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Am J Physiol Regul Integr Comp Physiol 281: R855-R860, 2001;
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Vol. 281, Issue 3, R855-R860, September 2001

Parathyroid hormone-related protein: a calcium regulatory factor in sea bream (Sparus aurata L.) larvae

Pedro M. Guerreiro1,2,*, Juan Fuentes1,*, Deborah M. Power1, Patricia M. Ingleton3, Gert Flik2, and Adelino V. M. Canario1

1 Centre of Marine Sciences, University of Algarve, Campus de Gambelas, 8000 - 810 Faro, Portugal; 3 Division of Musculo-Skeletal Medicine, Institute of Endocrinology, University of Sheffield, Beech Hill Road, Sheffield S10 2RX, United Kingdom; and 2 Department of Animal Physiology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of an N-terminal peptide (amino acids 1-38) of Fugu parathyroid hormone-related protein (PTHrP 1-38) on calcium regulation of larval sea bream were investigated in seawater (36per thousand ) and after transfer to dilute seawater (12per thousand ). Exposure to PTHrP 1-38 evoked a 1.5-fold increase in calcium influx in both full-strength and dilute seawater. Calcium influx in dilute seawater-adapted larvae was roughly one-half that observed in full-strength seawater controls. PTHrP 1-38 also reduced drinking of fish in seawater but, at all concentrations tested, was without effect in dilute seawater. The amount of water imbibed was 55% lower in dilute seawater than in seawater. PTHrP 1-38 exposure affected the calcium influx route: the main contribution of calcium uptake shifted from intestinal absorption to extraintestinal uptake, probably by the induction of a dose-dependent increase in branchial (active) transport. Moreover, seawater-adapted fish exposed to 1 nM and 10 mM PTHrP 1-38 experienced a 2.5-fold reduction in overall calcium efflux. Overall, the calciotropic action of PTHrP 1-38 resulted in a dose-dependent increase in net calcium balance.

hypercalcemia


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

UNLIKE TERRESTRIAL VERTEBRATES, fish generally live surrounded by a readily available supply of calcium, and organs such as gills, opercular epithelium, skin, and intestine, which are in permanent contact with water, are important surfaces for active transport of Ca2+ (10). Although these iono/osmoregulatory tissues become only fully developed in the adult, the embryos and larvae of several species of both freshwater and marine teleost fish show osmotic and mineral homeostasis independent of ambient salinity and thus must have regulatory mechanisms (2).

The endocrine factors involved in calcium regulation in marine fish are still poorly understood. Pituitary hormones, such as prolactin, growth hormone, and somatolactin (11, 33), and interrenal hormones, such as cortisol (8), have been reported as having a function in Ca2+ homeostasis. Similarly, calcitonin, produced in the gills and ultimobranchial gland, appears to participate in Ca2+ regulation, but its precise physiological role requires clarification (21, 32, 33). Only stanniocalcin, produced by the corpuscles of Stannius, shows marked hypocalcemic actions in fish (31, 33). Hypercalcemic effects have been attributed to an as yet unidentified protein present in fish pituitary extracts (22). In tetrapods, parathyroid hormone (PTH) is the classic hypercalcemic hormone, but PTH-related protein (PTHrP), a factor found in association with tumor cells (20), also has hypercalcemic functions. Their amino acid composition is similar at the N-terminus, and both bind to a common PTH/PTHrP receptor (16).

PTH has not been isolated in fish, but PTH-like immunoreactivity has been described in the pituitary of several teleosts using human antisera (11, 18). PTHrP has recently been demonstrated by radioimmunoassay in the plasma, pituitary, and saccus vasculosus of sea bream (Sparus aurata) using antisera to the human peptide (4, 17). Immunoreactivity was also detected in several tissues of dogfish Scyliorhinus canicula (5), stingray Dasyatis akajei (1), and in the urophysis and corpuscles of Stannius of the euryhaline flounder Platichthys flesus (16).

The recent cloning of the PTHrP gene in the pufferfish Fugu rubripes (24) and the isolation of PTHrP cDNA in sea bream (6) have permitted functional studies of this hormone in fish. Piscine and tetrapod sequences of PTH/PTHrP show the highest homology at the N-terminus, which has been implicated in Ca2+ transport, thus suggesting a conserved role in Ca2+ metabolism (6, 24).

