RESUMO
BACKGROUND: The incidence of cancer diagnosed during pregnancy is increasing. Data relating to investigation and management, as well as maternal and foetal outcomes is lacking in a United Kingdom (UK) population. METHODS: In this retrospective study we report data from 119 patients diagnosed with cancer during pregnancy from 14 cancer centres in the UK across a five-year period (2016-2020). RESULTS: Median age at diagnosis was 33 years, with breast, skin and haematological the most common primary sites. The majority of cases were new diagnoses (109 patients, 91.6%). Most patients were treated with radical intent (96 patients, 80.7%), however, gastrointestinal cancers were associated with a high rate of palliative intent treatment (63.6%). Intervention was commenced during pregnancy in 68 (57.1%) patients; 44 (37%) had surgery and 31 (26.1%) received chemotherapy. Live births occurred in 98 (81.7%) of the cases, with 54 (55.1%) of these delivered by caesarean section. Maternal mortality during the study period was 20.2%. CONCLUSIONS: This is the first pan-tumour report of diagnosis, management and outcomes of cancer diagnosed during pregnancy in the UK. Our findings demonstrate proof of concept that data collection is feasible and highlight the need for further research in this cohort of patients.
Assuntos
Cesárea , Neoplasias , Gravidez , Humanos , Feminino , Estudos Retrospectivos , Neoplasias/diagnóstico , Neoplasias/epidemiologia , Neoplasias/terapia , Reino Unido/epidemiologia , Nascido VivoRESUMO
BACKGROUND AND PURPOSE: During the bladder filling phase, the volume of the urinary bladder increases dramatically, with only minimal increases in intravesical pressure. To accomplish this, the smooth muscle of the bladder wall must remain relaxed during bladder filling. However, the mechanisms responsible for the stabilization of bladder excitability during stretch are unclear. We hypothesized that stretch-dependent K(+) (TREK) channels in bladder smooth muscle cells may inhibit contraction in response to stretch. EXPERIMENTAL APPROACHES: Bladder tissues from mouse, guinea pig and monkey were used for molecular, patch clamp, mechanical, electrical, Ca(2+) imaging and cystometric responses to methionine and its derivatives, which are putative blockers of stretch-dependent K(+) (SDK) channels. KEY RESULTS: SDK channels are functionally expressed in bladder myocytes. The single channel conductance of SDK channels is 89pS in symmetrical K(+) conditions and is blocked by L-methionine. Expressed TREK-1 currents are also inhibited by L-methioninol. All three types of bladder smooth muscle cells from mouse, guinea pig and monkey expressed TREK-1 genes. L-methionine, methioninol and methionine methyl ester but not D-methionine increased contractility in concentration-dependent manner. Methioninol further increased contractility and depolarized the membrane in the presence of blockers of Ca(2+)-activated K(+) conductance. L-methionine induced Ca(2+) waves that spread long distances through the tissue under stretched conditions and were associated with strong contractions. In cystometric assays, methioninol injection increased bladder excitability mimicking overactive bladder activity. CONCLUSIONS AND IMPLICATIONS: Methioninol-sensitive K(+) (SDK, TREK-1) channels appear to be important to prevent spread of excitation through the syncitium during bladder filling.
