Different ghrelin localisation in adult human and rat endocrine pancreas.

Ghrelin is an endocrine peptide that has been identified in gastric oxyntic glands and that induces growth hormone secretion in the pituitary gland. This growth hormone secretagogue is expressed in many tissues such as stomach, pituitary gland, thyroid, testis, placenta and pancreas. Initial studies of ghrelin focused on its role as a circulating orexigenic signal. However, ghrelin has also been found to be involved in the modulation of glucose homeostasis. Although a number of studies have reported ghrelin expression in developing pancreas, the location of ghrelin-immunoreactive cells in adult pancreas (epsilon cells) remains controversial. In this study, we have analysed the distribution of pancreatic epsilon cells in adult human and rat islets by immunohistochemistry and in situ hybridisation. In humans, our immunohistochemical analysis has shown that ghrelin is expressed in glucagon-secreting cells, whereas in rats, it is present in insulin-secreting cells. Similar observations have been revealed by in situ hybridisation.

Chronic sciatic nerve injury impairs the local cutaneous neurovascular interaction in rats.

Most studies of chronic nerve compression focus on large nerve function in painful conditions, and only few studies have assessed potential changes in the function of small nerve fibers during chronic nerve compression and recovery from compression. Cutaneous pressure-induced vasodilation is a neurovascular phenomenon that relies on small neuropeptidergic fibers controlling the cutaneous microvasculature. We aimed to characterize potential changes in function of these small fibers and/or in cutaneous microvascular function following short-term (1-month) and long-term (6-month) nerve compression and after release of compression (ie, potential recovery of function). A compressive tube was left on one sciatic nerve for 1 or 6 months and then removed for 1-month recovery in Wistar rats. Cutaneous vasodilator responses were measured by laser Doppler flowmetry in hind limb skin innervated by the injured nerve to assess neurovascular function. Nociceptive thermal and low mechanical thresholds were evaluated to assess small and large nerve fiber functions, respectively. Pressure-induced vasodilation was impaired following nerve compression and restored following nerve release; both impairment and restoration were strongly related to duration of compression. Small and large nerve fiber functions were less closely related to duration of compression. Our data therefore suggest that cutaneous pressure-induced vasodilation provides a non-invasive and mechanistic test of neurovascular function that gives direct information regarding extent and severity of damage during chronic nerve compression and recovery, and may ultimately provide a clinically useful tool in the evaluation of nerve injury such as carpal tunnel syndrome.

Autocrine proliferative effects of hGH are maintained in primary cultures of human mammary carcinoma cells.

CONTEXT: Empirical evidence suggests that autocrine human GH (hGH) may possess a proliferative and oncogenic role in human mammary carcinoma. However, this concept is largely derived from studies using cultured human mammary carcinoma cell (HMCC) lines. OBJECTIVE: We investigated the expression and functionality of hGH and the hGH receptor in isolated cultures of primary HMCC. DESIGN: Epithelial cell adhesion molecule-positive primary HMCC were isolated from surgical biopsies of patients with mammary carcinoma and cultured in vitro. Expression of hGH and hGH receptor was determined by RT-PCR, immunofluorescence microscopy, and ELISA. The proliferative response of the cultured primary HMCC to hGH stimulation or hGH inhibition with a hGH antagonist was determined. RESULTS: One hundred percent of cultured primary HMCC expressed the hGH receptor, and 52% expressed hGH at the mRNA level. hGH-positive primary HMCC produced hGH protein within the cell and secreted hGH to the media. Both hGH-negative and hGH-positive HMCC responded to hGH stimulation with large increases in cell number. hGH-positive HMCC responded to inhibition of hGH by a hGH antagonist with a decrease in cell number, whereas hGH-negative HMCC did not. CONCLUSION: Primary HMCC proliferate in response to hGH, and the proliferation of hGH-positive HMCC is inhibited by hGH antagonism. Inhibition of hGH in patients with mammary carcinoma may therefore limit tumor growth.

Growth hormone internalization in mitochondria decreases respiratory chain activity.

