Invited review: Dairy intake and bone health: a viewpoint from the state of the art.

The aim of this review was to focus on the complex relationships between milk and dairy products intake and bone health, with particular emphasis on osteoporosis. The literature was extensively examined to provide an objective overview of the most significant achievements on the subject. Osteoporosis can be defined as a disease characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk. Although the major determinants of peak bone mass and strength are genetic, major factors during childhood and adolescence may affect the ability to achieve peak bone mass. These include nutrition, particularly calcium and protein intake, physical activity, endocrine status, as well as exposure to a wide variety of risk factors. The role of calcium intake in determining bone mineral mass is well recognized to be the most critical nutritional factor to achieve optimal peak bone mass. The greatest amount of dietary calcium is obtained from milk and dairy foods, which also provide the human diet with vitamin D (particularly for products fortified with vitamin D), potassium, and other macro- and micronutrients. Although studies supporting the beneficial effects of milk or calcium on bone health are predominant in the literature, perplexity or discordance on this subject was expressed by some authors. Discordant data, mainly on the risk of fractures, provided limited proof of the unfavorable effect of dairy intake. More often, discordant works indicate no effect of dairy consumption on bone safety. Some considerations can be drawn from this viewpoint. Milk and dairy products are an optimal source of calcium as well as of other limiting nutrients (e.g., potassium and magnesium), with important effects on bone health. Bioactive components occurring in milk and dairy products may play an essential role on bone metabolism, as shown by in vivo and in vitro studies on colostrum acidic proteins and milk basic proteins. Calcium intake positively affects bone mass and is crucial in childhood and youth for correct bone development. In elderly people, calcium intake as well as vitamin D availability should be carefully checked. As a general conclusion, calcium is essential for bone health, although it will not prevent bone loss due to other factors; in this context, milk and dairy foods are bioavailable, relatively inexpensive sources of calcium for the human diet.

Improving patient waiting time of centralized front office service in a regional hub hospital using the discrete event simulation model.

BACKGROUND: Today, hospital rankings are based not only on basic clinical indicators, but even on quality service indicators such as patient waiting times. Improving these indicators is a very important issue for hospital management, so finding a solution to achieve it in a simple and effective way is one of the greatest goals. OBJECTIVES: The aim of this article is to evaluate the use of a discrete event simulation model to improve healthcare processes and reduce waiting time of patients and hospital costs. METHODS: The case study proposed in this paper is the reorganization of non-clinical front office operation for the patients (i.e. booking of exams, delivering medical reports, etc.) of the Careggi University Hospital of Florence, to optimize the utilization of the human resources and to improve performances of the process. RESULTS: The development and validation of the model was made according to an analysis of real processes and data, pre and post implementation of model outcomes. The new organization shows a decrease of waiting times from an average value of 10 minutes and 37 seconds to 5 minutes and 57 seconds (-44%). CONCLUSIONS: This paper shows that discrete event simulation could be a precise, cost-limited tool to optimize hospital processes and performance.

Feeding behavior during long-term hexarelin administration in young and old rats.

Ghrelin, a 28-amino-acid peptide isolated from the stomach, is the natural ligand of the GH-secretagogues receptor-1a (GHS-R1a) and, so far, the only discovered circulating appetite-stimulating hormone. Similarly to ghrelin, many synthetic compounds belonging to the GHS family stimulate both GH secretion and feeding, whereas some stimulate GH secretion only. In the past years, studies have focused on the potential of the GHS to stimulate GH release during long-term treatment in humans and experimental animals. Few data are available about the extraendocrine effects of the GHS during several weeks of treatment, particularly in old rats. The aim of the present study was first to identify the lowest dose of hexarelin giving maximal stimulation of food intake both in young (3-month-old) and old rats (24-month-old). A dose-response study (80-320 microg/kg, s.c.) revealed that hexarelin at the dose of 80 microg/kg gave reproducibly maximal stimulation of food consumption in young as well as in old rats. Second, we evaluated the effect of 8-week daily sc treatment with hexarelin in young and old male rats. The outcome of the chronic study was that hexarelin (80 microg/kg, s.c., once daily) maintained a persistent significant orexigenic action throughout the treatment period, both in young and old rats. Interestingly, hexarelin treatment did not affect body weight gain either in young or old rats. We conclude that hexarelin is endowed with long-lasting orexigenic activity and might represent a potential therapeutic approach for pathological conditions characterized by a decline in food intake.

