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Intracellular Monovalent Ions as Second Messengers
S.N. Orlov
1,2
, P. Hamet
1
1
Centre de recherche, Centre hospitalier de lÕUniversite
´de Montre
´al, (CHUM)-Hoˆ tel-Dieu, Montreal, Quebec, Canada
2
Laboratory of Pathophysiology of Ion Transport Disorders, Centre de recherche, CHUM - Hoˆ tel-Dieu, 3850 rue St-Urbain, Montreal,
Quebec H2W 1T7, Canada
Received: 13 November 2005/Revised: 8 February 2006
Abstract. It is generally accepted that electrochemical
gradients of monovalent ions across the plasma
membrane, created by the coupled function of
pumps, carriers and channels, are involved in the
maintenance of resting and action membrane poten-
tial, cell volume adjustment, intracellular Ca
2+
han-
dling and accumulation of glucose, amino acids,
nucleotides and other precursors of macromolecular
synthesis. In the present review, we summarize data
showing that side-by-side with these classic functions,
modulation of the intracellular concentration of
monovalent ions in a physiologically reasonable
range is sufficient to trigger numerous cellular re-
sponses, including changes in enzyme activity, gene
expression, protein synthesis, cell proliferation and
death. Importantly, the engagement of monovalent
ions in regulation of the above-listed cellular re-
sponses occurs at steps upstream of Ca
2+
i
and other
key intermediates of intracellular signaling, which
allows them to be considered as second messengers.
With the exception of HCO
3
)
-sensitive soluble ade-
nylyl cyclase, the molecular origin of sensors involved
in the function of monovalent ions as second
messengers remains unknown.
Key words: Sodium — Potassium — Proton —
Bicarbonate — Chloride — Intracellular signalling
Introduction
In accordance with Earl SutherlandÕs signal trans-
duction hypothesis [113], any intracellular molecule
can be considered a potential second messenger in the
signal transduction pathway if it satisfies 3 major
criteria. i) Modulation of intracellular concentration
of the potential second messenger triggered by
external stimuli precedes cellular responses and nor-
malizes after removal of these stimuli. ii) The tran-
sient modulation of intracellular second messenger
concentration per se is sufficient to trigger cellular
responses in the absence of investigated external
stimuli. iii) Cellular responses triggered by external
stimuli are mediated by the interaction of second
messengers with their intracellular sensors.
In pioneering studies performed more than
40 years ago, it was shown that cAMP satisfies the
above-listed criteria and provides coupling between
excitation of plasma membrane receptors by cate-
cholamines and peptide hormones with gluconeo-
genesis and lipolysis in hepatocytes and adipocytes,
respectively [105]. Later on, the list of second mes-
sengers was broadened in experiments demonstrating
a key role for cGMP, Ca
2+
and lipid molecules, such
as diacyl glycerol, inositol 1,4,5-triphosphate etc., in
the regulation of other cellular functions, such as
myocyte contraction and relaxation, hormone and
neurotransmitter release, light sensing, cell
proliferation and apoptosis, etc. [10, 18, 63, 119].
Similarly to the above-listed second messengers,
the intracellular concentration of monovalent cations
is transiently affected by diverse extracellular stimuli
and normalized to baseline values via feedback acti-
vation of the pumps, carriers and channels shown in
Fig 1. Thus, transient activation of Na
+
/H
+
ex-
change and [Na
+
]
i
elevation appear to be a universal
response of quiescent cells to growth-promoting
stimuli [15, 39, 44, 70, 90, 101, 125]. In neurons, short
periods of synaptic activity produce large increases of
[Na
+
]
i
, from 10 to 30 and 100 mMin apical den-
drites and dendritic spines, respectively, mainly due
to Na
+
influx via N-methyl-D-aspartate (NMDA)
receptor channels [106]. In erythrocytes and other
cells with low resting potential, transient activation of
Correspondence to: S.N. Orlov; email: sergei.n.orlov@umontreal.
ca
J. Membrane Biol. 210: 161–172 (2006)
DOI: 10.1007/s00232-006-0857-9
Topical Review
K
+
channels results in 5- to 8-fold attenuation of
[K
+
]
i
[9]. The regulation of these and other mono-
valent ion transporters by extracellular stimuli is
considered in detail in several comprehensive reviews
[1, 31, 33, 36, 45, 108, 125]. Here, we just summarize
data showing that modulation of the intracellular
concentration of monovalent ions triggered by
external stimuli is sufficient to affect cellular re-
sponses by acting upstream or even independently of
the signaling pathways evoked by well-defined second
messengers. We would like also to underline that the
goal of our mini-review is to support the concept of
monovalent ions as second messengers, to consider
the possible pathophysiological implications of these
signaling cascades, and to provoke the search for
intracellular monovalent ion sensors rather than to
provide a complete list of monovalent ion-dependent
cellular functions documented so far.
