Malignant gliomas display altered plasma membrane potential and pH regulation--interaction with Tc-99m-MIBI and Tc-99m-Tetrofosmin uptakes

Two radiopharmaceuticals, Tc-99m-MIBI (MIBI) and Tc-99m-Tetrofosmin (Tfos), are currently used for in vivo non-invasive monitoring of the MultiDrug Resistant (MDR) status of tumours. As gliomas are highly multidrug resistant, it is expected that the tracers would be poorly retained in those cells, but the in vivo and in vitro studies to date have shown that Tfos was highly retained in malignant gliomas. The high degree of malignancy of tumour cells is linked to alterations of physiological parameters as plasma membrane potential and intracellular pH. In order to elucidate the contribution of those parameters to Tfos and MIBI uptakes in malignant gliomas, we used several glioma cell lines--G111, G5, G152, and 42 MG-BA. These cells showed to be chemoresistant with a high level of expression and activity of the Multidrug Resistant associated Protein 1 (MRP1). They also had an alkaline intracellular pH (pHi) related to the Na+/H+ antiporter (NHE-1) expression and depolarised plasma membranes (-45 to -55 mV). In spite of their chemoresistance, we have found a high accumulation of both radiotracers in gliomas, more important for Tfos than MIBI, related to the presence and activity of NHE-1. In conjunction, the uptakes of the tracers were only partially dependent upon the plasma membrane potential of the glioma cell lines, again Tfos uptake being less dependent on this parameter than MIBI uptake. In conclusion, the evidence accumulated in this study suggests that Tfos could be a suitable glioma marker in vivo.     

NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells

Na(+)/H(+)-exchangers (NHE) mediate acid extrusion from duodenal epithelial cells, but the isoforms involved have not previously been determined. Thus we investigated 1) the contribution of Na(+)-dependent processes to acid extrusion, 2) sensitivity to Na(+)/H(+) exchange inhibitors, and 3) molecular expression of NHE isoforms. By fluorescence spectroscopy the recovery of intracellular pH (pH(i)) was measured on suspensions of isolated acidified murine duodenal epithelial cells loaded with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Expression of NHE isoforms was studied by RT-PCR and Western blot analysis. Reduction of extracellular Na(+) concentration ([Na(+)](o)) during pH(i) recovery decreased H(+) efflux to minimally 12.5% of control with a relatively high apparent Michaelis constant for extracellular Na(+). The Na(+)/H(+) exchange inhibitors ethylisopropylamiloride and amiloride inhibited H(+) efflux maximally by 57 and 80%, respectively. NHE1, NHE2, and NHE3 were expressed at the mRNA level (RT-PCR) as well as at the protein level (Western blot analysis). On the basis of the effects of low [Na(+)](o) and inhibitors we propose that acid extrusion in duodenal epithelial cells involves Na(+)/H(+) exchange by isoforms NHE1, NHE2, and NHE3.     

Critical role for NHE1 in intracellular pH regulation in pancreatic acinar cells

The primary function of pancreatic acinar cells is to secrete digestive enzymes together with a NaCl-rich primary fluid which is later greatly supplemented and modified by the pancreatic duct. A Na+/H+ exchanger(s) [NHE(s)] is proposed to be integral in the process of fluid secretion both in terms of the transcellular flux of Na+ and intracellular pH (pHi) regulation. Multiple NHE isoforms have been identified in pancreatic tissue, but little is known about their individual functions in acinar cells. The Na+/H+ exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride completely blocked pHi recovery after an NH4Cl-induced acid challenge, confirming a general role for NHE in pHi regulation. The targeted disruption of the Nhe1 gene also completely abolished pHi recovery from an acid load in pancreatic acini in both HCO3--containing and HCO3--free solutions. In contrast, the disruption of either Nhe2 or Nhe3 had no effect on pHi recovery. In addition, NHE1 activity was upregulated in response to muscarinic stimulation in wild-type mice but not in NHE1-deficient mice. Fluctuations in pHi could potentially have major effects on Ca2+ signaling following secretagogue stimulation; however, the targeted disruption of Nhe1 was found to have no significant effect on intracellular Ca2+ homeostasis. These data demonstrate that NHE1 is the major regulator of pHi in both resting and muscarinic agonist-stimulated pancreatic acinar cells.     

