Otolith growth in trout Oncorhynchus mykiss: supply of Ca2+ and Sr2+ to the saccular endolymph
uf3
1 Laboratoire R.O.S.E. (Réponses des Organismes aux Stress
Environnementaux), UMR 1112, INRA-UNSA, Université de Nice-Sophia
Antipolis, Faculté des Sciences, Parc Valrose, 06108 Nice Cedex 2,
France
2 IFREMER, DRV, RH, Laboratoire de Sclérochronologie des Animaux
Aquatiques, BP 70, 29280 Plouzane, France
3 Observatoire Océanologique, Laboratoire Arago, Université de
Pierre et Marie Curie, CNRS 639, BP 44, 66651, Banyuls sur Mer Cedex,
France
* Author for correspondence (e-mail: payan{at}unice.fr)
Accepted 15 May 2002
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Summary |
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Key words: trout, Oncorhynchus mykiss, calcium, flux, endolymph, otolith, perfusion, inner ear
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Introduction |
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The pathway of ions from the environment to the otolith is a multi-stage
process involving successive barriers and compartments, e.g. gill/intestine
epithelia blood saccular epithelium endolymph
otolith. Unlike most calcifying systems, e.g. vertebrate bones, enamel,
mollusc shells and coral skeletons, otolith mineralization takes place in an
acellular medium, the endolymph, which contains all the precursors for otolith
formation. The endolymph is secreted by the saccular epithelium, which is
composed of many cell types arranged in two zones relative to the position of
the otolith. (1) A proximal zone bathing the proximal face of the otolith and
composed of the macula (sensory cells, supporting cells and secretory cells)
and a crown of large ionocytes (mitochondriarich cells) arranged in a meshwork
around the macula. (2) On the opposite side, a distal zone bathing the distal
side of the otolith is composed of squamous cells and small ionocytes. Between
these two zones there is a transitional epithelium
(Mayer-Gostan et al., 1997;
Pisam et al., 1998
;
Takagi, 1997
).
Compared with plasma, the endolymph fluid is characterized by a higher
concentration of K+ and total CO2 corresponding to an
alkaline pH, whereas the calcium level is rather low
(Mugiya and Takahashi, 1985;
Payan et al., 1997
,
1999
). Payan et al.
(1999
) reported the presence
of proximodistal (PD) gradients of proteins, K+ and
total CO2 within the endolymph of trout and turbot and proposed
that this lack of chemical uniformity is involved in the otolith calcification
process. Although many studies have been done on the composition of the
endolymph (see review by Campana,
1999
) there is little knowledge of the mechanisms of transport
across the saccular epithelium for the ionic precursors of otolith formation,
i.e. Ca2+ and HCO3-. Most of the studies on
the transport of Ca2+ were carried out by Mugiya and coworkers, who
used an isolated preparation of the otolith-containing sacculus from trout
(Mugiya, 1984
) and concluded
that Ca2+ was transported by a transcellular route
(Mugiya and Yoshida, 1995
;
Toshe and Mugiya, 2000). Few studies have dealt with the analysis of the
concentrations of strontium in the endolymph (see review by
Campana, 1999
) and no published
data concerning Sr2+ movement across the saccular epithelium are
available.
In the present study, the kinetic parameters of Ca2+ and Sr2+ transport across the saccular epithelium of trout were studied on the inner ear using a perfusion technique. This original approach avoids the difficulties of an in vivo study and improves the isolated saccule technique by maintaining a better perfusion of the saccular epithelium and allowing for endolymph sampling at different places inside the saccule. The relationships between blood and endolymph calcium levels were investigated in particular.
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Materials and methods |
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In vivo experiments
Hypercalcemia was induced in nine trout by injection of 100 µl of 0.5
mol l-1 CaCl2 solution into the intraperitoneal cavity.
Successive blood and endolymph samples were then taken and the calcium and
protein content analyzed (see above).
Collection of plasma and endolymph
The techniques of plasma and endolymph sampling have already been described
(Payan et al., 1997,
1999
). Briefly, blood was
sampled from caudal vessels, centrifuged to obtain plasma, and kept on ice
until analysis. The experiments reported in this paper complied with the
Principles of Animal Care of the National Institute of Health (publication No.
86, revised 1985) and the French laws for experiments on animals (decree No.
87-848). After decapitation of the trout, 4-5 µl samples of endolymph were
removed from each side of the otolith (proximal near the macula and distal at
the opposite side) with calibrated capillary tubes connected to a withdrawal
pump. The endolymph contained in the capillaries was kept on ice until
analysis.
