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PHYSIOLOGY 27: 259 –275, 2012; doi:10.1152/physiol.00014.2012
The Combinatorial Nature of
Osmosensing in Fishes
Dietmar Kültz
Department of Animal Science, Physiological Genomics Group,
University of California, Davis, Davis, California
[email protected]
Organisms exposed to altered salinity must be able to perceive osmolality
change because metabolism has evolved to function optimally at specific
intracellular ionic strength and composition. Such osmosensing comprises a
complex physiological process involving many elements at organismal and
cellular levels of organization. Input from numerous osmosensors is integrated
to encode magnitude, direction, and ionic basis of osmolality change. This
combinatorial nature of osmosensing is discussed with emphasis on fishes.
1548-9213/12 ©2012 Int. Union Physiol. Sci./Am. Physiol. Soc.
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For fishes and other aquatic animals, environmental osmolality is equal to the salinity (the concentration of inorganic ions) of their aquatic habitat.
Estuaries, the intertidal zone, and desert lakes all
represent habitats in which salinity (and accordingly osmolality) fluctuates greatly over different
temporal scales (daily, tidally, and seasonally). An
increase in water salinity causes hyperosmotic
stress because it leads to passive water loss from
fish. Thus fish become dehydrated not unlike terrestrial animals that become dehydrated due to
evaporative and excretory water loss. Tissue dehydration also occurs in ectothermic polar animals as a
result of freezing at subzero environmental temperatures (effectively leading to aqueous solvent removal due to ice formation). These animals have
evolved mechanisms of freeze avoidance and freeze
tolerance to cope with adverse effects of severe dehydration (24). Conversely to hyperosmotic stress,
hypo-osmotic stress occurs when environmental salinity decreases and fish gain water passively and,
thus, become overly hydrated.
Because animal cell membranes are semipermeable (i.e., having high water but low ion permeability), a change in hydration state of fish is
reflected in an altered cellular hydration state. A
deviation of cellular hydration state from the optimal set point interferes with macromolecular and
cellular structure and function. The reason for this
deleterious effect of osmotic stress is that cell metabolism and biochemical properties of macromolecules
have been selected for functioning optimally at a
specific and highly stable intracellular ionic milieu
(195, 196). This internal milieu presumably reflects
the conditions under which cells, the smallest units
of life, have evolved.
Fishes are able to perceive and compensate for
changes in environmental salinity of their aquatic
habitat. From a mechanistic perspective, they utilize
1) osmosensors for perceiving osmotic stress, 2) osmosensory signaling mechanisms for transducing
information about osmotic stress, and 3) osmoregulatory effector mechanisms for alleviating osmotic
stress (57). Osmosensing is the physiological process of perceiving a change in environmental osmolality. In this review, the term osmosensor refers
to molecules that 1) directly sense changes in osmolality and 2) generate an intracellular signal that
contributes to the alleviation of osmotic stress. In
contrast, the term osmoreceptor is used for cells
that are unique to osmoregulating animals (fishes
and other vertebrates) and are capable of producing a specific para-, neuro-, or endocrine signal
that contributes to the maintenance of body water
and fluid homeostasis. The concept of a combinatorial nature of osmosensing that is developed in this
review refers to the multitude of sensory elements
involved in the perception of environmental osmolality, their different effective operating ranges, and
the convergence of specific combinations of signals
emanating from those osmosensors. Such combinatorial osmosensing allows for finely tuned, qualitatively and quantitatively appropriate responses to
changes in environmental osmolality.
What osmosensors do fishes utilize and how do
they work? This question is not easy to answer. The
problem is not the lack of knowledge of potential
osmosensors, but, on the contrary, there are too
many proteins (and other macromolecules) that
fulfill the criteria for an osmosensor. As outlined in
what follows, essentially every protein is affected in
its structure/function by osmotic stress (169), and
a large number of intracellular signaling pathways
are involved in controlling osmoregulatory effector
mechanisms (47, 48, 57, 101, 102). What sets them
apart is their relative sensitivity toward osmotic
stress and, hence, their effective operating range
(see below). Therefore, I had previously proposed
that animal cells integrate input from multiple osmosensors with different sensitivity ranges to accurately quantify environmental osmolality and
osmotic stress (97). Here, I will review the available
evidence supporting such a combinatorial nature
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of osmosensing and what we know about some of
the key elements that are involved in the osmosensing network. Although the emphasis will be on
fishes, the discussion presented here has broader
applicability to other organisms as well, and some
supporting evidence is borrowed from organisms
other than fish if necessary to illustrate the general
concept.
Evolutionary Driving Forces for
Combinatorial Osmosensing
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Principles of Cellular Osmosensing
The Threat of Osmotic Stress and Its Effects
on Animal Cells
The solute composition of cells has been subject to
stringent selection during evolution, and responses
to any form of osmotic stress (dessication, salinity
fluctuations, freezing) are evolutionarily highly
conserved (169, 196). Osmotic stress (i.e., water
stress) is caused by changes in intracellular water
activity (solvent capacity), which can be due to
1) changes in water (solvent) content or 2) changes
in electrolyte (charged solute) content. Both of
these scenarios alter the rates of biochemical reactions because the activities of macromolecules are
exquisitely sensitive to water activity and depend
on the dynamics of the hydrogen bonding network
of water (51, 63).
Cells of fishes and other animals are enclosed by
semi-permeable membranes but lack cell walls
and thus are unable to maintain a turgor pressure
or osmotic difference between extracellular and
intracellular fluid. However, intracellular solute
composition is very different from extracellular solute composition, with high intracellular K⫹ being a
key variable for physiological homeostasis (169,
196). Cells rigorously defend the intracellular inorganic ion composition and overall electrolyte concentration during osmotic stress to maintain
proper metabolism and function. From an evolutionary perspective, mechanisms for maintaining
an intracellular milieu that is compatible with cell
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Bacteria use simple two-component systems consisting of histidine kinases and a transcriptional
response regulator for osmosensing (133). Orthologous histidine kinases are also used as osmosensors by some unicellular eukaryotes such as slime
molds, yeast, and some plants (143, 158, 182).
