suggest that in the rat long-term uptake blockade leads to an impaired activity of the Na(+)/K(+)-ATPase, which increases intracellular ATP and consequently closes K(ATP) channels. In the mouse, however, short-term uptake blockade seems to already reduce the activity of the Na(+)/K(+)-ATPase and thereby the consumption of ATP. Blockade of 5-HT transporters thus may close K(ATP) channels through increased intracellular ATP. The following slight depolarisation seems to facilitate 5-HT release. These results may contribute to a better understanding of the mechanisms involved in the clinical time latency of antidepressant efficacy of monoamine uptake blockers.     

Allosteric Interaction Between the Site Labeled by [3H]Imipramine and the Serotonin Transporter in Human Platelets

The nature of interaction between the site labeled by [3H]imipramine (IMI) and the 5-hydroxytryptamine (5-HT, serotonin) transporter in human platelets was examined. The sulfhydryl characterizing agent N-ethylmaleimide (NEM) differentially affected [3H]5-HT uptake and [3H]IMI binding in human platelet preparations. Concentrations of NEM that completely abolished [3H]5-HT uptake only minimally reduced [3H]IMI binding. Examining the effect of IMI on the kinetics of human platelet [3H]5-HT uptake revealed significant reductions in maximal velocity (Vmax) without altering affinity (Km). IC50 values for selected uptake blockers on [3H]IMI binding and [3H]5-HT uptake were determined. IC50 values of these compounds for uptake and binding revealed that agents such as IMI, chlorpromazine, amitriptyline, and nisoxetine were preferential inhibitors of [3H]IMI binding whereas fluoxetine, CL 216, 303, pyrilamine, and bicifadine were preferential [3H]5-HT uptake blockers. 5-HT was a weak displacer of [3H]IMI binding (IC25 = 3.0 microM) and exhibited a rather low Hill coefficient (nH app = 0.46). Results reported herein support the notion of an allosteric interaction between the [3H]IMI binding site and the 5-HT transporter complex in human platelets.     

Uptake and release of [3H]-serotonin in photophores of the midshipman fish, Porichthys notatus

A kinetic analysis of [3H]-5-HT uptake in the photocytes of the photophores of Porichthys notatus revealed a high affinity (Km: 1.71 X 10(-7] and low affinity component (Km: 1.10 X 10(-5) M). The high affinity uptake was sodium- and potassium-dependent but largely insensitive to temperatures between 0 and 20 C. Ouabain (5 X 10(-3) M) and dinitrophenol (10(-3) M) reduced uptake significantly. DMI, imipramine and fluoxetine, in that order of potency, greatly inhibited [3H]-5-HT uptake. Noradrenaline and adrenaline reduced uptake in a non-competitive manner, while dopamine, tryptophan, 5-hydroxytryptophan and Cypridina luciferin had little or not effect on uptake. Non-facilitated luminescent responses to electrical stimulation were accompanied by release of [3H]-5-HT accumulated in the photocytes. Facilitatory luminescence excitation consistently failed to induce the release of [3H]-5-HT. Electrical and adrenaline (10(-5) M) stimulation of photophores after [3H]-5-HT release has occurred, failed to elicit any additional luminescent response. The photophores were responsive to KCN (10(-3) M) under these conditions. The results indicate that a specific carrier-mediated transport system is responsible for photocytic [3H]-5-HT uptake, and that release of photocytic [3H]-5-HT is stringently regulated and followed by inhibition of luminescence excitability.     

Heterogeneity,position, and functional capability of the macrophages in Peyer's patches

Summary Radioenzymatic assays and light microscope radioautographic studies performed on photophores of Porichthys notatus demonstrated (1) significant amounts of catecholamines (dopamine, noradrenaline, adrenaline) and 5-hydroxytryptamine (serotonin) in these organs; (2) selective uptake and storage of [³H]noradrenaline ([³H]NA) by axon terminals innervating the photocytes, and (3) strong accumulation of [³H]5-hydroxytryptamine ([³H]5-HT) within the photocytes. Uptake and storage of [³H]NA in the nerve fibers were seemingly unaffected by the addition of ten-fold molar concentrations of unlabelled serotonin. Accumulation of [³H]5-HT by the photocytes was dose-dependent and diminished markedly in the presence of ten-fold molar concentrations of non-radioactive noradrenaline. Neither neuronal uptake of [³H]5-HT or [³H]A, nor photocytic accumulation of [³H]A were detectable under the conditions of the present experiments. This information should provide a framework for further investigations of the regulation of photophore luminescence by the biogenic amines.Supported by grants from the National Research Council and Medical Research Council of CanadaJacques de Champlain and Lise Farley provided facilities and expertise with the radioenzymatic techniques. The technical assistance of Sylvia Garcia and Marie-Hélène Parizeau was also appreciated     