Sodium
In cells abundant with Na
+
/Ca
2+
exchanger, eleva-
tion of [Na
+
]
i
is sufficient to activate this carrier and
to elicit diverse Ca
2+i
-mediated responses, including
positive inotropic effect in cardiomyocytes and neu-
rotransmitter release in nerve terminals treated with
low doses of Na
+
/K
+
-ATPase inhibitors, such as
ouabain and other cardiotonic steroids (CTS) [11].
Data obtained in these studies should probably be
considered as first evidence for the involvement of
Na
+i
in the regulation of cellular function as a sec-
ond messenger. This section is focused on data
showing that Na
+i
can modulate cellular function
independently of [Ca
2+
]
i
elevation and of activation
of other Na
+
-coupled ion carriers.
In the late 1990s, we observed that almost com-
plete Na
+
,K
+
pump inhibition with ouabain pro-
tects rat vascular smooth muscle cells (VSMC) from
apoptosis triggered by growth factor withdrawal,
staurosporin or inhibitors of serine-threonine phos-
phatases and potentiated by transfection with c-myc
or its functional analogue E1A adenoviral protein
[86]. Suppression of apoptosis in CTS-treated VSMC
can be mediated by membrane depolarization, accu-
mulation of Na
+
or loss of K
+
. Conformational
transition of the Na
+
/K
+
-ATPase a-subunit trig-
gered by interaction with CTS may be sufficient per se
to generate the antiapoptotic signal. In addition, CTS
interaction with target(s) distinct from the Na
+
/K
+
-
ATPase a-subunit can not be excluded. We found
that apoptosis in VSMC is sharply suppressed by
incubation of VSMC in K
+
-free medium. Keeping in
mind an obligatory role of K
+o
in the activation of
Na
+
/K
+
-ATPase, this observation strongly suggests
the antiapoptotic action of ouabain is mediated by
inhibition of the Na
+
/K
+
pump. To further examine
the role of monovalent cations, we treated VSMC
with ouabain in high-K
+
medium. Sustained depo-
larization in high-K
+
, low-Na
+
medium did not
affect apoptosis. In contrast, dissipation of the
transmembrane gradient of monovalent cations
occurring in this medium sharply diminished the
effect of ouabain on Na
+i
and K
+i
content and
completely abolished its antiapoptotic action [86].
These data led us to conclude that Na
+
/K
+
pump
inhibition protects VSMC against apoptosis via ele-
vation of the [Na
+
]
i
/[K
+
]
i
ratio.
Later on, the antiapoptotic action of ouabain and
K
+
-depleted medium was detected in a cultured renal
proximal tubule cell line [131], in freshly-isolated rat
cerebellar granule cells [49] and in human umbilicial
vein endothelial cells [120]. It should be underlined
that relatively low concentrations of ouabain were
used in these studies, and its action on the [Na
+
]
i
/
[K
+
]
i
ratio was not examined. More recently, we
[Na+]o~140 mM; [K+]o~4 mM; [Cl-]o~100 mM; [HCO3-]o~20 mM; [H+]o~60 nM
out
in 123 567 8 9 104
3Na+
ATP ADP
2K+
K+K+
Cl-
Cl-
Cl-
Na+
H+HCO3-
Na+K+
H+
ATP ADP
2(1)HCO3-
CO2+H2O
CA
Na+Na+
2Cl-
K+
[Na+]i~10 mM; [K+]i~150 mM; [Cl-]o~20 mM; [HCO3-]i~30 mM; [H+]i~80 nM
a) b) c)
Fig. 1. Major plasma membrane ion transporters involved in intracellular Na
+
/K
+
(a), Cl
)
(b) and H
+
/HCO
3
)
(c) handling. 1: Na
+
/K
+
-
ATPase; 2:Na
+
channels; 3: K
+
channels; 4:Na
+
,K
+
, 2Cl
)
cotransport; 5:K
+
,Cl
)
cotransport; 6:Cl
)
channels; 7: Na
+
/H
+
exchange;
8: anion exchanger; 9:Na
+
,NaHCO
3
)
cotransport; 10: H
+
/K
+
-ATPase; CA: carbonic anhydrase. The representative values of extra- and
intracellular concentrations of monovalent ions are shown.
162 S.N. Orlov and P. Hamet: Monovalent Ions as Second Messengers
noted that K
+
-free, Na
+
-containing medium rescues
vascular endothelial cells from apoptosis triggered by
[
3
H]-decay-induced DNA damage. Because this pro-
tection was absent in K
+
-free, low-Na
+
medium, we
concluded that the antiapoptotic signal triggered by
Na
+
/K
+
pump inhibition is mediated by [Na
+
]
i
elevation rather than by loss of K
+i
[87].
To further explore the novel Na
+i
-mediated
antiapoptotic pathway, we treated cells with actino-
mycin D or cycloheximide. Both inhibitors of mac-
romolecular synthesis abolished protection against
apoptosis documented in VSMC pretreated with
ouabain [84]. Deploying a rat multi-probe template
set, we failed to detect, in ouabain-treated VSMC,
elevation of mRNA species encoding major pro- and
antiapoptotic proteins such as Bcl-2, Bcl-xL, Bcl-xS,
Bax, and caspases 1-3 [82]. With these negative data
in mind, we took a proteomics approach to identify a
set of Na
+i
-sensitive genes. Twelve soluble proteins,
including mortalin, whose expression is triggered by
ouabain, were identified by mass spectrometry [117].
