Amine Uptake Research Articles

Besipirdine (HP 749) reduces schedule-induced polydipsia in rats

The aim of the present paper is to report on the adrenergic and serotonergic effects of besipirdine (HP 749) in vivo and to discuss its potential use in the treatment of obsessive compulsive disorder. Besipirdine inhibited biogenic amine uptake in vitro. It prevented tetrabenazine-induced ptosis in mice and potentiated the 5-hydroxytryptophan-induced serotonin syndrome in rats. Furthermore, it decreased schedule-induced polydipsic behavior in rats. Schedule-induced polydipsia may be a model for obsessive compulsive disorder. Previous results from our group have shown that certain selective serotonin reuptake inhibitors decrease schedule-induced polydipsia after 14–21 days of treatment. Besipirdine reduced schedule-induced polydipsic behavior immediately and this reduction lasted throughout the duration of the experiment (29 days).

Na(+)- and Cl(-)-dependent neurotransmitter transporters in bovine retina: identification and localization by in situ hybridization histochemistry.

The physiological actions of biogenic amine and amino-acid neurotransmitters are terminated by their removal from the synaptic cleft by specific high-affinity transport proteins. The members of the Na(+)- and Cl(-)-dependent neurotransmitter transporter family expressed in bovine retina and responsible for the uptake of biogenic amine and amino-acid neurotransmitters were identified using a reverse transcriptase-polymerase chain reaction-based approach. cDNA clones encoding bovine homologues of glycine (GLYT-1), gamma-aminobutyric acid (GAT-1), creatine (CreaT), and orphan (NTT4) transporters were identified using this strategy. The expression pattern of mRNAs encoding these proteins in the retina was determined by in situ hybridization histochemistry. GLYT-1, CreaT, NTT4, and GAT-1 mRNAs were expressed in the retina by cells in the inner nuclear, inner plexiform, and ganglion cell layers. They were not expressed at detectable levels in the photoreceptor cells whose cell bodies are in the outer nuclear layer and are the most abundant cell type in the retina. GLYT-1 mRNA was present exclusively in the proximal inner nuclear layer. GAT-1 mRNA was localized to both the inner nuclear and ganglion cell layers. CreaT mRNA was expressed in all cell types in the retina, except photoreceptors, and NTT4 mRNA was expressed by a subpopulation of cells in the ganglion cell layer. Elucidation of the expression pattern of these neurotransmitter transporter mRNAs in the retina provides a basis for studies of the role of glycine, gamma-aminobutyric acid, and creatine transporters in retinal function.

Benzodiazepines as antidepressants: Does GABA play a role in depression?

Benzodiazepines, the most widely prescribed psychotropic drugs, are often used in patients with depressive disorders, either alone or in combination with standard antidepressants. This review evaluates the efficacy of benzodiazepines (alprazolam, diazepam, chlordiazepoxide) as established in acute-phase, randomized controlled trials (RCTs) in major depressive disorder. Metaanalyses using intent-to-treat, as well as adequate treatment exposure samples, revealed an overall efficacy of 47-63% and a drug-placebo difference of 0-27% for all benzodiazepines. Alprazolam, the best studied of the benzodiazepines, had a 27.1% (sd = 6.1%) greater response than placebo, which is comparable to standard antidepressants. Alprazolam, in particular, may be a useful treatment option for patients in whom standard antidepressant medications are contraindicated, poorly tolerated, or possibly ineffective. Alprazolam may have a more rapid onset of action for some patients. Benzodiazepines do not primarily affect biogenic amine uptake or metabolism, although they do augment gamma-amino butyric acid (GABA) activity. The antidepressant efficacy of benzodiazepines, which are GABAA receptor agonists, is consistent with the GABA theory of depression.

FDG PET imaging of paragangliomas of the neck: comparison with MIBG SPET

Two patients with cervical paragangliomas underwent positron emission tomography (PET) with 2-[18F]-fluoro-2-deoxy-D-glucose (FDG). There was marked tumor uptake and retention of FDG. Adjacent salivary gland accumulation of FDG was minimal, though quite prominent with meta-iodobenzylguanidine. FDG PET offers another potentially useful approach to functional imaging of these uncommon tumors, independent of the presence of specific amine uptake mechanisms or cell surface receptors required by other scintigraphic techniques.

