Physiology and pathophysiology of Na+/H+ exchange and Na+-K+-2Cl(-) cotransport in the heart, brain, and blood

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DOI: 10.1152/ajpregu.00782.2005 · Source: PubMed
Maintenance of a stable cell volume and intracellular pH is critical for normal cell function. Arguably, two of the most important ion transporters involved in these processes are the Na+/H+ exchanger isoform 1 (NHE1) and Na+ -K+ -2Cl- cotransporter isoform 1 (NKCC1). Both NHE1 and NKCC1 are stimulated by cell shrinkage and by numerous other stimuli, including a wide range of hormones and growth factors, and for NHE1, intracellular acidification. Both transporters can be important regulators of cell volume, yet their activity also, directly or indirectly, affects the intracellular concentrations of Na+, Ca2+, Cl-, K+, and H+. Conversely, when either transporter responds to a stimulus other than cell shrinkage and when the driving force is directed to promote Na+ entry, one consequence may be cell swelling. Thus stimulation of NHE1 and/or NKCC1 by a deviation from homeostasis of a given parameter may regulate that parameter at the expense of compromising others, a coupling that may contribute to irreversible cell damage in a number of pathophysiological conditions. This review addresses the roles of NHE1 and NKCC1 in the cellular responses to physiological and pathophysiological stress. The aim is to provide a comprehensive overview of the mechanisms and consequences of stress-induced stimulation of these transporters with focus on the heart, brain, and blood. The physiological stressors reviewed are metabolic/exercise stress, osmotic stress, and mechanical stress, conditions in which NHE1 and NKCC1 play important physiological roles. With respect to pathophysiology, the focus is on ischemia and severe hypoxia where the roles of NHE1 and NKCC1 have been widely studied yet remain controversial and incompletely elucidated.


Invited Review
Physiology and pathophysiology of Na
exchange and Na
cotransport in the heart, brain, and blood
S. F. Pedersen,
M. E. O’Donnell,
S. E. Anderson,
and P. M. Cala
Department of Biochemistry, Institute of Molecular Biology and Physiology, University of Copenhagen, Copenhagen, Denmark;
Department of Physiology and Membrane Biology, School of Medicine, University of California Davis, Davis, California
Pedersen, S. F., M. E. O’Donnell, S. E. Anderson, and P. M. Cala. Physiol-
ogy and pathophysiology of Na
exchange and Na
cotransport in
the heart, brain, and blood. Am J Physiol Regul Integr Comp Physiol 291: R1–R25,
2006. First published February 16, 2006; doi:10.1152/ajpregu.00782.2005.—Mainte-
nance of a stable cell volume and intracellular pH is critical for normal cell function.
Arguably, two of the most important ion transporters involved in these processes are the
exchanger isoform 1 (NHE1) and Na
cotransporter isoform 1
(NKCC1). Both NHE1 and NKCC1 are stimulated by cell shrinkage and by numerous
other stimuli, including a wide range of hormones and growth factors, and for NHE1,
intracellular acidification. Both transporters can be important regulators of cell volume,
yet their activity also, directly or indirectly, affects the intracellular concentrations of
, and H
. Conversely, when either transporter responds to a
stimulus other than cell shrinkage and when the driving force is directed to promote
entry, one consequence may be cell swelling. Thus stimulation of NHE1 and/or
NKCC1 by a deviation from homeostasis of a given parameter may regulate that
parameter at the expense of compromising others, a coupling that may contribute to
irreversible cell damage in a number of pathophysiological conditions. This review
addresses the roles of NHE1 and NKCC1 in the cellular responses to physiological and
pathophysiological stress. The aim is to provide a comprehensive overview of the
mechanisms and consequences of stress-induced stimulation of these transporters with
focus on the heart, brain, and blood. The physiological stressors reviewed are meta-
bolic/exercise stress, osmotic stress, and mechanical stress, conditions in which NHE1
and NKCC1 play important physiological roles. With respect to pathophysiology, the
focus is on ischemia and severe hypoxia where the roles of NHE1 and NKCC1 have
been widely studied yet remain controversial and incompletely elucidated.
ischemia; hypoxia; intracellular pH; intracellular sodium concentration
exchanger isoform 1 (NHE1)
and the Na
cotransporter isoform 1 (NKCC1) are
ubiquitous, electroneutral Na
-dependent transporters with
major roles in regulation of cellular volume and intracellular
ion concentrations. NHE1 and NKCC1 are major Na
pathways in essentially all cell types studied and exhibit
multiple similarities with respect to regulation and physiolog-
ical roles, and, consequently, in the pathophysiological conse-
quences of their inappropriate function. Yet there are also
important differences in their roles in specific tissues and
processes. Mounting evidence indicates that both transporters
are central to the cell damage induced by ischemia/hypoxia in
various organs and thus of substantial clinical interest. An
integrated view of the relative roles of NHE1 and NKCC1 in
the cellular response to physiological and pathophysiological
stress is therefore of major importance in understanding the
physiology and pathophysiology of these transporters. How-
ever, such analyses have so far not been available in the
literature. The aim of this review is to provide a comprehensive
comparative view of the mechanisms and consequences of
physiological and pathophysiological stimulation of these
transporters in the heart, brain, and blood. In each of these
tissues, we will examine the general relevance of the following
chain of events in cell damage: stimulation of NHE1 and/or
NKCC1, increases in intracellular Na
concentration ([Na
and cell volume, Na
exchange-mediated increases in
intracellular free Ca
concentration ([Ca
), and finally,
decreased ATP levels and altered intracellular pH (pH
Maintenance of a low [Na
is critical for normal cell
function. Under steady-state conditions, [Na
is generally
maintained below 20 mM, whereas the extracellular Na
concentration ([Na
)is140 mM. This sevenfold concen-
tration difference, together with the negative membrane poten-
tial (V
) constitutes a substantial inward driving force for Na
which is used ubiquitously to drive a wide variety of transport
Cell volume, and/or net intracellular osmolyte concentration
plays a pivotal role in a wide range of cellular processes, and
consequently must be tightly controlled to maintain normal cell
function. Osmotic cell shrinkage, resulting in vivo from either
a decrease in intracellular osmolarity or an increase in extra-
cellular osmolarity (106), therefore, generally activates com-
pensatory ion (and osmotically obliged water) uptake, which
persists until volume recovery is complete. The most important
acutely shrinkage-activated ion transport mechanisms are 1)
Address for reprint requests and other correspondence: S. F. Pedersen, Dept.
of Biochemistry, August Krogh Bldg., Institute of Molecular Biology and
Physiology, Univ. of Copenhagen, 13 Universitetsparken, Dk-2100 Copenha-
gen Ø, Denmark (e-mail:
Am J Physiol Regul Integr Comp Physiol 291: R1–R25, 2006.
First published February 16, 2006; doi:10.1152/ajpregu.00782.2005.
0363-6119/06 $8.00 Copyright
2006 the American Physiological Society R1
NHE1 functioning in parallel with a Cl
(AE), and 2) NKCC1 (106). In most cells, the activity of these
transporters leads to a full or partial volume recovery, also
known as the process of regulatory volume increase (RVI).
Regulation of volume by NHE1 and/or NKCC1 after cell
shrinkage can be associated with an incipient increase in
, and stimulation of NHE1 or NKCC1 in nonshrunken
cells by, e.g., hormones and growth factors, will result in both
water and Na
uptake. Yet in healthy cells, [Na
is main
tained relatively constant because of the activity of the Na
Another parameter fundamental to cell function and requir-
ing tight homeostasis is intracellular pH (pH
) (28, 48). In most
mammalian cells, steady-state pH
is maintained in the range of
7.0 –7.2, although this may vary widely (28, 48). Even though
extracellular pH (pH
)is7.4, given the negative V
, there is
a substantial net inward electrochemical driving force for H
per se. Additionally, metabolic processes create a net cellular
load, and H
must be actively extruded from the cyto
plasm. In most cases, acid-stimulated, H
extruding transport
ers utilize the inward Na
gradient, the most important players
being Na
exchange, Na
-coupled Cl
and Na
cotransport (28). Thus regulation of pH
an acid load may increase both [Na
and cell volume. On the
other hand, stimulation of NHE1 or NKCC1 may lead to
changes in pH
, the magnitude of this effect being dependent
on the activity of AE; NHE1 is functionally coupled to AE at
a ratio of 1 by changes in pH
and therefore [HCO
(for a
discussion, see Ref. 40). At 1:1 coupling, HCO
influx via AE
perfectly buffers H
efflux via NHE1, and NHE1 activity is
without effect on pH
. However, when the activity of NHE1
exceeds that of AE, intracellular alkalinization results (see Ref.
40). Although NKCC1 activity per se is without effect on pH
it may be noted that this transporter may be indirectly coupled
to pH
changes through [Cl
, that is, an increase in [Cl
causes AE to operate in the direction of recycling Cl
out of
the cell, resulting in HCO
influx and a intracellular alkalin
ization in cells with a robust AE.
Furthermore, because of the interplay of these transporters
with AE and the Na
-ATPase, stimulation of NHE1, as
well as of NKCC1, can affect [Cl
and [K
, both essential
modulators of multiple cell functions from effects on V
excitability to cell death and proliferation (283, 342). With
respect to cations, the eventual result of both NHE1 and
NKCC1 stimulation under conditions of optimal Na
ATPase activity is increased [K
, rather than increased
(see Ref. 40). However, as will be discussed below,
-ATPase activity is frequently compromised in
stressed cells, such that both NHE1 and NKCC1 can elicit
sizable increases in [Na
. Finally, whereas either NHE1
coupled with AE, or NKCC1 by itself, can increase [Cl
should be noted that functional coupling of AE with these
transporters may have opposite effects on [Cl
, i.e., 1:1
coupling of NHE1 and AE will tend to increase [Cl
, whereas
1:1 coupling of NKCC1 with AE is more likely to limit
increases in [Cl]
The overall aim of this review is to provide the reader with
the knowledge required to appreciate the respective roles of
NHE1 and NKCC1 in the detrimental events resulting from
ischemia and severe hypoxia in the heart, brain, and blood.
Thus we will first (section 2) provide an update on the defining
characteristics of these two transporters. This will be followed
(section 3) by an overview and comparative analysis of the
roles of NHE1 and NKCC1 in the response to a variety of
physiological stressors and, finally, (section 4) by an in-depth
description of the involvement of NHE1 and NKCC1 in ische-
mia and severe hypoxia in the heart, brain, and blood, respec-
tively. A brief overview (section 5) of the relatively scarce
evidence regarding the roles of NHE1 and NKCC1 in apoptosis
and necrosis in ischemia/hypoxia concludes the review.
2 NHE1 and NKCC1: Defining Characteristics
2.1 NHE1
2.1.1 Tissue and cellular localization. NHE1 (SLC9A1, Fig.
1A) belongs to the family of Na
exchangers of which nine
are identified to date (195, 209). NHE1 is expressed in virtually
all cell types studied (209, 222, 238) with avian red blood cells
(RBCs) as a notable exception in that they lack volume-
sensitive Na
exchange while exhibiting robust NKCC1
activity (140). In polarized cells, NHE1 is generally located in
the basolateral (abluminal) membrane (see Ref. 209). In the
Fig. 1. Na
exchanger isoform 1 (NHE1) and Na
porter isoform 1 (NKCC1). Predicted topology and major regions of impor-
tance for regulation. A: NHE1 (SLC9A1). PtdIns(4,5)P
, phosphatidyl-
inositol(4,5)bisphosphate; CAII, carbonic anhydrase II; PP1, protein phospha-
tase 1; NIK, Nck-interacting kinase; CHP, calcineurin homolog protein. B:
NKCC1 (SLC12A2). SPAK, ste20-related proline- and alanine-rich kinase.
Invited Review
AJP-Regul Integr Comp Physiol VOL 291 JULY 2006
microvascular endothelial cells of the blood-brain barrier
(BBB), NHE1 appears to contribute to secretion of Na
and water into the brain during ischemia (as discussed in
section 4); however, it is as yet unknown whether NHE1
localizes to the luminal or abluminal membrane of the BBB
cells (68, 307). At least in some cells, NHE1 localizes to
discrete plasma membrane subdomains, examples being the
preferential localization to focal adhesions in fibroblasts (97)
and to intercalated discs and transverse tubules in cardiomyo-
cytes (227).
2.1.2 Energetics, kinetics, and control. The driving force for
exchange via NHE1 is ⌬␮(Na
) ⫽⌬Na
RT ln ([Na
) RT ln ([H
where R is the gas constant, T is the temperature in degrees
Kelvin, and ln is natural logarithm. Given the substantial
inwardly directed gradient for Na
and modest outward gra
dient for H
prevalent under normal steady-state conditions,
there is a substantial driving force for net Na
uptake and H
efflux. Even after prolonged cardiac ischemia, where [Na
may be increased to about 30 mM and pH
and pH
decreased, there is still a large driving force favoring net Na
uptake and H
efflux via NHE1 (G ⫽⫺3.6 J/mmol). NHE1
exhibits simple Michaelis-Menten dependence on extracellular
with a reported apparent K
of 5–50 mM. NHE1 is
inhibited by extracellular H
, in part by competition with
extracellular Na
(see Ref. 209). In contrast, dependence on
intracellular H
is allosteric with a Hill coefficient of 2. This
phenomenon, which has been attributed to the presence of a
modifier site,” ensures that NHE1 is essentially silent in
most cell types at normal pH
, but efficiently stimulated by
acidic deviations from this “set point pH
(see Ref. 209).
