(Hypertension. 1998;31:259.)
© 1998 American Heart Association, Inc.
Scientific Contributions |
Na+/H+ Exchange in Vascular Smooth Muscle Cells Is Controlled by GTP-Binding Proteins
From the Research Center, Centre Hospitalier Universitaire de Montréal-(CHUM), Montreal, Quebec, Canada (S.N.O., J.T., P.H.), and the Laboratory of Biomembranes, Faculty of Biology, Moscow State University, Moscow, Russia (S.N.O., S.L.A., N.I.P.)
Correspondence to Pavel Hamet, Research Center, CHUM, Pavillon Hôtel-Dieu, Laboratory of Molecular Medicine, 3850 St. Urbain St., Montréal, Québec H2W 1T8, Canada. E-mail hamet{at}ere.umontreal.ca
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Abstract |
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This study examines the involvement of GTP-binding proteins (Gps) in the regulation of Na+/H+ exchange and Ca2+ influx, which are increased in vascular smooth muscle cells from spontaneously hypertensive rats. Gp activity was modulated by fluoride, GTP
S, GDPßS, and antisense oligodeoxynucleotides complementary to conserved regions of the 
- and ß-subunits of Gps (-comm and ß-comm, respectively). ß-Adrenergic-induced Gs-mediated cAMP production was used as a positive control to estimate the efficiency of these compounds. Na+/H+ exchange, measured as ethylisopropyl amiloride-sensitive 22Na influx, was activated by 5- to 6-fold by a 30-minute preincubation of cells with 10 mmol/L NaF with a K0.5 for NaF of _13 mmol/L. In contrast, no activation of 45Ca influx was observed under preincubation of vascular smooth muscle cells with NaF in Ca2+ -free medium, whereas at [Ca2+]o>0.5 mmol/L, simultaneous addition of 45Ca and 10 mmol/L NaF led to sharply increased isotope uptake. NaF-induced 45Ca influx did not reach saturation up to 3 mmol/L [Ca2+]0 and 20 mmol/L NaF and was correlated with the formation of calcium-fluoride complexes measured by light scattering. GTPS increased basal cAMP production and Na+/H+ exchange, whereas GDPßS decreased isoproterenol-induced cAMP production and Na+/H+ exchange, -comm reduced whereas ß-comm augmented isoproterenol-induced cAMP production by 70%. Both oligodeoxynucleotides decreased basal Na+/H+ exchange by 40% to 50%. NaF-induced Na+/H+ exchange was not sensitive to -comm but was inhibited by 60% in ß-comm-loaded cells. Neither basal nor NaF-induced 45Ca uptake was affected by GTPS, GDPßS, and the oligodeoxynucleotides. Our results show that 45Ca uptake is activated by NaF in vascular smooth muscle cells by nonspecific accumulation of calcium-fluoride complexes and is not related to modification of Gps. On the contrary, the Na+/H+ exchanger is controlled by Gps, and Gp ß-subunits are involved in [Ca2+]0-independent activation of this carrier by NaF.
Key Words: vascular smooth muscle cells • Na+/II+ exchange • Ca2+ uptake • fluoride • antisense oligodeoxynucleotides • GTP-binding proteins
Abbreviations: -comm, ß-comm = antisense oligodeoxynucleotides complementary to the conserved part of DNA encoding - and ß-subunits of GTP-binding proteins • DMEM = Dulbecco’s modified Eagle’s medium • EIPA = ethylisopropyl amiloride • Gp = GTP-binding protein • LDH = lactate dehydrogenase • NHE-1 = ubiquitous form of Na+/H+ • ODN = oligodeoxynucleotides • SHR = spontaneously hypertensive rat • VSMC = vascular smooth muscle cells
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Introduction |
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In the last decade, several laboratories have reported heightened Na+/H+ exchange and Ca2+ influx in SHR VSMCs. Increased Na+/H+ exchange has also been observed in blood cells and immortalized lymphoblasts from patients with essential hypertension, suggesting that this intermediate phenotype is caused by intrinsic factors rather than by a hypertensive milieu that might influence the carrier in vivo.1–4 The molecular mechanisms of these ion transport abnormalities remain unclear. Thus, neither Northern blot5,6 nor Western blot7 analysis has revealed overexpression of NHE-1 in SHR VSMCs. Using single-stranded conformational polymorphism analysis of cDNA from SHR and stroke-prone SHR VSMCs, we also did not find any mutation in the NHE-1 coding sequence (unpublished data). These observations are in agreement with the negative results of linkage analysis in affected sib-pairs, arguing against mutation of the NHE-1 in essential hypertension8 and suggesting that increased Na+/H+ exchanger activity in primary hypertension is caused by its enhanced turnover number due to abnormalities of intracellular signaling pathways involved in the regulation of this carrier.
