Antioxidant activity of hyaluronic acid investigated by means of chemiluminescence of equine neutrophil bursts and electron paramagnetic resonance spectroscopy.

Activated neutrophils (PMNs), the ROS/RNS released by PMNs and the derived inflammatory processes are involved in the pathogenesis and progression of human inflammatory airway diseases. Similar diseases are also present in horses which suffer from recurrent airway obstruction (RAO), exercise-induced pulmonary haemorrhage (EIPH) and inflammatory airway diseases (IAD). Hyaluronic acid (HA) plays numerous roles in modulating inflammatory processes. The aim of this study was to examine whether a preparation of HA (MW 900 000 Da) interferes with ROS/RNS during the course of equine PMN respiratory bursts, and to establish the lowest concentration at which it still has antioxidant activity by means of luminol-amplified chemiluminescence (LACL). Electron paramagnetic resonance (EPR) spectroscopy was also used to investigate the direct antiradical activity of HA. The hydroxyl radical was significantly scavenged in a concentration-dependent manner at HA concentrations ranging from 2.5 to 0.16 mg/mL. Superoxide anion, Tempol radical and the ABTS(•+) were significantly inhibited at concentrations ranging from 2.5 to 0.62 mg/mL. The LACL of stimulated equine neutrophils showed that HA induced a statistically significant concentration-effect reduction from 5 mg/mL to 1.25 mg/mL. These findings were confirmed also when l-Arg was added to investigate the inhibition of the resulting peroxynitrite anion. Our findings indicate that, in addition to the human use, HA can also be used to antagonize the oxidative stress generated by free radicals in horses peripheral blood mononuclear cells (PBMCs). In order to achieve therapeutic concentrations, a direct aerosol administration to horses with horse respiratory diseases can be considered, as this route of application is also recommended in human medicine.

Diclofenac-Choline Antioxidant Activity Investigated by means of Luminol Amplified Chemiluminescence of Human Neutrophil Bursts and Electron Paramagnetic Resonance Spectroscopy.

A new diclofenac salt called diclofenac-choline (DC) has recently been proposed for the symptomatic treatment of oropharyngeal inflammatory processes and pain because its greater water solubility allows the use of high concentrations, which are useful when the contact time between the drug and the oropharyngeal mucosa is brief, as in the case of mouthwashes or spray formulations. The antioxidant activity of DC has not yet been investigated, and so the aim was to use luminol-amplified-chemiluminescence (LACL) to verify whether various concentrations of DC (1.48, 0.74 and 0.37 mg/mL for incubation times of 2, 4 and 8 min) interfere with oxygen and nitrogen radicals during the course of human neutrophils respiratory bursts; electron paramagnetic resonance (EPR) spectroscopy was used to investigate its direct antiradical (scavenger) activity. The EPR findings showed that DC has concentration-dependent scavenging activity against the ABTS, the DPPH, and the hydroxyl radicals, but no activity on superoxide anion, as has been previously reported in the case of other NSAIDs. LACL revealed an inhibitory effect that was statistically significant after only 2 min of incubation, and similar after 4 and 8 min. The effects on the peroxynitrite radical paralleled those observed in the previous test. High concentrations and short incubation times showed that there is no interference on PMN viability, and so the inhibitory findings must be attributed to the effect of the drug. The anti-inflammatory effects of DC cannot be attributed solely to the inhibition of prostaglandin synthesis, but its effects on free radicals and neutrophil bursts suggest that they may contribute to its final therapeutic effect.

In vitro and in vivo pharmacological role of TLQP-21, a VGF-derived peptide, in the regulation of rat gastric motor functions.

BACKGROUND AND PURPOSE: Vgf gene expression has been detected in various endocrine and neuronal cells in the gastrointestinal tract. In this study we investigated the pharmacological activity of different VGF-derived peptides. Among these, TLQP-21, corresponding to the 556-576 fragment of the protein was the unique active peptide, and its pharmacological profile was further studied. EXPERIMENTAL APPROACH: The effects of TLQP-21 were examined in vitro by smooth muscle contraction in isolated preparations from the rat gastrointestinal tract and, in vivo, by assessing gastric emptying in rats. Rat stomach tissues were also processed for immunohistochemical and biochemical characterization. KEY RESULTS: In rat longitudinal forestomach strips, TLQP-21 (100 nmol x L(-1)-10 micromol x L(-1)) concentration-dependently induced muscle contraction (in female rats, EC(50) = 0.47 micromol.L(-1), E(max): 85.7 +/- 7.9 and in male rats, 0.87 micromol x L(-1), E(max): 33.4 +/- 5.3; n = 8), by release of prostaglandin (PG)E(2) and PGF(2a) from the mucosal layer. This effect was significantly antagonized by indomethacin and selective inhibitors of either cyclooxygenase-1 (S560) or cyclooxygenase-2 (NS398). Immunostaining and biochemical studies confirmed the presence of VGF in the gastric neuronal cells. TLQP-21, injected i.c.v. (2-32 nmol per rat), significantly decreased gastric emptying by about 40%. This effect was significantly (P < 0.05) blocked by i.c.v. injection of indomethacin, suggesting that, also in vivo, this peptide acts in the brain stimulating PG release. CONCLUSIONS AND IMPLICATIONS: The present results demonstrate that this VGF-derived peptide plays a central and local role in the regulation of rat gastric motor functions.