The present study investigated the role of PTHrP in Ca2+ balance in sea bream larvae and shows that the N-terminus PTHrP acts as a potent calciotropic factor. This action is achieved in two different ways: by increasing integumental Ca2+ uptake and by decreasing Ca2+ loss from the fish into the water.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fish. Sea bream larvae 50 days posthatch considered to be in the late larval stage when the majority of the skeleton is calcified and the hypothalamus-hypohyseal system present were used in the experiments. They were obtained from local commercial sources (Algarve, Portugal) and stocked in 200-l rearing tanks in a semiclosed full-strength seawater system (salinity 36per thousand , 1,100 mosmol/kgH2O, temperature 20°C). For experiments in diluted seawater (salinity 12per thousand , 380 mosmol/kgH2O, temperature 20°C), larvae were preadapted for 1 wk before experimental manipulations.

Peptides and chemicals. The N-terminal (1-38 amino acid) fragment of Fugu rubripes PTHrP 1-38 (24) was synthesized by Sigma-Genosys (Pampisford, UK). 51Cr-labeled EDTA (96.2 MBq/µmol) and 45CaCl2 (14.8 MBq/µmol) in aqueous solution were obtained from New England Nuclear Life Science Products (Lisbon, Portugal).

Calcium influx time response. Preliminary experiments tested the time dependence of tracer uptake in sea bream larvae to account for interference of tracer backflux. Fish (20-30 mg) were transferred to 20-ml vessels with aerated seawater and after 30 min, enough 45CaCl2 was added to the water to give an activity of 3.7 KBq/ml; groups of fish were sampled at 2, 4, 6, 8, and 16 h after tracer addition. Water samples were collected, and fish were killed by an excess of 2-phenoxyethanol (0.5% vol/vol; Sigma, Madrid, Spain), rinsed twice with double-distilled water, weighed to the nearest 0.1 mg, and digested overnight with 35% H2O2 (Fluka, Sigma). Seawater Ca2+ content was measured using a colorimetric endpoint assay (Sigma, assay No. 587). 45Ca2+ in water and in digested larval samples was determined by liquid-scintillation counting (model LS6000IC, Beckman Instruments, Fullerton, CA) after addition of OptiPhase HiSafe II liquid-scintillation cocktail (Wallac, Pharmacia, Lisbon, Portugal). Calcium influx rate (IR) was calculated according to the following expression: IR = (AfCw)/(Awtw), where Af is the total 45Ca2+ activity in fish, Cw is the total Ca2+ concentration in water, Aw is total activity in water, t is duration of exposure (h), and w is fish weight (mg). Ca2+ influx rates are expressed as picomoles per kilogram per hour. A linear increase of Ca2+ uptake was found over the 16-h time course studied (data not shown), and the 4-h exposure chosen in all further experiments was considered to reflect initial velocity of Ca2+ influx.

Relationship between size and Ca2+ influx in seawater. To determine the effect of body size on Ca2+ IR, sea bream larvae (5-150 mg wet wt) were placed in 20-ml vessels with seawater and 45CaCl2 (3.7 KBq/ml) was added and processed as earlier described. This experiment showed that fish weighing 30 mg or more had the smallest variation on Ca2+ influx and that no significant differences were found among larvae ranging from 30 to 60 mg. Subsequently, all experiments were carried out with larvae in this size interval.

Effects of PTHrP on whole body calcium influx. Sea bream larvae were placed in 20-ml aerated vessels containing either full-strength seawater (36per thousand , 11 mM Ca2+) or diluted seawater (12per thousand , 4 mM Ca2+). Piscine PTHrP 1-38 dissolved in water was added to the vessels, in which larvae were held, to a final concentration of 0 (control group), 0.1, 1, and 10 nM. After 30-min exposure to peptide, 45CaCl2 was added to the water and the larvae were left to swim freely for 4 h. They were then killed and processed, and Ca2+ influx rates were calculated as described above.

