Expression and functional analysis of mussel taurine transporter, as a key molecule in cellular osmoconforming
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Author for correspondence (e-mail:
toyohara{at}kais.kyoto-u.ac.jp)
Accepted 1 September 2005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: taurine, taurine transporter, osmolyte, osmoconforming, adaptation, Mediterranean blue mussel
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Whereas information on the osmotic adaptation and regulation at both the
cellular and whole-body levels, and the related molecular mechanisms in
teleost fish, has been collected (Karnaky
et al., 1998; Wood et al., 1995), osmotic adaptation at a cellular
level in aquatic invertebrates, which is probably a more vital process than
that in teleost fish or mammals, has almost been ignored and is still poorly
understood. Although the studies dealing with the biochemical analysis of
enzymes related to osmolyte metabolism
(Nomura et al., 2001
;
Yoshikawa et al., 2002
),
change in cell volume on exposure to salinity change (Neufeld and Wright,
1998
,
1996a
,b
),
and transport activity of osmolytes
(Neufeld and Wright, 1995
;
Petty and Lucero, 1999
;
Silva and Wright, 1992
) have
been reported, the related molecular mechanisms have never been studied.
Here, we report the molecular cloning, function and expression analysis of
a taurine transporter involved in taurine uptake in the cell of the
Mediterranean blue mussel. Most molluscs cannot entirely regulate their
internal environments (Somero and Bowlus,
1983). Among these, brackish species, including mussels, have to
adapt to a wide range of osmolality (almost 0-1000 mOsm kg-1).
Taurine is found to be the most abundant and thus, is an important osmolyte
not only in mussels (Deaton et al.,
1985a
,b
;
Gills, 1972
;
Livingstone et al., 1979
;
Potts, 1954
;
Toyohara and Hosoi, 2004
;
Zurburg and DeZwaan, 1981
) but
also in numerous other invertebrates, fishes and mammals
(Huxtable, 1992
).
Intracellular taurine concentration in one species of mussels was estimated to
be approximately 200 mmol l-1, corresponding to one-fifth of the
total intracellular osmolality (Neufeld
and Wright, 1995
). The intracellular taurine content is mainly
regulated via a transmembrane transport
(Huxtable, 1992
). Taking these
facts into consideration, the taurine transporter in animals such as mussels,
which mainly use taurine as an osmolyte, could be a key molecule of the
cellular osmoconforming process. In some mammals and fishes, taurine
transporters have already been cloned (Han
et al., 1998
; Jhiang et al.,
1993
; Liu et al.,
1992
; Miyamoto et al.,
1996
; Ramamoorthy et al.,
1994
; Smith et al.,
1992
; Takeuchi et al.,
2000a
,b
;
Uchida et al., 1992
;
Vinnakota et al., 1997
) and
the involvement of some of them in cellular osmotic adaptation were confirmed
(Takeuchi et al.,
2000a
,b
;
Uchida et al., 1992
), although
the taurine content and its contribution to the intracellular osmolality and
osmotic adaptation in these animals are relatively lower than that in
mussels.
In 1937, Baldwin summarized the regulation of the internal environment,
namely the solute composition and the consequent osmotic pressure, in aquatic
animals (Baldwin, 1937). Even
though the detailed mechanisms were unclear, it was already known that some
brackish invertebrates that were not equipped with advanced mechanisms for
regulating their internal environment, could adapt to significant changes in
environmental salinity. This present study is a major contribution to our
understanding of the mechanism of osmotic adaptation at a cellular level in
aquatic invertebrates, which has been known about since the beginning of the
last century, but is only now being elucidated.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cloning and sequencing
Total RNA was prepared from the mantle of mussels by the guanidine
isothiocyanate/cesium chloride method, and poly(A)+ RNA was
purified using Oligotex-dT30 (Takara Bio Inc., Shiga, Japan). cDNA fragments
were amplified by reverse transcription-polymerase chain reaction (RT-PCR). To
obtain cDNAs as PCR templates, 1 µg of poly(A)+ RNA was
reverse-transcribed with oligo(dT)20 primer. For PCR, the
degenerate primers with EcoRI sites were prepared on the basis of the
conserved amino acid sequence in the putative fourth extracellular loop and
ninth transmembrane domain of mammalian and fish taurine transporters
(Han et al., 1998;
Jhiang et al., 1993
;
Liu et al., 1992
;
Miyamoto et al., 1996
;
Ramamoorthy et al., 1994
;
Smith et al., 1992
;
