Physiological implications of biased signaling at histamine H2 receptors

Histamine mediates numerous functions acting through its four receptor subtypes all belonging to the large family of seven transmembrane G-protein coupled receptors. In particular, histamine H2 receptor (H2R) is mainly involved in gastric acid production, becoming a classic pharmacological target to treat Zollinger–Ellison disease and gastric and duodenal ulcers. H2 ligands rank among the most widely prescribed and over the counter-sold drugs in the world. Recent evidence indicate that some H2R ligands display biased agonism, selecting and triggering some, but not all, of the signaling pathways associated to the H2R. The aim of the present work is to study whether famotidine, clinically widespread used ligand acting at H2R, exerts biased signaling. Our findings indicate that while famotidine acts as inverse agonist diminishing cAMP basal levels, it mimics the effects of histamine and the agonist amthamine concerning receptor desensitization and internalization. Moreover, the treatment of HEK293T transfected cells with any of the three ligands lead to a concentration dependent pERK increment. Similarly in AGS gastric epithelial cells, famotidine treatment led to both, the reduction in cAMP levels as well as the increment in ERK phosphorylation, suggesting that this behavior could have pharmacological relevant implications. Based on that, histidine decarboxylase expression was studied by quantitative PCR in AGS cells and its levels were increased by famotidine as well as by histamine and amthamine. In all cases, the positive regulation was impeded by the MEK inhibitor PD98059, indicating that biased signaling toward ERK1/2 pathway is the responsible of such enzyme regulation. These results support that ligand bias is not only a pharmacological curiosity but has physiological and pharmacological implications on cell metabolism.Fil: Alonso, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Zappia, Carlos Daniel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cabrera, Maia Diana Eliana. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Davio, Carlos Alberto. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas (i); Argentina; ArgentinaFil: Shayo, Carina Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Monczor, Federico. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernandez, Natalia Cristina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Multidrug transporter MRP4/ABCC4 as a key determinant of pancreatic cancer aggressiveness

Recent findings show that MRP4 is critical for pancreatic ductal adenocarcinoma (PDAC) cell proliferation. Nevertheless, the significance of MRP4 protein levels and function in PDAC progression is still unclear. The aim of this study was to determine the role of MRP4 in PDAC tumor aggressiveness. Bioinformatic studies revealed that PDAC samples show higher MRP4 transcript levels compared to normal adjacent pancreatic tissue and circulating tumor cells express higher levels of MRP4 than primary tumors. Also, high levels of MRP4 are typical of high-grade PDAC cell lines and associate with an epithelial-mesenchymal phenotype. Moreover, PDAC patients with high levels of MRP4 depict dysregulation of pathways associated with migration, chemotaxis and cell adhesion. Silencing MRP4 in PANC1 cells reduced tumorigenicity and tumor growth and impaired cell migration. Transcriptomic analysis revealed that MRP4 silencing alters PANC1 gene expression, mainly dysregulating pathways related to cell-to-cell interactions and focal adhesion. Contrarily, MRP4 overexpression significantly increased BxPC-3 growth rate, produced a switch in the expression of EMT markers, and enhanced experimental metastatic incidence. Altogether, our results indicate that MRP4 is associated with a more aggressive phenotype in PDAC, boosting pancreatic tumorigenesis and metastatic capacity, which could finally determine a fast tumor progression in PDAC patients.Fil: Sahores, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Carozzo, A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: May, M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Gomez, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Di Siervi, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: de Sousa Serro, Maximiliano Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Yaneff, Agustín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Rodriguez Gonzalez, Angela Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Abba, Martín Carlos. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Inmunológicas Básicas y Aplicadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Shayo, Carina Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Davio, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; Argentin

Cyclic AMP efflux through MRP4 regulates actin dynamics signalling pathway and sperm motility in bovines

