Relationships between the Effects of Peripherally Administered Salmon Calcitonin on Calcaemia and Brain Biogenic Amines

Renato GAGGI, Rosaria DE IASIO and Anna Maria GIANNI Instituteof Pharmacology,University of Bologna,Via Irnerio 48, 40126 Bologna,Italy Accepted May18, 1989

Abstract-The effects of s.c. administered salmon calcitonin on the biogenic amine neurotransmitters and metabolites were studied in discrete brain sections in order to evaluate the relationships between these central effects and the fall of serum calcium and to investigate the mechanisms of calcitonin-induced behavioral changes. The biogenic amines and metabolites were simultaneously determined by high-performance liquid chromatography with electrochemical detection in tissue samples obtained from rats treated with 10-80 MRC U/kg of the peptide. Some rats were pretreated with calcium lactate to inhibit the calcitonin-induced hypo calcaemia. Most of the effects, consisting of signs of serotonergic system activation, have been observed in the hypothalamus, accounting for some behavioral effects of the peripherally administered salmon calcitonin. It was speculated that the acti vation of the brain serotonergic system could result from two indirect and separate mechanisms: the first, which would involve enhanced serotonin synthesis, seemed to be facilitated by calcium availability. A second possible mechanism, which would involve enhanced serotonin release from the neurons, seemed to be mediated by the fall of calcium levels in the blood.

It is well-known (1, 2) that the peripheral clarified. However, s.c. administered sCT administration of salmon calcitonin (sCT) does not have any effect on body temperature induces an increase in the brain levels of (R. Gaggi, unpublished observation). In serotonin (5HT) and its metabolite 5-hy regard to its effects on the noxious stimuli droxyindole-3-acetic acid (5HIAA), which response, a single sCT injection is ineffective, may be explained by activation of the seroton whereas repeated s.c. administrations in the ergic systems. mouse induce hyperalgesia (12). On the other hand, the serotonergic sys In any case, it is likely that all the central tems play an important role in several func effects of peripherally administered sCT are tions of the central nervous system including induced by an indirect mechanism. In fact, the release of hormones from the hypophysis, there are no convincing data suggesting the sensitivity to noxious stimuli, as well as the idea that this peptide passes the blood-brain regulation of food intake, body temperature barrier. Since the centrally active doses of sCT and locomotor activity. 5HT agonists adminis are high enough to produce hypocalcaemia tered to rats induce head-twitch behavior (13), and neuronal function has been shown (3, 4). These facts are consistent with the (14, 15) to be sensitive to physiological observations that sCT influences the release changes in calcium concentration, it is likely of hormones from the hypophysis (5-8), in that some sCT effects are mediated by the fall duces anorexia (9), depresses the amphet of calcium levels in the blood. amine-induced locomotor activity (10) and In fact, relationships between the hypo potentiates the head-twitch behavior elicited calcaemic and anorectic effects of various by 5-hydroxytryptophan (11). Notwithstand calcitonins have been demonstrated (13). ing, the role of the serotonergic system ac Moreover, it has been hypothesized that the tivation in some of these effects should still be effects mediated by hypocalcaemia also in

