Autophagy in normal pituitary and pituitary tumor cells and its potential role in the actions of somatostatin receptor ligands in acromegaly.

Autophagy is an evolutionary conserved process for the self-degradation and recycling of cellular components in the cytoplasm. It is involved in both physiological and pathological conditions. In detail, the term "autophagy" refers to intracellular degradative pathways that lead to packaging and deliver of cellular components to lysosomes or to plant and yeast vacuoles. Autophagy is triggered by a variety of stimuli like nutrient deprivation, hypoxia, mitochondrial dysfunction, endoplasmic reticulum stress, and is regulated by immune- and hormonal factors. The role of autophagy in tumor cells is complex. Indeed, autophagy may act as a tumor suppressor as well as a tumor survival factor, in a context-dependent manner. The research into autophagy in normal pituitary and pituitary tumors has not gained great consideration, yet. Nevertheless, some recent articles joint to previous case studies, suggest that this process plays a role in the modulation and fluctuation of normal pituitary cell functions and in the response of pituitary tumor cells to drug therapy, including the response to somatostatin receptor ligand (SRLs), the first-line medical therapy of acromegaly. Although it is not possible to draw any conclusion, the aim of this review was to highlight some considerations and perspectives in this research field. Reports on the effects of octreotide on autophagy induction and autophagic flux in extra-pituitary target tissues, have also been discussed.

International Union of Basic and Clinical Pharmacology. CV. Somatostatin Receptors: Structure, Function, Ligands, and New Nomenclature.

Somatostatin, also known as somatotropin-release inhibitory factor, is a cyclopeptide that exerts potent inhibitory actions on hormone secretion and neuronal excitability. Its physiologic functions are mediated by five G protein-coupled receptors (GPCRs) called somatostatin receptor (SST)1-5. These five receptors share common structural features and signaling mechanisms but differ in their cellular and subcellular localization and mode of regulation. SST2 and SST5 receptors have evolved as primary targets for pharmacological treatment of pituitary adenomas and neuroendocrine tumors. In addition, SST2 is a prototypical GPCR for the development of peptide-based radiopharmaceuticals for diagnostic and therapeutic interventions. This review article summarizes findings published in the last 25 years on the physiology, pharmacology, and clinical applications related to SSTs. We also discuss potential future developments and propose a new nomenclature.

Integrated or Independent Actions of Metformin in Target Tissues Underlying Its Current Use and New Possible Applications in the Endocrine and Metabolic Disorder Area.

Metformin is considered the first-choice drug for type 2 diabetes treatment. Actually, pleiotropic effects of metformin have been recognized, and there is evidence that this drug may have a favorable impact on health beyond its glucose-lowering activity. In summary, despite its long history, metformin is still an attractive research opportunity in the field of endocrine and metabolic diseases, age-related diseases, and cancer. To this end, its mode of action in distinct cell types is still in dispute. The aim of this work was to review the current knowledge and recent findings on the molecular mechanisms underlying the pharmacological effects of metformin in the field of metabolic and endocrine pathologies, including some endocrine tumors. Metformin is believed to act through multiple pathways that can be interconnected or work independently. Moreover, metformin effects on target tissues may be either direct or indirect, which means secondary to the actions on other tissues and consequent alterations at systemic level. Finally, as to the direct actions of metformin at cellular level, the intracellular milieu cooperates to cause differential responses to the drug between distinct cell types, despite the primary molecular targets may be the same within cells. Cellular bioenergetics can be regarded as the primary target of metformin action. Metformin can perturb the cytosolic and mitochondrial NAD/NADH ratio and the ATP/AMP ratio within cells, thus affecting enzymatic activities and metabolic and signaling pathways which depend on redox- and energy balance. In this context, the possible link between pyruvate metabolism and metformin actions is extensively discussed.

Effects of octreotide on autophagy markers and cell viability markers related to metabolic activity in rat pituitary tumor cells.

