RESUMO
Ghrelin is produced by gastric A-like cells and released in response to food deprivation. Interestingly, psychological stress also raises circulating ghrelin levels. This study compared plasma ghrelin levels in Sprague-Dawley (SPD) rats and high-anxiety Wistar Kyoto (WKY) rats. The two strains were also compared with respect to plasma gastrin, a gastric hormone with a pre- and postprandial release pattern opposite to that of ghrelin, and to the activity of the gastrin-dependent, histamine-forming ECL cells in the gastric mucosa. The rats were killed after being freely fed or after an over-night fast. The stomachs were weighed and tissue samples were collected for histological and biochemical analysis. Plasma ghrelin and gastrin levels were determined by RIA. While fasted SPD rats had higher plasma ghrelin levels than fasted WKY rats (P < 0.001), plasma ghrelin did not differ between freely fed rats of the two strains. Gastrin levels were higher in fed WKY rats than in fed SPD rats (P < 0.001). Despite the higher plasma gastrin level, the oxyntic mucosal histidine decarboxylase (HDC) activity (a marker of ECL-cell activity) in fed rats and the mucosal thickness did not differ between the two strains. In a subsequent study, rats were subjected to water-avoidance stress for 60 min, causing plasma gastrin to increase in WKY rats (P < 0.001) but not in SPD rats. In conclusion, high-anxiety WKY rats had lower circulating ghrelin and higher gastrin than SPD rats in both the fasted and fed state, while the ECL-cell activity (HDC activity) was only moderately affected.
Assuntos
Ansiedade/sangue , Privação de Alimentos/fisiologia , Mucosa Gástrica/metabolismo , Células Secretoras de Gastrina/metabolismo , Gastrinas/sangue , Hormônios Peptídicos/sangue , Animais , Jejum/fisiologia , Feminino , Mucosa Gástrica/química , Grelina , Histidina Descarboxilase/análise , Histidina Descarboxilase/metabolismo , Período Pós-Prandial , Ratos , Ratos Endogâmicos WKY , Ratos Sprague-Dawley , Privação de Água/fisiologiaRESUMO
The present study investigates the pharmacology of the cloned neurokinin 1 receptor from the gerbil (gNK(1)R), a species claimed to have human-like NK(1)R (hNK(1)R) pharmacology. The amino acid sequence of NK(1)R was cloned. The hNK(1)R, rat NK(1)R (rNK(1)R), gNK(1)R and mutants of the gNK(1)R were expressed in CHO cells. The affinity and potency of NKR agonists and the NK(1)R antagonists CP99994 and RP67580 (NK(1)R-selective) and ZD6021 (NK1/2R) were assessed in vitro by monitoring [(3)H]-SarMet SP binding and substance P-evoked mobilization of intracellular Ca(2+). The gerbil foot tap (GFT) method was used to assess the potency of the antagonists in vivo. The gNK(1)R coding sequence displayed an overall 95% and 97% homology with hNK(1)R and rNK(1)R, respectively. The affinity of the NK(1)R-selective agonist (3)H-SarMet SP for human and gerbil NK(1)R was similar (2.0 and 3.1 nM) but lower for rNK(1)R (12.4 nM). The rank order potency of the agonists for NK(1)R was SP > or = ASMSP > or = NKA >>> pro7NKB in all species. The NK(1)R antagonists, ZD6021 and CP99994, had comparable affinity and potency for gerbil and human NK(1)R, but were 1000-fold less potent for rNK(1)R. In contrast, RP67580 had comparable affinity and potency for all three species. Mutations in positions 116 and 290 did not affect agonist potency at the gNK(1)R while the potency of the antagonists ZD6021 and CP99994 were markedly decreased (10-20-fold). It is concluded that gNK(1)R has similar antagonist pharmacology as the human-like orthologue and that species differences in antagonist function depend on key residues in the coding sequence and antagonist structure.
