Phosphorylation and Activation of the Plasma Membrane Na+/H+ Exchanger (NHE1) during Osmotic Cell Shrinkage

The Na+/H+ Exchanger isoform 1 (NHE1) is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na+/H+ exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS). We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC). Na+/H+ exchange in atRBCs is mediated by an NHE1 homolog (atNHE1) that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA) increase Na+ transport capacity without affecting transport affinity (Km = 44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ 32P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na+ transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS) analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events

Effects of butyrate on active sodium and chloride transport in rat and rabbit distal colon

Short chain fatty acids, particularly butyrate, stimulate electroneutral NaCl absorption from the colon. Their effect in colonic epithelia lacking basal electroneutral NaCl absorption is unknown. Butyrate is also reported to inhibit active Cl− secretion in the colon. The present studies were undertaken to investigate the inter-relationships between the effects of butyrate on active Na+ and Cl− transport in the colon. Studies were carried out in rabbit distal colon (known to have predominant electrogenic Na+ absorption), rat distal colon (characterised by electroneutral Na+ absorption), and hyperaldosteronaemic rat distal colon (characterised by electrogenic Na+ absorption). The effect of cholera toxin (CT) was also noted. Potential difference, short-circuit current (ISC) and fluxes of Na+ and Cl− were measured in stripped mucosa under voltage-clamp conditions. Butyrate stimulated electroneutral Na+ and Cl− absorption in distal colon of normal and salt-depleted rats, and stimulated Na+ absorption in rabbit distal colon. Amiloride (10−4m) or CT did not inhibit this process. In rabbit distal colon, stimulation of Na+ absorption by butyrate was not dependent on the presence of Cl− in the medium. Butyrate significantly decreased conductance, decreased flux of sodium from serosa to mucosa (particularly in rabbit distal colon), and decreased ISC. Net Cl− secretion, induced by CT, was completely inhibited by butyrate. Stimulation of Na+ absorption was independent of exposure to CT. Bumetanide reversed net Cl− secretion to net absorption, but did not alter Na+ or Cl− fluxes in tissues exposed to butyrate. Thus butyrate stimulates active Na+ absorption in colonic epithelia, with or without expression of basal Na+-H+ exchange. Independently, butyrate inhibits active Cl− secretion induced by cAMP in these epithelia