Assuntos
Metionina/farmacologia , Músculo Liso/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Domínios Poros em Tandem/efeitos dos fármacos , Animais , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Feminino , Expressão Gênica , Cobaias , Macaca fascicularis , Masculino , Metionina/administração & dosagem , Metionina/análogos & derivados , Camundongos , Contração Muscular/efeitos dos fármacos , Músculo Liso/citologia , Técnicas de Patch-Clamp , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Especificidade da Espécie , Bexiga Urinária/citologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismoRESUMO
Although the cationic inward rectifiers (Kir and hyperpolarization-activated I(f) channels) have been well characterized in cardiac myocytes, the expression and physiological role of anionic inward rectifiers in heart are unknown. In the present study, we report the functional and molecular identification of a novel chloride (Cl(-)) inward rectifier (Cl.ir) in mammalian heart. Under conditions in which cationic inward rectifier channels were blocked, membrane hyperpolarization (-40 to -140 mV) activated an inwardly rectifying whole-cell current in mouse atrial and ventricular myocytes. Under isotonic conditions, the current activated slowly with a biexponential time course (time constants averaging 179.7+/-23.4 [mean+/-SEM] and 2073.6+/-287.6 ms at -120 mV). Hypotonic cell swelling accelerated the activation and increased the current amplitude whereas hypertonic cell shrinkage inhibited the current. The inwardly rectifying current was carried by Cl(-) (I(Cl.ir)) and had an anion permeability sequence of Cl(-)>I(-)>>aspartate. I(Cl.ir) was blocked by 9-anthracene-carboxylic acid and cadmium but not by stilbene disulfonates and tamoxifen. A similar I(Cl.ir) was also observed in guinea pig cardiac myocytes. The properties of I(Cl.ir) are consistent with currents generated by expression of ClC-2 Cl(-) channels. Reverse transcription polymerase chain reaction and Northern blot analysis confirmed transcriptional expression of ClC-2 in both atrial and ventricular tissues and isolated myocytes of mouse and guinea pig hearts. These results indicate that a novel I(Cl.ir) is present in mammalian heart and support a potentially important role of ClC-2 channels in the regulation of cardiac electrical activity and cell volume under physiological and pathological conditions.
Assuntos
Canais de Cloreto/metabolismo , Miocárdio/metabolismo , Animais , Ácido Aspártico/farmacologia , Northern Blotting , Canais de Cloro CLC-2 , Cádmio/farmacologia , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/genética , Cobaias , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Técnicas In Vitro , Meglumina/farmacologia , Camundongos , Técnicas de Patch-Clamp , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The bioavailability, biochemical effects, and safety of a slow-release preparation of sodium fluoride were examined. In 8 normal volunteers, a single administration of slow-release sodium fluoride (25 mg) caused a slow rise and gradual decline in serum fluoride concentration, thus avoiding sharp peaks produced by a rapid-release preparation. In 37 patients with postmenopausal osteoporosis, serum fluoride concentration was kept within the "therapeutic window" (95-100 ng/ml) during long-term intermittent sodium fluoride (slow-release) therapy (25 mg twice/day, given for 3 months in each 5-month cycle over five cycles). Serum fluoride was also kept within the therapeutic window in 64 patients who took sodium fluoride (slow release) continuously over 12 months. Serum osteocalcin concentration increased progressively during fluoride treatment (correlation coefficient of 0.88, p less than .001 for the relationship between serum osteocalcin and duration of therapy). Side effects to slow-release sodium fluoride therapy, assessed in 101 patients at two study sites, were minor and included diarrhea in 2 patients, nausea in 2 patients, abdominal pain and cramping in 2 patients, foot pain in 2 patients, and joint pain in 6 patients. Thus, slow-release sodium fluoride confers desired level of fluoride in serum, while providing safety of usage.