However, the genomes of fishes and other animals
do not contain homologous proteins. During the
evolution of metazoans, alternative mechanisms of
osmosensing have been selected, which include a
much larger number of proteins than osmosensory
bacterial two-component systems. As organismal
complexity and the number of differentiated cell
types with specialized functions within a single
organism increases, so does the complexity of
combinatorial logic underlying intracellular signaling networks, including those that are involved in
osmosensing. There are several strong evolutionary selection pressures that drive this phenomenon. First, the number of protein-coding genes in
simple vs. highly complex organisms does not increase proportionally to the number of differentiated cell types and states nor to the number of
specialized physiological functions. Thus the available genomic space for supporting increased cellular and tissue specialization and differentiation is
rather limited (89). Second, cells and cellular organelles such as nuclei have an optimal size and
volume, and their capacity for accommodating a
finite number of macromolecules (chiefly proteins
and nucleic acids) and micromolecules (metabolites, electrolytes, etc.) is limited (63). Third, the
risk of system failure increases with the number of
elements (e.g., proteins) constituting the system if
each protein has only one specialized function and
each function is only mediated by one specific
protein. These selection pressures have resulted in
the re-utilization of available proteins for novel
functions and increased redundancy and robustness of signaling networks and other biological
processes (64). Much innovation with regard to
physiological mechanisms appears to have evolved
via novel combinations and compartmentation of
protein/macromolecular interactions rather than invention of novel genes/proteins. Large multi-protein
complexes are common in higher organisms, and how
individual proteins function often depends on the
temporal-spatial dynamics of their interactions
with other macromolecules and their cellular compartmentation, which, in turn, is governed by environmental and developmental cues (32, 130, 156,
167, 183). This combinatorial nature of macromolecular dynamics in cells has profound physiological implications, for instance for the regulation of
transcriptional networks, neural circuits, and developmental pattern formation (70, 72, 163).
A combinatorial nature of osmosensing is suggested by the ubiquitous effects of osmotic stress on
many (signaling and other) proteins. Moreover,
fishes and other osmoregulators combine osmotic
stress signals that are generated in multiple tissues to
coordinate and integrate mechanisms for maintaining osmotic homeostasis of their extracellular body
fluid (ECF). In addition, differential sensitivity of osmoresponsive proteins allows for fine-grained physiological adjustments to be mounted to exactly match
environmental osmolality changes in quantity and
quality. I will discuss some specific examples of fish
osmosensors that have different sensitivity ranges after
briefly reviewing the general principles of osmosensing.
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(hyperosmolality prevails) rather than reestablishment of osmotic homeostasis. This circumstance
may have favored the evolution of mechanisms for
maintaining osmotic homeostasis in extracellular
body fluids (ECF) of osmoregulators. In these
highly evolved animals, including bony fishes and
all mammals, the ECF effectively dampens environmental osmolality fluctuations. Hypertonicity
not only resets the intracellular osmolality but also
causes cell shrinkage (volume stress) and crowding
of macromolecules and micromolecules (metabolites and inorganic ions) (FIGURE 1, scenario A).
Unlike alterations in the osmotic setpoint, cells
cannot tolerate permanent changes of cell volume
or intracellular inorganic ion strength. Therefore, a
two-phase adaptive response is mounted consisting of 1) cell volume regulation and 2) replacement
of excessive inorganic ions by compatible organic
osmolytes (FIGURE 1). Regulatory volume increase
(RVI) is achieved by active uptake of inorganic
ions, which are then replaced by either compatible
or counteracting osmolytes. Compatible osmolytes
are small uncharged or zwitter-ionic organic metabolites that, unlike inorganic ions, have no net
charge and do not alter macromolecular structure
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metabolism during osmotic stress have been subject to strong selection pressure. Homeostasis of
the intracellular milieu is absolutely essential for
1) adequate macromolecular structure and function
(stability) and 2) adequate macromolecular “regulatory responsiveness” (flexibility) (169). In particular,
proteins are highly responsive to changes in water
activity by changing their structure, interactions,
posttranslational states, and/or compartmentation,
all of which are properties that are critical for intracellular signal transduction.
Acute osmotic stress brought about by impermeable solutes (i.e., hyper-/hypotonicity) causes osmosis and changes cell volume because of the
semipermeable nature of the plasma membrane.
Hypertonicity and hypotonicity do not perturb
only cell volume but also intracellular inorganic
ion concentration. In the case of hypertonicity,
cells passively lose water due to osmosis, and the
intracellular osmolality equilibrates with extracellular osmolality. It is important to remember here
that animal cells are not able to maintain a constant intracellular osmolality setpoint. Any change
in extracellular osmolality is accompanied by a
shift in intracellular osmolality to a new setpoint
FIGURE 1. Effects of hyperosmotic stress on animal cells
A: acute, severe hyperosmotic stress caused by membrane-impermeable solutes leads to hypertoncity, cell shrinkage (volume stress), and macromolecular crowding. Hypertonic cell volume stress is relieved by regulatory volume increase resulting from active transport of inorganic ions. Subsequently, ionorganic ion stress is relieved by accumulation of compatible or counteracting osmolytes. B: less severe, gradual osmotic stress
caused by increased extracellular inorganic ion concentration can be isovolumetric, i.e., have no noticeable effect on cell volume. In this scenario,
inorganic ion stress still needs to be relieved by compatible or counteracting osmolyte accumulation. C: less severe, gradual osmotic stress caused
by increased extracellular compatible or counteracting osmolytes is isoionic when these osmolytes permeate cell membranes with similar efficiency as water. In this scenario, no volume or inorganic ion stress is imposed, but overall molecular density is still increased and water activity
(solvent capacity) is decreased. The dissimilar consequences of different types of osmotic stress provide a way for sensory perception of its nature. For hypo-osmotic stress, similar scenarios are applicable, except that the events proceed in the opposite direction. Yellow hexagons are
macromolecules; red pentagons are inorganic ions; green dots are water molecules; blue rectangles are compatible/counteracting osmolytes.
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(micromolecules ⫹ macromolecules) are still altered in
this scenario.
From this brief survey of osmotic effects on animal
cells, we can see that direct consequences of osmotic
stress differ depending on the direction (hyper- vs.
hypo-osmolality), acuteness, magnitude, and the
ionic nature of the osmolality change. In addition,
the state of the cell and its microenvironment (e.g.,
how readily compatible osmolytes can be recruited
to close the osmotic gap) modulates osmotic stress
effects. Differences in these parameters are utilized for the generation of intracellular signals,
the specific combination of which encodes the
quantity and quality of osmotic stress to produce
appropriate physiological responses.