Takeuchi et al., 2000a
;
Takeuchi et al., 2000b
;
Uchida et al., 1992
;
Vinnakota et al., 1997
). The
sequences of the forward and reverse primers were
5'-dCGGAATTCTTYATGGCNCARGARCARGGN-3' and
5'-dCGGAATTCCATNCCNCCYTCNGTNACCAT-3', respectively. The amplified
PCR product (324 base pairs) was subcloned into the EcoRI site of
pBluescript II KS+ (Stratagene, La Jolla, CA, USA) and the sequence
was determined. In order to identify the 5' and 3' ends of the
cDNA corresponding to this fragment, 5' and 3' rapid amplification
of cDNA ends (RACE) was performed using the SMARTTM RACE cDNA
Amplification Kit (BD Biosciences, San Jose, CA, USA). Amplified fragments
were directly subcloned into pGEM-T Easy vector (Promega Corporation, Madison,
WI, USA) and sequenced on both strands. The determined sequences were
assembled into one contig with an open reading frame, called muTAUT for the
mussel taurine transporter. For the expression of muTAUT, the sequence of the
complete open reading frame was amplified and subcloned into the
pcDNA3.1/myc-His B expression vector (Invitrogen Corporation, Carlsbad, CA,
USA) and the pCS2+ vector (Turner and
Weintraub, 1994
) for expression in HEK293T cells and Xenopus
laevis oocytes, respectively. The constructs were then verified by
sequencing. The nucleotide sequence for muTAUT has been deposited in the
DDBJ/EMBL/GenBank databases under accession number AB190909.
Expression in HEK293T cells
HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum, 25 mmol l-1 Hepes, 100
units ml-1 penicillin, and 100 µg ml-1 streptomycin
at 37°C in air containing 5% CO2. Cells were plated in 24-well
plates and transiently transfected with the muTAUT-pcDNA3.1/myc-His B vector
or the mock vector, using cationic liposomes (LipofectAMINE, Invitrogen
Corporation) in accordance with the manufacturer's instructions. The
transfection was carried out for 16-18 h, and the medium was then replaced.
Cells were assayed for taurine uptake activity 48 h post-transfection as
described previously by Takeuchi et al.
(2000b). Since the taurine
uptake was almost linear for at least the first 45 min of incubation (data not
shown), we used a 30 min uptake incubation period for the experiments.
Expression in Xenopus laevis oocytes
cRNA of muTAUT was obtained by in vitro transcription from
linearized muTAUT-pCS2+ vector using SP6 RNA polymerase. Stage IV-V
Xenopus laevis oocytes were isolated and the follicle removed using
Collagenase A (Roche Diagnostics, Mannheim, Germany). They were then injected
with 25 ng cRNA diluted with 50 nl diethyl pyrocarbonate-treated water per
oocyte and incubated in modified Barth's solution supplemented with 100 units
ml-1 penicillin and 100 µg ml-1 streptomycin for 2-3
days. The oocytes were briefly rinsed in a standard uptake buffer
(Utsunomiya-Tate et al., 1996)
and incubated in 24-well plates containing 1 ml of standard uptake buffer for
30 min at room temperature. Since a higher concentration of NaCl in the medium
was required to allow sufficient taurine uptake via muTAUT for
detailed functional analysis (described in Results and Discussion), the
taurine uptake activity of the oocytes was measured in NaCl-added uptake
buffer (standard uptake buffer containing 200 mmol l-1 NaCl). The
oocytes were incubated in 0.5 ml of NaCl-added uptake buffer containing 0.5
µCi ml-1 [3H]taurine (NEN Life Science Products,
Inc., Boston, MA, USA) for 30 min. Taurine and NaCl concentrations in the
buffer were varied and a portion or all of the Na+ and the
Cl- ions were replaced with choline ions and either gluconate or
NO3- ions, respectively, depending on the purpose of the
experiment. The oocytes were rinsed three times with standard uptake buffer
and each of them was solubilized by pipetting in 200 µl of water. The
radioactivity was measured by liquid scintillation counting.
The tilapia taurine transporter (tTAUT, accession no. AB033497) was also subcloned into pCS2+ vector and expressed in the oocytes using the same method as for the comparative analysis of the NaCl concentration requisite for taurine uptake activity.
Northern blot analysis
Twenty micrograms of total RNA purified from the mussels were
electrophoresed in 1% agarose gels containing 2 Mformaldehyde. RNAs were
transferred to a nylon membrane (Gene Screen Plus, NEN Life Science Products,
Inc.) and muTAUT mRNA was hybridized with the 324 base fragment of the RT-PCR
product labeled with [-32P]dCTP using a Megaprime DNA
labeling system (Amersham Biosciences Corp., Piscataway, NJ, USA) for 15 h at
65°C in 0.25 M Na2PO4, 1 mmol l EDTA, and 7% SDS
(Church and Gilbert, 1984
).
