Vol. 275, Issue 1, R69-R75, July 1998
Effect of calcium on development of amiloride-blockable
Na+ transport in axolotl in
vitro
Makoto
Takada1,
Hideko
Yai1, and
Shinji
Komazaki2
Departments of 1 Physiology and
2 Anatomy, Saitama Medical School,
Moroyama, Iruma-gun, Saitama 350-04 Japan
 |
ABSTRACT |
The axolotl, Ambystoma
mexicanum, which has no specific calcium-containing
sieve layer in the dermis, provides useful material for the study of
the effect of Ca2+ on the
development of amiloride-blockable active
Na+ transport across the skin of
amphibians. We raised axolotls in thyroid hormone or aldosterone or
cultured the skin with corticoid plus one of several
Ca2+ concentrations and found that
1) although the short-circuit
current (SCC) was increased by both aldosterone and
3,3',5-triiodo-L-thyronine in vivo, only corticoid was necessary for such an increase in vitro;
2) the development of the SCC in
vitro was both corticoid and Ca2+
dependent, because the SCC was well developed with over 100 µM Ca2+ but not with under 10 µM
Ca2+ in the presence of corticoid,
nor even with 300 µM Ca2+
without corticoid; and 3)
Ca2+, but not corticoid, was
necessary for the formation of cell-to-cell junctions, because the
resistance of the skin was well developed with 300 µM
Ca2+ without corticoid.
aldosterone; Ambystoma; cultured
epithelium; metamorphosis; cell-to-cell junctions
| |
INTRODUCTION |
AMILORIDE-BLOCKABLE ACTIVE
Na+ transport, measured as
short-circuit current (SCC), develops at climax stages in the
metamorphosis of Rana catesbeiana
(bullfrog) tadpoles (4, 10, 17). Metamorphosis can be artificially
induced by thyroid hormone in larval tadpoles, and the transport
develops at the same time (4, 17). Takada et al. (20), who devised a
way of culturing the skin of larval tadpoles, found that active
Na+ transport, one of the
adult-type characteristics of the skin, developed without thyroid
hormone when only corticoid was present in the medium. This was very
surprising, because active Na+
transport was once thought to develop under the action of thyroid hormone alone (18, 22).
The formation of cell polarity (resulting from the localization of
amiloride-blockable Na+ channels
on the apical side and of the ouabain-blockable
Na+ pump on the basolateral side)
and the tightening of cell-to-cell junctions are both necessary for the
proper functioning of transepithelial amiloride-blockable
Na+ transport (3, 13). The
development of active Na+
transport coincides with the appearance of adult features in the
epidermis in the bullfrog (20). Calcium participates in the formation
of cell-to-cell junctions and in the development of cell polarity in
epithelial Madin-Darby canine kidney (MDCK) cells; in fact, switching
the Ca2+ concentration from low to
normal stimulates the tightening of cell-to-cell junctions, whereas
switching in the other direction causes the opposite effect (2, 6, 23).
Calcium also participates in the proliferation of epidermal cells and
in the biosynthesis of adult-type keratin, a marker of adult-type
epidermis, in amphibians (14, 15). This led us to wonder whether
calcium might also be concerned with the development of
amiloride-blockable SCC across the skin of amphibians through both cell
proliferation and the establishment of cell-to-cell junctions and cell
polarity coincident with the appearance of adult features in epidermal
cells. Unfortunately, it is difficult to examine the effect of
Ca2+ on the development of this
type of transport in vitro using the skin of bullfrog tadpoles because
the sieve layer located in their dermis contains so much calcium (8,
11). In contrast, the axolotl, Ambystoma
mexicanum, has no sieve layer in its dermis, so this
type of analysis can be conducted without the confusing effect of the
calcium already present in the dermis.
The axolotl has an additional advantage as an experimental animal.
Although we now believe that active
Na+ transport does develop without
thyroid hormone in the bullfrog, the possibility cannot be excluded
that endogenous thyroid hormone in the larval skin may exert a
synergistic action with corticoid on the development of this type of
transport. The axolotl is a neotenic urodele that cannot secrete
thyroid hormone because of an inherently defective pituitary-thyroid
axis. However, treatment with thyroid hormone induces metamorphosis in
such animals (24, 25), showing that their tissues are sensitive to
thyroid hormone.
