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1.
J Gen Physiol ; 155(11)2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37702787

RESUMO

Pulmonary arterial (PA) smooth muscle cells (PASMC) generate vascular tone in response to agonists coupled to Gq-protein receptor signaling. Such agonists stimulate oscillating calcium waves, the frequency of which drives the strength of contraction. These Ca2+ events are modulated by a variety of ion channels including voltage-gated calcium channels (CaV1.2), the Tmem16a or Anoctamin-1 (ANO1)-encoded calcium-activated chloride (CaCC) channel, and Ca2+ release from the sarcoplasmic reticulum through inositol-trisphosphate receptors (IP3R). Although these calcium events have been characterized, it is unclear how these calcium oscillations underly a sustained contraction in these muscle cells. We used smooth muscle-specific ablation of ANO1 and pharmacological tools to establish the role of ANO1, CaV1.2, and IP3R in the contractile and intracellular Ca2+ signaling properties of mouse PA smooth muscle expressing the Ca2+ biosensor GCaMP3 or GCaMP6. Pharmacological block or genetic ablation of ANO1 or inhibition of CaV1.2 or IP3R, or Ca2+ store depletion equally inhibited 5-HT-induced tone and intracellular Ca2+ waves. Coimmunoprecipitation experiments showed that an anti-ANO1 antibody was able to pull down both CaV1.2 and IP3R. Confocal and superresolution nanomicroscopy showed that ANO1 coassembles with both CaV1.2 and IP3R at or near the plasma membrane of PASMC from wild-type mice. We conclude that the stable 5-HT-induced PA contraction results from the integration of stochastic and localized Ca2+ events supported by a microenvironment comprising ANO1, CaV1.2, and IP3R. In this model, ANO1 and CaV1.2 would indirectly support cyclical Ca2+ release events from IP3R and propagation of intracellular Ca2+ waves.


Assuntos
Cálcio , Hipertensão Pulmonar , Animais , Camundongos , Anoctamina-1 , Serotonina , Músculo Liso
2.
Pharm Biol ; 59(1): 1008-1015, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34362288

RESUMO

CONTEXT: Cucumber (Cucumis sativus Linn. [Cucurbitaceae]) is widely known for its purgative, antidiabetic, antioxidant, and anticancer therapeutic potential. However, its effect on gastrointestinal (GI) disease is unrecognised. OBJECTIVE: This study investigated the effect of C. sativus fruit extract (CCE) on intestinal chloride secretion, motility, and motor function, and the role of TMEM16A chloride channels. MATERIALS AND METHODS: CCE extracts were obtained from commercially available cucumber. Active fractions were then purified by HPLC and analysed by high resolution mass spectrometry. The effect of CCE on intestinal chloride secretion was investigated in human colonic T84 cells, ex vivo mouse intestinal tissue using an Ussing chamber, and the two-electrode voltage-clamp technique to record calcium sensitive TMEM16A chloride currents in Xenopus laevis oocytes. In vivo, intestinal motility was investigated using the loperamide-induced C57BL/6 constipation mouse model. Ex vivo contractility of mouse colonic smooth muscles was assessed by isometric force measurements. RESULTS: CCE increased the short-circuit current (ΔIsc 34.47 ± µA/cm2) and apical membrane chloride conductance (ΔICl 95 ± 8.1 µA/cm2) in intestinal epithelial cells. The effect was dose-dependent, with an EC50 value of 0.06 µg/mL. CCE stimulated the endogenous TMEM16A-induced Cl- current in Xenopus laevis oocytes. Moreover, CCE increased the contractility of smooth muscle in mouse colonic tissue and enhanced small bowel transit in CCE treated mice compared to loperamide controls. Mass spectrometry suggested a cucurbitacin-like analogue with a mass of 512.07 g/mol underlying the bioactivity of CCE. CONCLUSION: A cucurbitacin-like analog present in CCE activates TMEM16A channels, which may have therapeutic potential in cystic fibrosis and intestinal hypodynamic disorders.


