Expression of adrenomedullin and peptide amidation activity in human prostate cancer and in human prostate cancer cell lines.

After therapeutic hormone deprivation, prostate cancer (CaP) cells often develop androgen-independent growth through not-well-defined mechanisms. The presence of neuroendocrine (NE) cells is often greater in prostate carcinoma than in normal prostate, and the frequency of NE cells correlates with tumor malignancy, loss of androgen sensitivity, increase of autocrine-paracrine activity, and poor prognosis. In some CaPs, neuropeptides have been previously implicated as growth factors. Peptidylglycine alpha-amidating monooxygenase (PAM) is the enzyme producing alpha-amidated bioactive peptides from their inactive glycine-extended precursors. In the present work, we demonstrate that androgen-independent PC-3 and DU145 cell lines, derived from human CaP, express PAM in vitro and in xenografts implanted in athymic nude mice, indicating that they are able to produce alpha-amidated peptides. Contrarily, barely detectable levels of PAM were found in the androgen-sensitive LNCaP cell line. We also show that whereas PC-3 and DU145 cells produce and secrete adrenomedullin (AM), a multifunctional amidated peptide, no expression was found in LNCaP cells. We further demonstrate that AM acts as a growth factor for DU145 cells, which suggests the existence of an autocrine loop mechanism that could potentially drive neoplastic growth. PAM mRNA levels were found to be 3-fold higher in prostate adenocarcinomas compared with that of human benign prostate hyperplasia (BPH) as demonstrated by real-time quantitative reverse transcription-PCR. The analysis of AM message expression in BPH and CaP (Gleason's score, 6-9) shows a clear distinction between benign and CaP. The expression was detected only in adenocarcinomas tissues with a marked increase in samples with a high Gleason's score. Immunocytochemically, AM was localized in the carcinomatous epithelial compartment. NE phenotype, assessed after the immunocytochemical localization of neuron-specific enolase (NSE), was found in both the epithelial and the stromal compartments of cancers; in BPH, only some spare basal cells were NSE-labeled. Cancer progression could be accelerated by peptides secreted by a population of cells capable of inducing androgen-independent tumoral growth via autocrine-paracrine mechanisms.

Human somatostatin receptor subtypes in acromegaly: distinct patterns of messenger ribonucleic acid expression and hormone suppression identify different tumoral phenotypes.

Recently, studies using somatostatin (SRIF) analogs preferential for either the SRIF receptor 2 (SSTR2) or the SSTR5 subtype demonstrated a variable suppression of GH and PRL release from GH-secreting human adenomas. These data suggested the concept of SSTR subtype specificity in such tumors. In the present study the quantitative expression of messenger ribonucleic acid (mRNA) for the 5 SSTR subtypes and the inhibitory effects of SRIF14; SRIF28; octreotide; the SSTR2-preferential analog, BIM-23197; and the SSTR5-preferential analog, BIM-23268, on GH and PRL secretion were analyzed in cells cultured from 15 acromegalic tumors. RT-PCR analysis revealed a consistent pattern of SSTR2 and SSTR5 mRNA expression. SSTR5 mRNA was expressed at a higher level (1052 +/- 405 pg/pg glyceraldehyde-3-phosphate dehydrogenase) than SSTR2 mRNA (100 +/- 30 pg/pg glyceraldehyde-3-phosphate dehydrogenase). However, only SSTR2 mRNA expression correlated with the degree of GH inhibition induced by SRIF14, SRIF28, and BIM-23197. The SSTR5-preferential compound inhibited GH release in only 7 of 15 cases. In cells cultured from the 10 mixed adenomas that secreted both GH and PRL, RT-PCR analysis revealed a consistent coexpression of SSTR5, SSTR2, and SSTR1 mRNA. In all cases SRIF14, SRIF28, and the SSTR5-preferential analog, BIM-23268, significantly suppressed PRL secretion, with a mean maximal inhibition of 48 +/- 4%. In contrast, the SSTR2-preferential analogs, BIM-23197 and octreotide, were effective in suppressing PRL in only 6 of 10 cases. In cells cultured from adenomas taken from patients partially responsive to the SRIF analog, octreotide, partial additivity in suppressing both GH and PRL secretion was observed when the SSTR2- and SSTR5-preferring analogs, BIM-23197 and BIM-23268, were tested in combination. Our data show a highly variable ratio of the SSTR2 and SSTR5 transcripts, according to tumors. The SSTR2-preferring compound consistently inhibits GH release, whereas the SSTR5-preferring compound is the main inhibitor of PRL secretion. When both drugs are combined, the partial additivity observed in mixed GH- plus PRL-secreting adenomas may be of interest in the therapeutic approach of such tumors.