Earlier we had shown that NaF drastically increases the rate of 45Ca influx and EIPA-inhibitable 22Na entry into VSMCs and that this effect is potentiated by the addition of AlCl3.9 It is well documented that fluoride anions activate Gps via the formation of AIF4- complexes whose conformation resembles that of the terminal phosphate group in GTP.10,11 Data obtained in several laboratories indicate that Gp function is altered in SHR VSMCs.12–14 These results suggest that Gps could be involved in the abnormal functioning of Na+/H+ exchange and Ca2+ transport pathways via the cytosolic signaling cascade and modulation of the activity of protein kinases or directly by the interaction of Gp subunits with ion transporters. The latter hypothesis is supported by numerous data on the regulation of permeability of ion channels by purified Gp subunits.15,16 To test the involvement of Gp in the regulation of Na+/H+ exchange and Ca2+ influx in VSMCs, we studied the kinetic characteristics of 22Na and 45Ca uptake modulated by NaF and compared the results with those obtained using nonhydrolyzable analogues of GTP and GDP as well as antisense ODNs complementary to the conserved regions of - and ß-subunits of Gp. The effects of these substances on the well-characterized signaling cascade of adenylate cyclase activation by ß-adrenoceptors served as a control. Our results provide evidence of the nonspecific character of increases in 45Ca uptake under treatment of cells with NaF and suggest Gp participation in the regulation of Na+/H+ exchanger in VSMCs.
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Materials and Methods |
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Cell Culture
VSMCs were obtained by explant methods from the aortas of 10- to 13-week-old male Brown Norway (BN.lx) (Institute of Biology, Faculty of Medicine, Charles University, Czech Republic) and Wistar-Kyoto rats (Cardiology Center of the Ministry of Public Health, Moscow, Russia) as described in detail previously.17 They were seeded and grown in DMEM with 10% calf serum (Gibco), 100 U/ml penicillin, and 100 µg/ml streptomycin. When they reached confluency in 7 to 10 days, they exhibited a hill-and-valley pattern, which is typical of smooth muscle cells in culture and reacted positively to specific smooth muscle myosin antibodies, as verified by fluorescence microscopy.18 The VSMCs were passaged by treatment with 0.05% trypsin (Gibco) in Ca2+- and Mg2+ -free Dulbecco’s phosphate-buffered saline and incubated in 80-cm2 tissue culture flasks at a density of 105 cells/ml. This investigation was performed on VSMCs after 12 to 16 passages. Before the experiments, the cells were plated in 12-well (22Na influx and cAMP content study) or in 24-well dishes (45Ca and 86Rb influx measurements) and allowed to grow in DMEM containing 10% calf serum for 20 to 24 hours. Then, to establish quiescence, they were incubated for 48 hours in DMEM containing 0.2% fetal calf serum. This medium was aspirated and changed with a physiologically balanced salt solution immediately before the experiments. Cell protein content was determined by a modified Lowry method.19 LDH activity was measured by spectrophotometric assay (Sigma Chemical Co).