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.

Ticlopidine prevents the formation but delays the healing of ethanol-induced gastric lesions in the rat.

The effects of acute or long-term oral ticlopidine administration in normal rat gastric mucosa or on gastric lesions induced by ethanol 50% (EtOH, 1 ml/rat, os) were examined and compared with those of acetylsalicylic acid (ASA). Ticlopidine does not affect gastric mucosal integrity either after acute (100 and 300 mg kg(-1)) or 1-week (100 mg kg(-1), die) oral administration. Ticlopidine (30-300 mg kg(-1), os) administered 1h before EtOH dose-dependently prevented the development of gastric haemorragic lesions. When ticlopidine was administered 1h after EtOH, it significantly (p<0.05) delays gastric lesions healing. Acute ASA (50 and 100 mg kg(-1), os) administration causes a mild irritant activity similar to that observed after 1 week of ASA (50 mg kg(-1), os/die) administration. In condition of mucosal damage, ASA does not modify either the induction or the healing of EtOH-induced gastric lesions. To assess the possible involvement of endogenous nitric oxide (NO) or prostaglandins (PG) in the gastric protective activity of ticlopidine, the rats were pretreated with an inhibitor of NO-synthesis, L-NAME (70 mg kg(-1), s.c.) or the inhibitor of PG synthesis, indomethacin (Indo, 10 mg kg(-1), s.c.). Indo, but not L-NAME, was able to significantly counteract the gastroprotective activity of ticlopidine against EtOH injury. Furthermore, ticlopidine increases (47%) gastric PGE(2) content in normal mucosa compared to the one detected in control rats, thus suggesting that endogenous PGs contribute to enhanced mucosal resistance by ticlopidine. These results indicate that ticlopidine exerts dual effects during the development and healing of gastric lesions induced by EtOH.

Evidence for a role of the GHS-R1a receptors in ghrelin inhibition of gastric acid secretion in the rat.

Ghrelin, the endogenous ligand of the GH secretagogue receptor (GHS-R) has been previously shown to inhibit gastric acid secretion in pylorus-ligated rats. Two isoforms of GHS-R have been identified: GHS-R(1a) and GHS-R(1b). The present study aimed: (i) to characterise the type of GHS-R involved in the central gastric inhibitory activity of ghrelin by using des-octanoyl ghrelin, and synthetic GHS-R(1a) agonist (EP1572) and antagonist (D-Lys(3)-GHRP-6) and (ii) to investigate the relationship between ghrelin and cortistatin (CST) in the control of gastric acid secretion by using the natural neuropeptide CST-14 and the synthetic octapeptide CST-8. The specific interactions of all the compounds with GHS-R(1a) were determined by comparing their ability to displace labelled ghrelin or somatostatin from its receptors on rat hypothalamic membranes or on rat cardiomyocyte, respectively. Intracerebroventricular administration of 0.01 and 1 nmol/rat des-octanoyl ghrelin did not affect gastric acid secretion in pylorus-ligated rats, whereas EP1572 either i.c.v. (0.01-1 nmol/rat) or i.p. (10 and 20 nmol/kg) inhibited acid gastric secretion. Preteatment with D-Lys(3)GHRP-6 (3 nmol/rat, i.c.v.) was able to remove the inhibitory action of ghrelin (0.01 nmol/rat, i.c.v.) on gastric acid volume and acid output, thus indicating that the type 1a GHS-R likely mediates the gastric inhibitory action of ghrelin. This is supported by binding data showing that D-Lys(3)GHRP-6, but not des-octanoyl ghrelin, binds to hypothalamic GHS-R. CST-14 (1 nmol/rat, i.c.v.) did not affect either basal or ghrelin inhibition of gastric acid secretion. CST-8 (1 nmol/rat, i.c.v.) was able to counteract the gastric ghrelin response. The observation that CST-14 binds both GHR-S and somatostatin receptors, whereas CST-8 specifically displaces only ghrelin binding, indicates that CST-8 behaves as a GHS-R(1a) antagonist.