clude changes in the functional state of the dopamine (DA), serotonin (5HT), 5-hydroxy serotonergic system by which sCT poten indole-3-acetic acid (5HIAA), 3,4-dihydroxy tiates the head twitch response elicited by 5 benzylamine (DHBA) and octyl sulfate hydroxytryptophan (11). In this regard, it is sodium salt (OSA) were obtained from Sigma likely that many other neuronal systems, (St. Louis, MO, U.S.A.). Ascorbic acid and besides the serotonergic system, should be triethylamine were from Farmitalia (Milano, influenced by hypocalcaemia. This may con Italy) and methanol was from Inalco S.p.A. tribute to the central effects of sCT. (Milano, Italy). The other reagents were pro The aim of the present work was to in ducts of Merck (Darmstadt, F.R.G.). vestigate the effects of s.c. administered sCT Treatments: The rats were treated with sCT, on the biogenic amine neurotransmitters and by the subcutaneous route, and calcium its metabolites in discrete brain sections and lactate, by the intraperitoneal route, dissolved to evaluate the relationships between these in saline. The control animals received saline effects and the calcium levels in the serum. alone. Three experiments were performed. In the first experiment, 20 MRC U/kg of Materials and Methods sCT was administered to 5 groups of 4 rats Animals and general procedure: Male 8, 4, 2, 1 or 0.5 hr before the sacrifice. A con Sprague-Dawley rats (Nossan, Milano, Italy) trol group of 4 rats received saline 0.5 hr weighing 250-300 g were used. They were before the sacrifice. housed under controlled conditions of light In the second experiment, 10 groups of 4 (7:00 a.m.-7.00 p.m.), temperature (22±2°C) rats were treated with saline or sCT 10, 20, 40 and humidity (60%) with food and water ad and 80 MRC U/kg, 8 or 2 hr before the libitum. On the day of the experiment, all the sacrifice. rats were food deprived at 9:00 a.m. while For one part of the third experiment, 4 the water was kept ad libitum. All the rats groups of rats were used. The first two groups were sacrificed by means of decapitation in were treated with saline, while the third and the afternoon (4:00 p.m.-6:00 p.m.). A blood fourth groups received calcium lactate 100 or sample was collected and the brain of each 200 mg/kg. After 10 min, the first group animal rapidly removed and ice-cooled. The received saline, while the remaining 3 groups cerebellum and olfactory tubercles were were sCT-treated. All these rats were sacrific discarded, whereas the hypothalamus, hip ed 2 hr later. In the same day, another 4 pocampus, striatum, brainstem (pons plus groups of rats were treated in the same way, medulla oblongata) and cerebral cortex were but the calcium lactate dose for the third and dissected (16). Each sample was immediately the fourth group was 200 mg/kg. Four hours frozen with pulvered dry ice and kept at later, the same dose of the calcium salt was -25 °C until the time of analysis . The rem again administered to the fourth group; Thus nant cerebral tissue, consisting mostly of the the rats of this group received 400 mg/kg of thalamus and midbrain, was also collected calcium lactate (2 x 200 mg/kg at a 4 hr and frozen. In the present work, this brain interval). All these rats were sacrificed 8 hr section will henceforth be designated as the after the sCT administration. The dose of sCT "remnant" . was 20 MRC U/kg. The procedure was re Chemicals: Synthetic salmon calcitonin peated using 80 MRC U/kg of sCT. All (sCT) was kindly supplied by Sandoz together, 16 groups of 4 rats were used in the Farmaceutici S.p.A. (Milano, Italy) as com third experiment. mercial vials containing 100 MRC U=20,ug of Analytical procedures: The determination peptide, 2 mg of acetic acid, 2 mg of sodium of the calcium levels in rat serum was per acetate, 7.5 mg of sodium chloride and dis formed with a colorimetric method (Test tilled water to 1 ml volume. Calcium lactate combination Boehringer Mannheim GmbH, was obtained'from Fluka AG (Buchs, Switzer Mannheim, F.R.G.). The determinations of land). For the analytical procedure, norepi biogenic amines and its metabolites in the nephrine (NE), 3,4-d 1hydroxyphenyl -acetic brain sections were performed simultaneously acid (DOPAC), homovanillic acid (HVA), by high-performance liquid chromatography (HPLC) with electrochemical detection (17, 18). The HPLC system consisted of a P 330 Results pump (Violet, Roma, Italy) linked to a First experiment: Effects of the sCT ad Rheodyne 7125 injector. The column (OD51 ministered at the dose of 20 M RC U/kg during C18: 10 ,um, 25 cm x 4 mm i.d.) and the pre the 8 hr following treatment: As shown column (ODS1-C18: 10 ,um, 5 cm x 4 mm IA.) (Table 1), the treatment induced marked were also from Violet. The electrochemical hypocalcaemia (F(5,18)=5.85, P<0.01) that detector (Eldec 103; Chromatofield, Chateau reached its maximum in 30 min (t(5,18)= neuf-Les Martigues, France) was connected 4.00, P<0.01) and remained substantially with a TRIO integrator (Trivector Systems Int., unchanged during the whole period of obser Ltd., Sandy, England). The potential of the vation (after 8 hr: t(5,18)=4.63, P<0.01). glassy-carbon electrode versus silver-silver In regard to the levels of NE, DA, HVA and chloride reference electrode was maintained DOPAC (data not shown) in all the examined at +0.8 V. The mobile phase consisted of a brain sections, the sCT did not induce signifi 840:160 mixture of 0.1 M NaH2PO4 and cant changes with respect to control. On the methanol. The mixture was made 2.6.10-3 M other hand the levels of all these amines and in OSA, 1.0.10-4 M in EDTA and 2.5.10-4 M metabolites in the brain sections of the control in triethylamine, and the pH was adjusted to rats were in the normal range. The following 3.4 with 3 M H3 P04. The flow rate was 1.5 values were observed (the values in ,ag/g of ml/min. wet weight) : in the brainstem: NE (0.71± All H PLC standards were initially dissolved 0.075), DA (0.044±0.006) and DOPAC in deionized double-distilled water. Aliquots (0.020±0.006), in the hypothalamus: NE of the standard mixture were frozen at -80°C. (2.25±0.156), DA (0.46±0.038) and DOPAC At the time of the analysis, a frozen aliquot (0.055±0.008); in the hippocampus: NE was thawed and diluted 10 times with the (0.53±0.019); in the cortex: NE (0.35± medium used for the preparation of the tissue 0.050), DA (0.49±0.103), DOPAC (0.064± samples. This medium consisted of a freshly 0.015) and HVA (0.062±0.019), in the prepared solution of the following composi remnant: NE (0.55±0.036), DA (0.21 ±0.047) tion: 0.2 M HC104, 2.7.10-4 M EDTA and and DOPAC (0.058±0.021); and in the 1.6.10-4 M ascorbic acid. The frozen brain striatum, the detection of NE was uncertain, sections were weighed and immediately but the levels of DA, DOPAC and HVA were, ultrasound disrupted in 0.5-2.0 ml of the respectively, 9.69±0.31, 1.37±0.027 and described medium. After centrifugation for 10 0.63±0.014 ug/g of wet weight. min at 11,000 x g, 20 ,cl of the supernatant The 5HT levels (Table 2) were increased were injected into the HPLC apparatus. DH BA 8 hr after the treatment (t(5,18=4.05, P< was used as an internal standard. 0.01) in the hypothalamus alone (F(5,18)= Statistical analysis: The obtained data were 4.87, P<0.01). analyzed by ANOVAs, followed by the two The 5HIAA levels (Table 2) were increased tailed Dunnett's t-test to compare the in in the hypothalamus (F(5,18=3.93, P< dividual groups to the control. As needed, 0.05), hippocampus (F(5,18)=7.36, P< linear correlation ratios between a measured 0.05) and remnant (F(5,18)=3.93, P< effect and the log of the sCT doses were 0.05). Moreover, the hypothalamic increase computed. appeared 2 (t(5,18)=3.05, P<0.05), 4 (t

The values are means of 4 rats±S.E.M. Statistically significant difference from the control (°P<0.01, two tailed Dunnett's t-test). Values are means of 4 rats+S.E.M. Statistically different from the control by the two-tailed Dunnett's t test (**P<0.01, *P<0.05).