Deranged anterior pituitary responsiveness to hypothalamic hormones in depressed patients.

Abnormal anterior pituitary (AP) responsiveness to acute administration of thyrotropin-releasing hormone (TRH) and luteinizing hormone-follicle stimulating hormone-releasing hormone (LH-RH) was investigated in 14 patients (two men and 12 women) suffering from primary affective disorders. In ten, TRH, 500 microgram given intravenously, induced a rise in plasma growth hormone (GH) level, while in eight patients it induced a rise in plasma levels of FSH or LH or both. When LH-RH, 150 microgram was administered intravenously to ten patients, it induced a rise in plasma GH level in one patient and increased plasma prolactin level in three patients. Collectively, in only three of 14 patients was conventional AP responsiveness to hypothalamic neurohormones present. These findings demonstrate the existence of a profound derangement of AP responsiveness to hypothalamic neurohormones in depressed patients and suggest that a primary alteration in the physiologic links between the central nervous system and the AP may be at the origin of the neuroendocrine disturbance.

Role of AMP-activated protein kinase activators in antiproliferative multi-drug pituitary tumour therapies: effects of combined treatments with compounds affecting the mTOR-p70S6 kinase axis in cultured pituitary tumour cells.

AMP-activated protein kinase (AMPK) is activated under conditions that deplete cellular ATP levels and elevate AMP levels. We have recently shown that AMPK can represent a valid target for improving the medical treatment of growth hormone (GH)-secreting pituitary adenomas and the effects of its activation or inhibition in pituitary tumour cells are worthy of further characterisation. We aimed to determine whether AMPK may have a role in combined antiproliferative therapies based on multiple drugs targeting cell anabolic functions at different levels in pituitary tumour cells to overcome the risk of cell growth escape phenomena. Accordingly, we tried to determine whether a rationale exists in combining compounds activating AMPK with compounds targeting the phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR/p70S6K signalling pathway. AMPK down-regulation by specific small-interfering RNAs confirmed that activated AMPK had a role in restraining growth of GH3 cells. Hence, we compared the effects of compounds directly targeting the mTOR-p70S6K axis, namely the mTOR inhibitor rapamycin and the p70S6K inhibitor PF-4708671, with the effects of the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on cell signalling and cell growth, in rat pituitary GH3 cells. AICAR was able to reduce growth factor-induced p70S6K activity, as shown by the decrease of phospho-p70S6K levels. However, it was far less effective than rapamycin and PF-4708671. We observed significant differences between the growth inhibitory effects of the three compounds in GH3 and GH1 cells. Interestingly, PF-4708671 was devoid of any effect. AICAR was at least as effective as rapamycin and the co-treatment was more effective than single treatments. AICAR induced apoptosis of GH3 cells, whereas rapamycin caused preferentially a decrease of cell proliferation. Finally, AICAR and rapamycin differed in their actions on growth factor-induced extracellular signal regulated kinase 1/2 phosphorylation. In conclusion, the results of the present study suggest the increased efficacy of combined antiproliferative therapies, including rapamycin analogues and AMPK activators in GH-secreting pituitary tumours, as a result of complementary and only partially overlapping mechanisms of action.

Characterization of new selective somatostatin receptor subtype-2 (sst2) antagonists, BIM-23627 and BIM-23454. Effects of BIM-23627 on GH release in anesthetized male rats after short-term high-dose dexamethasone treatment.