PURPOSE: The aim of this work was to investigate possible direct effects of the somatostatin analog octreotide on autophagy markers and markers of cellular metabolic activity using in vitro cultured rat pituitary tumor cells (GH3 cell line). METHODS: We measured two markers of the autophagic flux in cell lysates by Western blot and MTT reductive activity, total cellular ATP levels, pyruvate dehydrogenase (PDH) complex activity in cells lysates as markers of cell viability related to metabolic activity. RESULTS: Octreotide (100 nM) treatment induced autophagy activation (increased LC3-I protein lipidation) and enhanced the autophagic flux (SQSTM1/p62 protein downregulation) in GH3 cells in different incubation media, in detail in Hank's balanced salt solution (HBSS) as well as in maintenance medium with serum. We did not observe any decrease of redox activity and energy production related to the induction of autophagy by octreotide. On the other hand, short-term treatments with octreotide in HBSS tended to enhance MTT reduction activity and to increase PDH complex enzymatic activity and ATP levels measured in GH3 cell lysates. CONCLUSIONS: We provided evidence that octreotide can affect autophagy in pituitary tumor cells. The observed effects of octreotide were not related to a decrease of cellular metabolic activity. Finally, the induction of autophagy was either short-lived or overshadowed by other factors in the long term and this limit does not help clarifying their real impact on the pharmacological activity of somatostatin analogs.

Role of Bioactive Peptide Sequences in the Potential Impact of Dairy Protein Intake on Metabolic Health.

For years, there has been an increasing move towards elucidating the complexities of how food can interplay with the signalling networks underlying energy homeostasis and glycaemic control. Dairy foods can be regarded as the greatest source of proteins and peptides with various health benefits and are a well-recognized source of bioactive compounds. A number of dairy protein-derived peptide sequences with the ability to modulate functions related to the control of food intake, body weight gain and glucose homeostasis have been isolated and characterized. Their being active in vivo may be questionable mainly due to expected low bioavailability after ingestion, and hence their real contribution to the metabolic impact of dairy protein intake needs to be discussed. Some reports suggest that the differential effects of dairy proteins-in particular whey proteins-on mechanisms underlying energy balance and glucose-homeostasis may be attributed to their unique amino acid composition and hence the release of free amino acid mixtures enriched in essential amino acids (i.e., branched-chain-amino acids) upon digestion. Actually, the research reports reviewed in this article suggest that, among a number of dairy protein-derived peptides isolated and characterized as bioactive compounds in vitro, some peptides can be active in vivo post-oral administration through a local action in the gut, or, alternatively, a systemic action on specific molecular targets after entering the systemic circulation. Moreover, these studies highlight the importance of the enteroendocrine system in the cross talk between food proteins and the neuroendocrine network regulating energy balance.

Interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK) and the estrogen receptor activities in regulating rat pituitary tumor cell (GH3) growth in vitro.

Estrogen receptor α has a role in regulating rat somatolactotroph tumor cell growth (GH3 cells). AMP-activated protein kinase (AMPK) is a metabolic checkpoint which is able to negatively regulate intracellular signaling downstream of growth factors receptors in conditions increasing cellular AMP levels. We have recently reported on the role of AMPK activation in affecting viability and proliferation of GH3 cells. In the present study, we investigated the interplay between ER- and AMPK-pathways. Results can be regarded as relevant to the development of novel multi-targeted pharmacological therapies against pituitary tumors. We confirmed that estradiol (E2) and the ER antagonist fulvestrant exert stimulatory and inhibitory effects, respectively on GH3 cell growth in a competitive manner. The upstream kinase LKB1 is known to phosphorylate and activate AMPK. Here we showed that neither E2 nor fulvestrant caused a downregulation of LKB1 expression and phospho-AMPK levels in GH3 cells. Actually, fulvestrant strongly reduced the phosphorylation of ACC, which is a direct target of AMPK and a known index of AMPK activity. 2-deoxyglucose, a compound reducing glucose utilization, caused an increase in AMPK activity vs baseline and was able to hinder the stimulatory effect of E2 on cell viability, confirming that the exposure of GH3 cells to estrogens does not prevent them from being responsive to the inhibitory activity of compounds activating AMPK. Finally, the AMPK activator AICAR (AMP analog) did not cause further decrease in cell viability in the course of co-treatments with fulvestrant versus fulvestrant alone, in agreement with impaired phospho-AMPK activity in the presence of the anti-estrogen.

Characterization of the mechanisms involved in the gastric antisecretory effect of TLQP-21, a vgf-derived peptide, in rats.

