RESUMO
Slc4a genes encode various types of transporters, including Na+-HCO3- cotransporters, Cl-/HCO3- exchangers, or Na+-driven Cl-/HCO3- exchangers. Previous research has revealed that Slc4a9 (Ae4) functions as a Cl-/HCO3- exchanger, which can be driven by either Na+ or K+, prompting investigation into whether other Slc4a members facilitate cation-dependent anion transport. In the present study, we show that either Na+ or K+ drive Cl-/HCO3- exchanger activity in cells overexpressing Slc4a8 or Slc4a10. Further characterization of cation-driven Cl-/HCO3- exchange demonstrated that Slc4a8 and Slc4a10 also mediate Cl- and HCO3--dependent K+ transport. Full-atom molecular dynamics simulation on the recently solved structure of Slc4a8 supports the coordination of K+ at the Na+ binding site in S1. Sequence analysis shows that the critical residues coordinating monovalent cations are conserved among mouse Slc4a8 and Slc4a10 proteins. Together, our results suggest that Slc4a8 and Slc4a10 might transport K+ in the same direction as HCO3- ions in a similar fashion to that described for Na+ transport in the rat Slc4a8 structure.
Assuntos
Potássio , Simportadores de Sódio-Bicarbonato , Animais , Camundongos , Bicarbonatos/metabolismo , Sítios de Ligação , Antiportadores de Cloreto-Bicarbonato/metabolismo , Antiportadores de Cloreto-Bicarbonato/genética , Cloretos/metabolismo , Transporte de Íons , Simulação de Dinâmica Molecular , Potássio/metabolismo , Sódio/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Simportadores de Sódio-Bicarbonato/genéticaRESUMO
Ae4 transporters are critical for Cl- uptake across the basolateral membrane of acinar cells in the submandibular gland (SMG). Although required for fluid secretion, little is known about the physiological regulation of Ae4. To investigate whether Ae4 is regulated by the cAMP-dependent signaling pathway, we measured Cl-/HCO3- exchanger activity in SMG acinar cells from Ae2-/- mice, which only express Ae4, and found that the Ae4-mediated activity was increased in response to ß-adrenergic receptor stimulation. Moreover, pretreatment with H89, an inhibitor of the cAMP-activated kinase (PKA), prevented the stimulation of Ae4 exchangers. We then expressed Ae4 in CHO-K1 cells and found that the Ae4-mediated activity was increased when Ae4 is coexpressed with the catalytic subunit of PKA (PKAc), which is constitutively active. Ae4 sequence analysis showed two potential PKA phosphorylation serine residues located at the intracellular NH2-terminal domain according to a homology model of Ae4. NH2-terminal domain Ser residues were mutated to alanine (S173A and S273A, respectively), where the Cl-/HCO3- exchanger activity displayed by the mutant S173A was not activated by PKA. Conversely, S273A mutant kept the PKA dependency. Together, we conclude that Ae4 is stimulated by PKA in SMG acinar cells by a mechanism that probably depends on the phosphorylation of S173.NEW & NOTEWORTHY We found that Ae4 exchanger activity in secretory salivary gland acinar cells is increased upon ß-adrenergic receptor stimulation. The activation of Ae4 was prevented by H89, a nonselective PKA inhibitor. Protein sequence analysis revealed two residues (S173 and S273) that are potential targets of cAMP-dependent protein kinase (PKA). Experiments in CHO-K1 cells expressing S173A and S273A mutants showed that S173A, but not S273A, is not activated by PKA.