We here report a pharmacological characterization of two new somatostatin (SS) receptor subtype-2 (sst2) selective antagonists by evaluating their GH-releasing activity when administered, by different routes, in anesthetized adult rats and in freely moving 10-d-old rats. Moreover, we describe the effect of these SS antagonists on the GH response to GHRH after short-term high-dose dexamethasone (DEX) treatment in young male rats. BIM-23454 and BIM-23627, given iv, were able to counteract the SS-induced inhibition of GH secretion occurring after urethane anesthesia in a dose-dependent manner. In DEX-treated animals, the GH response to GHRH was partially blunted (5-min peak values, 270 +/- 50 ng/ml in saline-treated vs. 160 +/- 10 ng/ml in DEX-treated, P < 0.05); however, the simultaneous administration of BIM-23627 (0.2 mg/kg, iv) restored higher amplitude GH pulse, leading to a significantly higher overall mean GH response (area under the curve, 4200 +/- 120 ng/ml/30 min vs. 2800 +/- 100 ng/ml/30 min after GHRH alone; P < 0.05). The SS antagonists showed a reduced GH-releasing effect when administered sc or ip, likely attributable to decreased bioavailability, as compared with the iv route. SS antagonist administration also increased plasma glucagon, insulin, and glucose levels. Based on prior reports that sst2 tonically suppresses glucagon secretion, the antagonist most likely increased glucagon secretion from the pancreatic alpha-cells, with resultant increases in plasma glucose and then insulin.

Different regulation of growth hormone-releasing factor-sensitive adenylate cyclase in the anterior pituitary of young and aged rats.

Low basal GH secretion and reduced GH responsiveness to different GH secretagogues, including GHRF, have been reported in aged animals and humans. Parallel to the in vivo findings, an impaired GH responsiveness to GHRF is evident in somatotropes from old rats of either sex. We report here that in anterior pituitaries (APs) from aged male and female rats GHRF-induced stimulation of adenylate cyclase (AC) activity was strikingly reduced (male rats, change from baseline 700% in young and 100% in old rats) or lacking (female rats, change from baseline 430% in young and 13% in old rats) when compared to that evoked by GHRF in the APs from young counterparts. Pretreatment with GHRH (5 micrograms/rat iv for 3 days) decreased the high basal AC activity of old male rats [from 33.38 +/- 3.60 to 15.99 +/- 5.75 (SEM) pmol cAMP/min.mg protein], did not alter the GHRF-stimulated rise in AC activity in old male rats, and induced a small but unequivocal rise in AC activity in old female rats (change from baseline 35% vs. 13%, respectively). Pretreatment with GHRF markedly reduced the acute effect of GHRF in the APs from young rats of both sexes (male rats, change from baseline 360% and 700%; female rats, change from baseline 230% and 430% in GHRF-pretreated and control rats, respectively). In parallel studies performed in female rats, it was shown that in vivo pretreatment with GHRF at the same schedule markedly reduced the effect of acute GHRF stimulation on GH secretion from cultured pituitary cells of young rats but left unchanged GHRF-induced stimulation of GH secretion from pituitary cells of old rats. In all, these data suggest that deficiency of endogenous GHRF synthesis and/or release may underlie defective GH secretion in old rats and that a GHRF replacement regimen that reduces the sensitivity of the young somatotrope cells does not alter the sensitivity of (male rats) or exerts a priming effect (female rats) on the old somatotrope cell.

A role for dopamine in growth hormone regulation in the dog.