Effect of haloperidol and its metabolites on dopamine and noradrenaline uptake in rat brain slices

The effects of haloperidol and its metabolites on dopamine (DA) and noradrenaline (NA) uptake were investigated. Both direct uptake of [3H]DA and [3H]NA into the rat striatal and hippocampus slices and binding of a specific DA uptake inhibitor [3H]GBR-12935 were employed in the present study. Haloperidol pyridinium (HP+), haloperidol 1,2,3,6-tetrahydropyridine (HTP), 4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine (CPTP) and reduced haloperidol (RHAL) are potent inhibitors of DA uptake. HTP N-oxide (HTPNO) exhibits a relatively weak effect on DA uptake. Other metabolites of haloperidol, i.e. 4-(4-chlorophenyl)-4-hydroxypyridine (CPHP) and haloperidol N-oxide (HNO), as well as haloperidol itself possess negligible inhibitory effect on DA uptake. HP+ has been shown to be an amine releaser. It is possible that HP+ may induce amphetamine-like neurotoxicity. The effects of the metabolites of haloperidol on [3H]NA uptake are similar to those on [3H]DA uptake. HP+ appears to be different from MPP+, which is a more potent [3H]NA uptake blocker than on [3H]DA uptake. Although haloperidol exhibits no DA uptake inhibitory effect, it has a high affinity for the [3H]GBR-12935 binding site. The possible pharmacological implications such inhibitory effects on amine uptake are discussed.

α1A‐Adrenoceptor‐mediated contractile responses of the human vas deferens

1. The predominant alpha 1-adrenoceptor mediating contractions of the human vas deferens has been characterised in vitro by use of subtype selective antagonists. 2. Responses of human epididymal vas deferens were obtained to phenylephrine in the presence of amine uptake inhibitors and propranolol. The effects of the alpha 1-adrenoceptor antagonists, 5-methylurapidil, oxymetazoline, WB4101, prazosin and chloroethylclonidine were examined and also the L-type calcium channel blocker, nifedipine. 3. 5-Methylurapidil, WB4101, oxymetazoline and prazosin acted as competitive antagonists of the responses to phenylephrine, yielding pA2 values of 8.8, 9.2, 7.7 and 8.8 respectively. All four antagonists produced Schild plots with slopes similar to unity and maximum responses to phenylephrine were not altered in the presence of any of the antagonists. 4. Tamsulosin (1 nM) caused rightward shifts of phenylephrine concentration-response curves yielding an apparent pKB value of 10.0. However, maximum responses were also reduced by 51% with this concentration of antagonist. 5. Incubation of tissues with chloroethylclonidine (100 microM for 40 min) failed to alter responses significantly but the presence of nifedipine (1 microM) reduced maximum responses to phenylephrine by 32%. 6. The high affinity of 5-methylurapidil, oxymetazoline and WB4101, together with the failure of chloroethylclonidine to antagonize responses, indicate that the predominant alpha 1-adrenoceptor mediating contraction of the human vas deferens has the characteristics previously described for the pharmacologically-defined alpha 1A-adrenoceptor. The data are also consistent with those described for the cloned alpha 1c-adrenoceptor subtype thereby supporting the hypothesis that the two receptors are identical. The human vas deferens therefore represents a readily accessible preparation for functional studies of the human alpha 1A-adrenoceptor.

Mice Transgenic for Copper/Zinc Superoxide Dismutase Exhibit Increased Markers of Biogenic Amine Function

Mice that are transgenic for and overexpress human copper/zinc superoxide dismutase were used to investigate the role of this enzyme in the pathophysiology of Down's syndrome (DS; trisomy 21). Previous studies have indicated that overexpression of copper/zinc superoxide dismutase leads to deficits in peripheral markers of neurochemical function, which are consistent with the hypothesis that this enzyme plays a role in the pathophysiology of DS. We have measured concentrations of amino acids and biogenic amines (catecholamines, serotonin, and their metabolites), uptake of biogenic amines into crude synaptosomes, and activities of synthetic enzymes in both control mice and mice transgenic for human copper/zinc superoxide dismutase that overexpress it by two- to fivefold above control values. We find that these transgenic mice exhibit higher concentrations of the biogenic amines in specific brain regions, with little or no change in amino acid concentration. Furthermore, tyrosine hydroxylase activity is increased in the striatum of the transgenics, whereas glutamic acid decarboxylase and choline acetyltransferase activities are unchanged in all but one brain region. These findings indicate that over-expression of copper/zinc superoxide dismutase, by itself, is not sufficient to cause the synaptic neurochemical deficits reported in DS.

