RESUMO
Mammary gland ion transport is essential for lactation and is regulated by prolactin and glucocorticoids. This study delineates the roles of prolactin receptors (PRLR) and long-term prolactin and dexamethasone (P-D)-mediation of [Ca(2+)](i) and Cl(-) transport in HC-11 cells. P-D (24 h) suppressed ATP-induced [Ca(2+)](i). This may be due to decreased Ca(2+) entry since P-D decreased transient receptor potential channel 3 (TRPC3) but not secretory pathway Ca(2+)-ATPase 2 (SPCA2) mRNA. ATP increased Cl(-) transport, measured by iodide (I(-)) efflux, in control and P-D-treated cells. P-D enhanced I(-) efflux response to cAMP secretagogues without altering Cl(-) channels or NKCC cotransporter expression. HC-11 cells contain only the long form of PRLR (PRLR-L). Since the short isoform, PRLR-S, is mammopoietic, we determined if transfecting PRLR-S (rs) altered PRLR-L-mediated Ca(2+) and Cl(-) transport. Untreated rs cells showed an attenuated [Ca(2+)](i) response to ATP with no further response to P-D, in contrast to vector-transfected (vtc) controls. P-D inhibited TRPC3 in rs and vtc cells but increased SPCA2 only in rs cells. As in wild-type, cAMP-stimulated Cl(-) transport, in P-D-treated vtc and rs cells. In summary, 24 h P-D acts via PRLR-L to attenuate ATP-induced [Ca(2+)](i) and increase cAMP-activated Cl(-) transport. PRLR-S fine-tunes these responses underscoring its mammopoietic action.
RESUMO
High levels of calcitonin (CT) observed in medullary thyroid carcinoma and other CT-secreting tumours cause severe diarrhoea. Previous studies have suggested that CT induces active chloride secretion. However, the involvement of CT receptor (CTR) and the molecular mechanisms underlying the modulation of intestinal electrolyte secreting intestinal epithelial cells have not been investigated. Therefore, current studies were undertaken to investigate the direct effects of CT on ion transport in intestinal epithelial cells. Real time quantitative RT-PCR and Western blot analysis demonstrated the expression of CTR in intestinal epithelial T84 cells. Exposure of T84 cells to CT from the basolateral but not from apical side significantly increased short circuit current (I(SC) ) in a dose-dependent manner that was blocked by 1 µM of CTR antagonist, CT8-32. CT-induced I(SC) was blocked by replacing chloride in the bath solutions with equimolar gluconate and was significantly inhibited by the specific cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor, CFTR(127inh). Further, biotinylation studies showed that CT increased CFTR levels on the apical membrane. The presence of either the Ca(2+) chelator, bis(2-aminophenoxy)ethane tetraacetic acid-acetoxymethyl (BAPTA-AM) ester or the protein kinase A (PKA) inhibitor, H89, significantly inhibited I(SC) induced by CT (â¼32-58% reduction). Response to CT was retained after permeabilization of the basolateral or the apical membranes of T84 cells with nystatin. In conclusion, the activation of CTR by CT induced chloride secretion across T84 monolayers via CFTR channel and the involvement of PKA- and Ca(2+) -dependent signalling pathways. These data elucidate the molecular mechanisms underlying CT-induced diarrhoea.
Assuntos
Calcitonina/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Receptores da Calcitonina/metabolismo , Biotinilação , Western Blotting , Membrana Celular/metabolismo , Proliferação de Células , Células Cultivadas , Quelantes/farmacologia , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Células Epiteliais/efeitos dos fármacos , Humanos , Intestinos/citologia , Intestinos/efeitos dos fármacos , Transporte de Íons , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores da Calcitonina/genética , Transdução de SinaisRESUMO
BACKGROUND: Lubiprostone, used clinically (b.i.d.) to treat constipation, has been reported to increase transepithelial Cl(-) transport in T84 cells by activating ClC-2 channels. AIM: To identify the underlying signaling pathway, we explored the effects of short-term and overnight lubiprostone treatment on second messenger signaling and Cl(-) transport. METHODS: Cl(-) transport was assessed either as I(sc) across T84 monolayers grown on Transwells and mounted in Ussing chambers or by the iodide efflux assay. [cAMP](i) was measured by enzyme immunoassay, and [Ca(2+)](i) by Fluo-3 fluorescence. Quantitation of apical cell surface CFTR protein levels was assessed by Western blotting and biotinylation with the EZ-Link Sulfo-NHS-LC-LC-Biotin. ClC-2 mRNA level was studied by RT-PCR. RESULTS: Lubiprostone and the cAMP stimulator, forskolin, caused comparable and maximal increases of I(sc) in T84 cells. The I(sc) effects of lubiprostone and forskolin were each suppressed if the tissue had previously been treated with the other agent. These responses were unaltered even if the monolayers were treated with lubiprostone overnight. Lubiprostone-induced increases in iodide efflux were ~80% of those obtained with forskolin. Lubiprostone increased [cAMP](i). H89, bumetanide, or CFTR(inh)-172 greatly attenuated lubiprostone-stimulated Cl(-) secretion, whereas the ClC-2 inhibitor CdCl(2) did not. Compared to controls, FSK-treatment increased membrane-associated CFTR by 1.9 fold, and lubiprostone caused a 2.6-fold increase in apical membrane CFTR as seen by immunoblotting following cell surface biotinylation. CONCLUSIONS: Lubiprostone activates Cl(-) secretion in T84 cells via cAMP, protein kinase A, and by increasing apical membrane CFTR protein.