This filter was rinsed three times with 2x standard saline citrate (SSC)
and washed three times with 0.2x SSC containing 0.1% SDS for 20 min at
65°C, it was then subjected to autoradiography.
Immunohistochemistry
The mantle and gill tissues of the mussels exposed to osmotic change were
fixed in Bouin's fixative. Fixed tissues were dehydrated in an ethanol series
and embedded in paraffin wax. Thin sections of approximately 7 µm were
prepared using a microtome PR-50 (Yamato Koki, Tokyo, Japan). The sections
were pretreated with 1.5% H2O2 in phosphate-buffered
saline (PBS; 137 mmol l-1 NaCl, 8.1 mmol l-1
Na2HPO4, 2.68 mmol l-1 KCl, 1.47 mmol
l-1 KH2PO4) and then washed several times
with 1x PBS and 1x PBST (PBS with 0.1% Triton X-100). Tissues were
blocked in 10% fetal bovine serum in 1x PBST for 1 h and then incubated
with the diluted muTAUT polyclonal antibody. The antibody was raised against
the synthetic peptide of (C)SMEYEKFLQKDSNV, which corresponded to the
carboxyl-terminal end of muTAUT with an additional amino-terminal cysteine.
The antibody was purified using an affinity column to which the synthetic
peptide was bound, and was then further diluted 1:1000 in 1% skimmed milk in
1x PBST. After 12 h incubation with the diluted antibody, sections were
washed in 1x PBST for 30 min (three changes) and incubated for 30 min at
room temperature with the second antibody labeled with horseradish peroxidase
(Nichirei Corporation, Tokyo, Japan). After 30 min of PBST washes (three
changes), the colorimetric reaction was initiated by adding diaminobentidine
substrate (0.5 mg ml-1 3,3-diaminobenzidine and 0.005%
H2O2 in 1x PBS). Stained sections were observed
using a light microscope (FX-PH-21, Nikon Corporation, Tokyo, Japan).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Functional analysis in Xenopus oocytes
Fig. 2 shows the kinetics of
the taurine transport of muTAUT expressed in Xenopus oocytes.
Eadie-Hofstee plots revealed an apparent Km of 41 µmol
l-1. The Na+ and Cl- stoichiometry of taurine
uptake estimated by the `activated method' is shown in
Fig. 3. In order to estimate
the Na+ stoichiometry, taurine uptake induced by muTAUT was
measured at various Na+ concentrations with a constant
Cl- concentration. The relationship between taurine uptake and
Na+ concentration was sigmoidal
(Fig. 3A). Hill-type analysis
of the data revealed a Hill coefficient of 2.1 with a K50
value of 158 mmol l-1 Na+. These data suggest two or
more Na+ ions are coupled with the transport of one taurine
molecule. By the same analysis, taurine uptake resulting from muTAUT exhibited
a hyperbolic dependence on Cl- concentration, and the coefficient
was 1.2 with a K50 value of 81 mmol l-1
Cl-, suggesting the involvement of one Cl- ion per
transport of one taurine molecule (Fig.
3B).
|
|
In order to analyze the substrate specificity of the muTAUT, various agents
were examined for their ability to inhibit the [3H]taurine uptake
activity (Fig. 4). Taurine
uptake of muTAUT-injected oocytes was markedly inhibited by unlabeled taurine,
hypotaurine and ß-alanine. Gamma aminobutyric acid (GABA) inhibited
taurine uptake to a smaller extent than these ß-amino acids. All the
other agents of -amino acids and glycinebetaine also inhibited taurine
uptake with a statistical significance.
|
Since sufficient taurine uptake from muTAUT had not been detected in the standard uptake medium, taurine uptake activity of muTAUT-injected oocytes was measured in various medium conditions in a preliminary analysis. As a result, it was revealed that taurine uptake due to muTAUT necessitated higher NaCl concentration. Fig. 5 shows the relationship between taurine uptake and NaCl concentration in muTAUT- and tilapia taurine transporter (tTAUT)-injected oocytes. muTAUT-injected oocytes showed hardly any activity in 50 mmol l-1 or lesser concentration of NaCl in the uptake medium, and only 30.6% uptake was exhibited in the standard uptake medium (100 mmol l-1 NaCl). By contrast, tTAUT-injected oocytes showed adequate activity in the standard uptake medium. Since the medium had NaCl concentration above 100 mmol l-1, the medium was hyperosmotic for the Xenopus oocytes. Therefore, the effect of the medium osmolality on taurine uptake activity was also measured in the hyperosmotic medium containing 100 mmol l-1 constant concentration of NaCl by adding various concentrations of glycerol. Uptake activities of both muTAUT-injected and tTAUT-injected oocytes were unaffected by a hyperosmolality below 400 mOsm kg-1·H2O. In medium with a higher osmolality than 500 mOsm kg-1·H2O, significant decrease in the uptake activity was observed, probably because of hyperosmotic stress on the oocytes and thus, the analysis was impossible.