Vesicular monoamine uptake by digitonin-permeabilized PC12 cells: inhibitory effect of neuromodulators and drugs on the amine transport.

Vesicular amine transport is crucial for activity of monoaminergic neurons since translocation of cytosolic amine to storage vesicles is required for both exocytotic release and reuptake of the neurotransmitter. We developed a convenient assay system for vesicular amine transport based on permeabilizing the plasma membrane of pheochromocytoma PC12 cells with digitonin at concentrations of 100 to 150 microM. Serotonin (5HT) is a better substrate than either epinephrine or norepinephrine in this assay system. In the presence of 2 mM ATP, 5HT uptake by the permeabilized cells increased linearly for at least 30 min at 25 degrees C, whereas without addition of exogenous ATP, 5HT uptake reached an apparent plateau after 20 min incubation. Reserpine (500 nM) completely blocked the ATP-dependent 5HT uptake in this system whereas nomifensine (10 microM) had no effect, indicating specificity for vesicular transport. Treatment of intact PC12 cells with AMP dose-dependently decreased 5HT uptake with an EC50 of 20-30 microM as measured after cell permeabilization. Treatment of the intact PC12 cells with forskolin and phorbol 12-myristate 13-acetate prior to permeabilization also down-regulated vesicular 5HT transport, whereas the addition of either of these agents into the reaction mixture for the amine uptake by already permeabilized cells did not alter the vesicular uptake activity. Thus, the system can be applied to studies on regulation of the vesicular amine transport by physiological signaling molecules, intracellular messengers, and drugs.

The effect of bile duct manipulation and pair-feeding on peripheral vascular neuroeffector mechanisms: In vitro studies

Investigators studying the pathophysiological sequelae of bile duct ligation use different species of laboratory animals at varying postoperative times. There is also considerable variation in the type of control animal used for these experiments. In this study, we have attempted to validate our choice of the 3-day bile-duct-manipulated rat as the most appropriate control to study peripheral vascular neuroeffector mechanisms in bile-duct-ligated rats. We have compared the in vitro contractile response to norepinephrine in the absence and presence of cocaine, and the accumulation of the amine using 3H-norepinephrine of arterial rings and portal veins prepared from three different types of control rats—unoperated control, the 3-day bile-duct-manipulated and the 3-day pair-fed, bile-duct-manipulated rats. In vitro arterial reactivity to norepinephrine in the sham-operated rats was significantly attenuated and was associated with a cocaine-sensitive increase in norepinephrine uptake. Portal veins from the same animals showed no changes in in vitro reactivity to norepinephrine, although bile-duct manipulation and pair-feeding enhanced amine uptake. This study has demonstrated that bile-duct manipulation and pair-feeding attenuate in vitro vascular reactivity and enhance norepinephrine uptake. These in vitro changes are more pronounced in arterial tissue than venous tissue. In conclusion, these data indicate that bile-duct manipulation is the control of choice when measuring in vitro vascular neuroeffector mechanisms in 3-day bile-duct-ligated rats. Furthermore, these data emphasize the need to validate the control when experiments involving bile-duct ligation are undertaken.

Effects of protein kinase inhibitors and protein phosphatase inhibitors on cyclic AMP-dependent down-regulation of vesicular monoamine transport in pheochromocytoma PC12 cells

Cyclic AMP down-regulates vesicular monoamine transport in PC12 cells and thereby decreased catecholamine reuptake from the extracellular fluid. We examined the effects of protein kinase inhibitors and protein phosphatase inhibitors on this cAMP action. Treatment of cells with a protein kinase inhibitor, K252a, increased vesicular amine transport and cellular amine uptake, thereby antagonizing the regulatory action of cAMP. In contrast, a protein phosphatase inhibitor, okadaic acid, had the opposite effect on the amine transport, i.e. it enhanced the cAMP action. These results suggest the involvement of a protein phosphorylation process in the cAMP-dependent modulation of vesicular monoamine transport.

