Gergely Kovacs

Surface Expression of ASIC2 Inhibits the Amiloride-sensitive Current and Migration of Glioma Cells

Gliomas are primary brain tumors with a complex biology characterized by antigenic and genomic heterogeneity and a propensity for invasion into normal brain tissue. High grade glioma cells possess a voltage-independent, amiloride-inhibitable, inward Na+ current. This current does not exist in normal astrocytes or low grade tumor cells. Inhibition of this conductance decreases glioma growth and cell migration making it a potential therapeutic target. Our previous results have shown that the acid-sensing ion channels (ASICs), members of the epithelial Na+ channel (ENaC)/degenerin (DEG) family of ion channels are part of this current pathway. We hypothesized that one member of the ENaC/DEG family, ASIC2, is retained intracellularly and that it is the lack of functional expression of ASIC2 at the cell surface that results in hyperactivity of this conductance in high grade gliomas. In this study we show that the chemical chaperone, glycerol, and the transcriptional regulator, sodium 4-phenylbutyrate, inhibit the constitutively activated inward current and reduce cell growth and migration in glioblastoma multiforme. The results suggest that these compounds induce the movement of ASIC2 to the plasma membrane, and once there, the basally active inward current characteristic of glioma cells is abolished by inherent negative regulatory mechanisms. This in turn compromises the ability of the glioma cell to migrate and proliferate. These results support the hypothesis that the conductance pathway in high grade glioma cells is comprised of ENaC/DEG subunits and that abolishing this channel activity promotes a reversion of a high grade glioma cell to a phenotype resembling that of normal astrocytes.

Na+/Ca2+ Exchanger Target for Oxidative Stress in Salt-Sensitive Hypertension

Abstract—The Na+/Ca2+ exchanger regulates intracellular calcium ([Ca2+]i), and attenuation of Na+/Ca2+ exchange by oxidative stress might lead to dysregulation of [Ca2+]i. We have shown that the Na+/Ca2+ exchanger differs functionally and at the amino acid level between salt-sensitive and salt-resistant rats. Therefore, the purpose of these studies was to determine how oxidative stress affects the activities of the 2 Na+/Ca2+ exchangers that we cloned from mesangial cells of salt-resistant (RNCX) and salt-sensitive (SNCX) Dahl/Rapp rats. The effects of oxidative stress on exchanger activity were examined in cells expressing RNCX or SNCX by assessing 45Ca2+ uptake (reverse mode) and [Ca2+]i elevation (forward mode) in the presence and absence of H2O2 and peroxynitrite. Our results showed that 45Ca2+ uptake in SNCX cells was attenuated at 500 and 750 &mgr;mol/L H2O2 (63±12% and 25±7%, respectively; n=16) and at 50 and 100 &mgr;mol/L peroxynitrite (47±9% and 22±9%, respectively; n=16). In RNCX cells, 45Ca2+ uptake was attenuated at only 750 and 100 &mgr;mol/L H2O2 and peroxynitrite (61±9% and 63±6%, respectively; n=16). In addition, the elevation in [Ca2+]i was greater in SNCX cells than in RNCX cells in response to 750 &mgr;mol/L H2O2 (58±5.5 vs 17±4.1 nmol/L; n=13) and 100 &mgr;mol/L peroxynitrite (33±5 vs 11±6 nmol/L; n=19). The enhanced impairment of SNCX activity by oxidative stress might contribute to the dysregulation of [Ca2+]i that is found in this model of salt-sensitive hypertension.The Na + /Ca 2+ exchanger regulates intracellular calcium ([Ca 2+ ] i ), and attenuation of Na + /Ca 2+ exchange by oxidative stress might lead to dysregulation of [Ca 2+ ] i . We have shown that the Na + /Ca 2+ exchanger differs functionally and at the amino acid level between salt-sensitive and salt-resistant rats. Therefore, the purpose of these studies was to determine how oxidative stress affects the activities of the 2 Na + /Ca 2+ exchangers that we cloned from mesangial cells of salt-resistant (RNCX) and salt-sensitive (SNCX) Dahl/Rapp rats. The effects of oxidative stress on exchanger activity were examined in cells expressing RNCX or SNCX by assessing 45 Ca 2+ uptake (reverse mode) and [Ca 2+ ] i elevation (forward mode) in the presence and absence of H 2 O 2 and peroxynitrite. Our results showed that 45 Ca 2+ uptake in SNCX cells was attenuated at 500 and 750 μmol/L H 2 O 2 (63±12% and 25±7%, respectively; n=16) and at 50 and 100 μmol/L peroxynitrite (47±9% and 22±9%, respectively; n=16). In RNCX cells, 45 Ca 2+ uptake was attenuated at only 750 and 100 μmol/L H 2 O 2 and peroxynitrite (61±9% and 63±6%, respectively; n=16). In addition, the elevation in [Ca 2+ ] i was greater in SNCX cells than in RNCX cells in response to 750 μmol/L H 2 O 2 (58±5.5 vs 17±4.1 nmol/L; n=13) and 100 μmol/L peroxynitrite (33±5 vs 11±6 nmol/L; n=19). The enhanced impairment of SNCX activity by oxidative stress might contribute to the dysregulation of [Ca 2+ ] i that is found in this model of salt-sensitive hypertension.

