Homotrimeric P2X3 and heteromeric P2X2/3 receptors are present in sensory ganglia and participate in pain perception. In order to develop pharmacological antagonists for these receptors, it is important to clarify which amino acid (AA) residues constitute the agonist binding pouch as well as to learn the stoichiometry of the receptor subunits forming a heteromeric receptor. We expressed the homomeric human (h)P2X3 receptor or its mutants in HEK293 cells and measured the ATP-induced responses by the whole-cell patch-clamp method. For the binding-site mutants, all conserved and some non-conserved AAs in the four nucleotide binding segments (NBSs) of the P2X3 subunit were sequentially replaced by alanine. Especially the positively charged AAs Lys and Arg appeared to be of critical importance for the agonist effects. We concluded that groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the P2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor. We were also interested to find out, whether two heteromeric receptors (P2X2/3 and P2X2/6), where P2X2 combines with two different partners, have an obligatory subunit stoichiometry of 1:2 or whether the subunit stoichiometry may be variable. For this purpose we used non-functional P2X2, P2X3 and P2X6 subunit-mutants to investigate the composition of heteromeric P2X2/3 and P2X2/6 receptors. The subunit stoichiometry of P2X2/3 and P2X2/6 was found to be 1:2 and 2:1, respectively. Thus, recognitions sites between P2X2 and its partners rather than random association may govern the subunit compositions of the receptor trimers.

Homotrimeric P2X3 and heteromeric P2X2/3 receptors are present in sensory ganglia and participate in pain perception. In order to develop pharmacological antagonists for these receptors, it is important to clarify which amino acid (AA) residues constitute the agonist binding pouch as well as to learn the stoichiometry of the receptor subunits forming a heteromeric receptor. We expressed the homomeric human (h)P2X3 receptor or its mutants in HEK293 cells and measured the ATP-induced responses by the whole-cell patch-clamp method. For the binding-site mutants, all conserved and some non-conserved AAs in the four nucleotide binding segments (NBSs) of the P2X3 subunit were sequentially replaced by alanine. Especially the positively charged AAs Lys and Arg appeared to be of critical importance for the agonist effects. We concluded that groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the P2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor. We were also interested to find out, whether two heteromeric receptors (P2X2/3 and P2X2/6), where P2X2 combines with two different partners, have an obligatory subunit stoichiometry of 1:2 or whether the subunit stoichiometry may be variable. For this purpose we used non-functional P2X2, P2X3 and P2X6 subunit-mutants to investigate the composition of heteromeric P2X2/3 and P2X2/6 receptors. The subunit stoichiometry of P2X2/3 and P2X2/6 was found to be 1:2 and 2:1, respectively. Thus, recognitions sites between P2X2 and its partners rather than random association may govern the subunit compositions of the receptor trimers.:Index of contents Introductory remarks „Wissenschaftlicher Anteil des Promovenden an der Publikation“ „Bibliographische Beschreibung“ I. Introduction Pain as a sensory quality Neuronal circuitry for pain processing and sensation in the PNS and CNS Transformation of thermal, mechanical and chemical stimuli into electrical activity by nociceptors; nociceptor-targeted therapeutic approaches Release mechanisms for nucleotides and their fate in the extracellular space Nucleotide receptor-types ATP-sensitive P2 receptors and pain-sensation References II. Scientific background and aims of my thesis ATP binding-sites of P2X3 receptors; subunit composition of P2X2/3 and P2X2/6 heteromeric receptors The aims of the present work III. Publications IV. Summary and conclusions Amino acid residues constituting the agonist binding site of the human P2X3 receptor ATP binding site mutagenesis reveals different subunit stoichiometry of functional P2X2/3 and P2X2/6 receptors „Eigenständigkeitserklärung“ Curriculum vitae Acknowledgements

