RESUMEN

Recent studies have shown that the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-binding cassette (ABC) transporter whose mutations are responsible for cystic fibrosis (CF), permeates ATP. However, little information is available concerning extracellular ATP concentrations in CF patients. Thus, the goal of this preliminary study was to determine the circulating levels of plasma ATP in CF patients. Circulating levels of plasma ATP were determined by the luciferin-luciferase assay in both CF patients and healthy volunteer control subjects. The two groups were compared using an analysis of variance. CF genotype and age, which ranged from 7 to 56 years, were also used to compare data by single-blind analysis. With comparable sample numbers, CF patients had statistically higher levels of circulating ATP (34%, P<0.01) when compared by analysis of covariance with the age of the subjects as the cofactor. The CF patients bearing the DeltaF508 genotype had a 54% (n=33, P<0.01) higher plasma ATP concentration compared to controls, while patients bearing other CF genotypes were similar to controls (n=10, P<0.4). We conclude that CF patients have higher circulating levels of ATP when compared to controls. Increased levels of plasma ATP, which is an important autocrine/paracrine hormone in many cell types, may be associated with chronic manifestations of the disease.

Asunto(s)

Adenosina Trifosfato/sangre , Fibrosis Quística/sangre , Adolescente , Adulto , Factores de Edad , Análisis de Varianza , Niño , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Genotipo , Homocigoto , Humanos , Persona de Mediana Edad , Mutación , Reproducibilidad de los Resultados

RESUMEN

The molecular mechanisms associated with intracellular ATP release by the heart are largely unknown. In this study the luciferin-luciferase assay and patch-clamp techniques were used to characterize the pathways responsible for ATP release in neonatal rat cardiac myocytes (NRCM). Spontaneous ATP release by NRCM was significantly increased after cAMP stimulation under physiological conditions. cAMP stimulation also induced an anion-selective electrodiffusional pathway that elicited linear, diphenylamine-2-carboxylate (DPC)-inhibitable Cl(-) currents in either symmetrical MgCl(2) or NaCl. ATP, adenosine 5'-O-(3-thiotriphosphate), and the ATP derivatives ADP and AMP, permeated this pathway; however, GTP did not. The cAMP-induced ATP currents were inhibited by DPC and glibenclamide and by a monoclonal antibody raised against the R domain of the cystic fibrosis transmembrane conductance regulator (CFTR). The channel-like nature of the cAMP-induced ATP-permeable pathway was also determined by assessing protein kinase A-activated single channel Cl(-) and ATP currents in excised inside-out patches of NRCM. Single channel currents were inhibited by DPC and the anti-CFTR R domain antibody. Thus the data in this report demonstrate the presence of a cAMP-inducible electrodiffusional ATP transport mechanism in NRCM. Based on the pharmacology, patch-clamping data, and luminometry studies, the data are most consistent with the role of a functional CFTR as the anion channel implicated in cAMP-activated ATP transport in NRCM.

Asunto(s)

Adenosina Trifosfato/metabolismo , Animales Recién Nacidos/metabolismo , AMP Cíclico/fisiología , Miocardio/metabolismo , Adenosina Trifosfato/fisiología , Animales , Anticuerpos/farmacología , Permeabilidad de la Membrana Celular , Células Cultivadas , Senescencia Celular/fisiología , Canales de Cloruro/fisiología , Cloruros/fisiología , AMP Cíclico/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/inmunología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Conductividad Eléctrica , Miocardio/citología , Ratas

RESUMEN

Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(-)]. cAMP stimulation of ABP(-) cells or cells genetically rescued with ABP-280 cDNA [ABP(+)] was without effect on whole cell Cl(-) currents. In ABP(-) cells expressing CFTR, cAMP was also without effect on Cl(-) conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell Cl(-) currents of ABP(+)/CFTR(+) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, however, resulted in a DPC-inhibitable increase in the whole cell currents of ABP(-)/CFTR(+) cells. At the single-channel level, protein kinase A plus ATP activated single Cl(-) channels only in excised patches from ABP(+)/CFTR(+) cells. Furthermore, filamin alone also induced Cl(-) channel activity in excised patches of ABP(-)/CFTR(+) cells. The present data indicate that an organized actin cytoskeleton is required for cAMP-dependent activation of CFTR.