|
Expression of muTAUT
Northern blot analysis with RNAs isolated from the mantle, gill and
adductor muscle of the mussel exposed to a change in ambient osmolality caused
by the salinity change is shown in Fig.
6. Abundance of muTAUT RNA increased in all analyzed tissues at
least 2 h after the exposure to 2x seawater and was maintained at a
level higher than that at 0 h (Fig.
6A). In the mussel exposed to 0.5x seawater, induction of
muTAUT RNA was also observed (Fig.
6B); this was depressed by the addition of 25 mmol l-1
taurine to the ambient seawater in the mantle of mussels exposed to 0.5x
seawater for 24 h. (Fig.
6C).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although muTAUT has the same transport mechanism as previously cloned
taurine transporters, i.e. coupling with Na+ and Cl-
ions and the stoichiometry of these ions
(Table 1,
Fig. 3), it has also become
apparent in this study that muTAUT, which has structural features distinct
from those of other cloned taurine transporters, has three characteristic
functional properties. The first is that the muTAUT Km
value for taurine transport is much higher than that of mammalian taurine
transporters. The muTAUT Km value for taurine transport is
41 µmol l-1 (Fig.
2), and that for taurine transport in mouse brain
(Liu et al., 1992), mouse
retina (Vinnakota et al.,
1997
), dog (MDCK cells; Uchida
et al., 1992
), human retina
(Miyamoto et al., 1996
) and
human placenta (Ramamoorthy et al.,
1994
) is 4.5, 13.2, 9.1, 2.0 and 5.9 µmol l-1,
respectively. Km values of taurine transporters in rat
(Smith et al., 1992
) and carp
(Takeuchi et al., 2000b
),
estimated using COS-7 cells, are excluded from the discussion because there is
some indication that the use of COS-7 cells affected the
Km values (Takeuchi et
al., 2000b
). It should be noted that the Km
value for taurine transport of muTAUT is in good agreement with the
Km value of taurine transport in the basolateral surface
of the excised gill cells (35.3 µmol l-1) of California mussel
Mytilus californianus (Neufeld
and Wright, 1995
).
The second distinct functional property is the competition of various
-amino acids and glycinebetaine to taurine transport
(Fig. 4). A much smaller
competitive effect from
-alanine and proline has been reported for the
taurine transporter of human placenta
(Ramamoorthy et al., 1994
) and
tilapia (Takeuchi et al.,
2000a
). However, other studies in human placental cell
(Miyamoto et al., 1988
),
rabbit jejunal cells (Miyamoto et al.,
1989
), brush-border membrane vesicle cells
(Miyamoto et al., 1989
), human
neuroblastoma x glioma hybrid cells
(Kurzinger and Hamprecht,
1981
), MDCK cells (Uchida et
al., 1991
) and cloned transporters
(Liu et al., 1992
;
Smith et al., 1992
;
Takeuchi et al., 2000b
;
Uchida et al., 1992
) have
reported competition only by hypotaurine, ß-alanine and GABA. In
addition, competition analysis of the tilapia taurine transporter under the
same conditions as those in this study revealed that the taurine transport
activity was completely unaffected by
-amino acids (data not shown).
Considering these facts, our results suggest that taurine specificity of
muTAUT is lower than that of other reported taurine transporters. However,
there is also evidence that in two species of mussel,
-amino acids had
no effect on epidermal taurine transport
(Wright and Secomb, 1984
).
The third characteristic is that muTAUT taurine transport requires a higher
NaCl concentration (Fig. 5).
Although the relationship between the NaCl concentration in the external
medium and the taurine transport activity of the cloned taurine transporter
expressed in Xenopus oocytes has never been determined, kinetic
analysis of epithelial taurine transport activity in Mytilus
californianus gill revealed an apparent K50 value of
200-400 mmol l-1 Na+
(Silva and Wright, 1992;
Neufeld and Wright, 1995
).