Assuntos
Menopausa/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Fluoreto de Sódio/farmacologia , Idoso , Idoso de 80 Anos ou mais , Disponibilidade Biológica , Ritmo Circadiano , Preparações de Ação Retardada , Feminino , Humanos , Menopausa/sangue , Pessoa de Meia-Idade , Osteoporose/sangue , Fluoreto de Sódio/sangue , Fluoreto de Sódio/farmacocinéticaRESUMO
Using the ambulatory protocol previously described, 241 patients with nephrolithiasis were evaluated. They could be categorized into 10 groups from the results obtained. Absorptive hypercalciuria type I (87 per cent male) comprised 24.5 per cent and was characterized by normocalcemia, normal fasting urinary calcium (less than 0.11 mg/100 ml glomerular filtration), an exaggerated urinary calcium following an oral calcium load (greater than 0.20 mg/mg creatinine), normal urinary cyclic adenosine monophosphate (AMP) (less than 5.4 nmol/100 ml glomerular filtration) and serum parathyroid hormone (PTH), and hypercalciuria (greater than 200 mg/day during a calcium- and sodium-restricted diet). Absorptive hypercalciuria type II (50 per cent male) accounted for 29.8 per cent; its biochemical features were the same as those for absorptive hypercalciuria type I, except for normocalciuria during a restricted diet and low urine volume (1.42 +/- 0.55 SD liter/day). Renal hypercalciuria (56 per cent male), disclosed in 8.3 per cent, was represented by normocalcemia and high values for fasting urinary calcium (0.160 +/- 0.054 mg/100 ml glomerular filtration), urinary cyclic AMP (6.80 +/- 2.10 nmol/100 ml glomerular filtration) and serum PTH. Primary hyperparathyroidism (57 per cent female), accounted for 5.8 per cent, typically included hypercalcemia, hypophosphatemia, hypercalciuria and high urinary cyclic AMP. Hyperuricosuric calcium urolithiasis (100 per cent male) comprised 8.7 per cent, and was characterized by hyperuricosuria (776 +/- 164 mg/day) and urinary pH exceeding pK for uric acid (5.91 +/- 0.33). In enteric hyperoxaluria (60 per cent female), encountered in 2.1 per cent of cases, urinary oxalate was increased (6.29 +/- 13.2 mg/day). Noncalcium-containing stones were found in 2.1 per cent of the patients with uric acid lithiasis (100 per cent male) and in another 2.1 per cent of the patients with infection lithiasis (60 per cent female). These conditions were typified by low urinary pH (5.29 +/- 0.12) and high urinary pH (6.69 +/- 1.16), respectively. Renal tubular acidosis was found in one patient (male, 0.4 per cent). In 10.8 per cent of the patients (81 per cent male), no metabolic abnormality could be found, although urine volume was low (1.41 +/- 0.51 liter/day). Hypercalciuria could not be differentiated between absorptive hypercalciuria and renal hypercalciuria in 5.4 per cent of the patients. Thus, this ambulatory protocol disclosed a physiologic disturbance in nearly 90 per cent of the cases and provided a definitive diagnosis in 95 per cent of the patients.
Assuntos
Assistência Ambulatorial , Cálculos Renais/diagnóstico , Adulto , Idoso , Cálcio/urina , AMP Cíclico/urina , Feminino , Taxa de Filtração Glomerular , Humanos , Cálculos Renais/etiologia , Cálculos Renais/fisiopatologia , Masculino , Pessoa de Meia-Idade , Oxalatos/urina , Hormônio Paratireóideo/sangue , Ácido Úrico/urinaRESUMO
We evaluated, in 128 patients with recurrent nephrolithiasis, the efficacy of special treatment programs for some of the common causes of nephrolithiasis, chosen on the basis of their ability to correct underlying physicochemical and physiologic derangements. Therapy included sodium cellulose phosphate for 18 patients with absorptive hypercalciuria, thiazide diuretics for 27 patients with absorptive hypercalciuria and for 10 with renal hypercalciuria, orthophosphate for eight patients with hypophosphatemic absorptive hypercalciuria, allopurinol for 21 patients with hyperuricosuric calcium oxalate nephrolithiasis, thiazide and allopurinol for 26 patients with absorptive hypercalciuria with hyperuricosuria, and high fluid intake and/or low calcium diet for 22 patients with normocalciuric nephrolithiasis. Patients in all seven groups had a significant reduction in stone formation during 1.70 to 3.37 years of treatment, as compared with the pretreatment period of three years. Remission was found in 70 to 91 percent of patients and reduced stone formation rate was encountered in 88 to 100 percent. Each treatment program produced a significant decline in stone formation rate from 1.90 to 2.28 stones per year to 0.09 to 0.55 stones per year. The actual number of stones formed during treatment was significantly lower than the number predicted from the pretreatment frequency of stone formation (less than 26 percent). The results provide evidence supporting a selective approach to therapy of nephrolithiasis.