Assuntos
Células Acinares/enzimologia , Antiportadores de Cloreto-Bicarbonato/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Glândulas Salivares/enzimologia , Animais , Células CHO , Antiportadores de Cloreto-Bicarbonato/química , Antiportadores de Cloreto-Bicarbonato/genética , Cricetulus , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Feminino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Mutação , Fosforilação , Conformação Proteica , Glândulas Salivares/citologia , Relação Estrutura-Atividade , CamundongosRESUMO
OBJECTIVE: To clarify clinical and genetic features of Japanese children with congenital chloride diarrhea (CCD). STUDY DESIGN: This was a multi-institutional, retrospective survey of 616 pediatric centers in Japan with identified patients with CCD between 2014 and 2018. Mutations involving SLC26A3 were detected by Sanger sequencing. RESULTS: Thirteen patients met all entry criteria including mutations in SLC26A3, and 14 patients satisfied clinical diagnostic criteria. Homozygous or compound heterozygous mutations in SLC26A3, including 6 novel mutations, were identified in 13 of these 14 patients (93%). The most common (detected in 7 of 13) was c.2063-1g>t. Median age at diagnosis was 1 day. Nine of the patients meeting all criteria were diagnosed as neonates (69%). Median follow-up duration was 10 years. When studied, 8 patients had <5 stools daily (62%), and all had fewer than in infancy. Only 1 patient had nephrocalcinosis, and 3 (23%) had mild chronic kidney disease. Neurodevelopment was generally good; only 1 patient required special education. Five patients (38%) received long-term sodium, potassium, and chloride supplementation. CONCLUSIONS: Early fetal ultrasound diagnosis and prompt long-term sodium, potassium, and chloride supplementation were common management features. Genetic analysis of SLC26A3 provided definitive diagnosis of CCD. In contrast with previously reported localities, c.2063-1g>t might be a founder mutation in East Asia.
Assuntos
Antiportadores de Cloreto-Bicarbonato/genética , DNA/genética , Diarreia/congênito , Previsões , Erros Inatos do Metabolismo/genética , Mutação , Vigilância da População , Transportadores de Sulfato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo , Análise Mutacional de DNA , Diarreia/epidemiologia , Diarreia/genética , Diarreia/metabolismo , Feminino , Seguimentos , Testes Genéticos , Humanos , Incidência , Recém-Nascido , Japão/epidemiologia , Masculino , Erros Inatos do Metabolismo/epidemiologia , Erros Inatos do Metabolismo/metabolismo , Estudos Retrospectivos , Transportadores de Sulfato/metabolismo , Taxa de Sobrevida/tendências , Fatores de TranscriçãoRESUMO
In obstructive cholestasis, there is an integral adaptive response aimed to diminish the bile flow and minimize the injury of bile ducts caused by increased intraluminal pressure and harmful levels of bile salts and bilirrubin. Canalicular bicarbonate secretion, driven by the anion exchanger 2 (AE2), is an influential determinant of the canalicular bile salt-independent bile flow. In this work, we ascertained whether AE2 expression and/or activity is reduced in hepatocytes from rats with common bile duct ligation (BDL), as part of the adaptive response to cholestasis. After 4 days of BDL, we found that neither AE2 mRNA expression (measured by quantitative real-time PCR) nor total levels of AE2 protein (assessed by western blot) were modified in freshly isolated hepatocytes. However, BDL led to a decrease in the expression of AE2 protein in plasma membrane fraction as compared with SHAM control. Additionally, AE2 activity (JOH-, mmol/L/min), measured in primary cultured hepatocytes from BDL and SHAM rats, was decreased in the BDL group versus the control group (1.9 ± 0.3 vs. 3.1 ± 0.2, p<0.005). cAMP-stimulated AE2 activity, however, was not different between SHAM and BDL groups (3.7 ± 0.3 vs. 3.5 ± 0.3), suggesting that cAMP stimulated insertion into the canalicular membrane of AE2-containing intracellular vesicles, that had remained abnormally internalized after BDL. In conclusion, our results point to the existence of a novel adaptive mechanism in cholestasis aimed to reduce biliary pressure, in which AE2 internalization in hepatocytes might result in decreased canalicular HCO3- output and decreased bile flow.
Assuntos
Bicarbonatos/metabolismo , Antiportadores de Cloreto-Bicarbonato/biossíntese , Cloretos/metabolismo , Colestase/metabolismo , Regulação para Baixo , Hepatócitos/metabolismo , Animais , Colestase/patologia , Modelos Animais de Doenças , Hepatócitos/patologia , Transporte de Íons , Masculino , Ratos , Ratos WistarRESUMO
OBJECTIVE: To determine the role of anion exchanger 3 (AE3) in dorsal root ganglion (DRG) in nerve injury-induced chronic nociception in the rat. METHODS: Spared nerve injury (SNI) was used to induce neuropathic pain. Von Frey filaments and Hargreaves test were used to assess tactile allodynia and thermal hyperalgesia, respectively. Drugs were given by intrathecal administration. Western blotting was used to determine AE3 expression in DRG. KEY FINDINGS: SNI produced long-lasting mechanical allodynia and thermal hyperalgesia. AE3 was found in DRG of sham-operated rats. SNI enhanced baseline AE3 expression in L4 and L5 DRGs at days 7 and 14, respectively. In contrast, SNI did not affect AE3 expression in L6 DRG. AE3 expression returned to baseline levels 21 days after SNI. Intrathecal 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) (5-50 µg) pretreatment prevented SNI-induced allodynia and, at a lesser extent, hyperalgesia. Moreover, DIDS (50 µg) reduced SNI-induced AE3 upregulation in L4, but not L5, DRGs. Intrathecal DIDS (5-50 µg) or anti-AE3 antibody (1 µg), but not vehicle, post-treatment (6 days) partially reversed SNI-induced allodynia and hyperalgesia. DIDS or anti-AE3 antibody post-treatment diminished SNI-induced AE3 upregulation in L4 and L5 DRGs. CONCLUSIONS: Data suggest that AE3 is present in DRG and contributes to mechanical allodynia and thermal hyperalgesia in neuropathic rats.