Impact of gsp oncogene on the expression of genes coding for Gsalpha, Pit-1, Gi2alpha, and somatostatin receptor 2 in human somatotroph adenomas: involvement in octreotide sensitivity.

The impact of the gsp oncogene on the expression of genes engaged in the somatotroph cell phenotype remains poorly understood in human somatotroph adenomas. As the gsp oncogene is associated with an increased octreotide (somatostatin agonist) sensitivity, a group of 8 somatotroph adenomas bearing the gsp mutation (gsp+) and another group of 16 adenomas without the mutation (gsp-) were analyzed, all of them presenting variable octreotide sensitivities. The expressions of genes encoding for G(s)alpha, Pit-1, G(i2)alpha, and SSTR2, involved in the regulation of secretory activity in somatotroph cells, were assessed by Northern blot. A decreased expression of the G(s)alpha gene was found in gsp + tumors, suggesting the existence of a negative feedback of the oncogenic protein upon its own messenger ribonucleic acid (mRNA). In contrast, G(i2)alpha, Pit-1, and GH messengers were not significantly different in the groups. A positive correlation between the in vitro and in vivo GH octreotide-induced secretory inhibition and the expression of SSTR2 mRNA was found. However, the expression of the gene for SSTR2 appeared not to be different between gsp + and gsp-, even when the octreotide sensitivity was significantly higher in the adenomas carrying the mutation. Interestingly, the SSTR2 gene expression was significantly correlated to those of G(i2)alpha and Pit-1. In the same way, the G(s)alpha mRNA expression was positively correlated with those of Gi2alpha and Pit-1. Such correlations strongly suggest a concerted dysregulation of the expression of these genes in both categories of adenomas. The loss of the octreotide sensitivity represents one aspect of the dysregulation process that partially results from the decreased SSTR2 expression. However, the improvement of the sensitivity associated with the presence of the gsp oncogene seems to proceed in a way different from SSTR2 expression.

Expression of functional growth hormone secretagogue receptors in human pituitary adenomas: polymerase chain reaction, triple in-situ hybridization and cell culture studies.

We examined the expression of functional growth hormone secretagogue receptors (GHS-R) in a series of 30 human pituitary adenomas-six secreting GH, three GH-PRL, six prolactin (PRL), five adrenocorticotrophic hormone (ACTH), one thyroid stimulating hormone (TSH), four gonadotroph and five non-secreting adenomas. By reverse transcriptase polymerase chain reaction (RT-PCR), the coexpression of the two GHS-R isoforms (Ia and Ib) was found in all the GH-, GH-PRL- and PRL-secreting adenomas, and only in two out of three corticotroph, two out of four gonadotroph and one out of five non-secreting tumours. They were absent in the TSH-secreting adenoma. The PCR products of GHS-R Ia and Ib were identical in size to those from two normal pituitaries. PCR cloning and sequencing of isoforms performed in two somatotroph adenomas revealed only two single, silent base mutations. Triple in-situ hybridization showed colocalization of GHS-R mRNA with messengers of GH and PRL, conjointly or separately, in individual cells of somatotroph, mammosomatotroph, and lactotroph adenomas. The presence of GHS-R mRNA in cells expressing PRL mRNA is emphasized. In cultured cells from six somatotroph and two mammosomatotroph adenomas, the powerful GHS MK-0677 stimulated GH release in a dose-dependent manner, with maximal effect at 6 h. Contrarily, when GHRH was applied, only three somatotrophs and two mamosomatotrophs were stimulated. In the two mammosomatotrophs, the PRL response to MK-0677 and to GHRH was similar to the GH response. An homologous desensitization of the GHS-R and the GHRH receptor was observed 24 h after a first stimulation by a single dose of the corresponding agonist. Heterologous desensitization was not observed. Interestingly, MK-0677 also stimulated, in a dose-dependent way, the hormone release of cells from all tested lactotroph and corticotroph adenomas. The existence of a functional expression of GHS-R in somatotroph, mammosomatotroph, lactotroph and corticotroph adenomas rises the question of the role played by GHS-R in pituitary adenomas, particularly those not engaged in GH secretion.