Assuntos
Cálculos Renais/terapia , Alopurinol/uso terapêutico , Benzotiadiazinas , Cálcio da Dieta , Celulose/análogos & derivados , Celulose/uso terapêutico , Diuréticos , Estudos de Avaliação como Assunto , Hidratação , Humanos , Planejamento de Assistência ao Paciente , Fosfatos/uso terapêutico , Recidiva , Remissão Espontânea , Inibidores de Simportadores de Cloreto de Sódio/uso terapêuticoRESUMO
BACKGROUND: Inflammatory responses can include recruitment of cells of hematopoietic origin to the tunica muscularis. These cells can secrete a variety of factors which can reset the gain of smooth muscle cells (SMC) and influence motor patterns. Histamine (H), a major mediator in inflammation, is released by mast cells and exerts diverse effects in SMC by binding to H receptors. The profiles of H receptor expression in animal models used to study inflammatory diseases are unknown. METHODS: Histamine receptor expression and electro-mechanical responses to H were tested in simian and murine colonic smooth muscle using qualitative and quantitative PCR, isometric force measurements, microelectrode recordings and patch clamp techniques. KEY RESULTS: H1, H2, and H4 receptor transcripts were expressed at similar levels in simian colonic tissue whereas only the H2 receptor transcript was detected in murine colonic tissue. Stimulation of simian colonic muscles with H caused depolarization and contraction in the presence of tetrodotoxin. Histamine activated non-selective cation channels in simian SMC. In contrast, H caused hyperpolarization and inhibited contractions of murine colon. The hyperpolarization was inhibited by the K(ATP) channel blocker, glibenclamide. Histamine-activated K(+) currents were inhibited by glibenclamide in murine colonic SMC. CONCLUSIONS & INFERENCES: Histamine receptor expression in simian SMC was similar to that reported in humans. However, H receptor profile and responses to H were considerably different in mice. Thus, monkey colon may be a more suitable model to study how inflammatory mediators affect the gain of smooth muscle excitability.
Assuntos
Colo/metabolismo , Histamina/metabolismo , Inflamação/metabolismo , Músculo Liso/metabolismo , Receptores Histamínicos/biossíntese , Animais , Colo/efeitos dos fármacos , Feminino , Histamina/farmacologia , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase em Tempo RealRESUMO
AIM: This study investigated the functional role of the ClC-3 amino-terminus in channel regulation in response to changes in cell volume. METHODS: Wild-type sClC-3 tagged with a green fluorescence protein (GFP) at the C-terminus was used as a template to construct a number of deletion mutants which were functionally expressed in NIH-3T3 cells. Whole cell and single channel patch-clamp electrophysiology was used to determine the functional properties of heterologously expressed channels. RESULTS: The first 100 amino acids of the ClC-3 N-terminus were removed and the truncated channel (sClC-3DeltaNT) was functionally expressed. Immunocytochemistry confirmed membrane expression of both wtsClC-3 and sClC-3DeltaNT channels in NIH/3T3 cells. sClC-3DeltaNT yielded constitutively active functional channels, which showed no response to protein kinase C or changes in cell volume. Deletion of a cluster of negatively charged amino acids 16-21 (sClC-3Delta16-21) within the N-terminus also yielded a constitutively active open channel phenotype, indicating these amino acids are involved in the N-type regulation. Intracellular delivery of a thiol-phosphorylated peptide corresponding to N-terminal residues 12-61 (NT peptide) markedly inhibited sClC-3DeltaNT whole-cell and single-channel currents, further confirming the essential role of the N-terminus in volume regulation of channel activity. CONCLUSIONS: These data strongly suggest the N-terminus of sClC-3 channels acts as a blocking particle inhibiting the flow of anions through the channel pore. This 'N-type' regulation of sClC-3 channels may be an important transducing mechanism linking changes in cell volume and channel protein phosphorylation to channel gating.