Assuntos
Antiportadores de Cloreto-Bicarbonato/biossíntese , Gânglios Espinais/metabolismo , Hiperalgesia/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Animais , Autoanticorpos/farmacologia , Antiportadores de Cloreto-Bicarbonato/efeitos dos fármacos , Feminino , Hiperalgesia/complicações , Hiperalgesia/prevenção & controle , Injeções Espinhais , Medição da Dor , Traumatismos dos Nervos Periféricos/complicações , RatosAssuntos
Antiportadores de Cloreto-Bicarbonato/genética , Diarreia/congênito , Regulação da Expressão Gênica no Desenvolvimento , Erros Inatos do Metabolismo/diagnóstico por imagem , Erros Inatos do Metabolismo/terapia , Transportadores de Sulfato/genética , Ultrassonografia Pré-Natal/métodos , Adulto , Cesárea/métodos , Colo/anormalidades , Colo/diagnóstico por imagem , Tratamento Conservador , Diarreia/diagnóstico por imagem , Diarreia/genética , Diarreia/terapia , Dilatação , Feminino , Seguimentos , Idade Gestacional , Humanos , Recém-Nascido , Imageamento por Ressonância Magnética/métodos , Erros Inatos do Metabolismo/genética , Gravidez , Resultado da Gravidez , Diagnóstico Pré-Natal/métodosRESUMO
The excitability of neuronal networks is strongly modulated by changes in pH. The origin of these changes, however, is still under debate. The high complexity of neural systems justifies the use of computational simulation to investigate mechanisms that are possibly involved. Simulated neuronal activity includes non-synaptic epileptiform events (NEA) induced in hippocampal slices perfused with high-K(+) and zero-Ca(2+), therefore in the absence of the synaptic circuitry. A network of functional units composes the NEA model. Each functional unit represents one interface of neuronal/extracellular space/glial segments. Each interface contains transmembrane ionic transports, such as ionic channels, cotransporters, exchangers and pumps. Neuronal interconnections are mediated by gap-junctions, electric field effects and extracellular ionic fluctuations modulated by extracellular electrodiffusion. Mechanisms investigated are those that change intracellular and extracellular ionic concentrations and are able to affect [H(+)]. Our simulations suggest that the intense fluctuations in intra and extracellular concentrations of Na(+), K(+) and Cl(-) that accompany NEA are able to affect the combined action of the Na(+)/H(+) exchanger (NHE), [HCO(-)(3)]/Cl(-) exchanger (HCE), H(+) pump and the catalytic activity of intra and extracellular carbonic anhydrase. Cellular volume changes and extracellular electrodiffusion are responsible for modulating pH.