Expression of the members of the Ptx family of transcription factors in human pituitary adenomas.

A number of putative transcription factors described in the pituitary have been implicated as key elements in the processes that direct pituitary development. Three recently described proteins, Ptx1, Ptx2, and Ptx3, define a new family of transcription factors, the Ptx subfamily, within the paired-like class of homeodomain factors. In mice, Ptx1 and Ptx2 gene expression has been detected in the area of the pituitary primordium and is maintained throughout development in Rathke pouch and adult pituitary. In the present study, the expression of the Ptx1, Ptx2, and Ptx3 genes was characterized in the normal human pituitary and in the different types of human pituitary adenomas. Although no Ptx3 gene expression could be detected in these tissues, Ptx1 presented with a quite ubiquitous pattern of distribution, being expressed at quite constant levels in normal tissues and in all 60 pituitary tumors analyzed. The pattern of expression of the Ptx2 gene among the different subsets of pituitary adenomas was even more varied. No Ptx2 expression could be detected in corticotroph tumors. In contrast, high levels of Ptx2 messenger ribonucleic acid were measured in the gonadotroph tumors, although no specific correlation to other markers of the gonadotroph lineage differentiation, such as alphaGsu, LHbeta, or FSHbeta, could be evidenced. Finally, Ptx2 was also expressed in pure lactotroph adenomas and not in somatotroph adenomas. Ptx2 is, therefore, the first paired homeodomain pituitary transcription factor differentially expressed in these two lineages, which derive from a common precursor. These results support a role for Ptx2 in the terminal differentiation of somatotroph and lactotroph cell phenotypes.

Ursodeoxycholate protects against ethanol-induced liver mitochondrial injury.

The purpose of this work was to examine whether ursodeoxycholate (UDC), a hydrophilic bile salt, could reduce mitochondrial liver injury from chronic ethanol consumption in rats. Animals were pair-fed liquid diets containing 36% of calories as ethanol or isocaloric carbohydrates. They were randomly assigned into 4 groups of 7 rats each and received a specific treatment for 5 weeks: control diet, ethanol diet, control diet + UDC, and ethanol diet + UDC. Respiratory rates of isolated liver mitochondria were measured using a Clark oxygen electrode with sodium succinate as substrate. Mitochondria from rats chronically fed ethanol demonstrated an impaired ability to produce energy. At the fatty liver stage, the ADP-stimulated respiration (V3) was depressed by 33%, the respiratory control ratio (RC) by 25% and the P/O ratio by 15%. In ethanol-fed rats supplemented with UDC, both the rate and efficiency of ATP synthesis via the oxidative phosphorylation were improved: V3 was increased by 35%, P/O by 8%. All the respiratory parameters were similar in control group and control + UDC group. On the other hand, the number and size of mitochondria were assessed by electron microscopy and computer-assisted quantitative analysis. The number of mitochondria from ethanol-treated rats was decreased by 29%, and they were enlarged by 74%. Both parameters were normalized to control values by UDC treatment. These studies demonstrate that UDC has a protective effect against ethanol-induced mitochondrial injury by improving ATP synthesis and preserving liver mitochondrial morphology. These UDC positive effects may contribute to the observed decrease in fat accumulation and may delay the progression of alcoholic injury to more advanced stages.