Growth hormone (GH) is a signaling molecule regulating cell proliferation, differentiation and metabolism via activation of specific cell surface receptors and subsequent triggering of signal transduction pathways. This is associated with GH/GH receptor internalization and accumulation of GH in several subcellular compartments, including mitochondria. To assess the functional relevance of such mitochondrial accumulation, we first confirmed the occurrence of mitochondrial GH uptake ex vivo as early as 10 min after (125)I-GH injection to the rats. We next showed that intact (125)I-GH accumulates in mitochondrial fractions in vitro in a specific, rapid and saturable manner with an apparent affinity (K(d)) of 1.44 nM. At the electron-microscopic level, immunoreactive GH density within mitochondria increased after in vitro hormone incubation, without any modification of the sub-mitochondrial distribution pattern. The presence of GH in the inter-membrane space and at the inner membrane seen by electron microscopy was confirmed by SDS-PAGE and autoradiography after mitochondrial fractioning thus suggesting the involvement of GH in the respiration control. To test this hypothesis further, we performed polarographic and spectrophotometric assays on isolated mitochondria. These assays pointed to a direct, selective and dose-dependent effect of GH on the inhibition of succinate dehydrogenase and cytochrome C oxidase activities. The latter inhibition was in contrast with indirect, GH receptor-initiated stimulation of cytochrome C oxidase activity observed in GH-treated whole BRL cells transfected to express this receptor. Altogether, these data show that GH is specifically imported in mitochondria, where it operates a direct metabolic effect, independently of cell surface receptors and signal transduction.

Pulmonary superinfection by trichomonads in the course of acute respiratory distress syndrome.

The finding of trichomonads in bronchoalveolar lavage fluid (BALF) samples from an acute respiratory distress syndrome (ARDS) patient, never previously reported, incited us to search for these parasites retrospectively in the BALF of patients with ARDS or related pathologies. Eighty-four consecutive BALF samples have been reviewed. Results were compared with data from clinical files of patients included in this study. Detection and identification of trichomonads were based on cytologic characteristics. Subsequently, immunocytochemistry and in situ hybridization were performed in the last case of the series. Our results were as follows: (1) Trichomonads were detected in 25/84 BALFs (20/77 patients). Among the patients testing positive, 17 suffered from ARDS, about 30% of the ARDS patients included in the study. (2) Trichomonads were detected more frequently at a late ARDS stage. (3) No correlation was found between trichomonad detection and other data. (4) Within the group of trichomonad-infected ARDS patients, density of infection correlated with higher mortality. The late detection of these microorganisms in the course of ARDS suggested that trichomonad development is a secondary event. As BALFs obtained early in the course of ARDS were also included in the study, trichomonad incidence could be underestimated. The significance of trichomonad lung colonization in ARDS and its potential pathogenic role are unclear. Nevertheless, the question of an active role of trichomonads in the extension of alveolar lesions or in the limitation of recovery is clearly raised.

C620R mutation of the murine ret proto-oncogene: loss of function effect in homozygotes and possible gain of function effect in heterozygotes.

Germline RET mutations are responsible for different inherited disorders: Hirschsprung disease (congenital aganglionic megacolon), caused by loss of function mutations, familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2, caused by gain of function mutations. Intriguingly, some RET mutations, including C620R, are associated with both types of diseases. To investigate the dual role of such RET mutations, a mouse model with a targeted mutation ret(C620R) was generated. ret(C620R/C620R) offspring die during the first postnatal day, and show kidney agenesis and intestinal aganglionosis. Decreased outgrowth of the Ret-positive cells was observed in ret(C620R/C620R) neuronal cell cultures, which is suggestive of an impaired migration, proliferation or survival of the Ret-expressing cells. Electronmicroscopy revealed the absence of membrane-bound Ret in ret(C620R/C620R) cells as compared to ret(+/+) and ret(+/C620R) cells. On the other hand, aged ret(+/C620R) mice develop precancerous lesions in the adrenal gland or in the thyroid. Our results suggest that the ret(C620R) mutation has a loss of function effect in homozygotes and exhibits a dominant gain of function effect with low penetrance causing hyperplasia in heterozygotes.

