Growth hormone is a weaker candidate than prolactin for the hormone responsible for the development of a larval-type feature in cultured bullfrog skin
Department of Physiology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 3500495, Japan
* Author for correspondence (e-mail: makokam{at}saitama-med.ac.jp)
Accepted 13 January 2003
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Summary |
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Key words: growth hormone, prolactin, amphibian metamorphosis, non-selective cation channel, bullfrog skin, Rana catesbeiana
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Introduction |
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During the climax stages of metamorphosis, these ligand-stimulated SCCs
disappear, while active Na+ transport, measured as an amiloride-SCC
(ASCC) across the skin, develops (Cox and
Alvarado, 1979; Hillyard et al., 1982a;
Takada, 1985
). The development
of this ASCC is due to the development of the epithelial Na+
channel (ENaC). The presence of ASCC and ENaC is a marker for adult-type
features in the skin.
Although the development of adult-type features is induced by thyroid
hormone in vivo (for reviews, see
Dodd and Dodd, 1976;
Kikuyama et al., 1993
), the
ASCC is not developed by thyroid hormone in vitro but by corticoids
(Takada et al., 1995a
). This
contrast between the in vitro and in vivo situations is an
important issue in the study of amphibian metamorphosis
(Takada et al., 1999
).
Whereas the development of adult-type features is induced by thyroid
hormone or corticoids, prolactin (PRL) is considered to maintain larval-type
features in amphibians since the regression of the isolated tail seen under
treatment with thyroid hormone is inhibited by PRL (for reviews, see
Dodd and Dodd, 1976;
Kikuyama et al., 1993
). If PRL
is indeed the `juvenile hormone', the serum concentration of PRL would be
expected to be higher in the larval stages than in the climax stages of
metamorphosis. However, it is actually lower in the larval stages and
increases during the climax stages of metamorphosis
(Yamamoto and Kikuyama, 1982
).
In addition, the mRNA for the PRL receptor increases during metamorphosis
(Yamamoto et al., 2000
). On
this basis, PRL seems unlikely to be the juvenile hormone, and other
hormone(s) have been proposed for this role (Huang and Brown,
2000a
,b
;
Yamamoto et al., 2000
).
The expression of the mRNA for growth hormone (GH) is higher in the larval
stages in Xenopus laevis and is downregulated in the climax stages of
metamorphosis or following treatment with thyroid hormone
(Buckbinder and Brown, 1993).
Therefore, GH may be the crucial juvenile hormone in amphibians. In fact,
overexpression of GH has been shown to stimulate growth in the tadpoles of
X. laevis (Huang and Brown,
2000a
).
To facilitate investigations of the development of larval-type and
adult-type features in bullfrog skin, Takada et al.
(1995a) developed a method for
culturing larval skin. The epidermis of the skin of the larval bullfrog is
composed of three types of cells: apical, skein and basal cells
(Robinson and Heintzelman,
1987
). EDTA treatment removes both apical and skein cells, leaving
only basal cells, and the amiloride- and acetylcholine-responses are not
present in such EDTA-treated skin (Takada et al.,
1995a
,b
,
1996
). EDTA-treated skin
cultured with corticoids develops an adult-type feature: a large SCC that is
blocked by amiloride. By contrast, skin cultured with corticoids supplemented
with prolactin (PRL) develops a larval-type feature: a small SCC that is
stimulated by amiloride and acetylcholine
(Takada et al., 1996
).
However, whether GH also induces the development of amiloride-, acetylcholine-
or ATP-stimulated SCC has not been investigated.
Here, we report that these types of ligand-stimulated SCC develop when EDTA-treated larval bullfrog skin is cultured with corticoids supplemented with GH or PRL. However, contrary to our expectations, some of the characteristics shown by the ligand-stimulated SCCs differed between skin cultured with corticoids supplemented with GH and intact tadpole skin.
