Proc. Nati. Acad. Sci. USAVol. 89, pp. 3635-3638, April 1992Physiology

Calcium uptake in the skin of a freshwater teleost(tilapia/ion transport/opercular membrane/caldum metabolism)

S. D. MCCORMICK*t*, S. HASEGAWAf, AND T. HIRANOttOcean Research Institute, University of Tokyo, Nakano, Tokyo 164, Japan; and *Department of Integrative Biology and Cancer Research Laboratory,University of California, Berkeley, CA 94720

Communicated by Howard A. Bern, January 8, 1992 (received for review October 20, 1991)

ABSTRACT The skin, particularly the opercular mem-brane of some teleosts, contains mitochondrion-rich "chlo-ride" cells and has been widely used as a model to studybranchial salt-extrusion mechanisms in seawater fish. Skinisolated from the operculum of the freshwater Nile tilapia(Oreochromis nilotcus) can transport Ca2' against an ionic andelectrical gradient. Adaptation of Nile tilapia to a low-Ca21environment increased the capacity ofthe opercular membraneto transport Ca2+. The density of mitochondrion-rich cellsincreased in parallel with Ca2' transport capacity. The resultsdemonstrate net Ca2+ uptake by vertebrate skin and stronglyimplicate mitochondrion-rich cells as the site of Ca2+ uptake infresh water.

Calcium is necessary for a variety of functions in animals,including bone and scale growth, muscle contraction, trans-mission of nerve impulses, hormone secretion, and intracel-lular signaling. In animals living in fresh water, calcium canbe obtained from food or from the surrounding medium.Freshwater fish have the capacity to maintain normal bloodCa2+ levels (2-4 mM) in a wide range of external Ca2+concentrations (<0.01 mM), even when food is withheld forlong periods (1-3). Although the gills (4-12) and skin (5, 13)have both been implicated as sites of Ca2` uptake in teleosts,net Ca2' uptake in isolated skin or gill tissue has not, to ourknowledge, previously been demonstrated. The presentstudy was undertaken to determine whether net Ca2' uptakeoccurs in fish skin in vitro and whether mitochondrion-rich(MR) cells are involved in Ca2+ uptake.

MATERIALS AND METHODSThe Nile tilapia Oreochromis niloticus is an Old World cichlidendemic to the freshwater lakes of the Rift Valley of Africa.Juvenile Nile tilapia, weighing 40-60 g and previously rearedin fresh water, were kept in 33-liter tanks maintained at 24 +1°C with charcoal filtration and aeration. Fish were kept ineither freshwater (FW: 0.77-1.04 mM Na+/0.54-0.58 mMCa2+) or low-Ca2+ freshwater (LCFW: deionized water sup-plemented with NaCl, 0.50-0.97 mM Na+, and 0.003-0.011mM Ca2+). Fish were maintained under experimental condi-tions for at least 2 weeks before sampling and were fed amaintenance ration of commercial trout chow (0.5% bodyweight per day). Water samples were taken intermittentlyfrom tanks containing fish to determine environmental [Na+]and [Ca2W]. Although LCFW solution contains normal, fresh-water concentrations of Na+ and Cl-, it has low levels ofother ions; we explicitly assume that the results obtained aredue to decreased Ca2+; absence of other divalent ions doesnot cause morphological changes in teleost gills (14). Envi-ronmental and total plasma [Na+] and [Ca2+] were measuredby atomic absorption spectrophotometry. Two-thirds of the

tank volume in each group was replaced every other day.Food was withheld for 24 hr before sampling of individualfish. Fish were killed by cranial concussion and pithing andwere bled from the caudal vessels into heparinized syringes.Plasma was isolated by centrifugation and stored at -80'C.