Plasma and endolymph analysis
Calcium and strontium contents were measured by spectrophotometry using
Sigma Ca kit. The strontium standard curve was done in the presence of calcium
(see Results). Protein content was measured by spectrophotometry, using
Coomassie Blue with BSA (bovine serum albumin) as a standard
(Bradford, 1976).
In vitro experiments
Heparin (5000 i.u. in 100 µl) was injected into the intraperitoneal
cavity 15 min before dissection. After blood sampling and decapitation, the
ventral aorta was cannulated with polyethylene tubing (Biotrol, 0.86-1.52 mm
diameter) and the isolated preparation perfused with a Ringer solution at a
flow rate of 1 ml min-1 using a peristaltic pump (P-3, Pharmacia).
After 5 min of perfusion to eliminate blood from the vascular space, the
dorsal aorta was cannulated with polyethylene tubing (Biotrol, 0.76-1.22 mm
diameter) and the preparation retroperfused via the dorsal aorta at a
flow rate of 1 ml min-1. A diagram of the retroperfusion technique
is shown in Fig. 1. At various
times after the start of retroperfusion, endolymph was sampled as described
above. In some experiments, after endolymph sampling from the first saccule,
the composition of the Ringer solution was changed (see below) before sampling
the second saccule. Experiments were performed at room temperature.
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The Ringer solution consisted of: 135 mmol l-1 NaCl, 2.5 mmol l-1 KCl, 1 mmol l-1 MgCl2, 1.5 mmol l-1 NaH2PO4, 0.4 mmol l-1 KH2PO4, 0-4 mmol l-1 CaCl2, 5 g l-1 albumin, buffered to pH 7.4 by 5 mmol l-1 NaHCO3. In strontium experiments, CaCl2 was replaced by SrCl2. The solution also contained freshly added glucose (1 g l-1) and was aerated before use. Pharmacological products (verapamil and cyanide) were purchased from Sigma.
The influx of Ca2+ through the saccular epithelium was brought about by retroperfusing the preparation with a Ringer solution containing 1.1 mmol l-1 of CaCl2 supplemented by 45Ca2+ (NEN) at a concentration of approximately 10 kBq ml-1. At different times after radioactive perfusion, 4-5 µl samples of proximal and distal endolymph were taken. Radioactivity in endolymph and Ringer solution samples was measured in a vial containing 5 ml of scintillation liquid using a ß counter (Kontron BETAmatic). The unidirectional Ca2+ influx across the saccular epithelium was evaluated by dividing the radioactivity appearing in the endolymph (cts min-1 ml-1) by the specific radioactivity of the 45Ca in the Ringer solution (cts min-1 nmole-1).
Statistics
Results are expressed as means ± S.E.M. (N specified for
individual experiments) and analyzed statistically by Statview application.
Comparison of the means was done using a oneway ANOVA. Differences were
considered significant at P<0.05.
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Results |
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Ca2+ influx across the saccular epithelium
Unidirectional influx of Ca2+ across the saccular epithelium was
measured by using 45Ca2+ tracer in equilibrium
conditions, where the inner ear was perfused with the same concentration of
Ca2+ as that found in the plasma (1.2 mmol l-1).
45Ca2+ accumulation within the endolymph reached a
plateau approximately 15 min after starting the radioactive perfusion
(Fig. 3). A rough estimation of
the Ca2+ influx was evaluated from the initial slope of the
45Ca2+ accumulation and was approximately 2.4 and 0.6
nmoles µl-1 endolymph h-1 for proximal and distal
compartments, respectively.
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Net flux of Ca2+ across the saccular epithelium during
calcium loading
In a first set of experiments, the inner ear was perfused with a Ringer
solution containing different calcium concentrations at time t=0 min.
Samples were taken from proximal and distal endolymphs in the left saccule at
t=35 min, and in the right saccule at t=70 min. Increased
calcium concentration provoked increased calcium accumulation in both proximal
and distal endolymphs, although the effect was less pronounced in the distal
sample with 4.4 mmol l-1 calcium in the Ringer solution
(Fig. 4A,B). Equilibrium was
reached approximately 30 min after the beginning of the perfusion in both
compartments. The use of a low-Ca Ringer (0.19 mmol l-1 Ca)
partially emptied the proximal compartment whereas the calcium level in the
distal sample did not vary within the 70 min of perfusion
(Fig. 4A,B).
