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

Carbachol regulation of rabbit ileal brush border Na(+)–H(+) exchanger 3 (NHE3) occurs through changes in NHE3 trafficking and complex formation and is Src dependent

The epithelial brush border membrane (BBM) Na(+)–H(+) exchanger 3 (NHE3) is the major transport protein responsible for ileal electroneutral Na(+) absorption. We have previously shown that ileal BBM NHE3 activity is rapidly inhibited by carbachol, an agonist that mimics cholinergic activation in digestion. In this study, we investigated the mechanisms involved in this NHE3 inhibition. Carbachol decreased the amount of ileal Na(+) absorptive cell BBM NHE3 within 10 min of exposure. Based on OptiPrep gradient centrifugation, carbachol increased the amount of NHE3 in early endosomes and decreased the amount of NHE3 in BBM, consistent with effects on NHE3 trafficking. The decrease in BBM NHE3 occurred in the detergent-soluble BBM fraction with no change in the amount of NHE3 in the BBM detergent-resistant membranes. The size of BBM NHE3 complexes increased in carbachol-exposed ileum, as studied with sucrose gradient centrifugation. The NHE3 complex size increased in the total BBM, but did not change in the detergent-soluble fraction. This suggests that carbachol treatment enhanced the association of proteins with NHE3 complexes specifically in the detergent-resistant fraction of ileal BBM. NHERF2, α-actinin-4 and protein kinase C were among those NHE3-associated proteins because they were more efficiently coimmunoprecipitated from total BBM after carbachol treatment. Moreover, Src was involved in the carbachol-mediated inhibition since: (1) c-Src was rapidly activated in the detergent-resistant membranes by carbachol; and (2) carbachol inhibition of ileal Na(+) absorption was completely abolished by the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, the carbachol-induced increase in the size of NHE3-containing complexes was reversed by PP2. These data demonstrate that regulation of NHE3 activity by carbachol can be achieved at several interrelated levels: (1) the subcellular level, at which NHE3 is rapidly endocytosed from BBM to endocytic vesicles upon treatment with carbachol; (2) multiple BBM pools, in which carbachol selectively decreases the amount of NHE3 in the BBM detergent-soluble fraction but not the detergent-resistant membrane; and (3) the molecular level, at which NHE3 complex-associated proteins can be changed upon carbachol treatment, with carbachol leading to larger BBM NHE3 complexes and increased co-IP of NHERF2 with α-actinin-4 and activated PKC. The study further describes NHE3 presence simultaneously in multiple dynamic BBM pools in which NHE3 distribution and associated proteins are altered as part of carbachol-induced and Src-mediated rapid signal transduction, which decreases the amount of BBM NHE3 and thus inhibits NHE3 activity

Inhibition of Na(+)–H(+) exchange by cariporide reduces inflammation and heart failure in rabbits with myocardial infarction

1. The aim of this study was to assess the effects of the Na(+)–H(+) exchange inhibitor cariporide on left ventricular (LV) morphology and function as well as inflammation in rabbits with heart failure. 2. Rabbits with myocardial infarction (MI) and sham controls were randomized to receive either standard chow or chow supplemented with cariporide for 9 weeks. LV morphology was determined by echocardiography. LV systolic and diastolic function was assessed under load-dependent and -independent conditions by analysis of LV pressure–volume loops using piezo-electric crystals. 3. Plasma concentrations of C-reactive protein and aldosterone were measured. 4. Rabbits with MI developed LV dilatation that was reduced by cariporide. Systolic and diastolic LV function was impaired in rabbits with MI when compared to sham, as indicated by a decreased dP/dt(max) (MI: 3537±718 mmHg s(−1), sham: 5839±247 mmHg s(−1), P<0.05), the load-independent preload recruitable stroke work (PRSW)(MI: 22±7 mmHg, sham: 81±23 mmHg, P<0.05) and a reduction in the time constant of relaxation tau (τ) (MI: 27±1 ms, sham: 17±1 ms, P<0.05), and significantly improved by cariporide (dP/dt(max): 4586±374 mmHg s(−1), PRSW: 67±18 mmHg, τ: 20±2 ms; P<0.05 vs MI/control). 5. Induction of MI was associated with an increase in aldosterone and CRP, indicating activation of the neurohormonal and the inflammatory system that were largely reduced by cariporide. 6. Cariporide improves LV morphology and function post MI and suppresses inflammation and neurohormonal activation in congestive heart failure (CHF). Na(+)–H(+) exchange inhibition may represent a new pharmaceutical approach for the treatment of CHF