Opercular membranes (the epithelium lining the buccalside of the opercular bone) were removed and placed intilapia Ringer's solution (140 mM NaCl/10 mM NaHCO3/4mM KCI/2 mM NaH2PO4/1 mM MgSO4/1 mM CaCl2/5.5mM glucose, pH 7.8), and isolated as described in ref. 15.Paired membranes (removed from both right and left sides)were used to avoid any variation among individuals. Mem-branes were attached to stainless steel pins on a circularPlexiglas ring that was then mounted on a Plexiglas chip andplaced in the center of an Ussing-style chamber (described indetail in ref. 16). Seals were made watertight with siliconegrease. The exposed membrane was 0.5 cm in diameter, andthe total chamber volume was 14 ml. Tilipia Ringer's solutionwas added to the basal (blood) side, and artificial freshwatersolution (1 mM NaCl/0.5 mM NaH2PO4/0.2 mM CaCl2, pH7.0) was added to the apical side. Two polyethylene 4%agar/3 M KCI bridges were placed 6 mm apart on either sideof the membrane; each bridge terminated in 3 M KCI con-nected to a voltmeter by Ag/AgCl electrodes. Electrodeasymmetry was nullified, and liquid junction potentials werecorrected (17) immediately before each experiment. After astable transepithelial potential (TEP) was achieved (changeof <0.2 mV/min), 45CaC12 (New England Nuclear) was addedto the apical side of one chamber and the basal side of theother chamber to attain a final activity of 2 ,Ci/ml (1 Ci = 37GBq). Air was bubbled into each side of the chamber topromote mixing and oxygenation. After a 45-min isotopeequilibration period, samples of 250-500 ,ul from each side ofthe chambers were removed every 45-90 min. Scintillationfluid was added to samples from the unlabeled bath at eachinterval, and radioactivity was counted for 20 min. An initialand a final sample from the labeled side was measured toverify the specific activity. Unidirectional fluxes were cal-culated from the linear rate of45Ca2+ movement (45-225 min)based on the known specific activity with appropriate cor-rections for volume changes. After each experiment, leakageacross the membrane was tested by removing the solutionfrom one side of the chamber; if fluid movement into theempty half of the chamber was detected, the preparation wasconsidered leaky, and the results were discarded.

In an effort to determine the site of Ca2+ transport, weexamined the skin for MR cells, which are known to be thesites of active ion transport in a variety of transport epitheliain fish. After dissection, opercular membranes were affixedto lens paper and placed in tilapia Ringer's solution with 2 ,uM

Abbreviations: FW, freshwater solution; LCFW, low-calcium FWsolution; MR, mitochondrion rich; TEP, transepithelial potential;DASEPI, 2-(4-dimethylaminostyryl)-1-ethylpyridium iodide.tTo whom reprint requests should be addressed at: Anadromous FishResearch Center, U.S. Fish and Wildlife Service, P. 0. Box 796,Turners Falls, MA 01376.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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2-(4-dimethylaminostyryl)-1-ethylpyridium iodide (DASEPI,K & K Laboratories), a mitochondrion-specific stain, whichstains only active mitochondria (18-20). After 1 hr the tissuewas rinsed several times with tilapia Ringer's solution andthen examined with an epifluorescent microscope (NikonDiaphot) with a 100-W mercury light source containing a450-490 bandpass excitation filter, a 510 chromatic beamsplitter, and a 520 longwave pass filter. Images were recordedwith a silicon intensified target camera (Hamamatsu Pho-tonics, Hamamatsu, Japan) and a video cassette recorder(Sony, Tokyo; 30 frames per s) by using several fields fromeach preparation. MR cell density was measured by countingthe number of DASEPI-positive cells in at least 10 fields of0.46 mm2 for each of five individuals per group. MR cell sizewas determined by measuring the minimum cross-sectionaldistance (cell diameter) from video cassette recorder stillframes. Use of the minimum cross-sectional distance mini-mized errors introduced by cells that were not perpendicularto the focal plain. Measurements were made only on imagesin sharp focus that had clear distinctions in cell outline. Videocassette recorder images were averaged over a 3.3-s period,and relative fluorescent (DASEPI) intensity per unit area wasmeasured in the central portion (excluding cell boundaries) ofMR cells.

RESULTSPlasma [Na'] (FW tilapia: 159 ± 2 mM, LCFW tilapia: 157± 2 mM, n = 13) and plasma [Ca2+] (FW tilapia: 3.1 ± 0.1mM, LCFW tilapia: 3.0 ± 0.1 mM, n = 14) were not affectedby exposure to low environmental calcium (P > 0.2, Mann-Whitney U test).TEP of opercular membranes was serosal-side positive and

not significantly affected by low environmental calcium (FWtilapia: +10.5 ± 0.8 mV, n = 8; LCFW tilapia: +11.5 ± 0.8mV, n = 8; P = 0.3). Under these conditions net Ca2+ uptakewould have to occur against both an electrical and chemicalgradient (5-fold higher [Ca2+] on serosal side, serosal-sidepositive).

In all preparations, Ca2+ influx and efflux were linearthroughout the 3-hr period of measurement (see Fig. 1). Ca2+influx of paired membranes was seven to eight times greaterthan Ca2+ efflux (Figs. 1 and 2), indicating that net uptakeoccurred under the imposed conditions.Ca2+ influx was >2-fold higher in opercular membranes of

tilapia from LCFW relative to those in FW (P < 0.05).Although mean Ca2+ efflux was higher in the LCFW group,there was no statistically significant difference between the

TILAPIA FROM FRESH WATER

-

E4-C)

