RESUMO
The cell volume regulation of the lower segment cells of the Malpighian tubule of Rhodnius neglectus in anisosmotic media was evaluated by using video-optic techniques. When the medium osmolality was increased with addition of 100 mM mannitol the cells shrank to a minimum of 16.84 +/- 2.62% and subsequently swelled towards their initial volume undergoing a typical regulatory volume increase (RVI). Replacement of either K+ or Cl- or HCO(3-) by Na+, gluconate and phosphate, respectively, abolished the RVI response. Furthermore, the substitution of Na+ by tetramethylammonium (TMA+) in isosmotic conditions led to cellular swelling and death. Addition of either amiloride 10(-4) M, anthracene-9-COOH 5 x 10(-4) M, furosemide 5 x 10(-4) M or ethacrynic acid 5 x 10(-5) M, also abolished RVI. On the other hand, addition of either Ba2+ 10(-3) M, SITS 5 x 10(-4) M, ouabain 10(-3) M or vanadate 10(-3) M, did not change the RVI response. When the tubules were incubated in hyperosmotic media with EGTA 2 mM or verapamil 10(-6) M, the RVI response was abolished. In contrast, a decrease of NaCl concentration from 129 to 79 mM induced a cell swelling to a maximum of 33.11 +/- 1.73%, but the cells maintained swollen, only partially regulating their volume. These results show that the proximal cells of Malpighian tubule of R. neglectus are able to regulate their volume in hyperosmotic but only partially regulating in hyposmotic solutions. The mechanisms in RVI involve Na+, K+, Cl-, Ca2+ and HCO(3-) transport pathways and a ouabain-insensitive ATPase stimulated by Na+.
Assuntos
Túbulos de Malpighi/fisiologia , Rhodnius/fisiologia , Animais , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Ácido Etacrínico/farmacologia , Furosemida/farmacologia , Masculino , Manitol/farmacologia , Microscopia de Vídeo , Concentração OsmolarRESUMO
Thin ascending limb cells of Henle's loop from Wistar rats were studied with in vitro microperfusion and video-optical techniques to investigate their ability in regulating cell volume during osmotic shock and to identify mechanisms of ion transport involved in the process. These cells showed a clear volume regulatory decrease (VRD) response in hyposmotic medium, but no volume regulatory increase in hyperosmotic medium. The presence of barium in the bath abolished VRD. Removal of K+ from bath and perfusate also inhibited the VRD response. Reintroduction of K+ in hyposmotic conditions reestablished cell volume regulation. Introduction of anthracene-9-COOH to the basolateral medium blocked cell volume regulatory response. Cl- removal from perfusate and bath solutions also inhibited VRD, probably because of a significant intracellular Cl- depletion. Exposure of cells to ethylene glycol-bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid in perfusate and bath solutions reduced significantly Ca2+ concentration and impaired VRD. Reintroduction of Ca2+ in hyposmotic conditions restored volume regulation. The presence of ouabain in basolateral medium also inhibited VRD. These data suggest that the following mechanisms in the basolateral membrane are involved in VRD response: K+ and Cl- conductive pathways, which might be Ca2+ dependent for activation, and an Na(+)-K(+)-adenosinetriphosphatase.
Assuntos
Alça do Néfron/citologia , Animais , Antracenos/farmacologia , Bário/farmacologia , Cloretos/farmacologia , Meios de Cultura , Ácido Egtázico/farmacologia , Gluconatos/farmacologia , Alça do Néfron/efeitos dos fármacos , Alça do Néfron/metabolismo , Concentração Osmolar , Osmose , Potássio/farmacologia , Ratos , Ratos WistarRESUMO
Thin ascending limb cells from Henle's loop were studied with optical and video techniques to evaluate cell volume regulation in response to anisoosmotic media and its ionic dependence. Cell volume regulation was observed when these cells were exposed to hypoosmotic solutions. Under hyperosmotic conditions only an osmometric response was found, with no volume regulatory increase (VRI). The removal of Cl- or HCO3- abolished the volume regulatory decrease (VRD) normally observed during exposure to hypoosmotic solutions. Re-addition of these ions did not elicit the VRD response. The removal of K+ from hypoosmotic solutions abolished VRD but its re-introduction restored the volume regulatory response. In the absence of Na+, a partial inhibition of VRD was found; re-addition of Na+ completely restored the regulatory response. These indicate that cells from the thin ascending limb of Henle's loop regulate their volume under hypoosmotic conditions, and that this process is dependent upon Cl-, HCO3-, Na+ and K+, with different patterns of response being observed upon addition or deletion of these ions.