Consequently, the axolotl is an ideal species for the investigation
both of the effect of Ca2+ and of
the interaction between corticoid and thyroid hormone on the
development of an amiloride-blockable SCC. Use of the axolotl in the
present study also allowed us to determine whether the development of
an amiloride-blockable SCC, which can be induced by corticoid alone in
R. catesbeiana (Anura), can be induced
in the same way in the axolotl (Urodela).
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MATERIALS AND METHODS |
Animals.
Larval A. mexicanum were purchased
from a local animal supplier in Hamamatsu, Shizuoka, Japan, and
maintained for 3 wk in tap water (control larvae) or in tap water
supplemented either with
3,3',5-triiodo-L-thyronine
(T3)
(10
8 M;
T3-treated larvae) or with
aldosterone (Aldo; 5 × 107 M; Aldo-treated
larvae). The water in the aquarium was changed every 3 days. Animals
were fed with Limnodrilus (Annelida)
or Profit (food substance for eels made by Nippon Haigoshiryo,
Yokohama, Japan).
Culture of skin.
Intact larvae were anesthetized with ice water containing 0.05% MS-222
(Sankyo, Tokyo, Japan), and a portion of the body or head skin was
dissected out. The culture method used for larval skin was similar to
that described by Takada et al. (20). In brief, the skin was washed
with 70% ethanol and then with
Ca2+- and
Mg2+-free saline (CMFS) and
transferred to CMFS containing 2.5 mM EDTA; this treatment leaves only
the innermost layer intact. Next, the skin was washed with normal
saline and transferred to tissue culture medium (see
Culture
medium). The skin was cultured in a
humidified atmosphere of 5% CO2
and 95% room air at 24°C for a number of weeks (see
RESULTS).
Culture medium.
One of two kinds of RPMI-1640 solution (Gibco, Grand Island, NY),
containing either 424 µM Ca2+ or
0.45 µM Ca2+, was used as the
starting solution for the culture medium. Both solutions were diluted
to 70% with distilled water and supplemented with 16.7 mM
NaHCO3, 10 mM HEPES (pH 7.4), 100 IU/ml penicillin, and 100 µg/ml streptomycin. Glutamine (1.4 mM) was
also added to the RPMI-1640 solution containing 0.45 µM
Ca2+ so that its overall
concentration of solutes was the same as that of the RPMI-1640 solution
containing 424 µM Ca2+ (which
contained 1.4 mM glutamine). Although it is conceivable that
development of an amiloride-blockable SCC is affected by the
concentration of glutamine in the medium, we know of no evidence that
this is the case in the axolotl. As a result of the dilution to 70%,
the Ca2+ concentration in the
first medium was ~300 µM (normal RPMI) and that in the second
medium was 0.32 µM (low-Ca RPMI). To produce 10 µM
Ca2+-, 100 µM
Ca2+-, and 1,000 µM
Ca2+-containing media,
Ca(NO3)2
was added to the low-Ca RPMI or to normal RPMI. The skin was cultured
in the above media with or without
1) 5 × 107 M Aldo,
2) Aldo + 5 × 107 M hydrocortisone + 5 × 107 M
corticosterone (Mix), and 3)
108 M
T3.
Measurement of PD, SCC, and R of skin.
Freshly dissected skin from whole animals (taken under anesthesia
induced with ice water supplemented with MS-222) or cultured skin
samples were mounted in a Ussing-type chamber using silicone gaskets
(inner diameter 5 mm) to minimize edge damage. Both sides of the skin
were bathed in aerated Ringer solution containing (in mM) 110 NaCl, 2 KCl, 1 CaCl2, 10 glucose, and 10 Tris at pH 7.2 and allowed to equilibrate for 1 h. To produce
an Na+-free solution, all NaCl was
replaced by choline-chloride. The potential difference (PD) across the
skin, the SCC, and the skin resistance
(R) were measured using the method
of Takada et al. (20, 21). The fluid R
was compensated. When required, amiloride was applied to the apical
side and ouabain to the basolateral side of the skin.
Light microscopy.
After the culture period, the skin was fixed with 10% formalin,
embedded in paraffin, and sectioned at 8-µm thickness. The sections
were stained with hematoxylin and eosin and viewed under a light
microscope.
Statistical analysis.
Values are expressed as means ± SE. Differences were analyzed using
Dunnett's one-tailed t-test,
Student's t-test, or Welch's test;
they were taken as significant when P < 0.05.
| |
RESULTS |
SCC across the skin.