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.

GH-releasing peptides and bone.

It has been extensively demonstrated that GH secretagogues (GHS) play a role in the regulation of bone metabolism in animals and humans. Unlike GH, administration of GHS does not increase bone resorption markers, suggesting that a mechanism exclusively linked to GH release cannot account for the effect of these compounds. On this line, we investigated the effect of GHS and ghrelin, the endogenous ligand of GHS receptors, on bone cells. We found that both hexarelin and ghrelin significantly stimulated cell proliferation and increased alkaline phosphatase and osteocalcin production in primary cultures of rat calvaria osteoblasts. In the same cells, we were able to detect the mRNA for the GHS receptor by RT-PCR and the corresponding protein by Western blot, indicating that ghrelin and GHS may bind and activate this receptor. Two isoforms of GHS receptors (GHS-R), which are presumably the result of alternate processing of pre-mRNA, have been identified and designed receptors 1a (R1a) and 1b (R1b). Ghrelin, the endogenous ligand of the GHS receptors, binds with high affinity GHS-R1a only. Unlike fetal calvaria cells, osteoblasts derived from adult rat tibia did not express the GHS-R1 a, but only the biologically inactive isoform GHS-R1b. The latter isoform was present in only one of the three specimens of human osteoblasts obtained from the iliac crest or the upper femur of patients during surgery. These results would indicate that only osteoblasts from fetal bone express functional receptors responsive to ghrelin and GHS.

Ghrelin in gastroenteric pathophysiology.

Ghrelin, an acylated peptide produced predominantly by the stomach, has been discovered to be a natural ligand of the growth hormone secretagogue receptor 1a (GHS-R1a). It is localized in distinct cells of the gastric mucosa, mainly distributed in the mid portion of the oxyntic gland characterized by P/D1 granules in man and X/A-like granules in rodents. The ghrelin cell represents the second most frequent endocrine cell type after the enterochromaffin-like cells in gastric oxyntic mucosa, pointing to a potentially relevant role in the physiology of the stomach. Ghrelin has no relevant homology with any known gastrointestinal peptide and displays strong GH-releasing activity both in animals and in humans. However, in addition to stimulating GH secretion, ghrelin possesses several other endocrine and extraendocrine biological activities that are explained by the widespread distribution of ghrelin and GHS-R1a expression. In the rat, ghrelin exerts a control in gastric acid secretion and motility: the gastric acid secretion is stimulated by peripheral administration of high doses of ghrelin, but inhibited by very low doses of ghrelin delivered into the central nervous system. Moreover, ghrelin provides a potent and dose-related gastroprotective action against ethanol- and stress-induced gastric ulcers. The integrity of both nitric oxide (NO) system and capsaicin afferent nerves are required for the gastroprotective effect of ghrelin, whereas the vagus nerve might be involved in conveying ghrelinergic signal from periphery to the brain. In addition, prostaglandins derived by the constitutive cyclooxygenase (COX) activity are essential for the protective activity of ghrelin in ethanol and stress-induced gastric lesions. Given its prevailing role in physiological and pathophysiological gastric function, the discovery of ghrelin will open new perspectives and potential clinical implications in the gastroenteric field.

Effects of hexarelin against acid-independent and acid-dependent ulcerogens in the rat.

The effects of intracerebroventricular (icv) or subcutaneous (sc) hexarelin (Hexa) administration, against gastric ulcers induced by ethanol (50%, 1 ml/rat/os) or Indomethacin (20 mg/kg/os) were examined in conscious rats. Hexa at 1 nmol/rat, icv or 10 nmol/kg, sc reduced ethanol-induced ulcers by 47% and 32% respectively. Hexa, but not ghrelin significantly worsened (+40%) Indomethacin-induced ulcers when injected sc. Hexa-gastroprotection against ethanol-induced ulcers was removed by the GHS-R antagonist (D-Lys3)-GRPR-6 and by the inhibitor of NO-synthase (NOS) Nomega-nitro-L-arginine methyl ester. Semiquantitative RT-PCR assay of gastric NOS mRNA isoforms revealed that the reduction in iNOS-derived NO and the increase of constitutive-derived NO are relevant for the gastroprotection of Hexa against ethanol-induced gastric damage.