Loading of VSMCs with GTPS, GDPßS, and ODN
To modulate Gp activity, we loaded VSMCs with nonhydrolyzable GTP and GDP analogues (GTPS and GDPßS, respectively) and with antisense or nonsense ODNs for - and ß-subunits of Gp under reversible permeabilization with streptolysin O.20 The sequence of -comm antisense ODN (5'-TCATYTGCTTCACAATGGT-3', where Y is T or C) corresponds to nucleotides 133 to 151 of the identical strand of the 02 gene sequence.21 The sequence of ß-comm ODN (5'-TTGCAGTTGAAGTCGTCRTA-3', where R is G or A) corresponds to nucleotides 825 to 844 of the identical strand of the ß1 gene sequence.22 -comm, ß-comm antisense and nonsense 20-mer ODN (5' -GTGATGTCGAACTATTGTGC-3') were synthesized using a Gene Assembler (Pharmacia). The cells were washed twice with 2 mL of medium A containing 130 mmol/L KCl, 8 mmol/L NaCl, 5 mmol/L glucose, 0.1 mmol/L EGTA, 1 mmol/L MgCl2, 1 mmol/L ATP Na2, 10 mmol/L HEPES-Tris (pH 7.4), and 0.15% bovine serum albumin and then incubated at room temperature in the same medium (vehicle) or in medium A containing 0.7 U/ml streptolysin O with or without 0.6 mmol/L GDPßS, GTPS, or 75 µmol/L ODNs. To estimate the efficiency of loading, GTP[35S] was added to part of the samples containing nonlabeled GTPS and streptolysin O. In 10 minutes, these media were aspirated, and the VSMCs were washed twice with DMEM containing 10% calf serum and used for the determination of ion fluxes, cAMP production, or GTP[35S] uptake. To study the effect of ODNs, after the first 10 minutes of permeabilization with ODNs, the VSMCs were washed twice with DMEM and incubated for 24 hours in the presence of 10% calf serum. The permeabilization procedure was repeated, and the cells incubated for 48 hours in DMEM containing 0.2% calf serum.
To estimate the effect of permeabilization on the properties of VSMCs, LDH release and passive permeability of intact and permeabilized cells to potassium were compared. As can be seen from Table 1, permeabilization with streptolysin O led to a 3-fold increase of GTP[35S] uptake. The intracellular concentration of GTPS, calculated as an increment of GTP[35S] uptake per VSMC water volume (3.85 µL/mg of protein),23 was increased up to 0.3 mmol/L in permeabilized cells. Neither passive permeability to K+ nor LDH release was affected by the permeabilization procedure (Table 1).
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45Ca Influx
VSMCs prepared in 24-well dishes were washed twice with 2-mL aliquots of 150 mmol/L NaCl, 10 mmol/L HEPES-Tris (pH 7.4) and then preincubated for 30 minutes in 0.5 mL of medium B containing 140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 5 mmol/L D-glucose, and 20 mmol/L HEPES-Tris (pH 7.4). In part of the experiments, the preincubation medium was also supplied with compounds mentioned in the figure and table legends. This medium was then aspirated, and 0.25 mL of medium B was added with different concentrations of CaCl2 and the compounds mentioned in the figure and table legends. 45Ca uptake was initiated by adding 0.25 mL of medium B with 2 µCi/ml 45CaCl2. Previously, it was demonstrated that the kinetics of 45Ca uptake by cultured VSMCs are linear up to 7 minutes.24 In NaF-treated VSMCs, these kinetics were linear up to 15 minutes (data not shown). In this study, 45Ca uptake was terminated after 5 minutes by adding 2 mL of ice-cold medium C containing 100 mmol/L MgCl2 and 10 mmol/L HEPES-Tris (pH 7.4). The VSMCs were washed four times with 2 mL of ice-cold medium C and lysed with 1 mL of a 1% sodium dodecyl sulfate/4 mmol/L EDTA mixture. 45Ca uptake (V, nmol mg protein-1 5 minutes-1) was determined as V = A/am, where A is radioactivity of the cell lysate (cpm), a is specific radioactivity of the incubation medium (cpm/nmol), and m is protein content of the cell lysate (mg).