Electrophysiological characterization of left ventricular myocytes from obese Sprague-Dawley rat.

OBJECTIVE: Obesity is a complex multifactorial disease that is often associated with cardiac arrhythmias. Various animal models have been used extensively to study the effects of obesity on physiological functions, but, to our knowledge, no study related to ionic membrane currents has been performed on isolated cardiac myocytes. Therefore, we examined the electrophysiological characteristics of four ionic currents from isolated left ventricular myocytes of a high-energy (HE)-induced obesity rat model. RESEARCH METHODS AND PROCEDURES: Male Sprague-Dawley rats were fed with either a control diet or a diet containing 33% kcal as fat (HE) for 14 weeks starting at 6 weeks of age. Voltage-clamp experiments were performed on ventricular myocytes. Leptin receptor (ObR) expression was measured using ObR enzyme-linked immunosorbent assay. RESULTS: In the HE group, rats designated as obese did not develop a cardiac hypertrophy, either at the organ level or at the cellular level. Densities and kinetics of the L-type calcium current, the transient outward potassium current, the delayed rectifier potassium current, and the sodium-calcium exchange current (I(NCX)) were not significantly different between control and obese rats. A down-regulation of ObR expression was evidenced in the heart of obese rats compared with controls. Acute exposure (5 minutes) of leptin (100 nM) did not induce a significant modification in the current densities either in control or in obese rats, except for I(NCX) density measured in control rats. DISCUSSION: The absence of effect of leptin on I(NCX) in obese rats could be a potential arrhythmogenic substrate in obesity.

Cardiac NGF and GDNF expression during Trypanosoma cruzi infection in rats.

In rats, autonomic nerve endings are damaged during Trypanosoma cruzi-induced myocarditis. Gradual recovery occurs after the acute phase. The present work shows the cardiac levels of glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF), and their cellular sources during T. cruzi infection in rats. Atrial and ventricular NGF levels (ELISA) increased significantly at day 20 post inoculation, the time-point of maximal sympathetic denervation. ELISA failed to show significant increase of cardiac GDNF levels. However immunohistochemistry showed a significant increase of anti-GDNF gold particles over atrial granules at day 20. Light microscopy showed stronger NGF immunostaining in atrial cardiomyocytes and several blood capillaries. In situ hybridization showed NGF and GDNF mRNAs in atrial and ventricular myocytes of both infected and uninfected animals. Endothelial cells exhibited NGF mRNA and protein only in infected rats. No evidence of neurotrophic factor expression by the infiltrating mononuclear cells was found. This is the first report on neurotrophic factor expression during T. cruzi infection. Our findings indicate an important role for NGF in the regenerative phenomena subsequent to a myocarditis able to damage sympathetic nerve endings, with preservation of preterminals and nerve trunks. GDNF could have a minor or a more transient participation.

Ghrelin localization in rat and human thyroid and parathyroid glands and tumours.

Ghrelin, the endogenous ligand of the growth hormone secretagogue receptor (GHS-R), is a 28 amino acid peptide originally isolated from rat stomach that has been primarily involved in the central neuroregulation of GH secretion and food intake. Previous studies demonstrated that ghrelin has a widespread expression in different normal cells and tissues, as well as in gastric, thyroid, testicular, breast and lung neoplasias. In the current study, we use molecular biology to detect ghrelin transcripts expression in rats, and immunohistochemical techniques to investigate the cellular distribution of this peptide in rat and human thyroid and parathyroid glands and tumours. Ghrelin was localized in thyroid C cells and in parathyroid cells. Thyroid carcinomas (medullar, follicular and papillary) and parathyroid adenomas also showed intense and diffuse immunostaining for ghrelin. These data provide direct morphological evidence that ghrelin may well be acting in a paracrine fashion in the regulation of thyroid follicular cell function. The diffuse ghrelin immunostaining found in the parathyroid gland opens up the possibility of its secretion to the bloodstream or its involvement in the regulation of the parathyroid function. Overall, expression of ghrelin in human and rat thyroid and parathyroid glands is highly suggestive of a conserved role of this molecule in the regulation of thyroid and parathyroid cell function.