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In a second set of experiments, the inner ear was perfused at time 0 with a Ringer solution containing 3.23 mmol l-1 of Ca. 35 min later proximal and distal endolymphs of the left saccule were sampled, then the calcium levels in the Ringer solution were changed (to 0.13 mmol l-1 or 4.27 mmol l-1) and at t=70 min, endolymphs were sampled in the right saccule. This experimental design allowed an individual preparation to be its own control. The results, summarized in Fig. 5A,B, confirm that increased calcium levels in the Ringer solution provoke an accumulation of Ca2+ within the endolymph and that decreased calcium levels empty the endolymph pool of Ca. These effects are less pronounced in the distal endolymph than in the proximal one.
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Net flux of Sr2+ across the saccular epithelium during
strontium-loading
The inner ear was perfused with a Ringer solution containing different
concentrations of SrCl2 (Ca-free). After 35 min of perfusion, the
proximal and distal endolymphs were sampled and the sum of calcium and
strontium contents were measured using a Sigma kit for calcium analysis. The
strontium level was estimated from a SrCl2 standard curve done in
the presence of 1.4 and 1 mmol l-1 of CaCl2
(corresponding to the levels of calcium in proximal and distal endolymphs
respectively, after 35 min perfusion with a Ca-free Ringer solution). The
presence of increasing concentrations of strontium in the perfusing medium
provoked an accumulation of increasing amounts of strontium in both proximal
and distal endolymphs (Table
1).
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Effects of inhibitors on endolymph Ca2+ supply
The preparation was perfused at t=0 min with a Ringer solution
containing 2.8-3.2 mmol l-1 CaCl2 and the endolymph was
sampled 35 min later. 10-5 mol l-1 verapamil (a blocker
of voltage-dependent calcium channels) or 1 mmol l-1 CN (a blocker
of ATP production by the mitochondria) were added to the Ringer solution at
t=0 and their effects on Ca2+ accumulation into the
endolymph studied. Neither inhibitor modified Ca2+ entry into
either proximal or distal endolymph (Table
2).
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Heterogeneous distribution of calcium within the saccule
The microtechnique described here permitted measurement of calcium and
protein concentrations in each sample and thus a study of their
interrelationships. In these experiments with trout, the protein content of
the control samples was: 16.0±1.62 g l-1 (N=20) for
proximal and 3.7±1.00 g l-1 (N=20) for distal
zones, (P<0.001), and the calcium content was 1.23±0.10
mmol l-1 (N=10) for proximal and 0.98±0.05 mmol
l-1 (N=10) for distal zones (P=0.0265). These
results can also be presented by plotting [calcium] as a function of [protein]
(Fig. 6), showing a significant
positive relationship between the two parameters.
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In one set of experiments performed with turbot, 4-5 µl samples of endolymph were withdrawn at different sites inside the saccule. The results show a significant positive relationship between the concentrations of calcium and protein (y=0.05x+0.76, N=64, r2=0.137, P=0.0024; data not illustrated) similar to that found in the trout, although the protein range in turbot endolymph (1-7 g l-1) was smaller than in trout (1-20 g l-1).
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Discussion |
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Dynamics of the endolymph Ca2+ pool
Very few studies have been concerned with the relationship between plasma
and endolymph Ca2+ levels and the present work is the first
evaluation of the unidirectional Ca2+ influx through the saccular
epithelium in electrochemical conditions resembling those in vivo.
The sum of the Ca2+ influxes into the proximal and distal spaces is
about 3-4 nmoles µl-1 endolymph h-1, which
corresponds to a global turnover rate of 230 % h-1 of the endolymph
pool of calcium (equal to 30 nmoles, calculated for a saccule of 20 µl
volume and a concentration of 1.5 mmol l-1 Ca). It must be noted
that this value may be an underestimation because it does not take into
account the Ca2+ incorporated into the otolith during the
experiments.
This very high turnover rate suggests that the perfusion of the inner ear with different concentrations of Ca2+ could rapidly influence the calcium level within the endolymph, a hypothesis confirmed by the results of the hyper (in vivo and in vitro) and hypo (in vitro) calcemia experiments. Indeed, the presence of a positive chemical gradient of calcium between Ringer solution and endolymph provoked the accumulation of Ca2+ in both proximal and distal endolymphs (Figs 2A, 4A,B) and a negative chemical gradient produced an emptying of the endolymph pools (Figs 4A, 5A,B). Surprisingly, perfusion with a Ca-free solution did not empty the distal compartment (Fig. 4B), suggesting that the distal calcium was strongly bound and thus not rapidly exchangeable.