In the present study, the gills of animals raised in
T3
(108 M) for 3 wk
(T3-treated larvae) were reduced
to 2.2 ± 0.86 from 10.8 ± 0.58 mm in length (mean ± SE,
n = 5, significantly different at
P < 0.05), showing that
this treatment had triggered metamorphosis. On the other hand, the
gills of animals raised in Aldo (5 × 107 M; Aldo-treated larvae)
did not show any detectable regression. In both the
T3-treated larvae and the
Aldo-treated larvae, the SCC across the skin was significantly higher
than in the control larvae, indicating that an increase in SCC can be
induced by either T3 or Aldo in
vivo (Table 1).
To test whether the development of the SCC can be induced by corticoid
alone in vitro, EDTA-treated axolotl skin was cultured under a variety
of hormonal conditions for 3 wk. As shown in Table 1, in skin cultured
in normal RPMI supplemented with Aldo, Mix, or Mix + T3, the SCC was significantly
higher than in the control larvae. By contrast, in skin cultured in
normal RPMI supplemented with T3,
the SCC was not significantly different from that of the controls, nor
was it significantly different from the SCC in EDTA-treated skin or in
skin cultured in normal RPMI without any hormone.
The conclusion that can be drawn from the above results is similar to
that reported for cultured larval bullfrog skin, that is, that an
amiloride-blockable SCC can develop under the influence of corticoid
alone (20). It is clear that a synergistic action of corticoid and
endogenous thyroid hormone is not necessary for the development of the
SCC because the axolotl has no endogeneous thyroid hormone.
Time course of development of SCC.
The SCC of skin cultured for 2 wk with Mix + T3 was greater than that of skin
cultured with Mix alone (Fig. 1). However,
after a 3-wk culture, the skin had developed about the same SCC
regardless of the culture medium used (see also Table 1). Thus Mix
alone eventually produced the same enhancement of the SCC as Mix + T3, but its effect occurred more
slowly.
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Fig. 1.
Effect of corticoids and
3,3',5-triiodo-L-thyronine
(T3) on development of
short-circuit current (SCC). EDTA-treated skin was cultured in normal
RPMI supplemented with corticoid mixture (Mix, 5 × 107 M aldosterone + 5 × 107 M
hydrocortisone + 5 × 107 M corticosterone) or
Mix + T3
(108 M).  , Skin cultured
with Mix;  , skin cultured with Mix + T3. Values are means ± SE.
Numbers in parentheses are numbers of experiments.
* P < 0.05 vs. Mix (Welch's
test).
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Effects of amiloride, ouabain, and
Na+-free medium
on SCC.
Table 2 shows the effects of amiloride
(104 M), ouabain
(105 M), and
Na+-free Ringer
1) on the SCC across skin taken from
control larvae, T3-treated larvae,
and Aldo-treated larvae (no data for
Na+-free Ringer) and
2) on the SCC across skin cultured
with Mix for 3 wk or with Mix + T3
for 3 wk. The results suggest that active Na+ transport was well developed
in those skins, because amiloride, ouabain, and
Na+-free Ringer all reduced the
SCC across the skin of T3-treated larvae and Aldo-treated larvae and the SCC across skin cultured with
Mix or with Mix + T3 (no data for
Na+-free Ringer on Aldo-treated
larvae). Although the SCC of control skin was not decreased
significantly by ouabain, this may have been due to the lower baseline
SCC value. However, Na+ transport
had actually started, because amiloride reduced the SCC across such
skin.
Effect of calcium on development of SCC and R in vitro.
Next, the effect of Ca2+ on the
development of amiloride-blockable active
Na+ transport in axolotl skin in
vitro was investigated. For this purpose, EDTA-treated axolotl skin
cultured with various combinations of calcium and corticoid for 3 wk
was used.
In the presence of corticoid, an SCC did not develop in 0.3 µM
Ca2+ or in 10 µM
Ca2+, but in over 100 µM
Ca2+ the SCC and
R developed well (Fig.
2). The effect of
Ca2+ on the development of SCC and
R was analyzed by means of
Lineweaver-Burk plots (Fig. 2, B and
C). Because the SCC did not develop
with under 10 µM Ca2+, SCC
values obtained at 100, 300, and 1,000 µM
Ca2+ were used for this analysis.
The R at under 10 µM
Ca2+ should be that of the dermis,
because the epidermis did not develop under such conditions (data not
shown). To analyze the relationship between
Ca2+ and
R during the development of the
epidermis, we subtracted the mean value of
R at 0.32 µM and 10 µM
Ca2+ (namely, 0.25 k
· cm2)
from the value of R at 100, 300, and
1,000 µM Ca2+ and used the
result for the analysis. Inspection of Fig. 2,
B and
C, shows that the maximum SCC
(SCCmax) and the
Ca2+ concentration required for a
one-half-maximum effect
(Km) on SCC
were 16.1 µA/cm2 and 503 µM,
respectively, and that the maximum R
and the Km for R were 2.71 k · cm2 and
359 µM, respectively.