Crystal structure of CHP2 complexed with NHE1-cytosolic region and an implication for pH regulation

The plasma membrane Na+/H+ exchangers (NHE) require calcineurin B homologous protein (CHP) as an obligatory binding partner for ion transport. Here, we report the first crystal structure of CHP (CHP2 isoform) in complex with its binding domain in NHE1. We show that the cytoplasmic alpha-helix of NHE1 is inserted into the hydrophobic cleft formed by N- and C-lobes of CHP2 and that the size and shape of this crevice together with hydrogen bond formation at multiple positions assure a high degree of specificity for interaction with NHE members. Structure-based mutagenesis revealed the importance of hydrophobic interactions between CHP/NHE1 for the function of NHE1. Furthermore, the crystal structure shows the existence of a protruding CHP-unique region, and deletion of this region in CHP2 inhibited the NHE1 activity by inducing the acidic shift of intracellular pH dependence, while preserving interaction with NHE1. These findings suggest that CHP serves as an obligatory subunit that is required both for supporting the basic activity and regulating the pH-sensing of NHE1 via interactions between distinct parts of these proteins.     

Functional importance of charged residues within the putative intracellular loops in pH regulation by Na+/ H+ exchanger NHE1

The plasma membrane Na+/H+ exchanger 1 is activated in response to various extrinsic factors, and this process is regulated by an intracellular pH-sensing mechanism. To identify the candidate residues responsible for intracellular pH regulation, we analyzed the functional properties of engineered Na+/H+ exchanger 1 mutants with charge-reversal mutations of charged residues located in the intracellular loops. Na+/H+ exchanger 1 mutants with mutations at 11 positions were well expressed in the plasma membrane, but that with E247R was not, suggesting that Glu247 is important for the functional expression of Na+/H+ exchanger 1. Charge-reversal mutations of Glu131 (E131R, E131K) and Arg327 (R327E) resulted in a shift in the intracellular pH dependence of the exchange activity measured by 22Na+ uptake to the acidic side, and it abolished the response to growth factors and a hyperosmotic medium; however, mutations of Asp448 (D448R) and Arg500 (R500E) slightly shifted it to the alkaline side. In E131R, in addition to the change in intracellular pH dependence, the affinities for extracellular Na+, Li+ and the inhibitor 5-(N-ethyl-N-isopropyl)amiloride significantly increased. Furthermore, charge-conserved mutation of E131 (E131D) was found to have no effect, whereas charge neutralization (E131Q) resulted in a slight acidic shift of exchange. These results support the view that the multiple charged residues identified in this study, along with several basic residues reported previously, participate in the regulation of the intracellular pH sensing of Na+/H+ exchanger 1. In addition, Glu131 may also be important for cation transport.     

Hormonal regulation of chicken intestinal NHE and SGLT-1 activities

The effects of aldosterone and arginine vasotocin (AVT) on intestinal Na(+)/H(+) exchange (NHE) and Na(+)-sugar cotransport (SGLT-1) activities have been investigated using brush-border membrane vesicles isolated from Hubbard chicken small and large intestines, and they were compared with those induced by either Na(+) depletion or dehydration. Na(+) depletion was induced by feeding the chickens with either a low- or a high-Na(+) diet for either 0.5, 1, 2, 4, or 8 days. Ileal and colonic NHE2 activity increased with the duration of the Na(+) depletion, whereas that of intestinal SGLT-1 decreased, reaching a plateau after 2 days of treatment. Three-hour incubation of the intestine with aldosterone produced the same effects on NHE activity as does Na(+) depletion, without altering SGLT-1 activity. However, 3-h incubation of the intestine with AVT increased intestinal SGLT-1 activity, without affecting intestinal NHE activity. It is concluded that aldosterone regulates apical ileal and colonic NHE2 activity, whereas that of SGLT-1 is regulated by AVT.     

Mice lacking the dopamine transporter display altered regulation of distal colonic motility

The mechanisms by which dopamine (DA) influences gastrointestinal (GI) tract motility are incompletely understood and complicated by tissue- and species-specific differences in dopaminergic function. To improve the understanding of DA action on GI motility, we used an organ tissue bath system to characterize motor function of distal colonic smooth muscle segments from wild-type and DA transporter knockout (DAT -/-) mice. In wild-type mice, combined blockade of D(1) and D(2) receptors resulted in significant increases in tone (62 +/- 9%), amplitude of spontaneous phasic contractions (167 +/- 24%), and electric field stimulation (EFS)-induced (40 +/- 8%) contractions, suggesting that endogenous DA is inhibitory to mouse distal colonic motility. The amplitudes of spontaneous phasic and EFS-induced contractions were lower in DAT -/- mice relative to wild-type mice. These differences were eliminated by combined D(1) and D(2) receptor blockade, indicating that the inhibitory effects of DA on distal colonic motility are potentiated in DAT -/- mice. Motility index was decreased but spontaneous phasic contraction frequency was enhanced in DAT -/- mice relative to wild-type mice. The fact that spontaneous phasic and EFS-induced contractile activity were altered by the lack of the DA transporter suggests an important role for endogenous DA in modulating motility of mouse distal colon.     