Asunto(s)

Actinas/fisiología , AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Citoesqueleto/fisiología , Aniones/farmacocinética , Bromo/farmacocinética , Cloro/farmacocinética , Proteínas Contráctiles/genética , Proteínas Contráctiles/farmacología , Reactivos de Enlaces Cruzados/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Diálisis , Filaminas , Expresión Génica/fisiología , Gluconatos/farmacocinética , Humanos , Yodo/farmacocinética , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Melanoma , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/farmacología , Técnicas de Placa-Clamp , Transfección , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/enzimología

RESUMEN

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR), and of at least one other member of the ATP-binding cassette family of transport proteins, P-glycoprotein, is associated with the electrodiffusional movement of the nucleotide ATP. Evidence directly implicating CFTR expression with ATP channel activity, however, is still missing. Here it is reported that reconstitution into a lipid bilayer of highly purified CFTR of human epithelial origin enables the permeation of both Cl- and ATP. Similar to previously reported data for in vivo ATP current of CFTR-expressing cells, the reconstituted channels displayed competition between Cl- and ATP and had multiple conductance states in the presence of Cl- and ATP. Purified CFTR-mediated ATP currents were activated by protein kinase A and ATP (1 mM) from the "intracellular" side of the molecule and were inhibited by diphenylamine-2-carboxylate, glibenclamide, and anti-CFTR antibodies. The absence of CFTR-mediated electrodiffusional ATP movement may thus be a relevant component of the pleiotropic cystic fibrosis phenotype.

Asunto(s)

Adenosina Trifosfato/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Animales , Transporte Biológico , Bloqueadores de los Canales de Calcio/metabolismo , Línea Celular , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Difusión , Conductividad Eléctrica , Humanos , Membrana Dobles de Lípidos/metabolismo , Magnesio/metabolismo , Modelos Moleculares , Proteínas Recombinantes/metabolismo , Spodoptera , ortoaminobenzoatos/metabolismo

RESUMEN

The actin cytoskeleton is an important contributor to the integrity of cellular shape and responses in neurons. However, the molecular mechanisms associated with functional interactions between the actin cytoskeleton and neuronal ion channels are largely unknown. Whole-cell and single channel recording techniques were thus applied to identified retinal bipolar neurons of the tiger salamander (Ambystoma tigrinum) to assess the role of acute changes in actin-based cytoskeleton dynamics in the regulation of voltage-gated ion channels. Disruption of endogenous actin filaments after brief treatment (20-30 min) with cytochalasin D (CD) activated voltage-gated K+ currents in bipolar cells, which were largely prevented by intracellular perfusion with the actin filament-stabilizer agent, phalloidin. Either CD treatment under cell-attached conditions or direct addition of actin to excised, inside-out patches of bipolar cells activated and/or increased single K+ channels. Thus, acute changes in actin-based cytoskeleton dynamics regulate voltage-gated ion channel activity in bipolar cells.

Asunto(s)

Actinas/fisiología , Citoesqueleto/fisiología , Canales Iónicos/fisiología , Células Fotorreceptoras/fisiología , Células Ganglionares de la Retina/fisiología , Ambystoma , Animales , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/ultraestructura , Homeostasis , Técnicas In Vitro , Potenciales de la Membrana , Técnicas de Placa-Clamp , Faloidina/farmacología , Células Fotorreceptoras/citología , Células Fotorreceptoras/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Células Ganglionares de la Retina/ultraestructura , Factores de Tiempo

RESUMEN

Gluteal aneurysms, whether true or false, are exceptional. They represent less than 1% of all aneurysms and develop within the superior or inferior gluteal arteries, being branches of the internal iliac artery. We report here the case of a 35-year-old patient with Marfan syndrome in whom annuloaortic ectasia and Barlow's disease with mitral valve insufficiency successively developed followed by a gluteal false aneurysm, which led us to investigate the etiologic mechanism of the patient's conditions. The gluteal aneurysm was successfully treated by selective embolization, which would appear to be the elective therapeutic approach for these lesions.

Asunto(s)

Aneurisma Falso/terapia , Nalgas/irrigación sanguínea , Embolización Terapéutica , Síndrome de Marfan/complicaciones , Adulto , Aneurisma Falso/complicaciones , Aneurisma Falso/diagnóstico por imagen , Humanos , Masculino , Radiografía