Assuntos
Canais de Cloreto/metabolismo , Fibroblastos/metabolismo , Ativação do Canal Iônico , Células 3T3 , Animais , Tamanho Celular , Canais de Cloreto/genética , Códon , Eletroporação , Deleção de Genes , Engenharia Genética , Proteínas de Fluorescência Verde/genética , Cobaias , Imuno-Histoquímica/métodos , Canais Iônicos/metabolismo , Camundongos , Concentração Osmolar , Técnicas de Patch-Clamp , FosforilaçãoRESUMO
The effect of treatment of renal stone formation with 5 to 20 mg./kg. per day oral disodium ethane-1-hydroxy-1,1-diphosphonate for up to 30 months was examined in 12 patients with active renal (calcium) stone disease. The over-all incidence of stone passage decreased from 17.8 stones per year per patient before treatment to 7.7 stones per year per patient during therapy. Of the 12 patients 7 passed fewer stones or no stones during treatment. However, the incidence of stone passage was not changed substantially by disodium ethane-1-hydroxy-1,1-diphosphonate in 5 patients. Symptoms of muscle weakness and pain in the back, hips and shoulders occurred in 3 patients during treatment, 2 patients had an increase in serum alkaline phosphatase and 1 patient had a decrease in bone density. Although disodium ethane-1-hydroxy-1,1-diphosphonate may be clinically useful to manage calcium urolithasis in certain patients its over-all use is limited because of its ineffectiveness in some patients and owing to its potential to induce osteomalacia.
Assuntos
Cálcio/metabolismo , Ácido Etidrônico/uso terapêutico , Cálculos Urinários/tratamento farmacológico , Administração Oral , Adulto , Idoso , Fosfatase Alcalina/sangue , Osso e Ossos/efeitos dos fármacos , AMP Cíclico/urina , Avaliação de Medicamentos , Ácido Etidrônico/administração & dosagem , Ácido Etidrônico/efeitos adversos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fósforo/metabolismoRESUMO
The long-term effects of potassium citrate therapy (usually 20 mEq. 3 times daily during 1 to 4.33 years) were examined in 89 patients with hypocitraturic calcium nephrolithiasis or uric acid lithiasis, with or without calcium nephrolithiasis. Hypocitraturia caused by renal tubular acidosis or chronic diarrheal syndrome was associated with other metabolic abnormalities, such as hypercalciuria or hyperuricosuria, or occurred alone. Potassium citrate therapy caused a sustained increase in urinary pH and potassium, and restored urinary citrate to normal levels. No substantial or significant changes occurred in urinary uric acid, oxalate, sodium or phosphorus levels, or total volume. Owing to these physiological changes, uric acid solubility increased, urinary saturation of calcium oxalate decreased and the propensity for spontaneous nucleation of calcium oxalate was reduced to normal. Therefore, the physicochemical environment of urine following treatment became less conducive to the crystallization of calcium oxalate or uric acid, since it stimulated that of normal subjects without stones. Commensurate with the aforementioned physiological and physicochemical changes the treatment produced clinical improvement, since individual stone formation decreased in 97.8 per cent of the patients, remission was obtained in 79.8 per cent and the need for surgical treatment of newly formed stones was eliminated. In patients with relapse after other treatment, such as thiazide, the addition of potassium citrate induced clinical improvement. Thus, our study provides physiological, physicochemical and clinical validation for the use of potassium citrate in the treatment of hypocitraturic calcium nephrolithiasis and uric acid lithiasis with or without calcium nephrolithiasis.
