M. Fouchereau-Peron

Distribution of calcitonin gene-related peptide and calcitonin-like immunoreactivity in trout.

Radioimmunoassay and chromatography were used to study the occurrence of calcitonin gene-related peptide in various tissues of the rainbow trout, Salmo gairdnerii. The highest concentrations of the peptide were found in gill (1.68 +/- 0.09 ng/mg protein) and in intestine (1.06 +/- 0.4 ng/mg protein). Significant concentrations were also found in heart and stomach. The level in brain was very low. In trout, the plasma concentration accounted for 283 +/- 82 pg/ml. Chromatographic analysis of the calcitonin gene-related peptide (CGRP)-like immunoreactivity occurring in gills showed that two molecular forms cross-reacted with the anti-human CGRP antibody, one co-eluting with the synthetic human CGRP. In addition, calcitonin in fish is not confined to the ultimobranchial organ but is also present in organs as heart, intestine, kidney, spleen and stomach. The evidence of CGRP in fish emphasizes the role of this hormone in evolution and leads us to investigate its physiological role in this species.

Characterization of the vasoactive intestinal polypeptide (VIP) in the gut of fishes

Abstract 1. 1. The presence of VIP was investigated in the dogfish, Scyliorhinus canicula, the ballanwrasse. Lubrus berggylta and the bib. Trisopterus luscus, using a specific radioreceptorassay. 2. 2. Pure porcine VIP and gut extracts of fishes yielded similar dilution curves. 3. 3. In the dogfish, the highest concentration of VIP was found in the hindgut. In contrast, in the two teleostei studied, the highest levels of VIP were in the first part of the gut. 4. 4. The biologically active VIP measured by radioreceptorassay correlated well with the molecule determined using a specific radioimmunoassay. 5. 5. Our results support the hypothesis of the appearance of VIP early in evolution.

Characterization of target organs for calcitonin in lower and higher vertebrates.

The bindings of calcitonin was investigated in trout bone, kidney and gill and rat bone and kidney. Specific binding of calcitonin was observed in all tissues tested except fish kidney membranes. The affinity constants for the sites of high affinity-low capacity (in trout bone and rat kidney) or for the unique site (in trout gill and rat bone) were of the same order of magnitude (2.0-9.0 x 10(9) M-1), the number of binding sites per mg of protein being higher in rat bone homogenates than in other tissues. These studies strongly support the theory that the gill in fishes is likely to perform some of the functions of the kidney in mammals.

Binding sites of calcitonin gene related peptide (CGRP) to trout tissues

We localized specific binding sites for human calcitonin gene related peptide (hCGRP) in different organs of the trout using labelled human CGRP. Maximal binding was observed in gill and spleen membranes. The binding of 125I-hCGRP was time and temperature dependent. Scatchard analysis of binding data for the spleen and the gills disclosed two binding sites. The constants for the site of high affinity and low capacity (KAM-1 and Bmax (fmol/mg of proteins] were 2.9 x 10(9) for the spleen and 70 and 3.5 x 10(9) for the gill. Salmon calcitonin (sCT) inhibited the binding of 125I-hCGRP to spleen membranes with the same order of potency as hCGRP. In contrast sCT was less effective than hCGRP in suppressing the specific binding of 125I-hCGRP to gill membranes.

Immunoreactive salmon calcitonin-like molecule in crustaceans: High concentrations in Nephrops norvegicus

A salmon calcitonin (CT)-like peptide was characterized in various crustaceans by radioimmunological and radioreceptor assays. The highest levels of the molecule were found in the anterior part of the gut and the hepatopancreas of the Norway lobster: Nephrops norvegicus. Molecular sieving of this molecule suggested an apparent molecular weight of 4500 Da, that is higher than that of calcitonin. This peptide can be referred to as crustacean calcitonin as it inhibits the binding of labeled salmon CT to rat kidney membranes. The high concentration found in crustaceans suggested that this molecule could have an important role in this class of arthropods.

Adaptation of rainbow trout (Salmo gairdnerii) to sea water: Changes in calcitonin levels

1. 1. The calcitonin content of the ultimobranchial body (UBB) and plasma levels of calcitonin, calcium and phosphate were measured in rainbow trout (Salmo gairdnerii) following their transfer from fresh to sea water. 2. 2. The plasma calcium level remained unchanged throughout the experiment while the UBB calcitonin content, plasma calcitonin and plasma phosphate rose significantly during the hours immediately following transfer. 3. 3. The levels of all three subsequently fall so that, 8–15 days later, a new equilibrium was established with lower than control (fresh water) levels of UBB calcitonin, plasma calcitonin and plasma phosphate. 4. 4. It would appear, from these data, that calcitonin plays some part in the endocrine regulation of sea water transfer.

Calcitonin-like peptide in the shrimpPalaemon serratus (Crustacea, Decapoda) during the intermolt cycle

A molecule immunologically related to salmon calcitonin has been detected in the hemolymph of the shrimpPalaemon serratus. Its concentration varies inversely with the calcium level during the molt cycle; a maximum (14 ng/ml) is found in the post-molt stage and a minimum (0.5 ng/ml) during the premolt stage.

Calcitonin induces hypercalcemia in grey mullet and immature freshwater and sea-water adapted rainbow trout

1. Changes in plasma calcium levels, in response to salmon calcitonin injections, were studied in freshwater and sea-water adapted trout (Salmo gairdnerii) and in grey mullet (Chelon labrosus). 2. Low doses (0.1 ng sCT/100 g body weight) elicited hypercalcemia in the two species studied. 3. High doses (0.5 microgram) provoked hypocalcemia only in freshwater and sea-water adapted trout. 4. An hypercalcemic response appears as the primordial effects of CT injections, higher doses of CT leading to hypocalcemic effects.

Variations of calcitonin-like immunoreactivity in the crustacean Orchestia cavimana during a molt cycle

The investigation of calcitonin-like immunoreactivity performed in whole extracts of the terrestrial crustacean Orchestia cavimana using radioimmunoassay revealed large amounts of these peptides varying in concentration according to the stages of the molt cycle. Their level is maximum at the time of the exuviation. The results are then discussed regarding the particular calcium metabolism of this species in function of its terrestrial conditions of life.

CALCITONIN GENE-RELATED PEPTIDE STIMULATES ADENYLATE CYCLASE ACTIVITY IN TROUT GILL CELL MEMBRANES

The physiological significance of calcitonin gene-related peptide (CGRP) was investigated by assessing the CGRP stimulated adenylate cyclase activity in various tissues of trout. The highest enzyme concentration was found in gill and stomach membranes. The maximal activity (190% of the basal value) was observed for a concentration of 53.3 nM CGRP I or II. In the presence of 58 nM sCT, the maximal enzyme activity represented 120% of the basal value. No additive effect was observed; this suggests that both CGRP and sCT activities are mediated through the same receptor. The present data are in favour of a role for this neuropeptide operating in branchial cell functions such as calcium transfer from the external to the internal milieu.