Acidosis and proton homeostasis in cells and tissues
Acidosis in the brain may severely impair a variety of functions, including synaptic
transmission, metabolic energy supply, membrane transport and other processes (Ruusuvuori
and Kaila, 2014).
Transport of acid–base equivalents across the cell membrane of neurons and glial cells
also results in pH changes in the extracellular spaces. Cytosolic and extracellular
buffer capacity and the activity of carbonic anhydrases contribute to shape pH changes,
which can be elicited by neuronal activity, neurotransmitters and neuromodulators,
metabolic processes, active cellular pH regulation, and secondary transporters carrying
acid–base equivalents, and in turn these pH changes can affect neuronal functions
(Deitmer and Rose, 1996; Chesler, 2003). The free H+ concentration in cells is in
the nanomolar range, and the high buffer capacity of cells provides a reservoir of
acid equivalents in the millimolar range. In other words, there is a pool of protons
in rapid exchange between buffer sites and free solution, with 105 or more protons
being buffered for each proton in solution. At a blood pH of 7.4, and 7.2–7.3 in the
extracellular spaces of brain tissue (Cragg et al., 1977; Ruusuvuori and Kaila, 2014),
and with a negative membrane potential of between −50 and −90 mV in mammalian brain
cells, H+ has to be continuously extruded to maintain a physiological cytosolic pH
of 7.0–7.3. Nevertheless, pH changes may peak well outside this range, at least for
short time periods, and may be considered as H+ signals, sometimes even with neurotransmitter
function (Deitmer and Rose, 1996; Du et al., 2014). The net extrusion of acid from
neurons and glial cells is accomplished by secondary active transport, wherein the
efflux of H+ or the influx of HCO−
3 is coupled to Na+ influx, utilizing energy stored in the transmembrane Na+ gradient.
pH regulation in these cells involves a variety of membrane acid–base carriers, including
sodium–hydrogen exchange, sodium–bicarbonate cotransport, and sodium-dependent and
sodium-independent chloride–bicarbonate exchange. In addition, there are a number
of acid/base-coupled carriers, which are linked to the transport of metabolites, such
as lactate and amino acids. The lactate transport via monocarboxylate transporters
(MCTs) has been suggested to play a major role for the supply of energy to neurons,
and led to the “Astrocyte-to-Neuron Lactate Shuttle Hypothesis” (ANLSH; Pellerin and
Magistretti, 1994).
Lactate shuttle and acid/base transport metabolon
Lactate, pyruvate, and ketone bodies are transported into and out of cells via MCTs
(SLC16), of which 14 isoforms have been described. The first four of these 14 isoforms
(MCT1-4) have been shown to transport monocarboxylates together with H+ in a 1:1 stoichiometry.
MCT1 is the ubiquitous isoform that is found in nearly all tissues, where it could
either operate as a lactate importer or exporter, and has an intermediate Km
value of 3–5 mM for L-lactate (Bröer et al., 1998). MCT2, the high-affinity carrier,
is mainly found in neurons, and MCT4, the low-affinity, high-capacity carrier, has
been reported for glial cells in the brain.
The lactate shuttle hypothesis suggests that lactate is produced and exported by glial
cells, in particular astrocytes, under normoxic conditions, and taken up by neurons
for further metabolization (Pellerin and Magistretti, 1994). The ANLSH infers that
astrocytes help to supply energetic substrates for neurons to meet their energy requirements,
especially during enhanced neuronal activity. There is substantial evidence, both
in vitro and in vivo, that lactate indeed can substitute for glucose to maintain neuronal
functions, such as e.g., synaptic transmission and memory formation (Schurr et al.,
1988; Suzuki et al., 2011). During energy deprivation, the addition of monocarboxylates
has been shown to restore synaptic function and to be neuroprotective in vivo, in
acute rodent brain slices, isolated optic nerve and neuronal cultures (Izumi et al.,
1997; Schurr et al., 1997; Cater et al., 2001; Wyss et al., 2011). The finding that
glucose is preferentially taken up by astrocytes and at higher rates than by neighboring
neurons (Barros et al., 2009; Jakoby et al., 2014), implying that some energetic substrate
has to be passed on to neurons, as they are the main energy consumers, also supports
the ANLSH. More recently, lactate production and supply to neuronal axons have been
suggested also for oligodendrocytes in the mammalian central nervous system (Fünfschilling
et al., 2012; Lee et al., 2012), indicating that astrocytes and oligodendrocytes form
a metabolic network with neurons to maintain neuronal function.
A transport metabolon has been defined as a supramolecular complex of sequential metabolic
enzymes and cellular structural elements in which metabolites are passed from one
active site to another without complete equilibration with the bulk cellular fluids
(Srere, 1985). First evidence for a transport metabolon, formed between carbonic anhydrase
(CA) and an acid/base transporter was found for CAII and the Cl−/HCO−
3 exchanger AE1 (Kifor et al., 1993; Vince and Reithmeier, 1998). Since then, various
acid/base transporters have been reported to interact with different isoforms of carbonic
anhydrase: For the electrogenic Na+/HCO−
3 cotransporter, NBCe1, both functional (Becker and Deitmer, 2007; Schüler et al.,
2011) and physical (Gross et al., 2002; Alvarez et al., 2003; Pushkin et al., 2004)
interaction with different CA isoforms has been suggested. All of these interactions
have in common that CA-mediated augmentation of transport activity requires the catalytic
activity of the different CA isoforms.