Previously we demonstrated that multidrug resistance-associated protein 4 transporter (MRP4) mediates cAMP efflux in bovine spermatozoa and that extracellular cAMP (ecAMP) triggers events associated to capacitation. Here, we deepen the study of the role of MRP4 in bovine sperm function by using MK571, an MRP4 inhibitor. The incubation of spermatozoa with MK571 during 45 min inhibited capacitation-associated events. MRP4 was localized in post-acrosomal region and mid-piece at 15 min capacitation, while at 45 min it was mainly located in the acrosome. After 15 min, MK571 decreased total sperm motility (TM), progressive motility (PM) and several kinematic parameters. The addition of ecAMP rescued MK571 effect and ecAMP alone increased the percentage of motile sperm and kinematics parameters. Since actin cytoskeleton plays essential roles in the regulation of sperm motility, we investigated if MRP4 activity might affect actin polymerization. After 15 min capacitation, an increase in F-actin was observed, which was inhibited by MK571. This effect was reverted by the addition of ecAMP. Furthermore, ecAMP alone increased F-actin levels while no F-actin was detected with ecAMP in the presence of PKA inhibitors. Our results support the importance of cAMP efflux through MRP4 in sperm capacitation and suggest its involvement in the regulation of actin polymerization and motility.Fil: Chiarante, Nicolás Agustín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Alonso, Carlos A. I.. McGill University; CanadáFil: Plaza, Jessica. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; ArgentinaFil: Lottero Leconte, Raquel María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Arroyo Salvo, Camila Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Yaneff, Agustín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Osycka Salut, Claudia Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Davio, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Miragaya, Marcelo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; ArgentinaFil: Perez Martinez, Silvina Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentin

Multidrug transporter MRP4/ABCC4 as a key determinant of pancreatic cancer aggressiveness

Recent fndings show that MRP4 is critical for pancreatic ductal adenocarcinoma (PDAC) cell proliferation. Nevertheless, the signifcance of MRP4 protein levels and function in PDAC progression is still unclear. The aim of this study was to determine the role of MRP4 in PDAC tumor aggressiveness. Bioinformatic studies revealed that PDAC samples show higher MRP4 transcript levels compared to normal adjacent pancreatic tissue and circulating tumor cells express higher levels of MRP4 than primary tumors. Also, high levels of MRP4 are typical of high-grade PDAC cell lines and associate with an epithelial-mesenchymal phenotype. Moreover, PDAC patients with high levels of MRP4 depict dysregulation of pathways associated with migration, chemotaxis and cell adhesion. Silencing MRP4 in PANC1 cells reduced tumorigenicity and tumor growth and impaired cell migration. Transcriptomic analysis revealed that MRP4 silencing alters PANC1 gene expression, mainly dysregulating pathways related to cell-to-cell interactions and focal adhesion. Contrarily, MRP4 overexpression signifcantly increased BxPC-3 growth rate, produced a switch in the expression of EMT markers, and enhanced experimental metastatic incidence. Altogether, our results indicate that MRP4 is associated with a more aggressive phenotype in PDAC, boosting pancreatic tumorigenesis and metastatic capacity, which could fnally determine a fast tumor progression in PDAC patients.Facultad de Ciencias MédicasCentro de Investigaciones Inmunológicas Básicas y Aplicada

Atrial natriuretic factor stimulates exocrine pancreatic secretion in the rat through NPR-C receptors

Increasing evidence supports the role of atrial natriuretic factor (ANF) in the modulation of gastrointestinal physiology. The effect of ANF on exocrine pancreatic secretion and the possible receptors and pathways involved were studied in vivo. Anesthetized rats were prepared with pancreatic duct cannulation, pyloric ligation, and bile diversion into the duodenum. ANF dose-dependently increased pancreatic secretion of fluid and proteins and enhanced secretin and CCK-evoked response. ANF decreased chloride secretion and increased the pH of the pancreatic juice. Neither cholinergic nor adrenergic blockade affected ANF-stimulated pancreatic secretion. Furthermore, ANF response was not mediated by the release of nitric oxide. ANF-evoked protein secretion was not inhibited by truncal vagotomy, atropine, or Nω-nitro-L-arginine methyl ester administration. The selective natriuretic peptide receptor-C (NPR-C) receptor agonist cANP-(4-23) mimicked ANF response in a dose-dependent fashion. When the intracellular signaling coupled to NPR-C receptors was investigated in isolated pancreatic acini, results showed that ANF did not modify basal or forskolin-evoked cAMP formation, but it dose-dependently enhanced phosphoinositide hydrolysis, which was blocked by the selective PLC inhibitor U-73122. ANF stimulated exocrine pancreatic secretion in the rat, and its effect was not mediated by nitric oxide or parasympathetic or sympathetic activity. Furthermore, CCK and secretin appear not to be involved in ANF response. Present findings support that ANF exerts a stimulatory effect on pancreatic exocrine secretion mediated by NPR-C receptors coupled to the phosphoinositide pathway.Fil: Sabbatini, María E.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; ArgentinaFil: Villagra, Alberto. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Davio, Carlos Alberto. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Vatta, Marcelo Sergio. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Fernandez, Belisario Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; ArgentinaFil: Bianciotti, Liliana Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; Argentin

