Neuropeptidergic Control of Feeding: Focus on the Galanin Family of Peptides.

Obesity/overweight are important health problems due to metabolic complications. Dysregulation of peptides exerting orexigenic/anorexigenic effects must be investigated in-depth to understand the mechanisms involved in feeding behaviour. One of the most important and studied orexigenic peptides is galanin (GAL). The aim of this review is to update the mechanisms of action and physiological roles played by the GAL family of peptides (GAL, GAL-like peptide, GAL message-associated peptide, alarin) in the control of food intake and to review the involvement of these peptides in metabolic diseases and food intake disorders in experimental animal models and humans. The interaction between GAL and NPY in feeding and energy metabolism, the relationships between GAL and other substances involved in food intake mechanisms, the potential pharmacological strategies to treat food intake disorders and obesity and the possible clinical applications will be mentioned and discussed. Some research lines are suggested to be developed in the future, such as studies focused on GAL receptor/neuropeptide Y Y1 receptor interactions in hypothalamic and extra-hypothalamic nuclei and sexual differences regarding the expression of GAL in feeding behaviour. It is also important to study the possible GAL resistance in obese individuals to better understand the molecular mechanisms by which GAL regulates insulin/glucose metabolism. GAL does not exert a pivotal role in weight regulation and food intake, but this role is crucial in fat intake and also exerts an important action by regulating the activity of other key compounds under conditions of stress/altered diet.

The involvement of the substance P/neurokinin 1 receptor system in viral infection: focus on the gp120 fusion protein and homologous dipeptide domains.

The human immunodeficiency virus (HIV) envelope, via a key extracellular amino acid sequence, may simulate the functionality of native undecapeptide substance P (SP) acting through the host's neurokinin 1 (SP preferring) receptor (NK-1R). Human monocytes and macrophages express both NK-1Rs and SP. In HIV/AIDS the NK-1R may function as a chemokine-like G-protein coupled co-receptor that: 1) fuses to the outer envelope of HIV; 2) enables intracellular entry of the envelope-capsid-NK-1R complex; 3) co-opts immune defence via its physiological interaction with the SP-like envelope; 4) may contribute to resistance of CD4/chemokine entry inhibitor type drugs; 5) relaxes the blood-brain barrier to support entry of the HIV into the central nervous system, and 6) mediates most of the common clinical sequelae of HIV/AIDS (encephalopathy and AIDS dementia complex). The data support the idea that NK-1R antagonists could be useful to treat HIV/AIDS. Keywords: human immunodeficiency virus; NK-1 receptor; NK-1 receptor antagonist; aprepitant; fusion protein; virus.

Neuroanatomical relationship between the cholinergic and tachykininergic systems in the adult human brainstem: An immunohistochemical study.

The cholinergic system plays an important role in brain homeostasis and interacts with the neuropeptidergic systems, and the functional relationships between both systems are well known. However, in the brainstem the possible physiological interactions between neurokinins and acetylcholine are unknown, although both substances have been detected in the same brainstem nuclei and have been implicated in similar functions controlled from brainstem regions such as some cranial motor nuclei. The aim of this work is to determine whether these possible physiological interactions might have a neuroanatomical basis by means of the double immunohistochemical detection of neurokinins (NK) and the enzyme choline acetyl-transferase (ChAT) in the human brainstem. No double-labelled structures were detected, although both NK and ChAT were observed in cell bodies and fibers of the same brainstem nuclei. The distribution of immunoreactive fibers is widespread, and NK and ChAT were observed in several motor cranial nerves as well as in the substantia nigra. The results obtained in the present work provide a neuroanatomical basis for possible physiological interactions between NK and ChAT that may be carried out by volume-transmission mechanisms. These interactions might participate in motor regulation or in limbic pathways as well as influence on other neurotransmitter systems such as the dopaminergic system.

Distribution of somatostatin-28 (1-12), calcitonin gene-related peptide, and substance P in the squirrel monkey brainstem: an immunocytochemical study.