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.

Ghrelin protects against ethanol-induced gastric ulcers in rats: studies on the mechanisms of action.

Ghrelin, the endogenous ligand for GH secretagogue receptors, has been reported to influence acid gastric secretion and motility, but its potential gastroprotective effect is unknown. The aims of this study were 1) to examine the effects of central and peripheral administration of ghrelin on ethanol-induced gastric ulcers in conscious rats, and 2) to investigate the possible roles of nitric oxide (NO), vagal nerve, and sensory fibers in the gastric effects of ghrelin. Ghrelin was administered either intracerebroventricularly or sc 30 min before ethanol, and mucosal lesions were examined macroscopically. Additionally, rats were either treated with the inhibitor of NO synthesis N(omega)-nitro-L-arginine methyl ester (L-NAME) or underwent bilateral cervical vagotomy or capsaicin-induced sensory denervation. Conventional histology and immunohistochemistry for ghrelin, gastrin, and somatostatin were performed on gastric specimens from representative rats. Central ghrelin (4-4,000 ng/rat) dose-dependently reduced ethanol-induced gastric ulcers by 39-77%. Subcutaneous ghrelin administration (80 micro g/kg) reduced ulcer depth only. L-NAME and capsaicin, but not vagotomy, prevented the gastroprotective effect of central ghrelin (4000 ng/rat). This is the first evidence that ghrelin exerts a potent central gastroprotective activity against ethanol-induced lesions. The gastroprotective effect of ghrelin is mediated by endogenous NO release and requires the integrity of sensory nerve fibers.

Bone effects of hexarelin, a GH-releasing peptide, in female rats: influence of estrogen milieu.

OBJECTIVE: The present study was performed to evaluate the potential influence of the estrogen milieu in modulating the effects of GH/IGF stimulation by a GH-releasing peptide, hexarelin (HEXA), on bone metabolism and mineral density in middle-aged female rats. METHODS: HEXA was administered for 60 days (50 microg/kg s.c. twice a day) to intact and ovariectomized (OVX) 11-month-old female rats and changes in bone parameters were evaluated with respect to those of the same rats under baseline conditions and with those of control rats (intact and OVX) administered isovolumetric amounts of physiological saline. Serum total alkaline phosphatase (ALP) and urinary deoxypyridinoline (Dpd) were measured before and at various times during HEXA treatment. Bone mineral content (BMC) and density of lumbar vertebrae and femoral mid-diaphyses were measured by dual energy X-ray absorptiometry before and after treatment. In all groups, serum IGF-I levels were determined before and during treatment and the GH secretory response to HEXA was assessed at the end of the experiment. RESULTS: In intact rats, HEXA did not modify Dpd urinary excretion, induced a trend toward an increase of serum ALP activity and significantly increased BMC (+6.5%) and bone area (+4.1%) only at lumbar vertebrae. In OVX rats, HEXA did not modify the OVX-induced increase in bone turnover markers (Dpd and ALP) and did not affect the OVX-induced vertebral bone loss, but significantly increased BMC (+7.2%) and bone area (+5.3%) at femoral mid-diaphyses. HEXA significantly increased serum IGF-I levels at day 14, but not at day 60, in both intact and OVX rats, whereas the GH secretory response to HEXA was higher in the former than in the latter. CONCLUSIONS: Overall, the present data demonstrate that chronic HEXA treatment increases BMC and bone area at lumbar vertebrae in intact rats and at femoral diaphyses in OVX rats. The different sensitivity to HEXA of the skeletal districts examined is related to the estrogen milieu and may reflect a complex interplay between estrogens and GH/IGF function.

Evidence for a central inhibitory role of growth hormone secretagogues and ghrelin on gastric acid secretion in conscious rats.