RESUMEN

Cystic fibrosis (CF) airway epithelia exhibit enhanced Na+ reabsorption in parallel with diminished Cl- secretion. We tested the hypothesis that actin plays a role in the regulation of a cloned epithelial Na+ channel (ENaC) by the cystic fibrosis transmembrane conductance regulator (CFTR). We found that immunopurified bovine tracheal CFTR coreconstituted into a planar lipid bilayer with alpha,beta,gamma-rat ENaC (rENaC) decreased single-channel open probability (Po) of rENaC in the presence of actin by over 60%, a significantly greater effect than was observed in the absence of actin (approximately 20%). In the presence of actin, protein kinase A plus ATP activated both CFTR and rENaC, but CFTR was activated in a sustained manner, whereas the activation of rENaC was transitory. ATP alone could also activate ENaC transiently in the presence ofactin but had no effect on CFTR. Stabilizing short actin filaments at a fixed length with gelsolin (at a ratio to actin of 2:1) produced a sustained activation of alpha,beta,gamma-rENaC in both the presence or absence of CFTR. Gelsolin alone (i.e., in the absence of actin) had no effect on the conductance or Po of either CFTR or rENaC. We have also found that short actin filaments produced their modulatory action on alpha-rENaC independent of the presence of the beta- or gamma-rENaC subunits. In contrast, CFTR did not affect any properties of the channel formed by alpha-rENaC alone, i.e., in the absence of beta- or gamma-rENaC. These results indicate that CFTR can directly downregulate single Na+ channel activity, which may account for the observed differences between Na+ transport in normal and CF-affected airway epithelia. Moreover, the presence of actin confers an enhanced modulatory ability of CFTR on Na+ channels.

Asunto(s)

Actinas/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Sistema Respiratorio/metabolismo , Canales de Sodio/metabolismo , Actinas/farmacología , Adenosina Trifosfato/farmacología , Animales , Bovinos , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/farmacología , Epitelio/metabolismo , Gelsolina/farmacología , Isomerismo , Membrana Dobles de Lípidos/metabolismo , Conejos , Ratas , Canales de Sodio/efectos de los fármacos

RESUMEN

Protein kinase A (PKA)- and G protein-mediated regulation of immunopurified adult rabbit alveolar epithelial type II (ATII) cell proteins that exhibit amiloride-sensitive Na+ channel activity was studied in planar lipid bilayers and freshly isolated ATII cells. Addition of the catalytic subunit of PKA + ATP increased single channel open probability from 0.42 +/- 0.05 to 0.82 +/- 0.07 in a voltage-independent manner, without affecting unitary conductance. This increase in open probability of the channels was mainly due to a decrease in the time spent by the channel in its closed state. The apparent inhibition constant for amiloride increased from 8.0 +/- 1.8 microM under control conditions to 15 +/- 3 microM after PKA-induced phosphorylation; that for ethylisopropylamiloride increased from 1.0 +/- 0.4 to 2.0 +/- 0.5 microM. Neither pertussis toxin (PTX) nor guanosine 5'-O-(3-thiotriphosphate) affected ATII Na+ channel activity in bilayers. Moreover, PTX failed to affect amiloride-inhibitable 22Na+ uptake in freshly isolated ATII cells. In vitro, ADP ribosylation induced by PTX revealed the presence of a specifically ribosylated band at 40-45 kDa in the total solubilized ATII cell protein fraction, but not in the immunopurified fraction. Moreover, the immunopurified channel was downregulated in response to guanosine 5'-O-(3-thiotriphosphate)-mediated activation of the exogenous G alpha(i-2), but not G(oA), G alpha(i-1), or G alpha(i-3), protein added to the channel. This effect occurred only in the presence of actin. These results suggest that amiloride-sensitive Na+ channels in adult alveolar epithelia regulated by PKA-mediated phosphorylation also retain the ability to be regulated by G alpha([i-2), but not G alpha([i-1) or G alpha(i-3), protein.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al GTP/fisiología , Alveolos Pulmonares/metabolismo , Canales de Sodio/metabolismo , Amilorida/farmacología , Animales , Bovinos , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Células Epiteliales , Epitelio/metabolismo , Membrana Dobles de Lípidos/metabolismo , Masculino , Toxina del Pertussis , Fosforilación , Alveolos Pulmonares/citología , Conejos , Canales de Sodio/efectos de los fármacos , Factores de Virulencia de Bordetella/farmacología