Assuntos
Simulação por Computador , Hipocampo/citologia , Hipocampo/fisiologia , Modelos Neurológicos , Rede Nervosa/citologia , Rede Nervosa/fisiologia , Tamanho Celular , Antiportadores de Cloreto-Bicarbonato/metabolismo , Cloro/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Modelos Biológicos , Neurônios/citologia , Neurônios/fisiologia , Potássio/metabolismo , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
La clorhidrorrea congénita es un raro desorden autosómico recesivo, causado por un defecto en el intercambio de cloruro/bicarbonato en el íleon y colon. En este trabajo se reporta el caso de un niño de 1 año de edad con características patognomónicas de esta condición, consistentes en antecedentes prenatales de polihidramnios, diarreas acuosas desde el nacimiento, poca ganancia de peso, alcalosis metabólica y deshidratación. El diagnóstico fue confirmado por el elevado contenido de cloruro en heces, y es el segundo caso reportado en la literatura cubana(AU)
Congenital chloride diarrhea is a rare autosomal recessive disorder caused by a defective exchange of chloride and bicarbonate in the ileum and the colon. This article reported the case of one-year old child with pathognomonic characteristics of this disease including prenatal history of polyhydramnios, watery diarrheas since birth, low weight gain, metabolic alkalosis and dehydration. The diagnosis was confirmed on the basis of the high contents of chloride in stools. He is the second case of this disease reported in the Cuban literature(AU)
Assuntos
Humanos , Lactente , Poli-Hidrâmnios/diagnóstico , Vipoma/complicações , Antiportadores de Cloreto-Bicarbonato/efeitos adversos , Relatos de CasosRESUMO
La clorhidrorrea congénita es un raro desorden autosómico recesivo, causado por un defecto en el intercambio de cloruro/bicarbonato en el íleon y colon. En este trabajo se reporta el caso de un niño de 1 año de edad con características patognomónicas de esta condición, consistentes en antecedentes prenatales de polihidramnios, diarreas acuosas desde el nacimiento, poca ganancia de peso, alcalosis metabólica y deshidratación. El diagnóstico fue confirmado por el elevado contenido de cloruro en heces, y es el segundo caso reportado en la literatura cubana.
Congenital chloride diarrhea is a rare autosomal recessive disorder caused by a defective exchange of chloride and bicarbonate in the ileum and the colon. This article reported the case of one-year old child with pathognomonic characteristics of this disease including prenatal history of polyhydramnios, watery diarrheas since birth, low weight gain, metabolic alkalosis and dehydration. The diagnosis was confirmed on the basis of the high contents of chloride in stools. He is the second case of this disease reported in the Cuban literature.
Assuntos
Humanos , Poli-Hidrâmnios/diagnóstico , Vipoma/complicações , Antiportadores de Cloreto-Bicarbonato/efeitos adversos , Relatos de CasosRESUMO
We determined the role of chloride-bicarbonate anion exchanger 3 in formalin-induced acute and chronic rat nociception. Formalin (1%) produced acute (first phase) and tonic (second phase) nociceptive behaviors (flinching and licking/lifting) followed by long-lasting evoked secondary mechanical allodynia and hyperalgesia in both paws. Local peripheral pre-treatment with the chloride-bicarbonate anion exchanger inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid prevented formalin-induced nociception mainly during phase 2. These drugs also prevented in a dose-dependent fashion long-lasting evoked secondary mechanical allodynia and hyperalgesia in both paws. Furthermore, post-treatment (on day 1 or 6) with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid reversed established hypersensitivity. Anion exchanger 3 was expressed in dorsal root ganglion neurons and it co-localized with neuronal nuclei protein (NeuN), substance P and purinergic P2X3 receptors. Furthermore, Western blot analysis revealed a band of about 85 kDa indicative of anion exchanger 3 protein expression in dorsal root ganglia of naïve rats, which was enhanced at 1 and 6 days after 1% formalin injection. On the other hand, this rise failed to occur during 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid exposure. These results suggest that anion exchanger 3 is present in dorsal root ganglia and participates in the development and maintenance of short and long-lasting formalin-induced nociception.
Assuntos
Antiportadores de Cloreto-Bicarbonato/metabolismo , Formaldeído/efeitos adversos , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Dor/induzido quimicamente , Dor/metabolismo , Animais , Feminino , Formaldeído/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Nociceptividade/efeitos dos fármacos , Dor/fisiopatologia , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Wistar , Ácidos Sulfônicos/farmacologiaRESUMO
Boron is an important micronutrient in plants and animals. The role of boron in living systems includes coordinated regulation of gene expression, growth and proliferation of higher plants and animals. There are several well-defined genes associated with boron transportation and tolerance in plants and these genes show close homology with human anion exchanger genes. Mutation of these genes also characterizes some genetic disorders. We investigated the toxic effects of boric acid on HEK293 cells and mRNA expression of anion exchanger (SLC4A1, SLC4A2 and SLC4A3) genes. Cytotoxicity of boric acid at different concentrations was tested by using the methylthiazolyldiphenyl-tetrazolium bromide assay. Gene expression profiles were examined using quantitative real-time PCR. In the HEK293 cells, the nontoxic upper concentration of boric acid was 250 µM; more than 500 µM caused cytotoxicity. The 250 µM boric acid concentration increased gene expression level of SLC4A2 up to 8.6-fold and SLC4A3 up to 2.6-fold, after 36-h incubation. There was no significant effect of boric acid on SLC4A1 mRNA expression levels.
