RESUMEN
Earlier studies have suggested a role for Ca2+ in regulatory volume decrease (RVD) in response to hypotonic stress through the activation of Ca2+-dependent ion channels (Kotera & Brown, 1993; Park et al., 1994). The involvement of Ca2+ in regulating cell volume in rat lacrimal acinar cells was therefore examined using a video-imaging technique to measure cell volume. The trivalent cation Gd3+ inhibited RVD, suggesting that Ca2+ entry is important and may be via stretch-activated cation channels. However, Fura-2 loaded cells did not show an increase in [Ca2+]i during exposure to hypotonic solutions. The absence of any changes in [Ca2+]i resulted from the buffering of cytosolic Ca2+ by Fura-2 during hypotonic shock and therefore inhibition of RVD. The intracellular Ca2+ chelator, BAPTA, also inhibited the RVD response to hypotonic shock. An increase in [Ca2+]i induced by either acetylcholine or ionomycin, was found to decrease cell volume under isotonic conditions in lacrimal acinar cells. Cell shrinkage was inhibited by tetraethylammonium ion, an inhibitor of Ca2+-activated K+ channels. On the basis of the presented data, we suggest an involvement of intracellular Ca2+ in controlling cell volume in lacrimal acinar cells.
Asunto(s)
Calcio/metabolismo , Aparato Lagrimal/citología , Animales , Calcio/farmacología , Agonistas de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , Tamaño de la Célula , Células Cultivadas , Agonistas de los Canales de Cloruro , Canales de Cloruro/metabolismo , Fura-2 , Aparato Lagrimal/metabolismo , Microscopía por Video , Presión Osmótica , Bloqueadores de los Canales de Potasio , Canales de Potasio/metabolismo , Ratas , Transducción de Señal , Espectrometría de FluorescenciaRESUMEN
The volume of acinar cells isolated from rat lacrimal glands was measured during hypertonic shock. Cells shrank in hypertonic solutions, but a regulatory volume increase (RVI) was only observed under certain conditions. In HEPES-buffered solutions at 37 degrees C, an RVI was observed. This was inhibited by 20 microM bumetanide, an inhibitor of Na(+)-K(+)-2Cl- cotransport. RVI did not occur in HEPES-buffered solutions at 20 degrees C suggesting that Na(+)-K(+)-2Cl- cotransport is inactive at this temperature. In HCO3- buffered solutions however, an RVI was observed at 20 degrees C. In these conditions, the RVI was inhibited by 500 microM 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2-DIDS) and 10 microM 5-(N-methyl-N-isobutyl)-amiloride (MIBA) indicating the involvement of Cl(-)-HCO3- exchange and Na(+) -H+ exchange respectively. RVI was also supported by a mixture of neutral amino acids, and by the nonmetabolizable amino acids 5 mM alpha-(methylamino)isobutyric acid (MeAIB) and 5 mM alpha-aminoisobutyric acid (AIB). These data suggest that the accumulation of amino acids, possibly by the system A Na(+)-coupled amino acid cotransporter, contributes to RVI in these cells. In conclusion, rat lacrimal gland acinar cells are capable of undergoing RVI following shrinkage by hypertonic shock.
Asunto(s)
Aparato Lagrimal/citología , Aminoácidos/metabolismo , Animales , Bicarbonatos/farmacología , Tampones (Química) , Tamaño de la Célula , HEPES/farmacología , Soluciones Hipertónicas/farmacología , Aparato Lagrimal/efectos de los fármacos , Aparato Lagrimal/metabolismo , RatasRESUMEN
The volumes of acinar cells isolated from rat lacrimal gland were measured on computer by video-imaging. Cells were found to swell on exposure to hypotonic solutions; they subsequently exhibited a regulatory volume decrease (RVD). RVD was inhibited in the absence of extracellular Ca2+, and by the K+ channel blocker tetraethylammonium chloride (2 mM TEA+). The possible involvement of K+ channels in RVD was further investigated in cell-attached patches. Exposing the cells to a hypotonic solution activated channels with a conductance of 141 +/- 6 pS (n = 11). These channels were partially blocked by 0.5 mM TEA+, and channel activation was not observed in the absence of extracellular Ca2+. Experiments in the inside-out patch configuration demonstrated that the channels activated by hypotonic stress were "maxi" Ca(2+)-activated K+ channels. It is concluded that the opening of these channels plays an important role in RVD, by facilitating K+ loss from the cell.