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Binding studies for rat amylin (AMY) and salmon calcitonin (sCT) were performed on rat membranes prepared from pons and medulla oblongata of rats. The aim was to see whether specific binding sites for AMY and/or for sCT present in these areas could be relevant to some of the biological activities of the two peptides. Binding sites specific for [125I]AMY are present in the pons-medulla of rat brain as AMY, but not sCT, was able to displace radiolabeled AMY binding with an IC50 = 3.7+/-0.5x10(-10) M. In contrast, binding of [125I]sCT was displaced by both sCT and AMY, although with different potencies, the IC50 for sCT being 1+/-0.1x10(-11) M, and for AMY, 1.8+/-0.08x10(-7) M. The functional significance of the presence of these binding sites was evaluated in two different nociceptive tests, hot-plate and tail-flick. In the tail-flick test neither AMY (5-10 microg/rat, i.c.v.) nor sCT (10 microg/rat i.c.v.) showed antinociceptive activity, whereas in the hot-plate test AMY (10 microg/rat, i.c.v.) significantly increased the response latencies as did sCT (250 ng/rat, i.c.v.). These results demonstrated that a 40-fold greater dose of AMY is necessary to produce a comparable antinociceptive effect to that exerted by sCT. These findings are in accordance with the low affinity of AMY for sCT binding sites in rat pons-medulla. It is therefore suggested that the central inhibitory activity of AMY on pain perception involves interaction with sCT receptors whereas the selective AMY binding sites subserve other (as yet unknown) functions.

In this study we examined the possible interplay of amylin (AMY) and salmon calcitonin (sCT) in the central control of growth hormone (GH) and prolactin (PRL) secretion in male rats. For this purpose we first compared effects of central intracerebroventricular (i.c.v.) admininstration of various doses of AMY (2.5-2,500 ng/rat) and sCT (2.2-220 ng/rat) on beta-endorphin (beta-END, 0.5 microg/rat)-induced GH and PRL secretion. AMY and sCT dose-dependently inhibited beta-END-induced GH secretion, whereas only sCT was able to inhibit beta-END-induced PRL secretion. To examine whether the GH inhibitory effect of AMY was due to the possible cross-reactivity of AMY and sCT on the same receptors in the CNS, we pretreated some rats with the AMY antagonist (AMY8-37, 2. 5 microg/rat, i.c.v.). AMY8-37 significantly enhanced the GH-stimulatory action of beta-END. AMY8-37, administered prior to AMY and sCT, significantly removed the inhibitory effect of both AMY and sCT on beta-END-induced GH release, suggesting that both peptides mediate their response on GH through a common receptor. In vitro competition binding studies on rat hypothalamic membranes have shown that both AMY and sCT compete with [125I]rAMY binding with half inhibition (IC50) values of 3.6 x 10(-11) and 1.6 x 10(-10) M, respectively. Binding of [125I]sCT was inhibited by sCT with an IC50 of 1.09 x 10(-10) M and to a lesser extent by AMY with an IC50 of 1. 3 x 10(-6) M. Thus it is possible that the two peptides recognize a common hypothalamic receptor but with different affinities (sCT > AMY). Overall these data indicate that AMY behaves as a mimic of sCT in the central control of GH secretion. The failure of AMY, at variance with sCT, to modify the PRL-releasing activity of beta-END indicates that different receptor subtypes for sCT are involved in the endocrine effects of sCT and only those mediating the modulatory action of GH respond to AMY.

In order to investigate the effects of ageing on the cerebral receptors for calcitonin (CT), we used an in vitro autoradiographic method to study the distribution of the binding sites for eel CT (eCT) in young and old rat brain. The inhibitory action of eCT on adenylyl-cyclase (AC) activity upon isolated brain cell membranes was also evaluated. The results show area-specific reduction of binding particularly in the hypothalamus and pons medulla of the old rat. The inhibitory action of eCT on AC activity was significantly reduced in the same areas, whereas in the striatum and mesencephalon no changes were observed. The parallel decrease of binding of eCT and of the inhibitory action of eCT on AC in ageing may represent a functional decline of neuronal activities during ageing.

This study examined the possible peripheral activity of eel calcitonin in the modulation of the response to noxious pressure on inflamed paws in rats (Randall and Selitto test). The intraplantar injection of eel calcitonin (20-200 ng/rat) but not the subcutaneous administration (200 ng and 2 micrograms/rat, s.c.), was able to significantly inhibit hyperalgesia induced by intraplantar injection of carrageenin. The development of oedema on the other hand was not inhibited. The intraplantar administration of eel calcitonin (200 ng/rat) in a non-inflamed paw did not modify paw pressure thresholds. Eel calcitonin (200 ng/rat, intraplantar, i.pl.) was also able to elicit an antinociceptive effect on formalin-induced hyperalgesia, both when the peptide was injected before or after (60 min) formalin. This effect, at difference with morphine (80 micrograms/rat, i.pl.), was not blocked by naloxone (10 micrograms/rat, i.pl.). These results demonstrate the local antinociceptive effect of eel calcitonin in inflammatory pain and might indicate a new way of using calcitonin in the control of pain.