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Materials and methods |
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Solutions
RPMI solution, prepared as follows, was used as the culture medium. RPMI
1640 (GIBCO, Grand Island, NY, USA) was diluted to 70% with distilled water
and supplemented with corticoids (106 mol
l1 aldosterone, 5x107 mol
l1 each of hydrocortisone and corticosterone), 16.6 mmol
l1 NaHCO3, 10 mmol l1 Hepes (pH
7.4), 100 i.u. ml1 penicillin and 100 µg
ml1 streptomycin. The skin was cultured in the above medium
with or without prolactin (PRL), growth hormone (GH) or T3
(3,3',5-triiodo-L-thyronine). The composition of the Ringer's solution
used for the measurement of the SCC was 110 mmol l1 NaCl, 2
mmol l1 KCl, 1 mmol l1 CaCl2,
10 mmol l1 glucose and 10 mmol l1 Tris at
pH 7.2. Ovine PRL and porcine GH, T3, ATP and amiloride were
purchased from Sigma Chemicals (St Louis, MO, USA).
Statistical analyses
Statistical significance was assessed using a one-way analysis of variance
(ANOVA) followed by Scheffé's test (for three groups), Student's
t-test or Welch's test (for two groups).
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Results |
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Fig. 2 shows typical
examples and Fig. 3 a summary
of the results. EDTA-treated tadpole skin was cultured with corticoids
supplemented with PRL (1 µg ml1) or GH (1 µg
ml1), since this concentration of PRL or GH is sufficient
for the development of larval-type features
(Takada et al., 1995b;
Fig. 1B). In this experiment,
the SCC and skin resistance (R) of intact tadpole skin were
2.7±0.4 µA cm2 and 3.0±0.3 k
cm2, respectively (N=26). Amiloride, acetylcholine
and ATP all induced a transient increase in SCC in intact (control) tadpole
skin; i.e. the SCC increased rapidly then declined, as other investigators
have shown (Hillyard and Van Driessche,
1989
; Cox, 1992
,
1993
,
1997
;
Takada et al., 1996
;
Fig. 2).
|
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The SCC and R recorded from skin cultured with corticoids
supplemented with PRL were 2.1±0.5 µA cm2 and
2.4±0.4 k cm2, respectively (N=18).
Three characteristics of the responses induced by amiloride, acetylcholine and
ATP (the
SCC, the time from the onset of the response to its peak and
the percentage of the normalized peak
SCC remaining at 20 min after
peak) were not significantly different between skin cultured with PRL and
intact tadpole skin (Fig.
3AC).
The SCC and R recorded from skin cultured with corticoids
supplemented with GH were 2.6±0.5 µA cm2 and
2.7±0.3 k cm2, respectively (N=22).
The acetylcholine- and ATP-induced responses were not significantly different
from those seen in intact tadpole skin (Figs
2,
3). However, the
amiloride-induced response did differ from that seen in intact tadpole skin
insofar as both the time from onset to peak and the relaxation kinetics were
significantly slower in skin cultured with GH than in intact tadpole skin
(Fig. 3B,C).
Thyroid hormone indirectly inhibits the action of GH in vivo, since it
downregulates the expression of the mRNA for GH in X. laevis
(Buckbinder and Brown, 1993).
Whether thyroid hormone directly inhibits the action of GH on the development
of a larval-type feature was investigated in vitro in the present
study. To this end, EDTA-treated tadpole skin was cultured with corticoids
supplemented with GH plus T3
(Fig. 4). The SCC and
R recorded from skin cultured with corticoids supplemented with GH
plus T3 were 2.3±0.5 µA cm2 and
1.9±0.4 k
cm2, respectively (N=16).
Thyroid hormone did not inhibit the development of the amiloride-,
acetylcholine- and ATP-induced responses in skin cultured with GH (i.e. there
are no P-values below 0.06 in Fig.
4).
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Discussion |
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The same year, Khakh et al.
