(Hypertension. 2000;35:1062.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
Inversion of the Intracellular Na+/K+ Ratio Blocks Apoptosis in Vascular Smooth Muscle Cells by Induction of RNA Synthesis
From Centre de Recherché du CHUM, Campus Hotel-Dieu, University of Montreal, Montreal, PQ, Canada (S.N.O., S.T., N.T.-T., J.T., P.H.), and the Department of Pharmacology, University of Illinois at Chicago (N.O.D.).
Correspondence to Dr S.N. Orlov, Laboratory of Pathophysiology of Ion Transport Disorders, Centre de Recherché du CHUM, Campus Hotel-Dieu, 3850 rue St-Urbain, Montreal, Quebec H2W 1T8, Canada. E-mail sergei.n.orlov{at}umontreal.ca
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Abstract |
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Abstract—This study examines the involvement of RNA and protein synthesis in the modulation of apoptosis in vascular smooth muscle cells (VSMC) by intracellular monovalent cations. In VSMC transfected with E1A adenovirus (VSMC-E1A), inversion of the [Na+]i/[K+]i ratio by an inhibitor of the Na+,K+ pump, ouabain, prevented the development of apoptosis triggered by serum withdrawal. Inhibition of apoptosis by ouabain was abolished by inhibitors of RNA and protein synthesis, actinomycin D, and cycloheximide, respectively. In VSMC-E1A, incubation with ouabain for 4 and 24 hours augmented RNA synthesis by 20% to 50% and 3-fold to 4-fold, respectively. In quiescent VSMC, the effect of ouabain and serum on RNA synthesis was additive. Ouabain did not affect the level of phosphorylation of ERK, JNK, and p38 MAP kinases and blocked apoptosis independent of the presence of the MAPK kinase inhibitors PD98059 and SB 202190. Equimolar substitution of NaCl with KCl in the incubation medium abolished the effect of ouabain on intracellular Na+ and K+ concentration, apoptosis, and RNA synthesis. Thus, our results demonstrate that the antiapoptotic effect of the inverted [Na+]i/[K+]i ratio is mediated by MAPK-independent induction of de novo synthesis of RNA species encoding inhibitor(s) of programmed cell death.
Key Words: muscle, smooth, vascular • apoptosis • potassium • sodium • RNA
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Introduction |
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Both vascular smooth muscle cell (VSMC) growth and programmed cell death contribute to the altered geometry of vessels in hypertension.1 2 3 Much is known about the pharmacological regulation of VSMC replication (for review see References 4 and 5 ), which is in contrast with the lack of systematic studies on the pharmacology of VSMC apoptosis. As in the majority of cells investigated so far, apoptosis of VSMC can be triggered by serum withdrawal2 6 or by treatment with antisense oligodeoxynucleotides against VSMC-derived growth factors.7 Apoptosis in serum-deprived VSMC is potentiated by overexpression of c-myc or its functional analogue, E1A adenovirus, and coexpression of p53 with c-myc/E1A and is inhibited by overexpression of bcl-2 (for recent review, see Reference 8 ). Several lines of evidence suggest that the antiapoptotic action of serum in VSMC is caused by the presence of growth factors such as IGF and EGF and is mainly mediated by receptor-coupled polyphosphoinositide-3-kinase.8 However, the intracellular mechanisms controlling the expression and functional activity of proapoptotic and antiapoptotic gene products in VSMC are poorly understood.
Recently, we reported that inversion of the intracellular [Na+]/[K+] ratio caused by sustained inhibition of the Na+,K+ pump with ouabain delays development of the apoptotic machinery in serum-deprived VSMC as well as in VSMC transfected with c-myc or E1A-adenovirus.9 This study examines whether inhibition of programmed cell death by ouabain is caused by de novo synthesis of inhibitors of apoptosis or by posttranslational modification of preexisting intermediates of the apoptotic machinery.
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Methods |
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Materials
VSMC were obtained by explant methods from the aortas of 10- to 13-week-old male Brown Norway (BN.lx) rats in accordance with previously described methods.1 VSMC transfected with E1A adenovirus (VSMC-E1A), prepared as outlined in detail elsewhere,6 10 were kindly provided by Dr M.R. Bennett (University of Cambridge, UK). Chromatin cleavage assay and caspase-3 activity were used for the measurement of apoptosis in accordance with previously described methods.9 11 12 RNA and protein synthesis were estimated by incorporation of [3H]-uridine and [3H]-leucine, respectively.13 14 The intracellular concentration of exchangeable Na+ and K+ was measured as the steady-state distribution of extracellular and intracellular 22Na and 86Rb, as described previously in detail.15 The volume of intracellular water was determined as [14C]-urea available space.16 MAPK phosphorylation was determined by Western blot analysis with antibodies against phospho-ERK, phospho-JNK, and phospho-p38, according to recently described protocol.17 Protein content in the cell lysate was measured by the Bradford method.
