I. Villa

Eel calcitonin binding site distribution and antinociceptive activity in rats

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.

Central effects of histamine H2-receptor agonists and antagonists on nociception in the rat

The effects of intracerebroventricular injection of histamine H2-receptor agonists (4-methylhistamine, 4-MeH; dimaprit, DIM), H2-antagonists (cimetidine, CIM; ranitidine, RAN; famotidine, FAM) and of the DIM chemical analogue SK&F 91487 on hot-plate latency in rats were examined. Both DIM (0.4–0.8 μmol/rat) and 4-MeH (0.4–0.8 μmol/rat) significantly enhanced the pain threshold, whereas SF&F 91487 (0.8 μmol/rat) had no effect, indicating that DIM antinociception is specifically due to its activity on histamine (HA) receptors. The H2-antagonists CIM (0.8 μmol/rat) and RAN (0.6 μmol/rat) also enhanced the pain threshold, while FAM (0.03 μmol/rat) did not modify pain latency. When injected before 4-MeH, FAM reduced the antinociceptive effect of 4-MeH. These findings suggest that the antinociceptive activity of CIM and RAN is not related to specific blockade of H2-receptors and that the activation of HA-H2-receptors is inhibitory to nociception.

Evidence for different classes of calcitonin binding sites in rat CNS: an autoradiographic study with carbo-calcitonin

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.

Involvement of brain histamine in basal and stress-induced release of prolactin in the rat

We investigated whether inhibition of brain histamine (HA) synthesis by α-fluoromethylhistidine (α-FMH) can influence basal or stimulated prolactin (PRL) release in male rats. α-FMH was administered either into the carotid (i.a., 20 and 100 mg/kg) or intracerebroventriculary (i.c.v., 200 μg/rat) into freely moving rats with indwelling catheters. Plasma PRL levels were measured 90, 120, 180 min later. Both i.a. and i.c.v. administration of α-FMH significantly inhibited basal PRL secretion at 120 and 180 min. When PRL secretion was stimulated by exposing rats to restraint stress, α-FMH administered 3 h before the stress (20 and 100 mg/kg, i.a.; 200 μg/rat, i.c.v.) was able to prevent the PRL surges at 10 and 20 min after stress. These results suggest that endogenous brain HA has a facilitatory role in the control of PRL secretion in rats.

Treatment with pertussis toxin does not prevent central effects of eel calcitonin

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.

Inhibitory effects of centrally administered |ASU1–7| eel calcitonin on basal and stimulated prolactin release in rats

We investigated the effects of /ASU1–7/eel calcitonin (ASU1–7 elCT) 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 μ/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 μ/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 μ/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.

Effects of selective histamine H3-receptor ligands on prolactin and growth hormone secretion in the rat

The effects of intracarotid (i.a.) administration of the histamine (HA) H3-receptor agonist (R)-α-methyl-histamine (αMeHA) and of the H3-antagonist thioperamide, (THIO) on basal or morphine (M)-induced prolactin (PRL) and growth hormone (GH) secretion were studied in male rats. M was administered 3 h after the H3-drugs. Neither THIO (2.5 mg/kg) nor αMeHA (10 mg/kg) changed basal PRL levels and only THIO enhanced the PRL-releasing effect of M (6 mg/kg). Basal GH secretion was not modified by THIO. αMeHA slightly increased GH secretion. THIO significantly decreased M-stimulated GH secretion (1 mg/kg, i.a.) and αMeHA slightly increased it. These results, in agreement with previous evidence obtained after central HA administration, indicate that endogenous brain HA facilitates PRL and inhibits GH secretion.

A selective role for brain histamine in prolactin release induced by opiates

We studied the effects of histamine (HA) antagonists on the facilitatory action of morphine (M) andβ-endorphin (βE) on prolactin (PRL) release and the effect of α-fluoromethylhistidine (α-FMH, inhibitor of HA synthesis) onβE-induced PRL secretion. Male rats were injected intracerebroventricularly (i.c.v.) with mepyramine (MEP, H1-antagonist, 0.8 μmol/rat) or ranitidine (RAN, H2-antagonist, 0.4 μmol/rat) 10 min before M (6 mg/kg, intracarotid, i.a.) orβE (0.25 μg/rat, i.c.v.). α-FMH (200 μg/rat, i.c.v.) was administered 3 h beforeβE. Plasma PRL levels were measured at various times before and after drug treatment. RAN but not MEP significantly reduced PRL release induced by M whereas neither HA-antagonists nor α-FMH modifiedβE-induced PRL release. The results obtained show that brain HA contributes through activation of H2-receptors to the PRL facilitatory action of M but not ofβE.

Effects of brain histamine depletion on stimulated prolactin release in rats.

We examined the effects of an irreversible inhibitor of brain histamine (HA) synthesis, α-fluoromethyl-histidine (α-FMH), on prolactin (PRL) release induced by an opiate agonist (morphine, M) or by a serotonergic agonist (MK212). α-FMH was administered intracerebroventricularly (i.c.v., 200 μg/rat) into freely moving rats with indwelling catheters in the carotid. M (6 mg/kg, intracarotid, i.a.) was administered simultaneously with or 3 h after α-FMH. MK212 (2.5 mg/kg, i.a.) was administered 3 h after α-FMH. Blood samples for assay for PRL were drawn at 0, 10, 20, 40 min after M or MK212 administration. α-FMH (3 h before) significantly reduced the PRL-releasing effect of M and MK212 but did not modify PRL release by M when administered simultaneously. The present results showing that the facilitatory actions of the opiate and serotonergic systems on PRL are impaired when brain HA synthesis is reduced, suggest that there is an HA-dependent step in opiate and serotonergic control of PRL.

Involvement of the catecholaminergic system in the inhibitory effect of brain histamine on growth hormone secretion

The effects of intracerebroventricular (icv) injection of histamine (HA, 0.4 μmol/rat) or of the inhibitor of HA synthesisα-fluoromethylhistidine (αFMH, 100 mg/kg, intracarotid (ia)) on growth hormone (GH) secretion induced by GH-releasing hormone (hGHRH1–40, 2 μg/kg, ia) in freely moving rats were examined. HA significantly inhibited GH release induced by GHRH whereas pretreatment (3 h before) with αFMH enhanced the GH response to GHRH.The inhibitory effect of HA on GHRH-induced GH release is still evident in somatostatin-depleted rats with cysteamine (300 mg/kg, sc, 4 h before) but is completely overcome by the inhibitor of catecholamine synthesis (α-methyl-p-tyrosine, 200 mg/kg, ip, 2 h before) suggesting that HA influences GH secretion by modulation of catecholaminergic neurons. This suggestion is supported by the biochemical data showing an increase in noradrenaline and dopamine contents in the hypothalamus and striatum of rats pretreated with αFMH.