Further Studies on the Nature of Postsynaptic Dopamine Uptake and Metabolism in Rat Striatum: Sodium Dependency and Investigation of a Possible Role for Carrier-Mediated Uptake into Serotonin Neurons

The nature of postsynaptic sites involved in the uptake and metabolism of striatal 3,4-dihydroxyphenylethylamine (dopamine, DA) was investigated. The accumulation of [3H]DA (10(-7) M) into slices of rat striatum was found to be greatly dependent (greater than 99%) on the presence of sodium ion in the incubation medium. However, the formation of the [3H]dihydroxyphenylacetic acid (DOPAC) and [3H]homovanillic acid (HVA) was only partially reduced in the absence of sodium (DOPAC, 27% of control; HVA, 47% of control). Inhibition of carrier-mediated DA neuronal uptake with nomifensine (10(-5) M) significantly decreased DA accumulation (18% of control) and [3H]DOPAC formation (62% of control), but enhanced [3H]HVA production (143% of control). Inhibition of the 5-hydroxytryptamine (5-HT, serotonin) neuronal uptake system with fluoxetine (10(-6) M) or selective 5-HT neuronal lesions with 5,7-dihydroxytryptamine (5,7-DHT) had no effect on [3H]DOPAC or [3H]HVA formed from [3H]DA in the presence or absence of nomifensine. These results demonstrate that the uptake and subsequent metabolism of striatal DA to DOPAC and HVA is only partially dependent on carrier-mediated uptake mechanism(s) requiring sodium ion. These data support our previous findings suggesting a significant role for synaptic glial cell deamination and O-methylation of striatal DA. Further, experiments with fluoxetine or 5,7-DHT suggest that 5-HT neurons do not significantly contribute in the synaptic uptake and metabolism of striatal DA.     

Uptake, Release, and Subcellular Localization of l-Methyl-4-Phenylpyridinium in Blood Platelets

The neurotoxic compound 1-[methyl-3H]-4-phenylpyridinium ([3H]MPP+) was actively taken up by human, rabbit, and guinea pig platelets incubated in plasma. In human platelets, the apparent Km of this uptake (22.6 microM) was 50 times higher than that for serotonin [5-hydroxytryptamine (5-HT]). The uptake of [3H]MPP+ by human platelets was inhibited by selective 5-HT uptake blockers [cianopramine, (-)-paroxetine, and clomipramine], by metabolic inhibitors (KCN and ouabain), and by drugs that interfere with amine storage in the 5-HT organelles (reserpine, mepacrine, and Ro 4-1284). Impairment of the transmembrane proton gradient by ionophores (monensin and nigericin) induced a marked release of radioactivity from platelets preincubated with [3H]MPP+. Fractionation of homogenates of rabbit platelets preincubated with [3H]MPP+ showed that the drug was concentrated to a great extent in the 5-HT organelle fraction. MPP+ competitively inhibited [14C]5-HT uptake by human platelets and reduced the endogenous 5-HT content of human, rabbit, and guinea pig platelets. These investigations show that MPP+ is transported into the platelets via the 5-HT carrier and is accumulated predominantly in the subcellular organelles that store 5-HT and other monoamines. It is suggested that an accumulation of MPP+ in amine storage vesicles of neurons may be involved in the effects of the drug in the CNS, e.g., by protecting other subcellular compartments from exposure to high concentrations of MPP+, by sustaining a gradual release of the toxin, or both.     