Foi descrita a presença do receptor P2X2 no sistema nervoso entérico e foi constatado que a desnutrição afeta os neurônios mioentéricos. Este projeto analisou os neurônios dos plexos mioentérico (PM) e submucoso (PS) imunorreativos ao receptor P2X2(ir), calbindina (Calb-ir), calretinina (Calr-ir), colina acetil transferase (ChAT-ir) e ao óxido nítrico sintase (NOS-ir) do íleo de ratos submetidos à desnutrição protéica pré e pós-natal e a renutrição pós-natal. Foram utilizados íleos de animais nutridos (N42), desnutridos (D42) e renutridos (RN42). Os resultados do PM e PS demonstraram 100% de neurônios Calb-ir, Calr-ir, ChAt-ir e NOS-ir expressavam o receptor P2X2-ir nos grupos. As densidades neuronais do PM e PS demonstraram aumento de 30% e 25% respectivamente, dos neurônios receptor P2X2-ir, Calr-ir, ChAT-ir e NOS-ir no grupo D42 e, recuperação no grupo RN42, os neurônios Calb-ir aumentaram 60% no PS. O perfil neuronal do PM mostrou diminuição de 26% nos neurônios receptor P2X2-ir e Calr-ir no grupo D42 e, no PS houve diminuição nos neurônios receptor P2X2-ir. Concluiu que a desnutrição afetou os neurônios entéricos e houve recuperação na renutrição, isto pode influenciar as funções do trato gastrintestinal. / It is showed the expression of the P2X2 receptor in the enteric nervous system; it was observed that undernutrition affect the myenteric neurons. This project analyzed the ileum myenteric (MN) and submucous (SN) neurons immunoreactive to P2X2 receptor, calbindin (Calb-ir), calretinin (Calr-ir), coline acetil transferase (ChAT) and nitric oxide sintase (NOS) of the animals submitted to pre- and postnatal protein deprivation and postnatal refeeding. Ileum was used from nourished (N42), undernourished (D42) and Refeeding (RN42) animals. The results showed 100% coexpression myenteric and submucous Calb-ir, Calr-ir, ChAt-ir e NOS-ir neurons with P2X2-ir receptor. The myenteric and submucos neuronal density showed increase of the 30% and 25% respectively, of the P2X2-ir, Calr-ir, ChAT-ir e NOS-ir neurons of the D42 group, the Calb-ir neurons increase 60% in the SN. It was observed in the MN neuronal profile decrease of the 26% of the P2X2-ir and Calr-ir in the RN42 group and recover in the RN42, there was decrease of the P2X2-ir receptor neurons in the submucous plexus. The conclusion demonstrated that undernutrition affects the enteric neurons and there was recuperation in the refeeding, this can influence the gastrointestinal functions.

Este projeto, analisou a distribuição dos neurônios nos plexos mioentérico (PM) e submucoso (PS) imunorreativos aos receptores P2X2 (ir) e P2X7 (ir), calbindina (Calb-ir), calretinina (Calr-ir), colina acetil transferase (ChAT-ir) e ao óxido nítrico sintase (NOS-ir) do colo distal de ratos submetidos à desnutrição a renutrição protéica. Utilizaram-se colos distais de ratos nutridos (N42), desnutridos (D42) e renutridos (RN42). Os resultados do plexo PM, demonstraram que 100% dos neurônios Calb-ir, Calr-ir, ChAT-ir e NOS-ir, expressavam os receptores P2X2-ir e P2X7-ir nos três grupos. A densidade neuronal no PM, demonstrou um aumento de 20% a 97% dos neurônios receptores P2X2-7-ir, Calr-ir, ChAT-ir e NOS-ir e no PS foi de 29% a 75%, ambos D42 e recuperação no RN42. O perfil neuronal P2X7-ir, Calb-ir, Calr-ir e ChAT-ir do PM demonstrou diminuição de 28% a 40% e no PS os neurônios P2X2-7-ir, Calb-ir e ChAT-ir de 19% a 47% no D42. Concluí-se que, a desnutrição afeta os neurônios entéricos havendo recuperação na renutrição, podendo influenciar nas funções gastrintestinais. / The aim of the work was to analyze the distal colon myenteric (MN) and submucous (SN) neurons immunoreactive for P2X2-7 receptors, calbindin (Calb-ir), calretinin (Calr-ir), choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) of the animals submitted to undernutrition and refeeding proteic. Distal colon was used from nourished (N42), undernourished (D42) and refeeding (RN42) rats. The results have shown 100% coexpression of the myenteric and submucous Calb-ir, Calr-ir, ChAt-ir e NOS-ir neurons with P2X2-7-ir receptors. The MN density have shown increase of the 20% and 97% of the P2X2-7-ir, Calr-ir, ChAT-ir e NOS-ir neurons of the D42 group, and the SN have been increased 29% a 75% in the D42 group. In the MN neuronal profile have shown decrease P2X7-ir, Calb-ir, Calr-ir and ChAT-ir neurons of the 28% to 40% and in the PS P2X2-7-ir, Calb-ir and ChAT-ir of the 19% a 47% neurons in the D42 group. I concluded that, the undernutrition affects the enteric neurons and there was recuperation in the refeeding, this can influence the gastrintestinal functions.