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Fig. 2.
Effect of Ca2+ on development of
SCC and skin resistance (R).
EDTA-treated skin was cultured with Mix + 0.3, 10, 100, 300, or 1,000 µM Ca2+ for 3 wk.
A: effect of
Ca2+ on SCC and
R. B:
Lineweaver-Burk plot of Ca2+ vs.
SCC. C: Lineweaver-Burk plot of
Ca2+ vs.
R. In Lineweaver-Burk plots, maximum
value of SCC or R is given by
reciprocal of intercept on y-axis
(i.e., intercept represents 1/maximum SCC or 1/maximum
R).
Ca2+ concentration required for a
one-half-maximum effect
(Km) is
obtained by taking the reciprocal of the intercept on the
x-axis and ignoring the sign (i.e.,
intercept represents
 1/Km).
Values are means ± SE. Numbers in parentheses show number of
experiments. * P < 0.01 vs. 10 µM Ca2+ (Dunnett's 1-tailed
t-test).
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Involvement of calcium and corticoid in development of SCC and
R in vitro.
Although the results presented so far seem to indicate that both
Ca2+ and corticoid are necessary
for the development of SCC and R, actually only Ca2+ was necessary
for the development of R and epidermal
cells, as shown in Table 1 and Fig.
4C. One interpretation is that both Ca2+ and corticoid are required
for the development of Na+
channels and the Na+ pump as
components of amiloride-blockable
Na+ transport, whereas the
development of cell-to-cell junctions can proceed to some extent
with Ca2+ alone (without
corticoid). If this were true, 1) an
SCC would develop when skin is cultured first with 300 µM
Ca2+ without corticoid and
subsequently with 300 µM
Ca2+ + corticoid and
2) neither SCC nor
R would develop when skin is cultured
first with 300 µM Ca2+ without
corticoid and subsequently with 10 µM
Ca2+ + corticoid.
To investigate in more detail the relationship between the effects of
Ca2+ and Mix on the development of
an SCC and R, EDTA-treated skin was
cultured for 6 wk or more under a variety of conditions (i.e., in
various Ca2+ concentrations with
or without Mix; Fig. 3). As stated above, an SCC did not develop when EDTA-treated skin was cultured with 0.3 µM Ca2+ + Mix for 3 wk (Fig. 2).
After an additional 3-wk culture with 300 µM
Ca2+ + Mix, the skin still did not
develop an SCC or R (total 6 wk; Fig.
3, A,
B, and
C). On the other hand, although an
SCC did not develop in skin cultured with 10 µM
Ca2+ + Mix for 3 wk (Fig. 2), an
additional 3-wk culture with 300 µM
Ca2+ + Mix resulted in a
considerable SCC and R (total 6 wk;
Fig. 3, A,
B, and
C). Although only
R developed (and SCC did not) without Mix, even with 300 µM Ca2+
present for 3 or 6 wk (Table 1 and Fig. 3,
A,
B, and
C), an additional 3-wk culture with
300 µM Ca2+ + Mix led to the
development of a large SCC and a large
R (total 6 or 9 wk; Fig. 3,
A, B
and C). Furthermore, neither SCC nor
R developed in skin that was cultured
first with 300 µM Ca2+ without
Mix and then with 10 µM Ca2+
with Mix (total 6 wk; Fig. 3, A,
B, and
C).
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Fig. 3.
Interaction between effects of
Ca2+ and effects of corticoid on
development of SCC. A: culture
conditions imposed on EDTA-treated skin. +Mix, cultured with Mix;
Mix, cultured without Mix; 0.3, 10, and 300 µM
Ca2+ show
Ca2+ concentration in medium; 0, 3, 6, and 9 show culture period in weeks.
a, b,
c1,
c2,
d, and
e, measurements taken at times
indicated by arrows. B and
C: development of SCC
(B) and
R
(C) under various culture
conditions. Values are means ± SE. Numbers in parentheses show
number of experiments. * P < 0.01, b vs.
a and
d vs.
c1 (Welch's
test); ** P < 0.05, b vs.
a and
d vs.
c1 (Welch's
test).
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Light microscopic observation of the skin.