(2001) succeeded in expressing
P2X2 or P2X3 receptors from hippocampal neurons on
Xenopus oocytes. The P2X2 receptors opened rapidly and
remained open as long as ATP was present, whereas the P2X3 receptor
opened rapidly but then desensitized rapidly. Following co-expression of the
P2X2/P2X3 receptors, the response to ATP was almost the
same as the sum of the responses seen when P2X2 and P2X3
receptors were expressed separately; i.e. rapid opening then desensitization,
with some current remaining as long as ATP was present.
In the present study, the SCC and desensitization kinetics seen in
the response to ATP were similar between intact (control) tadpole skin and
skin cultured with PRL, and between intact tadpole skin and skin cultured with
GH (Figs 2,
3). Hence, similar
ATP-sensitive non-selective cation channel(s) may be developed in these skins.
It is possible that two kinds of channels destined to mediate the ATP response
develop in these skins, since desensitization was slow and the response
resembled that exhibited by an oocyte in which P2X2/P2X3
receptors were co-expressed (see fig. 1 in
Khakh et al., 2001
).
In our experiment, the acetylcholine-induced response was similar between intact (control) tadpole skin and skin cultured with PRL, and between intact tadpole skin and skin cultured with GH (Figs 2, 3). Hence, similar acetylcholine-responsive channels may be developed in these skins, although whether there are two kinds of acetylcholine-responsive channels, or just one, is unknown as yet.
Hillyard and Van Driessche
(1989) have suggested that (1)
there are two kinds of binding sites for amiloride, a high-affinity site for
activation and a low-affinity site for inhibition or (2) at least two kinds of
NSCCs (nonselective cation channels) mediate the amiloride response, one for
activation and the other for inhibition, since a low concentration of
amiloride (<50100 µmol l1) simply stimulates,
and does not inhibit, the SCC across larval bullfrog skin, whereas higher
concentrations (>100 µmol l1) first stimulate then
block the SCC. Recently, Takada et al.
(2001
) showed that amiloride
simply blocked, and did not stimulate, the SCC across larval bullfrog skin in
the presence of Cu2+, even when the concentration of amiloride was
low (106 mol l1), suggesting that the
high-affinity site for amiloride, or the NSCC that is stimulated by amiloride,
is blocked by Cu2+.
The amiloride-induced response seen in skin cultured with GH differed in its kinetics from that seen in intact (control) tadpole skin (Figs 2, 3). Growth hormone thus apparently induced the development of a different amiloride-responsive channel than that developed in intact tadpole skin. The present results seem to show mainly activation by amiloride in skin cultured with GH. However, it is possible that such skin does contain some amiloride-blockable (low-affinity) NSCC or some low-affinity (amiloride-blockable) binding sites (mediating inhibition). To explain these possibilities will require further experiments comparing amiloride responses in the presence and absence of Cu2+ (which blocks stimulation by amiloride; see above).
Previously, our group showed that adult-type features are developed by
bullfrog skin in vitro under the influence of aldosterone but not
thyroid hormone (Takada et al.,
1995a). However, in vivo, such features are developed
with thyroid hormone but not with aldosterone (Takada et al.,
1997
,
1999
). We therefore
hypothesized a few years ago that aldosterone is the crucial hormone for the
development of adult-type features but that the effect of aldosterone is
suppressed by some means until the suppression is removed by thyroid hormone
during the climax stages of metamorphosis, so allowing the development of
adult-type features to proceed (Takada et
al., 1999
). We now feel that growth hormone is an attractive
candidate for a factor participating in the suppression of the
aldosterone-induced development of adult-type features, since (1) the
expression of the mRNA for GH is higher in the larval stages, (2) this
expression is inhibited by thyroid hormone during the climax stages of
metamorphosis and (3) GH inhibits the development of at least one adult-type
feature by corticoids (Buckbinder and
Brown, 1993
; Fig.
1). However, PRL seems a stronger candidate than GH for the role
of juvenile hormone since the characteristics of the amiloride-responsive
channel developed in the presence of PRL were closer to those of the channel
found in intact tadpole skin than those of the channel developed in the
presence of GH. How PRL and/or GH might act as juvenile hormone(s) in
amphibians is a matter for future consideration.
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Acknowledgments |
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References |
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