Effects of PTHrP on drinking rate. To calculate intestinal and extraintestinal contributions to whole body Ca2+ uptake, drinking rates were measured using 51Cr-labeled EDTA as a volume marker (12). In brief, larvae were placed in 20-ml vessels and left to settle. PTHrP 1-38 was added to water; 30 min later, 51Cr-labeled EDTA (3.7 KBq/ml) was also added and left to mix by aeration. Four hours later, water samples were collected, and larvae were killed and processed as described earlier. Radioactivity was counted on a Wallac 1470 Wizard gamma counter (Pharmacia). Drinking rates (DR) were calculated as DR = Af /(Awtw), where Af is the total activity of 51Cr-EDTA in the fish, Aw is the tracer-specific activity in water, t is the duration of exposure (h), and w is fish weight (mg). Results are expressed as nanoliter per hour per milligram. The combination of results of whole body Ca2+ influx and the amount of water imbibed (i.e., drinking rate) allowed estimation of the contribution of intestinal and extraintestinal uptake route to whole body Ca2+ uptake.

Effects of PTHrP on whole body Ca2+ efflux. Seawater-adapted larvae were preloaded with 45Ca2+ by leaving them in 3.7 KBq/ml 45CaCl2 for 48 h in a 10-l tank to achieve a constant specific activity of the readily available Ca2+ pool in the larvae. Larvae were then randomly housed in 5-ml vessels and separated into four groups (n = 14-16). PTHrP 1-38 was added to the water to give a final concentration of 0 (control), 0.1, 1, and 10 nM; 0.5-ml water samples were taken after 30 min and hourly thereafter for 4 h. Larvae were then killed and processed as before. Both water and fish samples were measured for 45Ca2+ radioactive decay. Whole body Ca2+ content was measured by colorimetric assay as described before. Calcium efflux rate (ER) was calculated as follows: ER = -(AwCf)/(Aftw), where Aw is the final specific activity in water, Cf is the total Ca2+ in fish (pmol), Af is the total specific activity of 45Ca2+ in the fish, t is time after addition of peptide (h), and w is fish weight (mg). Results are expressed as -picomoles per milligram per hour (the negative sign indicates loss of Ca2+ from the fish).

Statistical analysis. Data were analyzed by one-way ANOVA followed by the Bonferroni multiple-comparison t-test to identify groups that were significantly different from controls. Normality and equal variance of the data were tested using, respectively, the Kolmogorov-Smirnov test and the Levene Median test. The significance level was established as P < 0.05, unless otherwise stated. Results are presented as means ± SE unless otherwise specified.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Relationship between fish size and whole body calcium influx. A highly significant log-log relationship of the form log (calcium influx) = 3.52 - 0.626 log (body wt) was found (r = 0.79, n = 109, P < 0.0001), indicating that whole body calcium uptake (i.e., intestinal + extraintestinal) in sea bream larvae is highly dependent on body size.

Effect of salinity on whole body calcium influx and drinking rate. Sea bream larvae, when transferred from seawater (36per thousand ) to diluted seawater (12per thousand ), showed 100% survival for the duration of the experiments. In larvae kept at reduced salinity for over 1 wk, whole body Ca2+ uptake decreased significantly (P < 0.01) by 46% (see controls in Fig. 1). These larvae also had a significant (P < 0.05) 54% reduction in water intake (i.e., drinking rate) to 14 nl · mg-1 · h-1 compared with 26 nl · mg-1 · h-1 in seawater-adapted larvae (Fig. 2).


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  		Fig. 1.   Whole body calcium influx rate in response to different concentrations of parathyroid hormone-related protein (PTHrP 1-38) measured over a 4-h period in sea bream larvae (30-60 mg) adapted to full-strength seawater (A) and dilute seawater (B). Each group represents the means ± SE of 20-25 fish. *Significant difference from the respective control (P < 0.05, 1-way ANOVA). Note that scale in dilute seawater is one-half that in full-strength seawater.



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  		Fig. 2.   Drinking rates in sea bream larvae (30-60 mg) exposed during a 4-h period to different concentrations of PTHrP 1-38 adapted to either full-strength seawater (A) or dilute seawater (B). Each bar represents the means ± SE of 20-25 fish. *Significant difference from the respective control (P < 0.05, 1-way ANOVA).