Molecular Osmosensors
Molecular osmosensors are directly altered by at
least one of the consequences of osmotic stress
outlined above. In response to such alteration, they
produce a signal that contributes to the regulation
of osmoregulatory effector mechanisms. In fishes
(and other highly complex animals), signals that emanate from numerous osmosensors appear to be logically integrated in a combinatorial fashion that
allows highly specific outcomes despite the lack of
exclusively specific osmosensors (95). Osmosensors
include enzymes whose activity is stringently
correlated with intracellular electrolyte concentration, proteins that are regulated by ion concentration or osmotically altered membrane
properties, proteins associated with cytoskeletal
organization, ion channels, and proteins that
monitor the degree of macromolecular (protein
and DNA) damage (FIGURE 2).
Osmolality changes directly affect the reaction
rates of enzymes by altering their catalytic efficiency (kcat) and Michaelis-Menten constant (Km)
(169). The osmolality threshold at which such effects manifest themselves is enzyme specific. Theoretically, any protein kinase, protein phosphatase,
or other intracellular signaling protein could contribute to osmosensing via direct osmotic effects
on their catalytic rates and enzymatic efficiency. It
is, therefore, not surprising that many signaling
proteins such as numerous protein kinases and
phosphatases have been implicated in intracellular
osmosensing. Since most intracellular signaling may
be mediated via the dynamic assembly and disassembly of multimeric protein complexes, the ubiquitous osmotic effects outlined above are of fundamental
importance (see The Role of Protein Phosphorylation for
Osmosensing below). They could explain why other
sensory modalities, e.g., conspecific recognition,
are altered when fishes are faced with environmental salinity change (75). Nonetheless, convergence
of a multitude of intracellular signaling pathways
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and function over a very wide concentration range
(196). Counteracting osmolytes are pairs of organic
osmolytes whose effect on macromolecular structure and function cancels each other out over a
wide range of concentrations and at a given ratio.
An example for a pair of counteracting osmolytes is
the combination of urea and trimethyl-amine oxide (TMAO), whose strong individual effects on
macromolecules cancel each other out at a ratio of
⬃2:1 (196).
Less acute and more moderate hyperosmotic
stress can be isovolumetric (non-hypertonic), not
causing a change in cell volume or macromolecular crowding (FIGURE 1, scenario B). Isovolumetric
regulation in various cell types depends on the rate
and magnitude of osmolality change (25, 116, 151,
155, 170). Because in fishes and other osmoregulators the ECF greatly dampens environmental salinity fluctuations (with regard to both acuteness
and magnitude), isovolumetric regulation may be
more common for most fish tissues in vivo than
hypertonicity. Possible exceptions include epithelial cells that are directly exposed to external salinity, e.g., gill epithelial cells and epithelial cells
lining the GI tract. During isovolumetric regulation, the signals generated by volume changes and
macromolecular crowding are absent, and only intracellular inorganic ion strength has to be restored
to the homeostatic setpoint.
A third possibility for responding to osmotic
stress is by isoionic regulation, which could occur
in circumstances of mild and gradual osmotic
stress when intracellular compatible or counteracting osmolyte concentrations can be adjusted very
rapidly to offset water movement by osmosis
(FIGURE 1, scenario C). For instance, urea and glycerol are organic osmolytes that cross plasma membranes with similar ease as water. These organic
osmolytes and water have long been assumed to
freely permeate cell membranes, but it is questionable whether this assumption holds true under all
circumstances. If it did, then the presence of aquaporins and urea transporters in cell membranes
would seem counterintuitive. In any case, isoionic
regulation is similar to isovolumetric regulation except that compatible or counteracting osmolytes are
utilized right away to close the osmotic gap instead of
first relying on inorganic electrolytes. Isoionic regulation requires compatible osmolytes to be rapidly
adjustable, e.g., during hypo-osmotic stress via rapid
osmolyte release or during hyperosmotic stress via
rapid uptake (e.g., uptake of urea from elasmobranch
ECF or mammalian renal medullary interstitium, or
glycerol uptake from teleost ECF). Even though no
volume stress, macromolecular crowding, or inorganic ion stress occurs during isoionic regulation, water
activity (solvent capacity) and overall molecular density
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the open/closed state of apical crypts of gill chloride cells also depends on the actin cytoskeleton
(26). In addition, osmotic stress-induced actin remodeling invokes changes in the morphology of
tight junctions. For instance, in primary cultures of
trout gill cells, tight-junction adjustments to osmotic stress require claudin 28b and the supporting F-actin ring (162).
Acute and severe osmotic stress triggers changes in
cell volume and membrane organization. Changes in
membrane organization, including lipid packing
density and membrane tension, are important parameters that regulate the activity of membraneassociated proteins. An example is phospholipase A2
(PLA2), which is activated by membrane lipid rearrangement (109). PLA2 catalyzes the hydrolysis of
membrane glycerophospholipids, resulting in liberation of arachidonic acid (AA), an important signaling
molecule in cells (82, 142). One function of AA is to
activate AA-regulated Ca2⫹ selective (ARC) channels
in the plasma membrane to increase intracellular
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into unique combinations could produce stressorspecific output even if each pathway is also involved in numerous other processes.
The formation of multi-protein complexes depends on electrostatic interactions that are highly
sensitive to water activity and intracellular inorganic ion strength (169). For instance, the selfassembly of major cytoskeletal proteins (actin,
tubulin) is sensitive to intracellular inorganic ion
concentration (150, 177). Cytoskeleton strain has
been proposed to be one of the main inputs into
combinatorial osmosensing, but, based on the evidence outlined above, it is conceivable that cytoskeletal effects of osmotic stress also manifest themselves
in the absence of changes in cell volume or shape
(95, 102). The actin-based cytoskeleton has been
shown to participate in the salinity-dependent regulation of Na⫹/K⫹/2Cl⫺ (NKCC) cotransporters
(114). Furthermore, changes in ion transport
across intestinal epithelium of euryhaline eels require intact F-actin and microtubules (113), and
FIGURE 2. Molecular osmosensors that contribute to the perception of osmotic stress in fish cells
A few intracellular messenger compounds and a few effector mechanisms are depicted, but no detail of the osmotic stress signaling network, which
is still insufficiently understood, is shown. Please refer to the text for a discussion of the role of each of the depicted elements in fish osmosensing.