TLQP-21, a vgf-derived peptide modulates gastric emptying and prevents ethanol-induced gastric lesions in rats. However, it remains to be studied whether or not TLQP-21 affects gastric acid secretion. In this study, we evaluated the effects of central (0.8-8 nmol/rat) or peripheral (48-240 nmol/kg, intraperitoneally) TLQP-21 administration on gastric acid secretion in pylorus-ligated rats. The mechanisms involved in such activity were also examined. Central TLQP-21 injection significantly reduced gastric acid volume and dose-dependently inhibited total acid output (ED(50) = 2.71 nmol), while peripheral TLQP-21 administration had no effect. The TLQP-21 antisecretory activity was prevented by cysteamine (300 mg/kg, subcutaneously), a depletor of somatostatin, by indomethacin (0.25 mg/rat, intracerebroventricularly), a non-selective cyclooxygenase inhibitor, and by functional ablation of sensory nerves by capsaicin. We conclude that TLQP-21 could be considered a new member of the large group of regulatory peptides affecting gastric acid secretion. The central inhibitory effect of TLQP-21 on gastric acid secretion is mediated by endogenous somatostatin and prostaglandins and requires the integrity of sensory nerve fibres.

Pharmacological characterization of the ghrelin receptor mediating its inhibitory action on inflammatory pain in rats.

Recent research suggests a role for ghrelin in the modulation of inflammatory disorders. However, the type of ghrelin receptor (GHS-R) involved in both the anti-inflammatory and anti-hyperalgesic actions of ghrelin remains to be characterized. In this study, we examined whether the inhibitory effect of ghrelin in the development of hyperalgesia and edema induced by intraplantar carrageenan administration depends on an interaction with GHS-R1a. Both central (1 nmol/rat, i.c.v.) and peripheral (40 nmol/kg, i.p.) administration of the selective GHS-R1a agonist EP1572 had no effect on carrageenan-induced hyperalgesia measured by Randall-Selitto test and paw edema. Furthermore, pre-treatment with the selective GHS-R1a antagonist, D-lys(3)-GHRP-6 (3 nmol/rat, i.c.v.) failed to prevent the anti-hyperalgesic and anti-inflammatory effects exerted by central ghrelin administration (1 nmol/rat), thus indicating that the type 1a GHS-R is not involved in these peptide activities. Accordingly, both central (1 and 2 nmol/rat, i.c.v.) and peripheral (40 and 80 nmol/kg, i.p.) administration of desacyl-ghrelin (DAG), which did not bind GHS-R1a, induced a significant reduction of the hyperalgesic and edematous activities of carrageenan. In conclusion, we have shown for the first time that DAG shares with ghrelin an inhibitory role in the development of hyperalgesia, as well as the paw edema induced by carrageenan and that a ghrelin receptor different from type 1a is involved in the anti-inflammatory activities of the peptide.

AMP-activated protein kinase regulates normal rat somatotroph cell function and growth of rat pituitary adenomatous cells.

AMP-activated protein kinase (AMPK) is activated under conditions that deplete cellular ATP and elevate AMP levels such as glucose deprivation and hypoxia. The AMPK system is primarily thought of as a regulator of metabolism and cell proliferation. Little is known about the regulation and the effects of AMPK in somatotroph cells. We present results from "in vitro" studies showing that AMPK activity has a role in regulating somatotroph function in normal rat pituitary and is a promising target for the development of new pharmacological treatments affecting cell proliferation and viability of pituitary adenomatous cells. In parallel, we show "in vivo" data obtained in the rat suggesting that AMPK is an intracellular transducer that may play a role in mediating the effects of the pharmacological treatment with dexamethasone on somatotrophs. In rat pituitary cell cultures, the AMP analog AICAR induced a rapid and clear-cut activation of AMPK. AICAR decreased GH release and total cellular GH content. An appropriate level of AMPK activation was essential for GH3 adenomatous cells. Remarkably, over-activation by AICAR induced apoptosis of GH3 whereas the AMPK inhibitor compound C was more effective at reducing cell proliferation. The role of endocrine or paracrine factors in regulating AMPK phosphorylation and activity in GH3 cells has been also studied. As to "in vivo" studies, western blot analysis revealed a significant decrease of phosphorylated AMPK alpha-subunit in pituitary homogenates of DEX-treated rats versus controls, suggesting reduced AMPK activity. In conclusion, our studies showed that AMPK has a role in regulating somatotroph function in normal rat pituitary and proliferation of pituitary adenomatous cells.

How treatments with endocrine and metabolic drugs influence pituitary cell function.