Assuntos
Antagonistas dos Receptores de Neurocinina-1 , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Clonagem Molecular , Cricetinae , Cricetulus , Primers do DNA , Gerbillinae , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ratos , Receptores da Neurocinina-1/química , Receptores da Neurocinina-1/genética , Homologia de Sequência de AminoácidosRESUMO
The enteric nervous system is of great importance for maintenance and proper function of the gastrointestinal tract. The aim of this study was to quantify myenteric neuronal subpopulations expressing calcitonin gene-related peptide (CGRP), galanin, neuropeptide Y (NPY), somatostatin, vasoactive intestinal peptide (VIP) and nitric oxide synthase (NOS) in rat colon in vivo and after culturing. Further we investigated if culturing in the presence of CGRP, galanin, VIP, S-nitroso-N-acetyl-D,L-penicillamine (SNAP, a NO donor) or N-nitro-L-arginine methyl ester (L-NAME, a NOS inhibitor) affect neuronal survival. After 4 days of culturing the proportions of neurons expressing CGRP, NPY, somatostatin or VIP increased as compared to in vivo, while the proportions of neurons expressing galanin or NOS did not change. Neuronal survival was unaffected after culturing in media enriched with CGRP, galanin, VIP, SNAP or L-NAME. Neither did addition of CGRP, galanin nor VIP to the cultures affect the relative numbers of neurons expressing CGRP, galanin or VIP respectively. Addition of SNAP or L-NAME did not change the percentage of neurons expressing NOS. In conclusion, cultured rat colonic myenteric neurons increase their expression of CGRP, NPY, somatostatin and VIP, suggesting that these neuropeptides are of importance for neuronal survival.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Colo/citologia , Plexo Mientérico/citologia , Plasticidade Neuronal , Neurônios/metabolismo , Neurotransmissores/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Feminino , Plexo Mientérico/metabolismo , Neurônios/classificação , Neurônios/efeitos dos fármacos , Neuropeptídeos/metabolismo , Neuropeptídeos/farmacologia , Neurotransmissores/farmacologia , Doadores de Óxido Nítrico/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Intracerebroventricular (i.c.v.) administration of tachykinin NK(1) receptor agonists induces tapping of the hind legs in gerbils, so-called gerbil foot tapping, which is thought to reflect a fear-related response. The aim of the present study was to examine how ligands selective for NK(1), NK(2) and NK(3) receptors affect the gerbil foot tap response. Agonists selective for NK receptor subtypes were administered i.c.v. and the gerbil foot tap response was monitored. The effect of systemically administered antagonists was also studied. The interaction of ligands with gerbil NK(1) receptors was evaluated using autoradiography on gerbil brain slices with [(3)H]-Sar,Met(O(2))-substance P or [(3)H]GR205171 as radioligand. The effects of ligands on NK(1) and NK(3) receptor-mediated increases in intracellular calcium in vitro were studied in Chinese hamster ovary cells expressing the cloned gerbil receptors. The selective NK(1) receptor agonist ASMSP and the selective NK(3) receptor agonist senktide induced dose-dependent increases in gerbil foot tapping with similar potency. The maximal effect of senktide was approximately 40% of the maximal response evoked by ASMSP. The effects of ASMSP and senktide were blocked by administration of the selective NK(1) receptor antagonist CP99,994 (10 micromol/kg s.c.). The effects of senktide, but not ASMSP, were blocked by administration of the selective NK(3) receptor antagonist SB223412 (50 micromol/kg i.p.). Senktide did not displace NK(1) receptor radioligand binding and was >1000-fold less potent than ASMSP at activating gerbil NK(1) receptors. The selective NK(3) receptor agonist senktide evokes fear-related gerbil foot tapping, an effect which probably involves indirect enhancement of NK(1) receptor signalling.