To determine whether or not the CNS inhibitory activity of eel calcitonin (eCT) on adenylyl cyclase is the endocellular mechanism underlying the antinociceptive effect of the peptide, as shown for morphine analgesia, we administered Bordetella pertussis toxin (PTX) by intracerebroventricular (ICV) injection (0.5 microgram/rat) to block the receptor-mediated inhibition of adenylyl cyclase. In PTX-treated rats there was no change in eCT (2.5 micrograms/rat, ICV)-induced antinociceptive activity (hot-plate test) nor in eCT (100 ng/rat, ICV) inhibition of gastric acid secretion (Shay test) whereas morphine (5 micrograms/rat, ICV) analgesia was significantly reduced. In vitro studies showed no reduction of eCT binding in the CNS of rats treated with PTX in vivo. Moreover, PTX treatment did not change the inhibitory effect of eCT on adenylyl cyclase in isolated membranes from rat striatum in contrast with opiates (DAME and morphine) whose effects were lost. As PTX is known to inactivate the guanidine binding inhibitory protein Gi, these data suggest that a G protein, distinct from the Gi protein involved in the coupling of opiate receptors into a functional response, could be responsible for regulating the intracellular pathways resulting in eCT-induced antinociceptive effect and inhibition of gastric acid secretion.

A new peptide, eel calcitonin (eCT), synthesized to the sequence of calcitonin (CT) extracted from the ultimo branchial bodies of eels, is now on the market in some countries for clinical use in the treatment of Paget's disease of bone and in the prophylaxis or treatment of certain osteoporoses. We have now studied the effect of eCT on the inhibition of gastric acid secretion and protection against experimentally induced gastric ulcers in rats as other CTs have previously been shown to have such effects. EelCT shows a dose dependent inhibition of gastric acid secretion, both volume and concentration of acid - dose range 100-900 ng/Kg injected subcutaneously. Central administration is also effective at a dose of 100 ng/rat. There is no published evidence for a direct effect of CTs in the stomach and there is considerable speculation on possible mediating pathways. Somatostatin may be involved in the inhibitory effect of eCT on gastric acid secretion because when administered both centrally or peripherally eCT is ineffective in rats depleted of somatostatin by pretreatment with cysteamine. However, other mechanisms must also be involved as eCT has no preventive effect against gastric ulcers induced by ethanol but has a high index of protection against ulcers induced by cold restraint stress or indomethacin.

We investigated the effects of /ASU1-7/eel calcitonin (ASU1-7eelCT) on basal and stimulated prolactin (PRL) release in male rats. /ASU1-7/eelCT was administered intracerebroventricularly (icv) into freely moving rats with indwelling catheters. The administration of /ASU1-7/eelCT (2.5 micrograms/rat, icv) significantly inhibited basal PRL secretion. When PRL secretion was stimulated by exposing rats to restraint stress, /ASU1-7/eelCT (250 ng; 800 ng; 2.5 micrograms/rat, icv) dose-relatedly inhibited the PRL surges at 10 min after stress. The same doses of icv /ASU1-7/eelCT were effective in inhibiting morphine (6 mg/kg, intracarotid, ia-induced PRL release. No effect on stress-induced PRL secretion was observed when the peptide was administered intracarotid at the dose of 10 micrograms/rat. These results demonstrate that /ASU1-7/eelCT, as we previously observed with salmon calcitonin (sCT), has central inhibitory activity on PRL secretion, probably through enhancement of hypothalamic inhibitory pathways involved in the control of PRL.

It is well established that salmon calcitonin (sCT), given either by central (intracerebroventricular) or peripheral (parenteral) injection is a potent inhibitor of gastric acid secretion. It is generally believed that this effect of sCT is mediated by the central nervous system based on reports that the effective peripheral dose is up to 1000 times greater than the effective dose administered centrally. We have reexamined this hypothesis by carrying out a number of independent experiments in two laboratories on the effect of sCT, given either by intracerebroventricular or by subcutaneous injection, on basal gastric acid secretion in unanaesthetized rats. Statistical evaluation of the data showed the reproducibility of the effective inhibitory dose ranges of sCT despite the inherent variability of the pylorus ligated rat system (Shay test). The effective dose range for sCT given centrally was between three- and ten-fold lower than that for peripherally administered sCT. There is no published evidence that a significant amount of peripherally administered sCT passes through the blood-brain barrier to relevant areas of the brain and this is confirmed in our study by autoradiography of serial sections of brain following intravenous administration of radioiodinated sCT. It therefore appears that the inhibitory effect of sCT, given centrally or peripherally, may not necessarily be mediated by a common pathway in the central nervous system. Our results also show that the inhibitory effect of peripherally administered sCT is lost when rats are treated with cysteamine to deplete somatostatin, thus implicating somatostatin as a peripheral mediator.