Subcellular distribution of receptor sites in human brain: Differentiation between heavy and light structures of high and low density

Studies of the subcellular localization of neuroreceptors in the rat brain have shown that most of them are associated with light and low density subcellular fractions. In two human brain areas, quite different subcellular distributions were observed. After fractionation by differential centrifugation of frontal cortex homogenates, benzodiazepine and serotonin 5-HT 2 receptors were mainly found in the heavy mitochondrial (M) fraction, whereas μ-opiate and muscarinic cholinergic receptors were mainly concentrated in the microsomal (P) fraction. In human putamen, the presynaptic markers of dopaminergic nerve terminals (neurotensin receptors, dopamine uptake sites and amine vesicular transporter-binding sites), benzodiazepine receptors and serotonin uptake sites were recovered both in the high and low density fractions, whereas the muscarinic, opiate and, to a lesser extent, dopamine D 2 receptors were mostly concentrated in the microsomal fraction. In the cerebral cortex, after isopycnic centrifugation in sucrose gradients, neuroreceptors were found in the high density fractions where the peaks of cytochrome oxidase and that of nerve endings, as identified by amine uptake and by means of electron microscopy were also found. A single peak of benzodiazepine receptors was observed in high density (1.15–1.17 g/ml) fractions suggesting that these receptors are much more concentrated in the nerve terminals or dendrites rather than in the dendritic spines or vesicles. The fact that muscarinic and opiate receptors were recovered in the P fraction with plasma membrane constituents and also in M and L fractions, which is confirmed by a bimodal distribution in sucrose gradient, suggests that they are localized in both the nerve terminals or dendrites and in the small vesicles or dendritic spines. In the putamen, much of the specific binding to uptake sites for dopamine and serotonin was recovered in the high density fractions, but the existence of another peak at a lower density indicates the presence of microsomal uptake sites. The results indicate that differential and isopycnic fractionation methods performed on human brain samples, make it possible to separate tissue fractions enriched in nerve endings, dendrites, dendritic spines, plasma membranes or vesicles.

In vitro effects on monoamine uptake and release by the reversible monoamine oxidase-B inhibitors Lazabemide and N-(2-aminoethyl)- p-chlorobenzamide: A comparison with l-deprenyl

To investigate whether the reversible monoamine oxidase-B (MAO-B) inhibitors lazabemide and Ro 16–6491 have any additional effect on monoamine uptake and release, in vitro experiments were performed on rat forebrain synaptosomes and blood platelets. The effects of the two drugs were compared with those of l-deprenyl, the well-known irreversible MAO-B inhibitor which is reported to affect amine uptake. Both lazabemide and Ro 16–6491 behaved as weak inhibitors of [ 3H]monoamine uptake by synaptosomes, with a similar rank order of potency for amine uptake inhibition (noradrenaline (NA) ⩾ 5-hydroxytryptamine (5 HT) dopamine (DA)). The ic 50 values for lazabemide and Ro 16–6491, respectively, were: 86 μM and 90 μM for NA uptake; 123 μM and 90 μM for 5HT uptake; > 500 μM and > 1000 μM for DA uptake. l-Deprenyl (rank order of inhibitory potency: NA > DA > 5 HT) was four to 10 times more potent than either compound in inhibiting [ 3H]catecholamine uptake (IC 50 = NA 23 μM, DA 109 μM), and two to three times less potent in inhibiting 5 HT uptake ( ic 50 233 μM). Lazabemide and Ro 16–6491 also differed from l-deprenyl in their ability to induce release of endogenous monoamines from synaptosomes. Thus, Ro 16–6491 (500 μM) induced a greater 5 HT release than did l-deprenyl, but was less effective than l-deprenyl in releasing DA. On the contrary, lazabemide was almost completely inactive on either 5 HT and DA release. The differential effect of the three MAO-B inhibitors on synaptosome 5 HT uptake and release was confirmed by [ 14C]5HT uptake and liberation experiments with isolated rat platelets. The data indicate that the reversible MAO-B inhibitors lazabemide and Ro 16–6491 at relatively high concentrations possess amine uptake-inhibiting properties. With regard to the effects examined, lazabemide markedly differs from l-deprenyl since it does not interfere with DA uptake nor induce amine release from synaptosomes.