Este projeto, analisou a distribuição dos neurônios nos plexos mioentérico (PM) e submucoso (PS) imunorreativos aos receptores P2X2 (ir) e P2X7 (ir), calbindina (Calb-ir), calretinina (Calr-ir), colina acetil transferase (ChAT-ir) e ao óxido nítrico sintase (NOS-ir) do colo distal de ratos submetidos à desnutrição a renutrição protéica. Utilizaram-se colos distais de ratos nutridos (N42), desnutridos (D42) e renutridos (RN42). Os resultados do plexo PM, demonstraram que 100% dos neurônios Calb-ir, Calr-ir, ChAT-ir e NOS-ir, expressavam os receptores P2X2-ir e P2X7-ir nos três grupos. A densidade neuronal no PM, demonstrou um aumento de 20% a 97% dos neurônios receptores P2X2-7-ir, Calr-ir, ChAT-ir e NOS-ir e no PS foi de 29% a 75%, ambos D42 e recuperação no RN42. O perfil neuronal P2X7-ir, Calb-ir, Calr-ir e ChAT-ir do PM demonstrou diminuição de 28% a 40% e no PS os neurônios P2X2-7-ir, Calb-ir e ChAT-ir de 19% a 47% no D42. Concluí-se que, a desnutrição afeta os neurônios entéricos havendo recuperação na renutrição, podendo influenciar nas funções gastrintestinais. / The aim of the work was to analyze the distal colon myenteric (MN) and submucous (SN) neurons immunoreactive for P2X2-7 receptors, calbindin (Calb-ir), calretinin (Calr-ir), choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) of the animals submitted to undernutrition and refeeding proteic. Distal colon was used from nourished (N42), undernourished (D42) and refeeding (RN42) rats. The results have shown 100% coexpression of the myenteric and submucous Calb-ir, Calr-ir, ChAt-ir e NOS-ir neurons with P2X2-7-ir receptors. The MN density have shown increase of the 20% and 97% of the P2X2-7-ir, Calr-ir, ChAT-ir e NOS-ir neurons of the D42 group, and the SN have been increased 29% a 75% in the D42 group. In the MN neuronal profile have shown decrease P2X7-ir, Calb-ir, Calr-ir and ChAT-ir neurons of the 28% to 40% and in the PS P2X2-7-ir, Calb-ir and ChAT-ir of the 19% a 47% neurons in the D42 group. I concluded that, the undernutrition affects the enteric neurons and there was recuperation in the refeeding, this can influence the gastrintestinal functions.

Foi descrita a presença do receptor P2X2 no sistema nervoso entérico e foi constatado que a desnutrição afeta os neurônios mioentéricos. Este projeto analisou os neurônios dos plexos mioentérico (PM) e submucoso (PS) imunorreativos ao receptor P2X2(ir), calbindina (Calb-ir), calretinina (Calr-ir), colina acetil transferase (ChAT-ir) e ao óxido nítrico sintase (NOS-ir) do íleo de ratos submetidos à desnutrição protéica pré e pós-natal e a renutrição pós-natal. Foram utilizados íleos de animais nutridos (N42), desnutridos (D42) e renutridos (RN42). Os resultados do PM e PS demonstraram 100% de neurônios Calb-ir, Calr-ir, ChAt-ir e NOS-ir expressavam o receptor P2X2-ir nos grupos. As densidades neuronais do PM e PS demonstraram aumento de 30% e 25% respectivamente, dos neurônios receptor P2X2-ir, Calr-ir, ChAT-ir e NOS-ir no grupo D42 e, recuperação no grupo RN42, os neurônios Calb-ir aumentaram 60% no PS. O perfil neuronal do PM mostrou diminuição de 26% nos neurônios receptor P2X2-ir e Calr-ir no grupo D42 e, no PS houve diminuição nos neurônios receptor P2X2-ir. Concluiu que a desnutrição afetou os neurônios entéricos e houve recuperação na renutrição, isto pode influenciar as funções do trato gastrintestinal. / It is showed the expression of the P2X2 receptor in the enteric nervous system; it was observed that undernutrition affect the myenteric neurons. This project analyzed the ileum myenteric (MN) and submucous (SN) neurons immunoreactive to P2X2 receptor, calbindin (Calb-ir), calretinin (Calr-ir), coline acetil transferase (ChAT) and nitric oxide sintase (NOS) of the animals submitted to pre- and postnatal protein deprivation and postnatal refeeding. Ileum was used from nourished (N42), undernourished (D42) and Refeeding (RN42) animals. The results showed 100% coexpression myenteric and submucous Calb-ir, Calr-ir, ChAt-ir e NOS-ir neurons with P2X2-ir receptor. The myenteric and submucos neuronal density showed increase of the 30% and 25% respectively, of the P2X2-ir, Calr-ir, ChAT-ir e NOS-ir neurons of the D42 group, the Calb-ir neurons increase 60% in the SN. It was observed in the MN neuronal profile decrease of the 26% of the P2X2-ir and Calr-ir in the RN42 group and recover in the RN42, there was decrease of the P2X2-ir receptor neurons in the submucous plexus. The conclusion demonstrated that undernutrition affects the enteric neurons and there was recuperation in the refeeding, this can influence the gastrointestinal functions.