Growth hormone activity in mitochondria depends on GH receptor Box 1 and involves caveolar pathway targeting.

Growth hormone (GH) binding to its receptor (GHR) initiates GH-dependent signal transduction and internalization pathways to generate the biological effects. The precise role and way of action of GH on mitochondrial function are not yet fully understood. We show here that GH can stimulate cellular oxygen consumption in CHO cells transfected with cDNA coding for the full-length GHR. By using different GHR cDNA constructs, we succeeded in determining the different parts of the GHR implicated in the mitochondrial response to GH. Polarography and two-photon excitation fluorescence microscopy analysis showed that the Box 1 of the GHR intracellular domain was required for an activation of the mitochondrial respiration in response to a GH exposure. However, confocal laser scanning microscopy demonstrated that cells lacking the GHR Box 1 could efficiently internalize the hormone. We demonstrated that internalization mediated either by clathrin-coated pits or by caveolae was able to regulate GH mitochondrial effect: these two pathways are both essential to obtain the GH stimulatory action on mitochondrial function. Moreover, electron microscopic and biochemical approaches allowed us to identify the caveolar pathway as essential for targeting GH and GHR to mitochondria.

Cloning, expression and regulation of chicken ovalbumin upstream promoter transcription factors (COUP-TFII and EAR-2) in the rat anterior pituitary gland.

Chicken ovalbumin upstream promoter transcription factors (COUP-TF)-II (NR2F2) and EAR-2 (NR2F6) are structurally related orphan members of the nuclear receptors superfamily. There are growing evidences that these factors play important roles during processes of differentiation and proliferation of several tissues. To better understand their role in the differentiated adult rat pituitary gland, we cloned COUP-TFII and EAR-2 cDNAs from an anterior pituitary cDNA library. Subsequently, we raised and characterized specific antibodies to the N-terminal domain of both nuclear receptors. We next examined their cellular and subcellular distribution in the pituitary gland and determined their regulation during pregnancy. COUP-TFII and EAR-2 pituitary genes display, respectively, 90 and 100% homologies with their human and mouse homologues. Cellular expression of both nuclear receptors was mainly detected in the lactotropes of male and female rats, with a prominent distribution in the nuclear compartment for EAR-2, and interestingly both proteins were significantly upregulated in pituitaries of pregnant vs. cycling female rats. Thus, our results have characterized cloning of rat pituitary COUP-TFII and EAR-2 genes, demonstrated that they are both specifically expressed in lactotropes, and strongly suggested that they may play an important role in modulating prolactin (PRL) gene expression during pregnancy.

Sympathetic and brain monoaminergic regulation of energy balance in obesity-resistant rats (Lou/C).

In contrast to the Wistar rat, the Lou/C rat does not develop obesity with age. To determine the role of sympathetic output and brain monoamines in the different energy balance of Lou/C rats, the monoamine contents and activity of rate-limiting enzymes in catecholamine and serotonin biosynthesis were assessed in brain structures involved in energy balance regulation, i.e., brainstem noradrenergic (A6, A5, A2) and serotonergic cell groups (dorsal raphe, and median raphe), and two hypothalamic nuclei (ventromedial nucleus and paraventricular nucleus). In vivo tyrosine hydroxylase activity and noradrenaline content were measured in sympathetic target organs storing fuel substrates, the liver, white adipose and brown adipose tissues in the Lou/C rat and compared to the Wistar rat. In Lou/C rats, indirect calorimetric measurements showed a higher energy expenditure despite a reduced food intake. The Lou/C rat displayed selective monoamine features. The catecholaminergic activity was higher in the white adipose tissue and interscapular brown adipose tissue but lower in the liver and adrenal gland of Lou/C rats. The noradrenergic activity in A2, A6 and ventromedial nucleus, and the serotonergic pattern in A6, dorsal raphe and median raphe were lower in Lou/C. The metabolic particularities of Lou/C rats are associated with (i) a selectively enhanced sympathetic activity restricted to the white adipose tissue and brown adipose tissue, (ii) a reduced noradrenergic activity in selective brainstem and hypothalamic areas, which control the energy expenditure and food intake.

