Expression Of Vesicular Monoamine Transporter Research Articles

The dopamine mesocorticolimbic pathway is affected by deficiency in n−3 polyunsaturated fatty acids

Several findings in humans support the hypothesis of links between n-3 polyunsaturated fatty acid (PUFA) status and psychiatric diseases. The involvement of PUFAs in central nervous system function can be assessed with the use of dietary manipulation in animal models. We studied the effects of chronic dietary n-3 PUFA deficiency on mesocorticolimbic dopamine neurotransmission in rats. Using dual-probe microdialysis, we analyzed dopamine release under amphetamine stimulation simultaneously in the frontal cortex and the nucleus accumbens. The messenger RNA (mRNA) expression of vesicular monoamine transporter(2) and dopamine D(2) receptor was studied with the use of in situ hybridization. The protein expression of the synthesis-limiting enzyme tyrosine 3-monooxygenase (tyrosine 3-hydroxylase) was studied with the use of immunocytochemistry. Dopamine release was significantly lower in both cerebral areas in n-3 PUFA-deficient rats than in control rats, but this effect was abolished in the frontal cortex and reversed in the nucleus accumbens by reserpine pretreatment, which depletes the dopamine vesicular storage pool. The mRNA expression of vesicular monoamine transporter(2) was lower in both cerebral areas in n-3 PUFA-deficient rats than in control rats, whereas the mRNA expression of D(2) receptor was lower in the frontal cortex and higher in the nucleus accumbens in n-3 PUFA-deficient rats than in control rats. Finally, tyrosine 3-monooxygenase immunoreactivity was higher in the ventral tegmental area in n-3 PUFA-deficient rats than in control rats. Our results suggest that the mesolimbic dopamine pathway is more active whereas the mesocortical pathway is less active in n-3 PUFA-deficient rats than in control rats. This provides new neurochemical evidence supporting the effects of n-3 PUFA deficiency on behavior.

Time-course changes of ECL cell markers in acetic acid-induced gastric ulcers in rats.

Enterochromaffin-like (ECL) cells are the major source of histamine for the regulation of gastric acid secretion, and also contain histidine decarboxylase (HDC), vesicular monoamine transporter 2 (VMAT2), and chromogranin A (CgA). Although gastric acid secretion is suppressed during ulcer healing, the role of ECL cells in that process is not yet fully understood. In the present study, we investigated the changes in ECL cell number during healing of experimental ulcers in rats. Seven-week-old male Wistar rats were used. Acetic acid-induced ulcers were caused by an application of 100% acetic acid to the serosal surface of the rat stomachs. At different time points following the induction (12 h-15 days), time-course changes of HDC, VMAT2, and CgA mRNA expression were investigated by Northern blot analysis. The expressions of HDC, VMAT2, and CgA were immunostained on gastric mucosal sections with ulcers. HDC, VMAT2, and CgA mRNA in gastric mucosa each showed an initial marked transient decrease, followed by an increase on day 10 back to the initial value. HDC, VMAT2, and CgA-immunoreactive cells at the ulcer margin were reduced in number on day 3, compared with those in distant areas. On day 10, however, they returned to levels similar to those in distant areas. The present study revealed a local down-regulation of HDC, VMAT2, and CgA in ECL cells at the ulcer margin. As a result, we concluded that a suppression of ECL cell activity during ulcer healing may be involved in suppressed gastric acid secretion.

Amine uptake and peptide hormone secretion: APUD cells in a new landscape.

