Prolactin increases open-channel density of epithelial Na+ channel in adult frog 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 28 January 2003
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Summary |
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Key words: frog skin, current-fluctuation (noise) analysis, prolactin, active Na+ transport, epithelial Na+ channel (ENaC), tree frog, Hyla arborea japonica
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Introduction |
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Transcellular active Na+ transport across the skin, measured as
the amiloride-blockable short-circuit current (SCC), has been identified in a
wide variety of anuran and urodelan genera, including Rana, Leptodactylus,
Bufo, Cynops and Ambystome species
(Bentley and Yorio, 1977;
Rabito et al., 1978
;
Bentley and Baldwin, 1980
;
Hillyard et al., 1982
;
Takada, 1985
;
Takada and Komazaki, 1986
;
Takada and Hara, 1988
). This
transport develops during the climax stages of metamorphosis in the bullfrog,
and is due to the development of an epithelial Na+ channel (ENaC).
Therefore, the appearance of transcellular active Na+ transport as
SCC and/or ENaC is a marker of the development of adult-type features by the
skin (Cox and Alvarado, 1979
;
Hillyard et al., 1982
;
Takada, 1985
).
If prolactin does indeed have a positive role in the progression of
metamorphosis, the SCC across the skin should be stimulated by it. In fact, a
prolactin-induced increase in the SCC of adult amphibian skin has already been
reported (Eddy and Allen, 1979; Takada,
1986).
The activity of the epithelial Na+ channel (ENaC) located at the
apical membrane is the rate-limiting step in transcellular active
Na+ transport in epithelia such as A6 cells, MDCK cells, toad
urinary bladder and frog skin. An increase in SCC is considered to reflect an
increase in the single-channel current (i) and/or an increase in the channel
density (M). Blocker-induced current-fluctuation analysis (noise
analysis) is a useful method for determining the effect of a hormone on the
values of i and M when studying ENaC in the epithelium of frog skin,
for example (Lindemann and Van Driessche,
1977; Hillyard et al.,
1982
; Helman et al.,
1983
; Baxendale-Cox et al.,
1997
; Els and Helman,
1997
). Here, in a study involving current-fluctuation analysis by
a two-state model (use of a three-state model proving unsatisfactory with our
data), we report that the prolactin-induced increase in the SCC across the
skin of the adult tree frog is due to an increase in the open-channel density
of ENaC.
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Materials and methods |
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Electrical measurements
Dissected skin samples were mounted in an Ussing-type chamber fitted with
silicone gaskets (5 mm i.d.) to minimize edge damage. Both sides of the skin
samples were bathed in aerated Ringer's solution containing (in mmol
l-1) 110 NaCl, 2 KCl, 1 CaCl2, 10 glucose and 10 Tris,
pH 7.2. The short-circuit current (SCC) was measured continuously under
voltage-clamp conditions.
Current-fluctuation analysis was performed before and after application of
ovine prolactin (10 µg ml-1; Sigma Chemicals, St Louis, MO,
USA). The details of the method used for current-fluctuation (noise) analysis
were as previously described (Takada et
al., 1999). In brief, the fluctuations in SCC were high-pass
filtered (0.05 Hz), amplified (x500) and low-pass filtered (1,024 Hz) to
prevent aliasing errors. The signal was sampled at 2,048 Hz. Then, a
power-density spectrum (PDS) was calculated for these records using a Digital
Spectrum Analyzer (R-9211 A; Advantest, Tokyo). Analysis of the PDS yields the
Lorentzian parameters So (plateau) and
fc (corner frequency) as follows:
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Statistical analysis
Statistical significance was assessed using a one-way analysis of variance
(ANOVA) followed by Scheffé's test (for three groups) or by a Student's
t-test or Welch's test (for two groups).
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Results and Discussion |
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Basolateral application of prolactin increased the SCC (Fig. 1); however, apical application had no effect on the SCC (data not shown). This increase in SCC following basolateral application of prolactin was confirmed in SO4- and gluconate-Ringer's solutions (data not shown). Prolactin increased the SCC rapidly and the effect was typically maintained for more than 2 h (see example shown in Fig. 1); however, it took 20-30 min for the response to reach maximum in most cases (Figs 1, 2). In 6/42 cases, the SCC increased rapidly but then desensitized within 10-20 min. We did not use such responses for noise analysis since the SCC needs to be more or less stable for at least 20-30 min for this analysis. As yet, we are not sure why the prolactin-induced increase in SCC desensitized within 10-20 min in these six cases.
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Whether the increase in SCC was caused by an increase in the single-channel current (i) and/or by an increase in the open-channel density (M) of the epithelial Na+ channel (ENaC) was investigated by current-fluctuation analysis.
Fig. 2 shows a typical example of a staircase experiment in which CDPC was applied before and after an application of prolactin. When the SCC had stabilized after prolactin application, skins were again subjected to a staircase increase in CDPC. Current-noise power-density spectra were measured at each concentration of CDPC, before and after prolactin application.
Prolactin increased SCC 2.6±0.4-fold and M 6.1±1.2-fold (Fig. 3), but it decreased i to 0.4±0.1 times the control value (Figs 3, 4B). Although K10 increased 1.6±0.3-fold, Km was not significantly different before and after prolactin treatment (Figs 3, 4A).
|
The decrease in i induced by prolactin will presumably have been
due at least in part to depolarization of the apical membrane caused by the
increase in M, since depolarization decreases the single-channel
current of ENaC in MDCK cells (Ishikawa et
al., 1998). This idea is supported by the effect of steroid
hormones and forskolin: both induce an increase in SCC but a decrease in
i and, in frog skin, the latter is thought to be due to
depolarization of the apical membrane
(Helman et al., 1983
;
Baxendale-Cox et al.,
1997
).
There are two possibilities for the increase in M: (1) activation
of quiescent ENaC located at the apical membrane or (2) increases in
ENaC-trafficking and the cell-surface stability of ENaC. Kemendy et al.
(1992) showed that in A6
cells, aldosterone does not increase M but does increase Po
(open channel density). However, other investigators have shown that steroid
hormones, antidiuretic hormone (ADH) and forskolin all increase M in
A6 cells or frog skin, resulting in an increased SCC
(Els and Helman, 1997
;
Helman et al., 1983
;
Baxendale-Cox et al., 1997
;
Rosa et al., 2002
).
Mineralocorticoids increase the activity of serine-threonine kinase in A6
cells and in proximal tubule cells in rats
(Loffing et al., 2001
;
Wang et al., 2001
), resulting
in an increase in Na+ transport across the epithelium. Activation
of protein kinase C decreases the expression of the ß and
subunits of ENaC in A6 cells (Stockand et al., 2000). These results suggest
the possibility that the numbers of ENaC on the apical membrane are regulated
by protein phosphorylation. Further research will be needed on whether and how
prolactin-signal transduction leads to protein phosphorylation and an increase
in M.
In conclusion, prolactin has not only a counteracting effect on metamorphosis but also a stimulatory effect on sodium-transport activity (amiloride-blockable SCC) in adult frog skin. The present results suggest an increase in the open-channel density of ENaC, at least following short-term treatment with prolactin.
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Acknowledgments |
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