Sympathectomy alters bone architecture in adult growing rats.

Sympathetic nervous system (SNS) fibres and alpha- and beta-receptors are present in bone, indicating that the SNS may participate in bone metabolism. The importance of these observations is controversial because stimulation or inhibition of the SNS has had various effects upon both anabolic and catabolic activity in this tissue. In this study we evaluated the effects of pharmacological sympathectomy, using chronic treatment of maturing male rats with 40 mg of guanethidine/kg i.p., upon various parameters in bone. Double labelling with tetracycline injection was also performed 20 and 2 days before sacrifice. Bone mass, mineral content, density and histomorphometric characteristics in different skeletal regions were determined. Bone metabolic markers included urinary deoxypyridinoline and serum osteocalcin measurements. Guanethidine significantly reduced the accretion of lumbar vertebral bone and of mineral content and density, compared to controls. Femoral bone mineral content and density were also significantly reduced, compared to controls. Histomorphometric analyses indicated these effects were related to a reduction of cortical bone and mineral apposition rate at femoral diaphysials level. Both markers of bone metabolism were reduced in controls as they approached maturity. Guanethidine significantly decreased serum osteocalcin compared to controls, while urinary deoxypyridinoline was unchanged. These data indicate that guanethidine-induced sympathectomy caused a negative balance of bone metabolism, leading to decreased mass by regulating deposition rather than resorption during modeling and remodeling of bone.

Central ghrelin gastroprotection involves nitric oxide/prostaglandin cross-talk.

BACKGROUND AND PURPOSE: Ghrelin, a gut-brain peptide, is considered a gastroprotective factor in gastric mucosa. We investigated the role of prostaglandins (PG) and the possible interplay between PGs and nitric oxide (NO) in ghrelin gastroprotection against ethanol (EtOH)-induced gastric lesions. EXPERIMENTAL APPROACH: We examined the effects of (1) central ghrelin (4 mug per rat) injection on PGE(2) accumulation in normal or EtOH-lesioned gastric mucosa, (2) pretreatment with indomethacin (10 mg kg(-1), p.o.), a non-selective cyclooxygenase (COX) inhibitor, and with a selective COX-1, SC560 (5 mg kg(-1), p.o.) or COX-2 inhibitor, celecoxib (3.5 mg kg(-1), p.o.) on ghrelin gastroprotection against 50% EtOH (1 mL per rat)-induced gastric lesions, (3) the NO synthase inhibitor, L-NAME (70 mg kg(-1), s.c), on gastric PGE(2) content in ghrelin-treated rats and (4) central ghrelin on the expression of constitutive and inducible NOS and COX mRNA and on the localization of the immunoreactivity for COX-2 in the gastric mucosa exposed to EtOH. KEY RESULTS: Ghrelin increased PGE(2) in normal mucosa, whereas, it reversed the EtOH-induced PGE(2) surge. Ghrelin had no effect on mucosal COX-1 expression but reduced the EtOH-induced increase in COX-2 expression and immunoreactivity. Indomethacin and SC560, but not celecoxib, removed ghrelin gastroprotection. L-NAME prevented the PGE(2) surge induced by ghrelin and, like indomethacin, reduced EtOH-induced PGE(2) increase. Ghrelin enhanced eNOS expression and reduced iNOS mRNA. CONCLUSIONS AND IMPLICATIONS: This study shows that COX-1-derived PGs are mainly involved in ghrelin gastroprotection and that the constitutive-derived NO together with PGE(2) are involved in ghrelin gastroprotective activity.

Growth hormone stimulates osteoprotegerin expression and secretion in human osteoblast-like cells.

It is presently thought that osteoprotegerin (OPG) is a cytokine involved in the regulation of osteoblast/osteoclast crosstalk and maintenance of bone mass. Recent studies showed that GH replacement therapy in GH-deficient patients was able to induce a significant increase of OPG in the plasma, as well as in the cortical and the trabecular bone. In order to determine whether GH could directly modulate OPG secretion, the effect of GH on human osteoblast-like cells (hOB) in primary culture was studied. After detecting the presence of the mRNA for the GH receptor (GHR) by RT-PCR, hOB were exposed to increasing concentrations of GH, from 0.1 to 25 ng/ml, for 24 h. The results showed that GH exposure was able to stimulate OPG secretion in a concentration-dependent manner. In addition, the OPG mRNA levels were increased, indicating that the hormone has a stimulatory effect on gene expression. The stimulatory effect on OPG expression and production was prevented by exposing the cells to tyrphostin AG490 (10 muM), an inhibitor of Janus kinase 2, which is one of the kinases involved in the intracellular pathway activated by the binding of GH to its receptor. Similar results were obtained when the cells were exposed to a receptor antagonist of GH, pegvisomant at 50 nM. GH exposure neither induced an increase in IGF-I expression nor secretion in hOB. These results suggest that the stimulation of OPG production induced by GH in hOB is specific and receptor mediated and further support the view that GH is able to modulate bone remodeling by directly influencing osteoblast-osteoclast crosstalk.