Differential regulation of neuregulin 1 expression by progesterone in astrocytes and neurons

Glial-neuronal interactions are crucial processes in neuromodulation and synaptic plasticity. The neuregulin 1 family of growth and differentiation factors have been implicated as bidirectional signaling molecules that are involved in mediating some of these interactions. We have shown previously that neuregulin 1 expression is regulated by the gonadal hormones progesterone and 17beta-estradiol in the CNS, which might represent a novel, indirect mechanism of the neuromodulatory actions of these gonadal hormones. In the present study, we sought to determine the effects of progesterone and 17beta-estradiol on neuregulin 1 expression in rat cortical astrocytes and neurons in vitro. We observed that progesterone increased the expression of neuregulin 1 mRNA and protein in a dose-dependent manner in cultured astrocytes, which was blocked by the progesterone receptor antagonist RU-486. In contrast, 17beta-estradiol did not increase either neuregulin 1 mRNA or protein in astrocytes. We observed no effect of either progesterone or 17beta-estradiol on neuregulin 1 mRNA and protein in rat cortical neurons in vitro. Finally, we observed that treatment of cortical neurons with recombinant NRG1-beta1 caused PSD-95 to localize in puncta similar to that observed following treatment with astrocyte-conditioned medium. These results demonstrate that progesterone regulates neuregulin 1 expression, principally in astrocytes. This might represent a novel mechanism of progesterone-mediated modulation of neurotransmission through the regulation of astrocyte-derived neuregulin 1.     

NHE1 mediates MDA-MB-231 cells invasion through the regulation of MT1-MMP

Na⁺/H⁺ exchanger 1 (NHE1), an important regulator of intracellular pH (pH_i) and extracellular pH (pH_e), has been shown to play a key role in breast cancer metastasis. However, the exact mechanism by which NHE1 mediates breast cancer metastasis is not yet well known. We showed here that inhibition of NHE1 activity, with specific inhibitor Cariporide, could suppress MDA-MB-231 cells invasion as well as the activity and expression of MT1-MMP. Overexpression of MT1-MMP resulted in a distinguished increase in MDA-MB-231 cells invasiveness, but treatment with Cariporide reversed the MT1-MMP-mediated enhanced invasiveness. To explore the role of MAPK signaling pathways in NHE1-mediated breast cancer metastasis, we compared the difference of constitutively phosphorylated ERK1/2, p38 MAPK and JNK in non-invasive MCF-7 cells and invasive MDA-MB-231cells. Interestingly, we found that the phosphorylation levels of ERK1/2 and p38 MAPK in MDA-MB-231 cells were higher than in MCF-7 cells, but both MCF-7 cells and MDA-MB-231 cells expressed similar constitutively phosphorylated JNK. Treating MDA-MB-231 cells with Cariporide led to decreased phosphorylation level of both p38 MAPK and ERK1/2 in a time-dependent manner, but JNK activity was not influenced. Supplementation with MAPK inhibitor (MEK inhibitor PD98059, p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125) or Cariporide all exhibited significant depression of MDA-MB-231 cells invasion and MT1-MMP expression. Furthermore, we co-treated MDA-MB-231 cells with MAPK inhibitor and Cariporide. The result showed that Cariporide synergistically suppressed invasion and MT1-MMP expression with MEK inhibitor and p38 MAPK inhibitor, but not be synergistic with the JNK inhibitor. These findings suggest that NHE1 mediates MDA-MB-231 cells invasion partly through regulating MT1-MMP in ERK1/2 and p38 MAPK signaling pathways dependent manner.     

Role of astrocytes and altered regulation of spinal glutamatergic neurotransmission in stress-induced visceral hyperalgesia in rats

Glutamate (Glu) is the primary excitatory neurotransmitter in the central nervous system and plays a critical role in the neuroplasticity of nociceptive networks. We aimed to examine the role of spinal astroglia in the modulation of glutamatergic neurotransmission in a model of chronic psychological stress-induced visceral hyperalgesia in male Wistar rats. We assessed the effect of chronic stress on different glial Glu control mechanisms in the spinal cord including N-methyl-d-aspartate receptors (NMDARs), glial Glu transporters (GLT1 and GLAST), the Glu conversion enzyme glutamine synthetase (GS), and glial fibrillary acidic protein (GFAP). We also tested the effect of pharmacological inhibition of NMDAR activation, of extracellular Glu reuptake, and of astrocyte function on visceral nociceptive response in naive and stressed rats. We observed stress-induced decreased expression of spinal GLT1, GFAP, and GS, whereas GLAST expression was upregulated. Although visceral hyperalgesia was blocked by pharmacological inhibition of spinal NMDARs, we observed no stress effects on NMDAR subunit expression or phosphorylation. The glial modulating agent propentofylline blocked stress-induced visceral hyperalgesia, and blockade of GLT1 function in control rats resulted in enhanced visceral nociceptive response. These findings provide evidence for stress-induced modulation of glia-controlled spinal Glu-ergic neurotransmission and its involvement in chronic stress-induced visceral hyperalgesia. The findings reported in this study demonstrate a unique pattern of stress-induced changes in spinal Glu signaling and metabolism associated with enhanced responses to visceral distension.     