Data obtained in the present study was interpreted as confirmation, at the
molecular level, of previous results obtained at a tissue level.
Why does muTAUT have these distinctive functional properties? The most
obvious answer to this question is because of differences in the primary
structure: there is relatively lower percentage identity between the deduced
amino acid sequence of muTAUT and other cloned vertebrate taurine transporters
than there is among the vertebrate taurine transporters themselves. In
particular, some mutations exist on the residues that are completely conserved
among vertebrates. Of these, mutations in the regions important for the
transport activity, such as the first extracellular loop and the adjacent
domain involved in the reaction with Na+ and Cl- ions
(Nelson and Lill, 1994) and
the transmembrane region involved in the recognition and selectivity of the
transport substrate (Palacin et al.,
1998
), possibly influence the functional properties. Even though
the elucidation of this question requires further studies, such as
site-directed mutagenesis, to analyze the structure-function relationship,
muTAUT provides useful information on the functional features of taurine
transporters.
Molecular evolution of muTAUT that causes these structural and functional
differences should be considerably influenced by the internal environment of
the mussel where muTAUT is functioning. The mussel mainly utilizes taurine as
an osmolyte, as described above, and therefore, taurine is accumulated at a
high concentration in the mussel cells. Taurine in hemolymph is also highly
concentrated, for example, in the common mussel, taurine concentration in the
hemolymph is approximately 500 µmol l-1
(Zurburg and DeZwaan, 1981),
which is one order of magnitude higher than that in mammals (approximately
50-80 µmol l-1 in plasma;
Cuisinier et al., 2002
;
Delaney et al., 2003
;
Pacioretty et al., 2001
). In
addition, taurine is dominant and accounts for approximately 80% of the total
amino acid pool in the mussel (Toyohara
and Hosoi, 2004
), suggesting that the concentration and proportion
of taurine is much higher than in mammals. It is assumed that this
concentrated taurine condition is reflected in the lower taurine affinity and
specificity of muTAUT. Furthermore, because the mussel is an osmoconformer,
Na+ and Cl- concentration of the hemolymph is at the
same level as in seawater. This means that internal Na+ and
Cl- concentrations of the mussel are three times as high as those
in mammals and fishes. Considering these facts, the requisite for higher NaCl
concentration by muTAUT is also in good agreement with the internal
environment of the mussel.
Northern blot analysis demonstrated that the expression of the muTAUT gene
at the transcriptional level was induced not only by the hyperosmotic
condition but also by the hypoosmotic condition
(Fig. 6). Hyperosmotic
responsive expression of the taurine transporter has been reported in tilapia
(Takeuchi et al., 2000a) and
cell lines established from dog (Uchida et
al., 1992
) and carp (Takeuchi
et al., 2000b
), and therefore, one of the important roles of the
taurine transporter is considered to be the modulation of intracellular
osmolality by taurine uptake when the osmolality of the extracellular medium
increases (Uchida et al.,
1992
). It is assumed that the muTAUT gene is induced in conditions
of hyperosmolality to increase intracellular osmolality thus preventing the
cell from shrinking and the intracellular concentration of Na+ and
Cl- ions from increasing.
Hypoosmotic responsiveness of the taurine transporter, on the other hand,
has never been reported. It has not been analyzed in studies on mammalian
taurine transporters, but only in the EPC (Epithelioma papulosum
cyprini) cell line established from carp epithelium. It has been
confirmed in this cell line that mRNA abundance of carp taurine transporter
was not increased by hypoosmotic stress
(Takeuchi et al., 2000b).
Considering that the primary function of the taurine transporter is uptake and
accumulation of taurine in the cells, it was an unexpected finding that the
muTAUT gene is induced under hypoosmotic conditions, in which the mussel cells
should release taurine to decrease the intracellular osmolality. In fact, it
had been revealed by our previous study
(Toyohara and Hosoi, 2004
)
that taurine content in the mantle tissue decreased considerably from 8 to 24
h after exposure to hypoosmolality. Subsequently, we analyzed the relationship
between the hypoosmotic induction of muTAUT and taurine concentration, and it
was revealed that the induction of the muTAUT gene in hypoosmolality was
depressed by the addition of 25 mmol l-1 taurine to the ambient
seawater. This suggests that the expression of the muTAUT gene is not directly
regulated by hypoosmolality, but by a consequent decrease in the taurine
content caused by the decrease in ambient osmolality
(Fig. 6C). It also supports the
hypothesis that the pattern of muTAUT induction in the mantle, revealed by
northern blot analysis in this study, was in good agreement with the pattern
of decrease in taurine content in the mantle, under similar hypoosmotic
condition, in our previous study (Toyohara
and Hosoi, 2004
). In addition, the regulation at the
transcriptional level of the taurine transporter by taurine concentration has
been demonstrated in some mammals (Han et al.,
1997a
,b
,
1998
).