Different skeletal regional response to continuous brain infusion of leptin in the rat.

This study was designed to evaluate whether or not continuous intracerebroventricular infusion of leptin (1.5 microg/rat/24 h, for 28 days) produced different regional response on the skeleton of growing rats. Leptin reduce the accretion of total femoral bone mineral content (BMC) and density (BMD). This effect was related to a reduction of metaphyseal femur as no changes were detected in the diaphysis. Despite the reduced accretion in the volumetric of both femur and tibia compared to controls, leptin had no significant effects on the lumbar vertebrae. Urine deoxypyrydinoline and serum osteocalcin remained more elevated in the leptin-treated group as compared to controls. The results demonstrate that long-term central infusion of leptin activates bone remodeling with a negative balance. Leptin induces distinct responses in the different structure of bone and in the axial and appendicular skeleton.

Intracerebroventricular acute and chronic administration of obestatin minimally affect food intake but not weight gain in the rat.

We studied the effect of the acute central administration of obestatin on food intake and body weight in short-term starved male rats, and those of 28-day continuous intracerebroventricular (icv) infusion of obestatin in free feeding rats. In 16-h starved rats, obestatin induced a trend toward a reduction of food intake that did not reach statistical significance. In fed rats, the icv infusion of obestatin significantly decreased food consumption in the first day of treatment; but the anorexigenic effect of obestatin vanished thereafter. Interestingly, the body weight of rats infused for 28 days with obestatin was superimposable to that of the respective control at all time intervals. In all, our results indicate that the anorexigenic effect of obestatin is of little account and that the peptide does not modify energy metabolism in the long-term administration.

High sensitivity to calcitonin of prolactin-secreting control in lactating rats.

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.

Comparison of the effects of histamine H2-receptor antagonists on prolactin secretion in the rat.

Various compounds with different blocking potencies on histamine H2-receptors were administered to rats both into the carotid (ia) and into the brain ventricles (icv) and their effects on PRL release were evaluated. The drugs were cimetidine, as reference compound, ranitidine, 5 to 7 times as potent, and oxmetidine, 8 to 10 times as potent. PRL was measured in blood samples collected at -15, 0 and +5, +10, +20 and +40 min after treatment. Cimetidine (80 mg/kg) and ranitidine (30 mg/kg), when injected ia as a single bolus, induced prompt increases (P less than 0.01) in PRL. On the contrary, oxmetidine (the most potent H2 antagonist), even at the high dose of 80 mg/kg ia, had no effect on PRL release. When the drugs were given icv (0.2 mumol/rat or 0.8 mumol/rat), none of them caused any significant PRL release. These results suggest that histamine H2-blocking potency is not correlated with PRL release. Furthermore, the PRL-releasing activity of the drugs seems not to be due to any action on the central neural control of PRL secretion. This comparative study shows that it is possible (as with oxmetidine) to achieve complete dissociation of the H2-receptor blocking action from the unwanted PRL-release stimulating effect.

Antinociceptive activity of eel calcitonin, injected into the inflamed paw in rats.

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.

Evidence for an inhibitory role of central histamine on carrageenin-induced hyperalgesia.

The effects of intracerebroventricular (i.c.v.) injection of histamine, the H1 agonist 2-methyl-histamine and the H2 agonist dimaprit were tested on carrageenin induced hyperalgesia by the Randall-Selitto paw pressure test in the rat. Treatment with histamine (0.1, 0.2, 0.4 mumol/rat, i.c.v.) 150 min after intraplantar carrageenin (0.1 ml of 1% solution) caused a significant increase of paw pressure thresholds in inflamed (but not in non-inflamed) paws. The magnitude and the duration of the antinociceptive effects of histamine were dose-dependent. Administration of 2-methyl-histamine (0.2, 0.4, 0.8, 1.0 mumol/rat, i.c.v.) and dimaprit (0.1, 0.2, 0.4, 0.8 mumol/rat, i.c.v.) also displayed dose-dependent blockade of carrageenin-induced hyperalgesia. Antinociceptive ED50 values calculated 30 min after drug treatments were: histamine 0.18 mumol/rat; 2-methyl-histamine 0.65 mumol/rat; dimaprit 0.33 mumol/rat. These data indicate that histamine through central H1 and H2 receptors exerts an inhibitory role in the control of nociception in pain resulting from inflammation.

Investigation on the mechanisms involved in the central protective effect of amylin on gastric ulcers in rats.

