Amine Uptake Research Articles

Major dopamine innervation of the cortical motor areas in the Cynomolgus monkey. A radioautographic study with comparative assessment of serotonergic afferents

Dopamine (DA) and serotonin (5-HT) innervations were revealed by radioautography in primary motor, premotor and supplementary motor (SMA) cortex in Cynomolgus monkeys, using uptake of tritiated amines in vibratome sections under specific conditions that were previously established. DA and 5-HT axons were distributed throughout all cortical layers. A denser DA innervation was found in layers 1, III and to a lesser extent layer V with a striking cluster-like arrangement in layer III, particularly in the SMA. 5-HT axons appeared less numerous; their lower density in comparison with DA axons was especially apparent in layer III, particularly in the premotor and motor areas. A DA-5-HT complementarity was thus suggested in this layer. These results suggest that in addition to the motor control exerted through the nigrostriatal pathway, the cortical DA projections could directly modulate the neuronal activity in motor areas. This could be of major importance in the pathophysiology of motor disorders such as Parkinson's disease.

Amine uptake into intact mast cell granules in vitro.

Histamine, the principal amine of rat peritoneal mast cells, is taken up into isolated granules with intact membranes. Uptake is pH- and concentration-dependent and is not stimulated by the addition of Mg2+-ATP. The saturable uptake has a Km of 91.1 microM and a Vmax of 95.4 pmol (mg of protein)-1 min-1. Uptake is abolished by 5 mM ammonium ion. 5-HT, the other endogenous amine of the granules, and dopamine and tyramine, which do not occur naturally in rat mast cells, each competitively inhibits [3H]-histamine uptake with Ki's close to 1 microM. Reserpine, a putative amine carrier blocker, inhibits uptake at nanomolar concentrations. At high concentrations, uptake of [3H]-5-HT is nonsaturable; at low concentrations, a saturable component is observed with a Km of 1.6 microM. Uptake of [3H]-5-HT is not enhanced by Mg2+-ATP. It is pH-dependent but with a lower apparent pKa than that of histamine. [3H]-5-HT uptake can be completely inhibited by ammonium ions. Amine inhibition of [3H]-5-HT gives nonlinear Dixon plots, and high concentrations of the competing amines or reserpine cannot completely block uptake. We propose a model consistent with these results in which amine uptake occurs by several distinct saturable transport systems. According to the model, histamine is transported by a single system, which also transports 5-HT and dopamine. 5-HT and dopamine are transported by one or more other systems.

Amine trapping: Physical explanation for the inhibitory effect of gastric acidity on the postprandial release of gastrin: Studies on rats and dogs

We investigated the relationship between the pH dependence of meal-stimulated gastrin release and the permeability of the antral mucosa to dietary amines. This study was undertaken after previous work from our laboratory had demonstrated that dietary amines are potent in vivo and in vitro stimulants of gastrin release and that it is well established that amines are trapped in acidic environments. Three contrasting experimental model systems were employed to investigate the association of these two pH-dependent properties. In the first in vivo study, it was demonstrated that ingestion of standard rat Chow resulted in an increase in circulating gastrin and ammonia levels, whereas the postprandial increases in both properties were abolished if the rats ingested Chow that was preacidified to a pH of 2.4. Second, the antral uptake of the fluorescent cyclic amine, quinacrine, from the gastric lumen of pylorus-ligated rats was monitored by fluorescent microscopy and spectrophotometry and was demonstrated to be inhibited in a step-wise fashion as the luminal pH was decreased. Lastly, our in vitro studies suggested that the transport of [14C]methylamine into canine antral mucosa mounted in Ussing chambers was pH-dependent, as was gastrin release into the incubation medium. Thus, all the data are consistent with the possibility that meal-stimulated gastrin release is inhibited at low pH, due (in part) to the protonation of dietary amines, preventing their diffusion into the G cell to activate hormone secretion.