Chemicals
[3H]-thymidine,
[3H]-uridine,
[3H]-leucine, and
[14C]-urea were from Amersham;
86RbCl and 22NaCl were from
Dupont; PD98059 and SB 202190 were from Calbiochem Novabiochem;
ouabain, cycloheximide, and actinomycin D were from Sigma;
anti–phospho-ERK and anti–phospho-p38 antibodies were from New
England Biolab Inc; anti–phospho-JNK was from Santa Cruz
Biotechnology; 7-amino-4-methylcoumarin (AMC) was from
Molecular Probes; and DEVD-AMC
(N-acetyl-Asp-Glu-Val-Asp-AMC) and DEVD-CHO were from BIOMOL
Research Laboratories. The remaining chemicals were obtained from
Sigma, Gibco BRL, and Anachemia.
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Results |
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Kinetics of Apoptosis Modulation by Ouabain
Figure 1 shows that serum withdrawal resulted in rapid accumulation of chromatin fragments in VSMC-E1A. Indeed, after 2 hours of serum deprivation, the amount of intracellular chromatin fragments was increased in VSMC-E1A from 2.2±0.2% to 16.3±2.4% of total [3H]-labeled DNA and reached maximal values (25% to 27%) in 4 to 6 hours. Recently, we reported that 2-hour preincubation of VSMC-E1A with 1 mmol/L ouabain led to
10-fold elevation of the intracellular content
of exchangeable Na+ and decreased the content of
intracellular K+ by 4-fold.9 A
2-hour preincubation with ouabain completely abolished the effect of
serum deprivation on the accumulation of chromatin
fragments for the next 12 hours; after 24 hours of serum
deprivation, the amount of intracellular chromatin
fragments was still 2-fold less than in serum-deprived control cells
(Figure 1). Previously, we confirmed the antiapoptotic
effect of ouabain by analysis of DNA laddering,
caspase-3 activity, and phase-contrast
microscopy.9
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Effects of Actinomycin D and Cycloheximide on Apoptosis
Modulation by Ouabain
We did not observe any effect of 4-hour incubation with 2 µg/mL
of actinomycin D or cycloheximide on the baseline intracellular content
of Na+ and K+ and its
modulation by ouabain, whereas after 24 hours of incubation with
actinomycin D, baseline
[K+]i was declined by
2-fold, accompanied by 3- to 4-fold elevation of
[Na+]i (data not shown).
Considering these results, we took advantage of the rapid induction of
apoptosis in serum-deprived VSMC-E1A to study the effect of
inhibitors of RNA and protein synthesis.
Figure 2a shows that 30-minute preincubation with actinomycin D completely blocked RNA synthesis and decreased protein synthesis by 65%, whereas cycloheximide reduced RNA and protein synthesis by 85% and 80%, respectively. Neither actinomycin D nor cycloheximide significantly affected the increment of chromatin fragmentation triggered by 3-hour incubation of VSMC-E1A in serum-deprived medium (Figure 2b). This observation is in accordance with the lack of effect of actinomycin D and cycloheximide on the development of apoptosis in human VSMC transfected with p53.18 19 In contrast to baseline apoptosis, the antiapoptotic action of ouabain was completely blocked by actinomycin D and sharply attenuated by cycloheximide.
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Effects of Ouabain on RNA and Protein Synthesis
In light of the suppression of the antiapoptotic action of
ouabain by actinomycin D and cycloheximide, we examined the effects of
ouabain on RNA and protein synthesis in serum-supplied VSMC-E1A. After
2 and 6 hours of application, ouabain augmented RNA synthesis by 40%
to 50% and increased it up to 3- to 4-fold in 24 hours (Figure 3a). In contrast to RNA, protein
synthesis was attenuated by 40% and 30% after 2 and 6 hours of
ouabain addition but recovered in 24 hours (Figure 3b).