As atividades intestinais são coordenadas pelo sistema nervoso entérico e, disfunções na motilidade intestinal são observadas em indivíduos obesos. O ATP é um co-transmissor e seus receptores estão distribuídos nos neurônios entéricos constituindo a família P2X e P2Y. O presente estudo tem como objetivo analisar a expressão do receptor P2X2 e o código químico nos neurônios mioentéricos na obesidade. Foram utilizados íleo de camundongos obesos machos (OBM) e fêmeas (OBF) (C57BL/6J ob/ob), e controles (CF; CM) (+/+), que foram submetidos à técnica imuno-histoquímica de duplas marcações em neurônios imunomarcados ao receptor P2X2 com NOS (inibitórios), ChAT (excitatórios) ou Calr (excitatórios e sensoriais). Resultados dos grupos CF e OBF: Foi verificado a imunomarcação ao receptor P2X2, NOS, ChAT e Calr no citoplasma e nas membranas celular e nuclear dos neurônios mioentéricos de ambos os grupos. As duplas marcações dos neurônios imunorreativos (-ir) ao receptor P2X2 com NOS, ChAT ou Calr em CF foram 24±4%, 24±2% e 24±4%, e em OBF foram 19±4%, 25±4% e 22±1%, respectivamente. As duplas marcações dos neurônios NOS-ir, ChAT-ir ou Calr-ir com os neurônios receptor P2X2-ir foram 100% nos dois grupos. A densidade dos neurônios P2X2-ir aumentou em 62%, enquanto que a dos neurônios NOS-ir e ChAT-ir reduziram em 49% e 57%. A morfometria demonstrou aumento na área dos neurônios NOS-ir (CF 234±63, OBF 312±67); ChAT-ir (CF 210±24, OBF 253±14) e Calr-ir (CF 203±41, OBF 315±47), no entanto, neurônios P2X2-ir não apresentaram alterações (CF 325±23, OBF 336±67). A histoquímica demonstrou não haver diferença estatística entre os grupos quanto á densidade e a morfometria dos neurônios NADH-diaforase positivos. Resultados dos grupos CM e OBM: A expressão do receptor P2X2 foi identificada somente em CM e a imunomarcação a NOS, ChAT e Calr nos dois grupos. Em CM as duplas marcações dos neurônios P2X2-ir com NOS-ir, ChAT-ir ou Calr-ir foram 23±3%, 34±5% e 32±6%, respectivamente, e nos neurônios NOS-ir, ChAT-ir e Calr-ir com o receptor P2X2 foram 100%. A densidade dos neurônios NOS-ir e Calr-ir reduziram em 31% e 16%, enquanto que a densidade da população ChAT-ir aumentou 31%. A morfometria demonstrou que neurônios NOS-ir (CM 390±49, OBM 350±22); ChAT-ir (289±18, OBM 312±44), Calr-ir (CM 375±49, OBM 360±38) e P2X2-ir (CM 437±190) não apresentaram diferenças entre os grupos. A expressão protéica pela técnica do Western Blotting mostrou que houve uma redução de 36,5% no grupo OBM. Concluímos que neste modelo experimental tanto a obesidade (C57BL/6J ob/ob) como os gêneros podem contribuir com alterações: na expressão do receptor P2X2, na densidade e na morfologia dos neurônios NOS-ir, ChAT-ir e Calr-ir, promovendo mudanças nas atividades intestinais. / The intestinal activities are coordinated by enteric nervous system, and dysfunctions in intestinal motility were observed in obese individuals. The enteric neurons have been demonstrated to express different purinergics receptors. This study aims to analyze the expression of P2X2 receptor and the chemical code in the myenteric neurons in obesity. Sample from ileum of obese male (OBM) and females (OBF) (C57BL/6J ob/ob) and controls mice (CF, CM) (+/+) were used. Immunohistochemistry for double labelling in neurons P2X2 receptor immunostained with NOS (inhibitory), ChAT (excitatory) or Calr (excitatory and sensory) was performed. Results of CF and OBF groups: immunostaining was verified for P2X2 receptor, NOS, ChAT and Calr in the cytoplasm and in cell and nuclear