An in situ hybridization histochemistry technique allowing simultaneous visualization by the use of confocal microscopy of three cellular mRNA species in individual neurons.

We present a specific and sensitive method for simultaneous detection of three mRNA species in individual neurons. The method relies on the use of riboprobes labeled with [35S]-UTP, digoxigenin-UTP, or biotin-UTP. The nonradioactive probes were sequentially revealed by incubation with anti-digoxigenin immunoglobulins or streptavidin conjugated to peroxidase, followed by the use of fluorochrome-labeled tyramides as peroxidase substrates. The radioactive probe was revealed by conventional autoradiography. There was no interaction among the different probes or the various detection systems. We demonstrate the use of this method by illustrating on laser scanning confocal microscopy the co-localization of the mRNAs coding for corticotropin-releasing factor (CRF), arginine vasopressin (AVP), or peptidylglycine alpha-amidating monooxygenase (PAM) in rat hypothalamic paraventricular nucleus (PVN) and its modulation by endogenous glucocorticoids. Our results suggest that this method could be used not only to study the regulation of the hypothalamo-pituitary-adrenal axis but also in various models in which mRNAs are present at low concentrations.

Pronostic and therapeutic consequences of Gs alpha mutations in somatotroph adenomas.

Human pituitary somatotroph adenomas can be associated with mutations of the s alpha-subunit of G proteins. However, the impact of the gsp mutations on the tumoral phenotype is not well understood at present. This study aims to determine whether the detection of this mutation could impact on the management of acromegalic patients. We examined 30 acromegalic patients; 8 were gsp positive, and 22 were gsp negative. The gsp-positive adenomas appeared to secrete significantly more when the ratio of basal GH level/tumor size was considered. A better octreotide sensitivity of mutated adenomas was clearly shown under in vivo (short and long term) and in vitro conditions. During the acute octreotide test, the GH nadir was significantly lower in the gsp-positive adenomas (85% of maximal inhibition vs. 52%). Eighteen patients were treated with octreotide (300 micrograms/day) for at least 3 months before surgery: the percent inhibition of GH hypersecretion was higher in gsp-positive adenomas (76% vs. 47%). In cell culture, the octreotide-induced inhibition of GH release was significantly higher in gsp-positive adenomas (71% vs. 30%). Finally, during 2 yr of postoperative follow-up, GH hypersecretion was controlled in all patients with gsp mutation even in those in whom tumoral tissue remained after surgery. On the contrary, in the gsp-negative group, octreotide treatment was unable to control hypersecretion in 4 patients bearing tumoral remnants. The Gs alpha mutation could, therefore, be a new marker to foresee the susceptibility of the tumor to be controlled by somatostatin analogs, which improves prognosis.

Expression of angiotensin II receptor subtypes AT(1A) and AT(1B) in enriched fractions of dispersed rat pituitary cells.

Differential evaluation of angiotensin II (Ang II) receptors (AT1A, AT1B and AT2) expression was performed in dispersed adenohypophyseal cells fractionated by unit gravity sedimentation. Binding of [125I-Sar1-Ile8]-Ang II and its displacement by specific nonpeptidic AT1 (DuP753) and AT2 (PD123319) antagonists was monitored throughout the gradient. Quantification of mRNA levels corresponding to both AT1 receptor subtypes (AT1A and AT1B) was achieved by reverse transcriptase polymerase chain reaction (RT-PCR) amplification in the presence of an AT1 receptor mutant cRNA as internal standard. Fractions were characterised by radioimmunoassay for the five major anterior pituitary hormones and by counting immunocytochemically labelled cells. Quantification of AT1 receptor subtype mRNA levels was also performed in four hypophyseal cell lines secreting prolactin, growth hormone, corticotropin and a gonadotropin subunit. As already described for the whole pituitary, AT1B receptor mRNA is predominantly expressed (80% of total AT1A + AT1B receptor mRNA content), whereas AT1A is expressed at lower level (20%) in dispersed pituitary cells. Most AT1 receptor mRNA and binding co-elute with fractions enriched in lactotropes and corticotropes. In contrast to AT1B, AT1A receptor mRNA is not present in heavier populations of lactotropes or in somatomammotropes. Low AT1B mRNA levels are detected in GH4C1 and in GC cells, two clones which secrete respectively prolactin and growth hormone. In contrast, no AT1 receptor mRNA expression was found in two other cell lines, AtT20 and alphaT3-1, which produce pro-opiomelanocortin and gonadotropin. It is concluded that expression of AT1 receptor subtypes is heterogeneous in different populations of lactotropes and corticotropes.