Our results should be compared with those of Toshe and Mugiya
(2001), who studied the
translocation of 45Ca2+ from the Ringer solution to the
endolymph using an isolated preparation of the sacculus. By incubating the
sacculus with 3 mmol l-1 CaCl2 for 2 h they found that
Ca2+ incorporation into the endolymph was about 2 nmoles
µl-1 endolymph 2 h. In fact, if the excised sacculus behaves
like the perfused sacculus, after 2 h of incubation the specific
radioactivites of 45Ca2+ in the Ringer solution and
endolymph should be identical and the results of 2 nmoles µl-1
endolymph 2 h-1 correspond to a calcium level of 2 mmol
l-1. This is in agreement with the value of 2.4-1.7 mmol
l-1 (in proximal and distal spaces, respectively) obtained by
perfusing the inner ear with 3.2 mmol l-1 Ca2+
concentrations (Fig. 4A,B).
The analysis of the ratio between the endolymph pool of calcium and its
daily incorporation into the otolith offers a dynamic vision of the calcium
needs involved in the process of otolith growth. For a 12 month-old trout the
otolith mass is about 8 mg, with 0.2 % being total organic matrix and 99.8 %
CaCO3 (Borelli et al.,
2001). In such trout, the daily otolith growth necessitates 0.23
µmoles of Ca2+ corresponding to a daily incorporation of eight
endolymph calcium pools. These calculations indicate that the saccular
epithelium must transfer huge amounts of calcium for the daily otolith growth
and this reflects the high turnover rate of the endolymph calcium pool (about
55 times a day).
The endolymph Ca2+ is supplied mainly at the proximal area
of the saccular epithelium via a paracellular pathway
During the hypercalcemia experiments (in vivo and in
vitro) calcium levels were systematically higher in the proximal area of
the endolymph than in the distal one (Figs
2A,
4A,B). This suggests that
Ca2+ entry into the sacculus occurs mainly through the macula area
into the proximal compartment. This is confirmed by the kinetics of calcium
appearance in the endolymph during in vivo hypercalcemia, which
showed that calcium levels increased more rapidly in the proximal than in the
distal zones (Fig. 2A). Thus
our results confirm the conclusions of Mugiya
(1974) based on the analysis
of an autoradiogram of the saccular epithelium in
45Ca2+-injected trout.
The experiments of Ca2+ net flux are summarized in
Fig. 7, in which the chemical
gradients of calcium between the Ringer solution and endolymph at the
beginning of the perfusion are plotted as functions of the resulting
Ca2+ accumulation over a 30 min period. It is clear that these
relationships (including positive and negative net fluxes) are linear in both
the proximal and distal compartments, which suggests a passive mechanism for
Ca2+ transfer across the saccular epithelium, i.e. a paracellular
pathway. This is confirmed by the absence of any effect of verapamil or CN on
Ca2+ accumulation in the endolymph in the presence of a positive
chemical gradient of calcium (Table
2). Calculation of ECa by the
Nernst equation applied to the results of
Fig. 4A gives +4 mV (endolymph
positive) if the calcium levels are used for both compartments. Mugiya
(1966) found 72% of
ultrafiltrable calcium in the endolymph of trout and flatfish. Assuming that
the ultrafiltrable calcium and ionized calcium are comparable, the Nernst
potential is +8 mV, which is close to the only published value for teleosts,
namely a saccular potential of about +10 mV
(Enger, 1964
). It must be noted
that in vertebrates the endolymph side has always been found to be positive
with respect to the perilymph, and in mammals the voltage varies from +80 mV
in the cochlea to +5 mV in the utriculus
(Sterkers et al., 1988
).
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The main conclusion to be drawn from our study is that Ca2+
crosses the saccular epithelium via a paracellular route. This is not
in agreement with the model of Mugiya and Yoshida
(1995), who suggested that
Ca2+ entry into the endolymph is by a transcellular route involving
a combination of a receptor-operated Ca2+ channel,
Na+Ca2+ exchanger and ATP-dependent
Ca2+ pump. This proposal was based on the amounts of
Ca2+ incorporated into the otolith under different experimental
conditions. Recently, Toshe and Mugiya
(2001
) observed that the
trans-saccular transport of Ca2+ to endolymph was not
affected by the usual inhibitors of the pH-regulating mechanisms (amiloride,
acetazolamide, DIDS, SITS and thiocyanate). This lack of effect of inhibitors
known to disrupt the acidobasic homeostasis of the intracellular medium
also favours a paracellular pathway for Ca2+.