Although acidification-induced stimulation increases net
NHE1 flux as a consequence of the combined effects of
increased substrate availability and [H
interaction with an
allosteric regulatory site, stimulation of NHE1 by hormones
and growth factors is generally found to be associated with an
alkaline shift in the set point pH
(e.g., a reduced K
intracellular H
), resulting in stimulation at normal pH
. This
process appears to be at least partially dependent on protein
phosphorylation/dephosphorylation events, although not nec-
essarily involving direct phosphorylation of NHE1. A full
discussion of this is outside of the scope of the present review
(for recent reviews, see Refs. 222 and 238).
2.1.3 Molecular structure. The NHE1 protein consists of
780 820 amino acids with a calculated molecular mass of
87–91 kDa. Hydropathy analyses in conjunction with studies
of protease cleavage and cysteine accessibility support a topol-
ogy model with 12 transmembrane (TM) domains, a reentrant
loop between TM 9 and TM 10, and a long cytoplasmic-
COOH-terminal region consisting of about 300 amino acids
(272, 320) (Fig. 1A). The mammalian NHE1 contains consen-
sus sites for both N- and O-linked glycosylation in the first
extracellular loop, and N-linked glycosylation was proposed to
play a role in the basolateral sorting of NHE1 (51). High-
resolution structures are not available for the vertebrate
NHE1s; however, recent studies of two-dimensional crystals of
the Escherichia coli NHE, NhaA, also suggest a 12 TM
structure (326). Furthermore, these studies indicated that NhaA
may assemble as a dimer (326), which is in agreement with
studies of the mammalian NHE1 (104).
The NH
-terminal domain with the 12 TM regions (500
amino acids) is highly conserved between vertebrate species
and is responsible and sufficient for ion translocation by NHE1
(319). The COOH-terminal cytoplasmic domain (300 amino
acids) is the main site of regulation of NHE1 function and
exhibits a considerable sequence variation between species.
This COOH-terminal domain contains binding sites for a large
number of ancillary proteins and lipid mediators, primarily in
the region closest to the TM domains (Fig. 1A). Although not
all of these will be further discussed in this review, it should be
mentioned that NHE1 binding partners shown to interact with
the COOH-terminal domain include the plasma membrane-
cytoskeleton linker ezrin [of the ezrin/radixin/moesin (ERM)
protein family], calcineurin homolog protein (CHP), the ste20-
related kinase Nck-interacting kinase, protein phosphatase 1
(PP1), calmodulin, carbonic anhydrase, and the phospholipid
phosphatidyl-inositol(4,5)bisphosphate. The most COOH-ter-
minal part of this region contains multiple protein kinase
consensus sites, the roles of which in NHE1 regulation have
been extensively studied (see Refs. 181, 209, 222, and 238).
2.1.4 General physiological roles. By far the best character-
ized physiological functions of NHE1 are the regulation of
cellular volume and pH, although other roles have also been
described (see Refs. 209, 221, and 238). After cell shrinkage,
rapid NHE1-mediated Na
influx in conjunction with Cl
influx via the AE restores cell volume, while after intracellular
acidification, the NHE1-mediated H
efflux efficiently restores
(see, e.g., Refs. 209 and 222). Under normal physiological
conditions, i.e., as long as such deviations from steady state are
moderate and transient, this has the effect of normalizing cell
volume or pH
, whereas after dramatic or prolonged deviations
from steady-state pH
or volume, NHE1-mediated effects on
and pH
are important in the cellular damage associated
with such conditions, as will be discussed below.
An important aspect of NHE1 regulation is the effect of
interactions between different stimuli. In Amphiuma tridacty-
lum RBCs, when stimulated by intracellular acidification,
NHE1 remained active until pH
reached its set point, and was
not turned off by the cell swelling resulting from NHE1
activity (40). Conversely, when stimulated by cell shrinkage,
NHE1 remained active until cell volume was restored, in spite
of a substantial increase in pH
(40), which, if acting alone,
would otherwise deactivate NHE1. Similarly, a wide range of
cell culture studies show that after stimuli, such as hormones,
growth factors, and inhibitors of ser/ser phosphatases, which
activate NHE1 at normal pH
and volume, the exchanger can
remain active in spite of substantially increased cell volume,
, and [Na
(224 –226, 260, 318). Such increases in cell
volume and pH
may modulate, e.g., cell proliferation (see Ref.
221). This has led to the proposal that increased NHE1 activity
is a prerequisite for tumor cell proliferation, a notion which
has, however, been disputed by other workers in the field (for
a discussion, see Refs. 221 and 274). Again, a detailed discus-
sion of this is beyond the scope of this review, but it should be
noted that in recent years, the physiological roles of NHE1
have been suggested to include modulation of, e.g., cell mor-
phology, migration, and invasion (see Refs. 209 and 238).
Interestingly, these roles of NHE1 appear in some cases to be
at least partly unrelated to ion transport, and rather involving
direct interactions of NHE1 with, e.g., ERM proteins (see Ref.
Invited Review
AJP-Regul Integr Comp Physiol VOL 291 JULY 2006
Consistent with the fundamental roles of NHE1 in normal
cell function, NHE1 knockout mice exhibit multiple abnormal-
ities, including locomotor problems, growth retardation, abnor-
mal membrane excitability, and Na
permeability of hip
pocampal CA1 neurons, and 70% of the knockout animals die
before weaning (20, 99). On the other hand, these mice exhibit
significant protection from ischemic damage in both the heart
and the brain (section 4).
2.2 NKCC1
2.2.1 Tissue and cellular localization. The Na
cotransporter NKCC1 (SLC12a2, also known as BSC2; Fig.
1B) is expressed in the great majority of cell types studied,
whereas the other cloned isoform, NKCC2 (SLC12a1, BSC1),
is restricted to the kidney (see Ref. 254). RBCs of certain
species, such as teleost fishes and amphibians, however, do not
appear to exhibit volume-sensitive Na
yet exhibit robust NHE activity (37, 108, 141). NKCC1 is
found on the basolateral membrane of secretory epithelial cells
(254). An exception to this is the microvascular endothelial
cells of the BBB, which secrete Na
and Cl
from the blood
into the brain. In this case, NKCC1 is located in the apical
(luminal) membrane (205) and secretion (i.e., transport from
blood into tissue) occurs in an apical-to-basolateral direction.
Also, in the choroid plexus, NKCC1 appears to be located in
the apical membrane (235).
2.2.2 Energetics, kinetics, and control. The driving force for
cotransport via NKCC1 is ⌬␮(Na
) ⫽⌬Na
2 ⌬␮Cl
RT ln ([Na
) RT ln ([K
) 2{RT ln ([Cl
RT ln ([Na
)/ ([Na
). Under
normal physiological conditions, the outwardly directed K
gradient tends to be slightly greater than the inwardly directed
gradient, such that [Cl
becomes an important determi
nant of the direction of transport via NKCC1. In most cells, the
direction of transport will be inward, although near-equilib-
rium situations are found in some cells (254). After a few
minutes of ischemia-induced NHE1-mediated Na
tion, however, the driving force for transport via NKCC1 may,
in some cases, be directed out of the cell (7, 150). In RBCs,
outward transport by NKCC1 has been proposed to contribute
to the cell shrinkage involved in reticulocyte maturation (sec-
tion 3.3.2). Increased NKCC1 activity is associated with, and
appears to be dependent on, an increase in the direct phosphor-
ylation of NKCC1 on ser and thr residues (see Refs. 78, 101,
and 254). Only a few studies have addressed the molecular
mechanism of altered transport rates via NKCC1 on regulation.
Studies in intestinal epithelial cells have suggested that at least
in some cases, regulation of Na
cotransporter ac
tivity is associated with a change in the number of transporters
in the plasma membrane, i.e., an effect on V
, rather than on
substrate affinity (83, 170). On the other hand, in HeLa cells,
ATP depletion reduced NKCC1 activity with no effect on V
but with reduced affinity for extracellular Rb
2.2.3 Molecular structure. The NKCC1 protein consists of
about 1,200 amino acids and has a calculated molecular mass
of about 130 kDa (see Refs. 101 and 254). On the basis of
hydropathy analyses, a membrane topology of 12 central TM
domains and cytosolic NH
- and COOH-terminal regions has
been proposed (88) (Fig. 1B). NKCC1 contains two N-linked
glycosylation sites in the fourth extracellular loop. Both the
- and COOH-terminal cytosolic domains contain protein
kinase consensus sites, and, in addition, the NH
cytosolic domain has been shown to bind PP1 (57) and the
ste20-related proline- and alanine-rich kinase (SPAK) (231;
see Fig. 1B). NKCC1 has been proposed to form homodimers,
although a functional role for this has yet to be established
(184). The highest degree of conservation between species is
found in the central TM and COOH-terminal regions, although
three consensus sites for thr phosphorylation in the NH
terminal region are conserved across a broad range of species
(see Refs. 101 and 254).
2.2.4 General physiological roles. NKCC1 is stimulated by
a wide range of hormones and growth factors, as well as by
osmotic shrinkage (161, 192; see also Refs. 78 and 254),
laminar stress (296), and hypoxia or deoxygenation (79, 81,
131, 192). In contrast to the marked stimulation of NHE1 in
response to acidic pH
, NKCC1 is inhibited at pH
values below
7.0 (215, see also Ref. 254). The most well-defined physio-
logical roles of NKCC1 are in the regulation of [Cl
, epithe
lial Cl
secretion, and cell volume (see Ref. 254). NKCC1 is
stimulated by osmotic shrinkage in most cell types studied,
and, if the prevailing ion gradients permit, NKCC1 can and
does play an important role in the RVI process (see Ref. 254).
Studies in epithelial and nonepithelial cell types have indicated
that NKCC1 plays a major role as a Cl
influx pathway
maintaining [Cl
above equilibrium, i.e., the equilibrium
potential for Cl
more positive than V
(5, 95). Moreover,
astrocytes from NKCC1 knockout mice exhibited reduced
basal [Na
, suggesting a role for NKCC1 in regulating basal
astrocyte [Na
(289). Vectorial NaCl transport in secretory
epithelia is dependent on the functional coupling of the baso-
laterally located NKCC1 to apical Cl
channels, basolateral
channels and the Na
-ATPase (e.g., Ref. 95). In the
BBB, NKCC1 may function in vectorial transport of NaCl
from blood into brain, and as discussed below, increased
NKCC1 activity in the BBB contributes to cerebral edema
formation in ischemia (81, 203, 205). A role for NKCC1 in
regulation of cell proliferation has also been proposed, al-
though this is less well established (see Ref. 254).
Consistent with the proposed major physiological roles of
NKCC1, the phenotype of NKCC1 knockout mice includes
deafness, imbalance (Shaker/Waltzer phenotype), and de-
creased Cl
secretion in e.g., intestine and trachea (60, 77).
However, as will be discussed in section 4, they also exhibit
less brain damage in experimental stroke (47).
3 NHE1 and NKCC1 in the Response to Physiological Stress
As described above, NHE1 and NKCC1 play important roles
in reestablishing homeostasis after physiological perturbations
to cell volume, pH
, and [Cl
, and in the adaptive responses
to a variety of physiological stressors. We define physiological
stress in broad terms as the stimuli to which cells are exposed
under normal physiological conditions, resulting in temporary
deviations from the steady state. The relevant physiological
stressors are obviously numerous and will depend on the
species and cell type, but for the purpose of the present review,
we focus on three major categories: metabolic and exercise
stress, osmotic stress, and mechanical stress. All of these
conditions, if sufficiently severe, may of course become patho-
Invited Review
AJP-Regul Integr Comp Physiol VOL 291 JULY 2006
physiological; for instance, the ability of severe hypertonic
stress to elicit cell death is well established (29, 36, 201).
Metabolic stress in the form of a reduced cellular ATP level
may result from, e.g., exercise stress, mild hypoxia, or reduced
availability of nutrients, and is typically associated with re-
duced pH
, as well as with changes in the cellular level of
reactive oxygen species (ROS). Exercise stress exhibits several
similarities with hypoxic stress at the level of plasma hormone
and ion concentrations. In mammals, vigorous exercise is
associated with reductions in microvascular oxygen pressure
) (19, 125). Also similar to hypoxic conditions, exercise is
associated with catecholamine release and increased plasma
catecholamine levels, with elevated plasma [K
] because of
substantial K
loss from skeletal muscle, and with plasma
acidification (see, e.g., Refs. 153 and 172).