APUD cells were so named by Pearse (1969) as a result of his attempt to produce a synthesis relating a number of observations concerning the histochemistry and function of peptide-secreting endocrine cells, particularly those of the gastrointestinal tract. One of these properties was that peptide-secreting endocrine cells appeared to share an ability to take up precursors of biologically active amines, to produce active amine through subsequent intracellular decarboxylation and then store the amine product in secretory vesicles, hence APUD (amine precursor uptake and decarboxylation). This widespread phenomenon was initially described by Pearse himself (1966) for the calcitonin-secreting parafollicular C cells of dog thyroid using 5-hydroxytryptophan as the amine precursor. In that paper the author noted that such an uptake was also found in other peptide hormone-secreting cells, for example in the corticotrophs of the anterior pituitary gland and in the β-cells of the endocrine pancreas. However, these clear-cut observations became hidden beneath the much larger edifice that the APUD concept became, with APUD cells (eventually including more than 40 cell types) defined as a ‘diffuse neuroendocrine system … the third branch of the nervous system acting within the second, the autonomic nervous system in the control of all the intestinal organs’ and with a developmental lineage that was defined as necessarily being from neuroectoderm. This monumental hypothesis has not stood the rigour of experimental test using modern methods of cell and developmental biology. However, if the grand theory itself has been found wanting (see for example Andrew et al. 1998), the original observations that led to the idea of the APUD cell have certainly been confirmed and yet have remained neglected for much of the last decade. Why should cells that are undoubtedly peptide-secreting endocrine cells have the general property of handling amines in a way characteristic of neurones? In this issue of The Journal of Physiology, Blackmore et al. shed a fascinating new perspective on these early findings by showing that functionally the observed pattern of secreted peptide hormones (which emerge as processed fragments from larger polypeptide precursors) results from the ability of these cells to take up amines. The connection arises because of two phenomena which share a common molecular event - in one case the result of, in the other the cause of the link. Because peptide hormone processing is carried out by intracellular endopeptidases within secretory vesicles and because the enzymes responsible for site-specific activity (the pro-hormone convertases) are themselves highly pH sensitive, the pattern of peptide release will necessarily depend upon secretory vesicular pH. Blackmore et al. (2001) have now shown that the pH within this organelle is itself regulated by monoamines through a simple link. Amine transport across the vesicular membrane occurs through transporters that are proton-coupled antiporters. Hence addition of amine to the cell, by activating amine transport and accumulation within the vesicles reduces intravesicular acidification; this alters the processing of secreted peptide, thus establishing the link between amine uptake and peptide hormone secretion. The conclusion from this study is so unambiguous because of the imaginative combination of modern experimental strategies adopted by the authors. Using confocal microscopy to measure intravesicular pH (with a pH-sensitive green fluorescent protein targeted to secretory vesicles) in cells in situ and pharmacological tools to isolate the contribution of specific vesicular monoamine transporters, the authors make a combined attack using a genetically engineered cell line. A pancreatic β-cell line (a nice touch here is that the authors manage to acknowledge materials received from both Drs Banting and Best!) was stably transfected with cDNA constructs expressing both the vesicular monoamine transporter VMAT2 and the peptide hormone precursor progastrin, a peptide that is well known to be cleaved into a number of specific smaller fragments (e.g. G34 and G17) with differing biological effects and properties depending on the specific product released. Moreover gastrin is a peptide for which cleavage-product-specific antibodies have been developed, allowing peptide processing as a function of amine transport-dependent intravesicular pH to be monitored. The results are clear-cut: expression of the vesicular monoamine transporter alters peptide processing in these cells and the mechanism for this coupling does indeed appear to be altered intravesicular pH. Wilmer (1960) observed in his classic monograph Cytology and Evolution that ‘… in its evolutionary processes Nature tends to build on what is already present and does so by a continual process of modification. A plurality of purpose may arise as a consequence of some basic embryological processes which probably have an earlier and originally a functional origin' or as Jacob (see Crick, 1988) put it more succinctly, ‘evolution is a tinkerer’. The paper by Blackmore et al. (2001) exemplifies this particularly clearly by showing how a novel process linking neuronal and endocrine cell function emerges from such evolutionary tinkering with the cellular expression and properties of an intracellular vesicular monoamine transporter.

Transient expression of the vesicular monoamine transporter during development in the rat thalamus and cortex

The postnatal developmental pattern of the central vesicular monoamine transporter-2 (VMAT2) was analyzed in the rat brain by means of quantitative autoradiography with a specific and high affinity ligand [ 3H]dihydrotetrabenazine ([ 3H]TBZOH). We show a dense expression of VMAT2 in the cortex (especially area 17) and thalamus (particularly the dorsal lateral geniculate nucleus) at postnatal days 1 and 8. This pattern of VMAT2 distribution was transient since it was no longer observed at day 20 or in the adult rat brain where VMAT2 density was weak and uniform in these regions. These data suggest that monoamine vesicular storage participates in the early postnatal maturation of thalamus and cortex.