Assuntos
Cálcio/metabolismo , Citratos/uso terapêutico , Cálculos Renais/tratamento farmacológico , Ácido Úrico/metabolismo , Adulto , Benzotiadiazinas , Oxalato de Cálcio/metabolismo , Citratos/administração & dosagem , Citratos/efeitos adversos , Citratos/urina , Ácido Cítrico , Ensaios Clínicos como Assunto , Diuréticos , Gastroenteropatias/induzido quimicamente , Humanos , Concentração de Íons de Hidrogênio , Cálculos Renais/metabolismo , Potássio/metabolismo , Inibidores de Simportadores de Cloreto de Sódio/uso terapêutico , Fatores de TempoRESUMO
We investigated the regulation of cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by protein kinase C (PKC) in Xenopus oocytes injected with cRNA encoding the cardiac (exon 5-) CFTR Cl- channel isoform. Membrane currents were recorded using a two-electrode voltage clamp technique. Activators of PKC or a cAMP cocktail elicited robust time-independent Cl- currents in cardiac CFTR-injected oocytes, but not in control water-injected oocytes. The effects of costimulation of both pathways were additive; however, maximum protein kinase A (PKA) activation occluded further activation by PKC. In oocytes expressing either the cardiac (exon 5-) or epithelial (exon 5+) CFTR isoform, Cl- currents activated by PKA were sustained, whereas PKC-activated currents were transient, with initial activation followed by slow current decay in the continued presence of phorbol esters, the latter effect likely due to down-regulation of endogenous PKC activity. The specific PKA inhibitor, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), and various protein phosphatase inhibitors were used to determine whether the stimulatory effects of PKC are dependent upon the PKA phosphorylation state of cardiac CFTR channels. Intraoocyte injection of 1,2-bis(2-aminophenoxy)ethane-N,N, N,N-tetraacetic acid (BAPTA) or pretreatment of oocytes with BAPTA-acetoxymethyl-ester (BAPTA-AM) nearly completely prevented dephosphorylation of CFTR currents activated by cAMP, an effect consistent with inhibition of protein phosphatase 2C (PP2C) by chelation of intracellular Mg2+. PKC-induced stimulation of CFTR channels was prevented by inhibition of basal endogenous PKA activity, and phorbol esters failed to stimulate CFTR channels trapped into either the partially PKA phosphorylated (P1) or the fully PKA phosphorylated (P1P2) channel states. Site-directed mutagenesis of serines (S686 and S790) within two consensus PKC phosphorylation sites on the cardiac CFTR regulatory domain attentuated, but did not eliminate, the stimulatory effects of phorbol esters on mutant CFTR channels. The effects of PKC on cardiac CFTR Cl- channels are consistent with a simple model in which PKC phosphorylation of the R domain facilitates PKA-induced transitions from dephosphorylated (D) to partially (P1) phosphorylated and fully (P1P2) phosphorylated channel states.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Miocárdio/metabolismo , Proteína Quinase C/metabolismo , Animais , Sítios de Ligação , Fenômenos Biofísicos , Biofísica , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Humanos , Técnicas In Vitro , Potenciais da Membrana , Modelos Biológicos , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , RNA Complementar/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
The occurrence of calcareous renal stones in 12 members of a family was consistent with an autosomal dominant mode of inheritance. All 6 members with stones who were evaluated were shown to have absorptive hypercalciuria. The mother of 2 members with stones did not suffer stones but had biochemical evidence of absorptive hypercalciuria (increased intestinal calcium absorption, hypercalciuria and normal parathyroid function). Nephrolithiasis was encountered only in the progeny of members who had stones of biochemical absorptive hypercalciuria. The results suggest that physiological feature(s) of absorptive hypercalciuria may be an expression of the genetic trait.
Assuntos
Distúrbios do Metabolismo do Cálcio/genética , Cálcio/urina , Adulto , Distúrbios do Metabolismo do Cálcio/fisiopatologia , Aberrações Cromossômicas , Transtornos Cromossômicos , Feminino , Humanos , Absorção Intestinal , Cálculos Renais/genética , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
The molecular identification of cardiac chloride channels has provided probes to investigate their distribution and abundance in heart. In this study, the molecular expression and distribution of volume-regulated chloride channels ClC-2 and ClC-3 in cardiac tissues were analyzed and quantified. Total RNA was isolated from atria and ventricles of several species (dog, guinea pig, and rat) and subjected to a quantitative RT-PCR strategy. ClC-2 and ClC-3 mRNA expression were calculated relative to beta-actin expression within these same tissues. The transcriptional levels of ClC-3 mRNA were between 1.8 and 10.2% of beta-actin expression in atria and between 3.4 and 8.6% of beta-actin in ventricles (n = 3 for each tissue). The levels of ClC-2 in both atria and ventricles were significantly less than those measured for ClC-3 (n = 3; P < 0.05). ClC-2 mRNA levels were between 0.04-0.08% and 0.03-0.18% of beta-actin expression in atria and ventricles, respectively (n = 3 for each tissue). Immunoblots of atrial and ventricular wall protein extracts demonstrated ClC-2- and ClC-3-specific immunoreactivity at 97 and 85 kDa, respectively. Immunohistochemical localization in guinea pig cardiac muscle demonstrates a ubiquitous distribution of ClC-2 and ClC-3 channels in the atrial and ventricular wall. Confocal analysis detected colocalization of ClC-2 and ClC-3 in sarcolemmal membranes and distinct ClC-3 immunoreactivity in cytoplasmic regions. The molecular expression of ClC-2 and ClC-3 in cardiac tissue is consistent with the proposed role of these chloride channels in the regulation of cardiac cell volume and the modulation of cardiac electrical activity.