The ability of cells to regulate their volume is highly
conserved and often involves multiple osmolyte transport
mechanisms, reflecting the fundamental importance of cell
volume for cell function (see Ref. 106). As noted above, after
acute osmotic shrinkage, some, yet not all, cell types are
capable of regulating their volume in the process of RVI, and
NHE1 and NKCC1 are the most common mediators of this
process (107, 209, 254). Most of the cells in the body are not
exposed to major changes in extracellular osmolarity under
physiological conditions. Of the tissues of interest in this
review, RBCs are, however, an important exception, as they
are exposed to a major hypertonic challenge on their way
through the kidneys (see Ref. 106). Much more common are
changes in epithelial cell volume resulting from changes in
intracellular solute concentration, e.g., after secretion/absorp-
tion, and exercise- or hormone-mediated metabolic changes
(see Ref. 106). Notably, therefore, metabolic and osmotic
stress are in some cases functionally coupled. Similarly, me-
chanical stress (see below) elicits ion channel activation (e.g.,
Refs. 34 and 296), resulting in osmolyte loss and therefore cell
Mechanical stress in the form of, e.g., stretch or fluid shear
stress is experienced by many cell types. Cardiac myocytes,
cardiac and brain endothelial cells, and RBCs are exposed to
mechanical stress under physiological conditions (245, 256,
287), and neurons and glial cells can also experience a range of
mechanical stresses, ranging from physiological mechanical
stimuli to traumatic brain injury (160). Mechanical stress
elicits a wide range of signaling events, including activation of
integrins and growth factor receptors, G protein activation,
cytoskeletal rearrangement, ATP and ROS release, increases in
, and activation of mitogen-activated protein kinases
(MAPKs) and other protein kinases. These events, in turn,
elicit both acute activation of ion transporters, and activation of
transcription factors eliciting the induction of genes involved in
controlling, e.g., cell growth, morphology, and ion transport
(213, 220, 296; see also Ref. 76).
Tables 1 and 2 summarize the localization and major phys-
iological roles of NHE1 and NKCC1, respectively, in the heart,
brain, and blood. In the following sections, these roles will be
described, in turn, for those stimuli for which evidence is
available in the tissue in question.
3.1 Physiological Stress in the Heart
The heart depends primarily on aerobic metabolism under
physiological conditions, yet ATP produced through the gly-
colytic pathway appears to be necessary for some cardiac
functions, including Na
homeostasis (42). The proportion of
ATP produced glycolytically is further increased during exer-
cise and other physiological conditions of increased energy
demand, leading to an increased requirement for H
(42, 92). Cell volume perturbations in the heart occur predom-
inantly under pathophysiological conditions, such as diabetic
coma, septic shock, or ischemia (327). Hypertonic cell shrink-
age negatively affects cardiac contractility, and hypotonic
swelling shortens the cardiac action potential (110, 327). In
spite of these severe consequences of cardiomyocyte volume
perturbations, the extent to which cardiomyocytes are able to
volume regulate is a controversial issue, with most studies
concluding that these cells do not perform RVI (206, 327).
Shrinkage-induced stimulation of both NHE1 and NKCC1 has,
however, been reported in the heart, as will be discussed
3.1.1 Roles of NHE1. NHE1 appears to be the only, or by far,
the predominant NHE isoform in cardiac myocytes (80, 210,
310), where it has been shown to localize mostly to the
transverse tubules and intercalated disc regions (227). Consis-
tent with the high demand for metabolic acid extrusion under
Table 1. Localization and physiological roles of NHE1 in heart, brain, and blood
Tissue Localization Proposed physiological roles References
Heart Cardiomyocytes (t-tubules and intercalated discs) pH
regulation (metabolic acid extrusion)
(80, 183, 227, 325, 333)
Cell stretch response
Cell volume regulation?
Aortic endothelial cells pH
(18, 100)
Brain Neurons (all types studied) pH
regulation (metabolic acid extrusion)
(212, 223)
Glial cells (all types studied) pH
regulation (metabolic acid extrusion)
(173, 273)
Cell volume regulation
Brain microvascular endothelial cells Vectorial Na
transport pH
(68, 112, 281, 307)
Choroid plexus epithelial cells Vectorial ion transport? (127)
Blood Red blood cells Regulation of hemoglobin O
affinity and saturation
(lower vertebrates)
(37, 40, 123, 216)
Cell volume regulation
Platelets Platelet activation (247)
Leukocytes Cell volume regulation, pH
regulation, activation
(85, 96)
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steady-state conditions in the heart, the myocardial NHE1
exhibits high tonic activity and plays a major role in cardiac
homeostasis under physiological conditions (80). In con
gruence with the proposed importance of glycolytically derived
ATP for cardiac Na
homeostasis (42), NHE1 has been pro
posed to be specifically dependent on ATP produced by gly-
colysis rather than by oxidative phosphorylation (291). This
linkage has interesting implications, because NHE1 stimula-
tion may be preferentially coupled to ATP produced under
conditions (i.e., increased glycolysis) that necessitate increased
As in most cell types, NHE1 is also stimulated by hypertonic
stress in both mature and neonatal cardiac myocytes (183,
325). This may, however, not lead to an RVI response, as it has
been suggested that mature cardiomyocytes cannot perform
RVI (327).
NHE1 plays an important role in the physiological events
elicited in the heart by mechanical stress in the form of cell
stretch. In isolated rabbit myocardium, the slow force response
component of the muscle stretch response was dependent on
stretch-induced NHE1 activation, followed by increased cyto-
solic [Na
] and increased [Ca
because of Na
changer (NCX) reversal (316). Similar findings have been
reported in other cardiomyocyte preparations (333). Interest-
ingly, stimulation of NHE1 by cardiomyocyte stretch has been
found to be upstream of stretch-induced Raf-1 and MAPK
activation and subsequent hypertrophy (299, 333).
3.1.2 Roles of NKCC1. Evidence for the presence of NKCC1
in whole mammalian hearts, as well as in isolated ventricular
myocytes, has been obtained both at the functional level (7, 65,
252) and at the mRNA and protein level (6, 241, 314). NKCC1
has also been characterized in aortic endothelial cells (341).
Compared with the abundant literature on NHE1, relatively
little is known about the roles and regulation of NKCC1 in the
heart. NKCC1 likely plays a role in maintaining cardiac resting
and [Cl
(84, 114), yet we are not aware of direct
studies of the effects of physiological metabolic stress on
NKCC1 in the heart.
A role for NKCC1 in isotonic and anisotonic cell volume
regulation has been proposed in rabbit ventricular myocytes
(65) and in smooth muscle cells from rat aorta (208). Yet,
others failed to find detectable NKCC1 activity after hyper-
tonic stress in the heart (71). NKCC1 is also stimulated by
osmotic shrinkage of aortic endothelial cells in which RVI was
inhibited by bumetanide (202). The RVI was, however, not
complete by 30 min, suggesting that complete RVI is a slow
process or that the cells are unable to completely recover (202).
In other studies, an RVI response could not be demonstrated in
aortic endothelial cells (see Ref. 206). Finally, in bovine aortic
endothelial cells, mechanical stress in the form of steady
laminar shear stress activated and upregulated NKCC1, an
effect that was shown to involve shear stress-induced activa-
tion of K
and Cl
channels (296).
3.2 Physiological Stress in the Brain
In the brain, metabolic stress requiring regulation of pH
occurs under physiological conditions, because both neurons
and glial cells undergo significant shifts in pH
during in
creased neuronal activity. For instance, in cultured hippocam-
pal neurons (322) and brain stem slices (308), depolarizing
stimuli were shown to elicit intracellular acidification. This
decrease in pH
is, at least in part, dependent on Ca
entry and
in vertebrate neurons appears to mainly reflect metabolic acid
production (322, 344). Additionally, because of its permeabil-
ity to HCO
, activation of the GABA
receptor also decreases
, as shown in both cultured astrocytes (126) and acutely
isolated hippocampal neurons (219).
Under physiological conditions, the volume of both neurons
and glial cells is constantly challenged by the transmembrane
ion movements occurring during normal neuronal activity, and
these volume changes may, in turn, affect brain function (163,
218, 283). Brain volume regulation, associated with net cellu-
lar uptake of Na
, and water, and a concomitant
reduction in extracellular volume, has been measured in vivo in
rats exposed to acute hypernatremia (54). Glial cells are capa-
ble of cell volume regulation after acute osmotic swelling and
shrinkage (69, 175). With respect to neurons, this is more
controversial; in several neuronal preparations, no volume
regulation could be detected after acute osmotic stress (2, 11),
and it appears that more severe (nonphysiological) osmotic
challenges are required for neurons to be capable of acute RVI
(see e.g., Ref. 53). On the other hand, immature cortical
Table 2. Localization and physiological roles of NKCC1 in heart, brain, and blood
Tissue Localization Proposed physiological roles References
Heart Cardiomyocytes Cell volume regulation? (65, 71)
Regulation of basal [Na
and [Cl
(84, 114)
Aortic endothelial cells Cell volume regulation? (202, 206, 296, 341)
Responses to shear stress
Brain Neurons Regulation of [Cl
and GABAergic signaling
(265, 295)
Cell volume regulation?
Glial cells Cell volume regulation? (45, 175, 289)
Regulation of basal [Na
GABAergic trophic effects
Brain microvascular endothelial cells Vectorial ion transport (202, 204, 205)
Cell volume regulation?
Choroid plexus epithelial cells Vectorial ion transport (330)
Blood Red blood cells Regulation of plasma [K
(89, 158, 161, 165)
Cell volume regulation
Leukocytes Regulation of MAPK activity and proliferation (214)
, intracellular Na
concentration; [Cl
, intracellular Cl
concentration; [K
], K
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neurons exhibited RVI after a relatively modest increase in
extracellular osmolarity (265).
3.2.1 Roles of NHE1. NHE1 is widely expressed in the
central nervous system (CNS) and is the most abundant NHE
isoform in the cerebral cortex (162, 210). Na
activity has been found in virtually all neuronal and glial cell
types studied, although it was not always unequivocally estab-
lished whether the isoform in question was NHE1 (48). This is
an important question, since NHE2, NHE3, NHE4, and NHE5
are also found in the brain, although with more restricted
patterns of distribution (48). NHE1 message has also been
detected in choroid plexus (127; Praetorius J, personal com-
munication), which is known to exhibit amiloride-sensitive
exchange (191). Finally, message for NHE1 (and also
for NHE2, NHE3, and NHE4) was detected in BBB endothelial
cells (68, 127, 281, 281, 315), and there is evidence that NHE1
is an important mediator of steady-state Na
transport across
the BBB (68).
NHE1 plays a key role in pH
regulation in both neurons and
glial cells under steady-state conditions and after intracellular
acidification (48, 162, 173, 212, 223). Recovery of pH
metabolic acid production associated with neuronal activity
(see section 3.2) appears to be a major function of NHE1 in
brain neurons. Consistent with this view, both acutely dissoci-
ated CA1 pyramidal neurons from NHE1 null mice (339) and
astrocytes from NHE1 null mice (137) exhibit reduced steady-
state pH
, as well as reduced or virtually absent H
after intracellular acidification. Shrinkage-induced NHE1 stim-
ulation has been demonstrated in primary rat astrocytes (273).
No studies have, to our knowledge, directly demonstrated
osmotically induced NHE1 activity in neurons, neither in the
intact brain nor in culture.
3.2.2 Roles of NKCC1. NKCC1 mRNA has been detected in
choroid plexus, cerebellum, brainstem, and to a lesser extent,
cerebral cortex and hypothalamus (128, 235), reflecting
NKCC1 expression in a wide range of neuronal cell types (129,
235), in glial cells (oligodendrocytes and astrocytes) in various
regions of the brain (128, 150, 205, 323, 335), and in cerebral
microvascular endothelial cells (203, 205, 292, 293). In the
latter, functional and immunocytochemical evidence indicated
the presence of NKCC1 in the luminal membrane (202, 205),
although others have failed to detect brain microvessel NKCC1
mRNA in in situ hybridization studies (235). Interestingly, in
the choroid plexus epithelium, NKCC1 is extremely abundant
and is only expressed on the apical membrane (235).
The main physiological roles of NKCC1 in the brain appear
to be 1) the regulation of [Cl
and thus, -aminobutyric
(GABA)-ergic signaling (GABA being excitatory at high
, and inhibitory at low [Cl
) (59, 295); 2) ion transport
across the BBB (81, 203, 205) and choroid plexus (330); and
3) although this is less well established, in cell volume regu-
lation (see below). A role in regulating basal [Na
has also
been suggested, based on studies of NKCC1
NKCC1 expression is high in immature neurons and de-
creases with maturation, whereas the inverse is true for the
cotransporter KCC2 (see Ref. 59). These changes are
thought to underlie the decrease in neuronal [Cl
development and the shift from excitatory to inhibitory GABA
signaling (see Ref. 59). NKCC1 still contributes to regulation
of [Cl
in adult neurons, some of which, including, e.g.,
dorsal root ganglion neurons, exhibit a high NKCC1 expres-
sion, high [Cl
, and depolarizing responses to GABA (see
Ref. 59).