Previous studies demonstrated the pancytosolic and
mitochondrial/juxtanuclear localization of mortalin
in mortal and immortal cells, respectively [114, 122–
124]. Northern and Western blotting confirmed the
induction of mortalin expression in ouabain-treated
VSMC and documented its mitochondrial localiza-
tion. We established that, similarly to ouabain,
transfection with mortalin delayed the development
of apoptosis in serum-deprived VSMC-E1A. We also
found that transfection with mortalin inhibits
p53 translocation to the nucleus [117]. Viewed
collectively, these data suggest that elevated [Na
+
]
i
suppresses programmed cell death via augmented
mortalin expression that, in turn, blocks p53
nuclear translocation triggered by apoptotic stimuli
(Fig. 2).
In the last decade, it was found that, in several
types of cells, sustained inhibition of the Na
+
/K
+
pump triggers the expression of the a1- and b1-su-
bunits of the Na
+
/K
+
-ATPase, myosin light chain,
skeletal muscle actin, atrial natriuretic factor and
tumor growth factor-b(for recent review, see [116,
128]). These data together with augmented RNA
synthesis [85] and the appearance of numerous
protein spots, detected in ouabain-treated VSMC by
2-D electrophoresis [82, 117], suggest that this action
of Na
+
,K
+
pump inhibitors is at least partially
mediated by early response genes. Indeed, in VSMC,
we observed 10- and 4-fold elevations of immuno-
reactive c-Fos and c-Jun after 2- and 12-h treatment
with ouabain, respectively [115]. A 4-fold increment
of c-Fos mRNA content was detected in 30 min of
ouabain addition. Importantly, within this time
interval, [Na
+
]
i
was increased by 5-fold whereas
[K
+
]
i
was decreased by only 10–15%. This result
shows that [Na
+
]
i
augmentation rather than [K
+
]
i
attenuation generates a signal that leads to c-Fos
expression.
In accordance with known signaling pathways
triggered by CTS, gene expression, seen under
elevated [Na
+
]
i
, can be mediated by cell
volume modulation or/and activation of Na
+
/H
+
and Na
+
/Ca
2+
exchangers. The latter hypothesis is
also consistent with the presence of (Ca
2+
+ cAMP)
response element (CRE) within the c-Fos promoter
[107]. Indeed, we have demonstrated that K
+o
-in-
duced depolarization leads to c-Fos expression that is
completely abolished by the selective L-type Ca
2+
channel blocker nicardipine [115]. However, the data
listed below strongly indicate that c-Fos expression in
ouabain-treated VSMC is a Ca
2+
-independent phe-
nomenon. First, c-Fos expression in ouabain-treated
cells is not sensitive to nicardipine. Second, neither
ATP
3Na+
PROTEIN SYNTHESIS
[Na+]i
ADP
[K+]i
2K+
12
CTS
eEF-2 eEF-2~P
eEF-2 kinase
SK
?
pHRE 3
Apoptotic
stimuli
AP Gαβγ GαGβγ
+
ONCOSIS
NaRE mortalin
APOPTOSIS
p53
SNa
[Na+]i
[K+]i
?
SpH
caspase-3
Gp-mediated
responses
[H+]i
Fig. 2. Intracellular monovalent cations as second messengers. 1and 2: active and inactive conformation of the Na
+
/K
+
-ATPase a-subunit
triggered by its interaction with CTS; 3: H
+i
-sensitive gene(s) involved in the inhibition of oncosis; S
K
,S
Na
and S
pH
– hypothetical sensors
of intracellular K
+
,Na
+
and H
+
, respectively; AP: adapter protein(s) whose interaction with Na
+
,K
+
-ATPase a-subunit is affected by
CTS; pHRE and NaRE:H
+
- and Na
+
-sensitive elements implicated in the regulation of gene expression; eEF-2: elongation factor-2; Gabc::
a,band csubunits of GTP-binding proteins; ?: unknown intermediates; - fiand --| : activatory and inhibitory signals, respectively. For
more details, see text.
S.N. Orlov and P. Hamet: Monovalent Ions as Second Messengers 163
[Ca
2+
]
i
nor total exchangeable Ca content in VSMC is
affected by ouabain [115]. This observation is consis-
tent with negligible Na
+
/Ca
2+
exchanger activity
detected in VSMC [83]. Third, neither extracellular
(EGTA) nor intracellular (BAPTA-AM) Ca
2+
che-
lators abolish ouabain-induced c-Fos expression
[115]. These data as well as the lack of a significant
effect of ouabain on pH
i
and cell shrinkage on c-Fos
content allow us to hypothesize that gene expression
in ouabain-treated VSMC is mediated by a novel
Na
+i
-dependent, Ca
2+i
-insensitive mechanism of
excitation-transcription coupling.