22Na Influx
VSMCs were washed twice with 2-mL aliquots of 150 mmol/L NaCl, 10 mmol/L HEPES-Tris (pH 7.4) and preincubated for 30 minutes with 1 mL of medium B with the additions mentioned in the figure and table legends. This medium was then aspirated, and 0.5 mL of medium B containing 1 mmol/L ouabain and 20 µmol/L bumetanide was added. In part of the samples, this medium also contained 20 µmol/L EIPA and other additions listed in the figure and table legends. 22Na uptake was initiated by adding 0.5 mL of 150 mmol/L choline chloride with 4 µCi/ml 22NaCl. Previously, it was demonstrated that the kinetics of 22Na uptake by VSMCs are linear up to 5 minutes.23 In NaF-treated VSMCs, the kinetics were linear until 15 minutes (data not shown). In the present study, 22Na uptake was terminated after 5 minutes and calculated, using the same procedures as for 45Ca influx. The values of (ouabain+bumetanide)-resistant, EIPA-inhibited components of 22Na uptake were used as a measure of Na+/H+ exchange activity.
cAMP Production
Cells were washed twice with 2 mL of 150 mmol/L NaCl, 10 mmol/L HEPES-Tris (pH 7.4) and incubated in 1 mL of medium B. The other additions to the incubation medium are described in the table legends. After 1 hour of incubation, the medium was transferred into tubes containing 0.1 mL of 5.5 mmol/L 1-methyl-3-isobutylxantine and 110 mmol/L EDTA. Intracellular cAMP was extracted by treatment with 1 mL of 1 N perchloric acid. cAMP content was determined by radioimmunoassay as described earlier.25 The data in Tables 4 and 5 are presented as total (intracellular+extracellular) cAMP production.
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Light-Scattering Measurement
Three milliliters of medium was added in a standard cuvette (1 x 1 cm), and light scattering was measured at a right angle in a MPF-4 spectrofluorimeter (Hitachi) at an excitation and emission wavelength of 500 nm with 3-nm slits. All measurements were performed at 37°C with continuous stirring.
Chemicals
Isoproterenol bitartrate, bumetanide, deferoxamine, bovine serum albumin (fraction V, fatty acid-free), EGTA, streptolysin O were obtained from Sigma Chemical Co; GTPS and GDPßS were from Boehringer Mannheim GmBH; ouabain was from Aldrich Chemical Co; Ro 20-1724 (4-[3-butoxy-4-methoxybenzy1]-2-imidazolidinone) was from Gibco BRL; EIPA was from RBI; 45CaCl2, 22NaCl, and 86RbCl were from Amersham and Isotope; and GTP[35S] was from NEN. Salts, D-glucose and buffers were obtained from Sigma Chemical Co, Gibco BRL, Serva, and Anachemia.
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Results |
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Effect of NaF
Table 2 shows that preincubation of VSMCs in the presence of 20 mmol/L NaF resulted in a 3- to 4-fold increase of 45Ca influx. When NaF was added to the incubation medium 10 minutes before 45Ca addition, there was a 10-fold elevation of 45Ca uptake. A similar increment of 45Ca uptake was observed with simultaneous addition of NaF and isotope (data not shown). The decrease of [Ca2+]0 in the preincubation medium from 1 mmol/L to <1 µmol/L by the addition of 1.2 mmol/L EGTA did not modify 45Ca uptake measured at [Ca2+]0 = 1 mmol/L (data not shown) but completely abolished the effect of NaF on 45Ca (Table 2). These results suggest that potentiation of Ca2+ uptake by NaF was caused by accumulation of calcium-fluoride complexes rather than by accumulation of intracellular F- and subsequent activation of the Ca2+ transport system. To examine this hypothesis, we studied the dependence of 45Ca uptake by VSMCs on NaF and CaCl2 concentration and compared these results with the formation of calcium-fluoride complexes measured by light scattering of incubation media.