To examine whether morphological adult-type characteristics develop at
the same time as the functional ones, skin samples were examined
histologically. Leydig cells, which are large and granule rich and
characterize the larval epidermis of urodeles, were present in the
control skin of the axolotl (Fig.
4a) (5, 7,
24) and also in the skin of Aldo-treated larvae (data not shown).
However, few were present in the skin of
T3-treated larvae (Fig.
4f ). EDTA treatment left only
the innermost layer of the skin (Fig.
4b). It may be that the cells of this
layer are precursors of the other epidermal cells, as they are in the case of bullfrog skin. If this is so, we might expect that a stratified epidermis, such as that seen in the skin of
T3-treated larvae (but not Leydig
cells, such as are seen in the control skin), will develop when
EDTA-treated skin is cultured in normal RPMI supplemented with Mix,
because an amiloride-blockable SCC is well developed in such skin (see
Table 2).
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Fig. 4.
Light microscopic observation of skin. EDTA-treated skin was cultured
either in normal RPMI alone or in normal RPMI supplemented with Mix or
with Mix + T3.
a, Larval skin (showing many large
Leydig cells); b, EDTA-treated skin
(immediately after EDTA treatment); c,
skin cultured in normal RPMI alone for 3 wk;
d, skin cultured in normal RPMI
supplemented with Mix for 3 wk; e,
skin cultured in normal RPMI supplemented with Mix + T3 for 3 wk;
f, skin from a larva raised in
T3. Bar = 50 µm.
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In fact, Fig. 4d shows that when
cultured with Mix, EDTA-treated skin did indeed develop a stratified
epidermis and had no Leydig cells, as expected. In terms of its
morphology, skin cultured with Mix + T3 (Fig.
4e) was much the same as skin cultured
with Mix alone (Fig. 4d). We also
expected that skin cultured with normal RPMI alone might possess a
larval-type epidermis rather than the metamorphosed-type (stratified)
epidermis, because the SCC across such skin is much lower than that
across control skin. Actually, the skin was well maintained and showed
stratification, but no Leydig cells were observed (Fig.
4c). Thus axolotl skin cultured with
normal RPMI alone (without corticoid) developed cells that were
morphologically like the metamorphosed type, even though functionally
it was still of the larval type with only a low SCC. In contrast, skin
of Aldo-treated larvae, which was morphologically like the larval type,
had developed metamorphosed-type functions; that is, it had an
amiloride-blockable SCC of considerable size.
| |
DISCUSSION |
In the present study, amiloride attenuated the SCC of the control
axolotl skin. In addition, Aldo treatment in vivo induced an increase
in the SCC. Because the axolotl has no endogenous thyroid hormone, this
is in line with our hypothesis (based on a study of bullfrog tadpole
skin in vitro) that the development of an amiloride-blockable SCC
across bullfrog skin is not the direct result of an action of thyroid
hormone (20). Although, in the axolotl, in vivo
T3 treatment led to the
development of an amiloride-blockable SCC, this may be explained, as it
is in the bullfrog (12), by T3
stimulating the secretion of Aldo, which in turn induces the
development of the SCC.
Thyroid hormone enhanced the corticoid-induced SCC in the axolotl after
2 wk in culture but not after 3 wk in culture. Thyroid hormone also
enhances the hydrocortisone-induced biosynthesis of 63-kDa keratin, a
molecular marker of the metamorphosed-type epidermis in
Xenopus (14-16). However, the
development of a corticoid-induced amiloride-blockable SCC was not
enhanced by thyroid hormone in bullfrog skin in vitro (20). Possibly,
the synergy between the actions of
T3 and corticoid may vary with the
species and with the culture conditions.
A number of pieces of evidence indicate that
Ca2+ has some involvement in the
morphological and functional development of the epithelium in a variety
of species. The proliferation of epidermal cells in the mouse and in
amphibians was stimulated when they were cultured with less than 100 µM Ca2+, but differentiation
(such as cornification) was inhibited (9, 14, 15). In contrast,
proliferation was inhibited but differentiation was stimulated when
these cells were cultured with over 100 µM Ca2+. Furthermore,
Ca2+ is concerned with the
formation of cell polarity and cell-to-cell tightening in MDCK cells
(1, 2, 6, 23). These results led us to think that
Ca2+ may be involved in the
development of an amiloride-blockable SCC in amphibian skin. Thus
morphological development of the epidermis might be expected in
EDTA-treated axolotl skin cultured with under 100 µM
Ca2+, whereas the development of
an amiloride-blockable SCC might be expected in skin cultured with over
100 µM Ca2+. In fact, functional
development (an increase in the amiloride-blockable SCC) was stimulated
when EDTA-treated axolotl skin was cultured with
Ca2+ at concentrations of over 100 µM, as we expected. However, morphological development of the
epidermis was not stimulated in skin cultured with 0.3 µM or 10 µM
Ca2+, but it was stimulated with
concentrations of over 100 µM
Ca2+, just as functional
development was.