Gene expression is probably not the only cellular
function controlled by the Na
+i
-sensor. Indeed, it has
been shown in neuronal cells that elevation of [Na
+
]
i
is sufficient to activate heterotrimeric G-proteins via
the GTP-independent mechanism of dissociation of
their a- and bc-subunits [104]. Moreover, in these
cells, [Na
+
]
i
elevation modulates the activity
of NMDA receptors, K
+
and Ca
2+
channels by
G-protein-dependent and -independent mechanisms
[12, 104, 130]. Are these cellular responses and Na
+i
-
dependent gene expression, demonstrated in our
studies [115], mediated by the same Na
+i
-sensor? We
will address this question in future studies.
Potassium
More than 40 years ago, it was demonstrated that
protein synthesis in prokaryotes is sharply inhibited
in the absence of K
+
[65]. Later on, the requirement
of K
+
for protein synthesis was detected in animal
cells of different origins [56, 59, 64, 73, 100]. Using
human fibroblasts subjected to sustained inhibition of
Na
+
/K
+
-ATPase with ouabain, it was shown that
inversion of the [Na
+
]
i
/[K
+
]
i
ratio inhibits protein
synthesis without any impact on mRNA function,
ATP content and amino acid transport [59], thus
suggesting direct influence of [K
+
]
i
on the protein
synthesis machinery.
In reticulocytes, globin contributes to more than
90%of total protein synthesis. In these cells, it was
found that K
+i
depletion inhibits the elongation step
of globin synthesis without any impact on ribosome
subunit assembly [16]. The half-maximal activation of
globin synthesis by reticulocyte lysate in medium
containing 60, 90 and 125 mMNa
+
was observed at
[K
+
] of 15, 25 and 40 mM, respectively [16]. These
data indicate that elevation of [Na
+
]
i
diminishes the
efficacy of protein synthesis regulation by K
+i
via
attenuation of K
+
interaction with its hypothetical
sensor (Fig. 2). Intermediates of the protein synthesis
machinery involved in K
+i
sensing remain unknown.
It should be underlined that the effect of K
+i
loss
on protein synthesis is cell type-specific. Thus, in
contrast to the above-mentioned cells showing 2- to
4-fold attenuation of protein synthesis after sustained
inhibition of Na
+
/K
+
-ATPase in K
+
-free medium
or in the presence of CTS, we did not see any sig-
nificant effect on [
3
H]-leucine protein labelling after
24-h ouabain treatment of cultured VSMC from the
rat aorta [85]. Two hypotheses could explain these
data. First, the K
+i
-sensitive element of the protein
synthesis machinery is absent in VSMC. Second,
attenuation of protein synthesis in K
+
-depleted
VSMC is masked by augmented mRNA synthesis.
Indeed, we discerned a 6-fold elevation of total RNA
synthesis in VSMC treated with ouabain for 10 h [85]
that could be attributed to Na
+i
-mediated expression
of c-Fos and other early response genes detected in
VSMC after 1–2 h of ouabain addition [115].
Proton
The functioning of numerous proteins is affected by
cellular acidification. Thus, for example, acidification
inhibits TASK-3 K
+
channels [102] but activates
TREK-1 K
+
channels [67] and Ca
2+
-permeable acid-
sensitive ion channels [129]. It should be underlined,
however, that modulated activity of these ion trans-
porters was detected in pH ranges from 7.4 to 5.0,
which corresponds to 200-fold elevation of [H
+
]
i
concentration, thus suggesting the involvement of
these proteins in pH sensing under severe hypoxia
and/or HCO
3
)
depletion. A system with much higher
pH sensitivity has been detected in our studies of
oncosis in CTS-treated epithelial and endothelial cells.
In contrast to rat VSMC [85, 86], NIH 3T3
mouse fibroblasts, HEK-293, HeLa, human renal
carcinoma Caki cells [81], renal epithelial cells from
the Rhesus monkey [22, 23], human lymphocytes [32]
and rat astrocytes (unpublished data), 24-h exposure
to ouabain results in massive death of renal epithelial
cells from Madin-Darby canine kidney (MDCK) and
endothelial cells from the porcine aorta (PAEC).
Both types of ouabain-treated cells possess combined
markers of necrosis (cell swelling, negligible labelling
with nucleotides in the presence of terminal trans-
ferase, nuclei staining with cell-impermeable dyes,
such as propidium iodide) and apoptosis (nuclear
condensation, chromatin cleavage, caspase-3 activa-
tion) [23, 87, 96]. We also demonstrated that in
contrast to classical cell culture models of apoptosis,
death of ouabain-treated MDCK cells was insensitive
to the pan-caspase inhibitor z-VAD.fmk [96]. To
underline the striking difference in cell volume
behavior (swelling vs shrinkage detected in cells
undergoing apoptosis), revised terminology has been
proposed, claiming that necrosis was originally of-
fered as a concept to characterize any post-mortem
changes in cell morphology. In accordance with this
nomenclature, the label ‘‘oncosis’’, derived from the
Greek word for swelling, describes cell death that is
distinct from apoptotic shrinkage [68].