Effect of PTHrP on whole body calcium influx. Exposure of sea bream larvae to water-borne PTHrP 1-38 resulted in a pronounced and dose-related increase in whole body Ca2+ influx both in full-strength seawater (Fig. 1A) and in diluted seawater (Fig. 1B). In salt water, whole body Ca2+ influx was 283 ± 33 pmol · mg-1 · h-1 in the control group and 444 ± 35 pmol · mg-1 · h-1 in the group exposed to 10 nM PTHrP, resulting in an overall 1.6-fold increase (Fig. 1A). In larvae adapted to diluted seawater, exposure to the same concentrations of water-borne PTHrP 1-38 evoked an equivalent effect, causing a 1.5-fold increase in whole body Ca2+ influx in the 10 nM PTHrP 1-38 group compared with the control group (Fig. 1B).

Effect of PTHrP on drinking rates. Exposure to water-borne PTHrP 1-38 resulted in a significant decrease in drinking rates of larvae in full-strength seawater (Fig. 2A) with a maximum reduction of 27-35% in larvae exposed to 1 and 10 nM (P < 0.05). In fish adapted to diluted seawater, exposure to water-borne PTHrP 1-38 had no effect on drinking rates (Fig. 2B).

Effects of PTHrP on Ca2+ efflux rate and net flux in seawater. PTHrP 1-38 induced a significant dose-dependent reduction in whole body Ca2+ efflux rates. Total efflux was reduced 1.7-fold in the 0.1-nM PTHrP 1-38 group and 2.5-fold in the 1- and 10-nM groups compared with controls (Fig. 3). The reduction in efflux, combined with a positive and increased influx, resulted in a positive net gain in Ca2+, which, over the duration of the flux experiments (4 h), increased up to sevenfold from the untreated fish to the higher dosage group. Calcium content of larvae after 4 h treatment was not significantly different and was, respectively, 1.63 ± 0.094, 1.73 ± 0.103, 1.79 ± 0.106, and 1.38 ± 0.092 µmol/mg in the 0-, 0.1-, 1-, and 10-nmol PTHrP groups. Failure to detect significant differences in the calcium content of different groups is unsurprising when the sensitivity limits of the calcium assay are considered.


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  		Fig. 3.   Whole body calcium efflux in seawater-adapted larvae (30-60 mg) in response to different concentrations of 1-38 PTHrP measured over a 4-h period. Each group represents the means ± SE of 14-16 fish. *Significant difference from the respective control (P < 0.05, 1-way ANOVA).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study has identified for the first time that the N-terminal region of the recently identified piscine PTHrP (6, 24) is a factor that causes accumulation of calcium in larval sea bream. This calciotropic action was observed both in full-strength (36per thousand ) and in diluted (12per thousand ) seawater and was the result of stimulation of Ca2+ uptake (influx) from the surrounding medium and a reduction in Ca2+ efflux at the same time that drinking rates were maintained (at 12per thousand ) or substantially reduced (at 36per thousand ).

Effect of salinity on drinking and Ca2+ uptake. Previous studies have demonstrated that in juvenile and adult fish, environmental Ca2+ availability greatly affects how fish obtain Ca2+ from the surrounding water. Studies on tilapia larvae (15) and adults raised in fresh water (7) have shown that fish in low-Ca2+ fresh water (<= 0.2 mM) experience a 1.3- to 4-fold increase in Ca2+ influx relative to fish in normal fresh water (0.8 mM). However, when juvenile tilapia were raised in full seawater, Ca2+ influx rates were twice as high as those of tilapia raised in half-strength seawater and nearly three times more than tilapia raised in 25% seawater (30). The results of the present study are in agreement with those observed with tilapia, and sea bream larvae in full-strength seawater show a whole body Ca2+ uptake about twofold higher than that in larvae adapted to dilute seawater (Fig. 1).

There are no previous reports on Ca2+ balance in larvae of marine fish species such as the sea bream. The results from this study are indicative, however, of the existence of alternative strategies for Ca2+ regulation in marine and freshwater environments, as has previously been proposed (9, 29). Ca2+ uptake occurs in fish through intestinal and extraintestinal absorption (i.e., gills and skin), but the relative importance of the two routes in marine fish remains unclear. Whereas in freshwater fish, the gills are probably the main route of Ca2+ uptake (9), marine fish actively drink and take up salts, and a larger role for the intestinal route might be expected. Existing studies show that intestinal absorption accounts for 20% of Ca2+ uptake in tilapia (calculated from Ref. 27) and 30% in Atlantic cod (3, 28).