The role of dopamine (DA) in the secretion of GH in most animal species is still controversial. We examined in the dog the effect on GH release of nomifensine, a drug which activates both noradrenergic and dopaminergic neurotransmission. Nomifensine (0.4-2.8 mg/kg, iv), administered into 12 unanesthetized male and female beagles, induced short-dose-related rises in canine GH (cGH) levels. Blockade of alpha-adrenergic receptors by phentolamine prevented the GH stimulatory effect of 2.8 mg/kg nomifensine, and an iv bolus injection of haloperidol (a neuroleptic which antagonizes both norepinephrine and DA receptor function) given 45 min before was equally effective. Selective blockade of DA receptors by pimozide significantly reduced the GH-releasing effect of nomifensine. In sum, these data indicated that the effect of nomifensine was the consequence of an enhanced noradrenergic and dopaminergic-neurotransmission. Pretreatment with domperidone, a DA receptor blocker unable to cross the blood-brain barrier, failed to modify the GH-releasing effect of nomifensine, suggesting that the DA component subserving the neuroendocrine effect of the drug lies within the blood-brain barrier. Further evidence for a stimulatory role of DA on GH release was the fact that apomorphine, a direct stimulant of DA receptors, induced a rise in cGH levels when administered to dogs pretreated with domperidone. The latter drug was used to prevent emesis and distress due to activation of peripheral DA receptors by apomorphine. However, apomorphine was only active in the dog at doses (250 and 500 microgram/kg, sc) greatly exceeding those active in releasing GH in man, suggesting that the role of DA in cGH regulation is ancillary to that exerted by noradrenergic neurotransmission. In a final study, atropine, a muscarinic cholinergic receptor antagonist, abolished the neuroendocrine effect of nomifensine, a finding which suggests that cholinergic medication plays an important role in cGH regulation.

Role of central nervous system-derived or circulating gamma-aminobutyric acid on prolactin secretion in the rat.

To investigate the respective role in PRL secretion of gamma-aminobutyric acid (GABA), either derived from the central nervous system or circulating in plasma, experiments were performed using ethanolamine-O-sulfate (EOS), a specific inhibitor of GABA catabolism. Intracerebroventricular injection of EOS (2 mg/kg) induced in unanesthetized male rats 2-8 h post injection a clear-cut rise in hypothalamic, anterior pituitary (AP), and plasma GABA concentrations. Rises in GABA titers occurred earlier in the hypothalamus and AP (2 h) than in the plasma (4 h). Concomitant to alterations of GABA, there was a striking lowering of plasma PRL evident at 2 h and still present 24 h after EOS administration. In contrast, systemic administration of graded doses of EOS (200-400 mg/kg, iv) did not induce significant changes in plasma GABA concentrations 4 h post injection; only the 600 mg/kg dose of EOS increased GABA concentrations 4 h post injection in the hypothalamo-AP system and decreased plasma PRL concentrations. Finally, in hypophysectomized rats bearing ecotopic pituitaries, despite the occurrence of rises in the hypothalamic GABA after intracerebroventricular or systemic (600 mg/kg) administration of EOS, AP, plasma GABA, and plasma PRL concentrations were not altered. In all these findings indicate that: 1) changes in plasma PRL are best correlated to variations in the amino acid titers occurring in the hypothalamo-AP systems; and 2) circulating GABA does not play a functional role in the control of PRL secretion. Finally, since alterations in blood GABA levels after central or systemic administration of EOS appear to reflect primary changes occurring in the brain concentration of the amino acid, circulating GABA may be a reliable indicator of central nervous system GABAergic function.

Proof for histaminergic but not for adrenergic involvement in the growth hormone-releasing effect of an enkephalin analog in the dog.