Assuntos
Proteínas de Transporte de Ânions/genética , Antiporters/genética , Ácidos Bóricos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Sequência de Bases , Linhagem Celular , Antiportadores de Cloreto-Bicarbonato , Primers do DNA , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas SLC4ARESUMO
Iodine is a critical element involved in thyroid hormone synthesis. Its efflux into the follicular lumen is thought to occur, in part, through pendrin at the apical membrane of thyrocytes. This study attempted to investigate whether iodide administration affects SLC26A4 mRNA expression in rat thyroid and in PCCl3 cells. Rats and cells were treated or not with NaI from 30 min up to 48 h. One group was concomitantly treated with sodium perchlorate. SLC26A4 mRNA expression was also investigated in PCCl3 cells treated with actinomycin D prior to NaI treatment. Iodide administration significantly increased SLC26A4 mRNA content in both models. The simultaneous administration of NaI and perchlorate, as well as the treatment of PCCl3 cells with actinomycin D prevented this effect, indicating that intracellular iodide is essential for this event, which appears to be triggered by transcriptional mechanisms. These data show that intracellular iodide rapidly upregulates SLC26A4 mRNA expression.
Assuntos
Antiportadores de Cloreto-Bicarbonato/genética , Iodetos/metabolismo , Glândula Tireoide/metabolismo , Transcrição Gênica , Animais , Antitireóideos/farmacologia , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Antiportadores de Cloreto-Bicarbonato/metabolismo , Dactinomicina/farmacologia , Iodetos/farmacologia , Masculino , Metimazol/farmacologia , Percloratos/farmacologia , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transportadores de Sulfato , Glândula Tireoide/efeitos dos fármacos , Tireotropina/sangue , Tiroxina/sangue , Tri-Iodotironina/sangueRESUMO
It is well known that chemotactic agents active Na(+)/H(+) exchanger, increasing intracellular pH of neutrophils, but their effect on bicarbonate transporters have not been established yet. To study the effect of fMLP on the activity of Cl(-)/HCO(3)(-) exchange, the rate of pH recovery after acute Cl(-) readmission in cell subjected to an alkaline load by CO(2) washout in a Cl-free medium was measured. The activity of the exchanger was reduced to 72% of control when cells were pre-incubated for 5 min with 0.1 µM fMLP and reached 48% of control in steady state after acute exposure. After extracellular bicarbonate or TMA addition the rate recovery of intracellular pH was reduce at 72% and at 84%, respectively. The inhibitory effect on the intracellular pH recovery was not affected by blockers of Na(+)/H(+) exchange. We conclude from these studies that an increase of pH(i) produced for this chemotactic agent is facilitated by the simultaneous activation of Na(+)/H(+) exchange and inhibition of Cl(-)/HCO(3)(-) exchange in neutrophils.
Assuntos
Bicarbonatos/metabolismo , Cloretos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Receptores de Formil Peptídeo/agonistas , Células Cultivadas , Quimiotaxia , Antiportadores de Cloreto-Bicarbonato/metabolismo , Humanos , Hidrogênio/metabolismo , Neutrófilos/metabolismo , Sódio/metabolismoRESUMO
The functional versatility of the distal nephron is mainly due to the large cytological heterogeneity of the segment. Part of Na+ uptake by distal tubules is dependent on Na+/H+ exchanger 2 (NHE2), implicating a role of distal convoluted cells also in acid-base homeostasis. In addition, intercalated (IC) cells expressed in distal convoluted tubules, connecting tubules and collecting ducts are involved in the final regulation of acid-base excretion. IC cells regulate acid-base handling by 2 main transport proteins, a V-type H+-ATPase and a Cl/HCO3- exchanger, localized at different membrane domains. Type A IC cells are characterized by a luminal H+-ATPase in series with a basolateral Cl/HCO3- exchanger, the anion exchanger AE1. Type B IC cells mediate HCO3- secretion through the apical Cl-/HCO3- exchanger pendrin in series with a H+-ATPase at the basolateral membrane. Alternatively, H+/K+-ATPases have also been found in several distal tubule cells, particularly in type A and B IC cells. All of these mechanisms are finely regulated, and mutations of 1 or more proteins ultimately lead to expressive disorders of acid-base balance.