Using an immunocytochemical technique, we have studied the distribution of fibers and cell bodies containing somatostatin-28 (1-12) [SOM-28 (1-12)], calcitonin gene-related peptide (CGRP), and substance P (SP) in the brainstem of Saimiri sciureus. The distribution of the peptidergic cell bodies was very restricted: perikarya containing SOM-28 (1-12) were only observed in the substantia grisea centralis, while no immunoreactive cell bodies containing CGRP or SP were visualized. Fibers containing SOM-28 (1-12), CGRP, or SP were widely distributed in the brainstem: immunoreactive fibers containing SOM-28 (1-12) showed the most widespread distribution and were the most abundant. The distribution of SOM-28 (1-12)-, CGRP- or SP-immunoreactive fibers was very similar. Colocalization of immunoreactive fibers containing SOM-28 (1-12), CGRP or SP was observed in many brainstem nuclei. A neuroanatomical relationship between CGRP- and SP-immunoreactive fibers was observed, although this relationship was less marked for SOM-28 (1-12) and SP and lower still for SOM-28 (1-12) and CGRP. The widespread distribution of the peptidergic fibers suggests that the studied neuropeptides are involved in many physiological actions.

Region specific galanin receptor/neuropeptide Y Y1 receptor interactions in the tel- and diencephalon of the rat. Relevance for food consumption.

The aim of this work was to determine the interactions between NPY and GAL receptor (GALR) subtypes in the hypothalamus and the amygdala using quantitative receptor autoradiography to analyze the binding characteristics of NPY-Y1 and Y2 receptor subtypes in the presence and absence of GAL. Food intake in satiated animals was evaluated after intraventricular co-injections of GAL and NPY-Y1 or Y2 agonists. The expression of c-Fos IR in both regions was also investigated. GAL decreases NPY-Y1 agonist binding in the arcuate nucleus by about 15% (p<0.01), but increases NPY-Y1 agonist binding in amygdala (18%) (p<0.01). These effects were blocked with the GAL antagonist M35. Y2-agonist binding was not modified by GAL. GAL blocked the food intake induced by the Y1 agonist (p<0.01). Co-injections of Y1 agonist and GAL also reduced the c-Fos expression induced by the Y1 agonist in the arcuate nucleus and the dorsomedial hypothalamic nucleus but increased c-Fos expression in amygdala. These results indicate the existence of antagonistic interactions between GALR and NPY-Y1 receptors in the hypothalamus and their functional relevance for food intake. In contrast, a facilitatory interaction between GALR and Y1 receptors exists in the amygdala which may be of relevance for fear related behaviour.

Immunocytochemical visualization of D-glutamate in the rat brain.

Using highly specific antisera directed against conjugated d-amino acids, the distribution of d-glutamate-, d-tryptophan-, d-cysteine-, d-tyrosine- and d-methionine-immunoreactive structures in the rat brain was studied. Cell bodies containing d-glutamate, but not d-glutamate-immunoreactive fibers, were found. Perikarya containing this d-amino acid were only found in the mesencephalon and thalamus of the rat CNS. Thus, the highest density of cell bodies containing d-glutamate was observed in the dorsal raphe nucleus, the ventral part of the mesencephalic central gray, the superior colliculus, above the posterior commissure, and in the subparafascicular thalamic nucleus. A moderate density of immunoreactive cell bodies was observed in the dorsal part of the mesencephalic central gray, above the rostral linear nucleus of the raphe, the nucleus of Darkschewitsch, and in the medial habenular nucleus, whereas a low density was found below the medial forebrain bundle and in the posterior thalamic nuclear group. Moreover, no immunoreactive fibers or cell bodies were visualized containing d-tryptophan, d-cysteine, d-tyrosine or d-methionine in the rat brain. The distribution of d-glutamate-immunoreactive cell bodies in the rat brain suggests that this d-amino acid could be involved in several physiological mechanisms. This work reports the first visualization and the morphological characteristics of conjugated d-glutamate-immunoreactive cell bodies in the rat CNS using an indirect immunoperoxidase technique. Our results suggest that the immunoreactive neurons observed have an uptake mechanism for d-glutamate.