(5,18)=3.69, P<0.01) and 8 (t(5,18)=2.83, stantly higher in the treated rats than in the P<0.05) hr after the sCT administration, control. However, no significant changes were whereas the increases in the 5HIAA levels in observed. The 5HT and 5HIAA levels in the the hippocampus (t(5,18)=3.72, P<0.01) brainstem have shown a similar trend, al and in the remnant (t(5,18)=3.83, P<0.01) though to a lesser extent. were observed only after 8 hr. In the striatum, Second experiment: Effects of various the levels of HVA, 5HT and 5HIAA were con doses of sCT administered 8 or 2 hr before the sacrifice: As shown (Fig. 1), all the experi thalamus (Fig. 2) were increased (F(9,30)= mental doses produced marked hypo 2.94, P<0.05) in animals sacrificed 8 hr after calcaemia (F(9,30)=12.92, P<0.01 ). In the treatment. The doses of 20 (t(9,30)= particular, 2 hr after the sCT administration, 2.82, P<0.06), 40 (t(9,30)=2.86, P<0.05) the effect of 10 Pv1RCU/kg (t(9,30)=6.73, and 80 (t(9,30)=2.91, P<0.05) MRC U/kg P<0.01) was almost similar to those of 20, of sCT induced significant changes with 40 (t(9,30)=6.73, P<0.01) and 80 (t(9,30)= respect to the control. However, the linear 6.41, P<0.01) MRC U/kg of the peptide. The correlation between the logarithm of the sCT linear correlation between the hypocalcaemic dose and the hypothalamic levels of 5HT was effect and the logarithm of the sCT dose was not significant. Two hours after the treatment, not significant. In contrast, there was a linear the 5HT levels in the hypothalamus were correlation (r=0.812; P<0.01) when the unchanged. The hypothalamic concentra levels of serum calcium were evaluated 8 hr tions of 5HIAA were significantly changed after the treatment. In this case, the group (F(9,30)=4.81, P<0.01) by the treatments. treated at the dose of 10 MRC U/kg was not In the rats sacrificed after 8 hr, the linear log significantly different from the control, dose -.effect correlation was highly significant whereas the doses of 20 (t(9,30)=3.63, P< (r=0.663, P<0.01) because the doses of 20 0.01), 40 (t(9,30)=4.70, P<0.01) and 80 (t(9,30)=2.54, P<0.08), 40 (t(9,30)=3.35, (t(9,30)=6.84, P<0.01) MRC U,/kg of sCT P<0.05) and 80 (t(9,30)=3.53, P<0.01) induced significant changes. MRC U/kg of sCT induced a progressive in In regard to the various brain sections, the crease in the 5HIAA levels. After two hours, treatment did not produce significant changes the doses of 10 (t(9,30)=3.17, P<0.05), 20 in the NE, DA, HVA and DOPAC levels (data (t(9,30)=3.04, P<0.05), 40 (t(9,30)=3.18, not shown). The 5HT levels in the hypo

Fig. 2. Levels of 5HT (solid lines) and 5HIAA (dashed lines) in the hypothalamus 2 (A) or 8 (1*) Fig. 1. Rat calcaemia 2 (-A-) or 8 (-0-) hr hr after the s.c. administraticn of sCT. Control rats after the s.c. administration of various doses of sCT. (rectangular areas) were treated with saline. A Saline was administered to the control group (striped statistically significant coefficient of correlation was area). A linear correlation between calcium levels and computed only for the 5HIAA levels versus the logari the logarithm of the sCT doses was observed only 8 thm of the sCT doses 8 hr after the treatment (r= hr (r=0.812, d.f. 14, P<0.01) after the treatment. 0.663, d.f. 14, P<0.01). Each point represents the Each point represents the mean of 4 rats and vertical mean of four rats and vertical bars, the S.E.M. bars, the S.E.M. Statistically significant difference Statistically significant difference from the control from the control by the two-tailed Dunnett's t-test by the two-tailed Dunnett's t-test (*P<0.05, **P< ("P<0.01 ). 