An entirely different form of transport metabolon has first been detected, when expressing
MCT1 and CAII in Xenopus oocytes (Becker et al., 2005). The presence of CAII indeed
more than doubled the rate of lactate transport, and the CAII-induced augmentation
of MCT activity persisted in the absence of CO2/HCO−
3, and was insensitive to inhibition of CAII catalytic activity with EZA, and was
still present with the catalytically inactive mutant CAII-V143Y (Becker et al., 2005,
2011; Becker and Deitmer, 2008), suggesting that the augmentation of MCT activity
does not depend on the reversible conversion of CO2 and HCO−
3/H+ by CAII. No interaction between CAII and rat MCT2 could be detected, when the
enzyme was injected into oocytes co-expressing MCT2 together with its trafficking
protein embigin (Klier et al., 2011). Cytosolic CAII was shown to bind to the C-terminal
tail of MCT1, which presumably positions the enzyme close enough to the pore of the
transporter for efficient H+ shuttling (Stridh et al., 2012). The binding of CAII
to a glutamic acid cluster within the MCT C-terminal may also explain the isoform
specificity of the interaction between MCTs and CAII, since rat MCT4, but not MCT2,
possesses a similar cluster of three glutamate residues.
Augmentation of MCT activity by extracellular CAs has also been found in the brain:
By inhibition of extracellular CA activity with benzolamide and an antiserum against
CAIV, respectively, Svichar and Chesler (2003) could show a significant reduction
in lactate-induced intracellular acidification in rat hippocampal pyramidal neurons
and in cultured astrocytes.
CA activity mediates between different forms of metabolic acidosis
Carbonic anhydrases play a vital role in acid/base kinetics and mediate between acid
production by oxidative phosphorylation in form of CO2 and acid production by anaerobic
glycolysis. When CO2 increases in the cell, e.g., due to oxidative phosphorylation
in mitochondria, it can leave the cell by freely diffusing through the cell membrane,
or it can be converted to H+ and HCO−
3, with the rate of conversion depending on catalytic activity of cytosolic CA. Most
cells express CAII, which is the fastest isoform, and either CAIV and/or CAXIV, which
are fast extracellular isoforms in the brain. CAIV has recently been shown to display
intracellular activity in addition, which would further contribute to high intracellular
CA activity (Schneider et al., 2013). With this enzymatic equipment, neurons and glial
cells can produce considerable amounts of H+, which can be extruded by either NHE
or MCT. Extracellular CA activity can convert part of extracellular CO2 to H+ and
HCO−
3, the latter being substrate for NBC to be transported into and out of the cell.
Thus, additional HCO−
3 can be delivered to, or removed from, the cytosol, in particular in astrocytes,
which can have a robust expression of NBC, which mediates a high bicarbonate sensitivity
of the cells, to further compensate metabolically produced H+ (Theparambil et al.,
2014).
Furthermore, both extra- and intracellular CA isoforms, as e.g., CAIV, can form transport
metabolons with the bicarbonate- and proton-coupled carriers (see above). In mouse
retina, CAXIV co-localized with anion exchanger isoform 3 (AE3) in Müller and horizontal
cells, and physical and functional interaction between the CAXIV and AE3 was shown
(Casey et al., 2009). Disruption of transport metabolon function, as suggested to
occur after CAIV mutation, can interfere with photoreceptor maintenance and pH regulation
in the retina (Yang et al., 2005; Alvarez et al., 2007). Whether other extracellular
CA isoforms, which have been detected in brain tissue, also form functional metabolons
with MCT and/or NBC, is still unknown. Interestingly, cytosolic CAI and CAIII, which
are expressed by some cells, can enhance NBC activity in Xenopus oocytes (Schüler
et al., 2011), but not MCT transport activity (Becker and Deitmer, 2008). In addition,
by stabilizing the H+ gradient, NBC can support lactate transport via MCT, when expressed
together in oocytes (Becker et al., 2004).
From these and other results, it can be concluded that brain cells, and quite possibly
other cell types in other tissues, use a whole network of acid/base-coupled membrane
carriers and different CA isoforms to regulate intracellular pH, which links acid/base
status, H+ buffering, energy metabolism, and H+/HCO−
3-coupled membrane transport. Thus, acid/base-coupled metabolite transport is coupled
to pH regulation, and both are linked to CA activity and to non-catalytic functions
of CA.
Conclusions and perspectives
Regulation of metabolism in organisms is not only complex, but also involves a large
number of enzymes and membrane transporters, which may form networks to enhance their
efficacy. Lactate, as a metabolic intermediate from glucose or glycogen breakdown,
appears to play a major role as energetic substrate shuttled between cells and tissues,
both under hypoxic and normoxic conditions. The membrane transport of lactate via
monocarboxylate transporter occurs in cotransport with H+, which is a substrate, a
signal and a modulator of other metabolic processes. Lactate transporter form a “transport
metabolon” with carbonic anhydrases, which not only provide a rapid equilibrium between
CO2, HCO−
3, and H+, but in addition enhance lactate transport by a non-enzymatic interaction,
which requires physical binding as found in frog oocytes as expression system for
the proteins involved. Carbonic anhydrases mediate between different states of metabolic
acidosis, induced by glycolysis and oxidative phosphorylation, and play a relay function
in coupling pH regulation and metabolism.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.