Amine trapping: Physical explanation for the inhibitory effect of gastric acidity on the postprandial release of gastrin

Abstract We investigated the relationship between the pH dependence of meal-stimulated gastrin release and the permeability of the antral mucosa to dietary amines. This study was undertaken after previous work from our laboratory had demonstrated that dietary amines are potent in vivo and in vitro stimulants of gastrin release and that it is well established that amines are trapped in acidic environments. Three contrasting experimental model systems were employed to investigate the association of these two pH-dependent properties. In the first in vivo study, it was demonstrated that ingestion of standard rat Chow resulted in an increase in circulating gastrin and ammonia levels, whereas the postprandial increases in both properties were abolished if the rats ingested Chow that was preacidified to a pH of 2.4. Second, the antral uptake of the fluorescent cyclic amine, quinacrine, from the gastric lumen of pylorus-ligated rats was monitored by fluorescent microscopy and spectrophotometry and was demonstrated to be inhibited in a step-wise fashion as the luminal pH was decreased. Lastly, our in vitro studies suggested that the transport of [ 14 C]methylamine into canine antral mucosa mounted in Ussing chambers was pH-dependent, as was gastrin release into the incubation medium. Thus, all the data are consistent with the possibility that meal-stimulated gastrin release is inhibited at low pH, due (in part) to the protonation of dietary amines, preventing their diffusion into the G cell to activate hormone secretion.

Effect of diclofensine, a novel antidepressant, on peripheral adrenergic function.

Diclofensine is a novel antidepressant with equipotent inhibitive effects on the neuronal uptake of norepinephrine (NE), serotonin, and dopamine. It is devoid of monoamine-releasing or monoaminoxidase-inhibiting properties. The action of diclofensine on peripheral neuronal adrenergic function was studied through tests of the blood pressure response to NE, tyramine, and phenylephrine (PE). The blood pressure response to NE was enhanced and that to tyramine was decreased by diclofensine, as a result of its inhibitive action on peripheral neuronal amine uptake. PE sensitivity was also enhanced by diclofensine as well as after a single dose of desmethylimipramine (DMI). In contrast to the common opinion that PE does not interact with the neuronal uptake mechanism, the increase in PE-induced blood pressure response after diclofensine and DMI suggests that PE, a nonbiogenic amine, does indeed enter into the peripheral adrenergic neuron. This neuronal uptake may probably be unmasked only by powerful NE uptake inhibitors such as DMI or diclofensine.

Amine transport in isolated rat mast cells treated with betaadrenoceptor blocking drugs.

Histamine and serotonin uptake in isolated rat mast cells is decreased in the presence of betaadrenoceptor blocking (BAB) drugs. Such inhibition is dose-dependent and is evidently higher for the inhibition of serotonin uptake. The most potent drugs were the highly lipophilic compounds Kö 1124 and propranolol. No difference was found among the various BAB drugs in their ability to decrease histamine uptake. It is evident from the ratio between histamine and serotonin liberation on one side, to the uptake of both amines in mast cells on the other side, that with the exception of Kö 1124, all investigated BAB drugs possesses higher inhibition of extracellular amine uptake in comparison with histamine and serotonin liberation. Such an effect might be a result of a primary interaction of BAB drugs with mast cells at the plasma membrane.

Inhibition of mono amine oxidase in monoaminergic neurones in the rat brain by irreversible inhibitors

The irreversible inhibition of monoamine oxidase (MAO) inside and outside monoaminergic neurones in the rat brain by the suicide inhibitors clorgyline, selegiline ( l-deprenyl), pheniprazine, phenelzine, iproniazid, pargyline and the d- and l-enantiomers of tranylcypromine was determined. This was achieved by incubating crude synaptosomal preparations of hypothalamus and striatum from rats treated with the inhibitors 24 hr earlier, with low concentrations of [ 14C]serotonin (0.1 μM), [ 14C]-noradrenaline (0.25 μM) and [ 14C]dopamine (0.25 μM) in the absence and presence of selective uptake inhibitors (citalopram, maprotiline and amfonelic acid, respectively). It was found that all inhibitors inhibited the deamination of serotonin and noradrenaline outside the amine neurons at slightly lower doses than that within these neurones. This could at least in part be due to protection of MAO by the endogenous amines in these neurones. The deamination of dopamine was rather more strongly inhibited inside the neutrons than outside, particularly at higher doses. There was no indication that tranyl-cypromine or phenelzine was accumulated in the neurones by the membranous amine uptake mechanisms. The rate of the recovery of the deaminating activities inside and outside the serotonergic and noradrenergic neurones in hypothalamus after phenelzine and clorgyline inhibition was the same (50% recovery after 12–15 days), which indicates similar rate of synthesis of MAO in different cell types.

