RESUMEN
The effect of ANG II on pH(i), [Ca(2+)](i) and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pH(i) via the Na(+)/H(+) exchanger was examined in the first 2 min following the acidification of pH(i) with a NH(4)Cl pulse. In the control situation, the pH(i) recovery rate was 0.118 +/- 0.001 (n = 52) pH units/min and ANG II (10(-12) M or 10(-9) M) increased this value (by 106% or 32%, respectively) but ANG II (10(-7) M) decreased it to 47%. The control [Ca(2+)](i) was 99 +/- 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pH(i) recovery and [Ca(2+)](i). To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na(+)/H(+) exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10(-12) - 10(-7) M ANG II) and by increases of [Ca(2+)](i) in the lower range (at 10(-12) M ANG II) and 2) inhibition of the exchanger at high [Ca(2+)](i) levels (at 10(-9) - 10(-7) M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.
Asunto(s)
Angiotensina II/farmacología , Calcio/metabolismo , Tamaño de la Célula/efectos de los fármacos , Colon/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Amilorida/análogos & derivados , Amilorida/farmacología , Línea Celular Tumoral , Colon/metabolismo , Neoplasias del Colon , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Isoquinolinas/farmacología , Losartán/farmacología , Intercambiadores de Sodio-Hidrógeno/farmacología , Estaurosporina/farmacología , Sulfonamidas/farmacologíaRESUMEN
Potassium secretory flux (J(K)) by the distal nephron is regulated by systemic and luminal factors. In the present investigation, J(K) was measured with a double-barreled K(+) electrode during paired microperfusion of superficial segments of the rat distal nephron. We used control solutions (100 mM NaCl, pH 7.0) and experimental solutions in which Cl(-) had been replaced with a less permeant anion and/or pH had been increased to 8.0. J(K) increased when Cl(-) was replaced by either acetate ( approximately 37%), sulfate ( approximately 32%), or bicarbonate ( approximately 62%), and also when the pH of the control perfusate was increased ( approximately 26%). The majority (80%) of acetate-stimulated J(K) was Ba(2+) sensitive, but furosemide (1 mM) further reduced secretion ( approximately 10% of total), suggesting that K(+)-Cl(-) cotransport was operative. Progressive reduction in luminal Cl(-) concentration from 100 to 20 to 2 mM caused increments in J(K) that were abolished by inhibitors of K(+)-Cl(-) cortransport, i.e., furosemide and [(dihydroindenyl)oxy]alkanoic acid. Increasing the pH of the luminal perfusion fluid also increased J(K) even in the presence of Ba(2+), suggesting that this effect cannot be accounted for only by K(+) channel modulation of K(+) secretion in the distal nephron of the rat. Collectively, these data suggest a role for K(+)-Cl(-) cotransport in distal nephron K(+) secretion.