Assuntos
Equilíbrio Ácido-Base/fisiologia , Túbulos Renais Distais/metabolismo , Néfrons/metabolismo , Animais , Antiportadores de Cloreto-Bicarbonato/fisiologia , ATPase Trocadora de Hidrogênio-Potássio/fisiologia , Humanos , Transporte de Íons , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/fisiologia , ATPases Vacuolares Próton-Translocadoras/fisiologiaAssuntos
Diarreia Infantil/genética , Poli-Hidrâmnios/epidemiologia , Abdome , Antiporters/genética , Antiportadores de Cloreto-Bicarbonato , Cloretos/metabolismo , Consanguinidade , Diarreia Infantil/metabolismo , Diarreia Infantil/fisiopatologia , Feminino , Humanos , Hiponatremia/etiologia , Recém-Nascido , Recém-Nascido Prematuro , Doenças do Prematuro/fisiopatologia , Masculino , Peristaltismo , Gravidez , Transportadores de SulfatoRESUMO
Arginine-vasopressin (AVP) has been proposed to be involved in the modulation of acid-base transporters; however, the nature of the mechanisms underlying AVP direct action on intracellular pH (pH(i)) in the cortical collecting duct (CCD) is not yet clearly defined. The aim of the present study was to elucidate which are the proteins implicated in AVP modulation of pH(i), as well as the receptors involved in these responses using a CCD cell line (RCCD(1)); pH(i) was monitored with the fluorescent dye BCECF in basal conditions and after stimulation with basolateral 10(-8) M AVP. Specific V1- or V2-receptor antagonists were also used. RT-PCR studies demonstrated that RCCD(1) cells express V1a and V2 receptors. Functional studies showed that while V2-receptor activation induced a biphasic response (alkalinization-acidification), V1-receptor activation resulted in an intracellular acidification. The V2-mediated alkalinization phase involves the activation of basolateral NHE-1 isoform of the Na(+)/H(+) exchanger while in the acidification phase CFTR is probably implicated. On the other hand, V1-mediated acidification was due to activation of a Cl(-)/HCO(3)(-) exchanger. We conclude that in RCCD(1) cells AVP selectively activates, via a complex of V1 and V2 receptor-mediated actions, different ion transporters linked to pH(i) regulation which might have physiological implications.
Assuntos
Arginina Vasopressina/farmacologia , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/metabolismo , Receptores de Vasopressinas/metabolismo , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos , Linhagem Celular , Antiportadores de Cloreto-Bicarbonato/metabolismo , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Ratos , Receptores de Vasopressinas/genética , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
The Cl(-)/HCO3- exchanger (AE) is one of the mechanisms that cells have developed to adjust pH Despite its importance, the role of AE isoforms in controlling steady-state pH during alkalosis has not been widely investigated. In the present study, we have evaluated whether conditions simulating acute and chronic metabolic alkalosis affected the transport activity and protein levels of Cl-/HCO3- exchangers in a rat cortical collecting duct cell line (RCCD1). pH(i) was monitored using the fluorescent dye BCECF in monolayers grown on permeable supports. Anion exchanger function was assessed by the response of pH(i) to acute chloride removal. RT-PCR and immunoblot assays were also performed. Our results showed that RCCD1 cells express two members of the anion exchanger gene family: AE2 and AE4. Functional studies demonstrated that while in acute alkalosis pH(i) became alkaline and was not regulated, after 48 h adaptation; steady-state pH(i) reached a value similar to the physiological one. Chronic treated cells also resulted in a 3-fold rise in Cl(-)/HCO3- exchange activity together with a 2.2-fold increase in AE2, but not AE4, protein abundance. We conclude that RCCD1 cells can adapt to chronic extracellular alkalosis reestablishing its steady-state pH(i) and that AE2 would play a key role in cell homeostasis.
