Activation of Na/H exchanger in mesangial cells is associated with translocation of PKC isoforms

We investigated the potential coupling of the Na/H exchanger to protein kinase C (PKC) activation. Mesangial cells (MC), passage 3-8, were exposed to either serotonin (5-HT), arginine vasopressin (AVP), or fibroblast growth factor (FGF). We assessed the effect of these agonists on recovery from an acid load (NH₄/⁺/NH₃) in the nominal absence of CO₂/HCO₃. 5-HT, AVP, and FGF significantly enhanced the rate of recovery from 24.2 ± 4.5 x 10^-4 intracellular pH units/s to 67.7 ± 5.7, 70.5 ± 5.1, and 55.7 ± 6.8, respectively (n > 6, P < 0.0001). The increase in activity was abolished by ethylisopropylamiloride and removal of extracellular Na⁺, thereby suggesting that activation of Na/H exchanger activity was responsible for enhanced recovery by 5-HT, AVP, and FGF. MC were then pretreated with phorbol ester [phorbol 12-myristate 13-acetate (PMA); 10 μM for 40 h] and acid loaded. 5- HT and AVP did not enhance recovery of PMA-pretreated cells (23.3 ± 6.8 and 24.9 ± 4.7, n > 4, not significant), whereas FGF stimulated activity (48.2 ± 5.5, n > 4, P < 0.001). Similar results were found when MC were pretreated with the PKC antagonist, sphingosine. Specific antibodies were raised against α-, β(I)-, β(II)-, and γ-isoforms of PKC. We observed that MC express the α-, γ-, and β(I)-isoforms, but not the β(II)-isoform of PKC. When MC were exposed to 5-HT and AVP, translocation was evident immediately (within seconds) for the α-isoform, whereas maximal translocation occurred at 30 min for β(I)-PKC and at 24 h for γ-PKC (n > 6 for each agonist). FGF did not induce translocation of any of the PKC isoforms studied. These results indicate that in MC, PKC activation is required for the 5-HT and AVP stimulation of the Na/H exchanger. There appears to be a temporal correlation between the translocation pattern of the α-isoform of PKC and Na/H exchange activation.