0.01). P<0.05) and 80 (t(9,30)=3.66, P<0.01) the sCT dose was 20 (t(9,30) =5.22, P<0.01), MRC U/kg of sCT produced similar increases 40 (t(9,30)=5.58, P<0.01) or 80 (t(9,30)= in the 5HIAA levels. Therefore, the linear log 7.68, P<0.01) MAC U/kg. The coefficient of dose : effect correlation was not significant. linear log-dose : effect correlation was highly In the hippocampus, the 5HIAA levels significant (r=0.786, P<0.01). alone (Fig. 3A) were significantly changed Third experiment: Inhibition of the sCT (F(9,30)=4.89, P<0.01). A.significant coef induced effects by calcium administration: As ficent of linear log-dose : effect correlation shown in Fig. 4A, marked changes in the (r=0.767, P<0.01) was computed for data serum calcium levels were induced in the obtained from animals sacrificed 8 hr after the various groups of rats treated with 20 MAC treatment. In particular, 80 MRC U/kg of sCT U/kg of sCT (F(7,24)=18.99, P<0.01). In significantly increased the 5HIAA levels particular, 2 hr after the peptide administra (t(9,30)=4.07, P<0.01). No significant tion, marked hypocalcaemia was observed differences from the control were observed (t(7,24)=7.37, P<0.01). This effect was 2 hr after the treatment. reduced by 100 mg/kg of calcium lactate Almost similar effects were observed in the (t(7,24)=3.74, P<0.01) and abolished by striatum (Fig. 3B). The 5HIAA levels in 200 mg/kg of this salt. Equally, 8 hr after the creased (F(9,30)=6.80, P<0.01) in the 8 hr peptide treatment, 200 mg/kg of calcium treated rats, showing a linear correlation with lactate, administered once every 4 hr, abolish the logarithm of the sCT dose (r=0.695, P< ed the sCT induced hypocalcaemia (t(7,24)= 0.01). The highest dose of the peptide 7.37, P<0.01). A single administration of induced a significant change with respect to 200 mg/kg of the calcium salt did not the control (t(9,30)=5.92, P<0.01). (t(7,24)=5.28, P<0.01). In the hypothalamus Changes in the 5HIAA levels alone (Fig. 5) and in the remnant (Fig. 6) of these (F(9,30)=12.56, P<0.01) have also been ob animals the biogenic amines and their me served in the remnant of 8 hr treated rats (Fig. tabolites were determined. 3C). A marked increase was produced when As regards the hypothalamus, the 5HT

Fig. 3. Levels of 5HIAA in the hippocampus (A), the striatum (B) and the remnant brain tissue (C) 2 (A) or 8 (0) hr after the s.c. administration of various doses of sCT. Control rats (=) were treated with saline. A significant coefficient of linear correlation (d.f. 14) between 5H IAA levels anc] the logarithm of the sCT doses was computed for data obtained from animals sacrificed 8 hr after the peptide adminis tration (in A: r=0.760, P<0.01 ; in B: r=0.695, P<0.01 ; in C: r=0.786, P<0.01 ). Results represent the mean of 4 rats±S.E.M. Statistically significant difference from the control by the two-tailed Dunnett's t-test ('P<0.01). Fig. 5. Hypothalamic levels of DA, 5HT and SHIAA Fig. 4. Serum calcium in rats s.c. treated with 2 or 8 hr after the s.c. administration of saline (L) saline (=) or sCT ([~) at the dose of 20 (A) or or 20 MRC U/kg of sCT (F-6-1) in rats i.p. pretreated 80 (B) MRC U/kg 2 or 8 hr after the peptide admin with saline or calcium lactate at a low (tom) or a istration and i.p. pretreated with saline or calcium high (®) dose. In the rats sacrificed after 2 hr: lactate at a low ( ) or a high (M) dose. In the low dose=100 mg/kg, high dose=200 mg/kg. In rats sacrificed after 2 hr: lo~v dose=100 mg/kg, high the rats sacrificed after 8 hr: low dose=200 mg/kg, dose=200 mg/ka. In the rats sacrificed after 8 hr: high dose=400 mg/kg (2x200 at a 4 hr interval) of low dose=200 mg/kg, high dose=400 mg/kg the calcium salt. Results represent the mean of 4 rats (2x200 at a 4 hr interval). Results represent the ±S.E.M. Statistically significant difference from the mean of 4 rats+S.E.M. Statistically significant differ control by the two-tailed Dunnett's t-test ('P<0.05, **P<0 ence from the control by the two-tailed Dunnett's .01 ). t-test (*P<0.01 ).