SHR, steroid hormone receptor; ARC, arachidonic acid-regulated Ca2⫹ selective channels; ANXA11, annexin A11; AA, arachidonic acid; PLA2,
phopspholipase A2; CaSR, calcium sensing receptor; AC, adenylate cyclase; AQP, aquaporins; TRPV4, transient receptor potential vanilloid 4 cation
channel; CR, cytokine receptor.
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calcium concentration (FIGURE 2), which is a common response of cells exposed to acute and severe
osmotic stress (168).
The Role of Inorganic Ions for Osmosensing
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The Control of Fish Spermatocyte Motility
by Inorganic Ions
In fish species with external fertilization, spermatocyte motility is controlled primarily by osmolality and the intra- and extracellular concentration
of cations (2, 3, 23, 175). The molecular regulation
of motility/flagellar activity of fish spermatocytes
promises to be a valuable simple model for deciphering the logic of osmosensory signal transduction networks in highly complex animals because
the combination of sensors, signals, and effectors
involved in this system appears more tractable under physiological conditions than for other models.
In marine teleosts, spermatocyte motility increases
rapidly (within seconds) after release into a plasma-hyperosmotic environment (i.e., seawater). In
FW teleosts, spermatocyte motility increases with
comparable rapidity when released into a plasmahypoosmotic environment (FW) (175, 192). This
difference in osmotic effects on fish spermatocyte
motility is clearly a result of adaptation to living in
marine vs. limnic environments since the selection
pressure on spermatocyte motility is very strong.
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Multiple mechanisms for sensing concentrations
of inorganic ions and intracellular ionic strength
have evolved (95, 102). Because life originated in
the primordial ocean, evolution has favored mechanisms for monitoring concentrations of Na⫹, K⫹,
Ca2⫹, and Mg2⫹, which constitute the most prevalent cations in seawater and cells, having strong
effects on macromolecular function (131, 169).
Transient receptor potential (TRP) cation channels
have been shown to sense Na⫹, Ca2⫹, and osmolality in fish and other animals (8, 17, 19, 66, 110,
127, 154, 194, 199). Several isoforms of TRP channels, including osmosensory TRP vanilloid 4
(TRPV4), are expressed in many zebrafish tissues
(122, 134). TRPV4 is also expressed and salinityregulated in gill epithelial cells of euryhaline sea
bass (8). The osmosensory function of TRPV4 is
very highly evolutionarily conserved as evidenced by
functional complementation of nematode OSM-9
mutants (which lack a functional TRPV type channel)
with mammalian TRPV4 (111). TRPV channels are
highly sensitive to changes in osmolality and inorganic ions, and, like ARC channels (see above), their
activation modulates intracellular Ca2⫹ (110).
Calcium signaling pathways and intracellular
calcium concentration play a central role for controlling osmosensory signal transduction pathways
in fishes (FIGURE 2). For instance, hyposomotic
stimulation of prolactin secretion by rostral pars
distalis cells isolated from tilapia pituitary gland
(hypophysis) requires changes in intracellular calcium levels (166). In addition, the osmoregulatory
hormone cortisol inhibits prolactin secretion via reduction of free intracellular calcium (78), whereas
angiotensin II, another osmoregulatory hormone, increases free intracellular calcium in fish tissues (161).
A key role of intracellular Ca2⫹ for fish osmosensory
signal transduction independently emerged when
modeling a signaling network based on 20 immediate early genes that are rapidly induced during salinity stress in tilapia gill (55).
Extracellular Ca2⫹ concentration also plays a
critical role for osmosensing by directly modulating the activity of a transmembrane glycoprotein,
the calcium sensing receptor (CaSR) (FIGURE 2).
First cloned in the Hebert Lab in 1994, this protein
is important for Ca2⫹ homeostasis and osmoregulation in fishes and other vertebrates (1, 13, 74,
117). In a plasma-hyperosmotic environment, the
effective Ca2⫹ concentration for CaSR regulation
corresponds to the range found in the external
milieu, and this protein has been proposed to be a
salinity sensor in fish (145). CaSR is expressed in
osmoregulatory tissues of diverse fishes, including
elasmobranchs and teleosts (53, 54, 62, 83, 84, 118).
In fish gills, CaSR has been localized to mitochondria-rich cells (chloride cells) (117, 145). In fish
pituitary gland, CaSR has been localized to the
rostral pars distalis and the pars intermedia (118).
Pituitary CaSR mRNA abundance decreases rapidly
(by 50% within hours) in tilapia exposed to hyperosmotic stress, indicating that it is subject to feedback regulation during osmotic stress (184). Such
feedback regulation of CaSR mRNA abundance
was not observed in flounder gill and corpuscles of
Stannius (11, 71). However, CaSR undergoes a shift
in molecular mass when flounder is exposed to
osmotic stress, suggesting possible posttranslational modification, which is consistent with a
proximal osmosensory role of this protein (71).
CaSR has been shown to activate downstream signaling pathways in euryhaline tilapia in response to
salinity change, including phospholipase C-dependent and mitogen-activated protein kinase (MAPK)dependent signaling pathways (119). Because CaSR
is a trimeric G-protein-coupled receptor that acts
through modulation of adenyl cyclase activity, its
activation also affects intracellular cAMP levels (31)
(FIGURE 2). A role of adenyl cyclase in osmosensing of fishes is supported by electrophysiological
data on isolated opercular membranes of euryhaline killifish. Using these preparations, it has been
demonstrated that forskolin (an adenyl cyclase agonist) modulates transepithelial chloride secretion
(128). In addition, forskolin also markedly alters
osmoregulatory hormone/cytokine secretion from
tilapia pituitary gland (50).
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activation of spermatocytes in marine (140, 200,
202) and FW teleosts (141). In marine species, hypertonicity-induced spermatocyte shrinkage is intensified by aquaporins (201) and results in the
activation of adenyl cyclase and cAMP signaling
(200) even though cAMP is not necessary for initiation of spermatocyte motility in all species (159).
Alternative pathways (possibly with redundant
functionality to compensate for lack of cAMP signaling under certain conditions) may be involved
in some species.