In neurons capable of cell volume regulation after osmotic
shrinkage, NKCC1 may play a role in this process. In immature
cortical neurons, NKCC1 was activated by osmotic shrinkage,
and a bumetanide-sensitive RVI was demonstrated (265). Os-
motically induced Na
cotransport was also ob
served in squid giant axons (32) and in PC12 cells (151). In C6
glioma cells, NKCC1 appears to be important in both isotonic
volume homeostasis and RVI (45, 175). Similarly, RVI was
robust in wild-type (NKCC1
) astrocytes, but absent in
astrocytes (289). NKCC1 is also stimulated by
osmotic shrinkage in cerebral microvascular endothelial cells,
although it is not yet known whether this translates into an RVI
response in these cells (202).
3.3 Physiological Stress in the Blood
Exercise stress increases [K
, [Na
, and [Cl
in human
RBCs (153, 172), and as will be discussed below, in some
species, RBCs may play a role in buffering plasma K
, at least
during high-intensity exercise (153). RBCs from teleost fishes
have been extensively used as models for mild hypoxic stress,
given the often extremely high exercise level of these species,
frequent exposure to hypoxic conditions, and unique require-
ment for adaptation to large changes in P
, as well as the fact
that teleost RBCs are nucleated cells with protein synthesis,
mitochondria, etc., resembling those of mammalian non-RBC
cells (e.g., Refs. 49 and 198). One general finding, pioneered in
studies in RBCs from teleosts and other lower vertebrates, and
confirmed in some mammalian RBCs, is that a number of ion
transporters including, yet not limited to, NHE1 and NKCC1,
exhibit marked O
dependence (64; see Ref. 89). A role for
hemoglobin (Hb) as an O
sensor was suggested (89, 186);
however, findings in trout hepatocytes indicate that this phe-
nomenon is not limited to RBCs (311). A link with cell volume
is suggested in that swelling-activated transporters are gener-
ally found to be stimulated, and shrinkage-activated transport-
ers to be inhibited, by a decrease in O
pressure (PO
) (see Ref.
3.3.1 Roles of NHE1. NHE1 appears to be the only NHE
isoform in mammalian RBCs (e.g., Ref. 259). Although NHE1
transport capacity decreases with RBC maturation (41) and is
modest in most mammalian RBCs, including those of humans,
NHE1 is present and functional in mature RBCs from many
mammals, including dogs (216), rabbits (123), and humans
(30). Moreover, many nucleated RBCs, such as those of
teleosts (167, 225) and amphibians (174), exhibit robust
activity. In RBCs from both mammals and lower
vertebrates, NHE1 is activated by acidic pH
, as well as by
osmotic shrinkage (37, 123, 269). While RBCs are the focus
here, it should be noted that NHE1 is also the predominant or
only NHE isoform in platelets and at least some types of
leukocytes, in which it has been assigned important physiolog-
ical roles (85, 96, 247).
The teleost RBC NHE is denoted NHE for its -adrenergic activation, yet
it is much more homologous to NHE1 than to any other NHE isoform (in some
cases, the homology between a given NHE and an NHE1 is higher than
between NHE1 orthologs from different species). Hence, NHE will be
grouped as an NHE1.
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AJP-Regul Integr Comp Physiol VOL 291 JULY 2006
Exercise stress elicits an increase in circulating cat-
echolamines in teleosts, resulting in stimulation of NHE1, and,
consequently, increased RBC pH
and cell volume (159),
similar to what occurs in hypoxia (see section 4.1) or after
direct -adrenergic stimulation (225; see 222). In exercise-
stressed human RBCs, increased Na
following exercise stress has been described (1); however, the
possible identity of this transporter with NHE1 is controversial
In RBCs of teleosts and some amphibians, NHE1/-NHE is
stimulated under hypoxic conditions, by norepinephrine (NE)
via -adrenergic receptors (-ARs), via decreased pH
, and via
a still poorly understood deoxygenation effect on the RBC
NHE1 (see Refs. 89 and 222). The physiological consequence
is that in response to hypoxic or exercise stress, the O
of Hb is increased because of a combination of the NHE-
mediated limitation of intracellular acidification, and dilution
of Hb resulting from cell swelling (see Ref. 222). To the
knowledge of the authors, no such studies exist for NHE1 in
higher vertebrates, which, as discussed by Nikinmaa (197),
generally have Hbs with lower Bohr/Haldane effects and em-
ploy different strategies for optimizing RBC O
Osmotic stress potently stimulates NHE1 in RBCs from a
wide variety of species, with the interesting exception of some
teleosts (see Ref. 222). The first demonstration of the role of
exchange functionally coupled to Cl
change in RVI came from studies of A. tridactylum RBCs in
our laboratory (37). This is now known to be a general strategy
for RVI in a wide variety of cell types across the vertebrate
phylum (106, 222), and the Amphiuma Na
exchanger has
been cloned and found to be an NHE1 with high homology to
mammalian NHE1s (174).
3.3.2 Roles of NKCC1. There is functional evidence for the
presence of NKCC1 in RBCs from birds (161), as well as from
mammals, including humans (79, 153, 165). Similar to NHE1
in teleost RBCs, NKCC1 in avian RBCs is stimulated by
hypoxia and metabolic stress, by virtue of its potent stimulation
both by -adrenergic stimuli and by deoxygenation per se
(161, 192). As discussed by Gibson et al. (89), the driving force
for inward transport via NKCC1 in RBCs is markedly in-
creased by even slight elevations of plasma [K
], and a role for
NKCC1 as a dynamic buffer for plasma [K
] is conceivable.
This may play a role in birds (158), which exhibit robust
NKCC1 activity and experience extreme exercise stress during
migratory flight, but also in mammals with high hematocrits,
such as the horse (see Ref. 89). In humans, RBC K
content is
also elevated during exercise; however, this appears to be
primarily mediated by the Na
-ATPase, with little, if any,
contributions from NKCC1, except under conditions when the
-ATPase is inhibited (153). NKCC1 is also activated
by osmotic cell shrinkage in both avian (161) and mammalian
RBCs (165). It is interesting to note that as rat RBCs mature,
the net driving force for NKCC1 changes direction, being
inward in reticulocytes, yet outward in mature RBCs. This net
outward transport by NKCC1 may under some conditions
contribute to reducing the isotonic volume of the RBCs (166;
see also Refs. 158 and 165).
Finally, it may be noted that NKCC1 has also been found in
leukocytes, in which it has been proposed to play a role in
regulation of MAPK activity and proliferation (214).
4 NHE1 and NKCC1 in Pathophysiological Stress: Ischemia
and Severe Hypoxia
Under certain pathological conditions, stimulation of NHE1
or NKCC1 does not, as in the examples described in the
previous sections, lead to restoration of homeostasis, but rather
contributes significantly to the cellular damage resulting from
these conditions. Of these, ischemia (severely reduced perfu-
sion or blood flow), and severe hypoxia (including anoxia, i.e.,
zero or essentially zero oxygen supply, and prolonged hypoxia)
are by far the most studied with respect to the roles of NHE1
and NKCC1, yet many open questions and controversies re-
main. Some of the apparently conflicting findings are likely to
reflect differences in experimental protocols. For instance,
studies of ischemia have employed either only ischemia or
ischemia/reperfusion protocols and with greatly varying expo-
sure times. Moreover, with respect to studying hypoxia at the
cellular level, various labs have used either chemical “hyp-
oxia” (such as oligomycine or azide) in the presence of oxygen
or various degrees of true hypoxia, with or without glucose,
differences that can elicit very different responses. Finally, at
least in the brain, the majority of “hypoxia” studies have
actually evaluated the in vitro effects of oxygen-glucose de-
privation (OGD).
4.1 Ischemia and Severe Hypoxia in the Heart
4.1.1 General Aspects Energy status and ion homeostasis. Ischemia is
associated with a marked decrease in the cellular ATP level in
the heart (see Ref. 42). It should be noted that the reported rates
of decline of ATP levels in ischemic perfused hearts vary
considerably, from about one-third reduction within 45 min in
ferret hearts (66) to about 50% reduction within 15–20 min in
rat hearts (243, 298). Such differences in ATP levels are likely
to be due, at least in part, to differences in experimental
protocol (see Ref. 4) and may well account for some of the
controversy discussed below regarding transporter regulation
(sections 4.1.2 and 4.1.3).
The Na
-ATPase has generally been assumed to be
inhibited in ischemia because of ATP-depletion, a notion that
has been confirmed in several models of ischemia in the heart
(52, 98, 285). Consequently, the ischemia-induced increase in
in the heart was originally thought to reflect inhibition
of the Na
-ATPase (98, 285). On the other hand, others
have found no evidence of Na
-ATPase inhibition in early
ischemia (237), and more recent studies in perfused rabbit
hearts have indicated that in early ischemia, Na
activity is actually increased rather than decreased, presumably
as a consequence of the increases in [Na
and [K
(7). As
will be detailed below, the ischemia-induced increases in
and [Ca
(10, 271; see also Refs. 189 and 284) are
now known to largely reflect stimulation of NHE1 and conse-
quent uptake of Ca
via the NCX in response to increases in
. In agreement with the notion that NCX reversal/equilib
rium plays an important role in the ischemia/reperfusion-
mediated increase in [Ca
in the heart (268), NCX has been
shown to be near or at thermodynamic equilibrium during
ischemia (for a discussion, see Ref. 8), indicating that it is a
major Ca
influx pathway under these conditions. Increases in
during ischemia have also been demonstrated in the
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AJP-Regul Integr Comp Physiol VOL 291 JULY 2006
heart (242). Ischemia, moreover, elicits cardiac cell swelling,
because of a combination of net inward ion transport and
lactate production by anaerobic glycolysis (13, 327). Cardiac
pH homeostasis is also severely compromised by ischemia. On
exposure to ischemic conditions, pH
in the heart reaches about
6 6.5, and pH
falls rapidly, by about 0.5 pH unit in the first
5 min (155, 261, 286). Signaling events. Myocardial ischemia activates the
sympathetic nervous system and leads to local release of the
catecholamine NE in the ischemic heart (267). In early cardiac
ischemia and under normal physiological conditions, NE re-
lease is exocytotic and Ca
dependent, whereas in prolonged
ischemia it is carrier mediated and reflects reversal of the
driving force for the NE transporter (NET) (152). The renin-
angiotensin system also is activated in ischemic hearts, result-
ing in increased local angiotensin (ANG II) formation. Con-
sistent with a role for ANG II in ischemic damage, ANG II
type 1 (AT
) receptor blockade reduces cardiac damage after
ischemia, by mechanisms which are not fully elucidated (115,
122). Endothelin-1 (ET-1) contributes to myocardial damage in
ischemia (see Refs. 133 and 297), and at least some of the
cardiac effects of ANG II are mediated by ET-1 (185). Local
thrombin levels are elevated in intracoronary thrombosis, the
most frequent cause of acute myocardial ischemia (58). Fi-
nally, cytokines and inflammatory mediators play an important
role in myocardial ischemia, in which neutrophil accumulation
occurs, at least in part, as a result of increased IL-8 production
and contributes to ischemic damage (82).
MAPKs, including extracellular signal-regulated kinase
(ERK1/2) and the stress-activated kinases p38 MAPK and
c-Jun NH
-terminal kinase (JNK) are important intracellular
mediators after cardiac hypoxia and ischemia/reperfusion (12).
There are some discrepancies as to whether these kinases are
active in both ischemia and reperfusion. In some studies,
ERK1/2 appears to be inhibited rather than activated in ische-
mia, and only activated on subsequent reperfusion (182; see
Ref. 12). The ERK1/2 effector p90 ribosomal S kinase
(p90RSK) has been found to be activated in the heart during
both ischemia and reperfusion (182). Other classes of protein
kinases are also implicated, for instance, there is evidence that
at least some of the effects of G protein-coupled receptor
agonists in cardiac ischemia, including catecholamines (acting
-ARs), ANG II, ET-1, and thrombin, are mediated via
protein kinase C (PKC) (14).
Finally, reperfusion after ischemia/hypoxia is associated
with release of ROS, for which a major role has been clearly
established in the ensuing cell damage (91, 164). Notably, ROS
are also released during hypoxia per se and may be an impor-
tant signal in hypoxia sensing (102).
4.1.2 Mechanisms and Consequences of Stimulation
of NHE1
From studies in isolated cardiac myocytes, perfused hearts,
and in vivo models, it is widely acknowledged that NHE1
inhibitors exert protection against myocardial ische-
mic/hypoxic damage (138, 145, 155, 190, 286, 324). The
essential role of NHE1 inhibition in cardiac protection was
initially challenged by the fact that amiloride and amiloride
derivatives also exert a varying inhibitory effect on Na
channels (46) and on NCX (276). The finding that mice with an
NHE1 null mutation are protected from myocardial ischemic
damage (324) mitigates this concern. However, although a
detailed description of this is beyond the scope of the present
review, it should be kept in mind that noninactivating Na
channels, Na
cotransport, in some cells NKCC1 (sec
tion 4.1.3), and at least in late ischemia Na
inhibition, also have been found to contribute to cardiac ische-
mic damage (52, 132, 176, 312).