Synthesis, maturation and trafficking of human Na+-dicarboxylate cotransporter NaDC1 requires the chaperone activity of cyclophilin B

Renal excretion of citrate, an inhibitor of calcium stone formation, is controlled mainly by reabsorption via the apical Na+-dicarboxylate cotransporter NaDC1 (SLC13A2) in the proximal tubule. Recently, it has been shown that the protein phosphatase calcineurin inhibitors cyclosporin A (CsA) and FK-506 induce hypocitraturia, a risk factor for nephrolithiasis in kidney transplant patients, but apparently through urine acidification. This suggests that these agents up-regulate NaDC1 activity. Using the Xenopus lævis oocyte and HEK293 cell expression systems, we examined first the effect of both anti-calcineurins on NaDC1 activity and expression. While FK-506 had no effect, CsA reduced NaDC1-mediated citrate transport by lowering heterologous carrier expression (as well as endogenous carrier expression in HEK293 cells), indicating that calcineurin is not involved. Given that CsA also binds specifically to cyclophilins, we determined next whether such proteins could account for the observed changes by examining the effect of selected cyclophilin wild types and mutants on NaDC1 activity and cyclophilin-specific siRNA. Interestingly, our data show that the cyclophilin isoform B is likely responsible for down-regulation of carrier expression by CsA and that it does so via its chaperone activity on NaDC1 (by direct interaction) rather than its rotamase activity. We have thus identified for the first time a regulatory partner for NaDC1, and have gained novel mechanistic insight into the effect of CsA on renal citrate transport and kidney stone disease, as well as into the regulation of membrane transporters in general.

Amyloid Beta Peptide 1-40 Stimulates the Na+ / Ca2+ Exchange Activity of SNCX

The Na+/Ca2+ exchangers, RNCX and SNCX, were cloned from mesangial cells of salt sensitive and salt resistant Dahl/Rapp rats, respectively, and differ at amino acid 218 (RNCXi/SNCXf) and in the exons expressed at the alternative splice site (RNCXB, D/SNCXB, D, F). These isoforms are also expressed in myocytes, neurons, and astrocytes where they maintain cytosolic calcium homeostasis. We demonstrated that cells expressing SNCX were more susceptible to oxidative stress than cells expressing RNCX. Others demonstrated that amyloid beta peptide (Abeta) augments the adverse effects of oxidative stress on calcium homeostasis. Therefore, we sought to assess the effect of Abeta 1-40 on the abilities of OK-PTH cells stably expressing RNCX and SNCX and human glioma cells, SKMG1, to regulate cytosolic calcium homeostasis. Our studies showed that Abeta 1-40 (1 microM) did not affect RNCX activity, as assessed by changes in [Ca2+]i (Delta[Ca2+]i, 260+/-10 nM to 267+/-8 nM), while stimulating exchange activity 2.4 and 3 fold in cells expressing SNCX (100+/-8 to 244+/-12 nM) and in SKMG1 cells (90+/-11 nM to 270+/-18 nM), respectively. Our results also showed that Abeta 1-40, while not affecting the rate of Mn2+ influx in cells expressing RNCX, stimulated the rate of Mn2+ influx 2.8 and 2.9 fold in cells expressing SNCX and in SKMG1 cells. Thus, our studies demonstrate that Abeta-induced cytosolic calcium increase is mediated through certain isoforms of the Na+/Ca2+ exchanger and reveals a possible mechanism by which Abeta 1-40 can alter cytosolic calcium homeostasis.

Regulation of mesangial cell Na+/Ca2+ exchanger isoforms.

An isoform of the Na+/Ca2+ exchanger (SDNCX1.10) was cloned from mesangial cells of Sprague–Dawley rat. Regulation of this isoform was compared to two other clones that were derived from the Dahl/Rapp salt sensitive (SNCX) and salt resistant rat (RNCX). All isoforms differ at the alternative splice site and at amino acid 218 for SNCX. PKC activates RNCX but not SNCX while SDNCX1.10 was also activated by PKC. Regulation of exchanger activities by intracellular calcium ([Ca2+]i), pH, and kinases was assessed using Na‐dependent 45Ca2+ uptake assays in OK‐PTH cells expressing the vector, RNCX, SNCX, or SDNCX1.10. [Ca2+]i was elevated from 50 to 125 nM (n = 4) with thapsigargin (40 nM) and reduced from 50 to 29 nM (n = 4) and 18 nM (n = 4) with 10 or 20 μM BAPTA, respectively. RNCX was active at all three [Ca2+]i while SNCX and SDNCX1.10 were only active at lower [Ca2+]i. Varying extracellular pH (pHe, without nigericin) or pHe and intracellular pH (pHi, with 10 μM nigericin) from pH 7.4 to 6.2, 6.8, or 8.0 showed that SNCX activity was attenuated at both low and high pHs. SDNCX1.10 activity was attenuated only at pH 6.2 and 6.8 (with or without nigericin) while RNCX activity was attenuated at pH 6.2 (with or without nigericin) and pH 6.8 (with nigericin). Finally, only SDNCX1.10 activity was stimulated by 250 μM CPT‐cAMP or 250 μM DB‐cGMP treatment. Thus the differential regulation of [Ca2+]i by these exchangers is dependent upon the pattern of cellular Na+/Ca2+ exchanger isoform expression. J. Cell. Physiol. 199: 181–193, 2004© 2003 Wiley‐Liss, Inc.