A series of studies was performed in unanesthetized dogs to ascertain whether, in addition to cholinergic pathways, other neurotransmitter systems were involved in the GH-releasing effect of the potent enkephalin analog [D-Ala2, MePhe4, Met(o)5-ol]enkephalin (DAMME). DAMME at a dose of 8 microgram/kg iv elicited a striking rise in plasma canine GH (cGH), with peak levels at 30 min. Blockade of histaminergic H1 receptors by diphenhydramine (30 mg,iv, 15 min before) or clemastine (1 mg orally three times for 2 days and 2 mg orally 60 min before) completely suppressed the cGH release induced by DAMME without significantly altering baseline cGH levels. A slight reduction of the effect of DAMME was also induced by the histamine H2 receptor antagonist cimetidine (300 mg, iv, 15 min before). Pretreatment with the alpha-adrenergic inhibitor phentolamine (0.4 mg/min for 45 min) did not alter the neuroendocrine effect of DAMME, despite the occurrence of a rise in blood glucose (peak levels, 185 +/- 47 mg/dl). The administration of propranolol, a blocker of beta-receptors, did not potentiate the cGH release induced by a threshold dose of DAMME (4 microgram/kg, iv). An iv bolus injection of glucose (1 g/kg), which induced peak glucose levels of 296 +/- 29 mg/dl, completely suppressed the cGH release induced by DAMME or propranolol plus DAMME. These results indicate that histaminergic H1 receptors play an important role in the cGH release induced by DAMME, whereas this action occurs independently from adrenergic mediation. Based on these and previous findings, a neuromodulator role in GH-releasing mechanisms is suggested for opioid peptides.

Stimulation of growth hormone release by thyrotropin-releasing hormone in the hypophysectomized rat bearing an ectopic pituitary.

Hypophysectomized female rats which received renal grafts of anterior pituitary (AP) or weight-matched intact controls were sampled under urethane anesthesia. Plasma growth hormone (GH) in sequential samples from each rat was measured by radioimmunoassay to determine the effect of exogenous thyrotropin-releasing hormone (TRH) on GH release from ectopic or intact AP. In a first experiment, following a baseline sample, a pre-treatment sample was taken from each rat 30 min after urethane injection, after which TRH (0.3 or 0.6 mug) or isotonic saline was injected iv, and samples were taken at 10 and 30 min post-treatment. Baseline GH levels in hypophysectomized-transplanted rats were in the range of 4.0 to 8.0 ng/ml, and were not modified significantly by urethane. TRH caused a significantly greater increase in growth hormone at 10 min than did saline. Plasma GH tended to be higher at 30 min post-treatment only in the 0.6 mug TRH-treated group. In further experiments the above described protocol was followed except that four doses of TRH were used (0.15, 0.3, 0.6, and 1.2 mug) and post-TRH blood samples were taken at 5 and 10 min. TRH caused a clear-cut increase in plasma GH both at 5 and 10 min, although no dose-effect relationship was present. In intact controls, baseline GH levels were in the range 40.0 to 80.0 ng/ml and were drastically reduced by urethane. In these animals, only the 1.2 mug TRH dose induced a GH rise at 5 and 10 min. In similar experiments, iv administration of vasopressin (100, 200, or 400 mU) induced a rise in plasma GH when given to the hypohysectomized-transplanted rats, but was ineffective in intact controls; the administration of prostaglandin E2 (5.0 and 50.0 mug) increased plasma GH in both experimental conditions. The results indicate that TRH in the hypophysectomized-transplanted rat acts directly on the AP tissue to increase GH release and that the ectopic pituitary is more susceptible than the in situ pituitary to some GH-releasing stimuli.

Growth hormone releasing activity of thyrotropin-releasing hormone in rats with hypothalamic lesions.

The growth hormone (GH)-releasing effect of thyrotropin-releasing hormone (TRH) was investigated in rats in which central nervous system (CNS)-anterior pituitary (AP) connections had been experimentally interrupted. Sprague-Dawley (SD) female and male rats, underwent bilateral electrolytic lesions in the median eminence (ME) or the ventromedial nuclei (VMN) or were sham-operated (sham-op). Fifteen days after surgery, 0.9% NAACl or TRH was injected iv into sham-op rats or those with lesions in the CNS, anesthetized with urethane, and blood was drawn at 5 and 10 min posttreatment. In the rats with ME lesions, TRH at all the doses used (0.1, 0.4 and 0.8 microng/100 g BW) induced a marked, although not dose-related GH rise, which was not present in sham-op rats after TRH, or after NaCl administration to either rats with ME lesions or sham-op rats. In SD male rats lesioned in the VMN, TRH at doses of 0.4 and 0.8 microng/100 g BW induced significant GH rises, while the lowest TRH dose (0.1 microng/100 g BW) was ineffective; again, TRH was ineffective at all doses used in sham-op rats. Concomitant evaluation of the prolactin (PRL)-releasing effect of TRH (0.1-0.8 microng/100 g BW), showed a striking elevation of plasma PRL in both female and male sham-op controls, but no PRL rise in the rats with ME lesions. The results reveal that in the rat with surgical separation of the anterior pituitary from the CNS, a direct GH-releasing effect of TRH can be obtained, whereas its PRL-releasing effect is no longer observed, and suggest that, by analogy, the GH-releasing effect of TRH present in some disease states of the human may be due to an impairment of CNS-AP connections.

