Characterisation and distribution of calcitonin gene-related peptide in a primitive teleost, the eel, Anguilla anguilla and comparison with calcitonin.

Radioimmunoassay (RIA), radioreceptor assay and chromatography were used to study the occurrence of calcitonin gene-related peptide (CGRP) in a primitive teleost, the eel, Anguilla anguilla. Immunologically and biologically active CGRP-like molecules were found in brain, heart, kidney, liver, spleen and ultimobranchial body with the higher concentrations in brain, spleen and heart. Gel exclusion chromatography of heart and spleen extracts followed by SDS-PAGE showed that the eel CGRP-like molecules presented a molecular weight between 3.30 and 3.95 kDa similar to that of human CGRP. The wide distribution of CGRP reflects its multiple role as brain neuromediator and peripheral paracrine effector as described in mammals. In comparison, the distribution of calcitonin (CT) was much more restricted, immunologically and biologically active CT-like molecules being localised in the ultimobranchial bodies (UBB) that is the site of CT synthesis in non-mammalian vertebrates. In plasma, CGRP-like concentrations were 10 to 100 higher than those of CT. These high concentrations in a primitive teleost strengthen the possible endocrine role of CGRP in early vertebrates and emphasise the important role of this hormone in evolution.

Biomineralisation markers during a phase of active growth in Pinctada margaritifera.

To search for the biochemical parameters involved in calcium and carbonate transport during crystal formation and biomineralisation in nacreous molluscs, the carbonic anhydrase activity, the levels of calciotropic hormones in hemolymph and in tissues and the circulating concentration of calcium were measured in pearl oysters (Pinctada margaritifera) during a phase of active growth. Activity of carbonic anhydrase in gill tissue increased linearly with age of the animals, while no age variation in activity was noted for the mantle. The circulating level of total calcium increased during the growth of the animals. Calciotropic hormones were radioimmunoassayed in gill, mantle and hemolymph. Only a calcitonin-gene related peptide (CGRP) could be detected and its concentration decreased as a function of growth, both in hemolymph and mantle. No variation in CGRP concentration with age was observed in gill tissue. Our data demonstrate that carbonic anhydrase and a molecule biologically and immunologically related to CGRP are involved during growth of the animals. In addition, this study shows the presence of three main calcium compartments, gill, hemolymph and mantle, involved in the biomineralisation process.

TEM-89 beta-lactamase produced by a Proteus mirabilis clinical isolate: new complex mutant (CMT 3) with mutations in both TEM-59 (IRT-17) and TEM-3.

TEM-89 (CMT-3) is the first complex mutant beta-lactamase produced by a clinical strain of Proteus mirabilis (strain Pm 631). This new enzyme, which has a pI of 6.28, is derived from TEM-3 and has a single amino acid substitution also encountered in TEM-59 (inhibitor-resistant TEM beta-lactamase IRT-17): Ser-130 to Gly. TEM-89 hydrolyzed penicillins to the same extent that TEM-3 did but lost almost all hydrolytic activity for cephalosporins and, like TEM-59, was highly resistant to inhibitors.

Effect of calcitonin gene related peptide on the adenylate cyclase activity in abalone gill membranes.

The aim of the present study was to investigate and to compare the effect of calciotropic hormones, human calcitonin gene related peptide (CGRP) I and II, salmon calcitonin (CT) and human amylin on the adenylate cyclase activity in abalone gill membranes. In addition to human CGRPI, human CGRPII and salmon CT stimulated the adenylate cyclase activity. No effect was observed with amylin. The higher effect was observed with human CGRPI and II that induced a 160-170% increase of the enzyme activity. Fifty percent of the maximal activity was observed with 3 and 8 nM of CGRP I and II, respectively. Salmon CT induced a lower effect: the maximal activity was obtained with a hormone concentration of 266 nM and represented a 130% stimulation of the basal activity. In the presence of CGRP 8-37, a competitive antagonist of CGRP action, the stimulation observed with CGRPI was abolished and returned to the basal level. This study points out that, in invertebrates, CGRP receptors present in gill membranes are linked to an adenylate cyclase system similar to that described in vertebrates. In addition, these data are in favour of a role for CGRP in branchial function both in non-mammalian vertebrates and invertebrates

Interaction of cysteine proteases with calciotropic hormone receptors.