Overexpression of kynurenic acid in stroke: An endogenous neuroprotector?

It is known that kynurenic acid (KYNA) exerts a neuroprotective effect against the neuronal loss induced by ischemia; acting as a scavenger, and exerting antioxidant action. In order to study the distribution of KYNA, a highly specific monoclonal antibody directed against KYNA was developed. This distribution was studied in control rats and in animals in which a middle cerebral artery occlusion (stroke model) was induced. By double immunohistochemistry, astrocytes containing KYNA and GFAP were exclusively found in the ipsilateral cerebral cortex and/or striatum, at 2, 5 and 21days after the induction of stroke. In control animals and in the contralateral side of the stroke animals, no immunoreactivity for KYNA was found. Under pathological conditions, the presence of KYNA is reported for the first time in the mammalian brain from early phases of stroke. The distribution of KYNA matches perfectly with the infarcted regions suggesting that, in stroke, this overexpressed molecule could be involved in neuroprotective/scavenger/antioxidant mechanisms.

Thiamine-like fibers in the monkey brain: an immunocytochemical study.

The distribution of thiamine-immunoreactive structures was studied in the brain of the monkey using an indirect immunoperoxidase technique. Fibers containing thiamine, but no thiamine-immunoreactive cell bodies, were found. The highest density of fibers containing thiamine was observed in the pulvinar nucleus and in the region extending from the pulvinar nucleus to the caudate nucleus. In the mesencephalon, immunoreactive fibers containing thiamine were only found at rostral level close to the medial lemniscus (at the mesencephalic-diencephalic junction). In the thalamus, the distribution of thiamine-immunoreactive structures was more widespread. Thus, immunoreactive fibers were found in nuclei close to the midline (centrum medianum/parafascicular complex), in the ventrolateral thalamus (medial geniculate nucleus, inferior pulvinar nucleus), and in the dorsolateral thalamus (lateral posterior nucleus, pulvinar nucleus). Finally, in the anterior commissure and in the cerebral cortex a low density immunoreactive fibers was visualized. Thus, in the brainstem, no immunoreactive structures were visualized in the medulla oblongata, pons, or in the medial-caudal mesencephalon, and no immunoreactive fibers were observed in the cerebellum, hypothalamus and in the basal ganglia. The present report describes the first visualization and the morphological characteristics (thick, smooth and short, medium or long in length) of the thiamine-immunoreactive fibers in the primate central nervous system using an antiserum directed against this vitamin. The distribution of thiamine-immunoreactive structures in the monkey brain suggests that this vitamin could be involved in several physiological mechanisms.

Riboflavin-like inmunoreactive fibers in the monkey brain.

Using an antiserum directed against the vitamin riboflavin, we studied the distribution of riboflavin-like immunoreactive structures in the monkey brain. In the mesencephalon, at the level of the mesencephalic-diencephalic junction, single riboflavin-like immunoreactive fibers were observed in its dorsal part, whereas a low density of immunoreactive fibers was found below the surface of the section and close to substantia nigra, and a high density was observed above the substantia nigra and close to the medial geniculate nucleus. In the thalamus, single riboflavin-like immunoreactive fibers were found in the ventral regions of the lateral posterior and the medial geniculate nuclei; a low density in the region located above the medial and lateral geniculate nuclei and a high density in the ventral part of the pulvinar nucleus and in the region extending from this latter to the caudate nucleus. Immunoreactive fibers were not observed in the medulla oblongata, pons, cerebellum, hypothalamus, basal ganglia and cerebral cortex. Moreover, no riboflavin-like immunoreactive cell bodies were observed in the monkey brain. The distribution of riboflavin-like immunoreactive fibers in the monkey suggests that this vitamin could be involved in several physiological mechanisms.

Distribution of Neurotensin and Somatostatin-28 (1-12) in the Minipig Brainstem.