AGING AND THE CARDIOVASCULAR SYSTEM

What has preceded has been largely a listing of contradictory data: what follows is an attempt to gleam a pattern from all of this. In general, responsiveness is either reduced or unchanged by aging; reduced responsiveness has been demonstrated most frequently for cardiac beta-adrenoreceptors, cardiac muscarinic receptors, vascular beta-adrenoreceptors and vascular alpha 2-adrenoreceptors. A reduced ability of the amine uptake system has been demonstrated in some but not all studies: any reduction in re-uptake would tend to potentiate the effects of NA and counteract the reduced receptor responsiveness (or vice versa). This may explain why the most consistent cardiovascular alteration reported in the elderly is an increased plasma NA. While there are clinical reports that beta-blockers and converting enzyme inhibitors are less effective in elderly hypertensives at lowering blood pressure, this may reflect more the pathological development of hypertension with time rather than a true aging phenomenon. Overall, it appears that resting function of the cardiovascular system is near normal in the aged, but since the mode of control is somewhat altered, in particular with a blunted baroreflex, perturbations in the system produced by drugs may cause a higher incidence of adverse effects.

3H-5-hydroxytryptamine accumulation by rat brain synaptic vesicles in a membrane-impermeant medium, and selective reduction by 5,7-dihydroxytryptamine.

In order to examine possible selectivity of amine uptake by synaptic vesicles, the ATP-stimulated accumulation of 3H-5-hydroxytryptamine (5HT) by synaptic vesicles from rat whole brain was examined in a medium comprised largely of membrane-impermeant anions (d-tartrate). Such media have previously been shown to stabilize vesicular accumulation of several neurotransmitters. Accumulation of 3H-5HT did not occur in tartrate medium alone, but was increased biphasically with increasing concentrations of both potassium phosphate and potassium bicarbonate. At optimal concentrations of each anion (10 mM), stable accumulation of 3H-5HT was observed at 37 degrees (26.1 +/- 1.2 pmol/mg protein; Km 6 X 10(-7) M), which was reduced by greater than 95% in the absence of K2ATP, at 4 degrees C, in the presence of 10(-6) M reserpine, or in the presence of the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Uptake was significantly antagonized by millimolar concentrations of Na+, Mg++ or Cl-, but was unaffected by ouabain (10(-5) M). Pretreatment of animals with 5,7-dihydroxytryptamine (5,7-DHT) (200 micrograms, intraventricular) 10 days prior to sacrifice reduced endogenous 5HT levels by 70%, while levels of endogenous norepinephrine (NE) and dopamine (DA) were unaffected. Accumulation of 3H-5HT, examined in the presence of 10(-6) M NE to block 3H-5HT accumulation by vesicles from noradrenergic nerve endings, was reduced by 40% in vesicles from treated animals. Vesicular accumulation of 3H-(-)-NE and 3H-DA was unaffected by 5,7-DHT treatment. The data suggest the possibility of preferential accumulation of 3H-5HT by vesicles arising from serotonergic nerve endings.

Effects of various tricyclic antidepressants on amine uptake

A comparative study of the ability to block the amine pump was carried out on tricyclic antidepressants including dothiepin and northiaden in vivo. Dothiepin was found to prevent the 6-OHDA-induced depletion of cardiac noradrenaline but not the PCA-induced depletion of intracerebral serotonin. Northiaden, an active metabolite of dothiepin, also possessed the same ability with a greater potency than the parent drug. Neither compound affected the biosynthesis of catecholamines and indoleamine. However, neither dothiepin nor northiaden affected 5-HT uptake, as was also observed with imipramine and amitriptyline. These results suggest that the clinical of dothiepin may be due to inhibition of the amine pump, especially of the catecholamine uptake mechanism, which is qualitatively the same as for imipramine and amitriptyline.

Interaction of paraquat and amine uptake by rat lungs perfused in situ.