Role of vesicular dopamine in the in vivo stimulation of striatal dopamine transmission by amphetamine: Evidence from microdialysis and Fos immunohistochemistry

The role of vesicular and newly synthesized dopamine in the action of amphetamine was investigated by studying the effect of reserpine and alpha-methyl-p-tyrosine pretreatment on amphetamine-induced changes in extracellular dopamine and acetylcholine, estimated by brain microdialysis, and on c-fos expression, estimated by quantitative immunohistochemistry of the Fos antigene, in the dorsal caudate-putamen of rats. Blockade of dopamine synthesis by alpha-methyl-p-tyrosine pretreatment (1 or 2 h) only partially prevented the increase in extracellular dopamine concentrations elicited by 0.5 and 2 mg/kg s.c. of amphetamine. Inactivation of vesicular amine uptake by reserpine pretreatment (3 h) reduced the increase in extracellular dopamine by 2 mg/kg but not by 0.5 mg/kg of amphetamine. Combined pretreatment with reserpine (3 h) and alpha-methyl-p-tyrosine (1 h) drastically reduced the increase in extracellular dopamine by both doses of amphetamine (0.5 and 2 mg/kg s.c.). alpha-Methyl-p-tyrosine pretreatment reduced c-fos expression stimulated by amphetamine (2 mg/kg) in the dorsomedial and dorsolateral caudate-putamen while reserpine pretreatment reduced it only in the dorsolateral caudate-putamen. Amphetamine (2 mg/kg s.c.) stimulated acetylcholine release but this effect was not modified by reserpine or alpha-methyl-p-tyrosine pretreatment. The results indicate that blockade of dopamine synthesis, by itself, is insufficient to prevent the stimulation of dopamine transmission by amphetamine and, conversely, that inactivation of vesicular dopamine significantly reduces amphetamine effects at pre- and postsynaptic levels. Therefore, vesicular dopamine appears to contribute to the stimulation of dopamine transmission elicited by amphetamine in the dorsal caudate-putamen.

Platelet serotonin transport is altered in streptozotocin-induced diabetic rats

The present work was conducted to examine whether experimental diabetes (streptozotocin-induced) promotes changes in mean platelet volume, and platelet serotonin (5HT) uptake and content. These variables were measured in from four experimental groups: control, diabetic, diabetic+insulin, and non-diabetic+insulin. Animals treated fifteen days before with streptozotocin had platelets with higher 5HT uptake affinity, 5HT content, and volume. The insulin therapy reestablished the control values of all of these three variables. Non-diabetic animals treated one week with insulin did not show any variations. The effects of in vitro application of insulin, hyperglycaemic incubation medium, and streptozotocin on platelet amine uptake and release were also examined. Only those platelets incubated with streptozotocin showed an altered platelet 5HT uptake. No changes were observed for spontaneous 5HT release. The results are consistent with: a) an increase of platelet uptake capacity, as a consequence of an increase in platelet turnover, for explaining alterations of intraplatelet 5HT contents in experimental diabetes; b) a non-direct effect of insulin and glucose levels on platelet 5HT uptake -for explaining its dysfunctions in experimental diabetes-; c) the contribution of alterations in platelet 5HT transport for explaining the higher incidence of vascular complications in diabetic patients; d) the suitability of platelet as a model for investigating neuronal 5HT reuptake.

ATP enhances catecholamine uptake into PC12 cells

Catecholamine uptake into PC 12 cells, in the presence and absence of ATP, was measured in a bicarbonate-buffered Krebs Ringer. ATP enhanced significantly the uptake in a dose-dependent manner with maximal uptake at 1–3 mM. ATP increased the Vmax of uptake by 3–5 fold for both dopamine and norepinephrine, without a significant change in the Km. ATP-stimulated amine uptake was temperature- and Na +- dependent and robust in bicarbonate, but not in HEPES buffer. The ability to enhance uptake was not observed with metabolites of ATP. GTP and UTP were equally effective to ATP in enhancing CA uptake. This uptake was less sensitive to uptake inhibitors in bicarbonate buffered media than in phosphate-buffered media, and more so in the presence of ATP. It is suggested that ATP is an allosteric modulator of the transporter and that a stable ATP analog, with ATP-like enhancement of dopamine uptake, may be an effective cocaine antagonist.