Using an indirect immunoperoxidase technique, an in depth study has been carried out for the first time on the distribution of fibres and cell bodies containing neurotensin and somatostatin-28 (1-12) (SOM) in the minipig brainstem. The animals used were not treated with colchicine. The distribution of neurotensin- and SOM-immunoreactive fibres was seen to be quite similar and was moderate in the minipig brainstem: a close anatomical relationship between both neuropeptides was observed. The distribution of cell bodies containing neurotensin or SOM was quite different and restricted. Cell bodies containing neurotensin were found in four brainstem nuclei: nucleus centralis raphae, nucleus dorsalis raphae, in the pars centralis of the nucleus tractus spinalis nervi trigemini and in the nucleus ventralis raphae. Cell bodies containing SOM were found in six nuclei/regions of the brainstem: nucleus ambiguus, nucleus dorsalis motorius nervi vagus, formatio reticularis, nucleus parabrachialis medialis, nucleus reticularis lateralis and nucleus ventralis raphae. According to the observed anatomical distribution of the immunoreactive structures containing neurotensin or SOM, the peptides could be involved in sleep-waking, nociceptive, gustatory, motor, respiratory and autonomic mechanisms.

NO-tryptophan: a new small molecule located in the rat brain.

A highly specific monoclonal antibody directed against nitric oxide-tryptophan (NO-W) with good affinity (10-9 M) and specificity was developed. In the rat brain, using an indirect immunoperoxidase technique, cell bodies containing NO-W were exclusively found in the intermediate and dorsal parts of the lateral septal nucleus. No immunoreactive fibres were found in the rat brain. This work reports the first visualization and the morphological characteristics of cell bodies containing NO-W in the mammalian brain. The restricted distribution of NO-W in the rat brain suggests that this molecule could be involved in specific physiological mechanisms.

3-hydroxi-anthranilic acid is early expressed in stroke.

Using an immunohistochemical technique, we have studied the distribution of 3-OH-anthranilic acid (3-HAA) in the rat brain. Our study was carried out in control animals and in rats in which a stroke model (single transient middle cerebral artery occlusion) was performed. A monoclonal antibody directed against 3-HAA was also developed. 3-HAA was exclusively observed in the infarcted regions (ipsilateral striatum/cerebral cortex), 2, 5 and 21 days after the induction of stroke. In control rats and in the contralateral side of the stroke animals, no immunoreactivity for 3-HAA was visualized. Under pathological conditions (from early phases of stroke), we reported for the first time the presence of 3-HAA in the mammalian brain. By double immunohistochemistry, the coexistence of 3-HAA and GFAP was observed in astrocytes. The distribution of 3-HAA matched perfectly with the infarcted regions. Our findings suggest that, in stroke, 3-HAA could be involved in the tissue damage observed in the infarcted regions, since it is well known that 3-HAA exerts cytotoxic effects.

Mapping of methionine-enkephalin-arg6-gly7-leu8 in the human diencephalon.

Using an immunohistochemical technique, we mapped the immunoreactive structures containing methionine-enkephalin-Arg6-Gly7-Leu8 (Met-8) (a marker for the pro-enkephalin system) in the human diencephalon. Compared with previous studies, we observed a more widespread distribution of Met-8 in the human diencephalon. Met-8-immunoreactive cell bodies and fibers exhibited a more widespread distribution in the hypothalamus than in the thalamus. We observed six populations of Met-8-immunoreactive cell bodies. These perikarya were observed in the paratenial thalamic nucleus, ventromedial and dorsomedial hypothalamic nuclei, lateral hypothalamic area, pallidohypothalamic nucleus and in the paraventricular hypothalamic nucleus (posterior part). In the thalamus, Met-8-immunoreactive fibers were primarily observed in the midline region, whereas in the hypothalamus, these fibers were widely distributed. In general, a moderate/low density of Met-8-immunoreactive fibers was observed in the diencephalic nuclei. A moderate density was observed in the paraventricular thalamic nucleus, reuniens thalamic nucleus, lateral and medial geniculate nuclei, dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus (posterior part) and ventromedial hypothalamic nucleus. The present study is the first to demonstrate the presence of clusters of Met-8-immunoreactive cell bodies in the human thalamus and hypothalamus, the distribution of fibers containing neuropeptides in the hypothalamus and the presence of these fibers in several thalamic nuclei. This neuroanatomical study will serve to elucidate the physiological roles of Met-8 in future studies of the human diencephalon.