Anomalous growth hormone responses to thyrotropin-releasing hormone and glucose in cirrhotic patients: the effect of metergoline.

The anomalous GH response to TRH or glucose loading was evaluated in patients with severe liver disease untreated or pretreated with metergoline (MCE), a potent antiserotoninergic drug. In 9 patients, injection of 400 micrograms TRH as a bolus induced a clear-cut GH rise (> 8 ng/ml), with peak levels 15-90 min post injection. Pretreatment with MCE did not modify baseline GH levels but potentiated the TRH-induced GH rise in 4 patients. In addition, 2 of 8 TRH nonresponder patients developed the anomalous GH response after MCE pretreatment. Like MCE, methysergide, another antiserotoninergic drug, potentiated the TRH-induced GH rise in 2 of 4 patients. Glucose administration (100 g, orally) induced a paradoxical rise of GH in 9 of 10 patients; after MCE, the paradoxical GH response to glucose was potentiated in 6 patients. These data provide the first experimental evidence that a derangement in brain monoamine (serotonin) function is actually involved in the occurrence of anomalous GH responses in subjects with severe liver disease.

Growth hormone and prolactin responses to thyrotropin-releasing hormone in patients with severe liver disease.

Thyrotropin-releasing hormone (TRH) was administered iv to 10 patients with severe liver disease and 10 control subjects. Injection of 400 microgram TRH as a bolus induced in 7 out of 10 patients a clear-cut GH rise (larger than or equal 10 ng/ml) occurring 15-120 min after the injection, and no effect on GH levels in controls. Mean baseline GH levels wre higher in patients than in controls. An exaggerated and sustained PRL rise was present after TRH in the subjects with liver disease, whose mean baseline plasma PRL levels were within normal range.

Altered growth hormone and prolactin responses to dopaminergic stimulation in Huntington's chorea.

Seven patients affected by Huntington's chorea were given an acute administration of 2-Br-alpha-ergocryptine (CB 154, Sandoz), a direct agonist at dopamine receptor sites. Seven nonobese hospitalized patients were used as controls. Oral administration of CB 154 (2.5 mg) induced a more prompt and consistent rise in plasma growth hormone (GH) levels in patients than in controls. GH levels rose from baseline values of 0.3+/-0.1 ng/ml to mean peak values of 20.4+/-5.1 ng/ml (120-270 min) in choreic subjects and from baseline values of 1.0+/-0.4 ng/ml to mean peak values of 5.7+/-1.6 ng/ml (180-300 min) in control subjects (P less than 0.02). Baseline plasma prolactin (PRL) values were significantly higher in choreic than in control subjects (22.1+/-6.6 ng/ml vs. 8.1+/-1.4 ng/ml, respectively, P less than 0.02); administration of CB 154 induced a more consistent PRL decrease in control than in choreic subjects. Collectively, these results suggest the existence of an abnormal regulation of GH and PRL secretion in Huntington's chorea, probably due to alterations in central dopaminergic neurotransmission.

Alpha 2-adrenoceptor sensitivity in anorexia nervosa: GH response to clonidine or GHRH stimulation.