The effect of two cysteine proteases: papain and a cathepsin L-like enzyme purified from the oesophagus of Nephrops norvegicus (NCP) was studied on the specific binding of calcitonin (CT) and calcitonin gene related peptide (CGRP) to rat kidney and liver membranes, respectively. In addition, the response of adenylyl cyclase to increasing concentrations of these two enzymes was investigated. Each protease inhibited the initial CGRP and CT binding to rat liver and kidney membranes, respectively, in a manner not significantly different from that obtained in the presence of the unlabeled standard. The adenylyl cyclase activity in rat liver membranes was increased by the addition of each enzyme. The response was higher with papain that induced a fivefold increase of enzyme activity at a 4-microg/ml enzyme concentration. In rat kidney membranes, the magnitude of the response was identical with both enzymes. In contrast with NCP, papain induced a biphasic response. Leupeptin and E(64), two specific inhibitors of cysteine proteases, reversed the observed effects. Trypsin induced an inhibition of the liver membrane adenylyl cyclase activity and an activation in rat kidney membranes at low protease concentration. Thus, cysteine proteases are able to act, in vitro, at the receptor level in target organs specific for calciotropic hormones.

Characterization of binding sites for calcitonin gene related peptide in abalone gill.

Target organs for calcitonin gene related peptide were investigated in the abalone. To elucidate the function of this neuropeptide in the biomineralization process, we have localized, in different tissues from abalone, specific binding sites for human calcitonin gene related peptide (hCGRP). Highest binding was observed in gill membranes where two classes of affinity components were identified. The affinity constants and the number of binding sites per mg of proteins for the site I were 5 X 10(9)M(-1)and 8.8 x 10(10). For the site II, the affinity constant was 1.34 X 10(7) M(-1)and the number of binding sites per mg of proteins was 1.1 x 10(12). In contrast, no specific calcitonin binding could be detected in every tested tissue, but the similar displacement of the 125I-labeled CGRP binding with unlabeled hCGRP and sCT suggested that, in the abalone, the identified receptor could belong to a third class receptor subtype, that recognize both hCGRPalpha and sCT. These data suggest that, during evolution, the role of CGRP in gill function is particularly well conserved and that this neuropeptide is likely to participate in the control of hydromineral metabolism in aquatic species.

Isolation of an acid fraction from a fish protein hydrolysate with a calcitonin-gene-related-peptide-like biological activity.

The possibility of obtaining calcitonin and/or calcitonin-gene-related peptide (CGRP) immunorelated molecules from partly digested proteins was investigated with fish and shrimp hydrolysates. These two peptides were quantified by both radioimmunoassay and radioreceptor assay; the positive extracts were partly purified. Different hydrolysates were analysed: cod head, stomach and viscera hydrolysates, a shrimp hydrolysate and two sardine hydrolysates. Although each cod extract interacted in the CGRP radioimmunoassay, none of these extracts was able to displace the CT binding to its antibody. In contrast, shrimp and sardine hydrolysates interacted with both radioimmunoassays. Radioreceptor assays performed on the same extracts demonstrated that only three extracts contained the structural determinants that allowed them to interact in the CGRP radioreceptor assay. No interaction with the calcitonin radioreceptor assay could be demonstrated. Molecular sieving of the two sardine extracts showed that the immunoreactivity was resolved into two main fractions. The higher-molecular-mass fraction interacted only in the CGRP radioreceptor assay. The results obtained suggest the presence of a biologically related CGRP molecule in peptone hydrolysates and requires further investigation into the role of these peptide fragments in the regulation of intestinal function by partly digested proteins.

CGRP-like molecules and carbonic anhydrase activity during the growth of Pecten maximus.