Cross‐linking of poly(methyl methacrylate) by aminolysis of ester functions with diamines

AbstractThe cross‐linking of poly(methyl methacrylate) (PMMA) macromolecules by primary aliphatic diamines was carried out at 413–473 K, above the polymer glass transition temperature. The spherical grains of the suspension‐prepared polymer (0.25–0.8 mm) and the molar ratio to amine above 20 were used. The reaction is characterized by an induction period before the macroscopic gel was observed and by fast network formation. Both phenomena are temperature‐dependent (87± 2 kJ mol−1). The rate of gelation is dependent also on the type of diamine; the shorter induction period of gelation and the higher rate of cross‐linking were observed when amino groups were separated with longer alkyl chains. From the kinetics of amine uptake with PMMA, however, it can be concluded that gel formation is influenced also by the reactivity of amine in the transamination reaction and by the amount of bounded diamine. Aminolysis of PMMA ester groups proceeds from the very beginning of heating of the reaction charge. The polymer cross‐linking prevails when there is more than 60% of diamine attached to PMMA in dependence on the amine type used. © 1995 John Wiley & Sons, Inc.

In vitro perfusion studies of human resistance artery function in essential hypertension.

To simulate in vivo conditions as closely as possible to in vitro conditions, we examined the morphological and functional characteristics of isolated human subcutaneous small arteries from 17 essential hypertensive patients and 14 normotensive control subjects using a perfusion myograph. Vessel segments were cannulated and exposed to conditions of constant flow and pressure. The ratio of media thickness to lumen diameter in arteries from hypertensive patients increased significantly. With the endothelium intact, sensitivity to extraluminally applied norepinephrine was not different, and this was not affected by inhibition of neuronal amine uptake with cocaine. After removal of the endothelium, sensitivity to norepinephrine was augmented in normotensive vessels to a greater extent than in hypertensive vessels. Endothelium-dependent relaxation to acetylcholine was significantly reduced in arteries from hypertensive patients, but endothelium-independent relaxation to sodium nitroprusside was not different from that observed in vessels from normotensive control subjects. These data demonstrate that sensitivity to exogenous norepinephrine is not different in essential hypertension but that there is defective endothelium-dependent dilatation, suggesting a contributory role for endothelium dysfunction in human essential hypertension.

Discovery of potential antidepressant agents: Novel 3-arylpyrrolidines with dual mechanism of action on neurotransmission

Abstract

3-(Methyleneaminoxy)methylpiperidine derivatives as uptake inhibitors of biogenic amines in the brain synaptosomal fraction

A series of 3-(methyleneaminoxy)methylpiperidines ( 5a–h) and their corresponding N-methyl derivatives ( 6a–h) with a variety of substituents on the imino carbon were synthesized and tested for their potential antidepressant properties; their capacity to inhibit the re-uptake of biogenic amines (NA, 5-HT and DA) in rabbit brain synaptosomal fractions was also evaluated. The biological results obtained for the piperidine derivatives 5a–h and 6a–h and viloxazine 1, the reference drug, on the 3 re-uptake systems revealed that compounds 5 and 6 are generally able to inhibit biogenic amine uptake. The IC 50 values for 5 and 6 were often lower than that of viloxazine 1, in particular for the serotonin- and/or dopamine-uptake systems. A higher activity was found for compounds substituted with at least one phenyl ring on the imino carbon with respect to completely aliphatic systems, and for N-unsubstituted compounds with respect to N-methyl-substituted compounds.

The pharmacodynamic effects of antidepressant drugs. A short review

Resent research on the mechanisms of action of antidepressant treatment (ADT) has led to major revision of earlier concepts. The monoamine deficiency hypothesis seems to be too simplistic, as it does not account for the great temporal discrepancy between the rapid drug-induced biochemical effects of amine uptake and catabolism, which occurs within hours, and the antidepressant response, which occurs after at least 7–14 days. The pharmacodynamic effects of ADT are obviously complex involving a great number of interacting transmitter and receptor systems in the central nervous system. Secondary adaptive changes in neuronal systems seem to play an integral and possibly pivotal role in antidepressant drug action. Our knowledge about the details is sparse, primarily because most investigations on the subject are based on animal studies or in vitro technics, whereas the number of clinical investigations of the antidepressant drug effect on the living human organism is limited. This fact may explain the often-fo...