Ursodeoxycholate improves hepatobiliary dysfunction induced by valproate-carbamazepine treatment in the rat.

Carbamazepine (CBZ) and valproate (VPA) are commonly used antiepileptic drugs. These drugs, either alone or combined, may produce hepatotoxicity. We report results of a biochemical and histological study of the liver in rats treated for eight days with VPA (500 mg/Kg/day), CBZ (200 mg/Kg/day) and VPA plus CBZ. A hepatoprotective bile salt, ursodeoxycholate (UDC, 60 mg/Kg/day) was given as a supplement to rats treated with the VPA+CBZ combination. VPA strongly modified the biliary biochemical parameters inducing hypercholeresis and hyposecretion of phospholipids. Microscopically, hepatocytes showed intense vacuolation of the peripheral cytoplasm and alterations of the mitochondrial matrix. CBZ produced increased choleresis but had no effect on biliary lipid parameters. Ultrastructurally, CBZ induced marked proliferation of the smooth endoplasmic reticulum of hepatocytes. The VPA+CBZ association produced a combination of the alterations induced independently by each drug. In both bile and plasma, increased CBZ-epoxide and decreased VPA levels were observed. The addition of UDC restored the biliary phospholipid secretion, decreased cytoplasmic vacuoles and mitochondrial alterations, and diminished the hypertrophy of smooth endoplasmic reticulum, indicating a clear beneficial effect of UDC on hepatobiliary dysfunction induced by the VPA+CBZ combination. Furthermore, the supplementation with UDC did not significantly change the plasma levels of the antiepileptic drugs.

Respective effects of hindlimb suspension, confinement and spaceflight on myotendinous junction ultrastructure.

This study compares the effects of 14-day confinement and spaceflight with the respective effects of 8, 18 and 29-day hindlimb suspension on rat soleus and plantaris MTJ ultrastructure. Independently of the experimental situation, greater morphological changes were observed in the soleus as compared to the plantaris MTJ. 18 days of suspension and 14 days of confinement resulted in ultrastructural modifications of the digit-like processes in the soleus MTJ. Additional changes were observed in the myofibrils, microtendon and tendon after 29 days of suspension and 4 days of spaceflight. These results emphasize the influence of the intensity and duration of the muscle loading on the MTJ ultrastructure.

Mitochondria changes in human muscle after prolonged exercise, endurance training and selenium supplementation.

The functional and structural responses to acute exercise (E) and training, (T) with or without selenium supplementation (Sel), were investigated in a double-blind study on 24 young male subjects. The Sel or the placebo were given over 10 weeks of an endurance training programme. Prior to the programme and on its conclusion muscle biopsies were taken from the vastus lateralis muscle before and after an exhausting treadmill test of maximal endurance capacity (Capmax). The muscle samples were examined by electron microscopy to make a quantitative analysis of the mitochondria population in the muscle fibres. The number of mitochondria per area (QA) and the relative surface occupied by the total mitochondria profile area (AA) were estimated. The mean area per mitochondrion (â) was obtained by the quotient AA/QA. The effects of the isolated or combined independent variables T, E and Sel were analysed by nonparametric tests. Training induced significant increases in both QA (30%, P < 0.001) and AA (52%, P < 0.001), without changing â; T + Sel produced a slight rise of AA (27%, P < 0.001), which resulted in larger (24%, P < 0.001) â. The E produced an enlargement of â resembling swelling. This phenomenon was also found for the combinations E + T and E + T + Sel, but it was then far more pronounced in E + T. The training effects observed are in agreement with previous descriptions. In contrast, the changes observed after acute exercise seem to indicate a remarkable short-term plasticity of muscle mitochondria. The results in Sel would seem to suggest a dampening effect of the selenium on the mitochondria changes, both in chronic and acute exercise. The mechanism of this action on mitochondrial turnover is uncertain, but might be related to a higher efficiency of the selenium-dependent enzyme glutathione peroxidase.