A controversy has been whether NHE1 is active during the
ischemic phase, and consequently whether NHE1 inhibitors
need to be present throughout ischemia/reperfusion, or in the
reperfusion phase only, to be effective (4, 189). Thus some
studies have shown that although [Na
rose during the
subsequent reperfusion phase, there was no or only a very
small increase in [Na
during ischemia, suggesting that
NHE1 is inhibited during this phase (182, 331). In contrast,
numerous other studies have documented an immediate, robust
increase in [Na
during ischemia and have concluded that
NHE1 is active during both ischemia and reperfusion, although
generally most active in the latter (155, 190, 232, 324; see also
Ref. 189). Since pH
rapidly falls to 6 6.5 in ischemic hearts
(see section, inhibition of NHE1 during the ischemic
phase has been proposed to result from competition between
and H
for NHE1 binding (149). However, at the very
acidic pH
which is prevalent in ischemic hearts, NHE1 is only
modestly inhibited by acidic pH
(313; see also Ref. 4). NHE1
has a high effective half-maximal ATP concentration (2–5
mM) (43, 61) and has, accordingly, been found to be inhibited
by the reduced ATP levels associated with metabolic inhibition
(43, 329). Because the reported rate of decline of ATP levels in
ischemic hearts varies considerably (see section, dif-
ferences in ATP levels, in part, the result of differences in
experimental protocol (see Ref. 4), undoubtedly account for
some, if not all, of the controversy with regard to the activity
of NHE1 during ischemia. Mechanisms of stimulation of NHE1. Figure 2 sum-
marizes major pathways of NHE1 regulation in ischemia/
hypoxia. A major stimulus for maintaining high NHE1 activity
during ischemia/reperfusion is the decreased pH
. Consistent
with this notion, the rate and pharmacological profile of Na
uptake during hypoxia and during NH
Cl-induced acidification
were similar in perfused rabbit hearts (10). In addition to pH
many other mechanisms have been reported to modulate car-
diac NHE1 activity during ischemia and severe hypoxia. In the
mammalian heart, NHE1 is activated by catecholamines, such
as NE via
-ARs (146, 321), possibly the
A-subtype (see
Ref. 14). Consistent with the postulate that
NHE1 activation is a central event in NE-mediated myocardial
ischemic damage, NHE1 inhibition reversed the proarrhythmic
effect of an
-agonist in isolated rat hearts (340). With respect
to the -ARs, the picture is less clear, since both inhibition
(146) and stimulation (67) of NHE1 in response to stimulation
-AR have been reported.
ANG II (244, 332) has been proposed to stimulate NHE1 in
cardiac ischemia/reperfusion, although it is clear that ANG II is
not the sole activator of NHE1 after myocardial infarct (257).
ET-1 (which may, as noted above, be downstream from ANG
II) has also been found to be upstream of NHE1 in the adverse
effects of myocardial ischemia (35, 133).
ERK1/2 and/or the downstream effector p90RSK have been
found to be involved in the stimulation of NHE1 by ischemia/
Invited Review
AJP-Regul Integr Comp Physiol VOL 291 JULY 2006
reperfusion (182, 258). Supporting this notion, cardiac NHE1
stimulation by
-AR agonists, AT
, and thrombin were
attenuated by blocking the ERK1/2 pathway (see Ref. 14). In
rat hearts exposed to 30-min of no-flow ischemia and 30-min
reperfusion, NHE1 was proposed to be directly phosphorylated
by ERK1 following reperfusion, and by p90RSK during both
ischemia and reperfusion (182). In contrast, p38 MAPK was
found to negatively regulate NHE1 activity after ANG II
stimulation in vascular smooth muscle (143).
Not only MAPKs, but also PKC, is implicated in stimulation
of NHE1 by cardiac ischemia/hypoxia (243), as well as by a
number of G protein-coupled, receptor-mediated signaling
events characteristic of cardiac ischemia, including cat-
echolamines (acting via
-ARs), ANG II, ET-1, and thrombin
Finally, several studies (194, 249, 258) have implicated ROS
(specifically H
) as important mediators of NHE1 stimula
tion by cardiac ischemia/reperfusion, in some cases, via ROS-
mediated ERK1/2 activation (249, 258).
A number of ischemia-associated signaling events have also
been found to attenuate NHE1 activity. Estrogen stimulates the
release of nitric oxide (NO), a mechanism suggested to under-
lie the protection of premenopausal women against myocardial
ischemia (200). Recent evidence indicates that the protective
effect of NO in cardiac ischemia, at least in part, involves
inhibition of NHE1, an effect tentatively suggested to involve
cGMP-dependent kinase (PKG) and p38 MAPK (8, 119).
Histamine, acting via H
receptors, which are inhibitory recep
tors in cardiac adrenergic nerve endings, was also found to
inhibit NHE1, and this was proposed to be the mechanism of
-mediated protection in myocardial ischemia (280). Interest
ingly, hypertonic pretreatment appears to attenuate NHE1
activity and exert protection during hypoxia/ischemia (71,
105). This is consistent with the notion that signaling is
prioritized, such that when responding to a given stimulus (in
this case cell volume), NHE1 will no longer respond to a
second stimulus (in this case acidic pH
; see section 2.4.1). Consequences of stimulation of NHE1. Figure 2
illustrates some of the major consequences of NHE1 stimula-
tion by cardiac ischemia/hypoxia. Elevated [Ca
is generally
considered the major cause of cell damage associated with
myocardial ischemia (284). In 1985, Lazdunski and coworkers
(149) proposed the “coupled exchanger” hypothesis, which
stated that the ischemia-induced increase in [Ca
from influx, or decreased efflux, of Ca
via NCX as a
consequence of the increase in [Na
resulting from increased
Na uptake via NHE. The Lazdunski model, based on cells in
culture, has since been confirmed and extended by multiple
workers in the field (10, 39, 117, 155, 190, 286; see Ref. 264).
Among these, the authors of this review have verified the
model in both ischemia/reperfusion (7, 155, 156) and in hyp-
oxia (10, 38, 39). In perfused hearts, NHE1 inhibitors attenuate
the increases in [Na
and [Ca
during ischemia, and
improve [Na
and [Ca
recovery on reperfusion (155,
286). Moreover, in rat hearts, an inverse correlation between
accumulation during ischemia and functional recovery on
reperfusion was demonstrated, and Na
recovery on reperfu
sion was shown to be attenuated by low [Ca
or NCX
inhibition (117). This led to the conclusion that NCX-mediated
exchange of Ca
for Na
during ischemia is a major
mechanism of Ca
accumulation associated with reperfusion
injury (117; see Fig. 2).
The role of pH
in the downstream events after stress-
induced NHE1 activation is a complex issue. Recovery of pH
on reperfusion is attenuated by NHE1 inhibition in some (261,
286), although not all (155), studies of myocardial ischemia.
However, paradoxically, several studies have shown that inhi-
bition of NHE1 renders pH
unaltered or even less acidic in
myocardial ischemia, presumably because the NHE1-depen-
dent Na
and Ca
accumulation stimulates ATP hydrolysis
and thus H
production (8, 155, 232, 261, 286; see also Ref.
264). Moreover, both beneficial and deleterious effects of
acidic pH
during cardiac ischemia/reperfusion have been de
scribed. Elevation of pH
facilitates hypercontracture, and is
chemia-induced cardiomyocyte damage and hypercontracture
was inhibited both by blocking NHE1 and by inducing extra-
cellular acidification, which, as noted above, can also inhibit
NHE1 (26, 145). On the other hand, acidic pH
facilitates cell
death by apoptosis, at least in part, because many apoptotic
proteases and DNAses exhibit an acidic pH optimum (see also
section 5.2).
Inhibition of NHE1 by 5(N-ethyl-N-isopropyl)amiloride
(EIPA) or cariporide has repeatedly been found to reduce
ATP-depletion in cardiac ischemia (155, 232, 253, 261), and
mice exhibit better cardiac ATP preservation than
wild-type mice, both at the end of ischemia and at the end of
reperfusion (324). These findings strongly indicate that NHE1
activity contributes to ATP depletion in cardiac ischemia,
presumably to a large extent, reflecting the energetic cost of,
e.g., increased Na
-ATPase and Ca
-ATPase activity,
resulting from the NHE1-dependent increases in [Na
(for a discussion, see, e.g., Ref. 8).
In human neuroblastoma cells (a model for cardiac sympa-
thetic nerve endings), ANG II acting on AT
receptors was
found to stimulate NHE1 activity, and this led to NE release by
Fig. 2. Major mechanisms and consequences of ischemia/hypoxia-induced
activation of NHE1 in a cardiomyocyte. NO, nitric oxide; ET-1, endothelin 1;
PKC, protein kinase C; ERK1/2, extracellular signal-regulated kinase 1/2;
ROS, reactive oxygen species; NCX, Na
exchanger; ANG II, angio
tensin II.
Invited Review
AJP-Regul Integr Comp Physiol VOL 291 JULY 2006
NET reversal from reuptake to release mode (244, 280). Thus
it appears that excessive carrier-mediated NE release during
myocardial ischemia/infarction may, at least in part, reflect
reversal of the Na
-dependent NET in sympathetic nerve
endings because of the NHE1-mediated increase in [Na
Interestingly, there is evidence that NHE1 can be upstream,
rather than downstream, of MAPK activation in the heart also
under conditions similar to those occurring in ische-
mia/reperfusion. Thus ANG II- and 5-HT-mediated ERK1/2
activation in rat aortic smooth muscle cells were mediated via
NHE1-dependent Ras-Raf-MEK activation (187). In a rabbit
model of nonischemic heart failure, p38 MAPK phosphoryla-
tion, as well as apoptosis, fibrosis, myocyte cross-sectional
area, and intracellular nitric oxide synthase expression were all
significantly reduced by treatment with the NHE1 inhibitor
BIIB722 (3). In addition, NHE1 stimulation by cardiomyocyte
stretch is upstream of stretch-induced Raf-1 and MAPK acti-
vation and subsequent hypertrophy (299, 333; see also above,
section 3.1.1).
NHE1 inhibitors also attenuate cardiac myocyte swelling in
ischemia (13). Moreover, NO and OH release after cardiac
ischemia/reperfusion in rat hearts was secondary to increases in
NHE1 and NCX activity (164), suggesting that NHE1 stimu-
lation in the heart may also be upstream rather than down-
stream of the ROS formation known to contribute to ischemia/
reperfusion damage (section
Finally, NHE1-mediated cardiac endothelial cell swelling
could contribute to ischemic damage, e.g., by reducing capil-
lary diameter, potentially diminishing RBC and leukocyte
flow. Consistent with this postulate, in endothelial cells, NHE
inhibitors attenuated swelling induced by lactacidosis (18) and
by low-flow ischemia (171).
4.1.3 Mechanisms and Consequences of Stimulation
of NKCC1
Furosemide and bumetanide inhibit Na
influx and reduce
injury during hypothermic ischemia in rat hearts (252), and
findings in isolated perfused hearts from both rabbit and rat
indicate that NKCC1 is stimulated by ischemia and remains
active during reperfusion (7, 13, 252). Moreover, ischemic
preconditioning stimulated NKCC1 in hearts of newborn rab-
bits (9). It is important to note that in marked contrast to the
substantial driving force for Na
influx via NHE1, the force
driving NKCC1 can actually be directed out of the cell in
cardiac ischemia as a consequence of the robust Na
via NHE1. In rabbit hearts perfused with ouabain-containing
-free solution (i.e., outward directed driving force for
NKCC1), bumetanide augmented [Ca
elevation during is
chemia (1 h) and reperfusion, and inhibited [Na
recovery on
reperfusion. This indicates that under these conditions, NKCC1
contributes to Na
efflux during reperfusion (7). Under normal
conditions, data were consistent with the interpretation
that in the early phase of ischemia, the driving force for
NKCC1 is directed into the cell and NKCC1 contributes to
ischemia-induced [Na
elevation, whereas later in ischemia
the driving force for NKCC1 is directed outward, and NKCC1
functions as a Na
efflux pathway (7). Thus several lines of
evidence point to activation of NKCC1 by cardiac hypoxia/is-
chemia. On the other hand, although direct comparisons of the
relative roles of NHE1 and NKCC1 in myocardial ischemia/
severe hypoxia are essentially lacking, there is evidence to
suggest that at least in rat hearts, the role of NKCC1 may be
more modest than that of NHE1 (52, 241). Mechanisms of stimulation of NKCC1. The mecha-
nism(s) of ischemia-induced NKCC1 stimulation in the heart
have, to the knowledge of the authors, not been investigated
directly. As noted above (section 2.4.2), NKCC1 is generally
inhibited by acidic pH
. This would tend to limit its activity
under ischemic conditions, however, to our knowledge, the
potential effect of pH
on NKCC1 activity during cardiac
ischemia has not been studied directly.
Several signaling pathways known to be active in the ische-
mic heart (section stimulate NKCC1 and thus may
play a role in ischemic NKCC1 activation. In rat myocardium,
NKCC1 was phosphorylated and activated by catecholamines
-AR receptors in an ERK1/2 dependent manner (6).