A variety of endocrine and metabolic signals regulate pituitary cell function acting through the hypothalamus-pituitary neuroendocrine axes or directly at the pituitary level. The underlying intracellular transduction mechanisms in pituitary cells are still debated. AMP-activated protein kinase (AMPK) functions as a cellular sensor of low energy stores in all mammalian cells and promotes adaptive changes in response to calorie restriction. It is also regarded as a target for therapy of proliferative disorders. Various hormones and drugs can promote tissue-specific activation or inhibition of AMPK by enhancing or inhibiting AMPK phosphorylation, respectively. This review explores the preclinical studies published in the last decade that investigate the role of AMP-activated protein kinase in the intracellular transduction pathways downstream of endocrine and metabolic signals or drugs affecting pituitary cell function, and its role as a target for drug therapy of pituitary proliferative disorders. The effects of the hypoglycemic agent metformin, which is an indirect AMPK activator, are discussed. The multiple effects of metformin on cell metabolism and cell signalling and ultimately on cell function may be either dependent or independent of AMPK. The in vitro effects of metformin may also help highlighting differences in metabolic requirements between pituitary adenomatous cells and normal cells.

Differential effects of metformin on reductive activity and energy production in pituitary tumor cells compared to myogenic precursors.

PURPOSE: Given the multiple targets of metformin within cells, the mechanism by which it may exert a growth-inhibitory action on pituitary tumor cells in vitro remains to be explored. Previous research stressed metformin-induced changes in the activity of signaling pathways regulating cell growth and cell death. In this work, we investigated the effects of metformin on cell viability markers related to cell metabolic activity in rat pituitary tumor cells versus rat myogenic precursors as a model of normal proliferating somatic cells. METHODS: We designed our experiments in order to use the MTT reduction as a marker of cellular reductive activity and the total cellular ATP levels as a marker of energy supply during short incubations with different metabolic substrates (sodium pyruvate, D-glucose, L-glutamine, sodium citrate). Then, we extended the analysis to extracellular glucose consumption, extracellular medium acidification and pyruvate dehydrogenase (PDH) complex activity. RESULTS: Metformin was found to be effective in both cell types at the same concentrations, although the outcome of the treatment was quite the opposite. Unexpectedly, metformin increased the viability of subconfluent rat myoblasts. Rat pituitary tumor cells and myoblasts differed in the utilization of distinct metabolic substrates and the PDH complex activity. Metformin actions on reductive activity and ATP production were substrate-dependent. CONCLUSIONS: Overall, this work points out that metformin actions at the cellular level depend on metabolic features and metabolic requirements of cells. The pyruvate metabolic branch point is most likely to play a main role in the variability of cell response to metformin.

Chronic treatment with polychlorinated biphenyls (PCB) during pregnancy and lactation in the rat: Part 1: Effects on somatic growth, growth hormone-axis activity and bone mass in the offspring.

Polychlorinated biphenyls (PCBs) are pollutants detected in animal tissues and breast milk. The experiments described in the present paper were aimed at evaluating whether the four PCB congeners most abundant in animal tissues (PCB-138, -153, -180 and -126), administered since fetal life till weaning, can induce long-term alterations of GH-axis activity and bone mass in the adult rat. We measured PCB accumulation in rat brain and liver, somatic growth, pituitary GH expression and plasma hormone concentrations at different ages. Finally, we studied hypothalamic somatostatin expression and bone structure in adulthood, following long-term PCB exposure. Dams were treated during pregnancy from GD15 to GD19 and during breast-feeding. A constant reduction of the growth rate in both male and female offspring from weaning to adulthood was observed in exposed animals. Long-lasting alterations on hypothalamic-pituitary GH axis were indeed observed in PCB-exposed rats in adulthood: increased somatostatin expression in hypothalamic periventricular nucleus (both males and females) and lateral arcuate nucleus (males, only) and decreased GH mRNA levels in the pituitary of male rats. Plasma IGF-1 levels were higher in PCB-exposed male and female animals as compared with controls at weaning and tended to be higher at PN60. Plasma testosterone and thyroid hormone concentrations were not significantly affected by exposure to PCBs. In adulthood, PCBs caused a significant reduction of bone mineral content and cortical bone thickness of tibiae in male rat joint to increased width of the epiphyseal cartilage disk. In conclusion, the developmental exposure to the four selected PCB compounds used in the present study induced far-reaching effects in the adult offspring, the male rats appearing more sensitive than females.

Effects of olanzapine on glucose transport, proliferation and survival in C2C12 myoblasts.