In order to investigate the physiological role of calcitonin gene-related peptide (CGRP) in mollusc, both circulating CGRP-related molecules and gill or mantle carbonic anhydrase activity were analysed during the annual growth of Pecten maximus. CGRP like molecules measured by radioreceptor assay increased significantly during the annual cycle. Similarly, gill carbonic anhydrase activity increased and showed a maximum activity when growth is stimulated to the greatest extent. Correlation studies showed a significant relationship between the tissue weight and either the gill carbonic anhydrase activity or the CGRP-related molecules determined by radioreceptorassay. This observation suggests a possible interaction between carbonic anhydrase activity and CGRP. Accordingly, we searched for a direct effect of CGRP on the gill carbonic anhydrase activity. In gill membranes, CGRP stimulated the carbonic anhydrase activity. The maximum effect was obtained at a CGRP concentration of 50 nM.

Characterization of a calcitonin gene related peptide-like molecule in the abalone, Haliotis tuberculata.

Immunoreactive related CGRP molecules (ir-CGRP) were identified in the abalone, Haliotis tuberculata, mainly in mantle and cephalic part extracts. Ir-CGRP in both tissues accounted for 461 and 455.6 pg per mg of proteins, respectively. These CGRP-immunoreactive molecules were further analyzed for their ability to interact with the CGRP radioreceptor assay. In specific target tissues for CGRP (rat liver membranes), 50% inhibition of 125I-labeled CGRP specific binding was observed with 4.7 micrograms and 21.1 micrograms of proteins from mantle and cephalic part extract, respectively. These molecules were submitted to gel-filtration chromatography on a Sephacryl S-100 column and were further analyzed in the radioreceptor assay specific for CGRP. The elution position of these molecules suggested a molecular weight close to that of synthetic salmon calcitonin.

A lobster cysteine protease with immunoreactivity and activities of calcitonin and CGRP.

The high concentrations of molecules immunologically related to salmon calcitonin (CT) and/or to human calcitonin gene-related peptide (CGRP) in the oesophagus of the norway lobster Nephrops norvegicus have been examined. In the present study. We report the purification of these molecules by means of a specific radioimmunoassay for calcitonin and calcitonin gene related peptide. The immunoreactive molecules were tested for their functional similarities with CT and CGRP. This was investigated by measuring their ability to interact with CGRP and CT radioreceptor assays and to stimulate the adenylate cyclase activity in rat liver and kidney membranes, respectively. In addition, the purified product was injected in young rats in order to check for a CT-like biological activity of these molecules. The combination of these tests led us to purify a molecular form of 33 kDa. N-terminal sequence analysis of this protein revealed a considerable homology with the lobster cysteine proteases and the human cathepsin L. Control experiments performed with the highly purified American lobster cysteine protease I showed that crustacean cysteine proteases given in vivo to rats induce a fall in the plasma calcium and phosphate levels. This study therefore adds further documentation for a common ancestral origin of CT, CGRP and the much large cysteine proteases from invertebrates.

Characterization of binding sites for calcitonin gene-related peptide in the mollusc gill.

Target organs for calcitonin gene-related peptide (CGRP) were investigated in Pecten maximus using 125I-labelled human CGRP. CGRP was shown to interact specifically with mantle and gill tissue. Receptor studies using branchial membrane preparations indicated that the binding was time dependent. Scatchard analysis of binding data showed that there was a single class of binding sites. The affinity constant was found to be 0.7.10(8) M-1 and the number of binding sites 2600.10(8)/mg of protein. Salmon CT inhibited the binding of 125I-labelled CGRP to branchial membranes with a lesser efficiency than that of the unlabelled hormone. A 40% inhibition of the 125I-labelled CGRP binding was observed in the presence of 2.6 and 26 nM CGRP and salmon CT, respectively. In addition, 200 nM human CGRP inhibited 25 and 10% of the basal branchial and mantle adenylate cyclase activity, respectively. These data suggest that CGRP participates in the regulation of the branchial function in molluscs probably via a vasoconstrictor role.