The kinetics of [14C]paraquat (N,N-di[14C]methyl-4,4'-bipyridylium) uptake from the pulmonary circulation were investigated in rat lungs perfused in situ. During the 1st h of exposure to the herbicide paraquat entered the lungs primarily by diffusion; no evidence was obtained from concentrative uptake, saturation kinetics (1-3,500 microM paraquat), or inhibition by the amines methyl-glyoxal bis(guanylhydrazone) or spermidine, both of which were expected to compete for paraquat transport sites. In contrast, after 60 min of exposure, uptake rates increased two- to three-fold, and paraquat was accumulated to an apparent intracellular concentration greater than that in the perfusate. The latter phase of paraquat uptake was saturable and was inhibited by methylglyoxal bis(guanylhydrazone); it did not appear to reflect a progressive paraquat-induced alteration in cellular permeability but rather predominance of a rapid, carrier-mediated uptake pathway.

New dopaminergic terminal fields in the motor, visual (area 18b) and retrosplenial cortex in the young and adult rat. Immunocytochemical and catecholamine histochemical analyses

New dopaminergic terminal fields have been visualized in the rat cerebral neocortex, using two morphological methods based on distinct properties of the dopaminergic system: presence of the first synthetic enzyme, tyrosine hydroxylase, and high-affinity uptake of amines. Tyrosine hydroxylase was used as an immunocytochemical marker after destruction of the cortical noradrenergic system, induced either neonatally by 6-hydroxydopamine or later on by DSP4, and controlled by the absence of dopamine β-hydroxylase immunoreactivity. The uptake and storage of exogenous amines in tissue sections, in the presence of selective high-affinity transport inhibitors, enabled the specific visualization of the dopaminergic system with fluorescence histochemistry. A dopaminergic innervation of low density was observed along a dorsal sagittal strip which extended from the genu of corpus callosum until about 2 mm behind the splenium and encompassed several distinct cytoarchitectonic areas in the sensorimotor and visual cortex (medial and lateral agranular field, area 18b), as well as in discrete zones of the retrosplenial granular 29c,b. and agranular 29d areas. The distribution of these dopaminergic fields suggested a columnar organization. Several characteristics of the dopaminergic innervation were similar to that of the superficial anterior cingulate cortex (area 24): the laminar distribution to the superficial I–III layers, the secondarily developed varicose aspect in catecholamine fluorescence histochemistry and the delayed postnatal ingrowth in contrast with the early prenatal dopaminergic input to the prefrontal cortex. These similarities suggested that the subpopulation of dopaminergic neurons which provides projections to the anterior cingulate cortex could also contribute to the motor and visual cortex and thus play a role in sensorimotor integration. The predictive value of these results in the ascent of the phylogenetic scale are further considered.

The effects of ethanol on the efflux and release of norepinephrine and 5-hydroxytryptamine from slices of rat hypothalamus

Studies were done to determine the effects of ethanol on release of norepinephrine, dopamine and 5-hydroxytryptamine from nerve terminals in the central nervous system. Superfused slices of rat hypothalamus were used in these studies and endogenous amines in the superfusate were quantitated using HPLC with electrochemical detection. In these experiments ‘release’ of transmitters was studied in the presence of amitriptyline to block neuronal uptake of amines, whereas ‘efflux’ was measured in its absence. A highly intoxicating concentration of ethanol (69.6 mM, 320 mg%) increased the K+-evoked release of norepinephrine, dopamine and serotonin without affecting basal release. Since this concentration of ethanol increased the basal efflux but not the basal release of 5-hydroxytryptamine, it appeared that neuronal uptake of 5-hydroxytryptamine under basal conditions may also be inhibited by intoxicating levels of ethanol.