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Fig 1a shows that in the presence of 1 mmol/L CaCl2, 45Ca uptake by VSMCs was sharply increased at >5 mmol/L NaF and did not reach saturation up to 20 mmol/L. A similar dependence was observed earlier for 45Ca transport in endothelial cells.26,27 The addition of 10 mmol/L NaF to the incubation medium did not significantly affect 45Ca uptake at [Ca2+]0 <0.5 mmol/L. However, above this value, NaF-induced 45Ca uptake was increased sharply and reached 23.5±4.6 nmol (mg protein)-1 5 minutes-1 at [Ca2+]0= 3 mmol/L (Fig 1b). In both cases, dependence of 45Ca uptake was correlated with light scattering of media, suggesting that the NaF-induced increment of 45Ca uptake was caused by the formation of calcium-fluoride complexes.
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Because NaF evoked a sharp rise of Ca accumulation at [Ca2+]0 >0.5 mmol/L (Fig 1b), Na+/H+ exchange was studied in a Ca2+-free medium. As seen in Table 2, 1.2 mmol/L EGTA ([Ca2+]0 < 1 µmol/L) increased EIPA-resistant 22Na influx by 50% to 60% and had no effect on the EIPA-sensitive component (Na+/H+ exchange). A 30-minute preincubation of VSMCs with 10 mmol/L NaF did not change EIPA-insensitive 22Na influx but increased the rate of Na+/H+ exchange by 5-fold. When NaF was introduced directly into the incubation medium 5 minutes before measurement of 22Na entry at final concentration of 10 mmol/L, the Na+/H+ exchange rate was augmented 4-fold. Dependence of the Na+/H+ exchange rate on NaF concentration in the preincubation medium followed Michaelis-Menten kinetics (Fig 2a) with K0.5=13.3 mmol/L and Vmax=137 nmol (mg protein)-1 5 minutes-1 (Fig 2b).
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In the absence of NaF, neither AlCl3 nor deferoxamine, a chelator of polyvalent cations, affected the rate of Na+/H+ exchange (data not shown). In contrast, when these substances were added to the preincubation medium in combination with NaF, the introduction of AlCl3 increased the NaF-induced component by 30% to 40% whereas deferoxamine decreased it by a similar value (Table 3). Viewed collectively, these data suggest that potentiation of Na+/H+ exchange by NaF is mediated by penetration of F- in the cytoplasm and formation of AlF4- complexes mimicking the terminal phosphate group of GTP.
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Effect of GTPS and GDPßS
Cell permeabilization in the absence of nonhydrolyzable analogues of guanylnucleotides caused no change in basal or isoproterenol-induced cAMP production by VSMCs (Table 4). The addition of GTPS to permeabilized cells elevated basal cAMP production by 15-fold. Replacement of GTPS by GDPßS had no influence on basal levels of cAMP production compared with control values. Unlike basal cAMP, the isoproterenol-induced cAMP response was increased up to 2-fold by GTPS and decreased by 4-fold in VSMCs loaded with GDPßS (Table 4). The nonhydrolyzable analogues of guanylnucleotides had no effect on the rate of 45Ca uptake by VSMCs (Table 4). Unlike 45Ca uptake, the rate of Na+/H+ exchange was increased by 50% and decreased by 2-fold under cell permeabilization in the presence of GTPS and GDPßS, respectively (Table 4). EIPA-insensitive 22Na influx was not affected by permeabilization and did not depend on the presence of nonhydrolyzed analogues of guanylnucleotides (data not shown).
Effect of -comm and ß-comm ODNs
Neither basal cAMP production nor the rate of 45Ca uptake was affected by loading of VSMC with antisense ODNs against the - and ß-subunits of G-proteins (-comm and ß-comm, respectively) (Table 5). In contrast, isoproterenol-induced cAMP production was decreased by 2-fold in VSMCs permeabilized in the presence of -comm and was increased by 70% to 80% in VSMCs loaded with ß-comm (P<.05). Loading of VSMCs with -comm and ß-comm reduced the rate of EIPA-insensitive 22Na influx by 50% to 40%. Neither cAMP production nor ion fluxes were affected by permeabilization of VSMCs in the presence of nonsense ODNs. The NaF-induced increment of Na+/H+ exchange was insensitive to nonsense and -comm ODN but was decreased up to 3-fold in ß-comm-treated VSMCs (Fig 3b). In contrast to Na+/H+ exchange, ODN did not affect NaF-induced 45Ca uptake by VSMC (Fig 3a).