The development of higher SCC values in media containing higher levels
of Ca2+ may indicate that
tightening of cell-to-cell junctions is necessary for the proper
development of an amiloride-blockable SCC. Indeed, even if
Na+ channels and the
Na+ pump develop, the presence of
loose junctions would decrease the net flux of
Na+ through the low shunt
resistance
(RS)
of the skin, which would then fail to develop a full SCC (3, 13).
Actually, a high transepithelial R is
not formed in low-Ca2+
concentrations but is formed in
high-Ca2+ concentrations, the
one-half-maximum effect (in terms of the formation of cell-to-cell
junctions) being produced by 50 µM
Ca2+ in MDCK cells (23). In the
present study, the one-half-maximum effect of
Ca2+ on the development of
R (which is due to epidermal cells)
was 359 µM (Fig. 2C). This
discrepancy may reflect differences in the development of cell-to-cell
junctions between epithelial MDCK cells and the epidermal cells of the
axolotl. Development of amiloride-blockable Na+ channels would be expected to
lead to a decrease in total skin R; in
fact, R can actually increase.
Possibly, this may be due to an increase in
RS (17). Whether
the RS is
localized in cell-to-cell tight junctions or in other sites needs to be
clarified using vibrating microprobe analysis.
An amiloride-blockable SCC might be expected to develop simultaneously
with the adult features of the skin in the axolotl, because the SCC
develops together with the adult features of the epidermis in the skin
of the bullfrog (19, 20). Actually, an amiloride-blockable SCC was
already present to a small extent in the intact skin of the axolotl,
but an increase in the SCC would be expected to occur with the
morphological development of adult features, that is, the disappearance
of Leydig cells, which are a morphological marker of the larval-type
epidermis of the axolotl (5, 24). In fact, as shown in Fig. 4, few Leydig cells were observed in the skin of
T3-treated larva or in skin
cultured with corticoid (i.e., such skins were morphologically much the
same as adult skin), and these skins did indeed show an increased SCC
(Table 1). However, in skin cultured with normal RPMI, Leydig cells
were not observed, yet there was no increase in the SCC. Thus in the
axolotl, the disappearance of larval-type cells is not necessarily
accompanied by an increase in the SCC. This dissociation between the
development of an adult-type morphology and the development of an
amiloride-blockable SCC (two events that occur concomitantly in the
bullfrog) will clearly require further investigation.
The present study has confirmed that the development of an
amiloride-blockable SCC can be induced by corticoid alone in vitro in
the skin of the axolotl (Urodela), just as it can in the skin of
R. catesbeiana (Anura). Moreover, the
present results suggest that, at least in the axolotl, both calcium and
corticoid are necessary for the full development of an
amiloride-blockable SCC.
Perspectives
In bullfrog skin, the effect of Aldo on the development of an
amiloride-blockable SCC differs between the in vitro and in vivo
conditions (20). The SCC develops in the presence of Aldo alone in
vitro but not in vivo, suggesting the existence of an inhibitory
mechanism preventing Aldo from developing the transport in vivo. In
contrast, in the axolotl no such inhibitory mechanism seems to exist,
because Aldo is also effective in vivo. Our future work will include a
comparative study of this inhibitory mechanism in anurans and urodeles.
Calcium participates in the docking and fusion mechanisms by which
vesicles release transmitters at synaptic terminals. It is conceivable
that similar mechanisms are involved in the development of cell
polarity in epithelia. If so, Ca2+
may modulate the effect of Aldo by virtue of its induction of cell
polarity. This has already been explored in the
DISCUSSION.
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ACKNOWLEDGEMENTS |
The authors thank M. Suzuki for the constant supply of axolotls.
They also thank M. Shiibashi and M. Kasai for statistical analysis and
for drawing figures.
| |
FOOTNOTES |
Address for reprint requests: M. Takada, Dept. of Physiology, Saitama
Medical School, Moroyama, Iruma-gun, Saitama, 350-04 Japan.
Received 3 December 1997; accepted in final form 3 March 1998.
| |
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Abstract
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