Growth hormone receptor expression in atrophying muscle fibers of rats.

Biological actions of GH on muscle growth and metabolism are mediated through specific trans-membrane receptors. The aim of this study was to determine GH receptor (GHR) mRNA expression in muscle atrophy. GHR gene expression in the rat was investigated by in situ hybridization and RT-PCR in slow-twitch oxidative muscle [soleus (SOL)] and fast-twitch glycolytic muscle [extensor digitorum longus (EDL)] after 7 and 35 d of hindlimb unloading. In control rats, the RT-PCR mRNAs levels of GHR were greater (+34%) in EDL compared with SOL. At single fiber level, relative expression of GHR mRNA increases in the following order: IIb>IIa>I. After hindlimb unloading, GHR expression significantly increased in atrophied SOL muscle after 7 (+170%) and 35 (+220%) d, whereas no significant alterations appeared in the EDL muscle. At the individual fiber level, in situ hybridization demonstrated this increase was accounted for by an increase in type I fiber expression of GHR transcripts. This increase was also seen in the EDL, but the low content of type I fibers in EDL resulted in a nonsignificant increase in GHR transcript content. The present data suggest that muscle atrophy is associated with a muscle fiber type-specific GHR mRNA up-regulation mechanism that helps protect atrophying fibers in EDL but might be part of an attempt to repair in SOL.

Nuclear translocation and retention of growth hormone.

We have previously demonstrated that GH is subject to rapid receptor-dependent nuclear translocation. Here, we examine the importance of ligand activation of the GH-receptor (GHR)-associated Janus kinase (JAK) 2 and receptor dimerization for hormone internalization and nuclear translocation by use of cells stably transfected with cDNA for the GHR. Staurosporine and herbimycin A treatment of cells did not affect the ability of GH to internalize but resulted in increased nuclear accumulation of hormone. Similarly, receptor mutations, which prevent the association and activation of JAK2, did not affect the ability of the hormone to internalize or translocate to the nucleus but resulted in increased nuclear accumulation of GH. These results were observed both by nuclear isolation and confocal laser scanning microscopy. Staurosporine treatment of cells in which human GH (hGH) was targeted to the cytoplasm (removal of secretion sequence) or to the nucleus (addition of the nuclear localization sequence of SV40 large T antigen) resulted in preferential accumulation of hGH in the nucleus. We further investigated the requirement of receptor dimerization for GH nuclear translocation using the non-receptor-dimerizing hGH antagonist, hGH-G120R, conjugated to fluorescein isothiocyanate. Confocal laser scanning microscopy demonstrated efficient internalization of both hGH and hGH-G120R but lack of nuclear translocation of hGH-G120R. Thus, we conclude that activation of JAK2 kinase and the subsequent tyrosine phosphorylation is not required for nuclear translocation of GH but is pivotal for the removal of the hormone from the nucleus, and that GH translocates into the nucleus in a GHR dimerized-dependent fashion.

Visualization of gene expression of prolactin-receptor (PRL-R) by in situ hybridization in reproductive organs of Typhlonectes compressicauda, a gymnophionan amphibian.

The expression of the long form of prolactin receptor (PRL-R) mRNAs was studied in reproductive organs from both male and female Typhlonectes compressicauda, an amphibian, by quantitative in situ hybridization. These PRL-R mRNAs were expressed in all the organs studied. In ovarian tissue, mRNAs coding for the long form of PRL-R were strongly expressed in ovaries with compact and vascularized corpora lutea, i.e., during the middle and the end of pregnancy. During the other periods of cycle, sexual rest, vitellogenesis and beginning of pregnancy, expression of these mRNAs was lower than in other periods. In the testis, mRNAs coding for the long form of PRL-R were expressed in germ cells as well as in Leydig and Sertoli cells. In müllerian glands, important variations of expression in these mRNAs have been observed with a large increase during the breeding period and a decrease during sexual rest. Variations of gene expression of the long form of PRL-R for all tissues studied are correlated to synthesis of PRL in the pituitary in both male and female Typhlonectes compressicauda.