Acute regulation of Na/H exchanger NHE3 activity by protein kinase C: role of NHE3 phosphorylation

Acute hormonalmodulation of NHE3 activity is partly mediated by kinases, includingprotein kinase C (PKC). We examined the role of NHE3 phosphorylation inregulating its activity in response to PKC activation by phorbol12-myristate 13-acetate (PMA). In pooled NHE-deficient fibroblaststransfected with NHE3, PMA increased NHE3 activity and phosphorylation.When six potential PKC target serines were mutated, NHE3phosphorylation was drastically reduced and PMA failed to regulate NHE3phosphorylation or function. To examine whether NHE3 phosphorylation issufficient for functional regulation by PKC, we exploited theheterogeneous response of NHE3 activity to PMA in individual clones oftransfectants. Clones with stimulatory, inhibitory, or null responsesto PMA were observed. Despite the diverse functional response, changesin NHE3 phosphorylation as revealed by tryptic phosphopeptide maps weresimilar in all clones. We conclude that although phosphorylationappears to be necessary, it is insufficient to mediate PKC regulationof NHE3 function and factors extrinsic to the NHE3 protein must be involved.     

Acute regulation of Na+/H+ exchanger NHE3 by parathyroid hormone via NHE3 phosphorylation and dynamin-dependent endocytosis

Parathyroid hormone (PTH) is a potent inhibitor of mammalian renal proximal tubule Na(+) transport via its action on the apical membrane Na(+)/H(+) exchanger NHE3. In the opossum kidney cell line, inhibition of NHE3 activity was detected from 5 to 45 min after PTH addition. Increase in NHE3 phosphorylation on multiple serines was evident after 5 min of PTH, but decrease in surface NHE3 antigen was not detectable until after 30 min of PTH. The decrease in surface NHE3 antigen was due to increased NHE3 endocytosis. When endocytic trafficking was arrested with a dominant negative dynamin mutant (K44A), the early inhibition (5 min) of NHE3 activity by PTH was not affected, whereas the late inhibition (30 min) and decreased surface NHE3 antigen induced by PTH were abrogated. We conclude that PTH acutely inhibits NHE3 activity in a biphasic fashion by NHE3 phosphorylation followed by dynamin-dependent endocytosis.     

Cell cycle regulation by environmental pH

Purified populations of quiescent human tumour cells were isolated from plateau phase cultures of PMC-22 cells by centrifugal elutriation. Dilution into fresh medium resulted in these quiescent cells entering S phase exponentially with a t1/2 of 12 hr, after a 18-20-hr lag period during which cellular RNA content increased. Subsequent studies showed that recruitment of quiescent cells into the cell cycle could be regulated by extracellular pH. When exponentially growing PMC-22 cells were exposed to acidic extracellular pH levels, three growth patterns were observed: (1) Normal growth between pH 7.2 to pH 6.8; (2) A reduction in growth rate associated with accumulation of cells with a G₁ DNA content between pH 6.7 and 6.4 (this was also shown to occur in a number of other tumour cell lines); (3) Non-cell-cycle-phase-specific arrest of growth at pH levels less than 6.3. Further studies with purified quiescent cell populations showed the possible existence of a pH-dependent restriction point in the G₁ phase of these tumour cells. The implications of these observations to tumour biology are discussed.     

Proportion regulation in Dictyostelium is altered by oxygen

Abstract. Submerged cell aggregates of Dictyostelium form the anterior and posterior tissues seen in slugs, but the proportion of the tissues is altered [36]. The proportion of anterior tissue in such aggregates, about twice that found in normal slugs, is due to the elevated oxygen concentration used in submerged cultures. When the oxygen concentration in submerged cultures is reduced to levels similar to that encountered by slugs at an air-water interface, the percentage of anterior tissue is approximately halved. Conversely, raising the atmospheric oxygen concentration surrounding slugs from 21 % to 30% causes a doubling in the proportion of anterior cells. Higher oxygen concentrations cause no further increase. Changes in the pH of the medium, which occur when Dictyostelium is exposed to increased oxygen concentrations, are shown not to affect the proportion of anterior cells. As it is likely that oxygen affects tissue proportions through metabolism, it is suggested that the control of oxygen concentration may be useful in identifying proportion-regulating morphogens.