The aim of our study was to investigate the direct effects of atypical antipsychotics on muscle cell functions in order to ascertain the diabetic liability of these drugs. We investigated the effects of olanzapine, clozapine and alpha-methyl-5-hydroxytryptamine on basal glucose uptake and glucose uptake in response to insulin using in vitro cultures of mouse skeletal muscle satellite cells (C2C12). We extended our study to the effects of these compounds on cell proliferation, survival and differentiation into myotubes and on the growth of differentiated myotubes. Olanzapine and alpha-methyl-5-HT stimulated 2-deoxyglucose uptake in C2C12 myoblasts in a dose-dependent manner (minimal effective dose: 2 microM olanzapine and 10 microM alpha-methyl-5-HT). The treatment with clozapine had no effect on glucose transport. Insulin and olanzapine increased the plasma membrane (PM) abundance of glucose transporter GLUT4. We investigated whether protein kinase Akt (PKB) and AMP-dependent kinase may participate in mediating olanzapine effects on glucose transport. Clozapine and olanzapine did not induce DNA laddering in differentiating myoblasts and differentiated myotubes and did not affect myotube growth. Olanzapine-induced glucose disposal in vitro is consistent with the acute lowering of plasma glucose/insulin concentrations that occurs in rats before olanzapine-induced overeating [Albaugh, V.L., Henry, C.R., Bello, N.T., Hajnal, A., Lynch, S.L., Halle, B., Lynch, C.J., 2006. Hormonal and metabolic effects of olanzapine and clozapine related to body weight in rodents. Obesity 14, 36-50].

Effects of metformin on cell growth and AMPK activity in pituitary adenoma cell cultures, focusing on the interaction with adenylyl cyclase activating signals.

For a few years we have been investigating AMP-activated protein kinase (AMPK) as a target for drug therapy of GH-secreting pituitary adenomas. Aim of this study was to investigate the direct effects of metformin, which causes AMPK activation in different cell types, on rat pituitary adenoma cell growth and on related cell signalling pathways. Our results suggest that metformin can exert a growth-inhibitory activity in rat pituitary tumor cells mediated by AMPK activation, although multiple mechanisms are most likely involved. Membrane proteins, including growth factor receptors, are valuable targets of AMPK. The inhibition of the mTOR-p70S6 kinase signalling pathway plays a role in the suppressive effect of metformin on pituitary tumor cell growth. Metformin did not affect the MTT reduction activity in energetic stress conditions. Finally, metformin was still able to induce AMPK activation and to inhibit cell growth in cells treated with forskolin and in transfected cells overexpressing GHRH-receptor and treated with GHRH. Hence, adenylyl cyclase over-activation does not account for the lack of response of some human pituitary tumors to AMPK-activating compounds in vitro.

Novel insights in somatostatin receptor physiology.

The experimental data reviewed in the present paper deal with the molecular events underlying the agonist-dependent regulation of the distinct somatostatin receptor subtypes and may suggest important clues about the clinical use of somatostatin analogs with different pattern of receptor specificity for the in vivo targeting of tumoral somatostatin receptors. Somatostatin receptor subtypes are characterized by differential beta-arrestin trafficking and endosomal sorting upon agonist binding due, at least in part, to the differences in their C-terminal tails. Moreover, the subcellular expression pattern of somatostatin receptor subtypes and their activity in response to agonist treatment are affected by intracellular complements, such as proteins involved in intracellular vesicle trafficking. Different somatostatin analogs may induce distinct conformations of the receptor/ligand complex, preferentially coupled to either receptor signaling or receptor endocytosis.

Somatostatin receptor 2 is activated in cortical neurons and contributes to neurodegeneration after focal ischemia.

Somatostatin receptor 2 (SSTR2) mediates neuromodulatory signals of somatostatin and cortistatin in the cerebral cortex. Recently, SSTR2 has been shown to enhance conserved death ligand- and mitochondria-mediated apoptotic pathways in non-neuronal cells. Whether somatostatin receptors are activated in cerebrocortical neurons and contribute to neurodegeneration after experimental focal ischemia was unknown until now. Here we examined internalization of SSTR2 in a rat model of middle cerebral artery occlusion (MCAO) by confocal microscopy. At 3 and 6 hr after MCAO, SSTR2 was internalized excessively in cerebrocortical neurons adjacent to the infarct, which was prevented by intracerebroventricular application of the SSTR2-selective antagonist BIM-23627. SSTR2 internalization was associated with a transient depletion of somatostatin from axonal terminals and increased expression of SSTR2 mRNA. The initial loss of somatostatin was followed by an increase in somatostatin mRNA levels, whereas cortistatin mRNA expression was decreased. In SSTR2-deficient mice with lacZ under the control of the SSTR2 promoter, MCAO-induced upregulation of SSTR2 gene expression was less pronounced than in wild types. SSTR2-deficient mice exhibited a 40% reduction of infarct size after permanent distal MCAO and a 63% reduction after transient proximal MCAO. In summary, we provide direct evidence for activation of SSTR2 by an endogenous ligand after focal ischemia. Activation of functional SSTR2 receptors contributes to increased SSTR2 gene expression and postischemic neurodegeneration.