164 S.N. Orlov and P. Hamet: Monovalent Ions as Second Messengers
Surprisingly, we found that more than 500-fold
inhibition of the Na
+
/K
+
pump in K
+
-free medium
does not affect the survival of C7-MDCK cells [96].
As predicted, 6-h incubation of C7-MDCK cells in
K
+
-free medium led to a sharp [Na
+
]
i
elevation, and
the addition of ouabain only slightly altered this
parameter, whereas incubation in high-K
+
/low-Na
+
medium did not impact the baseline values of [Na
+
]
i
and [K
+
]
i
, but completely abolished the K
+i
loss
triggered by ouabain. However, similarly to control
medium, ouabain killed cells to the same extent in
K
+
-free and high-K
+
/low-Na
+
media [96]. More-
over, the same left-hand shift was noted in compari-
son to the dose-dependent action of ouabain on Na
+
/
K
+
pump activity and death of C7-MDCK and
PAEC [4, 87, 96]. These results strongly indicate that
in both cell types, CTS trigger Na
+i
,K
+i
-indepen-
dent oncosis via interaction with the Na
+
/K
+
-AT-
Pase a-subunit rather than with other potential K
+o
-
insensitive receptors. Considering these data, we
proposed that CTS-induced conformational transi-
tion of the Na
+
/K
+
-ATPase a-subunit is sufficient to
trigger its interaction with an unidentified adapter
protein(s), resulting in Na
+i
,K
+i
-independent
oncosis of renal epithelial and vascular endothelial
cells (Fig. 2). This adapter protein or downstream
intermediates of the Na
+i
,K
+i
-independent death
machinery are absent in CTS-resistant cells, including
VSMC.
Under analysis of the role of extracellular ions
in CTS-induced oncosis, we observed that decreases
of NaHCO
3
concentration from 44 to 11 mMshar-
ply attenuated the death of C7-MDCK cells trig-
gered by ouabain. Keeping in mind that total Na
+
concentration in control and NaHCO
3
-depleted
medium was the same, 2 hypotheses can explain this
finding. First, medium acidification caused by a
decreased HCO
3
)
/CO
2
ratio is sufficient to inhibit the
cell death machinery. Second, a decreased HCO
3
)
/
CO
2
ratio suppresses cell death independently of
medium acidification. Data obtained in additional
experiments do not support the latter hypothesis.
Indeed, cell death inhibition was also detected in
medium with high NaHCO
3
concentration and
acidified by HEPES, whereas alkalinization with
Tris abolished the protective action of NaHCO
3
depletion [5]. Finally, we used NaHCO
3
-free, HE-
PES-Tris-buffered medium and observed that the
death of ouabain-treated PAEC and C7-MDCK
cells is suppressed by acidification of the medium
from pH 7.4 to 7.0, i.e., under conditions when pH
i
was decreased from 7.2 to 6.9. The rescue of
ouabain-treated C7-MDCK cells was also detected
under selective intracellular acidification caused by
inhibition of the Na
+
/H
+
exchanger with ethyliso-
propyl amiloride [5].
Neither [
3
H]-ouabain binding nor ouabain-sen-
sitive
86
Rb influx was significantly affected by modest
acidification [5], showing that the H
+i
-sensitive ele-
ment of the cell death machinery is located down-
stream of Na
+
/K
+
-ATPase. It should be noted that
acidification from 7.2 to 5.0 activates rather than
inhibits caspases [71] and nucleases [97], excluding
these downstream intermediates as a potential H
+i
-
sensor involved in the suppression of death signaling
triggered by CTS.
Elongation factor-2 (eEF-2) is the most promi-
nently phosphorylated protein detected in mamma-
lian tissue extracts, and its phosphorylation by eEF-2
kinase leads to inactivation and inhibition of protein
synthesis [110]. By comparing liver homogenates
from wild-type and eEF-2 kinase knockout mice, it
was shown that eEF-2 phosphorylation is completely
blocked by pH elevation from 6.6 to 7.4 [30, 109], i.e.,
in the range where switch off/on regulation of the
CTS-induced cell death machinery is detected. Con-
sidering this, it may be proposed that acidosis sup-
presses the death signal via eEF-2 phosphorylation
that in turn abolishes eEF-2-mediated activation of
protein synthesis. However, the death of ouabain-
treated MDCK cells was noted in the presence of
RNA and protein synthesis inhibitors, whereas the
protective effect of acidification was sharply dimin-
ished by these compounds at modest non-toxic con-
centrations [5]. These results strongly suggest that the
rescue by modest intracellular acidification of renal
epithelial and vascular endothelial cells from Na
+i
,
K
+i
-independent oncosis triggered by CTS is medi-
ated by the de novo expression of gene(s) containing
pH
i
-response element.