The Role of Protein Phosphorylation for
Osmosensing
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Such evolutionary (genetic) adaptation illustrates
that the “interpretation” of osmotic stress can be
adjusted to generate a specific set of intracellular
signals for activation of appropriate effector mechanisms. Of particular interest in this regard are
euryhaline fishes that are capable of reproducing
in both marine and FW environments, e.g., tilapia
and sticklebacks (41, 138). These strongly euryhaline fish species employ physiological acclimation
mechanisms for changing the “interpretation” of
osmotic stress signals by spermatocytes. In such
species, osmosensory signal transduction is apparently reset to switch the trigger for initiation
of spermatocyte motility from hypo- to hyperosmolality after acclimation of fish to a plasmahyperosmotic environment (and vice versa). Not
much is known about the mechanisms associated
with such resetting, which seems to include differential phosphorylation of antioxidant proteins (138). In
addition to osmolality, fish spermatocyte motility
strongly depends on extracellular Ca2⫹ (106, 140).
The effect of extracellular Ca2⫹ on spermatocyte
motility varies somewhat between different species
of fish and is pH dependent (4, 139, 148). Uncoupling extracellular Ca2⫹ from osmolality has a
strong effect on spermatocyte motility in tilapia,
independent of which salinity the animals had
been acclimated to (112). These phenomena suggest that osmosensory input via CaSR may be critical for resetting the osmotic stress signaling
network during salinity acclimation of those euryhaline fish that can reproduce in marine habitat as
well as in FW. Interestingly, osmotic stimulation of
spermatocyte motility and flagellar beating is mediated via intracellular Ca2⫹ signaling in both marine and FW fish (3, 12). In euryhaline tilapia,
intracellular Ca2⫹ of spermatocytes increases during both hypotonic and hypertonic stress, but the
Ca2⫹ source for this increase is different. During
hypotonic stress, intracellular Ca2⫹ store depletion
occurs, whereas during hypertonic stress extracellular Ca2⫹ is taken up (140, 141). In common
carp, however, extracellular Ca2⫹ uptake may
also play a role during hypotonicity (91). In pufferfish, it has been demonstrated that an increase
in Ca2⫹/calmodulin interaction is required for
triggering spermatocyte motility (92). These data
strongly reinforce the notion that Ca2⫹ is of central importance for osmosensing (see The Role of
Inorganic Ions for Osmosensing above).
In addition to Ca2⫹, fish spermatocyte motility
also depends on extra- and intracellular K⫹ (and to
a lesser extent Na⫹ and possibly Cl⫺) in FW and
marine fishes (2, 3, 33, 136, 137, 174, 175). Thus
inorganic ion concentrations represent critical signals for osmosensory signal transduction in fish
spermatocytes. These signals mediate differential
phosphorylation of flagellar proteins during osmotic
Reversible phosphorylation/dephosphorylation of
proteins represents a major mechanism of intracellular signal transduction, which dynamically alters the composition and localization of protein
complexes and controls cell function and physiological responsiveness via adjustment of effector
protein abundance and activity (18, 171, 185). Any
search/screen for protein kinases and phosphatases involved in osmosensing is bound to be provoking, not because none can be identified but, on
the contrary, because too many of them are affected by one form of osmotic stress or another
(see general consideration of osmotic effects on
proteins discussed above). To illustrate the physiological significance of reversible, dynamic protein
phosphorylation for osmosensory signal transduction in fishes, I will now highlight some selected
examples of kinases, phosphatases, and protein
phosphorylation events (and the corresponding
osmoregulatory effectors) involved in this process.
A possible link between cytoskeletal proteins that
may sense osmotic stress (see discussion above) and
osmoregulatory effectors is focal adhesion kinase
(FAK), which is an enzyme that responds to altered
cytoskeleton dynamics by promoting stabilization of
the cytoskeleton (5, 49, 120, 121, 188). In gill and
opercular epithelia of euryhaline killifish exposed
to hypotonicity, FAK is dephosphorylated in an
integrin-dependent manner (125). Such FAK dephosphorylation has been shown to alter the activity of the Na⫹/K⫹/2Cl⫺ (NKCC) cotransporter and
the cystic fibrosis transmembrane conductance
regulator (CFTR) Cl⫺ channel in these tissues in
response to salinity change (124, 126). In primary
cultures of Japanese eel gill cells, hypertonic induction of the Na⫹/Cl⫺/taurine co-transporter, which
represents an important effector of osmotic stress
signaling pathways, depends on myosin light chain
kinase (MLCK) (21). In this tissue, MLCK-dependent
phosphorylation of a major cytoskeletal building
block that associates with tight junctions (MLC) is
accompanied by redistribution of F-actin to the cell
periphery (21).
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suggesting that its function during osmotic stress is
evolutionarily conserved in vertebrates (59).
The Role of Macromolecular Crowding and
Damage for Osmosensing
Macromolecular crowding (or dilution) and macromolecular damage are direct physical consequences of salinity stress that contribute to the
combinatorial signaling logic of osmosensing.
These effects are mostly encountered during severe/acute osmotic stress (FIGURE 1). Changes in
macromolecular density are intimately linked to
cell volume changes. Experiments with primary
fish cells isolated from a wide variety of tissues
have shown an abundance of molecular responses
to osmotically induced changes in cell volume, and
the corresponding literature is too extensive to be
reviewed here (6, 16, 40, 73, 88, 107, 108, 178, 193).
Some of these studies imply that membrane
stretch is a trigger for osmosensory signal transduction. However, it seems unlikely that changes
in cell volume lead to overall changes in membrane tension such as stretch because most cells
(in particular epithelial cells) are characterized by
basolateral and apical plasma membrane infoldings (at least in vivo). It is more likely that cell
volume changes are sensed via changes in macromolecular density in general or in certain localized
areas (such as specialized membrane lipid rafts).
Isolated fish cells respond to anisotonic osmotic
stress by activating a multitude of diverse signaling
pathways and adaptive effector mechanisms for
counteracting osmotic stress-induced volume changes
(6, 16, 88, 108, 178).