RESUMEN

The molecular mechanisms associated with ATP transport and release into the extracellular milieu are largely unknown. To assess the presence of endogenous ATP-conductive pathway(s) in shark rectal gland (SRG) cells, patch-clamp techniques were applied to primary cultures of SRG cells. Whole cell currents were obtained with either intracellular tris(hydroxymethyl)aminomethane (Tris) or Mg2+ salts of ATP (200 mM nominal ATP) and 280 mM NaCl bathing solution. Basal currents showed a sizable ATP permeability for outward movement of MgATP. Adenosine 3',5'-cyclic monophosphate (cAMP) stimulation significantly increased the whole cell conductance (with either intracellular Tris-ATP or MgATP). Symmetrical whole cell ATP currents were also observed after cAMP activation, thus consistent with ATP as the main charge carrier. The cAMP-inducible ATP currents were insensitive to the Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, diphenylamine-2-carboxylate, and anthracene-9-carboxylic acid but were readily blocked by nifedipine (400 microM) and glibenclamide (400 microM). The nature of the electrodiffusional ATP movement was further assessed by single-channel analysis of either MgATP or Tris-ATP currents in excised inside-out patches, both spontaneous and after activation with protein kinase A. Single-channel ATP currents were inhibited by either nifedipine or glibenclamide. Thus SRG cells express endogenous ATP-permeable pathways both before and after cAMP stimulation. Electrodiffusional ATP movement by SRG cells may play a significant role in the transport and delivery of cellular ATP to the extracellular milieu, which may help coordinate the dynamics of the epithelial secretory response in this cell model.

Asunto(s)

Adenosina Trifosfato/fisiología , AMP Cíclico/fisiología , Glándula de Sal/fisiología , Tiburones/fisiología , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Animales , Células Cultivadas , Canales de Cloruro/antagonistas & inhibidores , Conductividad Eléctrica , Canales Iónicos/fisiología , Masculino , Nifedipino/farmacología , Técnicas de Placa-Clamp , Glándula de Sal/citología , ortoaminobenzoatos/farmacología

RESUMEN

Cytoskeletal elements play an important role in the regulation of ion transport in epithelia. We have studied the effects of actin filaments of different length on the alpha, beta, gamma-rENaC (rat epithelial Na+ channel) in planar lipid bilayers. We found the following. 1) Short actin filaments caused a 2-fold decrease in unitary conductance and a 2-fold increase in open probability (Po) of alpha,beta,gamma-rENaC. 2) alpha,beta,gamma-rENaC could be transiently activated by protein kinase A (PKA) plus ATP in the presence, but not in the absence, of actin. 3) ATP in the presence of actin was also able to induce a transitory activation of alpha, beta,gamma-rENaC, although with a shortened time course and with a lower magnitude of change in Po. 4) DNase I, an agent known to prohibit elongation of actin filaments, prevented activation of alpha,beta,gamma-rENaC by ATP or PKA plus ATP. 5) Cytochalasin D, added after rundown of alpha,beta,gamma-rENaC activity following ATP or PKA plus ATP treatment, produced a second transient activation of alpha,beta,gamma-rENaC. 6) Gelsolin, a protein that stabilizes polymerization of actin filaments at certain lengths, evoked a sustained activation of alpha,beta,gamma-rENaC at actin/gelsolin ratios of <32:1, with a maximal effect at an actin/gelsolin ratio of 2:1. These results suggest that short actin filaments activate alpha, beta,gamma-rENaC. PKA-mediated phosphorylation augments activation of this channel by decreasing the rate of elongation of actin filaments. These results are consistent with the hypothesis that cloned alpha,beta,gamma-rENaCs form a core conduction unit of epithelial Na+ channels and that interaction of these channels with other associated proteins, such as short actin filaments, confers regulation to channel activity.

Asunto(s)

Actinas/metabolismo , Citoesqueleto/metabolismo , Activación del Canal Iónico , Canales de Sodio/metabolismo , Actinas/farmacología , Adenosina Trifosfato/farmacología , Animales , Epitelio/metabolismo , Membrana Dobles de Lípidos/metabolismo , Ratas , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Canales de Sodio/efectos de los fármacos , Canales de Sodio/genética

RESUMEN

Apx, the amphibian protein associated with renal amiloride-sensitive Na+ channel activity and with properties consistent with the pore-forming 150-kDa subunit of an epithelial Na+ channel complex initially purified by Benos et al. (Benos, D. J., Saccomani, G., and Sariban-Sohraby, S.(1987) J. Biol. Chem. 262, 10613-10618), has previously failed to generate amiloride-sensitive Na+ currents (Staub, O., Verrey, F., Kleyman, T. R., Benos, D. J., Rossier, B. C., and Kraehenbuhl, J.-P.(1992) J. Cell Biol. 119, 1497-1506). Renal epithelial Na+ channel activity is tonically inhibited by endogenous actin filaments (Cantiello, H. F., Stow, J., Prat, A. G., and Ausiello, D. A.(1991) Am. J. Physiol. 261, C882-C888). Thus, Apx was expressed and its function examined in human melanoma cells with a defective actin-based cytoskeleton. Apx-transfection was associated with a 60-900% increase in amiloride-sensitive (Ki = 3 microM) Na+ currents. Single channel Na+ currents had a similar functional fingerprint to the vasopressin-sensitive, and actin-regulated epithelial Na+ channel of A6 cells, including a 6-7 pS single channel conductance and a perm-selectivity of Na+:K+ of 4:1. Na+ channel activity was either spontaneous, or induced by addition of actin or protein kinase A plus ATP to the bathing solution of excised inside-out patches. Therefore, Apx may be responsible for the ionic conductance involved in the vasopressin-activated Na+ reabsorption in the amphibian kidney.