Growth hormone (GH) response to clonidine and growth hormone-releasing hormone (GHRH) stimulation, together with baseline somatomedin C (SmC) levels, were examined in parallel in a group of 21 patients with anorexia nervosa (AN) and in 10 controls. In addition, the Hamilton Rating Scale for Depression (HRS) was administered to the patients. Clonidine (2.5 micrograms/kg body weight, iv) induced GH elevations that were not significantly different between patients and controls. In contrast, GHRH (1 microgram/kg body weight, iv) produced a significantly higher GH response in anorectics than in controls. The ratio between GH responses (area under the curve, or AUC) to GHRH and to clonidine was significantly higher in patients than in controls. Baseline SmC levels (6 patients) were significantly lower in anorectics than in controls. Minor depressive symptomatology was present in all patients. When viewed in relation to the GH hyperresponsiveness to GHRH, the apparent normality of the response to clonidine in anorectics reflects the existence of an actual alpha 2-adrenoceptor subsensitivity. As clonidine reportedly acts via release of endogenous GHRH, an excessive, rather than a normal, GH response to clonidine was to be anticipated.

Feedback effects of growth hormone on growth hormone-releasing hormone and somatostatin are not evident in aged rats.

In 8- and 20-month-old male rats, treated or not with growth hormone (GH) for 4 days, simultaneous evaluation of hypothalamic GH-releasing hormone (GHRH) and somatostatin (SS) gene expression, GH secretion from anterior pituitaries (APs) in vitro (basal and GHRH-stimulated) and plasma IGF-I levels was performed. Twenty-month-old rats showed decreased GHRH mRNA levels, decreased GH secretion from APs in vitro (not responsive to GHRH stimulation) and reduced plasma IGF-I levels as compared to younger counterparts. SS mRNA levels were only slightly reduced in the hypothalamus of aged rats. Short-term administration of biosynthetic human GH (125 microgram/rat, twice daily, IP) to 8-month-old rats abolished the in vitro GHRH-stimulated GH release from APs and altered GH regulatory neuropeptides gene expression, i.e., reducing GHRH mRNA levels and increasing SS mRNA levels. In 20-month-old rats, hGH administration increased plasma IGF-I levels but did not change significantly GHRH and SS gene expression. These data indicate that the feedback effects exerted by circulating GH on GHRH and SS neurons, while evident in adult rats, are not detectable in aged rats.

Higher content of insulin-like growth factor-I in dystrophic mdx mouse: potential role in the spontaneous regeneration through an electrophysiological investigation of muscle function.

Insulin-like growth factor-I (IGF-I) is known to promote proliferation and differentiation of muscle cells during growth and regeneration. Both these conditions are characterized by acquisition of specialized muscle functions, such as a large macroscopic chloride conductance (GCl), a parameter that is a target of growth hormone (GH)/IGF-I axis action on skeletal muscle. The present study has been aimed at evaluating the role of IGF-I in the spontaneous regeneration occurring in hind limb muscle of dystrophic mdx mouse. IGF-I levels have been measured in hind limb muscles, plasma and liver of mdx and control mice of 8-10 weeks and 5 months of age by radioimmunoassay. In parallel the biophysical and pharmacological properties of muscle chloride channels of extensor digitorum longus (EDL) muscle fibers of mice belonging to the same age-group have been measured electrophysiologically in vitro. At 8-10 weeks of age, significantly greater amounts of IGF-I were found in plasma and hind limb muscles of mdx mice with respect to controls. Such a difference was only just detectable and no longer statistically significant at 5 months of age. No differences were found in hepatic IGF-I levels at either age. The EDL muscle fibers of mdx mice at 8-10 weeks of age were characterized by higher GCl values and by a different pharmacological sensitivity to the enantiomers of 2-(p-chlorophenoxy)-propionic acid (CPP), specific chloride channel ligands, with respect to age-matched controls. However, these differences were no longer detected at 5 months of age. Our results suggest a role of IGF-I in the high regenerative potential of muscles from mdx mice and support the hypothesis that the biophysical and pharmacological properties of chloride channels of EDL muscle fibers are sensitive indices of the action of regeneration-promoting factors on muscle function.