Macromolecular damage resulting from perturbed macromolecular density and intracellular
ionic strength represents an important signal for
perception of osmotic stress (35–37, 93, 94, 96, 103,
105). Thus mechanisms for protein, DNA, and RNA
stabilization are activated during osmotic stress in
fish. HuR is an mRNA stabilizing protein that is
induced during hyperosmotic stress in tilapia gill
epithelium (55). It is possible that HuR relays information about mRNA stability during hyperosmolality to the osmosensory signal transduction
network. Another protein that is rapidly upregulated in tilapia gill epithelium during hyperosmotic
stress is a ubiquitin E3 ligase (a Grail/Goliath homolog) (55). Tilapia Grail was localized to the tips
of secondary lamellae in fish gill epithelium and
shown to increase osmotic stress tolerance of
HEK293 cells when overexpressed in this human
cell line (60). A similar ubiquitin E3 ligase (an Rbx1
homolog) is also induced by hyperosmotic salinity
stress in salmon (153). Ubiquitin E3 ligases may
sense protein damage by quantifying the amount
of substrates and/or integrating signals transduced
via diverse proteins that are being tagged with
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Another link between osmotic effects on cytoskeletal proteins and phosphorylation-based signal
transduction is apparent for mitogen-activated
protein kinase (MAPK) cascades. All three major
MAPKs, in particular the stress-activated protein
kinases (SAPKs) p38 and jun-N-terminal kinase
(JNK), are activated by small GTPases of the Rho
family that are linked to the cytoskeleton (187).
Even though MAPKs are highly evolutionarily conserved and utilized for osmosensory signal transduction in all eukaryotic cells (98, 101), the
regulation of MAPKs by activators and inhibitors
and MAPK substrates are far less conserved between diverse groups of animals and other eukaryotes (57). MAPK cascades seem to have
evolved as a major cell signaling node capable of
integrating diverse combinations of inputs to produce relevant signaling output. We have shown
that the activity of p38, JNK, and extracellular signal-regulated (ERK) MAPK cascades is rapidly altered in gills of euryhaline killifish after exposure to
salinity stress in vivo (100). Osmotic regulation of
p38 and JNK phosphorylation was also observed in
killifish isolated opercular epithelium. In such in
vitro preparations, chloride secretion is inhibited
by a pharmacological p38 blocker (107). In addition, regulatory volume decrease of isolated hepatocytes from turbot (Scophthalmus maximus) depends
on p38 MAPK (132). Furthermore, an upstream regulator of MAPK cascades, mitogen-activated protein kinase kinase kinase 7 interacting protein 2
(TAK 1 binding protein 2 ⫽ TAB 2) is induced as an
immediate early gene during hyperosmotic stress
in tilapia gill epithelium (46). TAB2 induction is
very rapid (within 2 h at the mRNA level) and
transient, which is consistent with a role in osmotic
stress signaling. In euryhaline medaka, activation
of the JNK pathway is required for osmotic induction of the osmotic stress transcription factor 1b
(OSTF1) (160). OSTF1 has been discovered after a
suppression subtractive hybridization screen for
salinity-induced genes in Mozambique tilapia
gill epithelium (58). This protein is a homolog of
mammalian transforming growth factor ␤ (TGF␤)-stimulated clone 22 3b (TSC22–3b) (56, 59). A
role for OSTF1 in osmotic stress signaling also has
been demonstrated for other euryhaline fishes, including black porgy (20), Japanese eel (22, 179),
Nile tilapia (12), and medaka (180). A role of OSTF1
in osmosensory signal transduction is not limited
to fishes but is also evident in mammalian cells,
where we identified TSC22D2 as an OSTF1 ortholog that is activated and alternatively spliced in
response to hypertonicity with similar kinetics
as in fish gill cells (59). Overexpression of mammalian TSC22D2 increases osmotolerance of murine
inner medullary collecting duct (mIMCD3) cells,
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appropriate and specific response at the cellular
level.
Osmosensing by Multiple Tissues
In multicellular animals, the combinatorial nature
of osmosensing is compounded at higher levels of
organization. In particular osmoregulators have
multiple osmosensory tissues with specialized osmoreceptor cells that monitor ECF osmolality.
Some osmosensory tissues are also target tissues
for systemic osmoregulation (e.g., epithelia that are
directly exposed to the external environment)
(FIGURE 3). Although osmotic stress has consequences for all cell types, their sensitivity toward
osmotic stress and capacity for generating systemic signals in response to osmotic stimulation
differs (e.g., specialized osmoreceptor cells are
highly sensitive, whereas other cell types are less
sensitive). The combination of signals generated in
multiple tissues by osmoreceptor cells and other
osmosensory cell types (e.g., certain epithelial
cells) and their relative strengths provides a means
for integrating and coordinating osmotic stress responses at the whole organism level.
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ubiquitin (123). In most cases, such protein substrates are terminally damaged and destined for
proteolytic degradation and removal, but specific
signaling proteins also transduce information via
reversible mono-ubiquitination (15, 85, 189). Some
targets of ubiquitin E3 ligases are osmoregulatory
effector proteins. For instance, in mammalian kidneys, the interaction of Nedd4 E3 ubiquitin ligase
with epithelial Na⫹ channels (ENaC) is controlled
by osmolality, vasopressin and 14-3-3 proteins
(81). Whether these osmoregulatory effectors are also
targets of the osmotic stress-induced ubiquitin E3
ligases in fish remains to be determined. Ubiquitinmediated protein degradation prevents nonspecific
aggregation of terminally damaged proteins, i.e., proteins with exposed hydrophobic residues that cannot
be repaired/correctly refolded. Molecular chaperones (including heat shock proteins) are utilized and
induced during many forms of environmental stress
to either direct such proteins toward proteolytic degradation or, alternatively, to refold them into their
native state to prevent nonspecific protein aggregation (52, 77, 152, 160). Many examples (too many to
be mentioned here) for molecular chaperone activation in fish challenged by osmotic stress have
been documented (28 –30, 99, 197). The latter function of molecular chaperones resembles that of
compatible osmolytes in the context of osmotic
stress illustrating that macromolecules and micromolecules act in concert to maintain optimal protein conformation during osmotic stress. Notably,
molecular chaperones themselves are subject to
modulation by osmolytes (172). Because unfolding
and degradation of specific signaling proteins during osmotic stress will alter intracellular signal
transduction, it is conceivable that cells monitor
how much overall protein damage is accumulated
during osmotic stress via the activity of molecular
chaperones. A mechanism for such monitoring
and induction of appropriate responses is exemplified by transcriptional regulation via heat shock
factors (e.g., HSF1) and the regulation of these
proteins via sequestration by molecular chaperones (157, 186). HSF sequestration is in competition with binding of other substrates (i.e., unfolded
proteins) to molecular chaperones. As a result of
such competition, the transcriptional activity of
HSF represents a readout of the amount of protein
damage in cells. Similar regulatory circuits operate
to sense and quantify cellular DNA damage (115,
129). Macromolecular damage quantitation may
not be stressor-specific (unless certain types of
damage are associated with only one specific form
of stress), but it contains information about the
severity of stress. Combined with signals from
other osmosensors, macromolecular damage sensors contribute to encode an overall integrated signal that allows for a qualitatively and quantitatively
Cerebral, Hypophyseal, and Systemic
Osmoreceptor Cells
Most fishes are osmoregulators that maintain osmotic homeostasis of their ECF constant even
when environmental salinity/osmolality is altered.