Assuntos
Alprostadil/análogos & derivados , Carcinoma/metabolismo , Cloretos/metabolismo , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Alprostadil/farmacologia , Benzoatos/farmacologia , Transporte Biológico/efeitos dos fármacos , Bumetanida/farmacologia , Catárticos/farmacologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Colforsina/farmacologia , Neoplasias do Colo/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lubiprostona , Tiazolidinas/farmacologiaRESUMO
PURPOSE OF REVIEW: The 2009 review on small intestinal ion transport, in this series, focused on recent advances in duodenal bicarbonate secretion, the importance of scaffolding proteins and the pathophysiology of inflammation-associated diarrhea. The current review focuses on advances in ion-coupled solute transport, the dynamic role of the paracellular pathway in transepithelial-fluid transport and of elucidating the cellular basis of diarrheas associated with enteric infections. RECENT FINDINGS: In understanding the cellular pathophysiology underlying diarrheal diseases, there is increased focus on the role of altering Na absorptive mechanisms as well as the role of the paracellular pathway. This is not to minimize the role of Cl-secretory pathways, especially cystic fibrosis transmembrane conductance regulator (CFTR), which continues to have pleiotropic roles in modulating other transporters. The Na-glucose cotransporter (SGLT) was the first transporter ever to be cloned. Twenty-one years later, with another first, the crystal structure of the related Na-galactose transporter has been described and opens new avenues to understand structure-function relationships and intelligent drug design for transporters. SUMMARY: Progress continues to be made on integrating information obtained from reductionist models into more complex in-vivo animal models and where possible in human studies. Recognition of the coordinated regulation of cellular Na absorptive and Cl-secretory pathways together with the paracellular route in health and disease will help develop a more holistic picture of the multifaceted nature of small intestinal ion transport.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Intestino Delgado/fisiologia , Transporte de Íons/fisiologia , Via Secretória/fisiologia , Animais , Permeabilidade da Membrana Celular/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Modelos Animais de Doenças , Humanos , Intestino Delgado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Sensibilidade e Especificidade , Simportadores de Sódio-Bicarbonato/metabolismoRESUMO
OBJECTIVES: We have shown that Ca2+-dependent regulation of Cl- secretion in the mammalian colon exhibits age dependence. Because epidermal growth factor (EGF) has a well-established role in growth and can increase intracellular calcium [Ca2+]i, it is conceivable that its developmental influence may extend to the regulation of intestinal ion transport. In this study, we examined the role of EGF in the regulation of Cl- transport in the developing rabbit distal colon. MATERIALS AND METHODS: Because serum contains growth factors, which could have confounded our studies, we first established an optimal milieu for testing EGF in primary cultures of adult rabbit distal colonocytes by culturing them for 24 h in media containing 0%, 1%, 5%, and 20% serum. Chloride transport (millimoles per second) and [Ca2+]i were measured with use of the fluorescent indicator N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) and Fura-2AM, respectively. RESULTS: Serum depletion had no effect on cell number, DNA content, or basal Cl- transport, but it significantly affected cell viability. In media with 0%, 1%, or 20% serum, bethanechol, 8BrcAMP, taurodeoxycholate, and EGF stimulated Cl- transport to a similar extent. EGF maximally stimulated Cl- transport at 16.3 nmol/L and 20 minutes. Bethanechol, but not EGF, increased [Ca2+]i. EGF did not alter bethanechol-stimulated Cl- transport or [Ca2+]i. EGF acts via an EGF-receptor and mitogen activated protein kinase (MAPK) signaling pathway, since stimulation of Cl- transport was abolished by genistein, AG1478, and PD98059. Weanling and adult colonocytes, cultured in 1% serum, showed similar basal and EGF-stimulated Cl- transport. CONCLUSIONS: EGF stimulates rabbit colonic Cl- transport via a Ca2+-independent, tyrosine kinase- and MAPK-dependent pathway, and its effects are not age dependent.
Assuntos
Cloretos/metabolismo , Colo/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Transporte de Íons/fisiologia , Animais , Canais de Cálcio , Células Cultivadas , Colo/citologia , Proteínas Quinases Ativadas por Mitógeno , Proteínas Tirosina Quinases/metabolismo , Coelhos , Transdução de Sinais/fisiologiaRESUMO
Calcium-dependent secretagogues, such as neurotensin, stimulate age-dependent chloride transport in rabbit distal colonocytes, but their action in the proximal colon is unknown. This study examines the effect of neurotensin on chloride transport and its mechanism of action in rabbit proximal colonocytes. Our results show that neurotensin stimulates chloride transport only in adult, and not weanling or newborn, colonocytes. The calcium ionophore A23187 shows similar age dependence, while PGE2, which acts via cAMP, stimulates transport in all ages. The roles of phospholipase C, tyrosine kinases, and src tyrosine kinases were examined using specific inhibitors, i.e., U73122, genistein, and PP2, respectively. All three agents significantly inhibit neurotensin-stimulated chloride transport in adult colonocytes. In conclusion, this study reports for the first time that neurotensin stimulates chloride secretion in rabbit proximal colonocytes. This is also the first demonstration that neurotensin action exhibits age dependence and is dependent on phospholipase C and src tyrosine kinase activity.