The use of in situ hybridization to study the transgene pathway following cellular transfection with cationic phosphonolipids.

Gene therapy is a promising field of research and biotechnological development. Considering their safety and non-immunogenicity, cationic lipids are widely used for gene transfer in vitro and show promise for in vivo gene transfer applications. However, a better understanding of the mechanisms by which transfection occurs and the limiting steps in cellular transfer of foreign DNA are critical for significant improvements of gene transfer. In this work, we have traced the plasmid DNA into human hematopoietic cell line (K562) using the in situ hybridization method in order to define the main difficulties in transfection and to design new agents better adapted to cellular constraints. In this hematopoietic cell line, after showing the efficiency of our synthetic vectors and optimizing their formulation, we observed that only 5 h after transfection the nucleus to cytoplasm signal ratio was three to one, whereas at 24 h it was one to one. In parallel, the level of the reporter protein strongly increased between these times. Those results emphasize the rapidity of transfection and lead one to imagine chemical modifications adjusted to the environment.

Rat heart GDNF: effect of chemical sympathectomy.

Developmental studies indicate a role for GDNF in survival of motor, autonomic, and sensory neurons. However, no study attempted to demonstrate its participation in autonomic nerve regeneration. In this work, chemical sympathectomy by 6-hydroxydopamine provided the model for assessing heart GDNF expression during denervation and axonal regrowth. A glyoxylic acid-based histochemical technique evaluated the noradrenergic innervation. ELISA determined GDNF levels after concentrating heart homogenates. Light and ultrastructural in situ hybridization and immunocytochemistry were used for identifying cells expressing GDNF mRNA and protein. In control rats, the GDNF cardiac levels were significantly higher in 37-day-old animals in comparison with those aging 60 days. In sympathectomized rats, GDNF cardiac levels were significantly higher 7 days after sympathectomy and dropped to control levels at day 30. GDNF mRNA was expressed in atrial and ventricular myocytes from normal and sympathectomized rats. GDNF immunoreactivity occurred on atrial granules and quantitative analysis in electron micrographs confirmed ELISA-obtained data. In ventricular myocytes gold particles occurred sparsely. These findings constitute the first evidence for GDNF synthesis by cardiomyocytes and postulate a role for this factor soon after cardiac sympathetic denervation, probably in nerve regeneration. In atrial myocytes, GDNF is probably secreted by regulated pathway.

Localization and regulation of a functional GHRH receptor in the rat renal medulla.

To provide information about the kidney GHRH receptor (GHRH-R), we assessed its tissue and cellular localization, defined its pattern of expression in developing and aging rats, and studied the effects of GHRH on the regulation of GHRH-R mRNA levels and receptor internalization. In situ hybridization and ribonuclease protection assay demonstrated that GHRH-R mRNA is restricted to the Henle's loop (HL). GHRH-R mRNA levels were low in the medulla from 3- and 12-d-old male rats, increased significantly in that from 30- to 70-d-old rats, and decreased in that from 12- and 18-month-old animals. Compared with the GHRH-R mRNA profile obtained in the pituitary, these data support the concept of a tissue-specific regulation of GHRH-R. In HL cell cultures from 70-d-old rats, a 4-h incubation with 1-100 nM rat GHRH-(1-29)NH(2) reduced GHRH-R mRNA levels significantly. As anti-GHRH-R- (392-404) immunoreactivity was demonstrated in HL cells, internalization of [N(alpha)-5-carboxyfluoresceinyl-D-Ala(2),Ala(8), Ala(15),Lys(22)]hGHRH-(1-29)NH(2) in a time- and temperature-dependent manner and inhibition of this process by phenyl arsine oxide indicate that desensitization to GHRH involves both GHRH-R internalization and down-regulation of GHRH-R mRNA levels. Localization of a functional GHRH-R in HL and its regulation during development and aging suggest roles associated with cellular proliferation, differentiation, and/or water/electrolyte transport.