Vesicular Transport Regulates Monoamine Storage and Release but Is Not Essential for Amphetamine Action

To assess the role of exocytotic release in signaling by monoamines, we have disrupted the neuronal vesicular monoamine transporter 2 (VMAT2) gene. VMAT2−/− mice move little, feed poorly, and die within a few days after birth. Monoamine cell groups and their projections are indistinguishable from those of wild-type littermates, but the brains of mutant mice show a drastic reduction in monoamines. Using midbrain cultures from the mutant animals, amphetamine but not depolarization induces dopamine release. In vivo, amphetamine increases movement, promotes feeding, and prolongs the survival of VMAT2−/− animals, indicating that precise, temporally regulated exocytotic release of monoamine is not required for certain complex behaviors. In addition, the brains of VMAT2 heterozygotes contain substantially lower monoamine levels than those of wild-type littermates, and depolarization induces less dopamine release from heterozygous than from wild-type cultures, suggesting that VMAT2 expression regulates monoamine storage and release.

Ontogeny of Vesicular Amine Transporter Expression in the Rat: New Perspectives on Aminergic Neuronal and Neuroendocrine Differentiation

Publisher Summary The patterns of expression of vesicular monoamine transporter 1 (VMAT1), VMAT2, and vesicular acetylcholine transporter (VAChT) during ontogenesis of monoaminergic and cholinergic neurons and endocrine cells provide insight into pathways of lineage commitment leading to the final chemical coding of aminergic neurotransmission in the adult rat. Neuroepithelial cells expressing exclusively VMAT2 appear around embryonic day 12 (E12), and are the presumptive neuroblasts giving rise to the monoaminergic cell groups of the central nervous system (CNS). VMAT1 expression at both protein and mRNA levels is absent from CNS neurons at all stages of development examined in this chapter. Monoaminergic neuronal and endocrine cells of the central nervous and gastrointestinopancreatic systems appear to develop via an initial commitment to exclusive expression of one or the other VMAT isoform. In contrast, VMAT1 and VMAT2 are coexpressed during development in adrenomedullary and carotid body neuroendocrine cells and in principal ganglion cells of the sympathetic chain. A subpopulation of postganglionic neurons of the paravertebral sympathetic chain, responsible for innervation of skeletal muscle vasculature and sweat glands, is cholinergic. Cholinergic sympathetic differentiation within the sympathetic chain could conceivably occur at the same time as sympathetic noradrenergic differentiation, as reported in other vertebrate species, although this clearly remains to be determined.

Differential expression of two vesicular monoamine transporters

Specific transport proteins package classical neurotransmitters into vesicles so that their release can be regulated by neural activity. Previous studies have suggested that a single activity mediates the vesicular transport of monoamines in the adrenal gland, brain, and other tissues such as mast cells and platelets. However, molecular cloning has recently identified two vesicular transporters for monoamines. Although the predicted proteins are closely related in sequence, they show a range of differences in their physiologic and pharmacologic properties. To clarify further the biological significance of the observed functional differences, we have generated anti-peptide antibodies to the C-termini of the two transporters and used them to determine the distribution and localization of the proteins in the rat. We have detected expression of vesicular monoamine transporter 1 (VMAT1) in adrenal chromaffin cells but not in neural cells. Interestingly, some adrenal chromaffin cells also express VMAT2 but the amount of VMAT2 relative to VMAT1 appears much lower than in the bovine adrenal gland. In contrast, sympathetic ganglion cells express only VMAT2, as do enteric neurons and enterochromaffin-like cells of the stomach. Thus, although adrenal chromaffin cells, sympathetic and enteric neurons derive from the neural crest, they express different vesicular amine transporters. In the CNS, dopamine, norepinephrine, epinephrine, 5-HT, and histamine cell groups all express VMAT2. These findings are consistent with the functional characteristics of VMAT1 and VMAT2 and help to explain several classic pharmacological observations. VMAT2-immunoreactivity is generally stronger in cell bodies, proximal dendrites and axonal processes, indicating the potential for monoamine storage at each of these sites. Surprisingly, dopaminergic interneurons in the olfactory bulb show no detectable immunoreactivity for either VMAT1 or VMAT2.