Asunto(s)

Actinas/metabolismo , Citoesqueleto/metabolismo , Canales de Sodio/metabolismo , Proteínas de Xenopus , Amilorida/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Transporte Biológico , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Epitelio , Humanos , Riñón/metabolismo , Melanoma , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes/metabolismo , Canales de Sodio/efectos de los fármacos , Canales de Sodio/genética , Células Tumorales Cultivadas , Xenopus laevis

RESUMEN

Actin filaments are novel second messengers involved in ion channel regulation. Because cytoskeletal components interact with the nuclear envelope, the actin cytoskeleton may also control nuclear membrane function. In this report, the patch-clamp technique was applied to isolated nuclei from amphibian A6 epithelial cells to assess the role of actin filaments on nuclear ion channel activity under nucleus-attached or -excised conditions. The most prevalent spontaneous nuclear ion channel species, 76% (n = 46), was cation selective and had a maximal single-channel conductance of approximately 420 pS. Nuclear ion channels also displayed multiple subconductance states, including channel activity of 26 pS that was frequently observed. Nuclear ion channel activity on otherwise quiescent patches was induced by either addition of the actin cytoskeleton disrupter cytochalasin D (CD; 5 micrograms/ml, 60%, 3 of 5 patches) or actin (10-1,000 micrograms/ml) to the bathing solution of nucleus-attached patches (59%, 13 of 22 patches). Actin also induced ion channel activity in quiescent excised inside-out patches from the nuclear envelope (80%, 4 of 5 patches). In contrast, addition of bovine serum albumin (10-1,000 micrograms/ml) to the bathing solution of nucleus-attached patches was without effect on nuclear ion channel activity (5 of 5 patches). The monoclonal antibody MAb414, specific for nuclear pore complex proteins, completely prevented either spontaneous or cytosolic actin-induced nuclear ion channels under nucleus-attached conditions (4 of 4 patches) but not intranuclear actin-induced nuclear ion channels under excised inside-out conditions (3 of 3 patches). In nucleus-attached patches, channel activity was readily activated by addition of the G-actin-binding protein deoxyribonuclease I to nucleus-attached patches (56%, 5 of 9 patches) or further addition of the actin-cross-linker filamin in the presence of actin (57%, 4 of 7 patches). The data indicate that dynamic changes in actin filament organization may represent a novel mechanism to control nuclear function.

Asunto(s)

Actinas/fisiología , Núcleo Celular/metabolismo , Citoesqueleto/fisiología , Canales Iónicos/metabolismo , Anfibios , Animales , Línea Celular , Citocalasina D/farmacología , Células Epiteliales , Canales Iónicos/efectos de los fármacos , Proteínas de Microfilamentos/farmacología , Xenopus laevis

RESUMEN

Recent studies from our laboratory indicate that members of the ATP-binding cassette (ABC) family of transporters, including P-glycoprotein and cystic fibrosis transmembrane conductance regulator (CFTR), are ATP-permeable channels. The physiological relevance of this novel transport mechanism is largely unknown. In the present study, intra- and extracellular ATP content, cellular ATP release, and O2 consumption before and after adenosine 3',5'-cyclic monophosphate (cAMP) stimulation were determined to assess the role of CFTR in the transport of ATP under physiological conditions. The functional expression of CFTR by the stable transfection of mouse mammary carcinoma cells, C1271, with human epithelial CFTR cDNA resulted in a stimulated metabolism, since both basal and cAMP-inducible O2 consumption were increased compared with mock-transfected cells. The stimulated (but not basal) O2 consumption was inhibited by diphenyl-2-carboxylic acid (DPC), a known inhibitor of CFTR. CFTR expression was also associated with the cAMP-activated and DPC-inhibitable release of intracellular ATP. The recovery of intracellular ATP from complete depletion after metabolic poisoning was also assessed under basal and cAMP-stimulated conditions. The various maneuvers indicate that CFTR may be an important contributor to the release of cellular ATP, which may help modify signal transduction pathways associated with secretory Cl- movement or other related processes. Changes in the CFTR-mediated delivery of nucleotides to the extracellular compartment may play an important role in the onset and reversal of the cystic fibrosis phenotype.