The role of the serotonergic system in the antinociceptive effect of centrally administered salmon calcitonin (sCT) was studied in rats. The animals were given sCT either intracerebroventricularly (i.c.v.) or intrathecally (i.t.). I.c.v. administration of sCT (2,5 micrograms/rat) to animals depleted in CNS serotonin (5-HT) either by treatment with 25 mg/kg desmethylimipramine (DMI) i.p. plus 100 micrograms/rat 5,7 dihydroxytryptamine (5,7 DHT) i.c.v., ten days before or by 150 mg/kg p-chlorophenylalanine (pCPA) i.p., 72 and 24 h before, still significantly increased the hot-plate latencies comparable to those of non-depleted animals. The same result was obtained when the 5-HT receptors were blocked with metergoline. The i.t. administration of sCT (2 micrograms/rat) to animals with spinal cord 5-HT depleted by treatment with DMI plus 5,7 DHT, i.t., delayed but did not abolish the antinociceptive activity of i.t. injected sCT, which was of the same intensity as in non depleted animals. When 5,7 DHT was administered alone, either i.c.v. or i.t., without protection of the catecholaminergic neurons so that noradrenaline (NA) was greatly reduced, the antinociceptive effect of sCT was completely abolished even when NA had been depleted only in the spinal cord. We conclude that it is the catecholaminergic system, not the serotonergic, that plays a fundamental role in the anti-nociceptive effect of centrally administered sCT.

The distribution of binding site for [125I]-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing [125I]-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain.

The effects of synthetic human calcitonin gene-related peptide (CGRP) on nociceptive response were evaluated in rats by two behavioral tests (tail-flick and hot-plate) and by electrophysiological recording of the firing of thalamic neurons evoked by peripheral noxious mechanical stimuli. CGRP was administered intracerebroventricularly (i.c.v.) and its effects were compared with that of salmon calcitonin (sCT). In the tail-flick test, CGRP (0.25, 2.5 and 5 micrograms/rat) dose-dependently increased response latencies, whereas sCT (0.125, 2.5, 5 and 10 micrograms/rat) did not. Conversely, in the hot-plate test CGRP was effective in enhancing response latencies only at the highest dose of 10 micrograms/rat, while sCT (0.125, 0.25 and 2.5 micrograms/rat) inhibited the hot-plate response dose-dependently. In electrophysiological studies, CGRP (2.5 micrograms/rat, i.c.v.) completely inhibited the evoked neuronal thalamic firing and the same dose of sCT induced only a partial reduction. Furthermore, the antinociceptive effects of CGRP in the tail-flick test and in the electrophysiological studies were not prevented by naloxone. These results demonstrate that central administration of CGRP is effective in inhibiting nociceptive responses and its action like that of sCT does not involve an opioid mechanism. The differences in the antinociceptive profiles of CGRP and sCT suggest that the inhibitory effects of these peptides may involve different neuronal pathways.

The distribution of calcitonin (CT) binding sites in serial sections of the rat brain and spinal cord has been examined by an 'in vitro' autoradiographic technique using a radioisotope-sensitive sheet film and [125]salmon CT. Autoradiograms of the diencephalic region had the highest grain density throughout the entire hypothalamus, with the exception of the nuclei ventromedialis, posterior and mammillaris, which were not labeled at all. In the brainstem, large amounts of grains were found in the ventrolateral division of the periaqueductal gray, in the locus coeruleus, in the nucleus tractus spinalis nervi trigemini and in the raphe obscurus, pallidus and magnus, while a widespread and lower grain density was observed in the reticular formation. In the spinal cord the labeling was discretely localized in laminae IV, V and VI of the dorsal horn. The observed distribution of CT binding sites is closely related to the neuroendocrine and analgesic effects of exogenous CT and reinforces the concept of a possible neuromodulatory role proposed for the peptide at brain level.