increased levels of 5HT (t(7,24)=2.82, P< (F(7,24)=3.88, P<0.01), 5HIAA (F(7,24)= 0.05) were lacking in rats pretreated with the 5.81, P<0.01) and DA (F(7,24)=3.22, P< calcium salt. The 5HIAA levels in the hypotha 0.05) levels were significantly affected. The lamus were significantly affected (F(7,24)= DOPAC and NE (data not shown) levels 5.81, P-<0.01) by the various treatments. remained unchanged with respect to the The more important effects consisted of a control. Two hours after the treatment, the marked increase in 5HIAA both 2 (t(7,24)= sCT administered after 100 (t(7,24)=2.79, 3.07, P<0.05) and 8 (t(7,24)=4.70, P< P<0.05) or 200 (t(7,24)=3.83, P<0.01) 0.01) hr after the administration of sCT alone. mg/kg of calcium lactate increased the 5HT In the first case, the increased levels were levels, while the sCT alone did not. On the inhibited by both 100 and 200 mg/kg of contrary, 8 hr after its administration, the sCT calcium lactate. In the latter case, the sCT abolished by the high dose of calcium lactate. Calcium levels in the serum (Fig. 4B) were markedly affected when the rats were treated with 80 MRC U/kg of sCT (F(7,24)=19.11, P<0.01). The animals that were treated with peptide alone showed marked hypocalcaemia both 2 hr (t(7,24)=6.25, P<0.01) and 8 hr (t(7,24)=7.83, P<0.01) after treatment. The effect that appeared after 2 hr was reduced (t(7,24)=4.09, P<0.01) and, respectively, abolished by 100 and 200 mg/kg of calcium lactate, In the same way, 400 mg/kg of calcium lactate abolished the effect that ap peared after 8 hr, but 200 mg/kg of this salt did not (t(7,24)=6.37, P<0.01). The rats of these groups were used to determine the biogenic amines and their metabolites in the hippocampus and in the striatum. In the hippocampus, the 5HIAA levels alone were significantly affected (F(7,24)= 6.43, P<0.01) by the various treatments (Fig. 7). In particular, these levels were increased 8 hr after the treatment in rats which

Fig. 6. Levels of NE and 5HIAA in the remna received sCT alone (t(7,24)=4.98, P<0.01). brain tissue in the same rats of Fig. 5. For oth The high dose of calcium lactate abolished details, see Fig. 5 legend. this effect, but the low dose did not (t(7,24)= 3.73, P<0.01). In the striatum, the 5HIAA (F(7,24)= effect was reduced by 200 mg/kg (t(7,24) 11.07, P<0.01) and DOPAC (F(7,24)=2.58, 3.47, P<0.05) and abolished by 400 mg/l, P<0.05) levels were significantly affected of the calcium salt. As regards to DA, (Fig. 8), but the 5HT, DA and HVA levels were increase in the hypothalamic content w~ unchanged. The 5HIAA levels were increased observed in rats treated with 200 mg/kg , 8 hr after the treatment in rats that received calcium lactate and sCT at 2 hr before the were sacrificed (t(7,24)=3.33, P<0.05). In the remnant, NE (F(7,24)=3.92 P< 0.01) and 5HIAA (F(7,24)=6.28, P<0.01) levels were significantly affected, whereas the DA, DOPAC and 5HT concentrations were unchanged. In regard to the NE levels, signifi cant changes were observed only in rats that were sacrificed 8 hr after the sCT injection. The peptide administration reduced the NE levels (t(7,24)=3.07, P<0.05). Calcium lactate abolished this sCT effect at the dose of 400 mg/kg, whereas at the dose of 200 mg/kg, the sCT effect remained unchanged (t(7,24) = 3.51, P<0.01). The 5HIAA levels were Fig. 7. Levels of 5HIAA in the hippocampus 2 or increased in rats that had been treated 8 hr 8 hr after the s.c. administration cf saline (0) or before the sacrifice with sCT alone (F(7,24)= 80 M RC U/kg of sCT (ro ) in rat i.p. pretreated with 4.57, P<0.01). This effect was reduced by saline or calcium lactate. The other details are the the low dose (t(7,24) =3.24, P<0.05) and same as those in Fig. 5. to the diurnal variations of 5HT and 5HIAA levels because all the rats were sacrificed at the same time of day (4:00 p.m.