Abstract
We have examined the functional properties and growth factor responsiveness of the plasma membrane Na+/H+ exchanger in pluripotent P19 embryonal carcinoma (EC) cells and in a differentiated mesodermal derivative (MES-1) by analyzing the recovery of cytoplasmic pH (pHi) from an acute acid load under bicarbonate-free conditions. In the absence of exogenous growth factors, the mean steady-state pHi of undifferentiated P19 cells (7.49 +/- 0.03) is 0.55 unit higher than the value of differentiated MES-1 cells (6.94 +/- 0.01). In both cell types, recovery of pHi from an NH+4-induced acid load follows an exponential time course and is entirely mediated by the amiloride-sensitive Na+/H+ exchanger in the plasma membrane. Kinetic analysis indicates that the higher steady-state pHi in P19 EC cells is due to an alkaline shift in the pHi sensitivity of the Na+/H+ exchange rate, as compared to that in MES-1 cells. The Na+/H+ exchanger of MES-1 cells is responsive to epidermal growth factor, platelet-derived growth factor, serum, phorbol esters, and diacylglycerol, as shown by a rapid amiloride-sensitive rise in pHi of 0.15-0.35 unit. This mitogen-induced alkalinization is attributable to an alteration in the pHi sensitivity of the exchanger. In contrast, the Na+/H+ exchanger of P19 EC cells fails to respond to any of these stimuli. Similarly, hypertonic medium rapidly activates the Na+/H+ exchanger in MES-1, but not in P19 EC cells. We conclude that the Na+/H+ exchanger in undifferentiated P19 EC stem cells is maintained in a fully activated state which is unaffected by extracellular stimuli, as if signal pathways normally involved in growth factor action are constitutively operative.
Highlights
The Na+/H+Exchanger Is Constitutively Activated in P19 Embryonal Carcinoma Cells, but Not in a Differentiated Derivative*
The molecular mechanisms underlying embryonic growth and development are largely unknown, it seems likely that polypeptide growth factors andtheir receptorlinked signal pathways have a major role in mammalian embryogenesis [1].A suitable approachto studying embryonic growth control is to use in uitro model systems such as murine embryonal carcinoma (EC)’ cells, the undifferentiated stemcells of teratocarcinomas, media [8,9,10]. While both P19 EC and MES-1 cella have a normally functioning Na+/H+ exchanger in terms of pHi regulation, the exchanger of MES-1 cells ishighlyresponsive to growth factors and other agonists, whereas the Na+/H+ exchanger in P19 EC cells is in apermanently activated state, resulting in a much higher steady-state pHi value and in caomplete lack of responsiveness to extracellular
The curves are drawn according to the simplified Goldman equation as given in the legend to Fig. 1. From these graphs the intracellularK+ concentration is estimated at 146 mM for P19 EC and 149 mM for MES-1 cells, i.e. the value of [K+l0where V, becomes 0
Summary
The Na+/H+Exchanger Is Constitutively Activated in P19 Embryonal Carcinoma Cells, but Not in a Differentiated Derivative*. As pHi6'Y2 rnin shown, addition of FCS, EGF, phorbol esters, or diacylglycerols to MES-1 cells leads to a substantial cytoplasmic alkalinization, up to 0.3 pH unit above the initial steady-state pH;.
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