We suggest that, as in mammals, muTAUT expression at a transcriptional
level is regulated by the taurine concentration of the mussel. The remarkable
decrease in taurine content caused by the considerable decrease in hemolymph
osmolality seems to be specific for the osmoconforming animals such as
molluscs. Therefore, the hypoosmotic induction of the taurine transporter was
not observed in the carp (Takeuchi et al.,
2000b). In other words, these facts demonstrate that the extensive
decrease in intracellular taurine content occurs in hypoosmolality, suggesting
the importance of taurine as an `adaptive' osmolyte, which mediates the change
in intracellular osmolality.
Immunohistochemistry using anti-muTAUT antibody revealed the intense
expression of muTAUT in the basolateral region of the mantle epithelium and
the gill (Fig. 7), where the
cells are directly in contact with the external seawater. Considering that
taurine is apparently absent in the environmental seawater
(Braven et al., 1984) despite
its high concentration in the hemolymph (approximately 500 µmol
l-1; Zurburg and DeZwaan,
1981
) and cytoplasm (approximately 200 mmol l-1;
Neufeld and Wright, 1995
), the
cells exposed directly to environmental seawater will need to maintain a much
higher concentration gradient of taurine than the cells surrounded entirely by
hemolymph. Our results suggest that the high expression of muTAUT in these
cells corresponds to the accumulation of intracellular taurine from hemolymph
to cope with the steep taurine gradient between the cytoplasm and seawater,
and consequent high adaptability to environmental osmotic change. Although the
expression of muTAUT in the apical membrane of the mantle epithelium could not
be detected, it would seem to be appropriate to assume that apical taurine
transport resulting from muTAUT is likely to be insignificant regardless of
the apical expression of muTAUT, because the Km value for
taurine transport of muTAUT (41 µmol l-1) is much higher than
the taurine concentration in the seawater. However, a previous study by Wright
and Secomb (1989) demonstrated that the apical surface of the gill cells of
Mytilus edulis and M. californianus certainly show taurine
transport activity with an apparent Km value of
approximately 5 to 8 µmol l-1. They reached the conclusion that
apical taurine transport with a taurine affinity that is not suited to uptake
from much low concentrations of taurine in seawater may play a role in
recovering taurine lost from the gill surface. In vivo dynamics of
taurine and the involvement of muTAUT is one of the important future
assignments.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
Present address: Central Research Laboratory, Nippon Suisan Kaisha, Ltd.,
Tokyo 192-0906, Japan
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baldwin, E. (1937). An Introduction to Comparative Biochemistry. London: Cambridge University Press.
Braven, J., Evens, R. and Butler, E. I. (1984). Amino acids in sea water. Chem. Ecol. 2, 11-21.
Church, G. M. and Gilbert, W. (1984). Genomic
sequencing. Proc. Natl. Acad. Sci. USA
81,1991
-1995.
Cuisinier, C., Michotte De Welle, J., Verbeeck, R. K., Poortmans, J. R., Ward, R., Sturbois, X. and Francaux, M. (2002). Role of taurine in osmoregulation during endurance exercise. Eur. J. Appl. Physiol. 87,489 -495.[CrossRef][Medline]
Deaton, L. E., Hilbish, T. J. and Koehn, R. K. (1985a). Hyperosmotic volume regulation in the tissue of the mussel Mytilus edulis. Comp. Biochem. Physiol. 80A,571 -574.[CrossRef]
Deaton, L. E., Hilbish, T. J. and Koehn, R. K. (1985b). Protein as a source of amino nitrogen during hyperosmotic volume regulation in the mussel Mytilus edulis.Physiol. Zool. 57,609 -619.
Delaney, S. J., Kass, P. H., Rogers, Q. R. and Fascetti, A. J. (2003). Plasma and whole blood taurine in normal dogs of varying size fed commercially prepared food. J. Anim. Physiol. Anim. Nutr. Berlin 87,236 -244.[Medline]
Gills, R. (1972). Osmoregulation in three molluscs: Acanthochitona discrepans (Brown), Glycymeris glycymeris (L.) and Mytilus edulis (L.). Biol. Bull. Mar. Biol. Lab. 142,25 -35.