Calcitonin gene-related peptide stimulates carbonic anhydrase activity in trout gill membranes.

The effect of human calcitonin gene-related peptide (hCGRP) on carbonic anhydrase activity in trout branchial membranes was investigated. hCGRP stimulated the carbonic anhydrase activity, 5-fold over the basal value. This effect was specific: in the presence of sCT only a 2.4-fold increase was observed. Rat amylin that has a 43% homology with hCGRP had no effect on carbonic anhydrase activity. On the other hand hCGRP(8-37), which is a partial antagonist, inhibited the hCGRP-stimulated carbonic anhydrase activity. This effect was observed at low hormonal concentration and suggests the involvement of hCGRP in regulating carbonic anhydrase activity in this important target organ for CO2 exchange.

Mode of action of calcitonin-gene related peptide in trout gill membranes: evidence for a GTP coupling process.

To evaluate the functional relationship between the calcitonin-gene related peptide (CGRP) receptor in trout gills and guanine nucleotide-binding proteins, we investigated the effect of GTP not only on the CGRP stimulated adenylate cyclase activity but also on the human CGRP binding to trout gill membranes. In the presence of 1 microM GTP, the basal and the CGRP stimulated adenylate cyclase activity were increased by 1.8-fold. In addition, GTP decreased the CGRP binding to gill membranes and accelerated the dissociation of bound labeled hormone. Scatchard analysis of the data revealed that the reduction of human CGRP binding by GTP was mainly due to a decrease in the binding affinity with no significant change in the binding capacity. Thus, the binding of CGRP to fish gill membranes activates adenylate cyclase via a guanine nucleotide dependent mechanism, suggesting the involvement of a guanine nucleotide-binding stimulatory protein in the action of CGRP in fishes.

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.

Calcitonin gene-related peptide immunoreactivity in a crustacean Nephrops norvegicus and correlation with calcitonin.

Immunoreactive calcitonin-gene-related peptide (ir-CGRP) was detected in the crustacean Nephrops norvegicus. High levels of ir-CGRP were present in the foregut and hepatopancreas (3 +/- 0.7 and 4.6 +/- 1.0 micrograms eq per 100 mg of fresh organ, respectively). Molecular sieving of acidic extracts of anterior gut of Nephrops norvegicus showed a high molecular weight immunoreactive peptide in the range 15,000 to 25,000 Da. Immunoreactivity related to salmon calcitonin was present in the high molecular weight fraction.

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.

Calcitonin variations in male and female trout, Salmo gairdneri, during the annual cycle.

Calcitonin (CT) levels in the ultimobranchial body and in plasma were radioimmunoassayed in rainbow trout, Salmo gairdneri, as a function of the annual cycle. In male and female, there was an important increase in the CT levels in the ultimobranchial body and in plasma. These variations in CT levels in both male and female suggest that sexual maturity influences the synthesis and the secretion of calcitonin in fishes. A positive correlation was observed between plasma and ultimobranchial CT levels and the gonadosomatic index in both sexes, suggesting that CT has a role in the processes involved in gonadal development.

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.

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.

Partial characterization and amino acid composition of a high molecular weight peptide with salmon calcitonin immunoreactivity in a crustacean: Nephrops norvegicus.

Anti-salmon calcitonin antibodies were used to follow the purification of a high molecular weight peptide present both in the haemolymph and in the hepatopancreas of the Norway lobster Nephrops norvegicus. An apparent molecular weight of 22 kDa has been measured in electrophoresis on SDS gels and amino acid composition compared to salmon calcitonin. The amount determined by the immunoreactivity assay corresponds to about 1/40 and 1/140 of that which is based on direct protein measurement for the hepatopancreas and the haemolymph respectively. The total amount of this peptide could be estimated as 3.5 mg/g fresh weight for the hepatopancreas and 140 ug/ml for the haemolymph. The function of this peptide is still unknown.