LONG-TERM EFFECTS OF GROWTH-FACTORS ON PH AND ACID-BASE TRANSPORT IN RAT GLOMERULAR MESANGIAL CELLS

We investigated the long-term effects of exposing mesangial cells (MCs), previously quiescent, to platelet-derived growth factor, arginine vasopressin (AVP), epidermal growth factor, and serotonin (5-HT). MCs were exposed to the mitogens continuously for periods of 30-70 h. For each agent, we periodically measured intracellular pH (pH(i)), activities of Na+/H+, Na+-dependent Cl-/HCO3-, and Cl-/HCO3- exchangers, and cell number, pH(i) and transporter activity were assessed using the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in a population of cells attached to glass cover slips. The first two transporters were assayed at the single pH(i) of 6.6, whereas Cl-/HCO3- exchange was assayed at pH(i) 7.5. Cell number was determined using fluorescence-activated cell sorting (FACS). Incubations and assays were conducted in presence of CO2/HCO3-. No agent substantially altered pH(i), which generally ranged from 7.25 to 7.35. pH(i) in AVP-treated cells tended to be similar to 0.05 lower than in time-matched controls at the longer incubation times. All agents, when assayed minutes after mitogen application, caused large increases (70-130%) in activities of all three transporters. However, transporter activity gradually declined in continuous presence of each agent, first rapidly (e.g., 2-4 h) and then more slowly. Measurements of immunoreactive extracellular AVP ([AVP](0)) levels indicate that the decrease in Cl-/HCO3- exchange activity is not due to a decrease in [AVP](0). Minimum transporter activity (10-40% above baseline) was achieved similar to 18-35 h after mitogen application. Later, transporter activity slowly increased, although not to the high levels achieved minutes after mitogen addition. For each mitogen, activities of all three transporters tended to rise and fall together. FACS data showed that each agent produced a gradual but significant increase in the number of cells in each cell-cycle phase (G(0)/G(1), S, and M). There also was an increase in the total number of cells, confirming previous [H-3]thymidine incorporation data. The fraction of cells in G(0)/G(1), S, or M phase did not correlate well with the gradual decrease in transporter activity. However, the total number of cells peaked at the time of minimal transporter activity. These data demonstrate that, when studied at the cell-population level, mitogens produce a burst in acid-base transporter activity that gradually wanes as cell number increases. Conversely, transporter activity later increases after the peak in cell number.