Adaptation of sea bream larvae to decreased external salinity results in a 1.9-fold reduction in drinking rates, which is in keeping with a potentially less-dehydrating environment. The drinking rates observed for larval sea bream in full seawater (Fig. 2A) are similar to those observed for seawater-adapted 10-day-old tilapia (19). In dilute seawater, the reduction in Ca2+ influx was proportional to the reduction in drinking rates and the amount of imbibed water, indicating a lower contribution of intestinal Ca2+ to total Ca2+ uptake. An important role for the gut in Ca2+ balance in sea bream larvae can be proposed, because subtraction of imbibed Ca2+ from the whole body influx in seawater larvae would result in an unlikely negative net Ca2+ balance (considering the calculated Ca2+ efflux rates; Fig. 3).

The absence in larvae of data on the percentage of the imbibed Ca2+ that is absorbed in the gut does not allow an accurate assessment of the contribution of intestinal Ca2+ uptake to overall Ca2+ balance. However, if the intestine absorbed 70% of imbibed Ca2+ (values from adult southern flounder, see Ref. 14), the intestinal contribution to Ca2+ uptake in sea bream larvae would be 60% in full seawater and 30% in dilute seawater (Fig. 4). The use of small larvae makes it technically difficult to collect blood plasma, the actual compartment from which Ca2+ is lost to the environment (7). As a consequence, our calculations of efflux based on whole body tracer specific activity (rather than plasma specific activity) may give an overestimation of efflux when significant non- or slow-exchanging calcium pools develop in the fish. Therefore, results presented here are for whole body Ca2+ efflux and cannot be considered as direct estimates of integumental (i.e., skin and branchial) exchanges.


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  		Fig. 4.   Intestinal (filled bars) and extraintestinal (open bars) contributions to whole body calcium influx. Calculations were performed by combination of the data shown in Figs. 1 and 2. *Significant difference from the respective control (P < 0.05, 1-way ANOVA). Note that scale in dilute seawater (B) is one-half that in seawater (A).

Effect of PTHrP on drinking and Ca2+ balance. The biological activity of PTHrP has largely been characterized in mammals and birds, in which it acts as a multifunctional factor involved in Ca2+ transport, development, and maintenance of muscle tone (16, 23). In aquatic vertebrates, the biological activity of this hormone is unknown, although injections of high doses of heterologous hormone (human PTHrP 1-34) caused a reduction in whole body Ca2+ uptake (13) in a manner similar to that observed in freshwater-adapted tilapia exposed to bovine PTH 1-34 (34). However, the results of experiments with heterologous hormones should be carefully interpreted because the identity between the amino acid sequence of piscine and human N-terminal peptide is only 57% (6, 24). Recently, several zebrafish, Danio rerio, PTHrP/PTH receptors with differing affinity for human PTH and PTHrP peptides have been cloned (26). However, the peptide used in binding and transactivation studies (25, 26) was based on an incomplete 1-32 amino acid sequence of Fugu rubripes PTHrP, and the results should be also interpreted with caution.

The results from the present study suggest that PTHrP is a hypercalcemic factor in fish larvae. Exposure to 1 and 10 nM piscine PTHrP 1-38 significantly reduced drinking rates by 30% in sea bream larvae maintained in seawater (Fig. 2A) but not in dilute seawater (Fig. 2B). Furthermore, PTHrP 1-38 caused a significant dose-dependent increase in Ca2+ influx of up to 57% (10 nM) and 46% (1 nM) in seawater and diluted seawater, respectively. Moreover, in seawater, a large (up to 60% at 1 and 10 nM) and significant decrease in efflux was also observed. These effects suggest that in marine larvae, PTHrP may cause a shift in the way Ca2+ is taken up by the fish, because, despite a reduction in drinking rates, whole body Ca2+ uptake increased in response to increased concentrations of PTHrP 1-38, indicating a dose-dependent increment in branchial uptake over a reduced or unaltered intestinal contribution (Fig. 4). The observed reduction in drinking caused by PTHrP (20-25%, Fig. 2) would also reduce hormone uptake if the major route of peptide uptake is via the gut. However, at the range of peptide concentrations used in the experiments (1, 10, and 100 nM), a differential peptide dose would still be achieved, explaining the different dose effects observed. The overall result, therefore, of administering PTHrP 1-38 was a positive net gain in Ca2+ of up to sevenfold, generating a large pool of internal calcium in the larvae.