E

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+cs

N4a0

) TILAPIA FROM FRESH WATER- TILAPIA FROM LOW-CALCIUM FRESH WATER

INFLUX EFFLUX NET FLUX

FIG. 2. Ca2+ fluxes in isolated opercular membrane of Niletilapia. Open bars represent opercular membranes from fish adaptedto normal-Ca FW, filled bars represent opercular membranes fromfish adapted to LCFW. Four paired membranes were used tomeasure Ca2+ fluxes in each group. Values are expressed as mean +SEM. *, Significant difference from normal-Ca FW group (P < 0.05,Mann-Whitney U test).

groups. Net Ca2+ uptake was 2-fold higher in fish adapted toLCFW (P < 0.05, Fig. 2).MR cell density of the opercular membrane increased

2-fold from adaptation to LCFW (P < 0.01, Table 1, Fig. 3).MR cell size (diameter) ranged between 6 and 12 ,m;adaptation of fish to LCFW shifted MR cell-size distribution(Fig. 4) and slightly (6%) but significantly increased averageMR cell size relative to fish in FW (Table 1). Relativefluorescence (per unit area) of MR cells also increasedslightly (8%) but significantly after adaptation to LCFW (P <0.05, Table 1).

DISCUSSIONResults of the present study indicate that the skin (opercularmembrane) of Nile tilapia is capable of Ca2+ uptake againstan ionic and electrical gradient, thus demonstrating a netCa2+ uptake in isolated epithelia of a freshwater vertebrate.The kinetics of Ca2+ uptake were examined in trout gill byusing the perfused head technique (5); however, limitations inthis method have prevented measurement of Ca2+ efflux andnet flux. Net Ca2+ uptake has been demonstrated in theisolated intestine of a marine teleost (21). Ca2+ uptake wassuggested to occur in the skin of frogs (22) but was notsupported by subsequent studies (23, 24).The isolated opercular membrane of Nile tilapia in fresh

water could transport Ca2+ against an ionic and electrical

TILAPIA FROM LOW-CALCIUM FRESH WATER

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1influx

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60 120

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not flux: 0.76 nmol.cm-2h1

Influx

0 -- Oe fflux

0 60 120 180

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FIG. 1. Linear Ca2l fluxes in isolated, paired opercular membranes of Nile tilapia adapted to normal-calcium FW or LCFW. Measurementsbegan after a 45-min isotope equilibration period.

3,not flux: 0.31 nmoI~cm-2.h-1N

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Proc. Natl. Acad. Sci. USA 89 (1992) 3637

Table 1. Effect of adaptation of Nile tilapia to LCFW on MRcell density, size (diameter), and fluorescence intensity

Adaptation medium

Normal Ca2+ Low Ca2+Cell density, cells per cm2 7,598 ± 1274 17,588* + 3662Cell size, tim 8.4 ± 0.1 8.9* ± 0.1Fluorescence intensity,

units 1.92 ± 0.03 2.08* ± 0.04Preparations of opercular membrane were stained with DASEPI

and examined as detailed in text. MR cell size and relative fluores-cent intensity were measured on 20 cells in preparations from fiveindividuals in each group (n = 100 per group). Values are mean ±SEM.*Significant difference from fish adapted to normal-Ca2+ FW (P <0.05, Mann-Whitney U test).

gradient, even when the external Ca2+ concentration in vitro(0.2 mM) was lower than the levels the tilapia had experi-enced in vivo (0.5 mM). Adaptation of Nile tilapia to LCFWresulted in a >2-fold increase in net influx of Ca2+ by theopercular membrane. The present study supports the ideathat increases in net Ca2+ uptake in fish under conditions oflow environmental Ca2+ (25) are the result of increases in thecapacity of the gills and skin to actively transport Ca2+.

Estimates of whole-animal net Ca2+ uptake in teleosts infresh water range between 3 and 20 ,umol h-1-kg-l (25, 26),indicating that the basal Ca2+ uptake by the opercular mem-brane might contribute 1-7% to the total net Ca2+ uptake(calculations are based on an average 8 cm2 opercular mem-brane in a 50-g fish). This estimate represents a minimumcontribution of the opercular membrane, as the measure-ments in the present study represent a basal, unstimulated

40

G)

cL.

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C4-

a)EL

ED TILAPN FROM FRESH WATER- TILAPIA FROM LOW-CALCIUM FRESH WATER

30

20

10

6-7 7-8 8-9 9-10 10-11 11-12

Cell Diameter (jm)

FIG. 4. MR cell size distribution in isolated opercular membranesof Nile tilapia adapted to normal-calcium FW and LCFW. Prepara-tions of opercular membrane were stained with DASEPI and exam-ined as detailed in text. MR cell size and relative fluorescent intensitywere measured on 20 cells in preparations from five individuals ineach group (n = 100 per group). The density ofMR cells was >2-foldgreater in opercular membranes of Nile tilapia adapted to LCFW.

