RESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells that line the airway, gut, and exocrine glands; it is well established that CFTR plays a pivotal role in cholera toxin (CTX)-induced secretory diarrhea. Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in blood and foods, has been reported to play a vital role in a variety of conditions involving gastrointestinal wound repair, apoptosis, inflammatory bowel disease, and diarrhea. Here we show, for the first time, that type 2 LPA receptors (LPA2) are expressed at the apical surface of intestinal epithelial cells, where they form a macromolecular complex with Na+/H+ exchanger regulatory factor-2 and CFTR through a PSD95/Dlg/ZO-1-based interaction. LPA inhibited CFTR-dependent iodide efflux through LPA2-mediated Gi pathway, and LPA inhibited CFTR-mediated short-circuit currents in a compartmentalized fashion. CFTR-dependent intestinal fluid secretion induced by CTX in mice was reduced substantially by LPA administration; disruption of this complex using a cell-permeant LPA2-specific peptide reversed LPA2-mediated inhibition. Thus, LPA-rich foods may represent an alternative method of treating certain forms of diarrhea.
Assuntos
Toxina da Cólera/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Diarreia/tratamento farmacológico , Lisofosfolipídeos/farmacologia , Análise de Variância , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Toxina da Cólera/toxicidade , Cricetinae , AMP Cíclico/metabolismo , Proteínas do Citoesqueleto/metabolismo , Diarreia/induzido quimicamente , Proteína 4 Homóloga a Disks-Large , Células Epiteliais/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio , Proteína da Zônula de Oclusão-1RESUMO
Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized at apical cell membranes and exists in macromolecular complexes with a variety of signaling and transporter molecules. Here, we report that the multidrug resistance protein 4 (MRP4), a cAMP transporter, functionally and physically associates with CFTR. Adenosine-stimulated CFTR-mediated chloride currents are potentiated by MRP4 inhibition, and this potentiation is directly coupled to attenuated cAMP efflux through the apical cAMP transporter. CFTR single-channel recordings and FRET-based intracellular cAMP dynamics suggest that a compartmentalized coupling of cAMP transporter and CFTR occurs via the PDZ scaffolding protein, PDZK1, forming a macromolecular complex at apical surfaces of gut epithelia. Disrupting this complex abrogates the functional coupling of cAMP transporter activity to CFTR function. Mrp4 knockout mice are more prone to CFTR-mediated secretory diarrhea. Our findings have important implications for disorders such as inflammatory bowel disease and secretory diarrhea.
Assuntos
AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Mucosa Intestinal/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Animais , Compartimento Celular/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Canais de Cloreto/metabolismo , AMP Cíclico/análogos & derivados , Diarreia/induzido quimicamente , Células HT29 , Humanos , Camundongos , Camundongos Knockout , Modelos Biológicos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Propionatos/farmacologia , Ligação Proteica , Quinolinas/farmacologia , Fatores de TempoRESUMO
S-nitrosothiols (RSNOs) are important mediators of nitric oxide (NO) biology. The two mechanisms that appear to dominate in their biological effects are metabolism leading to the formation of NO and S-nitrosation of protein thiols. In this study we demonstrate that RSNOs inhibit uterine smooth muscle cell proliferation independent of NO. The antiproliferative effects of NO on vascular smooth muscle are well defined, with the classic NO-dependent production of cGMP being demonstrated as the active pathway. However, less is known on the role of NO in mediating uterine smooth muscle cell function, a process that is important during menstruation and pregnancy. The RSNOs S-nitrosoglutathione and S-nitroso-N-acetyl pencillamine inhibited growth factor-dependent proliferation of human and rat uterine smooth muscle cells (ELT-3). Interestingly, these cells reduced RSNOs to generate NO. However, use of NO donors and other activators of the cGMP pathway failed to inhibit proliferation. These findings demonstrate the tissue-specific nature of responses to NO and demonstrate the presence of a RSNO-dependent but NO-independent pathway of inhibiting DNA synthesis in uterine smooth muscle cells.