The sites at which PTHrP 1-38 acts to bring about the changes in Ca2+ described in this paper remain to be identified. However, a preliminary study to confirm the uptake of peptide from the water by juvenile sea bream using 125I-labeled PTHrP 1-38 or cold peptide (measured by RIA) indicates that 1-2% of administered peptide appears in the blood. This corresponds to estimated plasma values of 12, 30, and 200 pM at the dosage used (1, 10, and 100 nM), which fall within the range of plasma concentrations previously reported in sea bream for PTHrP (4). The distribution of abundant PTHrP mRNA expression in Ca2+-transporting tissues in sea bream larvae [epithelial cells in the gill region, ionocytes ("chloride cells"), the basal region of the hindgut epithelium, and in some but not all kidney tubules (6)] suggests they may be a direct target for PTHrP.

The reduction in drinking rates caused by PTHrP 1-38 is suggestive of a compensatory mechanism for excessive salt load (Ca2+ and/or other ions), which is particularly relevant in the full seawater group. Interestingly, PTHrP administration stimulates arginine-vasopressin secretion in rats, with resultant antidiuretic and pressor effects (35), and it is possible that this or a similar effect on water-/ion-regulating factors (i.e., the renin-angiotensin system) may be mediating this response in the sea bream.

Perspectives

In tetrapods, PTHrP and PTH have similar hypercalcemic effects that are explained by their binding to a common receptor (16, 23). The recent identification of PTHrP (6, 24), together with the failure to identify PTH in fish, raises intriguing questions about the evolution of calcium-regulating systems. The lack of PTH and the fact that fish generally inhabit water that contains a readily available pool of calcium have led to the generally accepted view that calcium regulation in fish is primarily under the control of hormones that downregulate, such as stanniocalcin. Our finding that PTHrP has a stimulatory action on calcium uptake, reducing simultaneously calcium efflux, is suggestive that it acts as a hypercalcemic factor in fishes. We hypothesize that calcium homeostasis in fish is achieved by the interplay of stanniocalcin and PTHrP with hormones, such as prolactin and cortisol (33), modulating this activity during specific life history events (e.g., salmon entering fresh water). It remains to be determined whether in fish, in common with tetrapods, PTHrP can also evoke the mobilization of internal calcium stores or whether it instead stimulates calcium uptake primarily from the environment. In sea bream larvae, despite PTHrP causing a reduction in drinking, calcium uptake increased, indicating that the site of action is extraintestinal. We hypothesize that in teleost fish, PTHrP acts on specific receptors at the level of the gill chloride cells and, to some extent, in the intestinal epithelium to enhance the uptake of calcium, probably via the Ca2+-ATPase and/or Na+/Ca2+ exchanger.


    ACKNOWLEDGEMENTS

The authors thank V. Vilanova, Timar, and Instituto das Pescas e do Mar for supplying the larvae and H. Viegas for assistance in rearing and maintenance.


    FOOTNOTES

* P. M. Guerreiro and J. Fuentes contributed equally to this work.

This research was supported by the Commission of the European Union, Agriculture and Fisheries specific Research and Technological Development and Demonstration program, CT96-1742. P. M. Guerreiro and J. Fuentes are funded by PRAXIS XXI BD/9207/96 and BPD/22033/99.

Address for reprint requests and other correspondence: A. V. M. Canario, Centre of Marine Sciences, Univ. of Algarve, Campus de Gambelas, 8000-810 Faro, Portugal (E-mail: acanario{at}ualg.pt).

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

Received 10 July 2000; accepted in final form 1 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Regul Integr Comp Physiol 281(3):R855-R860
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