Evolution of this physiological mechanism/trait in
ancestral bony fishes was an important prerequisite for tetrapods to become independent of
aquatic habitats and conquer land. During exposure of euryhaline fish to severe osmotic stress
(e.g., transfer from FW to SW or vice versa), the
internal osmolality setpoint of ⬃300 mosmol/kg
can deviate temporarily by as much as ⫾100
mosmol/kg (34, 86, 104, 184). Smaller environmental salinity changes lead to proportionally smaller
changes in ECF osmolality and to shorter periods
of deviation of ECF osmolality from the optimal
setpoint. Because the constancy of the “milieu
interieur” is pivotal and tighly regulated in all vertebrates, any deviations from the homeostatic optimum trigger compensatory responses that are
aimed at restoring osmotic homeostasis of ECF.
Special osmoreceptor cells have evolved in fishes
to monitor body fluid and electrolyte homeostasis
and provide sensitive regulatory feedback upon
deviation from the optimal setpoint. Their function
illustrates the importance of sensory feedback as a
general principle for maintaining homeostasis of
physiological systems (147). These specialized osmoreceptor cells are superior over other cell types
in converting osmotic signals into specific physiological outcomes. In the brain, such osmoreceptor
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extracellular osmolality changes in the range of
plasma osmolality variation experienced in teleosts exposed to salinity stress (69). These cells
swell in response to hypo-osmotic stress-induced
decrease of ECF osmolality. Prolactin cell swelling,
in turn, triggers release of prolactin, which is an
important hormone/cytokine for coordinating acclimatory responses to plasma-hypo-osmotic environments (191). The swelling-induced prolactin
release is mediated by stretch-activated ion channels, aquaporin 3, and the second messengers
Ca2⫹ and cAMP (164 –166, 190).
Endothelial ECF volume-/pressure-sensing neurons are located in the vasculature of gill arches in
several teleost and elasmobranch species (14). These
cells correspond to the mammalian baroreceptor
cells that are located in the carotid and aortic arches,
which are evolutionarily derived from fish gill arches
(132). In mammals, these baroreceptors contribute
(at least in the short term) to body fluid osmotic and
volume homeostasis by signaling to osmoregulatory
effector tissues that control blood pressure via adjustment of water intake and urinary excretion (27,
68, 90). Coordination of neurotransmitter signals
from cerebral, hypophyseal, and systemic osmoreceptor neurons contributes to synchronizing the
whole organism physiological response to salinity
stress in fishes. Another important element for coordination of whole organism responses are osmoregulatory hormones/cytokines.
Osmosensing by Osmoregulatory Epithelia
Epithelial cells directly facing the external milieu
such as gill epithelial cells and cells lining the gastrointestinal tract including the esophagus are
FIGURE 3. Multiplicity of osmosensory tissues in fishes
Different organs and tissues that are known to participate in osmosensing in fishes are shown color-coded in
relation to their overall body position. Fishes integrate osmosensory signals perceived by osmoreceptor neurons and epithelial cells in these tissues to coordinate whole organism osmoregulatory responses: brain (gray),
pituitary gland (blue), gills (carmine), GI tract including esophagus and oral cavity (orange), bulbus arteriosus/
branchial endothelium (red).
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cells are neurons whose action potentials, firing
rate, and neurotransmitter secretion are tightly
controlled by ECF osmolality (10). In mammals,
these neurons are localized to distinct circumventricular brain areas including the organum vasculosum laminae terminalis (OVLT), subfornical
organ, area postrema, and posterior pituitary (9).
In fishes, the OVLT is present and well developed
but has not been studied with regard to its role for
osmosensing (65). However, the area postrema and
subfornical organ are known to participate in the
control of osmoregulatory actions of atrial natriuretic peptide and renin-angiotensin hormone systems in euryhaline eels (146, 176, 181). Thus it
appears that osmoreceptor cells in the brain are
conserved in all vertebrate classes, consistent with
the conserved physiological capacity for maintaining a constant ECF osmolality.
The posterior pituitary (i.e., neurohypophysis derived
from neural ectoderm) representing an extension of the
brain containing a collection of hypothalamic neuronal
projections harbors mammalian osmoreceptor cells (9).
Of interest, however, in fishes, the most prominent
hypophyseal osmoreceptor cells have been identified in the adenohypophysis, specifically in the
pro-adenohypophysis (rostral pars distalis), which
is derived from oral ectoderm (7, 43, 69, 149). Of all
the brain structures mentioned above, the role of
the hypophyseal rostral pars distalis for osmosensing in fishes is best understood. This discrete tissue
consists to a large degree of prolactin-producing
cells, which can be isolated and cultured in vitro
(42). Experiments on such isolated cells provided
proof that they are functioning as osmoreceptors. Prolactin cells are capable of sensing subtle
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c-fos and osmotic stress transcription factor 1
(OSTF1) (56, 99). Moreover, cytokines such as interleukin 8 (IL-8) and tumor necrosis factor ␣
(TNF-␣) are part of the osmotic stress signaling
network in tilapia gill epithelium (55). In addition,
proteome analysis of euryhaline leopard sharks
suggested that TNF-␣ is an important element in
the osmotic stress response network of gill and
rectal gland epithelium (38). Therefore, cytokines
that act as para-, auto-, and/or endocrine messengers represent an additional avenue for transducing osmotic signals within and between cells and
tissues (95).
The term “cytokine” is used as a common denominator of a diverse array of soluble proteins
and peptides that regulate cell and tissue functions
at low concentrations (80). Important cytokines in
fishes include known osmoregulatory growth factors and peptide hormones (e.g., growth hormone,
insulin-like growth factor 1, atrial natriuretic peptide, and prolactin). TNF-␣ and ILs are cytokines
that can act in autocrine and paracrine fashions
and, therefore, reinforce and coordinate epithelial
responses to salinity change (FIGURE 4).