Asunto(s)

Adenosina Trifosfato/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Adenosina Trifosfato/antagonistas & inhibidores , Animales , Benzoatos/farmacología , Canales de Cloruro/antagonistas & inhibidores , AMP Cíclico/farmacología , Cinética , Ratones , Consumo de Oxígeno , Recombinación Genética , Células Tumorales Cultivadas/metabolismo

RESUMEN

From March 1992 through March 1995 we have performed 45 Ross procedures for total aortic root replacement in our institution. There were 32 males and 13 females with a mean age of 31 years (range: 3-49 years). Indications for surgery were: aortic stenosis (n = 20), aortic regurgitation (n = 16), native valve endocarditis (n = 6), replacement of prosthetic valve (n = 3). Of these 45 patients 13 (28%) had at least one prior repair. Additional procedures were Dacron graft extension of the autograft (n = 7), enlargement of aortic annulus (n = 3), mitral valve repair (n = 2), CABG (n = 1), closure of VSD (n = 1). The mean cross-clamp time was 132 minutes (76-187 minutes) and the mean bypass time 156 minutes (106-240 minutes). There were two postoperative cardiac deaths, not valve-related, and five non-lethal postoperative complications: right ventricular failure (n = 1), low cardiac output (n = 1), sternal re-entry for bleeding (n = 3). The follow up is complete (1.5-37 months) for the 43 survivors. There was one non-cardiac late death (acute fulminating hepatitis) in an eight years old boy eight months post-operatively. Discharge echo-Doppler studies showed normal autograft and homograft valve function except in one patient who had a grade two aortic regurgitation. Serial echo-Doppler studies showed no significant progression of aortic regurgitation, no significant pulmonary gradients, no dilatation of the autografts during the follow up. It is suggested in conclusion that aortic root replacement with a pulmonary autograft is a safe procedure in selected patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Válvula Aórtica/cirugía , Válvula Pulmonar/trasplante , Adolescente , Adulto , Insuficiencia de la Válvula Aórtica/fisiopatología , Insuficiencia de la Válvula Aórtica/cirugía , Estenosis de la Válvula Aórtica/cirugía , Niño , Preescolar , Ecocardiografía Doppler , Endocarditis/cirugía , Femenino , Estudios de Seguimiento , Enfermedades de las Válvulas Cardíacas/cirugía , Prótesis Valvulares Cardíacas , Humanos , Masculino , Persona de Mediana Edad , Válvula Mitral/cirugía , Tereftalatos Polietilenos , Complicaciones Posoperatorias , Prótesis e Implantes , Reoperación , Tasa de Supervivencia , Trasplante Autólogo

RESUMEN

Protein kinase A (PKA)-activation of epithelial Na+ channels requires actin filaments. Mouse mammary adenocarcinoma cells expressing the human cystic fibrosis transmembrane conductance regulator (CFTR) or mock transfectants were used to determine whether CFTR is also modulated by the actin cytoskeleton. The actin filament disrupter cytochalasin D (CD; approximately 5 micrograms/ml) readily activated whole cell currents in CFTR but not in mock-transfected (MOCK) cells. Addition of actin to the cytosolic side of quiescent excised inside-out patches of CFTR but not MOCK cells also activated CFTR. The actin-activated Cl- channels (symmetrical Cl-) had a linear conductance of 9.3 pS and were inhibited by diphenylamine-2-carboxylate and monoclonal antibodies raised against CFTR. Channel activity was also blocked by addition of the actin-binding proteins deoxyribonuclease I and filamin. Incubation of CFTR cells with CD (approximately 15 micrograms/ml) for > 6 h prevented CFTR activation by the addition of either 8-bromoadenosine 3',5'-cyclic monophosphate plus forskolin under whole cell conditions or PKA under excised inside-out conditions. However, CFTR activation was restored by subsequent addition of actin. The data indicate that CFTR is regulated by actin filaments whose effect may, in turn, be associated with the PKA-dependent pathway.