Heterogeneity of the endolymph Ca2+ pool and otolith
growth
The present study confirms the existence of the decreasing PD
gradient of proteins in endolymph that had already been observed by Payan et
al. (1999), and also brings to
light the presence of a lower but significant decreasing PD gradient of
calcium that had not been described. This raises the question as to whether a
PD gradient of Ca2+ occurs within the endolymph compartment.
Attempts to evaluate Ca2+ levels in proximal and distal endolymphs
by using a Ca2+-sensitive mini-electrode (KWIK-TIP-WPI) failed,
owing to difficulties in establishing a standard curve taking into account the
different parameters of these two media (pH, protein, Mg2+,
K+ contents). No data are available concerning the Ca2+
level in the endolymph of fishes and only Mugiya
(1966
) has measured the
ultrafiltrable fraction (72%) of calcium in this fluid. It should be noted
that in higher vertebrates, vestibular endolymph contains about 3 mmol
l-1 Ca, with one-tenth in the ionized form (Steckers et al.,
1988).
The fact that perfusing with a calcium-free solution did not decrease the
calcium level in the distal compartment
(Fig. 4B) supports the
hypothesis of a highly bound distal calcium. Recently, Borelli et al.
(2001) described an increasing
PD gradient of proteoglycans whose polyanionic nature could explain
this phenomenon. We therefore propose that the decreasing PD gradient
of calcium corresponds to a decreasing gradient of Ca2+ that is the
result of two factors: a predominantly proximal entry of Ca2+,
combined with the position of the otolith, which presents a physical barrier
to diffusion from proximal to distal compartments. It should be noted that the
decreasing PD gradient of Ca2+ and also that of protein
concentration are clearly related to the growth axis of the otolith, as the
proximal zone facing the macula corresponds to the convex shape of the otolith
where the increment is greater than on the concave distal side. This situation
reinforces the proposal that the proximal zone is of importance in the
CaCO3 deposition process as it is already held to be for the
formation of organic compounds (Gauldie and
Nelson, 1988
; Payan et al.,
1999
; Borelli et al.,
2001
).
Endolymph Sr2+ homeostasis in relation to otolith
strontium microchemistry
Because of their similar properties, a paracellular route across the
saccular epithelium might also be expected for Sr2+, as was found
for Ca2+. Subjecting a chemical gradient of SrCl2 (0.98,
2.39 and 3.93 mmol l-1) to the saccular epithelium provoked an
accumulation of Sr2+ within the sacculus in both proximal and
distal zones (Table 1). The
relationship between the strontium gradient and the resulting Sr2+
accumulation in the endolymph over 30 min was linear and the slope comparable
to that found for calcium in similar conditions
(Fig. 8). The values for the
strontium in endolymph may only be approximate, since we used a Ca-kit to
measure the strontium level, but the data relating to the slope should still
be significant. These results indicate that, under our experimental
conditions, the Sr2+ crosses the saccular epithelium via a
paracellular route and reinforces the hypothesis of a paracellular pathway for
Ca2+ endolymph supply. This hypothesis is supported by Kalish
(1991), who reached similar
conclusions after studying the correspondence between blood plasma and
endolymph strontium/calcium in bearded rock cod.
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In conclusion, our results suggest that variations of Ca2+ and
Sr2+ plasma levels rapidly induce corresponding variations in the
endolymph via a passive paracellular route. This may be relevant to
the calcification process as these two ions, together with
HCO3-, are the precursors of the aragonite (which is
predominant) and strontionite formations. However, within the calcification
process we have to consider Ca2+ and Sr2+ activities
rather than their total levels, and these depend on several parameters such as
total concentration, pH of the fluid, presence, nature and concentration of
binding proteins and presence of competitors (Mg2+) and inhibitors
(such as PO43-, proteoglycan). The composition of the
fish endolymph is not spatially uniform, showing PD gradients for most
of its components (Payan et al.,
1999). Furthermore, the levels of some calcifying parameters
(total CO2, pH, proteins) vary within the proximal and distal zones
as functions of environmental conditions such as circadian cycle
(Edeyer et al., 2000
), fasting
(Payan et al., 1998
) and
stress (P. Payan, H. De Pontnal, A. Edeyer, G. Borelli, G. Boeuf and N.
Mayer-Gostan, unpublished results), making it difficult to evaluate the ionic
activities of Ca2+ and Sr2+. These factors should be
taken into consideration when studying strontium otolith microchemistry, since
incorporation of strontium is directly dependent on its ionic activity at the
interface between endolymph and otolith.
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Acknowledgments |
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References |
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