Bicarbonate
Mammalian spermatozoa undergo activation pro-
cesses induced by bicarbonate and mediated by ele-
vation of intracellular cAMP content. It has been
assumed that this action of HCO
3
)
is caused by
alkalinization of the cytoplasm. However, several
laboratories have reported that spermatozoa are
highly abundant in soluble adenylyl cyclase (sAC)
[19, 37, 79] stimulated by HCO
3
)
in a pH
i
-independent
manner [19]. It was also shown that bicarbonate
activates purified and recombinant sAC with an EC
50
of 20 mM[19] relevant to the physiological range of
[HCO
3
)
]
i
(Fig. 1c). Amino acid residues involved in
the organization of the HCO
3
)
-binding site of sAC
and the role of these sites in the conformational
transition of this enzyme remain unknown.
Side-by-side with spermatozoa, sAC was also
detected in the kidney and choroid plexus [19, 95],
indicating involvement of HCO
3
)
as a second mes-
senger in the regulation of cAMP-dependent func-
tions of these tissues. Based on the identification of
sAC within nuclei, it has been proposed that this
enzyme contributes to [H
+
]
i
/[HCO
3
)
]
i
-dependent
S.N. Orlov and P. Hamet: Monovalent Ions as Second Messengers 165
modulation of gene expression via activation of CRE-
binding protein [132]. It was also suggested that pH
i
-
coupled modulation of sAC activity is responsible for
pH-dependent recycling of vacuolar H
+
-ATPase [95].
Chloride
The first data suggesting the role of Cl
)
i
in cellu-
lar signaling were probably obtained in the study
of regulation of the ubiquitous isoform of Na
+
,
K
+
, 2Cl
)
cotransporter (NKCC1). Several research
groups demonstrated that inwardly-directed
NKCC1 contributes to the accumulation of Cl
)
above the Nernst equilibrium potential. For exam-
ple, in secretory epithelial, renal epithelial, vascular
endothelial, mesangial and neuronal cells, inhibition
of NKCC1 with bumetanide decreases [Cl
)
]
i
by 2- to
6-fold [52, 69, 77]. In resting neuronal cells with high
permeability for K
+
(P
K
>> P
Cl
), NKCC1-medi-
ated elevation of [Cl
)
]
i
does not affect membrane
potential (E
m
) but leads to transient depolarization
under activation of GABA-sensitive anion channels
[29]. In VSMC, P
K
and P
Cl
values are somewhat
similar [21], indicating NKCC1 involvement in the
regulation of resting E
m
. Indeed, bumetanide de-
creases [Cl
)
]
i
[89], hyperpolarizes [28] and abolishes
differences in [Cl
)
]
i
and E
m
between VSMC from
normotensive and deoxycorticosterone-salt-hyper-
tensive rats [14]. In recent studies, we demonstrated
that in HCO
3
)
-depleted medium, NKCC1 inhibition
sharply suppresses smooth muscle cell contrac-
tion triggered by modest depolarization or by
a-adrenergic stimulation [7, 55].
Since the seminal studies of dialyzed squid axons
[13], it was shown that in all types of cells studied so
far NKCC1 activity is decreased by 5- to 10-fold
under elevation of [Cl
)
]
i
from 20 to 150 mM(108),
thus providing feedback regulation of this carrier. In
Cl
)
-depleted tracheal epithelial cells [42] and shark
rectal glands [66], NKCC1 activation is accompanied
by phosphorylation of the carrier, indicating the
presence of protein kinases whose activity is nega-
tively regulated by [Cl
)
]
i
. The presence of Cl
)
i
-sensi-
tive intermediates of signal transduction, including
protein kinases and phosphatases, was also proposed
in the study of activation of permeabilized neu-
trophils [38], GABA receptors [58], and phosphory-
lation of membrane-bound proteins in airway
epithelium, including nucleoside diphosphate kinase
[75, 118]. In contrast, we failed to detect any impact
of Cl
)
i
depletion on baseline phosphorylation of
proteins in MDCK cells or on protein phosphoryla-
tion triggered by activators of protein kinases A, C
and mitogen-activated protein kinases (unpublished
data).
Several laboratories have reported that growth
factors and other mitogens transiently activate
NKCC1 [91, 92], whereas chronic inhibition of the
carrier with bumetanide or furosemide suppresses the
growth of fibroblasts [91], VSMC [17], lymphocytes
[92], vascular endothelial cells [93] and airway smooth
muscle cells [51]. More recently, Panet and co-work-
ers reported that NKCC1 overexpression sharply
accelerates the proliferation of mouse Balb/c 3T3
cells in serum-free medium [94]. Since there is no
conclusive data on the involvement of NKCC in the
regulation of intracellular Na
+
and K
+
content un-
der baseline conditions [61, 89], elevation of [Cl
)
]
i
seen under NKCC activation in the above-listed cells
[52, 69, 77] seems to be crucial for proliferative
responses (Fig. 3).