The release of endogenous dopamine from rat striatum during incubation in vitro

In this study the conditions for quantitative estimation of the spontaneous and KCl-stimulated release of endogenous dopamine from rat striatum during in vitro incubation have been investigated. KCl concentrations of 15–35 mM produced increasing stimulation of dopamine release during 20 min of incubation in vitro, while KCl concentrations of 45 and 60 mM produced no further significant increase in release. In the presence of 4.7 or 20 mM KCl, dopamine release was linear throughout a 20 min period of incubation; in the presence of 25 or 60 mM KCl, however, dopamine release was not linear, reaching a maximum within 8 min of incubation. Three structurally unrelated inhibitors of monoamine uptake were shown to enhance the net spontaneous release of endogenous dopamine at low concentrations, but to inhibit release at high concentrations. The ethanol-induced enhancement of striatal dopamine release was used to demonstrate the importance of an appropriate concentration of an amine uptake inhibitor in the incubation media. It is concluded that the incubation of rat striatal tissues in vitro, coupled with amine analysis by high performance liquid chromatography with electrochemical detection, constitutes an effective technique for the quantitative estimation of endogenous dopamine release, providing appropriate concentrations of a reuptake inhibitor and a monoamine oxidase inhibitor are included in the incubation media. Where rates of KCl-stimulated amine release with time are to be determined, however, a low concentration of KCl must be used, or the tissues must be subjected to incubation for short periods of time.

Effects of amitriptyline and imipramine on brain amine neurotransmitter metabolites in cerebrospinal fluid.

The effects of amitriptyline (AMI) or imipramine (IMI) on levels of 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) (the major brain metabolites of the neurotransmitters norepinephrine [NE], serotonin [5-HT], and dopamine [DA]) in cerebrospinal fluid were determined in 66 subjects with unipolar and bipolar depression. There were significant reductions in MHPG and 5-HIAA levels for the depressed group taken as a whole, but levels of HVA did not change significantly. The changes were similar when subjects were grouped as treated with AMI and IMI and with unipolar and bipolar depression. Reductions in MHPG and 5-HIAA levels were greater in women than in men. In all subjects with depression and in those treated with AMI and IMI, amine metabolite changes did not differ significantly between those who had a positive clinical response to drug therapy and those who did not. Responders with bipolar depression had smaller reductions in MHPG levels than did responders with unipolar depression. The similar effects of AMI and IMI on MHPG and 5-HIAA differ from the dissimilar effects of the two drugs on NE and 5-HT amine uptake systems reported in animal and in in vitro studies. Results provide conclusive evidence of the effects of AMI and IMI on noradrenergic and serotonergic (but not dopaminergic) systems in patients with depression.

Biogenic amine uptake by nerve cords from the American cockroach and the influence of amidines on amine uptake and release

Uptake of 5-hydroxytryptamine (5-HT) by isolated whole nerve cords of the American cockroach, Periplaneta americana (L.), involved a dual component system, with one component consisting of rapid active uptake and the other of passive diffusion. Using high performance liquid chromatography with electrochemical detection, it was shown that nerve cords contained 5-HT levels of about 350 ng/g and an equivalent amount of 5-hydroxyindoleacetic acid (5-HIAA), a 5-HT metabolite not previously reported in cockroach nerve cords. Amidines had no discernable effect on uptake of 5-HT or octopamine by nerve cords or on endogenous levels of 5-HT and 5-HIAA in cords.

Microinjection of calmodulin antibodies into cultured chromaffin cells blocks catecholamine release in response to stimulation

Polyclonal monospecific antibodies raised in sheep against rat testis calmodulin demonstrated cross-reactivity with bovine adrenal medullary chromaffin cell calmodulin. This antibody immunoprecipitated a [ 35S]methionine-labelled protein from chromaffin cell extracts prepared from [ 35S]methionine prelabelled cells that comigrated on a sodium dodecylsulfate gel electrophoresis system with calmodulin. In addition, an excess of non-radioactive exogenous calmodulin was shown to readily compete with this labelled endogenous protein for the antibodies' binding sites. Erythrocyte ghosts were used as vehicles for microinjecting either preimmune immunoglobulin G or anti-calmodulin immunoglobulin G into chromaffin cells following a polyethylene glycol-induced cell fusion procedure. The efficiency of ghost cell fusion was monitored and found to be 43.6 ± 1% ( n = 33). Cell morbidity subsequent to fusion and microinjection was negligible (87.8 ± 0.6% of the total cell population were viable cells; n = 33) as determined by the Trypan Blue exclusion test. The delivery of intact antibodies raised against calmodulin directly into the cytoplasm of cultured chromaffin cells by erythrocyte ghost-mediated microinjection, inhibited catecholamine output in response to stimulation by either acetylcholine (10 −4M) or a depolarizing concentration of potassium (56 mM). However, under these conditions, the chromaffin cell's ability to accumulate exogenous catecholamines through a high affinity uptake system, as well as the kinetic parameters that characterize this uptake mechanism remained unaltered. Furthermore, microinjection of preimmune immunoglobulin G did not modify either catecholamine uptake or stimulation-induced amine release from chromaffin cells. It therefore appears that calmodulin may play a role in the process of stimulus-secretion coupling in the chromaffin cell in culture while it is of little significance to the high affinity amine uptake mechanism.