ANG II, aldosterone, and increases in [Ca
have also been
proposed to stimulate the myocardial NKCC1 (see Ref. 6), and
thrombin is a well-known activator of NKCC1 (215). Consequences of stimulation of NKCC1. In diabetic
rat hearts, in which basal NKCC1 activity is increased, NKCC1
was found to contribute to ischemic damage, whereas no
contribution of NKCC1 to ischemic damage could be detected
in nondiabetic rat hearts (241). Bumetanide inhibited the is-
chemia-induced increase in [Na
in both control and diabetic
hearts, but significantly more so in the latter, and only in
diabetic hearts was NKCC1 inhibition associated with reduced
ATP depletion and improved functional recovery on reperfu-
sion (241). This suggests that, at least in the rat, the role of
NKCC1 in cardiac ischemia may be modest; essentially no
influx was noted in another study of
rat hearts exposed to low-flow ischemia (52). Whether this is
particular to rats is, to our knowledge, unknown. Recent
findings in newborn rabbit hearts suggest that NKCC1, when
functionally coupled with Cl
exchange, can be linked
to changes in pH
during ischemia (that is, Cl
entering on
NKCC1 is recycled out of the cell via AE in exchange for
influx to elevate pH
, or vice versa, if the driving force
for NKCC1 is directed out of the cell) (9). Thus after precon-
ditioning, acidification during ischemia was relatively de-
creased in the presence of bumetanide (9). Similarly, inhibition
of NKCC1 was associated with increased pH
during ischemia
in the diabetic rat hearts (241). These results are consistent
with the hypothesis that during late ischemia, after [Na
have risen (7), the force driving NKCC1 is directed out
of the cell, such that NKCC1 inhibition limits Cl
and Na
loss via NKCC1 and thereby functionally coupled HCO
via AE.
4.2 Ischemia and Severe Hypoxia in the Brain
4.2.1 General Aspects Energy status and ion homeostasis. Similar to car-
diac ischemia, cerebral ischemia has been found to be associ-
ated with a marked decrease in cellular ATP levels (see Ref.
154). In cultured BBB endothelial cells on the other hand, ATP
levels were unaltered through4hofhypoxia (the approximate
time of edema formation in stroke), even at very low O
or in the absence of glucose, and after 24 h of hypoxia, ATP
levels fell by only 50% (81). Both pH
and pH
in the brain
of normoglycemic animals can fall by a full pH unit in hypoxia
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AJP-Regul Integr Comp Physiol VOL 291 JULY 2006
(see Refs. 154 and 338). However, transient elevation of pH
above steady state has also been described in brain cells
exposed to ischemia or simulated ischemia, both in culture and
in vivo (124, 279). Intracellular space comprises about 80% of
the total space occupied by the brain. In ischemia, major ionic
shifts occur between the intra- and extracellular spaces, includ-
ing, most notably, increased [Na
, [Ca
, and [Cl
, and
decreased [K
, accompanied by the corresponding inverse
changes in these ions in the extracellular space (103, 154, 248).
Similar to the situation in the heart, increases in [Na
ischemia favor NCX-mediated Ca
influx and consequent
-induced cell damage (111), and the rapid increases in
and [Ca
together with swelling of neurons, astro
cytes, and endothelial cells are major causes of brain damage
associated with limited perfusion (33, 90, 124, 135, 142, 177,
278, 288). The extent of [Na
elevation and cell swelling
varies depending on location in the ischemic region, with the
most pronounced effects occurring in the core of the ischemic
zone and more moderate slower onset effects in the penumbral
regions (130).
Under normal conditions, the BBB mediates Na
, and
water influx into the brain interstitial fluid and in so doing
secretes (i.e., transports from blood into brain) up to 30% of the
brain interstitial fluid (the remainder produced by the choroid
plexus) (55). During the early hours of cerebral ischemia, brain
edema forms by a process involving increased secretion of
and water across an intact BBB (135, 178, 262). At the
same time, ischemia stimulates NHE1- and/or NKCC1-depen-
dent uptake of Na
, and water into astrocytes, causing
cytotoxic edema (31, 135, 144), a process likely facilitated by
the increased BBB secretion of Na
, and water into the
brain interstitium (229). In other words, whereas BBB break-
down and consequent paracellular solute and water uptake does
not occur until after prolonged ischemia (4 6 h), the Na
water uptake, which is the basis for brain edema in the early
hours of ischemic stroke, occurs before BBB breakdown,
indicating that it is transcellular and thus transporter mediated
(22). As described below, both NHE1 and NKCC1 appear to
play a role in the ionic shifts and cell damage elicited by
cerebrovascular ischemia/hypoxia. Signaling events. With the important exception of the
elevated glutamate levels found in cerebral ischemia (154),
many of the signaling events elicited by ischemia, anoxia, and
prolonged hypoxia in the brain are similar to those described
above for the heart. ANG II release resulting from activation of
the brain renin-angiotensin system appears to be involved in
brain damage elicited by ischemic stroke (56), and ET-1
release has also been found to contribute to ischemic cell
damage in the brain (see Refs. 133 and 297). There is evidence
for the involvement of centrally released arginine vasopressin
(AVP) in brain ischemia and consequent cell damage (62, 275).
Brain thrombin levels have also been proposed to be elevated
after cerebral ischemia and to contribute to ischemic damage
(113). Cytokines and inflammatory mediators released, at least
in part, from astrocytes and brain microglia also play important
roles, and specifically both IL-1 and IL-6 appear to contribute
to ischemic brain damage (250, 343). As in the heart, changes
in the activity of the MAPKs ERK1/2, JNK, and p38 MAPK
(73), as well as of several other protein kinases, have been
reported in the ischemic brain (see Ref. 154). Finally, ROS
release in the brain is increased during both ischemia and
(more so) during reperfusion, and this appears to contribute to
ischemia/reperfusion-induced cell damage (see Ref. 154).
4.2.2 Mechanisms and Consequences of Stimulation
of NHE1
Ischemic/anoxic/hypoxic NHE1 stimulation and protective
effects of NHE1 inhibitors have been described in neurons
from many brain regions, including mouse neocortical neurons
(124), rat cortical neurons (317), and rat hippocampal CA1
neurons (270, 337), as well as in glial cells (27, 121, 234) and
in brain endothelial cells (23, 109, 307). Moreover, protective
effects of NHE1 inhibitors have been demonstrated in in vivo
models of brain ischemia (317). Similar to what has been found
in the heart, however, NHE1 is not the only culprit; NKCC1
(section 4.2.3), tetrodotoxin-sensitive Na
channels (86), and
influx via ionotropic glutamate receptors (90) have been
found to contribute to the ischemia-induced increase in [Na
in the CNS. Mechanisms of stimulation of NHE1. In rat hip-
pocampal CA1 neurons, NHE1 is stimulated by anoxia fol-
lowed by reoxygenation, yet is apparently inhibited during the
anoxia phase per se, possibly because of a reduced cellular
ATP level under these conditions (270, 337). Similar to the
situation in the heart, pH in the brain decreases very rapidly by
up to a full pH unit in response to hypoxia (section;
thus it appears likely that acidic pH
contributes to stimulation
of NHE1. On the other hand, in mouse astrocytes, in which
OGD is associated with intracellular acidification (137), NHE1
activation during in vitro ischemia in mouse astrocytes was
proposed to be independent of prolonged acidosis (136). Sim-
ilarly, in these cells, the rate of pH
recovery after intracellular
acid loading by NH
washout was increased by 80% after a
2-h exposure to OGD (137), suggesting that OGD-induced
stimulation of NHE1 involves other mechanisms than an acid-
ification-induced increase in activity.
Catecholamines, specifically NE, appear to play an impor-
tant role in ischemia-induced increases in NHE activity in the
brain via
- and
-ARs and the cAMP/PKA pathway (282);
however, at least in CA1 neurons, NE-mediated NHE activity
was found to be amiloride insensitive (282). Hence, although
NHE1 is also present in CA1 neurons and stimulated during
anoxia (99, 337), the -AR-stimulated NHE in these neurons
may be NHE5, which is found almost exclusively in the CNS
and which has been proposed to be the amiloride-insensitive
brain NHE (240).
NHE1 activation during in vitro ischemia in mouse astro-
cytes has been proposed to be partially dependent on ERK1/2,
although the upstream signal leading to ERK1/2 activation was
not identified (136). Cytokines released during ischemia and
severe hypoxia may also play a role in NHE activation in the
brain, as IL-1 and IL-6 have been shown to stimulate NHE1 in
astrocytes (21). In contrast to the proposed role of ROS in
NHE1 activation during cardiac ischemia/reperfusion, the few
available studies indicate that ROS may inhibit NHE activity in
the brain (188, 309). Finally, in brain microvascular endothe-
lial cells, NHE1 was reported to be stimulated by ET-1 (315). Consequences of stimulation of NHE1. Elevation of
is an important cause of the damage induced by
ischemia and severe hypoxia in the brain (154, 277, 284). As in
the heart, NCX reversal has been found to be involved in
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AJP-Regul Integr Comp Physiol VOL 291 JULY 2006
ischemia-induced Ca
influx and consequent damage in both
neurons and glial cells (27, 111, 150). The relative contribu-
tions of NHE1 and NKCC1 to the disruption of Ca
meostasis in the ischemic brain have, to our knowledge, not
been directly investigated. However, at least in mouse astro-
cytes, OGD-induced changes in Ca
homeostasis were
mainly dependent on NKCC1 (150; see also section 4.2.3);
hence, it appears that, whereas NHE1 is clearly a major player
in disruption of Ca
homeostasis in the ischemic heart,
NKCC1 may, in some cases, be more important in the brain. In
glial cells, stimulation of NHE1 by ischemia/hypoxia or lactic
acidosis and protective effects of NHE1 inhibition against glial
cell swelling and death, under these conditions, have been
demonstrated in a variety of preparations (27, 121, 234).
Consistent with the view that NHE1 contributes importantly to
the hypoxia-induced increase in Na
transport across the BBB,
NHE1 inhibition is protective against hypoxia-induced brain
endothelial cell dysfunction and BBB disruption (23, 109,
307), and intravenous HOE-642 and bumetanide to inhibit
BBB NHE1 and NKCC1 activity was recently found to de-
crease edema formation and cerebral infarct size in middle
cerebral artery occlusion (MCAO) in the rat, apparently in an
additive manner (307 and O’Donnell ME, unpublished
Stimulation of NHE1 during ischemia and severe hypoxia in
the brain appears to ameliorate the decrease in pH
during both
ischemia and reperfusion. In mouse neocortical neurons in
primary culture, NHE1 inhibition exacerbated acidification
during chemical anoxia and inhibited pH
recovery on “reper
fusion” (124). Similar to the findings in the heart, a “pH
paradox” of both protective and damaging effects of acidic pH
has been described in the brain. Mild acidosis is protective in
brain ischemia, at least partly, by attenuating Ca
influx via
D-aspartate (NMDA) and -amino-3 hydroxy-5-
methylisoxazole-4 propionic acid (AMPA) receptors and volt-
age-dependent Ca
-channels and by reducing energy demand
and ATP depletion (102, 305), but likely also because of the
inhibition of NHE1 by acid pH
(317). Acid pH
, which
inhibits NHE1 activity, was neuroprotective in chemical an-
oxia in dissociated cortical cultures, apparently, at least in part,
because of NHE1 inhibition (317). Consistent with the notion
that NHE1 exhibits little activity during the ischemic phase per
se, this effect of acidic pH
was only seen during reperfusion
(317). Similarly, in mouse neocortical neurons, in which an
EIPA-sensitive NHE is a major regulator of pH
after acidifi
cation, EIPA and acidic pH
both reduced pH
recovery (223),
and acidic pH
(EIPA not tested) attenuated the increase in
during chemical anoxia (124). Ischemia/reperfusion-
induced free fatty acid release is shown to be reduced by
inhibition of NHE1, perhaps because of a pH effect on phos-
pholipase A
(233). If, however, pH
is too acidic, this will
favor cell death by apoptosis (section 5.2). Acidic pH
has also
been demonstrated to potentiate ROS formation in the brain
directly, to inhibit mitochondrial metabolism (thus accelerating
ATP depletion), and to exacerbate neuronal death (338).
4.2.3 Mechanisms and Consequences of Stimulation
of NKCC1
NKCC1 knockout mice exhibit reduced brain damage in
stroke models (47), and, as described below, there is evidence
for roles for NKCC1 in ischemic damage in neurons, astro-
cytes, and brain endothelial cells. Early studies provided evi-
dence that a luminal Na
transporter, working in tandem with
abluminal Na
pump and Cl
efflux pathways, is rate
limiting in BBB Na
secretion (24, 262). More recent data
strongly indicate that NKCC1 serves as such a luminal Na
uptake pathway during ischemia. Thus NKCC1 resides pre-
dominantly in the luminal membrane of BBB endothelial cells
in situ (205) and is stimulated by AVP (203), hypoxia (81), and
aglycemia (81), prominent factors present during cerebral is-
chemia. Mechanisms of stimulation of NKCC1. In the brain,
mechanisms of NKCC1 activation in ischemia are better un-
derstood than in the heart, although far from fully elucidated.
Elevated extracellular K
and glutamate levels (154), both of
which occur in cerebral ischemia (section 4.2.1) have been
found to stimulate NKCC1 activity in neurons (Fig. 3A). Thus
Fig. 3. Major mechanisms and consequences
of ischemia/hypoxia-induced activation of
NKCC1 in neurons, glial cells, and brain en-
dothelial cells. A: neurons. B: glial cells. C:
brain microvascular endothelial cells (blood-
brain barrier). depol., Depolarization; EAA,
excitatory amino acid; ET-1, endothelin-1;
VSOR, volume-expansion sensing outward
rectifying anion channel; AVP, arginine vaso-
pressin; P, phosphorylation.