G-protein-coupled receptor 30/adenylyl cyclase/protein kinase A pathway is involved in estradiol 17ß-D-glucuronide-induced cholestasis

Estradiol-17ß-D-glucuronide (E17G) activates different signaling pathways (e.g., Ca21- dependent protein kinase C, phosphoinositide 3-kinase/protein kinase B, mitogenactivated protein kinases [MAPKs] p38 and extracellular signal-related kinase 1/2, and estrogen receptor alpha) that lead to acute cholestasis in rat liver with retrieval of the canalicular transporters, bile salt export pump (Abcb11) and multidrug resistanceassociated protein 2 (Abcc2). E17G shares with nonconjugated estradiol the capacity to activate these pathways. G-protein-coupled receptor 30 (GPR30) is a receptor implicated in nongenomic effects of estradiol, and the aim of this study was to analyze the potential role of this receptor and its downstream effectors in E17G-induced cholestasis. In vitro, GPR30 inhibition by G15 or its knockdown with small interfering RNA strongly prevented E17G-induced impairment of canalicular transporter function and localization. E17G increased cyclic adenosine monophosphate (cAMP) levels, and this increase was blocked by G15, linking GPR30 to adenylyl cyclase (AC). Moreover, AC inhibition totally prevented E17G insult. E17G also increased protein kinase A (PKA) activity, which was blocked by G15 and AC inhibitors, connecting the links of the pathway, GPR30-AC-PKA. PKA inhibition prevented E17G-induced cholestasis, whereas exchange protein activated directly by cyclic nucleotide/MAPK kinase, another cAMP downstream effector, was not implicated in cAMP cholestatic action. In the perfused rat liver model, inhibition of the GPR30-AC-PKA pathway totally prevented E17G-induced alteration in Abcb11 and Abcc2 function and localization. Conclusion: In conclusion, activation of GPR30-AC-PKA is a key factor in the alteration of canalicular transporter function and localization induced by E17G. Interaction of E17G with GPR30 may be the first event in the cascade of signaling activation.Fil: Zucchetti, Andrés E. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Barosso, Ismael R. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Boaglio, Andrea C. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Basiglio, Cecilia Lorena. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Miszczuk, Gisel Sabrina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Larocca, María Cecilia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); ArgentinaFil: Ruiz, M. Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Davio, Carlos A. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Laboratorio de Farmacología de Receptores; Argentina.Fil: Roma, Marcelo Gabriel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Crocenzi, Fernando A. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Sánchez Pozzi, Enrique J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina

A Novel Effect of β-Adrenergic Receptor on Mammary Branching Morphogenesis and its Possible Implications in Breast Cancer

Understanding the mechanisms that govern normal mammary gland development is crucial to the comprehension of breast cancer etiology. β-adrenergic receptors (β-AR) are targets of endogenous catecholamines such as epinephrine that have gained importance in the context of cancer biology. Differences in β -AR expression levels may be responsible for the effects of epinephrine on tumor vs non-tumorigenic breast cell lines, the latter expressing higher levels of β -AR. To study regulation of the breast cell phenotype by β -AR, we over-expressed β -AR in MCF-7 breast cancer cells and knocked-down the receptor in non-tumorigenic MCF-10A breast cells. In MCF-10A cells having knocked-down β -AR, epinephrine increased cell proliferation and migration, similar to the response by tumor cells. In contrast, in MCF-7 cells overexpressing the β -AR, epinephrine decreased cell proliferation and migration and increased adhesion, mimicking the response of the non-tumorigenic MCF-10A cells, thus underscoring that β -AR expression level is a key player in cell behavior. β-adrenergic stimulation with isoproterenol induced differentiation of breast cells growing in 3-dimension cell culture, and also the branching of murine mammary epithelium in vivo. Branching induced by isoproterenol was abolished in fulvestrant or tamoxifen-treated mice, demonstrating that the effect of β-adrenergic stimulation on branching is dependent on the estrogen receptor (ER). An ER-independent effect of isoproterenol on lumen architecture was nonetheless found. Isoproterenol significantly increased the expression of ERα, Ephrine-B1 and fibroblast growth factors in the mammary glands of mice, and in MCF-10A cells. In a poorly differentiated murine ductal carcinoma, isoproterenol also decreased tumor growth and induced tumor differentiation. This study highlights that catecholamines, through β-AR activation, seem to be involved in mammary gland development, inducing mature duct formation. Additionally, this differentiating effect could be resourceful in a breast tumor context