membranes of myenteric neurons of both groups. The double-labelling for P2X2 receptor/NOS, ChAT or Calr in CF were 24 ±4%, 24 ±2% and 24 ±4%, respectively, and in OBF were 19 ±4%, 25 ±4% and 22±1%. Inversely, NOS, ChAT or Calr/P2X2 receptor immunoreactives neurons were 100% in both groups. The density of P2X2 neurons showed an increase of 62%, whereas NOS and ChAT neurons decreased by 49% and 57%. In morphometric analysis showed an increase in NOS (CF 234 ± 63, 312 ± 67 OBF), ChAT (CF 210 ± 24, 253 ± 14 OBF) and Calr (CF 203 ± 41, 315 ± 47 OBF) neuronal area, however, P2X2 neurons no changed in this feature (FC 325 ± 23, 336 ± 67 OBF). The histochemistry for NADH-diaphorase showed no statistical difference to density and morphometry of neurons between the groups. Results of CM and OBM groups: The P2X2 receptor expression was identified only in CM. Immunostaining for NOS, ChAT and Calr were observed in both groups. The double-labelling observed in CM for P2X2 receptor and NOS, ChAT or Calr were 23±3%, 34 ±5% and 32 ±6%, and inversely, NOS, ChAT or Calr/P2X2 receptor immunoreactives neurons were 100%. The NOS and Calr neuronal density were reduced about 31% and 16%, while population ChAT neuronal density increased about 31%. The morphometry showed that NOS (CM 390 ± 49, 350 ± 22 OBM), ChAT (289 ± 18, 312 ± 44 OBM), Calr (CM 375 ± 49, 360 ± 38 OBM) and P2X2 (CM 437 ± 190) neurons no differ between groups. The P2X2 receptor protein expression by Western blotting showed that there was a reduction in 36.5% in OBM. We concluded that in this experimental model, both obesity (C57BL/6J ob/ob) and genders may contribute to changes in P2X2 receptor expression, density and morphology of neurons NOS, ChAT and Calr, promoting alterations in intestinal activities.

As atividades intestinais são coordenadas pelo sistema nervoso entérico e, disfunções na motilidade intestinal são observadas em indivíduos obesos. O ATP é um co-transmissor e seus receptores estão distribuídos nos neurônios entéricos constituindo a família P2X e P2Y. O presente estudo tem como objetivo analisar a expressão do receptor P2X2 e o código químico nos neurônios mioentéricos na obesidade. Foram utilizados íleo de camundongos obesos machos (OBM) e fêmeas (OBF) (C57BL/6J ob/ob), e controles (CF; CM) (+/+), que foram submetidos à técnica imuno-histoquímica de duplas marcações em neurônios imunomarcados ao receptor P2X2 com NOS (inibitórios), ChAT (excitatórios) ou Calr (excitatórios e sensoriais). Resultados dos grupos CF e OBF: Foi verificado a imunomarcação ao receptor P2X2, NOS, ChAT e Calr no citoplasma e nas membranas celular e nuclear dos neurônios mioentéricos de ambos os grupos. As duplas marcações dos neurônios imunorreativos (-ir) ao receptor P2X2 com NOS, ChAT ou Calr em CF foram 24±4%, 24±2% e 24±4%, e em OBF foram 19±4%, 25±4% e 22±1%, respectivamente. As duplas marcações dos neurônios NOS-ir, ChAT-ir ou Calr-ir com os neurônios receptor P2X2-ir foram 100% nos dois grupos. A densidade dos neurônios P2X2-ir aumentou em 62%, enquanto que a dos neurônios NOS-ir e ChAT-ir reduziram em 49% e 57%. A morfometria demonstrou aumento na área dos neurônios NOS-ir (CF 234±63, OBF 312±67); ChAT-ir (CF 210±24, OBF 253±14) e Calr-ir (CF 203±41, OBF 315±47), no entanto, neurônios P2X2-ir não apresentaram alterações (CF 325±23, OBF 336±67). A histoquímica demonstrou não haver diferença estatística entre os grupos quanto á densidade e a morfometria dos neurônios NADH-diaforase positivos. Resultados dos grupos CM e OBM: A expressão do receptor