RESUMEN
In previous studies we developed and introduced a method to examine the transport mechanisms of ions in primary cell cultures of human nasal epithelium. In the current study, substances, especially nicotine, that influence these mechanisms are investigated. Specimens of nasal and paranasal epithelium of patients treated by endonasal surgery because of chronic sinusitis (n = 217) were used as primary cell cultures. Cell cultures of smokers (n = 83) and non-smokers (n = 134) were differentiated. Transepithelial Ussing chamber measurements were performed to examine sodium channel functions and to evaluate the influence of nicotine. These examinations were accompanied by simultaneous continuous capacitance measurements. Whereas transepithelial parameters, such as short-circuit current, (Isc), potential (Vt) and resistance (Rt), in tissues derived from smokers and non-smokers showed no difference, the transepithelial conductance was reduced immediately in cell cultures with apical application of nicotine (2 mM). This decrease was accompanied by a marked reduction of epithelial surface area. In the presence of nicotine, amiloride (100 microM) completely lost its inhibitory capacity. Amiloride-insensitive sodium channels were unaffected by nicotine, as proved by Na+ substitution. Furthermore, the Na+ channel blocker was accompanied by an increase in intracellular Ca2+. We conclude that the nicotine-induced increase in intracellular calcium (Ca2+) has stimulated Ca2+-dependent protein kinase (PKC). PKC promotes endocytosis removing amiloride-sensitive Na+ channels from the cell membrane into the cell by means of vesicular transport.
Asunto(s)
Amilorida/farmacología , Endocitosis/efectos de los fármacos , Mucosa Nasal/efectos de los fármacos , Nicotina/farmacología , Canales de Sodio/efectos de los fármacos , Calcio/metabolismo , Células Cultivadas , Endocitosis/fisiología , Humanos , Potenciales de la Membrana/efectos de los fármacos , Mucosa Nasal/fisiopatología , Proteína Quinasa C/fisiología , Fumar/fisiopatología , Canales de Sodio/fisiologíaRESUMEN
Nitric oxide (NO) has been reported to activate Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) and inhibit epithelial Na+ absorption mediated by amiloride-sensitive epithelial Na+ channels (ENaC). These ion transport systems are defective in cystic fibrosis (CF): Cl- secretion by CFTR is impaired and Na+ absorption by ENaC is dramatically increased. By activating CFTR and depressing ENaC, NO is a potentially beneficial therapeutic agent for ion transport defects in human CF respiratory epithelia. To assess the effects of NO on human respiratory epithelial cells, the NO donors sodium nitroprusside (SNP) and spermine NONOate were applied to primary cultured nasal cells, surgically obtained from non-CF and CF patients. Measurements of transepithelial short-circuit current (ISC) showed that NO has no inhibitory potency against amiloride-sensitive nasal ENaC (nENaC) or amiloride-insensitive Na+-absorbing mechanisms in non-CF and CF epithelia. Furthermore, NO had no stimulatory effect on Cl- secretion by CFTR or any other Cl- conductance pathway in either tissue. Although NO elevated the intracellular Ca2+ concentration, we did not detect any activation of Ca2+-dependent Cl- channels. These results demonstrate that NO has no beneficial effect on CF epithelial cells of the upper airways.
Asunto(s)
Fibrosis Quística/metabolismo , Canales Iónicos/efectos de los fármacos , Canales Iónicos/fisiología , Mucosa Nasal/metabolismo , Óxido Nítrico/farmacología , Absorción , Calcio/metabolismo , Calcio/farmacología , Células Cultivadas , Canales de Cloruro/fisiología , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Conductividad Eléctrica , Humanos , Donantes de Óxido Nítrico/farmacología , Óxidos de Nitrógeno , Nitroprusiato/farmacología , Sodio/metabolismo , Espermina/análogos & derivados , Espermina/farmacologíaRESUMEN
Na+ and Cl- currents were studied in primary cultures of human nasal epithelium derived from non-cystic fibrosis (non-CF) and cystic fibrosis (CF) patients. We found that Na+ absorption dominates transepithelial transport and the Na+ current contains an amiloride-sensitive and amiloride-insensitive component. In non-CF tissue both components contribute about equally to the entire short-circuit current (ISC), whereas in CF tissues the major part of the current is amiloride-sensitive. Na+ removal reduced ISC to values close to zero. Several Cl- channel blockers were used to identify the remaining tiny Na+-independent current. Under unstimulated, physiological conditions in the presence of Cl- on both sides and amiloride on the apical side of the epithelium diphenylamine-2-carboxic acid (DPC), 4,4'-diisothiocyanatostilbene-2, 2'- disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) failed to induce clearcut inhibition of ISC. cAMP as well as ATP did not affect ISC either in CF or in non-CF epithelia. Reduction of apical Cl- increased ISC and depolarized transepithelial potential; however, the observed increase was insensitive to DIDS, DPC and NPPB. From these data we conclude that Cl- conductances in primary cultures of human nasal epithelium derived from CF patients as well as from non-CF patients are present only in low numbers or do not contribute significantly to transepithelial ion transport.
Asunto(s)
Cloruros/metabolismo , Fibrosis Quística/fisiopatología , Mucosa Nasal/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Adenosina Trifosfato/farmacología , Amilorida/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/fisiología , AMP Cíclico/farmacología , Humanos , Potenciales de la Membrana/fisiología , Mucosa Nasal/fisiología , Mucosa Nasal/fisiopatología , Nitrobenzoatos/farmacología , Valores de Referencia , Sodio/metabolismo , Canales de Sodio/efectos de los fármacos , Canales de Sodio/fisiología , ortoaminobenzoatos/farmacologíaRESUMEN
Cystic fibrosis (CF) is the most commonly inherited disease in Caucasians and is caused by a mutation in the gene encoding a membrane transport protein. This cystic fibrosis transmembrane conductance regulator (CFTR) is thought to be an apical Cl- channel activated by intracellular cAMP. Most recent findings suggest that CFTR is more than a pure Cl- channel and might be involved in the regulation of other transport systems. In the present study we show that CFTR as a Cl- channel plays only a minor role in primary cultured human nasal epithelium derived from non-CF and CF patients. These findings are especially of interest for non-CF human nasal epithelia in which CFTR is correctly inserted. In both tissues Cl- secretion is negligible as compared with Na+ absorption. We confirm and expand our previous observations that Na+ absorption in human nasal epithelium is the dominant ion transport process and that Cl- secretion is detectable in both CF and non-CF tissue. Moreover, we show that cAMP and ATP were not able to stimulate any silent Cl- channels in CF or non-CF human nasal epithelial cells. We further give evidence that in human nasal CF and non-CF epithelium Na+ absorption is mediated by epithelial Na+ channels (ENaC) that are either different from those of other epithelia or which exhibit altered regulation. These differences between Na+ channels of human nasal epithelium and "classical" epithelial Na+ channels include lack of activation by the intracellular second messenger cAMP and the steroid hormone aldosterone. We show further that human nasal Na+ channels are inhibited by Cl- channel blockers and exhibit a different pharmacology towards common Na+ channel blockers.