Glucose Uptake and Intracellular pH in a Mouse Model of Ductal Carcinoma In situ (DCIS) Suggests Metabolic Heterogeneity.

Mechanisms for the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma remain unclear. Previously we showed that the transition to invasiveness in the mammary intraepithelial neoplastic outgrowth (MINO) model of DCIS does not correlate with its serial acquisition of genetic mutations. We hypothesized instead that progression to invasiveness depends on a change in the microenvironment and that precancer cells might create a more tumor-permissive microenvironment secondary to changes in glucose uptake and metabolism. Immunostaining for glucose transporter 1 (GLUT1) and the hypoxia marker carbonic anhydrase 9 (CAIX) in tumor, normal mammary gland and MINO (precancer) tissue showed differences in expression. The uptake of the fluorescent glucose analog dye, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), reflected differences in the cellular distributions of glucose uptake in normal mammary epithelial cells (nMEC), MINO, and Met1 cancer cells, with a broad distribution in the MINO population. The intracellular pH (pHi) measured using the fluorescent ratio dye 2',7'-bis(2-carboxyethyl)-5(6)-155 carboxyfluorescein (BCECF) revealed expected differences between normal and cancer cells (low and high, respectively), and a mixed distribution in the MINO cells, with a subset of cells in the MINO having an increased rate of acidification when proton efflux was inhibited. Invasive tumor cells had a more alkaline baseline pHi with high rates of proton production coupled with higher rates of proton export, compared with nMEC. MINO cells displayed considerable variation in baseline pHi that separated into two distinct populations: MINO high and MINO low. MINO high had a noticeably higher mean acidification rate compared with nMEC, but relatively high baseline pHi similar to tumor cells. MINO low cells also had an increased acidification rate compared with nMEC, but with a more acidic pHi similar to nMEC. These findings demonstrate that MINO is heterogeneous with respect to intracellular pH regulation which may be associated with an acidified regional microenvironment. A change in the pH of the microenvironment might contribute to a tumor-permissive or tumor-promoting progression. We are not aware of any previous work showing that a sub-population of cells in in situ precancer exhibits a higher than normal proton production and export rate.

Structural modeling and electron paramagnetic resonance spectroscopy of the human Na+/H+ exchanger isoform 1, NHE1.

We previously presented evidence that transmembrane domain (TM) IV and TM X-XI are important for inhibitor binding and ion transport by the human Na(+)/H(+) exchanger, hNHE1 (Pedersen, S. F., King, S. A., Nygaard, E. B., Rigor, R. R., and Cala, P. M. (2007) J. Biol. Chem. 282, 19716-19727). Here, we present a structural model of the transmembrane part of hNHE1 that further supports this conclusion. The hNHE1 model was based on the crystal structure of the Escherichia coli Na(+)/H(+) antiporter, NhaA, and previous cysteine scanning accessibility studies of hNHE1 and was validated by EPR spectroscopy of spin labels in TM IV and TM XI, as well as by functional analysis of hNHE1 mutants. Removal of all endogenous cysteines in hNHE1, introduction of the mutations A173C (TM IV) and/or I461C (TM XI), and expression of the constructs in mammalian cells resulted in functional hNHE1 proteins. The distance between these spin labels was ∼15 A, confirming that TM IV and TM XI are in close proximity. This distance was decreased both at pH 5.1 and in the presence of the NHE1 inhibitor cariporide. A similar TM IV·TM XI distance and a similar change upon a pH shift were found for the cariporide-insensitive Pleuronectes americanus (pa) NHE1; however, in paNHE1, cariporide had no effect on TM IV·TM XI distance. The central role of the TM IV·TM XI arrangement was confirmed by the partial loss of function upon mutation of Arg(425), which the model predicts stabilizes this arrangement. The data are consistent with a role for TM IV and TM XI rearrangements coincident with ion translocation and inhibitor binding by hNHE1.

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 (K(m)=44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ(32)P 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.

Na/H exchange inhibition protects newborn heart from ischemia/reperfusion injury by limiting Na+-dependent Ca2+ overload.