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Discussion |
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The data obtained in the present study show that Na+/H+ exchange but not the activity of ion transporter(s) involved in Ca2+ uptake by VSMCs is under the control of Gps. Several lines of evidence support this conclusion. Thus, to activate Na+/H1 exchange, it was enough to preincubate cells with NaF in Ca2+ -free medium, whereas its subsequent introduction into the medium during measurement of 22Na influx had no additional influence (Table 2). Dependence of the Na+/H+ exchange rate on NaF concentration followed a saturation curve with half-maximal activation at 13 mmol/L NaF (Fig 2). The addition of AlCl3 to Ca2+-free medium potentiated the action of NaF on EIPA-inhibitable 22Na influx whereas chelation of endogenous Al3+ with deferoxamine decreased this component (Table 3). These results are in accordance with the hypothesis of activation of Na+/H+ exchange by NaF via AlF4- -induced dissociation of Gp heterotrimers.
Unlike Na+/H+ exchange, Ca influx was activated by preincubation of VSMCs with NaF in Ca2+ -containing medium only (Table 2). These results indicate that in contrast to Na+/H+ exchange, for activation of 45Ca influx, the presence of F- and Ca2+ in the incubation medium is sufficient. The extracellular effect of NaF on 45Ca uptake seems be associated with complex formation involving F-, Ca2+, and other polyvalent cations. The formation of these complexes was demonstrated directly by measuring the free Ca2+ concentration, giving Ksp=[Ca2+] x [F]2=0.3 mmol/L3 for monovalent iondepleted media.28,29 The involvement of calcium-fluoride complexes in NaF-induced Ca2+ uptake follows from a correlation of the dependence of 45Ca influx on NaF and CaCl2 concentration and complex formation measured in the same media by light scattering (Fig 1). This dependence took a hyperbolic shape that is also in accordance with the kinetics of complex formation at a concentration range close to the value of the solubility product. The measurement of light scattering demonstrated that both AlCl3 and deferoxamine modulated the formation of calcium-fluoride complexes (data not shown) which complicates the use of these compounds in studying the role of Gp in the regulation of Ca2+ uptake. To overcome this problem, we loaded VSMCs with nonhydrolyzable analogues of GTP and GDP and antisense ODNs for Gp subunits. Neither approach revealed an involvement of Gps in the regulation of Ca2+ uptake by VSMCs (Tables 4 and 5).
Unlike data on the activation of 45Ca influx by NaF observed in all types of nucleated cells studied so far, those on activation of amiloride-sensitive 22Na influx, apart from VSMC,9 are limited to fibroblasts,30 enteric endocrine cells,31 and L-6 myoblasts.9 In thymocytes32 and platelets,33 NaF did not change the Na+/H+ exchange rate while in renal brush border membrane vesicles34 an opposite inhibitory effect was noted, indicating a specific regulatory mechanism. To further evaluate the role of Gps in regulation of the activity of Na+/H+ exchanger in VSMCs, we treated cells with nonhydrolyzable guanine nucleotides. VSMC permeabilization in the presence of GTPS resulted in the activation of basal cAMP production and Na+/H+ exchange, whereas GDPßS decreased both the isoproterenol-induced component of cAMP production and Na+/H+ exchange (Table 4). Data on the modulation of cAMP production by nonhydrolyzable guanine nucleotides suggest that these compounds affected VSMC Na+/H+ exchange via modulation of Gp activity. Previously, it was shown that the nonhydrolyzed analogue of GTP caged guanosine-5'-(3-thiophosphate)-3-S-(4,5-dimethoxy-2-nitrobenzyl) thioester, loaded into enteric endocrine cells by reversible hypotonic shock, activated Na+/H+ exchange light dependently,31 also in accordance with the hypothesis of regulation of this carrier by Gp subunits. (We tested this method of VSMC permeabilization. Unlike streptolysin O (Table 1), this method produced a 2- to 3-fold increase in passive permeability of membranes to potassium and a 30% to 40% decrease in intracellular LDH content [data not shown].)