Another group of cytokines that are involved in
fish osmosensing are bone morphogenetic proteins (BMPs). A BMP receptor (BMPR2) increases
rapidly during hyperosmotic stress in tilapia gill
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prime candidates for sensing changes in environmental salinity and evoking rapid anticipatory responses even before disruption of physiological
homeostasis. In fishes, neuroepithelial cells are
embedded in the gill epithelium (39), and some of
these cells are in direct apical contact with the
external environment (198). Neuroepithelial cells
are known to produce and respond to cytokines
(144, 173), and they were suggested to have local,
paracrine functions in euryhaline fishes (67, 135).
Osmoregulatory cytokines that are synthesized locally in fish gills include endothelins and urotensins (46, 79). Nevertheless, the paradigm for
osmosensing in vertebrates emphasizes brain and
systemic osmoreceptor neurons discussed in the
preceding paragraph. These neurons convert an
osmotic stimulus into an electrical signal that can
be transduced along axons and then converted
into a neurotransmitter response at efferent nerve
endings (9). However, (at least some) osmotic
stress induced re-differentiation and re-programming of mitochondria-rich cells in tilapia gill and
yolk sac epithelia, and primary cultures occur independent of systemic (neuroendocrine) signals
(56, 76, 87). Experiments on isolated gill cells of
euryhaline fish have shown that they are capable of
responding directly to osmotic stress by activating
genes involved in cytokine production, including
FIGURE 4. Integration of auto-, para-, and endocrine cytokine and hormone signals with osmosensing in gill epithelial cells
during osmotic stress
Osmotic stress responses of different cell types are unequal because they have different molecular phenotypes (e.g., they express distinct quantities of cytokine receptors and osmosensors). Distinct responses of different cell types underlie extensive remodeling of gill epithelium in euryhaline
teleosts exposed to a reversal of the osmotic gradient between extracellular fluid and the environment. For clarity, general impacts of cytokines
and hormones are only indicated for a single mitochondria-rich cell. The same principle applies to the other cells, but the corresponding relationships are not depicted. Shown are pavement cells (green), mitochondria-rich (chloride) cells (orange), neuroepithelial cells (blue), and undifferentiated progenitor cells (white).
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integration of these signals into intracellular osmosensory signaling networks and contributes to the
generation of cell type-specific outcomes that result from osmotic stress.
Conclusions
Changes in environmental osmolality have profound effects on macromolecules, cells, and organisms that need to be compensated for to maintain
adequate physiological functionality at all levels of
organization. In fishes and other vertebrates, perception of altered environmental osmolality (osmosensing) is a highly complex physiological process
that involves many elements at multiple levels of
organization. To coordinate osmoregulatory responses of multiple tissues and cells and to mount
appropriate effector mechanisms that match the
magnitude, ionic nature, and other aspects of osmotic stress, many signals (none of which may be
stressor specific) are integrated in a combinatorial
manner. In theory, such combinatorial osmosensing allows for fine-grained transduction of information about the specific nature of osmotic stress,
which enables activation of appropriate effector
mechanisms. In praxis, we do not (yet) know the
code that governs how multifaceted osmosensory
input is integrated in signal transduction networks
to yield specific qualitatively and quantitatively accurate outcomes. To decipher this code, we need
to know and understand in detail the behavior of
FIGURE 5. Model for prolactin receptor (PRLR) regulation in gill epithelial cells of euryhaline fish during salinity stress
A: in plasma hypo-osmotic environments, functional (long) forms of both PRLRs are expressed. These receptors homo- or hetrodimerize and effectively bind PRL, which is present in high concentration in plasma. PRL binding activates PRLR dimers and induces the Jak2/Stat5 pathway, among
other pathways. B: when fish are transferred from a plasma hypo- to a plasma hyperosmotic environment, then alternative splicing leads to a rapid
increase in the short form of PRLR2, which lacks a large part of the extracellular domain. The short form sequesters functional (long form) PRLRs via
heterodimer formation, which prevents these dimers from binding PRL. Thus gill epithelial cells are rapidly rendered less responsive to circulating
PRL to suppress this cytokine’s stimulating effect on active NaCl absorption and to allow gill epithelial cells to rapidly switch to active NaCl
secretion.
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epithelium (55). In zebrafish gills and yolk sac epithelium, BMPs control the balance between DeltaNotch signaling and, correspondingly, the activity
of the transcription factors glial cell missing 2
(Gcm2) and Foxi3a (44, 45). When Notch function
is impaired by mutation, Foxi3a expression is increased, and differentiation of precursor cells is
shifted toward generation of H⫹-ATPase-rich mitochondria-rich cells (45). Conversely, overexpression of Notch drives precursor cell differentiation
toward the keratinocyte cell fate (45). Collectively,
these findings indicate that BMP cytokines contribute to remodeling of gill epithelium via mitochondria-rich cell fate determination by Delta-Notch and
Foxi3a signaling pathways, even though the source
of BMP (auto-, para-, or endocrine) is currently
unknown (FIGURE 4).
Responses of osmoregulatory target tissues to
systemic or local extracellular signals are likely
“primed” by the internal state of cells in those
tissues. For instance, cytokine responsiveness is
determined by the presence of corresponding receptors in target cells and thus can be modulated
via regulation of receptor abundance (see BMPR2
above). Another mechanism for such modulation
is the expression of decoy cytokine receptors. An
example is the osmotic induction of a short splice
variant of tilapia prolactin receptor 2 that lacks
most of its extracellular prolactin binding domain
(61) (FIGURE 5). Differential responsiveness of cells
to extracellular signals such as cytokines alters the
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the key elements that comprise the osmosensory
signaling network, not just in any given cell but
also at the whole organism level. Based on our
current knowledge, it appears that calcium is the
most critical intracellular messenger for transducing information about osmotic stress in fish. But
there are many additional elements, which have
been briefly outlined in this essay, that contribute
to osmosensory signaling. Defining the role and
relative importance for osmosensing of each of
these elements in various physiological contexts
and cell types represents a formidable challenge. 䡲
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This work was funded by the National Science Foundation (IOS-1049780).
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