Effects of chronic growth hormone treatment in aged rats on the biophysical and pharmacological properties of skeletal muscle chloride channels.

1. The effects of a 4-month daily treatment with recombinant human growth hormone (GH) (150 micrograms kg-1) to aged rats were evaluated on the passive and active membrane electrical properties of extensor digitorum longus (EDL) muscle fibres in vitro by means of a two intracellular microelectrode technique. 2. Chronic GH treatment completely restored the diameter and the membrane capacitance of aged EDL muscle fibres and significantly lowered the membrane resistance towards the adult value. There was also an increase of the threshold current, a shortening of the latency and an increase of the amplitude of the action potential and a significant amelioration of the membrane firing capability. 3. The effects were almost fully attributable to a significant 50% increase of resting conductance to chloride ions (GCl), although an observed restoration of potassium conductance and a possible effect on voltage-activated sodium channels could contribute to the effects. 4. EDL muscle fibres of untreated aged rats showed a different pharmacological response to 2-(p-chlorophenoxy) propionic acid (CPP) enantiomers from that seen in adult rats; the S-(-) isomer was less potent in blocking GCl and the R-(+) isomer always increased GCl instead of producing the typical biphasic effect observed in adult fibres (an increase of GCl at 1-10 microM and a decrease at higher concentrations). The 4-month-GH-treated aged rats showed a pharmacological sensitivity to CPP enantiomers similar to that of adults. 5. The in vitro application of insulin-like growth factor I (IGF-I), the peripheral mediator of GH, produced a significant and irreversible increase of GCl of EDL muscle of EDL muscle of untreated aged rats, an effect not observed in adults. This effect was completely inhibited by preincubation with 0.5 microM okadaic acid, suggesting that the IGF-I receptor transduction pathway can act on the phosphorylation state of the chloride channel through a serine-threonine protein phosphatase. 6. The results show that the skeletal muscle chloride channel is a target of the impairment of GH/IGF-I axis occurring in aged subjects. The acute and chronic effects observed on GCl of aged muscle fibres suggest that the GH/IGF-I stimuli act through a modulation of channel phosphorylation state and through the synthesis of 'adult'-like type chloride channels.

Age-related changes of growth hormone secretory mechanisms in the rat pituitary gland.

The mechanisms underlying the age-related decrease and increase in somatotroph responsiveness to growth hormone-releasing factor (GHRF) and somatostatin respectively were studied in rat pituitary membranes in vitro. Basal adenylate cyclase (AC) activity was similar in pituitary membranes from rats of 8 days (either sex) and male rats of 3 months, but it was almost threefold higher in membranes from male rats of 21-23 months. GHRF induced a lower percentage stimulation of AC activity in membranes from infant and old than adult rats. Somatostatin inhibited stimulation of AC induced by forskolin more effectively in membranes from adult than infant and old rats. In parallel experiments, since the tissue we used is formed by a mixed population of pituitary cells, we evaluated, for comparison, the effect on AC of neurohormones, i.e. vasoactive intestinal polypeptide (VIP) and dopamine which act primarily on lactotrophs. VIP induced a lower fold-stimulation of AC activity in membranes from infant and old than adult rats. Dopamine inhibited forskolin-induced stimulation of AC in the following rank order of magnitude: old, adult and infant rats, and was also more effective in inhibiting basal AC activity in old than in adult rats. The stimulatory and inhibitory G proteins (Gs and Gi) coupled to AC were measured indirectly by evaluating stimulatory and inhibitory effects of different concentrations of GTP on AC. GTP, at stimulatory concentrations, increased AC activity in membranes from infant and adult rats similarly whereas its effect was significantly greater in membranes from old rats.(ABSTRACT TRUNCATED AT 250 WORDS)