Effect of hypercapnia on intracellular pH regulation in a rainbow trout hepatoma cell line, RTH 149.

Fish exposed to elevated water CO(2) experience a rapid increase in blood CO(2) levels (hypercapnia), resulting in acidification of both intra- and extra-cellular compartments. While the mechanisms associated with extracellular pH regulation have been well explored, much less is known about intracellular pH (pH(i)) regulation. There is great interest in developing non-animal models for research. One such model is the rainbow trout hepatoma cell line (RTH 149), which has been used to study a wide range of topics; however, no studies have investigated its potential use in pH(i) regulation. Employing the pH-sensitive fluoroprobe BCECF, the present study examined pH(i) regulation in RTH 149 under normocapnia and during extracellular acidification induced by either elevated CO(2) or 1 M HCl. During exposure to hypercapnia, RTH 149 cells were acidified without recovery as long as the elevated CO(2) was maintained. In addition, rates of pH(i) recovery from NH(4)Cl-induced acidosis were significantly lower in cells exposed to hypercapnia or HCl compared to that in normocapnic cells, indicating that elevated CO(2) indirectly impeded pH(i) recovery through a reduction in pH(e) and/or pH(i). Moreover, pH(i) regulation in RTH 149 was EIPA-sensitive, suggesting that an NHE may be involved. Overall, RTH 149 may have the potential for identifying transporters likely to play a role in pH(i) regulation in fish. However, it should not be used as a complete replacement for in vivo studies, especially to quantify acid-base regulatory ability at whole animal level, since RTH 149 appeared to have enhanced pH(i) recovery rates relative to primary hepatocytes.

Capacity for intracellular pH compensation during hypercapnia in white sturgeon primary liver cells.

Fish, exposed to elevated water CO(2), experience a rapid elevation in blood CO(2) (hypercapnia), resulting in acidification of both intra- and extra-cellular compartments. White sturgeon, Acipenser transmontanus, are exceptionally CO(2) tolerant and can regulate tissue intracellular pH (pH(i)) in the presence of a pronounced hypercapnic acidosis (preferential pH(i) regulation). In this study, pH(i) regulatory capacity of sturgeon liver cells in primary culture was examined to assess the suitability of employing this in vitro system to understand in vivo CO(2) tolerance in sturgeon. Using the pH-sensitive fluoroprobe BCECF, real-time changes in resting pH(i) and rates of pH(i) recovery were investigated during exposure to hypercapnia (3 and 6% CO(2)) in the absence and presence of additional acid loads induced by (20 mM) ammonium prepulse. During short-term (10 min) exposure to hypercapnia (3 and 6% CO(2)), sturgeon cells were acidified and no pH(i) compensation was observed. However, when exposure to 6% CO(2) was extended to over 19 h, the CO(2)-induced intracellular acidosis was partially compensated by a pH(i) increase of over 0.2 pH unit despite the sustained extracellular acidosis, indicative of a capacity for preferential pH(i) regulation in vitro. Since this capacity in sturgeon liver is present both in vivo and in vitro, the transmembrane transporters involved may be the same. Therefore, cell culture may be a suitable tool to identify the transporters (i.e., the cellular mechanisms underlying in vivo CO(2) tolerance) in white sturgeon and possibly in other hypercapnia-tolerant species.