-6:00 p.m.). No other significant change in the brain sections was found in the first experi ment, although in the striatum of the sCT treated rats, the 5HT and 5HIAA levels were constantly higher than in their respective control. Therefore, the striatum was also ex amined in the second and third experiments. The second experiment was undertaken in order to verify if the supra-referred sCT effects were dose-related. The hypocalcaemic effect that was exhibited 8 hr after the treatment, unlike that exhibited after 2 hr, was linearly related to the logarithm of the sCT dose. This finding can be explained by assuming that all the supraphysiological sCT doses adminis tered to the rats have produced within 2 hr the maximum effect on calcaemia. Therefore, the fall in calcaemia was not dose-related because all the experimental doses have pro Fig. 8. Levels of DOPAC and 5HIAA in the striatum duced similar effects. Nevertheless, when the of the same rats in Fig. 7. For all the other details, see sCT doses were increased from 10 to 80 M RC Fig. 5 legend. U/kg, the length of the hypocalcaemic effect appeared proportionately increased. So after sCT alone (t(7,24)=5.70, P<0.01). The dose 8 hr, the hypocalcaemia was lacking in the rats of 400 mg/kg of calcium lactate completely treated with the low dose, whereas it was inhibited this effect, but the dose of 200 mg/ still marked in the rats that had received the kg did not (t(7,24)=3.69, P<0.01). high dose of the peptide. Therefore, 8 hr after Finally, an increase of the DOPAC levels in the treatment, the fall in calcaemia could be the striatum was observed in the rats that dose-related. I n the same way, the 5HIAA received sCT and calcium lactate at the dose levels in the hypothalamus were increased of 100 mg/kg (t(7,24)=3.68, P<0.01) 2 hr both 2 and 8 hr after the sCT injection: the before the sacrifice. The DOPAC levels were effect exhibited after 8 hr, unlike that ex unchanged when the peptide alone was hibited after 2 hr, was linearly related to the administered. log-dose of sCT. Similar effects have been found in the hippocampus, striatum and Discussion remnant. The 5HIAA levels linearly increased As expected, the sCT administered at the 8 hr after the treatment, but remained un dose of 20 M RC U/kg rapidly produced a changed after 2 hr. Therefore, it has been as marked and long-lasting hypocalcaemia. In sumed that the increase in 5HIAA levels in the various brain sections, the 5HIAA content discrete brain sections was related to the size began to increase shortly after the sCT ad and the lenght of the sCT induced hypocal ministration; and in the hypothalamus of caemia and that the hypothalamus is more treated rats, it became significantly greater sensitive that the other brain sections to the than the control in 2 hr. Six hours later, the fall in calcaemia. In particular, a period of 2 hr 5HIAA levels were significantly increased in appeared sufficient to produce the effect in the hippocampus and remnant also. The in the hypothalamus, but approx. 8 hr each were crease in 5HT levels was observed 8 hr after required for the hippocampus, striatum and the sCT administration in the hypothalamus remnant. The delay of the cerebral effects with alone. These changes could not be attributed respect to the onset of the hypocalcaemia could be bound to the time required for the mic effect, was not linearly related to the log rearrangement of the calcium levels in the ex dose of sCT and, therefore, could not be tracellular fluid and in the tissues of the brain. thought of as being mediated by the fall of the This hypothesis was consistent with the calcium levels in the blood. On the contrary, finding that the administration of calcium in the data of the third experiment have sug hibited both the sCT induced hypocalcaemia gested that the sCT effect on the 5HT syn and the increase in 5HIAA levels in all the thesis was facilitated by the inhibition of the tested brain sections. hypocalcaemia. In particular, 2 hr after the As regards to the 5HT, some workers (1) sCT administration, the enhancement of the have indicated that the content of this amine hypothalamic levels of 5HT was greater in the in the whole rat brain greatly increased rats treated with calcium lactate than in those shortly after s.c. administration of sCT. This treated with saline. Since the 5HIAA remained effect appeared unrelated to the