The results of the Guardian/Expedition trials demonstrate the need for more precisely controlled studies to inhibit Na/H exchange (NHE1) during ischemia/reperfusion. This is because overwhelming evidence is consistent with the hypothesis that myocardial ischemic injury results in part from increases in intracellular Na (Nai) mediated by NHE1 that in turn promote Na/Ca exchanger-mediated increases in intracellular Ca ([Ca]i) and Ca-dependent cell damage. We used a more potent and specific NHE1 inhibitor HOE 694 (HOE) to test whether inhibition of NHE1 during ischemia limits increases in Nai and [Ca]i in newborns. NMR was used to measure pHi, Nai, [Ca]i, and ATP in isolated newborn rabbit hearts. Perfusion pressure, left ventricular developed pressure, and creatine kinase were measured. HOE was added before global ischemia. Results are reported as mean +/- SE. Nai (mEq/kg dry weight) rose from 11.6 +/- 0.9 before ischemia to 114.0 +/- 16.1 at the end of ischemia and recovered to 55.2 +/- 11.8 in the control group. During ischemia and reperfusion, the corresponding values for Nai in the HOE group (63.1 +/- 8.4 and 15.9 +/- 2.5, respectively, P < 0.05) were lower than control. In the control group [Ca]i (nM/L) rose from 331 +/- 41 to 1069 +/- 71 and recovered to 814 +/- 51, whereas in the HOE group [Ca]i rose less (P < 0.05): 359 +/- 50, 607 +/- 85, and 413 +/- 40, respectively. Total creatine kinase release was significantly reduced in the HOE group. Perfusion pressure and left ventricular developed pressure also recovered significantly better in the HOE group than in the control. In conclusion, NHE1 inhibition diminishes ischemia-induced increases in Nai and therefore [Ca], and thus diminishes myocardial injury in neonatal hearts.

Coordinated control of volume regulatory Na+/H+ and K+/H+ exchange pathways in Amphiuma red blood cells.

The Na(+)/H(+) and K(+)/H(+) exchange pathways of Amphiuma tridactylum red blood cells (RBCs) are quiescent at normal resting cell volume yet are selectively activated in response to cell shrinkage and swelling, respectively. These alkali metal/H(+) exchangers are activated by net kinase activity and deactivated by net phosphatase activity. We employed relaxation kinetic analyses to gain insight into the basis for coordinated control of these volume regulatory ion flux pathways. This approach enabled us to develop a model explaining how phosphorylation/dephosphorylation-dependent events control and coordinate the activity of the Na(+)/H(+) and K(+)/H(+) exchangers around the cell volume set point. We found that the transition between initial and final steady state for both activation and deactivation of the volume-induced Na(+)/H(+) and K(+)/H(+) exchange pathways in Amphiuma RBCs proceed as a single exponential function of time. The rate of Na(+)/H(+) exchange activation increases with cell shrinkage, whereas the rate of Na(+)/H(+) exchange deactivation increases as preshrunken cells are progressively swollen. Similarly, the rate of K(+)/H(+) exchange activation increases with cell swelling, whereas the rate of K(+)/H(+) exchange deactivation increases as preswollen cells are progressively shrunken. We propose a model in which the activities of the controlling kinases and phosphatases are volume sensitive and reciprocally regulated. Briefly, the activity of each kinase-phosphatase pair is reciprocally related, as a function of volume, and the volume sensitivities of kinases and phosphatases controlling K(+)/H(+) exchange are reciprocally related to those controlling Na(+)/H(+) exchange.

Activation of Na+/H+ and K+/H+ exchange by calyculin A in Amphiuma tridactylum red blood cells: implications for the control of volume-induced ion flux activity.

Alteration in cell volume of vertebrates results in activation of volume-sensitive ion flux pathways. Fine control of the activity of these pathways enables cells to regulate volume following osmotic perturbation. Protein phosphorylation and dephosphorylation have been reported to play a crucial role in the control of volume-sensitive ion flux pathways. Exposing Amphiuma tridactylu red blood cells (RBCs) to phorbol esters in isotonic medium results in a simultaneous, dose-dependent activation of both Na(+)/H(+) and K(+)/H(+) exchangers. We tested the hypothesis that in Amphiuma RBCs, both shrinkage-induced Na(+)/H(+) exchange and swelling-induced K(+)/H(+) exchange are activated by phosphorylation-dependent reactions. To this end, we assessed the effect of calyculin A, a phosphatase inhibitor, on the activity of the aforementioned exchangers. We found that exposure of Amphiuma RBCs to calyculin-A in isotonic media results in simultaneous, 1-2 orders of magnitude increase in the activity of both K(+)/H(+) and Na(+)/H(+) exchangers. We also demonstrate that, in isotonic media, calyculin A-dependent increases in net Na(+) uptake and K(+) loss are a direct result of phosphatase inhibition and are not dependent on changes in cell volume. Whereas calyculin A exposure in the absence of volume changes results in stimulation of both the Na(+)/H(+) and K(+)/H(+) exchangers, superimposing cell swelling or shrinkage and calyculin A treatment results in selective activation of K(+)/H(+) or Na(+)/H(+) exchange, respectively. We conclude that kinase-dependent reactions are responsible for Na(+)/H(+) and K(+)/H(+) exchange activity, whereas undefined volume-dependent reactions confer specificity and coordinated control.

NHE1 inhibition by amiloride- and benzoylguanidine-type compounds. Inhibitor binding loci deduced from chimeras of NHE1 homologues with endogenous differences in inhibitor sensitivity.