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The low intensity of apoptosis in serum-deprived VSMC compared
with cells transfected with E1A6 20 allows us to establish
quiescence and to compare the effect of ouabain on RNA synthesis in the
presence and absence of growth factors. Figure 4a shows that 4 hours of incubation of
quiescent VSMC with ouabain slightly augmented RNA synthesis, whereas
after 24-hour incubation in serum-deprived medium, RNA synthesis was
increased in ouabain-treated cells by 3- to 4-fold. Approximately the
same increment of RNA synthesis was observed after 24-hour incubation
of VSMC with 10% calf serum (CS); at this time point, the combined
addition of serum and ouabain augmented RNA synthesis by 7-fold.
Four-hour incubation with ouabain did not affect protein synthesis in
serum-deprived VSMC. Four-hour incubation with 10% CS augmented
[3H]-leucine uptake by 94±12%, and this
increment was completely abolished by ouabain. Neither baseline nor
serum-induced protein synthesis was affected after 24-hour incubation
of VSMC with ouabain (data not shown).
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After 4 hours of serum withdrawal, the content of intracellular chromatin fragments in nontransfected VSMC was increased from 0.7±0.2% to 1.7±0.4% and 1.4±0.2% in the absence and presence of ouabain, respectively (Figure 4b). In VSMC treated with serum-deprived medium for 24 hours, the content of chromatin fragments was elevated up to 11% to 12%. Similar to VSMC-E1A, the increment of chromatin cleavage triggered by serum-deprivation was completely blocked by ouabain.
Effects of Extracellular Na+ and
K+
The addition of ouabain to the control medium (medium 1) led to
13-fold elevation of intracellular Na+
concentration and to a 10-fold decrease of
[K+]i in VSMC-E1A (Table 1). As predicted, equimolar
substitution of NaCl with KCl (medium 2) completely abolished the
effect of ouabain on intracellular Na+ and
K+ concentration. Neither apoptosis
triggered by serum withdrawal nor RNA synthesis were affected by 6-hour
equimolar substitution of NaCl with KCl (data not shown). Similar to
the results reported in Figures 1 and 3, 6-hour
incubation of VSMC-E1A in medium 1 with ouabain caused 10-fold
decrease of chromatin fragmentation triggered by serum
deprivation, augmented RNA synthesis by 50%, and
inhibited protein synthesis by 30% (Figure 5a). We did not observe any effect of
ouabain on chromatin cleavage and macromolecular synthesis in
Na+-depleted medium 2. The lack of
antiapoptotic action of ouabain after substitution of NaCl with
KCl was confirmed by phase-contrast microscopy (Figure 5b) and
by analysis of caspase-3 activity (Table 2).
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Role of MAP Kinase Signaling Cascade
An overwhelming number of stimuli triggering mRNA expression use
signaling cascade, which is terminated by
phosphorylation of 3 protein kinases from the MAPK
family: extracellular signal-regulated protein kinases (ERK1/2 or
p42/p44), JNK or stress-activated protein kinase (SAPK), and a
mammalian homologue of Hog, p38 protein.21 Considering
this, we explored the role of these kinases in the induction of RNA
synthesis revealed in ouabain-treated VSMC. Figure 6 shows that neither 2-hour incubation of
VSMC-E1A with ouabain in the presence of 10% CS nor additional 6-hour
incubation in the absence of growth factors affected the level of
phosphorylation of ERK1/2, JNK, and p38. Quiescent VSMC
treated with CS and anisomycin displayed drastic
phosphorylation of ERK and JNK/p38, respectively, and
were used as a positive control in this study.
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In additional experiments, we studied the effect of inhibitors of ERK- and p38-directed MAP kinase kinase (PD98059 and SB 202190, respectively) on apoptosis. Table 3 shows that neither baseline apoptosis in serum-deprived VSMC-E1A nor its inhibition by ouabain was significantly modified by these compounds.