To modulate the content of Gp subunits in VSMCs, we used -comm and ß-comm ODNs. As shown by Kleuss et al,35,36 -comm can be hybridized with mRNAs of all known rat variants of t, o, and s and with the -subunit of transducin, while ß-comm can hybridize with mRNAs of all known ß-subunits of Gps. Loading of streptolysin-treated VSMCs with -comm decreased the isoproterenol-induced increment of cAMP production by 2-fold, whereas ß-comm increased the cAMP response by 80% (Table 5). These results are in accordance with the well-documented role of the s-subunit in the activation of adenylate cyclase by ß-adrenoceptors37 and inactivation of this pathway by ß-subunit-dependent ß-adrenergic receptor kinase (ßADRK1).38 The augmented isoproterenol-induced cAMP production in ß-comm-treated VSMCs may also be due to increased availability of s for activation of adenylate-cyclase.
Based on these results, we applied ODNs to study the involvement of Gps in the regulation of Na+/H+ exchange by NaF. Both -comm and ß-comm decreased basal Na+/H+ exchange by 40% to 50% (Table 5). These results suggest that both - and ß- subunits of Gps are involved in the regulation of the basal activity of VSMC Na+/H+ exchange. This conclusion is supported by recent data on this activation of NHE-1 by 13 subunit39 and by transducin ß-dimer40 in the human embryonic kidney cell line and in Xenopus oocytes, respectively. In contrast to basal Na+/H+ exchange, the NaF-induced increment of carrier activity was drastically inhibited by ß-comm only (Fig 3b), thus indicating that NaF mainly stimulates this transporter via an increase of the content of activated ß-dimer.
In conclusion, our results show that VSMC Na+/H+ exchange is under the control of Gps and its activation by NaF is mediated via the Gp ß-subunit. Recently, it was reported that the transient expression of mutationally-activated 9, 12, and 13-subunits of Gps stimulates Na+/H+ exchange in HEK29339 and COS-141 cells. Stimulation of Na+/H+ exchange in these cells by 9 and 12 was completely abolished after downregulation of protein kinase C whereas the effect of 13 was protein kinase C independent. It should be also underlined that the relative contribution of Gp subunits in the regulation of Na+/H+ exchanger seems to be dependent on the cellular milieu. Thus, in COS-1 cells both 12 and 13 increased the basal activity of Na+/H+ exchanger.41 In contrast, in HEK293 cells and in CCL39 fibroblasts 12 did not modify basal activity of the carrier but abolished its activation by serum and 13.42 Further studies should clarify the relative contribution of different isoforms of Gps as well as protein kinase-mediated and direct membrane-delimited signal transduction pathways in the regulation of the activity of Na+/H+ exchanger in VSMCs. Both the ODN approach and the search for quantitative trait loci using F2 hybrids of normotensive and hypertensive rats can be used to study the role of Gps in the enhanced activity of VSMC Na+/H+ exchanger in primary hypertension.
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Acknowledgments |
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This work was supported by grants from the Medical Research Council of Canada (P.H.), the Heart and Stroke Foundation of Canada (S.O.), Servier Pharmacological Company (P.H.), Pfizer Canada (P.H., S.O.), and the Russian Foundation for Fundamental Research (S.O.), S.O. was the recipient of the Pfizer Award from the International Society of Hypertension. The technical assistance of Monique Poirier and Regis Tremblay, the secretarial help of Josée Bédard-Baker, and the editorial assistance of Ovid Da Silva are greatly appreciated.
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