P2X2 foi identificada somente em CM e a imunomarcação a NOS, ChAT e Calr nos dois grupos. Em CM as duplas marcações dos neurônios P2X2-ir com NOS-ir, ChAT-ir ou Calr-ir foram 23±3%, 34±5% e 32±6%, respectivamente, e nos neurônios NOS-ir, ChAT-ir e Calr-ir com o receptor P2X2 foram 100%. A densidade dos neurônios NOS-ir e Calr-ir reduziram em 31% e 16%, enquanto que a densidade da população ChAT-ir aumentou 31%. A morfometria demonstrou que neurônios NOS-ir (CM 390±49, OBM 350±22); ChAT-ir (289±18, OBM 312±44), Calr-ir (CM 375±49, OBM 360±38) e P2X2-ir (CM 437±190) não apresentaram diferenças entre os grupos. A expressão protéica pela técnica do Western Blotting mostrou que houve uma redução de 36,5% no grupo OBM. Concluímos que neste modelo experimental tanto a obesidade (C57BL/6J ob/ob) como os gêneros podem contribuir com alterações: na expressão do receptor P2X2, na densidade e na morfologia dos neurônios NOS-ir, ChAT-ir e Calr-ir, promovendo mudanças nas atividades intestinais. / The intestinal activities are coordinated by enteric nervous system, and dysfunctions in intestinal motility were observed in obese individuals. The enteric neurons have been demonstrated to express different purinergics receptors. This study aims to analyze the expression of P2X2 receptor and the chemical code in the myenteric neurons in obesity. Sample from ileum of obese male (OBM) and females (OBF) (C57BL/6J ob/ob) and controls mice (CF, CM) (+/+) were used. Immunohistochemistry for double labelling in neurons P2X2 receptor immunostained with NOS (inhibitory), ChAT (excitatory) or Calr (excitatory and sensory) was performed. Results of CF and OBF groups: immunostaining was verified for P2X2 receptor, NOS, ChAT and Calr in the cytoplasm and in cell and nuclear membranes of myenteric neurons of both groups. The double-labelling for P2X2 receptor/NOS, ChAT or Calr in CF were 24 ±4%, 24 ±2% and 24 ±4%, respectively, and in OBF were 19 ±4%, 25 ±4% and 22±1%. Inversely, NOS, ChAT or Calr/P2X2 receptor immunoreactives neurons were 100% in both groups. The density of P2X2 neurons showed an increase of 62%, whereas NOS and ChAT neurons decreased by 49% and 57%. In morphometric analysis showed an increase in NOS (CF 234 ± 63, 312 ± 67 OBF), ChAT (CF 210 ± 24, 253 ± 14 OBF) and Calr (CF 203 ± 41, 315 ± 47 OBF) neuronal area, however, P2X2 neurons no changed in this feature (FC 325 ± 23, 336 ± 67 OBF). The histochemistry for NADH-diaphorase showed no statistical difference to density and morphometry of neurons between the groups. Results of CM and OBM groups: The P2X2 receptor expression was identified only in CM. Immunostaining for NOS, ChAT and Calr were observed in both groups. The double-labelling observed in CM for P2X2 receptor and NOS, ChAT or Calr were 23±3%, 34 ±5% and 32 ±6%, and inversely, NOS, ChAT or Calr/P2X2 receptor immunoreactives neurons were 100%. The NOS and Calr neuronal density were reduced about 31% and 16%, while population ChAT neuronal density increased about 31%. The morphometry showed that NOS (CM 390 ± 49, 350 ± 22 OBM), ChAT (289 ± 18, 312 ± 44 OBM), Calr (CM 375 ± 49, 360 ± 38 OBM) and P2X2 (CM 437 ± 190) neurons no differ between groups. The P2X2 receptor protein expression by Western blotting showed that there was a reduction in 36.5% in OBM. We concluded that in this experimental model, both obesity (C57BL/6J ob/ob) and genders may contribute to changes in P2X2 receptor expression, density and morphology of neurons NOS, ChAT and Calr, promoting alterations in intestinal activities.