The interaction of the ubiquitous Na(+)/H(+) exchanger, NHE1, with its commonly used inhibitors, amiloride- and benzoylguanidine (Hoechst type inhibitor (HOE))-type compounds, is incompletely understood. We previously cloned NHE1 from Amphiuma tridactylum (AtNHE1) and Pleuronectes americanus (PaNHE1). Although highly homologous to the amiloride- and HOE-sensitive human NHE1 (hNHE1), AtNHE1 is insensitive to HOE-type and PaNHE1 to both amiloride- and HOE-type compounds. Here we generated chimeras to "knock in" amiloride and HOE sensitivity to PaNHE1, and we thereby identified several NHE1 regions involved in inhibitor interaction. The markedly different inhibitor sensitivities of hNHE1, AtNHE1, and PaNHE1 could not be accounted for by differences in transmembrane (TM) region 9. Replacing TM10 through the C-terminal tail of PaNHE1 with the corresponding region of AtNHE1 partially restored sensitivity to amiloride and the related compound 5'-(N-ethyl-N-isopropyl)amiloride (EIPA) but not to HOE694. This effect was not due to the tail region, but it was dependent on TM10-11, because replacing only this region with that of AtNHE1 also partially restored amiloride and EIPA but not HOE sensitivity. The converse mutant (TM10-11 of AtNHE1 replaced with those of PaNHE1) exhibited even higher amiloride and EIPA sensitivity and was also HOE-sensitive. Replacing an LFFFY motif in TM region 4 of PaNHE1 with the corresponding residues of hNHE1 (VFFLF) or AtNHE1 (TFFLF) greatly increased sensitivity to both amiloride- and HOE-type compounds, despite the fact that AtNHE1 is HOE694-insensitive. Gain of amiloride sensitivity appeared to correlate with increased Na(+)/H(+) exchange rates. It is concluded that regions within TM4 and TM10-11 contribute to amiloride and HOE sensitivity, with both regions imparting partial inhibitor sensitivity to NHE1.

Regulation of the Pleuronectes americanus Na+/H+ exchanger by osmotic shrinkage, beta-adrenergic stimuli, and inhibition of Ser/Thr protein phosphatases.

The ubiquitous Na+/H+ exchanger NHE1 is regulated by protein phosphorylation events, but the mechanisms involved are incompletely understood. We recently cloned NHE1 from the red blood cells of the winter flounder, Pleuronectes americanus (paNHE1), and demonstrated its activation by osmotic cell shrinkage, beta-adrenergic stimuli, and the Ser/Thr protein phosphatase PP1 and PP2A inhibitor calyculin A(CLA) (Pedersen et al. [2003] Am. J. Physiol. 284, C1561-C1576). Here, we investigate the mechanisms involved in paNHE1 activation by these stimuli. Osmotic shrinkage and CLA were only partially additive in their effects on paNHE1 activity, and CLA-mediated paNHE1 activation was inhibited by osmotic cell swelling. Activation by the beta-adrenergic agonist isoproterenol (IP) was fully additive to activation by osmotic shrinkage or CLA. IP-mediated, but neither shrinkage- nor CLA-mediated paNHE1 activation were associated with an increase in cellular cyclic adenosine monophosphate (cAMP) level. IP-mediated activation was partially blocked by the protein kinase A (PKA) inhibitor H89 (10 microM), whereas shrinkage- and CLA-mediated activation were unaffected. All three stimuli activated paNHE1 in a manner unaffected by inhibitors of protein kinase C (calphostin C, 5 microM) and protein kinase G (KT5823, 10 microM) as well as of myosin light chain kinase (ML-7, 10 microM). IP-mediated, but not shrinkage-mediated, paNHE1 activation was associated with an increase in serine phosphorylation of the paNHE1 protein. It is suggested that paNHE1 activation by osmotic shrinkage and by PP1/PP2A inhibition involves partially convergent signaling pathways, whereas activation of paNHE1 by beta-adrenergic stimuli is mediated by a separate pathway.

Effects of cold cardioplegia on pH, Na, and Ca in newborn rabbit hearts.