Assuntos
Anoctamina-1/metabolismo , Cloretos/metabolismo , Cucumis sativus/química , Intestinos/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Linhagem Celular , Constipação Intestinal/induzido quimicamente , Constipação Intestinal/tratamento farmacológico , Motilidade Gastrointestinal/efeitos dos fármacos , Humanos , Loperamida/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Músculo Liso/efeitos dos fármacos , Técnicas de Patch-Clamp , Xenopus laevis
3.
Biochem Biophys Rep ; 25: 100912, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33537462

RESUMO

TMEM16A (Transmembrane protein 16A or Anoctamin1) is a calcium-activated chloride channel. (CaCC),that exerts critical roles in epithelial secretion. However, its localization, function, and regulation in intestinal chloride (Cl-) secretion remain obscure. Here, we show that TMEM16A protein abundance correlates with Cl- secretion in different regions of native intestine activated by the Ca2+-elevating muscarinic agonist carbachol (CCH). Basal, as well as both cAMP- and CCH-stimulated Isc, was largely reduced in Ano1 ± mouse intestine. We found CCH was not able to increase Isc in the presence of apical to serosal Cl- gradient, strongly supporting TMEM16A as primarily a luminal Cl- channel. Immunostaining demonstrated apical localization of TMEM16A where it colocalized with NHERF1 in mouse colonic tissue. Cellular depletion of NHERF1 in human colonic T84 cells caused a significant reduction of both cAMP- and CCH-stimulated Isc. Immunoprecipitation experiments revealed that NHERF1 forms a complex with TMEM16A through a PDZ-based interaction. We conclude that TMEM16A is a luminal Cl- channel in the intestine that functionally interacts with CFTR via PDZ-based interaction of NHERF1 for efficient and specific cholinergic stimulation of intestinal Cl- secretion.

4.
Am J Physiol Cell Physiol ; 317(6): C1093-C1106, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31461344

RESUMO

This study explored the mechanism by which Ca2+-activated Cl- channels (CaCCs) encoded by the Tmem16a gene are regulated by calmodulin-dependent protein kinase II (CaMKII) and protein phosphatases 1 (PP1) and 2A (PP2A). Ca2+-activated Cl- currents (IClCa) were recorded from HEK-293 cells expressing mouse TMEM16A. IClCa were evoked using a pipette solution in which free Ca2+ concentration was clamped to 500 nM, in the presence (5 mM) or absence of ATP. With 5 mM ATP, IClCa decayed to <50% of the initial current magnitude within 10 min after seal rupture. IClCa rundown seen with ATP-containing pipette solution was greatly diminished by omitting ATP. IClCa recorded after 20 min of cell dialysis with 0 ATP were more than twofold larger than those recorded with 5 mM ATP. Intracellular application of autocamtide-2-related inhibitory peptide (5 µM) or KN-93 (10 µM), two specific CaMKII inhibitors, produced a similar attenuation of TMEM16A rundown. In contrast, internal application of okadaic acid (30 nM) or cantharidin (100 nM), two nonselective PP1 and PP2A blockers, promoted the rundown of TMEM16A in cells dialyzed with 0 ATP. Mutating serine 528 of TMEM16A to an alanine led to a similar inhibition of TMEM16A rundown to that exerted by either one of the two CaMKII inhibitors tested, which was not observed for three putative CaMKII consensus sites for phosphorylation (T273, T622, and S730). Our results suggest that TMEM16A-mediated CaCCs are regulated by CaMKII and PP1/PP2A. Our data also suggest that serine 528 of TMEM16A is an important contributor to the regulation of IClCa by CaMKII.


Assuntos
Anoctamina-1/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Regulação da Expressão Gênica , Proteínas de Neoplasias/genética , Proteína Fosfatase 1/genética , Proteína Fosfatase 2/genética , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Anoctamina-1/metabolismo , Benzilaminas/farmacologia , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cantaridina/farmacologia , Cloretos/metabolismo , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Células HEK293 , Humanos , Transporte de Íons/efeitos dos fármacos , Camundongos , Proteínas de Neoplasias/metabolismo , Ácido Okadáico/farmacologia , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Sulfonamidas/farmacologia
5.
Am J Physiol Gastrointest Liver Physiol ; 316(2): G229-G246, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30406698