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AJP-Regul Integr Comp Physiol VOL 291 JULY 2006
activation of both ionotropic and metabotropic NMDA recep-
tors, as well as of AMPA receptors, activated NKCC1 in
cortical neurons, as did high [K
-induced depolarization,
both in a Ca
-dependent manner (17, 266). Elevated [K
also increased NKCC1 activity in glial cells, similarly in a
-dependent manner (290; Fig. 3B).
AVP was found to activate NKCC1 in brain endothelial cells
-dependent manner associated with an increase in
NKCC1 phosphorylation (see Ref. 203), and ET-1 activated
NKCC1 in brain endothelial cells in a Ca
- and PKC-depen
dent manner (131; Fig. 3C). In this regard, it is interesting to
note that NKCC1 was activated by ketoacids, which can cause
the release of ET-1 and/or increases in [Ca
, in brain
microvascular endothelial cells, and this mechanism appears to
be important in the cerebral edema formation occurring in
diabetic ketoacidosis (147).
Cytokines, long known as mediators of ischemic brain
damage (section may be involved in ischemia-induced
increases in NKCC1 activity. Thus IL-6 released by astrocytes
was shown to activate NKCC1 in brain endothelial cells (293),
and the immunosuppressant FK506, which prevents IL-6 up-
regulation in astrocytes and microglia, reduced infarct volume
in a rat MCAO model (343).
Similar to other mechanisms of acute stimulation of
NKCC1, ischemia-induced increases in NKCC1 activity are
generally associated with increased NKCC1 phosphorylation
on ser
and ser
, as reported in rat cerebral cortex at 4 8 h
of reperfusion after MCAO (334) and in mouse cortical astro-
cytes after2hofOGD(150). Similarly, both hypoxia and AVP
increased the phosphorylation of NKCC1 in cultured bovine
cerebral microvascular endothelial cells (81). The protein ki-
nases that mediate ischemia-induced NKCC1 phosphorylation
in the brain are unknown; however, several candidates are
known to be activated in the ischemic brain (section
Not only posttranslational regulation appears to be important,
e.g., in rat model of focal cerebral ischemia/reperfusion (2-h
MCAO and 24-h reperfusion), NKCC1 protein levels were
significantly upregulated in the cortical neurons at the end of
reperfusion (336), and similar findings were reported in whole
rat cerebral cortex and striatum (334).
Interestingly, at least in BBB endothelial cells, a decrease in
cellular ATP is not necessary for hypoxia-induced stimulation
of NKCC1 (81). In these cells, hypoxia is a rapid and potent
stimulator of NKCC1, counter to the common assumption that
BBB cells are highly resistant to hypoxia (81). Simply remov-
ing glucose and pyruvate increases NKCC1 activity in the BBB
cells under normoxic conditions (81; Fig. 3C). In the presence
of pyruvate, glucose has no effect on the ability of hypoxia to
stimulate NKCC1; however, if pyruvate is removed, NKCC1
activity is increased 2.6-fold both in normoxia and in hypoxia
(81). Thus NKCC1 activity in BBB endothelial cells appears to
be very sensitive to changes in metabolic profile, rather than
just to a decrease in P
Finally, it may be noted that recent findings from O’Donnell,
et al. (204) indicate that a reduction in NKCC1 activity in
astrocytes and BBB endothelial cells, and consequent reduced
edema formation, contributes to the neuroprotective effects of
estradiol in stroke. Consequences of stimulation of NKCC1. Studies of
NKCC1 knockout mice (47), as well as studies employing
intracerebral bumetanide administration, strongly indicate an
important role of NKCC1 in ischemic brain damage. Bumet-
anide potently reduced neuron and astrocyte swelling and
infarct volume after focal cerebral ischemia in rats (2-h MCAO
followed by 24-h reperfusion) (336). Several studies have
specifically addressed the involvement of NKCC1 in neuronal
damage (Fig. 3A). NKCC1 inhibition was found to inhibit the
increase in [Na
, cell swelling, and cell death elicited by
OGD or glutamate in 14- to 15-day cortical neurons in vitro
(17). In rat hippocampus, furosemide and bumetanide en-
hanced ATP recovery after OGD and attenuated CA1 neuronal
injury (236). In astrocytes, NKCC1 is an important contributor
to the cell swelling and excitatory amino acid (EAA) release in
response to high [K
(a condition prevalent in the ischemic
brain, see section In accordance with this, astrocytes
from NKCC1
mice exhibit absence of swelling and de
creased EAA release after high [K
(289, 290). The main
mechanism in NKCC1-mediated EAA release appears to be
that NKCC1-induced cell swelling activates the volume-expan-
sion sensing outward rectifying anion channel (also known as
the volume-regulated anion channel), through which EAA are
released (255; Fig. 3B). In postnatal day 10 (P10), rat optic
nerve astrocytes, OGD-induced cell swelling, and necrotic cell
death were dependent on NKCC1 (302). In mouse cortical
astrocytes, Na
uptake by NKCC1 appeared to contribute to
NCX-mediated Ca
-loading of intracellular Ca
stores dur
ing ischemia (Fig. 3B). Thus 2 h of OGD followed by1hof
reperfusion elicited [Na
elevation and cell swelling, both of
which were attenuated by bumetanide and by genetic ablation
of NKCC1 (150). OGD, moreover, elicited an increase in
agonist-induced Ca
release, which was inhibited by bumet
anide, absent in astrocytes from NKCC1
mice, and inhib
ited by KB-R7943, an inhibitor of Ca
entry via NCX (150).
Increased NKCC1 activity in BBB endothelial cells also
appears to contribute importantly to the detrimental effects of
brain ischemia (Fig. 3C). In rat brain, NKCC1 was found
predominantly on the luminal side of the microvascular endo-
thelial cells, and administration of intravenous bumetanide to
inhibit the BBB cotransporter significantly attenuates edema
formation in the rat MCAO model of stroke, whether added 20
min before or after onset of MCAO (205, 306, 307). These
findings underscore the important role for NKCC1 in mediat-
ing solute uptake across the BBB and suggest that ischemia-
induced increases in NKCC1 activity elicits excessive solute
uptake, resulting in brain edema. In contrast to findings in rat
brains, where microdialysis of bumetanide into the cortices
was only protective if administered preischemia (334), bumet-
anide has also been shown to reduce edema and infarct size in
rat brain, even when administered after MCAO (306), i.e., the
remaining 20% perfusion during MCAO is sufficient to deliver
bumetanide to the BBB NKCC1. This is of obvious clinical
interest, as it indicates that bumetanide given after the onset of
stroke may still be beneficial in reducing edema and infarct
As noted above (section 3.2.2), [Cl
is a major determinant
of GABAergic signaling (increased [Cl
being associated
with attenuation or even reversal of the inhibitory effect of
GABA, and thus increased excitability). It is thus likely that the
detrimental effect of increased NKCC1 activity in the ischemic
brain may, at least in part, reflect elevation of [Cl
; however,
this has, to our knowledge, not been directly investigated.
Invited Review
AJP-Regul Integr Comp Physiol VOL 291 JULY 2006
Finally, another consequence of increased NKCC1 activity
may be modulation of the activity of stress-activated protein
kinases. NKCC1 associates directly with SPAK and the related
kinase oxidative stress response 1 (230, 231), members of the
germinal center kinase subfamily of the ste 20-related kinases,
both of which are expressed in the brain (230). In brain slices,
NKCC1, SPAK, and p38 MAPK were found to form a com-
plex from which p38 MAPK appeared to dissociate on ische-
mic stress, leading to the proposal that NKCC1 may play a
scaffolding role in regulating the cellular stress response
4.3 Ischemia and Severe Hypoxia in the Blood
4.3.1 General Aspects
In their capacity as O
transporters, RBCs play a
central role in the responses to hypoxic stress. Although this
review only deals with the possible role of RBCs, it should also
be noted that both platelets (180) and leukocytes contribute
importantly to the response to ischemia/hypoxia (see Refs. 139
and 154). Energy status and ion homeostasis. In mouse RBCs,
cellular ATP levels were unaffected by1hofhypoxia (0.5%
) (25). In RBCs with mitochondria (e.g., nucleated RBCs
from birds or teleosts or mammalian reticulocytes), low oxy-
gen conditions can elicit a shift from oxidative to glycolytic
metabolism and are associated with decreased ATP levels
(although this may, in fact, not be caused by decreased oxida-
tive metabolism, see e.g., Ref. 198). Even in nonnucleated
RBCs, glycolysis is increased by deoxygenation (179; see Ref.
In contrast to the pH
decrease induced by deoxygenation in
the heart and brain (see sections and, deoxy-
genation per se will lead to an increased pH
of RBCs because
of the Haldane effect (the effect of oxygenation on the H
binding properties of Hb) (197). Deoxygenation, moreover,
increases free cytoplasmic Mg
concentration purely as a
result of the Hb oxy-deoxy changes (79, 239). Finally, some
cell swelling will occur in hypoxic RBCs solely because of the
shift induced by deoxygenation (75). If NKCC1 and/or
NHE1 is stimulated under conditions in which the driving force
favors Na
influx via these transporters, deoxygenation is
expected to increase [Na
and [Cl
, and in some cases also
, and elicit further cell swelling. Precisely such effects
were reported in RBCs from several species, including trout
and turkey (75, 192). On the other hand, [Na
and [K
mouse RBCs were reported to be essentially unaffected by 45
min of hypoxia, as well as by 15-min hypoxia followed by
15-min reoxygenation (25). With respect to [Ca
, this was
reported to be elevated in RBCs for several weeks after
myocardial ischemia in human patients (118). Oxidative stress
or energy depletion of mammalian RBCs also appears to be
associated with increased [Ca
(148). On the other hand,
experimental deoxygenation of human RBCs had no detectable
effect on [Ca
(303). Signaling events. RBCs are obviously exposed to the
many extracellular mediators released to the bloodstream dur-
ing ischemia and severe hypoxia, including, e.g., cat-
echolamines (adrenaline, noradrenaline), ET-1, AVP, dopa-
mine, and atrial natriuretic peptide (ANP), as measured in
human patients following cerebral infarction or stroke (16, 70,
193). The extent to which the RBCs have functional receptors
for these mediators is likely to differ with species and RBC
age/developmental stage. For instance, it has been reported that
mature human RBCs lack functional receptor-coupled adenyl-
ate cyclase systems (72), although, in apparent contrast to this,
NHE1 in human RBCs was reported to be stimulated by
catecholamines, in part, via
-receptors (30).
Numerous studies point to important roles of protein kinases
in the oxygen-dependent regulation of NHE1 and NKCC1 in
RBCs (section 3.3); however, the molecular identity of these
putative hypoxia-activated kinase(s) is essentially unknown.
Changes in redox systems may also be important signaling
events in deoxygenated RBCs. In RBCs, ROS are formed to a
degree proportional to P
, because of the high levels of
ferrous iron and reducing enzymes (50). Therefore, hypoxia
should decrease ROS production and prevent oxidation of
reduced glutathione (GSH). Consistent with this, an increase in
GSH levels was noted in mouse RBCs exposed to hypoxia
4.3.2 Mechanisms and Consequences of Stimulation
of NHE1
As pointed out above (section 3.3.1), it is well established
that NHE1 is activated by hypoxic conditions in RBCs from
lower vertebrates (89). It seems highly probable that this is the
case also in mammalian RBCs, since multiple factors known to
stimulate NHE1 are released to the bloodstream during ische-
mia in mammals (section However, studies directly
addressing the effect of hypoxia on NHE1 in mammalian
RBCs appear to be lacking. In mammals, ischemia-induced
NHE1 activation may only be of real consequence in RBCs,
such as those of rabbits and dogs, or in reticulocytes, which
exhibit robust transporter activities, since NHE1 activity in
mature human RBCs is relatively minor (41, 123, 216; see also
Ref. 222). Mechanisms of stimulation of NHE1. In the RBCs of
lower vertebrates, NHE1 is activated by deoxygenation per se,
by a mechanism that has yet to be fully understood but may
involve changes in redox systems, membrane-bound Hb, and
protein kinase activity (section 3.2.3, and for an in-depth
discussion, see Ref. 89).
In addition to this, hypoxic stimulation of NHE1 in the
RBCs of most teleosts is also dependent on circulating cat-
echolamines (see section 3.3.1). In flounder RBCs, cat-
echolaminergic stimulation of NHE1 was, at least in part,
PKA-dependent, and was mediated by a pathway distinct from
that involved in stimulation of NHE1 by osmotic cell shrinkage
or by inhibition of PP1 and PP2A (108). The elevated plasma
level of catecholamines that results from ischemic stress in
mammals (section has also been reported to increase
NHE1 activity in human RBCs, possibly, via both
- and
-ARs (30), although this may be of minor consequence given
the low NHE1 activity in these cells. Similarly, ANP, the
plasma level of which is increased in ischemia (section
has been reported to stimulate NHE1 in human RBCs, an effect
proposed to be mediated via cGMP (228).