Assuntos
Antiportadores de Cloreto-Bicarbonato/metabolismo , Túbulos Renais Coletores/metabolismo , Adaptação Fisiológica , Animais , Linhagem Celular , Meios de Cultura , Concentração de Íons de Hidrogênio , Túbulos Renais Coletores/efeitos dos fármacos , Ratos , Bicarbonato de Sódio/farmacologiaRESUMO
We studied the participation of carbonic anhydrase (CA), V-H(+)-ATPase, and Cl(-)/HCO3- exchanger in electrogenic ion absorption through the gills of Chasmagnathus granulatus. CA activity was measured in anterior gills and posterior gills after acclimation to 2 per thousand, 10 per thousand, 30 per thousand (about seawater), and 45 per thousand salinity. The highest CA specific activity was detected in the microsomal fraction in anterior gills, and in the cytosolic fraction, in posterior ones. Both fractions were strongly induced by decreasing salinity only in posterior gills. Perfusion of posterior gills from crabs acclimated to either 2 per thousand or 10 per thousand with acetazolamide inhibited CA activity almost completely. In posterior gills from crabs acclimated to 2 per thousand and perfused with 20 per thousand saline (iso-osmotic for these crabs), acetazolamide reduced transepithelial potential difference (V(te)) by 47%, further addition of ouabain enhanced the effect to 88%. Acetazolamide had no effect in the same gills perfused with 30 per thousand saline (iso-osmotic for seawater acclimated crabs). Bafilomycin A1 and SITS (inhibitors of V-H(+)-ATPase and Cl(-)/HCO3-) reduced V(te) by 15-16% in gills perfused with normal 20 per thousand saline, and by 77% and 45%, respectively when they were applied in Na-free 20 per thousand saline, suggesting the participation of those transporters and cytosolic CA in electrogenic ion absorption.
Assuntos
Adenosina Trifosfatases/metabolismo , Braquiúros/metabolismo , Anidrases Carbônicas/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Brânquias/metabolismo , Acetazolamida/farmacologia , Animais , Transporte Biológico Ativo , Inibidores da Anidrase Carbônica/farmacologia , Eletrofisiologia , Transporte de Íons , Pressão Osmótica , Ouabaína/farmacologia , Cloreto de SódioRESUMO
Recently, our laboratory has reported the presence of one acidifying Cl-/HC exchange mechanism in human platelets. This paper demonstrates that this exchanger decreases its activity after inhibition of carbonic anhydrase. BCECF-loaded platelets, previously equilibrated in a bicarbonate/CO2 buffered solution, were resuspended in a Hepes-buffered, chloride-free (glucuronate) medium to produce a pHi increase. After addition of 50 mM NaCl, pHi fell rapidly reaching steady state in the succeeding 400 s. The recovery in chloride-containing solution was in contrast to the effect of a similar change in osmolarity by addition of 50 mM sodium glucuronate that produced a significantly slower variation of pHi. Alkali loads produced by 25 mM TMA were also counteracted by HC equivalent efflux via Cl-/HC exchange. The present study shows that the efflux of HC was slower when the platelets were previously incubated in 100 microM methazolamide. As a conclusion, the recovery of pHi from alkalosis by Na-independent Cl-/HC exchange is facilitated in platelets by the enzymatic activity of the carbonic anhydrase.
Assuntos
Bicarbonatos/metabolismo , Plaquetas/metabolismo , Anidrases Carbônicas/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Cloretos/metabolismo , Isoenzimas/metabolismo , Soluções Tampão , Anidrase Carbônica II/metabolismo , Inibidores da Anidrase Carbônica/metabolismo , Corantes Fluorescentes/metabolismo , HEPES/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Metazolamida/metabolismoRESUMO
We analyzed the effect of molecular iodine (I2), potassium iodide (KI) and a subclinical concentration of thyroxine (T4) on the induction and promotion of mammary cancer induced by N-methyl-N-nitrosourea. Virgin Sprague-Dawley rats received short or continuous treatment. Continuous I2 treated rats exhibited a strong and persistent reduction in mammary cancer incidence (30%) compared to controls (72.7%). Interruption of short or long term treatments resulted in a higher incidence in mammary cancer compared to the control groups. The protective effect of I2 was correlated with the highest expression of the I-/Cl- transporter pendrin and with the lowest levels of lipoperoxidation expression in mammary glands. Triiodothyronine serum levels and Na+/I- symporter, lactoperoxidase, or p53 expression did not show any changes. In conclusion continuous I2 treatment has a potent antineoplastic effect on the progression of mammary cancer and its effect may be related to a decrease in the oxidative cell environment.