level of Ca2+ uptake. Chemical mediators (e.g., hormones)present in the whole animal are likely to increase substan-tially the in vivo uptake of the opercular membrane. In someteleosts MR cells are located throughout the skin, particularlyin larvae and juveniles (27), indicating a potentially greateroverall contribution of the skin to total Ca2l uptake than thatestimated solely from the opercular membrane. Based onwhole-animal 45Ca2' exposures, Perry and Wood (5) deter-

FIG. 3. MR cells in opercular membranes of Nile tilapia adapted to normal-calcium freshwater (A and C) and low-calcium freshwater (B andD). (A and B) Photomicrograph of isolated opercular membrane stained with the mitochondrion-specific fluorescent dye DASEPI. (Bar = 50Am.) (C and D) Three-dimensional image of corresponding fluorescent image (A and B, respectively) showing fluorescent intensity as a functionof the planar image area.

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mined that up to 50% of nondietary Ca2+ uptake occursthrough the skin of rainbow trout (Oncorhynchus mykiss).The TEP of fish in fresh water can be slightly positive or

slightly negative (28), whereas euryhaline fish adapted to seawater are usually positive relative to the external medium,with increasing salinity causing a more positive TEP (28-30).Young et al. (30) reported that the whole-animal TEP in theclosely related tilapia Oreochromis mossambicus in freshwater was -1 to + 10 mV. In the present study, TEP of theisolated opercular membrane under "physiological" condi-tions ranged between 8 and 13 mV and was not affected byprior adaption to LCFW solution. The TEP of the isolatedhead of rainbow trout increased from -10.2 to +1.2 whensubjected to an increase in environmental calcium from 0.05to 2.52 mM (5). As in the present study, increased Ca2+uptake could not be accounted for by changes in the trans-epithelial potential.The "chloride secretory cell" was originally described by

Keys and Willmer (31) in the gills of seawater-adaptedteleosts. These cells are MR and have been shown to be thesite of Cl- secretion in the gill and skin of seawater-adaptedteleosts (32-34). However, MR cells are often termed "chlo-ride cells", even when found in freshwater-adapted teleost,where a Cl- secretory function is unlikely and a Cl- uptakefunction is uncertain (for reviews of MR cell structure andfunction, see refs. 35-37). Using the isolated, perfused headof rainbow trout, Payan et al. (4) implicated MR cells as thesite of Ca2+ uptake in the gill. Morphological studies havealso provided indirect evidence that MR cells are involved inCa2+ uptake in the gill offreshwater teleosts (6, 38), althoughconflicting evidence exists (14). Perry and Wood (5) foundthat Ca2+ influx and "chloride" cell density increased inparallel in the gills of rainbow trout adapted to low externalcalcium (25 mM Ca2+). Research on the scaleless skin ofrainbow trout (W. S. Marshall and C. M. Wood, personalcommunication) indicates that the magnitude of CaW+ influxis related to the density ofMR cells. Along with these studies,the present findings that Ca2+ uptake and MR cell densitydouble after adaptation of the Nile tilapia to low environ-mental calcium strongly implicates MR cells as the site ofCa2+ uptake in teleost skin. In fresh water, the "chloride"cell may be a "calcium" cell.

In addition to increases in MR cell density, there was aslight increase in MR cell size after adaptation to low envi-ronmental calcium, which was the result of a shift in the sizedistribution of MR cells. Though slight, this increase mayfunctionally relate to the increased Ca2+ transport capacity ofthe opercular membrane. It should be noted, however, thatthis hypertrophy is small relative to the increase in MR cellsize that occurs after adaptation of teleosts to seawater. Forexample, Foskett et al. (39) reported a 60% increase in MRcell diameter within 3 weeks of transfer of tilapia from freshwater to seawater.Net Ca2' uptake in the skin occurred against both an

electrical and ionic gradient, suggesting an active, energy-dependent transport system. Biochemical and vesiculartransport properties of gill tissue suggest that a high-affinityCa2+-ATPase is involved in Ca2+ uptake in this tissue (6-12),although the possibility of other active transport pathwayshas not been ruled out. Although the resent study does notaddress the mechanism(s) ofactive Ca + uptake, the methodsused should prove useful in determining both the mecha-nism(s) and the regulation of Ca2` transport.

We thank H. A. Bern and A. Urano for their interest and encour-agement during this study. S.D.M. was a Research Fellow of theUniversity of Tokyo on leave from the University of California atBerkeley during the conduct of these studies. This study wassupported, in part, by Grants-in-Aid for Scientific Research from the

Ministry of Education and from the Fisheries Agency, Japan, andalso by the National Sea Grant College Program, Department ofCommerce, under Grant NA80AA-D-00120, through the CaliforniaSea Grant College Program, and the California State ResourcesAgency, Project R/F-117.

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