The NK1 receptor is involved in the antitumoural action of L-733,060 and in the mitogenic action of substance P on neuroblastoma and glioma cell lines.

We have carried out an in vitro study to investigate the ability of substance P to activate cell growth and the NK1 receptor antagonist L-733,060 to inhibit cell growth in the SKN-BE(2) neuroblastoma and GAMG glioma cell lines. A coulter counter was used to determine viable cell numbers, followed by application of the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium], inner salt, colorimetric method to evaluate cell viability in this cytotoxicity assay. Nanomolar concentrations of substance P increased, and micromolar concentrations of L-733,060 inhibited the growth of both cell lines studied, with and without previous administration of substance P. In addition, we have demonstrated by immunoblot analysis that NK1 receptors are present in both cancer cell lines studied here. Thus, this study demonstrates that substance P acts as a mitogen in the SKN-BE(2) neuroblastoma and GAMG glioma cell lines, and that the antitumoural action of L-733,060 on both human cell lines occurs through the NK1 receptor. This action suggests that the NK1 receptor is a new and promising target in the treatment of human neuroblastoma and glioma.

Role of galanin and galanin(1-15) on central cardiovascular control.

Galanin and the N-terminal fragment Galanin(1-15) are involved in central cardiovascular regulation. The present paper reviews the recent cardiovascular results obtained by intracisternal injections of Galanin and Galanin(1-15) showing that: (A) the Galanin antagonist M40 blocks the central cardiovascular responses induced by Galanin(1-15) but not those elicited by Galanin; (B) both Galanin and Galanin(1-15) induce the expression of c-Fos in cardiovascular nuclei of the medulla oblongata with different temporal and spatial profiles; (C) the cardiovascular action of Galanin(1-15), but not Galanin, is mediated by peripheral beta-receptor stimulation; (D) and it is demonstrated an antagonistic Galanin/alpha2-adrenoceptors interaction as well as a differential modulation of central cardiovascular responses of Angiotensin II by Galanin or Galanin(1-15). All these data strengthen the involvement of both Galanin molecules as neuromodulators on central cardiovascular regulation.

Mapping of neurokinin b in the cat brainstem.

We studied the distribution of neurokinin B-immunoreactive cell bodies and fibers in the cat brainstem using an indirect immunoperoxidase technique. The highest density of immunoreactive fibers was found in the motor trigeminal nucleus, the laminar and alaminar spinal trigeminal nuclei, the facial nucleus, the marginal nucleus of the brachium conjunctivum, the locus coeruleus, the cuneiform nucleus, the dorsal motor nucleus of the vagus, the postpyramidal nucleus of the raphe, the lateral tegmental field, the Kölliker-Fuse nucleus, the inferior central nucleus, the periaqueductal gray, the nucleus of the solitary tract, and in the inferior vestibular nucleus. Immunoreactive cell bodies containing neurokinin B were observed, for example, in the locus coeruleus, the dorsal motor nucleus of the vagus, the median division of the dorsal nucleus of the raphe, the lateral tegmental field, the pericentral nucleus of the inferior colliculus, the internal division of the lateral reticular nucleus, the inferior central nucleus, the periaqueductal gray, the postpyramidal nucleus of the raphe, and in the medial nucleus of the solitary tract. This widespread distribution of neurokinin B in the cat brainstem suggests that the neuropeptide could be involved in many different physiological functions. In comparison with previous studies carried out in the rat brainstem on the distribution of neurokinin B, our results point to a more widespread distribution of this neuropeptide in the cat brainstem.

Mapping of tachykinins in the cat spinal cord.