It may be noted that SPAK is a regulator of NKCC1 (63, 87), and its role
in ischemia-induced NKCC1 activation is yet unclear.
Invited Review
AJP-Regul Integr Comp Physiol VOL 291 JULY 2006
In contrast to the prominent role of intracellular acidification
in ischemic/hypoxic NHE1 stimulation in the heart and brain,
acid pH
is unlikely to be a major pathway for NHE1 stimu
lation in ischemic/hypoxic RBCs. Although plasma pH
can be
decreased in extreme hypoxia (75), deoxygenation per se will
tend to increase pH
in RBCs because of the Haldane effect
(section Moreover, Hbs of mammalian RBCs exhibit
very high H
-buffering capacities; thus the effect of anoxia on
is likely to be limited (40). ROS may also be unlikely to
mediate hypoxic/ischemic stimulation of NHE1 in RBCs, since
was found to inhibit NHE1 in trout RBCs (199). Consequences of stimulation of NHE1. In teleosts,
hypoxic NHE1 activation in RBCs will elicit an elevation of
and cell swelling, both of which will increase the O
affinity and saturation of Hb by virtue of the Bohr and Root
effects (197). In humans, these effects could, in principle,
apply but are likely to be modest because of the limited net
NHE1 activity in mature human RBCs and the different Hb
properties in mammals compared with teleosts (see above, and
for a discussion, see Ref. 222). Reduced RBC deformability
reduces blood flow (217), an effect that has been proposed to
contribute to ischemic damage (304). The RBC swelling re-
sulting from hypoxic activation of NHE1 could therefore in
principle elicit detrimental effects on blood rheological prop-
erties, although this has yet, to our knowledge, to be directly
investigated. It may also be noted that RBC lysis, which could
be caused or facilitated by severe RBC swelling, contributes to
ischemic damage, e.g., as shown in rat brain (113).
4.3.3 Mechanisms and Consequences of Stimulation
of NKCC1
In RBCs from birds, ferrets, and humans, NKCC1 is stim-
ulated by deoxygenation per se (64, 79, 192; see Ref. 89). On
the other hand, in mouse RBCs, NKCC1 was reported to be
unaffected by hypoxia (25), pointing to the existence of spe-
cies-specific effects of hypoxia on NKCC1 activity.
Similar to that of NHE1, NKCC1 activity is modest in
mature mammalian RBCs compared with the activity in reticu-
locytes or in nucleated RBCs (64, 166). On the other hand, at
least under conditions of robust driving forces and high hemat-
ocrit, NKCC1 activity is high enough even in mammalian
RBCs to elicit significant physiological effects (see Ref. 89),
although little direct evidence is available for ischemia and
severe hypoxia, as discussed below (section Mechanisms of stimulation of NKCC1. NKCC1 in
RBCs is expected to be stimulated by the elevated plasma
levels of catecholamines and other NKCC1-activating factors
resulting from ischemic insults (section, as well as by
the above-described stimulation by deoxygenation per se. The
mechanism(s) of stimulation of NKCC1 by a reduction in P
per se are not fully elucidated. A role for Mg
seems possible,
since increased [Mg
stimulates NKCC1 (see Ref. 254) and
deoxygenation, as noted above, increases [Mg
(239). How
ever, neither the increase in [Mg
(79) nor the increase in
expected purely on the basis of Hb conformational changes
on deoxygenation of RBCs (section, are likely to be
sufficient to explain the marked stimulation of NKCC1 by
deoxygenation. Studies in both avian and mammalian RBCs
have strongly indicated a central role for a putative deoxygen-
ation-activated protein kinase(s). In avian RBCs, increased
NKCC1 phosphorylation was proposed to occur upon RBC
deoxygenation (192). In ferret RBCs, thr phosphorylation of
NKCC1 did not appear to be increased upon deoxygenation-
induced stimulation, suggesting a pathway different from that
mediating NKCC1 stimulation, by e.g., protein phosphatase
inhibitors (79). Consequences of stimulation of NKCC1. As de-
scribed in section 3.3.3, hypoxia-induced NKCC1 activation in
RBCs will, under conditions of an inwardly directed driving
force for this transporter, tend to elevate K
and may serve to
buffer increases in plasma [K
]. Similarly, RBC swelling
resulting from increased NKCC1 activity will increase the O
affinity and saturation of Hb but could also cause rheological
problems, as noted above for NHE1 (section Also, as
discussed above, the gradient for NKCC1 is not always in-
wardly directed in RBCs (see sections 3.3.2 and and
also Ref. 166), such that in ischemia and severe hypoxia, where
shifts in intra- and extracellular [Na
] and [K
] occur, the
direction of transport via NKCC1 is hard to predict. Under
conditions eliciting substantial outward flux via NKCC1, this
could, in principle, contribute to apoptotic RBC shrinkage and
ultimately death (section 5); however, to our knowledge, the
relevance of this phenomenon to ischemia and severe hypoxia
has not been directly studied.
5 NHE1- and NKCC1-Mediated Cell Death in Ischemia and
Severe Hypoxia
5.1 Cell death in ischemia and severe hypoxia—apoptosis
or necrosis?Apoptosis and necrosis are classically described as
two distinct forms of cell death with fundamentally different
characteristics. Cell death by apoptosis involves membrane
blebbing, outer membrane leaflet inversion, nuclear condensa-
tion, and cell shrinkage (74). In contrast, necrosis is character-
ized by cell swelling and loss of plasma membrane integrity,
resulting in the release of cell content to the surrounding milieu
(74). As described above, cell swelling is a characteristic
feature of ischemia/reperfusion damage in both the heart and
the brain (see Refs. 120, 327, and 345), and swelling of RBCs
on deoxygenation stress has also been reported (75). In part, for
this reason, cell death as a consequence of ischemia was
initially thought to be largely necrosis; however, accumulating
evidence indicates that both apoptosis (or apoptosis-like,
caspase-independent cell death) and necrosis contribute to
ischemic cell death in both the heart (211, 345) and brain (93,
196). Whether cells exposed to ischemia or severe hypoxia die
by apoptosis or necrosis, by a combination of both, or by some
pathway unique to this condition, is a point of some contro-
versy, and is probably dependent on multiple factors, such as
cell type and severity of the ischemic insult (see, e.g., Refs. 120
and 196). For instance, apoptosis is an ATP-dependent process,
and if ATP levels fall below a critical level, certain stimuli
normally leading to apoptotic cell death will instead cause
death by a necrotic pathway (see Ref. 74). The increase in
resulting from ischemia and severe hypoxia will acti
vate death effectors, including Ca
-dependent proteases and
initiate mitochondrial apoptotic death pathways. Similarly,
ROS are important mediators of apoptosis, in part, because of
mitochondrial damage (157). Studies in cardiomyocytes indi-
cate that the combination of hypoxia and low pH
prevalent in
the ischemic heart synergistically trigger a unique death path-
Invited Review
AJP-Regul Integr Comp Physiol VOL 291 JULY 2006
way involving the Bcl-2 family death-promoting protein
BNIP3 (94). Mammalian RBCs, which lack nuclei and mito-
chondria, do not undergo classical caspase-dependent apopto-
sis, yet die following energy depletion or oxidative stress by a
caspase-independent form of cell death exhibiting many char-
acteristics of apoptosis, and due, at least in part, to Ca
and increased [Ca
5.2 Roles of NHE1. Consistent with a role for NHE1 in
ischemia-induced cell swelling in both the heart and the brain,
amiloride inhibited cold ischemia-induced cell swelling in rat
hearts (13), and genetic ablation of NHE1 inhibited OGD-
induced astrocyte swelling (137). NHE1 inhibitors have also
been shown to attenuate apoptosis in a number of models of
myocardial hypoxia/ischemia and heart failure (3, 15, 44, 294).
In one study, cariporide was reported to inhibit ischemia/
reperfusion-induced cardiomyocyte death by necrosis, as well
as apoptosis (211). The NHE1-mediated necrosis is presum-
ably because of Na
influx and concomitant cell swelling (Fig.
2). Only a few studies have addressed the mechanisms by
which increased NHE1 activity leads to apoptotic cell death in
ischemia. In neonatal rat cardiomyocytes, the NHE1-mediated
increase in [Ca
was proposed to lead to mitochondrial Ca
overload and activation of a mitochondrial death pathway (294,
300). NHE1-mediated mitochondrial Ca
overload after in
vitro ischemia was also recently reported in mouse astrocytes
(136). In cerebellar granule neurons, inhibition of NHE1 alone
was found to elicit caspase-independent cell death with mor-
phological features most characteristic of apoptosis (parapto-
sis) (263). The role of NHE1 in apoptosis is thus not straight-
forward, and, additionally, antiapoptotic effects of NHE1 have
been described in numerous studies of nonischemia conditions
(246, 301, 328). An important protective effect of NHE1 in
such cases appears to be maintaining pH
above the acidic pH
optimum for many death effectors, including caspases and
cathepsins (169, 251, 263). Although this is in apparent con-
trast to the consistently protective effect of NHE1 inhibition in
ischemia, it should be kept in mind that inhibition of NHE1 in
ischemia may, in fact, somewhat counterintuitively lead to
increased pH
5.3 Roles of NKCC1.Very few studies to date have addressed
the potential involvement of NKCC1 in apoptotic vs. necrotic
death following ischemic/hypoxic insults. An important con-
sequence of NKCC1 stimulation by ischemia and severe hyp-
oxia in the brain is neuronal and glial swelling and edema
(section, pointing to a possible role of NKCC1 in
ischemia/hypoxia-induced brain cell necrosis (Fig. 3). To our
knowledge, only one study has specifically addressed this
issue. The findings indicated that NKCC1 inhibition reduced
necrosis, but not apoptosis, after focal ischemia in rat cerebral
cortex (334). A few nonischemia studies in tissues other than
heart, brain, or blood have implicated NKCC1 in apoptotic cell
death (134, 168). In RBCs, outward transport via NKCC1 and
concomitant isotonic cell shrinkage has been tentatively pro-
posed to play a role in the apoptotic process (158). As noted
above, the driving force for NKCC1 may reverse during
ischemia also in the heart (section, in principle, allow-
ing similar cell shrinkage. In light of the small extracellular
space of the brain, [K
is probably sufficiently high in
ischemia so that outward transport by NKCC1 seems unlikely.
In brain cells, ischemic activation of NKCC1 could, in princi-
ple, also lead to an apoptosis-like cell death, for instance, via
the above-described entry of Ca
via NCX and concomitant
changes in Ca
homeostasis (section, see also Fig. 3);
however, this possibility remains to be studied.
6 Perspectives and Open Questions
In this review, we have sought to document the considerable
progress made in recent years in understanding the physiolog-
ical and pathophysiological roles of NHE1 and NKCC1 in the
heart, the brain, and the blood. Although these transporters are
of pivotal importance in the return to tissue homeostasis
following various forms of physiological stress, their excessive
activity may, under certain pathophysiological conditions, be a
major cause of cell damage and death. The overall aim of this
review was to evaluate and compare the roles of NHE1 and
NKCC1 in the detrimental events resulting from ischemia and
severe hypoxia. Specifically, we addressed the similar roles of
these transporters in [Na
overload leading to NCX reversal,
increased [Ca
, and consequent cell death via Ca
dent mechanisms, including mitochondrial death pathways. As
discussed above, this scheme contributes importantly to the
detrimental roles of both of these transporters in ischemia and
severe hypoxia in the heart and brain, and there is also
evidence to suggest a role in the blood. However, in all of the
three tissues, there is increasing evidence for a major involve-
ment of a number of other mechanisms in NHE1- and NKCC1-
mediated damage, including changes in pH
and cell volume,
activation of MAPKs, and release of EAAs and ROS. More-
over, it appears that a complex interplay of multiple ischemia/
hypoxia-activated signaling events mediates the activation of
NHE1 and NKCC1 under these conditions, although the acti-
vation pathways are still incompletely described. The mecha-
nisms leading to NHE1 and NKCC1 activation and the mech-
anisms by which activation of these transporters lead to cell
damage and death in ischemia and severe hypoxia constitute
potentially interesting targets for therapeutic intervention and
should be a focus for future research in this clinically very
important field.
We apologize to those whose work, although relevant to this review, could
not be cited because of space restrictions. The members of our labs are
gratefully acknowledged for contributing unpublished data and stimulating
Work in our laboratories cited above was supported by the National
Institutes of Health Grants HL-21179 (to P. M. Cala), HL-56681 (to S. E.
Anderson), and NS039953 (to M. E. O’Donnell); an American Heart Associ-
ation Grant-in-Aid (to M. E. O’Donnell); External Research Program Grants
from Philip Morris (to M. E. O’Donnell and S. E. Anderson); the Carlsberg
Foundation Grant 0544/20 (to S. F. Pedersen); and the Danish Natural Science
Research Council Grant 22–01-0055 (to S. F. Pedersen). The nuclear magnetic
resonance spectrometer expense was funded, in part, by National Institutes of
Health Grant RR02511 and National Science Foundation Grant
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