Signalling pathways involved in hypertonicity- and acidification-induced activation of Na+/H+ exchange in trout hepatocytes

Abstract

SUMMARY In trout hepatocytes, hypertonicity and cytosolic acidification are known to stimulate Na+/H+ exchanger (NHE) activity, which contributes to recovery of cell volume and intracellular pH (pHi),respectively. The present study investigated the signalling mechanisms underlying NHE activation under these conditions. Exposing trout hepatocytes to cariporide, a specific inhibitor of NHE-1, decreased baseline pHi,completely blocked the hypertonicity-induced increase of pHi and reduced the hypertonicity-induced proton secretion by 80%. Changing extracellular pH (pHe)above and below normal values, and allowing cells to adjust pHi accordingly,significantly delayed alkalinization during hypertonic exposure, whereas following an acid load an enhanced pHi recovery with increasing pHe was seen. Chelating Ca2+, and thereby preventing the hypertonicity-induced increase in intracellular Ca2+ ([Ca2+]i), significantly diminished hypertonic elevation of pHi, indicating that Ca2+signalling might be involved in NHE activation. A reduction in alkalinization and proton secretion was also observed in the presence of the protein kinase A(PKA) inhibitor H-89 or the calmodulin (CaM) inhibitor calmidazolium. A complete inhibition of hypertonic- and acidification-induced changes of pHi concurrent with an increase in hypertonically induced proton efflux was seen with the protein kinase C (PKC) inhibitor chelerythrine. Recovery of pHi following sodium propionate addition was reduced by more than 60% in the presence of cariporide, was sensitive to PKA inhibition, and tended to be reduced by CaM inhibition. In conclusion, we showed that NHE-1 is the main acid secretion mechanism during hypertonicity and recovery following acid loading. In addition, Ca2+-, PKA- and CaM-dependent pathways are involved in NHE-1 activation for recovery of cell volume and pHi. On the other hand, PKC appeared to have an impact on NHE-independent pathways affecting intracellular acid-base homeostasis.

Ionic determinants of pH of acidic compartments under hypertonic conditions in trout hepatocytes

“…The existence of this exchange is supported by the pH L acidification upon release of Ca 2+ from acidic stores using A23187. Under hypertonic conditions, Ca 2+ influx from the extracellular medium as well as Ca 2+ release from cellular stores is the source of the measured increase in [Ca 2+ ] i (Ahmed et al, 2006). Interestingly, BAPTA exposure induced an increase in pH L .…”

“…In trout hepatocytes, exposure of cells to NHE-specific inhibitors (Ahmed et al, 2006) or removal of extracellular Na + (Krumschnabel et al, 2003) decreased steady-state pH i and abolished the hypertonicity-induced pH i increase. Intracellularly, NHE isoforms have been reported to reside in the membranes of acidic compartments (Numata and Orlowski, 2001;Brett et al, 2002;Nakamura et al, 2005); however, an involvement of NHE activity in maintaining pH L has not been detected (Demaurex et al, 1998;Llopis et al, 1998;Schapiro and Grinstein, 2000).…”

“…We have shown recently that removal of extracellular Ca 2+ or chelation of intracellular Ca 2+ affects pH i under steady-state conditions (Ahmed and Pelster, 2007) and during hypertonicity challenge (Ahmed et al, 2006). Thus, in the next set of experiments, we attempted to investigate possible changes in pH L in response to the above-mentioned conditions.…”

“…Fish were fed daily with trout pellets (AGRA TAGGER, Innsbruck, Austria) ad libitum. Hepatocytes were isolated following the collagenase digestion procedure described previously (Ahmed et al, 2006). In brief, fish were killed by a blow on the head, the liver was exposed, and the portal vein was cannulated.…”

“…As pH L changes induced by anisotonicity reflect movements of H + or acid equivalents between the cytosol and acidic luminal, it is reasonable to assume that such changes in pH L might contribute to overall pH i regulation. In fact, in a recent study on trout hepatocytes, the involvement of intracellular mechanisms in the regulation of pH i under hypertonicity (Ahmed et al, 2006) was postulated.…”