The somatostatin subtype-2 receptor antagonist, BIM-23627, improves the catabolic effects induced by long-term glucocorticoid treatment in the rat.

BIM-23627 is a synthetic peptide with "in vitro" and "in vivo" properties consistent with a pure sst2 antagonist. The aim of the present study was to evaluate the effects of long-term administration of BIM-23627 and the combined effects of BIM-23627 and dexamethasone (DEX) on the somatotropic axis, including growth, epididymal fat accumulation, glucose homeostasis and insulin activity, in young male rats. Beginning on day 23 of age, 16 animals were treated daily with saline or DEX (40 microg/kg/daily). Each group was subdivided into two paired groups and treated with either vehicle or BIM-23627 (0.5 mg/kg, t.i.d.). The treatment period lasted 31 days. The animals were killed by decapitation; trunk blood and pituitaries were collected for the determination of hormone concentrations and GH mRNA expression, respectively. Based on plasma GH and IGF-I concentrations and GH mRNA expression in the pituitary, BIM-23627 was able to counteract the inhibitory effects of DEX on the somatotropic axis; however, only a partial reversal of somatic growth inhibition was observed. DEX-treated rats remained euglycemic, but their insulin levels were significantly increased, indicating an incipient insulin resistance. Although BIM-23627 itself tended to increase insulin concentration in saline-treated rats, its administration to DEX-treated rats reduced insulin levels (saline: 25+/-3; DEX: 55+/-16*; DEX+BIM-23627: 34+/-5; BIM-23627: 38+/-7 microIU/ml; *P<0.05 vs. saline), apparently improving the degree of insulin sensitivity. DEX administration significantly reduced circulating ghrelin, whereas the sst2 antagonist had no significant effect. An inverse correlation was found between ghrelin concentrations and plasma insulin levels. Both rats receiving DEX and rats receiving BIM-23627 had decreased plasma concentration of total testosterone (P<0.05); however, the effects of DEX and BIM-23627 were not additive. In conclusion, BIM-23627 may represent a new pharmacological agent to reduce the suppression of the GH-IGF-I axis in long-term GC treated patients and enhance insulin sensitivity. Further studies are required in order to fully optimize the SSTR-2 antagonist-induced reversal of DEX-induced somatic growth inhibition.

MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line.

The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS.

Ghrelin plays a minor role in the physiological control of cardiac function in the rat.

We have previously reported that a 7-d pretreatment with hexarelin, a synthetic ligand of the GH secretagogue receptor (GHS-R), largely prevented damages induced by ischemia and reperfusion in isolated rat hearts. Our aim was to ascertain whether ghrelin, an endogenous ligand of the GHS-R, is physiologically endowed with cardioprotective activity. Hypophysectomized rats were treated in vivo for 7 d with either ghrelin (320 microg/kg) or hexarelin (80 microg/kg), and their hearts were subjected in vitro to the ischemia and reperfusion procedure. Ghrelin was far less effective than hexarelin in preventing increases in left ventricular end-diastolic pressure (15% and 60% protection for ghrelin and hexarelin, respectively), coronary perfusion pressure (10% and 45% reduction), and release of creatine kinase in the heart perfusate (15% and 55% reduction). In the second experiment, normal rats were passively immunized against ghrelin for 21 d before the ischemia and reperfusion procedure. In these isolated hearts, the ischemia-reperfusion damage was not significantly increased compared with control rats. After hypophysectomy, CD36 mRNA levels significantly increased, whereas those of atrial natriuretic factor significantly decreased. We conclude that: 1) ghrelin plays a minor role in the control of heart function; and 2) hexarelin effects are mediated in part by the GHS-R and largely by interactions with the CD36.