Asunto(s)

Actinas/farmacología , Canales de Cloruro/fisiología , AMP Cíclico/fisiología , Citoesqueleto/fisiología , Proteínas de la Membrana/fisiología , Actinas/química , Actinas/metabolismo , Adenocarcinoma , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas Portadoras/farmacología , Línea Celular , Proteínas Contráctiles/química , Proteínas Contráctiles/metabolismo , Proteínas Contráctiles/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Filaminas , Humanos , Cinética , Neoplasias Mamarias Experimentales , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/efectos de los fármacos , Ratones , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/farmacología , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , Transfección , Células Tumorales Cultivadas

RESUMEN

The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to a superfamily of proteins implicated in the transport of ions, proteins, and hydrophobic substances. Recent studies have demonstrated that CFTR is a protein kinase A-sensitive anion channel regulated by ATP. In the present study, patch-clamp techniques were used to assess the role of CFTR in the transport of Cl- and ATP. The stable transfection of mouse mammary carcinoma cells, C127i, with the cDNA for human CFTR resulted in the appearance of a diphenylamine-2-carboxylate-inhibitable Cl- channel, which was activated by cAMP under whole-cell and cell-attached conditions and by protein kinase A plus ATP under excised, inside-out conditions. CFTR expression was also associated with the electrodiffusional movement of ATP as indicated by the cAMP activation of ATP currents measured under whole-cell conditions. In excised, inside-out patches, it was demonstrated that ATP currents were mediated by ATP-conductive channels, which were also activated by protein kinase A and blocked by the Cl- channel blocker diphenylamine-2-carboxylate under excised, inside-out conditions. Single-channel currents observed in the presence of asymmetrical Cl-/ATP concentrations indicated that the same conductive pathway was responsible for both ATP and Cl- movement. Thus, CFTR is a multifunctional protein with more than one anion transport capability and may modify signal transduction pathways for Cl- or other secretory processes by the selective delivery of nucleotides to the extracellular domain.

Asunto(s)

Adenosina Trifosfato/metabolismo , Canales de Cloruro/metabolismo , Fibrosis Quística/metabolismo , Proteínas de la Membrana/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Canales de Cloruro/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Humanos , Transporte Iónico , Potenciales de la Membrana , Ratones , Transfección , Células Tumorales Cultivadas , ortoaminobenzoatos/farmacología

RESUMEN

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel activated by protein kinase A and regulated by ATP in a complex manner. We have applied patch-clamp techniques to C127i mouse mammary carcinoma cells transfected with human CFTR to assess the role of external ATP in the modulation of CFTR function. Extracellular ATP was sufficient to activate non-rectifying, Cl(-)-selective whole-cell currents in CFTR-transfected, but not mock-transfected cells. The ATP-mediated activation of CFTR was independent of protein kinase A since channel activation by ATP was preserved in cells that were (a) depleted of intracellular ATP, (b) incubated with the cAMP antagonist Rp-cAMPS, or (c) exposed to the protein kinase A inhibitor, 5-24 amide. In each of these conditions, 8-Br-cAMP was no longer capable of activating CFTR. The possibility that the extracellular ATP activation of Cl- currents in CFTR-expressing C127i cells was mediated by a P2-type purinergic receptor was supported by studies in which the effect of external ATP on the Cl- currents was mimicked by the ATP analogs, ATP gamma S and beta,gamma-methylene ATP, but not the uridine nucleotide, UTP. Single-channel analysis of ATP-activated Cl -currents under both cell-attached and excised, inside-out patch-clamp configurations indicated that this channel is only present in CFTR-transfected cells and indistinguishable from CFTR. External ATP also activated ATP currents in CFTR-transfected cells, a novel function of CFTR. These findings are consistent with the presence of a purinergic receptor signal transduction mechanism in C127i cells whose activation by external ATP is linked to the activation of CFTR in a cAMP-independent manner. The data provide additional support for the use of ATP and its analogs as alternative therapies in cystic fibrosis.

Asunto(s)

8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Canales de Cloruro/fisiología , AMP Cíclico/metabolismo , Proteínas de la Membrana/fisiología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Antimicina A/farmacología , Línea Celular , Canales de Cloruro/efectos de los fármacos , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Humanos , Cinética , Neoplasias Mamarias Experimentales , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/efectos de los fármacos , Ratones , Estereoisomerismo , Tionucleótidos/farmacología , Transfección , Células Tumorales Cultivadas , Uridina Trifosfato/farmacología