Pathophysiological implications
Augmented activity of NKCC1 has been detected in
blood cells and VSMC from spontaneously hyper-
tensive rats (SHR) and in some patients with essential
hypertension [80]. Two hypotheses explain the pos-
sible involvement of abnormal activities of this
carrier in the pathogenesis of hypertension. First,
NKCC1 activation leads to augmented contractile
responses of VSMC, as demonstrated by our group
[7, 55] and by OÕNeill and co-workers [2, 3]. Sec-
ond, NKCC1-mediated elevation of [Cl
)
]
i
causes
heightened proliferation of VSMC (Fig. 3), i.e., a
well-documented hallmark of vascular remodeling
(increased wall-to-lumen ratio) detected in hyperten-
sion and implicated in the development of cardio-
vascular complications of this disease [34, 88]. The
latter hypothesis is consistent with data showing
shortening of the G
0
/G
1
phase in cultured VSMC
[HCO3-]i
sAC
cAMP
ATP
cAMP-mediated responses
2Cl-
NKCC1
[Cl-]i
?
proliferation
K+
Na+
SCl-
ClRE COX-2
?
Fig. 3. Intracellular monovalent anions as second messengers. S
Cl
:
hypothetical sensor of intracellular Cl
)
;ClRE:Cl
)
-sensitive ele-
ment implicated in the regulation of gene expression; ?: unknown
intermediates; - fiand --| : activatory and inhibitory signals,
respectively. For more details, see text.
166 S.N. Orlov and P. Hamet: Monovalent Ions as Second Messengers
from SHR [43, 121] and lengthening of the same cell-
cycle phase in bumetanide-treated cells [51]. Impor-
tantly, vascular remodeling might be further en-
hanced by elevation of [Na
+
]
i
caused by augmented
production of endogenous CTS documented in
hypertension and several other extracellular fluid
volume-expanded disorders [81]. This hypothesis is
consistent with data on Na
+i
-dependent inhibition of
apoptosis in VSMC considered in the Sodium section.
So-called ‘‘kidney resetting’’, i.e., normal salt
and water excretion under elevated blood pressure,
is the most powerful servomechanism of the long-
term maintenance of severe hypertension indepen-
dently of the origin of this disease, including
hypertension caused by enhanced salt consumption
[40, 41]. Several lines of evidence strongly suggest
that elevated intake of Cl
)
rather than Na
+
triggers
the development of NaCl-induced hypertension [53,
111]. It may be assumed that similar to VSMC
(Chloride section) elevation of [Cl
)
] in the macula
densa, caused by augmented NaCl intake via renal
specific NKCC2 isoform [57, 78] leads to the in-
creased contraction of mesangial cells, thus provid-
ing an explanation for the altered tubuloglomerular
feedback regulation of kidney function. In addition,
luminal [Cl
)
] negatively correlates with renin pro-
duction in juxtaglomerular preparations [46, 54]. In
the macula densa, renin secretion is under the con-
trol of cyclooxygenase (COX) activity and prosta-
glandin production [35] (Fig. 3). More recent studies
show that COX-2 expression is augmented in Cl
)
-
depleted medium and in the presence of NKCC
inhibitors [20]. The mechanism of the involvement
of [Cl
)
] in the regulation of COX-2 expression
remains unknown.
As for the physiological significance of H
+i
-
sensing, it must be noted that acidosis with pH
i
< 6.5
is considered a hallmark of hypoxia and ischemia
[112]. In several tissues, including the heart [126],
brain [8] and kidney [50], short ischemic precondi-
tioning protects cells from death caused by a sub-
sequent severe ischemic event. Importantly, both
ouabain-treated cells (Proton section) and cells sub-
jected to severe ischemia [60] possessed combined
markers of apoptosis and necrosis. Moreover, simi-
larly to ouabain-treated cells, the protective action of
ischemic preconditioning on severe ischemia-induced
tissue damage was transient [62] and diminished
sharply in the presence of cycloheximide [8]. The
protective action of acidification was also demon-
strated in mouse macrophages subjected to UV-irra-
diation [98], serum-deprived bovine and human
umbilical vein endothelial cells [26, 27] and endothe-
lial cells from human pulmonary arteries treated with
staurosporin [25]. Viewed collectively, these data
suggest that intracellular signaling triggered by H
+i
sensor plays a universal role in modulation of efficacy
of the cell death machinery.