Metabolic and pharmacokinetic activity of the isolated sheep bronchial circulation

We sought to determine bronchial vascular metabolic and pharmacokinetic activity toward benzoyl-Phe-Ala-Pro (BPAP), ADP, adenosine, and prostaglandin E2 (PGE2) by developing an isolated sheep bronchial circulation preparation. We measured mean transit time (t), uptake, and metabolism by injecting 3H-labeled substrates with [14C]sucrose into the bronchial artery of sheep lungs stripped clean of parenchymal tissue. After [3H]BPAP the t for 3H was the same as for 14C. Thirty-six percent of the injected BPAP was converted to metabolite ([3H]benzoyl-Phe) in a single pass. An inhibitor of angiotensin-converting enzyme, SQ 20,881, depressed BPAP metabolism by 50%, while perfusion of the bronchial circulation with glutaraldehyde reduced metabolism to a basal level. After [3H]ADP the t for 3H was again the same as for 14C. 3H recovery after 40 pmol [3H]ADP was less (58%) than after 400 nmol [3H]ADP (79%). Twenty-two percent of the injected radioactivity emerged in the effluent as metabolites of ADP for either dose. Adenosine and PGE2 uptake was negligible, and most of the recovered radioactivity in each case was unchanged substrate. This study suggests that the bronchial circulation is pharmacokinetically and metabolically active with respect to vasoactive mediators like angiotensin I, bradykinin, and adenine nucleotides, and that the enzymes responsible for this metabolic activity line the vascular lumen.     

Identification of 5-Hydroxytryptamine1A Receptor Proteins in Bovine Frontal Cortex

5-Hydroxytryptamine1A (5-HT1A) receptor proteins were identified by a novel approach in which photoaffinity labeling technique was used in conjunction with affinity column chromatography. 5-HT1A receptors were solubilized from bovine frontal cortical membranes with 0.3% digitonin and 0.1% Nonidet P-40, and bound effectively to 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP)-coupled Affi-Gel 10 in a time-dependent manner. PAPP was shown previously to be a selective ligand for the 5-HT1A receptor. Two protein bands with molecular masses of approximately 55,000 and 38,000 daltons revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were eluted from the affinity column with either 1 mM 5-HT or 1 microM [3H]1-[2-(4-azidophenyl)ethyl]-4-(3-trifluoromethyl-phenyl)piperazine ([3H]p-azido-PAPP). [3H]p-Azido-PAPP is a selective photoaffinity labeling probe for the 5-HT1A receptor. The intensity of these two protein bands and the incorporation of [3H]p-azido-PAPP into these two proteins decreased significantly when the solubilized fraction was preincubated with excess 5-HT or PAPP (saturating all 5-HT1A receptors) prior to affinity column chromatography. These results suggest strongly that these two proteins are related to the 5-HT1A receptor protein. The isoelectric points of the photolabeled 5-HT1A receptor proteins were 6.0 and 6.5.     

5-Methoxytryptoline, a Competitive Endocoid Acting at [3H]Imipramine Recognition Sites in Human Platelets

5-Methoxytryptoline potently inhibits [3H]imipramine binding to membranes from the cerebral cortex and platelets. Since 5-methoxytryptoline, which appears to occur endogenously with particularly high levels in the human pineal gland, also inhibits 5-hydroxytryptamine (5-HT, serotonin) uptake, it should be considered as a putative endogenous ligand modulating 5-HT transport. As the 5-HT transporter complex comprises the imipramine and the substrate recognition sites, which interact allosterically, it was essential to define the mechanism of inhibition of [3H]imipramine binding by 5-methoxytryptoline. Human platelets show an active and saturable uptake of 5-HT and tryptamine. The uptake of both substrates appears to be mediated by the same carrier and it is inhibited by 5-methoxytryptoline at submicromolar concentrations. 5-HT and tryptamine inhibit [3H]imipramine binding in human platelets with a Hill slope for inhibition close to unity and IC50 values of 3,265 and 3,475 nM, respectively. This inhibition is, however, not competitive because both 5-HT and tryptamine significantly decrease the rate of [3H]imipramine-receptor dissociation. Although 5-methoxytryptoline potently inhibits [3H]imipramine binding (IC50 = 44 nM) in human platelets with a Hill slope of unity, it does not affect the receptor-ligand dissociation rate of [3H]imipramine even at concentrations up to 100 microM. The present experiments show that 5-methoxytryptoline, in spite of its chemical similarity to the indoleamine transporter substrates, interacts with the imipramine receptor through a mechanism of competitive inhibition. This conclusion is supported by a selective effect of 5-methoxytryptoline on the Kd of [3H]imipramine binding.(ABSTRACT TRUNCATED AT 250 WORDS)