We examined the possible central and peripheral effects of synthetic growth hormone secretagogues (GHS), hexarelin (Hexa) and EP 40737 (D-Thr-D-Trp (2-Me)-Ala- Trp-D-Phe-Lys-NH2), and of their endogenous counterpart, ghrelin, on gastric acid secretion. The compounds were administered intracerebroventricularly (i.c.v.) or subcutaneously (s.c.) in conscious male rats and the volume of gastric secretion and gastric acid output were examined 3 h after pylorus ligation (Shay-test). Central Hexa, EP 40737 and ghrelin administration (from 0.1 pmol to 1 nmol/rat, i.c.v.) significantly inhibited gastric acid secretion. The maximum inhibitory effect on gastric acid output was detected at the dose of 10 pmol/rat, i.c.v. for Hexa (-51.3%), of 100 pmol/rat, i.c.v. for EP 40737 (-70%) and of 1 pmol/rat, i.c.v. for ghrelin (-60%). All peptides were less effective at the highest dose used (1 nmol/rat, i.c.v.). Hexa, EP 40737 and ghrelin injected s.c. did not modify gastric acid secretion. The inhibitory action of Hexa on gastric acid secretion seems to involve brain somatostatinergic system since Hexa (10 pmol/rat, i.c.v.) did not inhibit gastric acid secretion in rats pretreated (4 h before) with cysteamine (300 mg/kg, s.c.), a depletor of endogenous somatostatin. These results show that synthetic GHS and ghrelin exert a central long-lasting inhibitory effect on gastric acid secretion in conscious pylorus-ligated rats. The fact that very low doses of ghrelin and GHS inhibit gastric secretion, provide evidence for a tonic inhibitory role of the peptides in the central control of gastric secretory function.

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.

Amylin compared with calcitonin: competitive binding studies in rat brain and antinociceptive activity.

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.

The influence of sex and gonadectomy on the growth hormone and corticosterone response to hexarelin in the rat.

The present study is designed to investigate the role of sex and gonadal status in the growth hormone (GH) and corticosterone response to hexarelin (HEXA), a GH-releasing peptide, which also causes a stimulatory action on the hypothalamic-pituitary-adrenal (HPA) axis. HEXA (80 microg/Kg) was administered intracarotid to anesthetized intact or gonadectomized male (ORC) and female (OVX) middle-aged rats. The GH stimulatory response to HEXA was gender-related since the GH increase was significantly (p < 0.001) higher in intact males (area under the curve, AUC= 12560 +/- 1784 ng/ml.45 min) than in females (AUC= 4628 +/- 257 ng/ml.45 min). This sex difference does not depend on circulating gonadal steroids since it persists in ORC (AUC = 11980 +/- 1136 ng/ml.45 min) and OVX (AUC = 5539 +/- 614 ng/ml.45 min) rats. The different effects of HEXA on corticosterone secretion detected in male and female rats are probably dependent on the prevailing activity of the HPA axis. In fact, in male rats that have low basal corticosterone levels, HEXA caused an increase in corticosterone secretion, which was significantly (p< 0.05) higher in ORC than in intact rats. The increase in corticosterone secretion by HEXA both in intact and OVX females was delayed, probably due to the elevated initial corticosterone levels, which could have activated the glucocorticoid negative feedback. We suggest that gender-specific patterns in the regulation of the hypothalamus-pituitary function could be responsible for the GH and corticosterone sexually differentiated responses to HEXA.

Hexarelin, a growth hormone - releasing peptide, counteracts bone loss in gonadectomized male rats.

The age-related decline in growth hormone (GH) secretion has been implicated in the pathogenesis of involutional bone loss. Whether restoration of GH secretion might be helpful in maintaining and/or improving bone mass during aging is still unsettled. The aim of the present study was to examine the effects of 30-day treatment with hexarelin (HEXA, 50 microg/kg subcutaneously b.i.d.), a highly effective GH-releasing compound, on bone metabolism and bone mineral density (BMD) in intact and osteopenic gonadectomized (GDX) mature male rats. Serum total alkaline phosphatase (ALP, bone formation marker) and bone resorption markers (lysylpyridinoline, LP and hydroxylysylpyridinoline, HP) were measured before and 7, 14 and 30 days after treatment. BMD was measured by dual-energy X-ray absorptiometry at lumbar vertebrae, femoral metaphysis and diaphysis before and at the end of the experiment. In intact rats, HEXA significantly (P<0.05) decreased LP (-36.3%) and HP (-22.8%) excretion at day 7, whereas it did not change serum ALP activity and BMDs. In GDX rats, HEXA completely prevented the significant (P<0. 01) increase in urinary excretion of both LP (+143.8%) and HP (+119. 4%), the early decrease in ALP activity (-26.5%) and the significant (P<0.05) decrease in BMDs in the femoral metaphysis (-7.9%) and lumbar vertebrae (-6.8%) caused by androgen deficiency. The bone-protective effects of HEXA could be attributed, at least in part, to its GH-releasing activity since chronic-treated rats maintained the GH response to an acute challenge with HEXA. The evidence that HEXA, unlike GH, inhibits bone resorption indicates that other mechanisms contribute to the bone sparing effect of HEXA.