Search for Intracellular Monovalent Ion Sensors
The ability of proteins to sense the modulation of
monovalent ion concentration is strongly supported
by cell physiology and molecular biology data ob-
tained in studies of superfamilies of monovalent ion
pumps, carriers and channels (Fig. 1). However, with
the exception of HCO
3
)
-sensitive sAC, the molecular
origin of monovalent ion sensors distinct from ion
transporters and involved in intracellular signaling is
still a mystery. This statement is in contrast with
rapid progress in the identification of Ca
2+i
sensors
and may be explained by several features of these
molecules. First, the high thermostability of several
Ca
2+
-binding proteins, such as calmodulin, and the
well-defined molecular origin of their targets, such as
phosphodiesterase and plasma membrane Ca
2+
-AT-
Pase, allowed researchers to purify them and to
identify their amino acid sequence, even before the
molecular biology era. As shown above, the down-
stream targets of monovalent cations and Cl
)
sensors
are still unknown. Second, high-affinity Ca
2+i
sensors
are almost completely saturated at [Ca
2+
]
i
of 1 lM,
and their Ca
2+
-binding sites are slightly affected by
the presence of monovalent cations and Mg
2+
. This
feature led to the identification of amino acid residues
by
45
Ca binding assay. In contrast to Ca
2+
,Na
+
,
K
+
, HCO
3
)
and Cl
)
affect cellular function in the
millimolar range. If monovalent ions bind with low-
affinity sensors, these interactions may be affected
by numerous factors, which complicates their
identification by screening with radioisotopes. Third,
high-affinity binding sites, initially detected in parv-
albumins and calmodulin, are formed by a highly
conservative linear amino acid sequence consisting of
14 amino acid residues (the so-called ‘‘EF-hand’’
domain). This knowledge led to the rapid identifica-
tion of more than 30 other Ca
2+i
sensors by the
screening of cDNA libraries [47]. In contrast,
monovalent ion sensors are probably formed by 3-D
protein structures and recruit space-separated amino
acid residues. This hypothesis is consistent with data
obtained by the identification of amino acid residues
in monovalent ion transporters performed with sin-
gle-point mutated constructs. Thus, it was shown that
Ala
330
, Glu
786
, Glu
796
, Asn
783
and Asp
815
contribute
to Na
+
binding within one of the 3 Na
+
-binding sites
of the Na
+
/K
+
-ATPase a-subunit [103].
Keeping these data in mind, we tried to identify
aNa
+
response element (NaRE) involved in c-Fos
expression triggered by the sustained inhibition of
Na
+
/K
+
-ATPase (Sodium section). In case of
positive results, this approach can lead to the iden-
tification of an upstream Na
+i
-sensor in a 2-hybrid
yeast system, i.e., by mating yeast transformed with
NaRE of the c-Fos promoter as a bait, with yeast
expressing an activation domain fusion cDNA li-
brary. This approach was supported by previous
S.N. Orlov and P. Hamet: Monovalent Ions as Second Messengers 167
data on mouse NIH 3T3 cells transfected with
human c-Fos showing the lack of an activatory ac-
tion of ouabain on human c-Fos mRNA content
after deletion of the )222 to )70 promoter region
[76]. To achieve our goal, we transfected HeLa cells
with the vector encoding the luciferase reporter gene
under the control of the human c-Fos )650 to +103
region, which contains all known transcription ele-
ments of c-Fos promoter. With this construct, we
failed to detect any significant elevation of luciferase
expression in HeLa cells subjected to 6-h inhibition
of Na
+
/K
+
-ATPase with ouabain or in K
+
-free
medium, which contrasted with massive accumula-
tion of endogenous c-Fos mRNA and immunore-
active protein in ouabain-treated HeLa cells
(unpublished data). We also did not observe any
positive impact of Na
+
/K
+
-ATPase inhibition on
luciferase expression driven by Elk-1, SRF, CREB
and AP1 transcription factors [115]. At least two
hypotheses could explain our negative results. (i)
Chromatin architecture involved in the formation of
NaRE is different in c-Fos and luciferase driven by
c-Fos promoter. Indeed, nuclease digestion assays
documented that chromatin transcription-sensitive c-
Fos sites are located in a region centered to )350
base pair as well as at a position )1900, i.e., up-
stream of the classical promoter [48]. (ii) Side-by-
side with 5¢-untranslated region, introns contribute
to organization of c-Fos NaRE. The role of introns
in transcriptional regulation has been proved in the
study of several genes, including c-Fos (24;72) and
WNK kinase [127].
In conclusion, we would like to stress that the
proposed models of monovalent ion sensors (Figs. 2
and 3) are based on the assumption that their
function occurs in a fixed environment. This is not a
case of the cytoplasm containing up to 0.4 g of
protein and 0.1 g of other macromolecules, such as
carbohydrates, lipids and nucleic acids, per ml of
intracellular water [6, 74]. Because of extreme mac-
romolecular crowding, cytoplasm functions as a gel
undergoing gel-sol phase transitions in response to
diverse stimuli, including modulation of the content
of monovalent ions [99]. We speculate that these
transitions per se can affect the functions of target
proteins, thus contributing to the mechanism sensing
monovalent ion concentrations. Further investiga-
tions should be performed to confirm or reject this
hypothesis and to design new approaches for iden-
tification of the molecular nature of monovalent ion
sensors.
This work was supported by grants from the Canadian Institutes of
Health Research, the Heart and Stroke Foundation of Canada and
the Kidney Foundation of Canada. The editorial help of Ovid Da
SilvaÕResearch Support Office, CHUM, is appreciated.
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