Han, X., Budreau, A. M. and Chesney, R. W. (1997a). Adaptive regulation of MDCK cell taurine transporter (pNCT) mRNA: transcription of pNCT gene is regulated by external taurine concentration. Biochim. Biophys. Acta 1351,296 -304.[Medline]
Han, X., Budreau, A. M. and Chesney, R. W. (1997b). Functional expression of rat renal cortex taurine transporter in Xenopus laevis oocytes: adaptive regulation by dietary manipulation. Pediatr. Res. 41,624 -631.[Abstract]
Han, X., Budreau, A. M. and Chesney, R. W. (1998). Molecular cloning and functional expression of an LLC-PK1 cell taurine transporter that is adaptively regulated by taurine. Adv. Exp. Med. Biol. 442,261 -268.[Medline]
Huxtable, R. J. (1992). Physiological actions
of taurine. Physiol. Rev.
72,101
-163.
Jhiang, S. M., Fithian, L., Smanik, P., McGill, J., Tong, Q. and Mazzaferri, E. L. (1993). Cloning of the human taurine transporter and characterization of taurine uptake in thyroid cells. FEBS Lett. 318,139 -144.[CrossRef][Medline]
Karnaky, K. J. J., Claiborne, J. B., Walsh, P. J., Bernstein, R. M., Schluter, S. F. and Marchalonis, J. J. (1998). Section 3. Homeostasis. In The Physiology of Fishes (ed. D. H. Evans). Boca Raton: CRC Press.
Krogh, A., Larsson, B., von Heijne, G. and Sonnhammer, E. L. (2001). Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305,567 -580.[CrossRef][Medline]
Kurzinger, K. and Hamprecht, B. (1981). Na+-dependent uptake and release of taurine by neuroblastoma x glioma hybrid cells. J. Neurochem. 37,956 -967.[Medline]
Liu, Q. R., Lopez-Corcuera, B., Nelson, H., Mandiyan, S. and
Nelson, N. (1992). Cloning and expression of a cDNA encoding
the transporter of taurine and beta-alanine in mouse brain. Proc.
Natl. Acad. Sci. USA 89,12145
-12149.
Livingstone, D. R., Widdows, J. and Fieth, P. (1979). Aspects of nitrogen metabolism of the common mussel Mytilus edulis: Adaptation to abrupt and fluctuating change in salinity. Mar. Biol. 53,41 -55.[CrossRef]
Lockwood, A. P. M. (1963). Animal Body Fluids and Their Regulation. London: Heinemann Educational Books.
Miyamoto, Y., Balkovetz, D. F., Leibach, F. H., Mahesh, V. B. and Ganapathy, V. (1988). Na++ Cl--gradient-driven, high-affinity, uphill transport of taurine in human placental brush-border membrane vesicles. FEBS Lett. 231,263 -267.[CrossRef][Medline]
Miyamoto, Y., Tiruppathi, C., Ganapathy, V. and Leibach, F. H. (1989). Active transport of taurine in rabbit jejunal brush-border membrane vesicles. Am. J. Physiol. 257,G65 -G72.
Miyamoto, Y., Liou, G. I. and Sprinkle, T. J. (1996). Isolation of a cDNA encoding a taurine transporter in the human retinal pigment epithelium. Curr. Eye Res. 15,345 -349.[Medline]
Nelson, N. and Lill, H. (1994). Porters and
neurotransmitter transporters. J. Exp. Biol.
196,213
-228.
Neufeld, D. S. and Wright, S. H. (1995). Basolateral transport of taurine in epithelial cells of isolated, perfused Mytilus californianus gills. J. Exp. Biol. 198,465 -473.
Neufeld, D. S. and Wright, S. H. (1996a).
Response of cell volume in Mytilus gill to acute salinity change.
J. Exp. Biol. 199,473
-484.
Neufeld, D. S. and Wright, S. H. (1996b).
Salinity change and cell volume: the response of tissues from the estuarine
mussel Geukensia demissa. J. Exp. Biol.
199,1619
-1630.
Neufeld, D. S and Wright, S. H (1998). Effect
of cyclical salinity changes on cell volume and function in Geukensia
demissa gills. J. Exp. Biol.
201,1421
-1431.
Nomura, T., Yamamoto, I., Morishita, F., Furukawa, Y. and Matsushima, O. (2001). Purification and some properties of alanine racemase from a bivalve mollusc Corbicula japonica. J. Exp. Zool. 289,1 -9.[CrossRef][Medline]
Pacioretty, L., Hickman, M. A., Morris, J. G. and Rogers, Q. R. (2001). Kinetics of taurine depletion and repletion in plasma, serum, whole blood and skeletal muscle in cats. Amino Acids 21,417 -427.[CrossRef][Medline]
Palacin, M., Estevez, R., Bertran, J. and Zorzano, A.