1. The mechanisms involved in the protective effect of amylin (administered into the brain ventricle, i.c.v.) on gastric ulcers induced by the oral administration of ethanol 50% (EtOH, 2 ml/rat) or indomethacin (indomethacin, 20 mg kg(-1), at a dosing volume of 5 ml) were investigated in rats. 2. The possible involvement of endogenous nitric oxide (NO) in the beneficial effect of amylin against EtOH-induced ulcers was examined. The inhibitor of NO-synthesis, NG-nitro-L-arginine methyl ester (L-NAME, 70 mg kg(-1), s.c.) was injected 30 min before amylin (2.2 microg/rat, i.c.v.) followed by EtOH after a further 30 min. Rats were sacrificed 1 h after EtOH. L-NAME completely removed the protective effect of amylin. 3. The interaction between amylin and gastric nonprotein sulfhydryl groups was studied. The rats were treated with N-ethyl-maleimide (NEM, 25 mg kg(-1), s.c.) 30 min before amylin (2.2 microg/rat, i.c.v.) followed by EtOH 30 min after or by indomethacin 5 min after amylin. Rats were sacrificed 1 h or 6 h respectively after EtOH or indomethacin. NEM counteracted the protective effect of amylin against EtOH-induced ulcers but not against those provoked by indomethacin. 4. To determine whether amylin was able to promote ulcer healing, the peptide was injected 5 min after EtOH or 1 h after indomethacin. In the case of EtOH, the beneficial effect of amylin was lost whereas it was still effective on indomethacin-induced ulcers. 5. The results indicate that: the mechanisms involved in the antiulcer effects of amylin are different in these two types of gastric lesions probably because of the different etiopathology of various types of ulcers. Endogenous NO and nonprotein sulfhydryl groups are involved in the mucosal protective effects of amylin on EtOH and not on indomethacin-induced ulcers. Furthermore the effectiveness of amylin against indomethacin-induced lesions when administered after the ulcerogenic process has started suggests that amylin is involved not only in the protection but also in the healing mechanisms in this type of ulcer.

Protection by amylin of gastric erosions induced by indomethacin or ethanol in rats.

1. The effect of amylin on gastric ulcers induced by oral administration of indomethacin (Indo, 20 mg kg-1 at a dosing volume of 5 ml) or ethanol 50% (EtOH, 1 ml/rat) was investigated in conscious rats. 2. Amylin given intracerebroventricularly (0.22, 0.66 and 2.2 micrograms/rat, i.c.v.) demonstrated a dose-dependent cytoprotective effect against both Indo and EtOH-induced ulcers. In contrast, amylin, given subcutaneously at doses effective in inhibiting acid gastric secretion (2.5, 10 and 40 micrograms kg-1, s.c.), did not show any cytoprotective effect. 3. The interaction between amylin and endogenous nitric oxide (NO) in the maintenance of gastric mucosal integrity was investigated by pretreating the rats with a selective inhibitor of NO-synthesis, NG-nitro-L-arginine methyl ester (L-NAME, 25 and 70 mg kg-1, s.c.). Administration of L-NAME to rats did not significantly increase the degree of the Indo-induced ulcer index and was not able to remove the protective effect of amylin on Indo-induced ulcers, thus excluding a role for endogenous NO in mediating the protective effect of this peptide. 4. To determine whether the cytoprotective effect of amylin was mediated by endogenous prostaglandins, we studied the effect of amylin (2.2 micrograms/rat, i.c.v.) on EtOH- induced ulcers in rats pretreated with Indo (10 mg kg-1, s.c.) to inhibit prostanoid biosynthesis; Indo was injected 30 min before amylin and EtOH after a further 30 min. Pretreatment with Indo did not significantly increase the ulcer index induced by EtOH but counteracted the ability of amylin to prevent the ulcer formation. 5. These findings suggest that amylin exerts a gastroprotective activity that is not strictly related to inhibition of acid gastric secretion and can be partly explained through a prostaglandin-dependent mechanism mediated by receptors for the peptide in the brain. Amylin might be considered as a new brain-gut peptide.

Long-term effects on bone of postnatal immunization against GHRH in female and male rats.

The effects of neonatal passive immunization against GHRH on bone was examined in male and female rats. Pups were treated subcutaneously with GHRH-antiserum (GHRH-Ab) from day 1 to day 10 of age. Bone mineral content (BMC) and bone mineral density (BMD) were evaluated at monthly intervals until 7 months. Markers of bone resorption (urinary lysylpyridinoline, LP), bone formation (serum osteocalcin, OC) and serum IGF-I were measured at 2, 3 and 7 months. In male rats, GHRH-Ab did not modify BMC and BMD when compared with controls. In contrast, female rats demonstrated lower whole body and femoral BMC and BMD from 2 to 7 months of age. Reduced bone growth in the females was associated with lower IGF-I levels than controls at 2 and 3 months of age, whereas in males IGF-I titers did not change during the period of the study. LP excretion was higher in GHRH-Ab-treated rats at 2 and 3 months in both sexes. In females, no difference in OC values was recorded, whereas in GHRH-Ab-treated males, there was an increase in OC levels at 2 and 3 months. These data indicate that transient GHRH deprivation induces an osteopenic effect in female rats which is not evident in male rats.