Assuntos
Canais de Cloreto/metabolismo , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Miocárdio/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Western Blotting , Canais de Cloro CLC-2 , Canais de Cloreto/genética , Cães , Cobaias , Imuno-Histoquímica , Peso Molecular , Especificidade de Órgãos , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
1. P2-purinoceptors couple extracellular ATP to the activation of a Cl- current (ICl,ATP) in heart. We studied the molecular mechanism and intracellular signalling pathways of ICl,ATP activation in mouse heart. 2. Extracellular adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS; 100 microM) activated ICl,ATP in both atrial and ventricular myocytes. A specific PKC inhibitor, bisindolylmaleimide blocked the effect of ATPgammaS while a PKC activator, phorbol 12, 13-dibutyrate (PDBu) activated a current with identical properties to ICl,ATP. Maximal activation of ICl,ATP by ATPgammaS or PDBu occluded further modulation by the other agonist, suggesting that they may activate the same population of Cl- channels. 3. Isoprenaline increased ICl,ATP pre-activated by ATPgammaS or PDBu, while isoprenaline or forskolin alone failed to activate any Cl- current in these myocytes. Adenosine 3',5'-cyclic monophosphothionate, a PKA inhibitor, prevented ATPgammaS or PDBu activation of ICl,ATP. Thus, ICl,ATP is regulated by dual intracellular phosphorylation pathways involving both PKA and PKC in a synergistic manner similar to cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. 4. Glibenclamide (50 microM) significantly blocked ICl,ATP activated by ATPgammaS or by the CFTR channel activator, levamisole. 5. The slope conductance of the unitary ICl,ATP in cell-attached patches was 11.8 +/- 0.3 pS, resembling the known properties of CFTR Cl- channels in cardiac myocytes. 6. The reverse transcription polymerase chain reaction and Northern blot analysis revealed CFTR mRNA expression in mouse heart. 7. We conclude that ICl,ATP in mouse heart is due to activation of CFTR Cl- channels through a novel intracellular signalling pathway involving purinergic activation of PKC and PKA.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Miocárdio/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Sequência de Bases , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Primers do DNA/genética , Ativação Enzimática , Expressão Gênica , Técnicas In Vitro , Isoproterenol/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Miocárdio/citologia , Dibutirato de 12,13-Forbol/farmacologia , Proteína Quinase C/metabolismo , Transdução de SinaisRESUMO
NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca2+-activated K+ channels and results in smooth muscle relaxation. However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels. The present study was designed to clarify PKG-mediated modulation of channels at the molecular level. The cDNA encoding the alpha-subunit of the large conductance Ca2+-activated K+ channel, cslo-alpha, was expressed in HEK293 cells. Whole cell and single channel characteristics of cslo-alpha exhibited functional features of native large conductance Ca2+-activated K+ channels in smooth muscle cells. The NO-donor sodium nitroprusside increased outward current 2.3-fold in whole cell recordings. In cell-attached patches, sodium nitroprusside increased the channel open probability (NPo) of cslo-alpha channels 3.3-fold without affecting unitary conductance. The stimulatory effect of sodium nitroprusside was inhibited by the PKG-inhibitor KT5823. Direct application of PKG-Ialpha to the cytosolic surface of inside-out patches increased NPo 3.2-fold only in the presence of ATP and cGMP without affecting unitary conductance. A point mutation of cslo-alpha in which Ser-1072 (the only optimal consensus sequence for PKG phosphorylation) was replaced by Ala abolished the PKG effect on NPo in inside-out patches and the effect of SNP in cell attached patches. These results indicate that PKG activates cslo-alpha by direct phosphorylation at serine 1072.