Using an indirect immunoperoxidase technique, the location of cell bodies and fibers containing substance P, neurokinin A and neurokinin B was studied in the cat spinal cord. The former two neuropeptides showed a widespread distribution throughout the whole spinal cord, whereas the distribution of neurokinin B was more restricted. Neurokinin A-immunoreactive structures showed a more widespread distribution and a higher density than the immunoreactive structures observed to contain substance P. In the cat spinal cord, we observed cell bodies containing neurokinin A, but no cell bodies containing neurokinin B or substance P were found. These cell bodies were located in laminae V (sacral 1 and 2 levels), VI (sacral 1 and 3), VII (lumbar 7, sacral 1 and 3, caudal 1) and X (sacral 1). Laminae I and II showed the highest density of immunoreactive fibers for each of the three tachykinins studied, being in general lamina IV who showed the lowest number of immunoreactive fibers containing substance P, neurokinin A or B. The anatomical distribution of the three tachykinins studied in the cat spinal cord indicates that the neuropeptides could be involved in the neurotransmission and/or in the neuromodulation of nociceptive information, as well as in autonomic and affective responses to pain. Moreover, the involvement of substance P, neurokinin A or B in other functions unrelated to the transmission of pain is also possible (autonomic and motor functions). The distribution of the neuropeptides studied in the cat is compared with the location of the same neuropeptides in the spinal cord of other species. The possible origin of the tachykinergic fibers in the cat spinal cord is also discussed.

The antiproliferative action of [D-Arg(1), D-Phe(5), D-Trp(7,9), LEU(11)] substance P analogue antagonist against small-cell- and non-small-cell lung cancer cells could be due to the pharmacological profile of its tachykinin receptor antagonist.

It is known that in human lung cancer samples, both small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cells express the neurokinin-1 (NK-1) receptor; that after binding to the NK-1 receptor the peptide substance P (SP) elicits tumour cell proliferation and an antiapoptotic effect. By contrast, it has been demonstrated that non-peptide NK-1 receptor antagonists, after binding to the NK-1 receptor and hence by blocking the SP action in SCLC/NSCLC, exert an antiproliferative action (inhibit lung cancer cell proliferation and induce the death of tumour cells by apoptosis). It is also known that SP peptide NK-1 receptor antagonists also called SP analogue antagonists (broad-spectrum GPCR antagonists, broad-spectrum neuropeptide antagonists or synthetic analogues of SP), also exert antiproliferative actions against SCLC/NSCLC. However, the underlying mechanisms involved in this antiproliferative action remain unknown. By using competition assays with SP, here we demonstrate that the antiproliferative action exerted by the [D-Arg(1), D-Phe(5), D-Trp(7,9), Leu(11)] SP analogue on human H-69 SCLC and COR-L23 NSCLC cell lines, occurs at least through the NK-1 receptor.

Developmental study of vitamin C distribution in children's brainstems by immunohistochemistry.

Vitamin C (Vit C) is an important antioxidant, exerts powerful neuroprotective brain effects and plays a role in neuronal development and maturation. Vit C is present in brain tissue at higher concentrations than in other organs, but its detailed distribution in brain is unknown. Immunohistochemical detection of this vitamin has been performed by using a highly specific antibody against Vit C. The aim of the present work was to analyze the distribution of Vit C in children's brainstems during postnatal development, comparing two groups of ages: younger and older than one year of life. In general, the same areas showing neurons with Vit C in young cases are also immunostained at older ages. The distribution of neurons containing Vit C was broader in the brainstems of older children, suggesting that brainstem neurons maintain or even increase their ability to retain Vit C along the life span. Immunohistochemical labeling revealed only cell bodies containing this vitamin, and no immunoreactive fibers were observed. The distribution pattern of Vit C in children's brainstems suggests a possible role of Vit C in brain homeostatic regulation. In addition, the constant presence of Vit C in neurons of locus coeruleus supports the important role of Vit C in noradrenaline synthesis, which seemed to be maintained along postnatal development.