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Discussion |
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Data obtained in the present study show that the recently discovered inhibition of apoptosis in VSMC by compounds that invert the intracellular [Na+]/[K+] ratio9 is abolished by RNA and protein synthesis inhibitors (Figure 2b). Moreover, prolonged incubation with ouabain sharply increased RNA synthesis (Figures 3 and 4a). Previously, it was shown that sustained inhibition of the Na+,K+ pump leads to activation of RNA synthesis in astroglial cells,22 in rat liver–derived ALP15 cells,23 and in HF cells derived from newborn foreskin.24 Viewed collectively, these results indicate that induction of RNA synthesis under long-term inhibition of the Na+,K+ pump is a universal rather than a VSMC-specific phenomenon. They also suggest that massive RNA synthesis is triggered by initial expression of early response genes (ERG) such as c-fos, c-jun, and c-myc, which further affect the expression of targeting genes. Indeed, transient expression of c-fos or c-jun during the initial 0.5 to 1 hour of treatment with ouabain or with K+-depleted medium has been demonstrated in isolated perfused rat kidney and renal epithelial cells,25 ARL15 cells,26 rat ventricular myocytes,27 28 HeLa cells,29 and lymphocytes.30 Golomb and coworkers31 reported that 1-hour application of ouabain induced 2- to 3-fold activation of c-fos and c-myc expression in VSMC from the rat aorta. It should be underlined, however, that 0.1 µmol/L ouabain was used in that study. We failed to observe any effect on [Na+]i and [K+]i in VSMC after their 7-hour incubation with ouabain at a concentration <10 µmol/L (data not shown), which is consistent with well-documented data on the lack of contribution of high-affinity ouabain binding sites to inhibition of the rodent Na+,K+ pump, including that of the rat aorta.32
In contrast to RNA synthesis, ouabain inhibited protein synthesis during the initial 4 to 6 hours, an effect that was not detectable at 24 hours (Figure 3b). This is in accordance with data obtained with other cell types studied so far.24 33 34 With the use of cell-free systems, it was shown that equimolar substitution of K+ with Na+ only slightly affected RNA synthesis; in contrast, protein synthesis was completely blocked in K+-free medium at a step of transfer of amino acid from aminoacyl tRNA to the polypeptide.35 We speculate that rapid suppression of protein synthesis by Na+,K+-pump inhibitors is mediated by the K+-dependent step of the ribosomal protein synthesis machinery, whereas under sustained Na+,K+ pump inhibition, this inhibitory effect is compensated by increased synthesis of a few [Na+]i/[K+]i-sensitive mRNA species, which cannot be detected under estimation of total [3H]-leucine-labeled protein content.
In thymocytes and Jurkat cells, apoptosis can be partially inhibited by substitution of extracellular Na+ with K+9 36 37 that is probably caused by involvement of K+ loss in cell volume decrease and induction of apoptosis.9 36 38 In contrast to immune system cells,39 apoptosis in VSMC is insensitive to cell volume modulation,11 9 and in these cells ouabain affects apoptosis and macromolecular synthesis through inversion of the [Na+]i/[K+]i ratio rather than by dissipation of transmembrane gradients of these cations, alteration of membrane potential, or by side effects unrelated to monovalent ion transport. Indeed, suppression of apoptosis in serum-deprived VSMC was observed under inhibition of the Na+,K+-pump by digoxin and digitoxin (unpublished data) or by incubation of cells in K+-depleted medium.9 Equimolar substitution of Na+ with K+ in the incubation medium abolished the effect of ouabain on the [Na+]i/[K+]I ratio (Table 1), on apoptosis (Figure 5, a and b, and Table 2), and on RNA and protein synthesis (Figure 5a). To estimate the relative contribution of [Na+]i versus [K+]i in these phenomena, we substituted NaCl with choline chloride, assuming that under these conditions ouabain does not significantly affect [Na+]i but causes loss of [K+]i. Indeed, as seen in Table 1, the intracellular Na+ concentration in control VSMC-E1A incubated in medium 1 and in ouabain-treated cells incubated in medium 3 is approximately the same. However, substitution of NaCl with choline chloride eventually increased baseline [K+]i and decreased the loss of K+i triggered by ouabain. Thus, another approach should be designed to evaluate the role of [Na+]i and [K+]i in the regulation of apoptosis and macromolecule synthesis.
In conclusion, the results obtained in the present study demonstrate that inhibition of apoptosis in VSMC by an inverted intracellular [Na+]i/[K+]i ratio is mediated by de novo synthesis of the inhibitor of apoptosis, which we call [Na+]i/[K+]i-regulated inhibitor(s) of apoptosis (NKRIA). These results also raise several questions. What is the origin of the sensor triggering the antiapoptotic signal under inversion of the [Na+]i/[K+]i ratio? Which mechanisms are involved in transduction of the signal and the induction of NKRIA expression? Does this transduction pathway use ERG? Which step upstream of caspase-3 is affected by NKRIA? Is an enhanced level of ouabain-like substances seen in hypertension40 41 involved in vascular remodeling through inhibition of VSMC apoptosis? We are addressing these unresolved issues in our ongoing studies.
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Acknowledgments |
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This work was supported by grants from the Medical Research Council of Canada (MT-10803), Bayer Canada, and Pfizer Canada. The assistance of Monique Poirier and Than-Vinh Dam and the editorial help of Ovid Da Silva are appreciated.
Received November 8, 1999; first decision November 23, 1999; accepted January 5, 2000.
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