The central GABAergic system and control of food intake under different experimental conditions.

Intracerebroventricular injections of gamma-aminobutyric acid (GABA) and of the GABA-transaminase inhibitor, ethanolamine-O-sulphate (EOS), decreased the food intake of freely-fed (GABA and EOS) and food-deprived rats (EOS). The effect, still evident 24 h after treatment, was not decreased by the GABA receptor-blocker bicuculline. In contrast, intracerebroventricular injections of the GABA receptor-agonist, muscimol, caused an increase in food intake of freely-fed rats that was antagonized by bicuculline. The eating of animals receiving only bicuculline was stimulated in free-feeding and depressed in food-deprived conditions. These opposite results suggest that muscimol binds preferentially to some GABA receptors, probably those within the satiety-controlling areas (i.e. ventromedial hypothalamus), and that bicuculline influences mainly those postsynaptic neurons where GABAergic inputs prevail. These observations and the data from EOS- and GABA-treated rats provide evidence for involvement of GABA neurons in the regulation of feeding behaviour. The balance of the different effects produced in each of these areas by this modulation appears to be a decrease in feeding behaviour.

Decrease in rat brain calcitonin binding sites and adenylyl-cyclase activity during ageing.

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.

Differential effects of CGRP on adenylyl cyclase in adult and embryonic rat brain.

The presence of functional receptors for calcitonin gene-related peptide (CGRP) in the brain of adult rats and on nerve cell cultures was investigated. Neuronal and glial cultures were obtained from mesencephalons of embryos at gestational day 16. The response to CGRP was tested by measuring the adenylyl cyclase (AC) activity on isolated membranes. CGRP binding in adult rat brains was ineffective in activating AC, whereas a dose-dependent stimulation of AC activity was induced by the peptide both in neuronal and glial cultures. This effect was more pronounced in the glial cells where high affinity binding sites for CGRP were detected. The presence of functional CGRP receptors in embryonic mesencephalic cells, suggests a role for CGRP in the development of rat mesencephalon.

Effects of amylin on human osteoblast-like cells.

Amylin has been reported to have bone-conserving effects. In the present study we evaluated the possible activity of the peptide on human osteoblast-like (hOB) cells in primary culture. Amylin between 10(-9) and 10(-6) M, dose-dependently stimulated cell proliferation with a maximal effect (200%) at 10(-6) M. In addition, amylin increased osteocalcin production when hOB cells were exposed to 1,25(OH)2D3 (10(-8)M) but there was a nonsignificant upward trend on alkaline phosphatase activity. The present results suggest that amylin could be included among the group of peptides endowed with osteogenic activity.

Amylin given by central and peripheral routes inhibits acid gastric secretion.

The effect of rat amylin on acid gastric secretion in the pylorus-ligated, unanesthetized rat system (Shay test) was examined. Amylin administered peripherally (2.5, 5, 10, 40, 100, or 160 micrograms/kg, SC) or intracerebroventricularly (1.5, 2.7, or 5 micrograms/rat, ICV) decreased acid gastric secretion in a dose-dependent manner. Central administration of amylin gave a stronger suppression of gastric secretion than peripheral injection. In addition, ICV-injected amylin inhibited insulin-stimulated acid gastric secretion and was effective in suppressing acid gastric secretion in rats depleted of somatostatin by pretreatment with cysteamine. This study suggests that amylin may participate in the central regulation of acid gastric secretion and indicates a possible biological function of amylin as a gastrointestinal peptide.

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.

The role of sensory neurons in the antiulcer effect of centrally injected amylin in rat.

Central administration of amylin (2.2 microg/rat, i.c.v.) reduces (from a minimum of 67% to 83%) indomethacin (Indo, 20 mg Kg(-1), orally) induced ulcers in rats. The anti-ulcer effect of the peptide is not removed by the administration of prokinetic drugs like domperidone or neostigmine but it is reduced by 35% in rats treated with capsaicin or with the CGRP antagonist, CGRP(8-37). These data indicate that amylin gastroprotection involves capsaicin-sensitive nerve fiber leading to CGRP-dependent gastric vasodilatory effect. Additional mechanisms could involve noradrenergic alpha(2) receptors as the peptide gastroprotective activity is reduced from 67% to 20% by the alpha(2) antagonist yohimbine.

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.