Many studies suggest myocardial ischemia-reperfusion (I/R) injury results largely from cytosolic proton (H(i))-stimulated increases in cytosolic Na (Na(i)), which cause Na/Ca exchange-mediated increases in cytosolic Ca concentration ([Ca]i). Because cold, crystalloid cardioplegia (CCC) limits [H]i, we tested the hypothesis that in newborn hearts, CCC diminishes H(i), Na(i), and Ca(i) accumulation during I/R to limit injury. NMR measured intracellular pH (pH(i)), Na(i), [Ca]i, and ATP in isolated Langendorff-perfused newborn rabbit hearts. The control ischemia protocol was 30 min for baseline perfusion, 40 min for global ischemia, and 40 min for reperfusion, all at 37 degrees C. CCC protocols were the same, except that ice-cold CCC was infused for 5 min before ischemia and heart temperature was lowered to 12 degrees C during ischemia. Normal potassium CCC solution (NKCCC) was identical to the control perfusate, except for temperature; the high potassium (HKCCC) was identical to NKCCC, except that an additional 11 mmol/l KCl was substituted isosmotically for NaCl. NKCCC and HKCCC were not significantly different for any measurement. The following were different (P < 0.05). End-ischemia pH(i) was higher in the CCC than in the control group. Similarly, CCC limited increases in Na(i) during I/R. End-ischemia Na(i) values (in meq/kg dry wt) were 115 +/- 16 in the control group, 49 +/- 13 in the NKCCC group, and 37 +/- 12 in the HKCCC group. CCC also improved [Ca]i recovery during reperfusion. After 40 min of reperfusion, [Ca](i) values (in nmol/l) were 302 +/- 50 in the control group, 145 +/- 13 in the NKCCC group, and 182 +/- 19 in the HKCCC group. CCC limited ATP depletion during ischemia and improved recovery of ATP and left ventricular developed pressure and decreased creatine kinase release during reperfusion. Surprisingly, CCC did not significantly limit [Ca]i during ischemia. The latter is explained as the result of Ca release from intracellular buffers on cooling.

Acute effects of 17beta-estradiol on myocardial pH, Na+, and Ca2+ and ischemia-reperfusion injury.

Evidence suggests that 1) ischemia-reperfusion injury is due largely to cytosolic Ca(2+) accumulation resulting from functional coupling of Na(+)/Ca(2+) exchange (NCE) with stimulated Na(+)/H(+) exchange (NHE1) and 2) 17beta-estradiol (E2) stimulates release of NO, which inhibits NHE1. Thus we tested the hypothesis that acute E2 limits myocardial Na(+) and therefore Ca(2+) accumulation, thereby limiting ischemia-reperfusion injury. NMR was used to measure cytosolic pH (pH(i)), Na(+) (Na(i)(+)), and calcium concentration ([Ca(2+)](i)) in Krebs-Henseleit (KH)-perfused hearts from ovariectomized rats (OVX). Left ventricular developed pressure (LVDP) and lactate dehydrogenase (LDH) release were also measured. Control ischemia-reperfusion was 20 min of baseline perfusion, 40 min of global ischemia, and 40 min of reperfusion. The E2 protocol was identical, except that 1 nM E2 was included in the perfusate before ischemia and during reperfusion. E2 significantly limited the changes in pH(i), Na(i)(+), and [Ca(2+)](i) during ischemia (P < 0.05). In control OVX vs. OVX+E2, pH(i) fell from 6.93 +/- 0.03 to 5.98 +/- 0.04 vs. 6.96 +/- 0.04 to 6.68 +/- 0.07; Na(i)(+) rose from 25 +/- 6 to 109 +/- 14 meq/kg dry wt vs. 25 +/- 1 to 76 +/- 3; [Ca(2+)](i) changed from 365 +/- 69 to 1,248 +/- 180 nM vs. 293 +/- 66 to 202 +/- 64 nM. E2 also improved recovery of LVDP and diminished release of LDH during reperfusion. Effects of E2 were diminished by 1 microM N(omega)-nitro-L-arginine methyl ester. Thus the data are consistent with the hypothesis. However, E2 limitation of increases in [Ca(2+)](i) is greater than can be accounted for by the thermodynamic effect of reduced Na(i)(+) accumulation on NCE.

Molecular cloning of NHE1 from winter flounder RBCs: activation by osmotic shrinkage, cAMP, and calyculin A.

In this report, we describe the cloning, cellular localization, and functional characteristics of Na(+)/H(+) exchanger 1 (NHE1) from red blood cells of the winter flounder Pseudopleuronectes americanus (paNHE1). The paNHE1 protein localizes primarily to the marginal band and exhibits a 74% similarity to the trout beta-NHE, and 65% to the human NHE1 (hNHE1). Functionally, paNHE1 shares characteristics of both beta-NHE and hNHE1 in that it is activated both by manipulations that increase cAMP and by cell shrinkage, respectively. In accordance, the paNHE1 protein exhibits both protein kinase A consensus sites as in beta-NHE and a region of high homology to that required for shrinkage-dependent activation of hNHE1. After shrinkage-dependent activation of paNHE1 and resulting activation of a Cl(-)/HCO(3)(-) exchanger, their parallel operation results in net uptake of NaCl and osmotically obliged water. Activation of paNHE1 by cAMP is at least additive to that elicited by osmotic shrinkage, suggesting that these stimuli regulate paNHE1 by distinct mechanisms. Finally, exposure to the serine/threonine phosphatase inhibitor calyculin A potently activates paNHE1, and this activation is also additive to that induced by shrinkage or cAMP.