The possibility that the catecholaminergic (CA) system might be involved in calcitonin (CT)-induced analgesia was examined. The administration of the neurotoxin for CA neurons, 6-hydroxydopamine (6-OHDA) significantly reduced salmon CT (sCT) analgesia as measured in rats by the hot-plate test. Pretreatment with an alpha- and beta-blocker (phentolamine and propranolol) was also effective in lowering significantly the activity of sCT. When the two drugs were administered alone, propranolol, but not phentolamine, reduced the analgesic effect of sCT. A more pronounced and long-lasting inhibitory effect on sCT-analgesia was obtained using atenolol (selective beta 1-receptor blocker). The present data support the role of the CA system in sCT-induced analgesic activity.

The distribution of binding sites in the rat CNS for a synthetic analogue of eel calcitonin, [Asu1-7]eel calcitonin (carboCT) was investigated. This distribution was compared to that for the natural peptide to see whether a modified molecule would also reveal different classes of binding sites for CT. The regional distribution of 125I-carboCT binding in coronal sections of rat CNS was examined by an in vitro autoradiographic technique. Non-specific binding was assessed after addition of excess cold carboCT or eel CT and the results showed that carboCT binding is specific and that it is displaced equally by cold carboCT and by eel CT. There was dense labelling in the nucleus accumbens, in the tractus striohypothalamicus, in the anterior and posterior part of the hypothalamus except for the nucleus ventromedialis, in the amygdala, in the pars medialis of the reticular formation, in the nucleus ruber, in the periventricular gray and in the raphe magnus. Grains were less dense in the hypothalamus lateralis, in the substantia nigra and in the nucleus interpeduncularis. In contrast to eel CT, carboCT did not bind in the spinal cord, nor did carboCT prevent eel CT binding in this area, whereas it was able to prevent it in the brain. These results are consistent with the existence of different classes of binding sites for CT in rat brain and in spinal cord, and indicate that the substitution of the S-S bond with a C-C bond in the eel CT molecule makes the peptide more selective for one class of binding sites.

The effects of intracerebroventricular or intracarotid injection of synthetic rat calcitonin gene-related peptide (CGRP) on growth hormone (GH) secretion induced by various stimuli in male rats were examined. CGRP (2.5 or 5 micrograms/rat intracerebroventricularly, i.c.v.) was administered 10 min before beta-endorphin (0.5 microgram/rat i.c.v.) or morphine (1 mg/kg intracarotidly, i.a.) or clonidine (0.25 mg/kg i.a.) or GH-releasing hormone (GHRH1-40; 2 micrograms/kg i.a.). When injected peripherally, CGRP (10 micrograms/rat, i.a.) was administered 5 min before morphine or GHRH. To investigate the possible involvement of somatostatin (SRIF) in the inhibition of GH secretion by CGRP, the effect of the peptide on GHRH-induced GH release was also examined in rats with hypothalamic SRIF depletion obtained by pretreatment (4 h before) with cysteamine (300 mg/kg subcutaneously). Blood samples for hormone determination were drawn from freely moving rats at various times before and after drug treatment. The intracerebroventricular administration of CGRP (5 micrograms/rat) significantly inhibited GH secretion induced by all the stimuli used. In rats with SRIF depletion CGRP did not modify the stimulation of GH by GHRH. When CGRP was administered intracarotidly, even the dose of 10 micrograms/rat did not reduce the GH release induced by GHRH or morphine. The effects of intracerebroventricular CGRP on basal or beta-endorphin-induced prolactin release were also examined. When given intracerebroventricularly, the peptide did not modify prolactin secretion. The present results indicate that CGRP has a central inhibitory role in the control of GH secretion, probably through a stimulation of SRIF release.

The effects of intracerebroventricular (icv;25ng/rat) or iv (10 micrograms/kg) salmon calcitonin (sCT) on PRL secretion were determined in lactating rats and compared to the effects in intact or ovariectomized-estrogen-treated female rats. The icv or iv injections of sCT 9 days after estrogen treatment did not significantly lower the PRL levels in intact rats. In ovariectomized-estrogen-primed animals, sCT (icv or iv) did not modify the afternoon surge of PRL secretion when injected during the surge, nor did it delay or attenuate the increasing secretory activity when administered (iv) before the afternoon surge had begun. On the contrary, 30 or 60 min after the icv or iv administration of sCT to lactating rats, suckling-induced PRL secretion was prevented. These results and our previous evidence that a greater dose of sCT is needed to decrease the less intense morphine- or stress-induced PRL secretion indicate differential hypoprolactinemic properties of sCT, which are particularly striking during lactation. While the mechanisms underlying these selective activities deserve further investigation, the proposed participation of PRL in the regulation of calcium metabolism during lactation suggests that the potent PRL inhibitory effect of CT in this condition should be regarded as one factor in the complex mechanism that prevents bone loss and protects the maternal skeleton.