Assuntos
Comportamento Animal/efeitos dos fármacos , Antagonistas dos Receptores de Neurocinina-1 , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Receptores da Neurocinina-3/antagonistas & inibidores , Substância P/análogos & derivados , Animais , Autorradiografia , Encéfalo/metabolismo , Células CHO , Cálcio/metabolismo , Clonagem Molecular , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Pé , Gerbillinae , Injeções Intraventriculares , Masculino , Neurocinina A/análogos & derivados , Neurocinina A/farmacologia , Piperidinas/farmacologia , Quinolinas/farmacologia , Receptores da Neurocinina-2/antagonistas & inibidores , Substância P/antagonistas & inibidores , Substância P/farmacologiaRESUMO
Total parenteral nutrition (TPN) is held to cause intestinal atrophy and weaken mechanical and immunological barriers. To monitor the degree of atrophy caused by TPN, female Sprague-Dawley rats were, for 8 days, maintained on TPN (n = 6) and compared to identically housed controls given food and water ad libitum (n = 6). Specimens from jejunum, ileum, and colon were taken for histology and morphometric analysis. Topographic distribution and presence of eosinophils, by eosinophil peroxidase (EPO) staining, were examined in the gastric fundus, jejunum, ileum, and colon. Atrophy in terms of a markedly reduced circumference was noted throughout the intestinal tract in all rats subjected to TPN. The width of jejunal and ileal villi was narrowed and the length of jejunal villi was decreased. Furthermore, submucosal thickness in the jejunum and ileum increased. The height of ileal enterocytes remained unaltered. The number of goblet cells decreased in jejunal but not in ileal villi. The Paneth cells, suggested to play important roles in innate defense, increased in size. In the gastric fundus a marked increase in eosinophils was revealed predominantly in the mucosa and submucosa. The number and distribution of jejunal and ileal eosinophils were identical to those of controls. In colon from TPN rats, a redistribution of eosinophils was noted, causing a "band-like" accumulation of eosinophils in the basal portion of the mucosa. In conclusion, TPN causes gut atrophy and an increase in Paneth cell size. Eosinophils increase in number in the gastric fundus and a topographic redistribution occurs in the colon.
Assuntos
Eosinófilos/patologia , Trato Gastrointestinal/patologia , Nutrição Parenteral Total/efeitos adversos , Animais , Atrofia , Colo/patologia , Feminino , Células Caliciformes/patologia , Íleo/patologia , Mucosa Intestinal/patologia , Jejuno/patologia , Celulas de Paneth/patologia , Celulas de Paneth/fisiologia , Ratos , Ratos Sprague-Dawley , Estômago/patologiaRESUMO
We compared the neurokinin 1 receptor (NK(1)R) antagonists aprepitant, CP-99994 [(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine], and ZD6021 [3-cyano-N-((2S)-2-(3,4-dichlorophenyl)-4-[4-[2-(methyl-(S)-sulfinyl)phenyl]piperidino]butyl)-N-methyl]napthamide]] with respect to receptor interactions and duration of efficacy in vivo. In Ca(2+) mobilization assays (fluorometric imaging plate reader), antagonists were applied to human U373MG cells simultaneously with or 2.5 min before substance P (SP). In reversibility studies, antagonists were present for 30 min before washing, and responses to SP were repeatedly measured afterward. The compounds were administered i.p. to gerbils, and the gerbil foot tap (GFT) response was monitored at various time points. The NK(1)R receptor occupancy for aprepitant was determined in striatal regions. Levels of compound in brain and plasma were measured. Antagonists were equipotent at human NK(1)R and acted competitively with SP. After preincubation, aprepitant and ZD6021 attenuated the maximal responses, whereas CP-99994 only shifted the SP concentration-response curve to the right. The inhibitory effect of CP-99994 was over within 30 min, whereas for ZD6021, 50% inhibition still persisted after 60 min. Aprepitant produced maximal inhibition lasting at least 60 min. CP-99994 (3 micromol/kg) inhibited GFT by 100% 15 min after administration, but the effect declined rapidly together with brain levels thereafter. The efficacy of ZD6021 (10 micromol/kg) lasted 4 h and correlated well with brain levels. Aprepitant (3 micromol/kg) inhibited GFT and occupied striatal NK(1)R by 100% for >48 h despite that brain levels of compound were below the limit of detection after 24 h. Slow functional reversibility is associated with long-lasting in vivo efficacy of NK(1)R antagonists, whereas the efficacy of compounds with rapid reversibility is reflected by their pharmacokinetics.