RESUMO

Whether zinc (Zn2+) regulates barrier functions by modulating tight-junction (TJ) proteins when pathogens such as Shigella alter epithelial permeability is still unresolved. We investigated the potential benefits of Zn2+ in restoring impaired barrier function in vivo in Shigella-infected mouse tissue and in vitro in T84 cell monolayers. Basolateral Shigella infection triggered a time-dependent decrease in transepithelial resistance followed by an increase in paracellular permeability of FITC-labeled dextran and altered ion selectivity. This led to ion and water loss into the intestinal lumen. Immunofluorescence studies revealed redistribution of claudin-2 and -4 to an intracellular location and accumulation of these proteins in the cytoplasm following infection. Zn2+ ameliorated this perturbed barrier by redistribution of claudin-2 and -4 back to the plasma membrane and by modulating the phosphorylation state of TJ proteins t hough extracellular signal-regulated kinase (ERK)1/2 dependency. Zn2+ prevents elevation of IL-6 and IL-8. Mice challenged with Shigella showed that oral Zn2+supplementation diminished diverse pathophysiological symptoms of shigellosis. Claudin-2 and -4 were susceptible to Shigella infection, resulting in altered barrier function and increased levels of IL-6 and IL-8. Zn2+ supplementation ameliorated this barrier dysfunction, and the inflammatory response involving ERK-mediated change of phosphorylation status for claudin-2 and -4. Thus, Zn2+ may have potential therapeutic value in inflammatory diarrhea and shigellosis. NEW & NOTEWORTHY Our study addresses whether Zn2+ could be an alternative strategy to reduce Shigella-induced inflammatory response and epithelial barrier dysfunction. We have defined a mechanism in terms of intracellular signaling pathways and tight-junction protein expression by Zn2+. Claudin-2 and -4 are susceptible to Shigella infection, whereas in the presence of Zn2+ they are resistant to infection-related barrier dysfunction involving ERK-mediated change of phosphorylation status of claudins.


Assuntos
Claudina-2/metabolismo , Claudina-4/metabolismo , Permeabilidade/efeitos dos fármacos , Zinco/farmacologia , Animais , Claudina-2/efeitos dos fármacos , Claudina-4/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Enteropatias/tratamento farmacológico , Enteropatias/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Zinco/metabolismo
6.
J Biol Chem ; 291(52): 26816-26836, 2016 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-27799301

RESUMO

Accessory cholera enterotoxin (Ace) of Vibrio cholerae has been shown to contribute to diarrhea. However, the signaling mechanism and specific type of Cl- channel activated by Ace are still unknown. We have shown here that the recombinant Ace protein induced ICl of apical plasma membrane, which was inhibited by classical CaCC blockers. Surprisingly, an Ace-elicited rise of current was neither affected by ANO1 (TMEM16A)-specific inhibitor T16A(inh)-AO1(TAO1) nor by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker, CFTR inh-172. Ace stimulated whole-cell current in Caco-2 cells. However, the apical ICl was attenuated by knockdown of ANO6 (TMEM16F). This impaired phenotype was restored by re-expression of ANO6 in Caco-2 cells. Whole-cell patch clamp recordings of ANO currents in HEK293 cells transiently expressing mouse ANO1-mCherry or ANO6-GFP confirmed that Ace induced Cl- secretion. Application of Ace produced ANO6 but not the ANO1 currents. Ace was not able to induce a [Ca2+]i rise in Caco-2 cells, but cellular abundance of phosphatidylinositol 4,5-bisphosphate (PIP2) increased. Identification of the PIP2-binding motif at the N-terminal sequence among human and mouse ANO6 variants along with binding of PIP2 directly to ANO6 in HEK293 cells indicate likely PIP2 regulation of ANO6. The biophysical and pharmacological properties of Ace stimulated Cl- current along with intestinal fluid accumulation, and binding of PIP2 to the proximal KR motif of channel proteins, whose mutagenesis correlates with altered binding of PIP2, is comparable with ANO6 stimulation. We conclude that ANO6 is predominantly expressed in intestinal epithelia, where it contributes secretory diarrhea by Ace stimulation in a calcium-independent mechanism of RhoA-ROCK-PIP2 signaling.


Assuntos
Cloretos/metabolismo , Toxina da Cólera/toxicidade , Cólera/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Anoctaminas , Sequência de Bases , Sistemas CRISPR-Cas , Células CACO-2 , Cálcio/metabolismo , Cólera/induzido quimicamente , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Células HEK293 , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Proteínas de Transferência de Fosfolipídeos/antagonistas & inibidores , Proteínas de Transferência de Fosfolipídeos/genética , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Vibrio cholerae/patogenicidade , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/genética
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