Indirect adrenergic effect of histamine in cat right atrium

1. 1. Superfused cat atrial fragments preincubated with [ 3H]noradrenaline showed an increased tritium release under the action of 10 −4 M histamine. 2. 2. Reserpine pretreatment of the animals, external calcium removal or the presence of 10 −6 M cocaine, 10 −6 M diphenhydramine or 10 −3 M colchicine in the superfusion medium significantly reduced this effect of histamine (10 −4 M). 3. 3. Diphenhydramine (10 −5 M) also decreased the release of tritium brought about by 10 −6 M tyramine. 4. 4. These results suggest that histamine may release neurotransmitter from cardiac sympathetic nerve endings by means of an exocytotic process and needs to enter the nerve terminals through the amine uptake system.

Histamine uptake in pig platelets and isolated dense granules

Histamine, a major constituent of the amine-storage organelles in pig platelets, is taken up by intact platelets in only trace amounts under conditions where 70% of 14C-serotonin is accumulated. Thrombin caused the release of 70–90% of endogenous histamine but only 5–10% of the newly absorbed 3H-amine; however, after 18 hr 30% of the 3H-amine could be specifically released by thrombin. Isolated storage organelles accumulated histamine in a reserpine-sensitive, ATP-dependent manner but at a rate 80–100-fold less than serotonin uptake. Incubation of intact platelets with 1 mM serotonin until amine uptake was saturated caused no changes in platelet histamine content. Similarly, loading of isolated storage organelles with 1 mM histamine or 1 mM serotonin did not affect the levels of the other amine. These results suggested that the storage of each amine is independent of the other. Histidine decarboxylase was not detected in platelet lysates. Since platelets have a short half-life (1–2 weeks) and pig plasma levels of histamine are higher than in other animals, it is concluded that most of the histamine in the storage organelles is probably accumulated in the platelet precursor, the megakaryocyte, either by slow uptake or by synthesis.

Incorporation of biogenic amines into phospholipids studied in two-phase-system.

Phospholipids (PL) from splenic nerves, splenic nerve granules, adrenal medullary granules and egg yolk were used as binding ligands for studies on uptake of various biogenic amines. Efficient uptake of catecholamine (CA) occurred at low ionic strength (about 2-5 mM) and at pH 6.8-7.7. This uptake was also a function of the amine concentration in the aqueous medium. At higher ionic strength (e.g. 128 mM K-phosphate buffer) an uptake still occurred, but to considerably less extent, apparently since the association/dissociation ability of the amines within the PL was a function of cation concentration in the aqueous media. Sucrose- and dextrose-induced increase in the osmolarity of the aqueous medium did not to any appreciable extent reduce the CA uptake. Scatchard plot indicated that only one type of binding sites was engaged when PL from splenic nerves was used. Using PL from splenic nerves the following affinity order was obtained for the amine tested: 5-HT greater than TA greater than DA greater than NA greater than IP greater than A. When PL from medullary granules was used the order was: DA greater than IP greater than A greater than or equal to NA. No or negligible uptake of Dopa and its metabolites Doma and Dopac occurred. When added to the aqueous medium, the cationic amphiphilic detergent CTAB induced some uptake of Doma and Dopac, but no a Dopa. However, CTAB considerably diminished the uptake of NA and the other amines. On the other hand, ionic (DOC) and anionic (SDS) detergents strongly increased the uptake of NA and other amines into PL. The nonionic Triton X-100 was without significant effect on uptake of NA. It is suggested that the binding ligands may be present in the dipolar head of the PL molecules, particularly in the lipid phosphate group. The lipid protein may also be partially involved since chromatographic experiments showed that the CA accompanied lipid phosphorus and lipid protein. Electrostatic or complex interaction of CA within the polar head group or in conjunction with lipid protein would constitute an amine storage with efficient association/dissociation capacity.