RESUMEN

To determine the molecular steps involved in the vasopressin-induced renal Na+ reabsorption, the patch-clamp technique was utilized to study the role of this hormone in the regulation of apical Na+ channels in renal epithelial A6 cells. Addition of arginine vasopressin (AVP) induced and/or enhanced Na+ channel activity within 5 min of addition under cell-attached conditions. The AVP-induced channel activity was a reflection of both an increase in the average apparent channel number (0.2-1.7) and the percent open time (2-56%). Addition of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, the adenosine 3',5'-cyclic monophosphate (cAMP) analogues, 8-(4-chlorophenylthio)-cAMP and 8-bromo-cAMP, or forskolin elicited a comparable effect to that of AVP. The induced channels had similar properties to Na+ channels previously reported, including a channel conductance of 9 pS, Na(+)-to-K+ selectivity of 3-5:1, and high amiloride sensitivity. The cAMP-dependent protein kinase A (PKA) in the presence of ATP induced and/or enhanced Na+ channel activity in excised inside-out patches with a change in average apparent channel number and percent open probability similar to those observed with either AVP or cAMP analogues in intact cells. Addition of activated pertussis toxin (100 ng/ml) completely blocked the AVP- or PKA-induced Na+ channel activity in excised inside-out patches, whereas incubation of intact cells with the toxin completely prevented the effect of both activators. The data indicate that AVP mediates its effect through a cAMP-dependent pathway involving PKA activation whose target is the G protein pathway that regulates apical epithelial Na+ channel activity.

Asunto(s)

Arginina Vasopresina/farmacología , Proteínas de Unión al GTP/fisiología , Riñón/metabolismo , Proteínas Quinasas/farmacología , Canales de Sodio/metabolismo , Amilorida/farmacología , Animales , Línea Celular , AMP Cíclico/farmacología , Electrofisiología , Células Epiteliales , Epitelio/metabolismo , Riñón/citología , Toxina del Pertussis , Canales de Sodio/efectos de los fármacos , Canales de Sodio/fisiología , Factores de Virulencia de Bordetella/farmacología

RESUMEN

We have recently demonstrated a novel role for "short" actin filaments, a distinct species of polymerized actin different from either monomeric (G-actin) or long actin filaments (F-actin), in the activation of epithelial Na+ channels. In the present study, the role of actin in the activation of apical Na+ channels by the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A (PKA) was investigated by patch-clamp techniques in A6 epithelial cells. In excised inside-out patches, addition of deoxyribonuclease I, which prevents actin polymerization, inhibited Na+ channel activation mediated by PKA. Disruption of endogenous actin filament organization with cytochalasin D for at least 1 h prevented the PKA-mediated activation of Na+ channels but not activation following the addition of actin to the cytosolic side of the patch. To assess the role of PKA on actin filament organization, actin was used as a substrate for the specific phosphorylation by the PKA. Actin was phosphorylated by PKA with an equilibrium stoichiometry of 2:1 mol PO4-actin monomer. Actin was phosphorylated in its monomeric form, but only poorly once polymerized. Furthermore, phosphorylated actin reduced the rate of actin polymerization. Thus actin allowed to polymerize for at least 1 h in the presence of PKA and ATP to obtain phosphorylated actin filaments induced Na+ channel activity in excised inside-out patches, in contrast to actin polymerized either in the absence of PKA or in the presence of PKA plus a PKA inhibitor (nonphosphorylated actin filaments). This was also confirmed by using purified phosphorylated G-actin incubated in a polymerizing buffer for at least 1 h at 37 degrees C. These data suggest that the form of actin required for Na+ channel activation (i.e., "short" actin filaments) may be favored by the phosphorylation of G-actin and may thus mediate or facilitate the activation of Na+ channels by PKA.

Asunto(s)

Actinas/fisiología , Riñón/metabolismo , Proteínas Quinasas/farmacología , Canales de Sodio/metabolismo , Animales , Línea Celular , Citocalasina D/farmacología , Desoxirribonucleasa I/farmacología , Electrofisiología , Células Epiteliales , Epitelio/metabolismo , Riñón/citología , Fosforilación , Polímeros/metabolismo , Canales de Sodio/efectos de los fármacos , Canales de Sodio/fisiología

RESUMEN

The cell volume regulatory response to a hypotonic stimulus is frequently initiated by activation of K+ and Cl- channels. We have characterized the hypotonic cell volume regulatory response of human melanoma cells devoid of actin-binding protein (ABP) and their genetically rescued counterpart transfected with the cDNA for ABP. ABP-deficient cells were unable to volume-regulate or activate K+ channels when exposed to a hypotonic stimulus. Genetic rescue with ABP resulted in recovery of both the cell volume regulatory response and the osmotically linked K+ channel activation. These data are consistent with a functional interaction between the actin cytoskeleton and osmotically sensitive ion transport.

Asunto(s)

Proteínas de Microfilamentos/metabolismo , Canales de Potasio/metabolismo , Tamaño de la Célula , Canales de Cloruro , Cloruros/metabolismo , Humanos , Melanoma , Potenciales de la Membrana , Proteínas de la Membrana/metabolismo , Concentración Osmolar , Células Tumorales Cultivadas