(1998). Molecular biology of mammalian plasma membrane amino acid
transporters. Physiol. Rev.
78,969
-1054.
Petty, C. N. and Lucero, M. T. (1999).
Characterization of a Na+-dependent betaine transporter with
Cl-channel properties in squid motor neurons. J.
Neurophysiol. 81,1567
-1574.
Potts, W. T. W. (1954). The inorganic composition of the blood of Mytilus edulis and Anodonta cygnea.J. Exp. Biol. 57,681 -692.
Ramamoorthy, S., Leibach, F. H., Mahesh, V. B., Han, H., Yang-Feng, T., Blakely, R. D. and Ganapathy, V. (1994). Functional characterization and chromosomal localization of a cloned taurine transporter from human placenta. Biochem. J. 300,893 -900.[Medline]
Silva, A. L. and Wright, S. H. (1992).
Integumental taurine transport in Mytilus gill: short-term adaptation
to reduced salinity. J. Exp. Biol.
162,265
-279.
Smith, K. E., Borden, L. A., Wang, C. H., Hartig, P. R.,
Branchek, T. A. and Weinshank, R. L. (1992). Cloning and
expression of a high affinity taurine transporter from rat brain.
Mol. Pharmacol. 42,563
-569.
Somero, G. N. and Bowlus, R. D. (1983). Osmolytes and metabolic end products of molluscs: the design of compatible solute systems. In Environmental Biochemistry and Physiology, The Mollusca, Vol. 2 (ed. P. W. Hochachka), pp. 77-100. London: Academic Press.
Takeuchi, K., Toyohara, H., Kinoshita, M. and Sakaguchi, M. (2000a). Ubiquitous increase in taurine transporter mRNA in tissues of tilapia (Oreochromis mossambicus) during high-salinity adaptation. Fish Physiol. Biochem. 23,173 -182.[CrossRef]
Takeuchi, K., Toyohara, H. and Sakaguchi, M. (2000b). A hyperosmotic stress-induced mRNA of carp cell encodes Na(+)- and Cl(-)-dependent high affinity taurine transporter. Biochim. Biophys. Acta 1464,219 -230.[Medline]
Toyohara, H. and Hosoi, M. (2004). The role of taurine in the osmotic adaptation in the marine mussel Mytilus galloprovincialis. Mar. Biotechnol. 6,S511 -S516.[CrossRef]
Turner, D. L. and Weintraub, H. (1994). Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev. 8,1434 -1447.[Abstract]
Uchida, S., Kwon, H. M., Preston, A. S. and Handler, J. S.
(1991). Expression of Madin-Darby canine kidney cell Na(+)-and
Cl(-)-dependent taurine transporter in Xenopus laevis oocytes. J.
Biol. Chem. 266,9605
-9609.
Uchida, S., Kwon, H. M., Yamauchi, A., Preston, A. S., Marumo,
F. and Handler, J. S. (1992). Molecular cloning of the cDNA
for an MDCK cell Na(+)- and Cl(-)-dependent taurine transporter that is
regulated by hypertonicity. Proc. Natl. Acad. Sci. USA
89,8230
-8234.
Utsunomiya-Tate, N., Endou, H. and Kanai, Y.
(1996). Cloning and functional characterization of a system
ASC-like Na+-dependent neutral amino acid transporter. J. Biol.
Chem. 271,14883
-14890.
Vinnakota, S., Qian, X., Egal, H., Sarthy, V. and Sarkar, H. K. (1997). Molecular characterization and in situ localization of a mouse retinal taurine transporter. J. Neurochem. 69,2238 -2250.[Medline]
Wood, C. M. and Shuttleworth, T. J. (1995). Cellular and Molecular Approaches to Fish Ionic Regulation: Fish physiology. San Diego: Academic Press.
Wright, S. H. and Secomb, T. W. (1984). Epidermal taurine transport in marine mussels. Am. J. Physiol. 247,R346 -R355.
Yoshikawa, N., Dhomae, N., Takio, K. and Abe, H. (2002). Purification, properties, and partial amino acid sequences of alanine racemase from the muscle of the black tiger prawn Penaeus monodon. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 133,445 -453.[CrossRef][Medline]
Zurburg, W. and DeZwaan, A. (1981). The role of amino acids in anaerobiosis and osmoregulation in bivalves. J. Exp. Zool. 215,315 -325.[CrossRef]