peptide simultaneously unchanged, an increase in the induced hypocalcaemia as well as both the synthesis, unlike an inhibition of the release contemporaneous increase of free tryptophan and metabolism, should account for the and the decrease of other large neutral amino enhanced amine levels. Nevertheless, 8 hr acids in the serum. It seems likely that these after the treatment with sCT, the hypothala changes in the serum levels of amino acids mic 5HT content was enhanced in rats could greatly enhance the brain synthesis of treated with saline, whereas it was unchanged 5HT, since (a) the free tryptophan competes with respect to the control in rats that had with the other large neutral amino acids in the received calcium lactate. Therefore, it seemed transport system across the blood-brain barrier that in these animals, the 5HT synthesis has (19) and (b) the tryptophan hydroxylase is been inhibited by the calcium administration. not normally saturated with its substrate, However, considering that the great enhance tryptophan. Nevertheless, in the first experi ment of the 5HT synthesis during the early ment, we have observed a significant en hours after the sCT and calcium administra hancement of 5HT levels only 8 hr after the tion should have consumed most of the sCT administration and in the hypothalamus tryptophan, we have assumed that after 8 hr, alone. The disagreement with the previous the hypothalamic 5HT content has been observations (1) could be attributed, at least limited by a lowered precursor availability. partly, to differences in the animals (e.g., body Therefore, these present findings agreed weight: 250-300 g instead of 100 g; cal with the previous observation (1) that the caemia: 8.95±0.38 instead of 9.9±0.2 mg% sCT induced increase in the synthesis of ml), in the experimental procedure (fasted cerebral 5HT is unrelated to hypocalcaemia rats for 8 instead of 16 hr) and/or in the and, moreover, suggested that this effect is methods of analysis (separation by HPLC and facilitated by calcium. electrochemical detection instead of separa In conclusion, we assumed that the s.c. ad tion by extraction and fluorimetric determi ministration of sCT activated the serotonergic nation). On the other hand, a moderate in system by two indirect and separate mech crease in the 5HT synthesis could be masked anisms. The first, likely involving changes in by an increased release and metabolism of the the blood levels of large neutral amino acids, amine. In fact, the ratio between 5HIAA and appears to be facilitated by calcium adminis the 5HT levels in the various brain sections of tration and enhances the synthesis of neuro treated rats was almost higher than those of transmitter. The latter, likely involving the fall the controls. Therefore, the increase in the of the calcium levels in the blood that balances 5HIAA levels could account for failure of the brain extracellular fluid, enhances the enhanced 5HT levels in some brain sections. release (15) and, consequently, the me For the same reasons, it seems unlikely that an tabolism of the neurotransmitter. The hypo enhanced 5HT synthesis could account for thalamus appeared to be the more sensitive the increased 5HIAA levels. However, in the brain section to the actions of sCT. Higher second experiment, the increase in 5HT levels doses of the peptide and/or longer periods of in the hypothalamus, unlike the hypocalcae hypocalcaemia were required to produce signs of activation of the serotonergic system thyroid hormone and prolactin secretion. In in the other brain sections. These signs con Hormonal Control of Calcium Metabolism, sisted of an increase of metabolite levels, Edited by Cohn, D.V., Talmage, R.V. and while neurotransmitter levels increased to a Mathews, J.L., p. 287-292, Excerpta Medica, lesser extent or remained unchanged. How Amsterdam (1981) ever, the 5HIAA/5HT ratio was generally in 8 Pecile, A., Olgiati, V.R., Luisetto, G., Guidobono, creased with respect to the control. The F., Netti, C. and Ziliotto, D.: Calcitonin and control of prolactin secretion. 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