Pathogenic and non-pathogenic T lymphocytes specific for the encephalitogenic epitope of myelin basic protein: functional characteristics and vaccination properties.

Activated CD4+ T lymphocytes specific for myelin basic protein (MBP) can cause experimental autoimmune encephalomyelitis (EAE) upon their inoculation into syngeneic recipients. In Lewis rats, most of the pathogenic T cell clones that develop following immunization with MBP are reactive against the 72-84 amino acid sequence of MBP, the major encephalitogenic region for Lewis rats. In this study, some MBP-specific T cell clones were found to be non-pathogenic, in spite of their strong reactivity against the encephalitogenic epitope. One of these non-pathogenic clones, designated Znp, and an encephalitogenic clone, Z1a-p, were derived from Z1a encephalitogenic line cells. These subclones were compared for epitope specificity, T cell receptor variable gene expression and for various functional activities, in order to delineate properties crucial for pathogenicity. The Z1a-p and Znp cells expressed comparable levels of the T cell receptor genes and shared strong reactivity against the 72-84 epitope of MBP. The pathogenic Z1a-p cells displayed MBP-specific cytolytic activity in vitro, provided an in-vivo 'help' for elicitation of MBP-specific antibodies, mediated a delayed type hypersensitivity (DTH) response to MBP, caused EAE and vaccinated against the disease, thus demonstrating that a single CD4+ T cell clone is capable of eliciting various functions. The non-pathogenic Znp cells could also carry out most of these various functions, but failed to mediate a DTH response to MBP in normal animals. However, when inoculated into sublethally (650 R) irradiated syngeneic recipients, the Znp cells became highly pathogenic and mediated DTH response to MBP. Local irradiation of the recipient facilitated a DTH response to MBP in the irradiated ear, indicating that Znp cells are equipped with the effector mechanisms required for pathogenicity, and that their failure to cause disease may be accounted for by their inability to migrate into extravascular target tissue. Similar data were obtained with an independently isolated non-pathogenic clone, LB-3, specific for the encephalitogenic epitope of MBP. The ability of these non-pathogenic cells to vaccinate against EAE mediated by pathogenic cells raises the possibility that such non-pathogenic cells may play a role in triggering downregulation of pathogenic T cells.

Corticotropin-releasing factor release from in vitro superfused and incubated rat hypothalamus. Effect of potassium, norepinephrine, and dopamine.

We have compared the release of CRF induced by potassium depolarization, noradrenaline or dopamine as monitored either during superfusion of mediobasal hypothalamus or during incubation of whole hypothalamus. The superfusion device was improved in order to prevent gas leakage and to keep constant pO2 and pCO2 in the superfusion chamber. Basal CRF secretion as well as KCl- and norepinephrine-induced CRF release were comparable in superfusion and incubation experiments. Pharmacological investigations suggest that the stimulatory effect of norepinephrine on CRF release is mediated mainly through alpha 1 and alpha 2 adrenergic receptors, and partially through beta receptors.

Vimentin and glial fibrillary acidic protein filaments in radial glia of the adult urodele spinal cord.

This work, based on Golgi impregnations, transmission electron microscopy and immunocytochemistry, demonstrates that the intermediate filaments found in the radial gliocytes of the adult newt spinal cord are both vimentin and glial fibrillary acidic protein (GFAP) structures. Gliocytes appeared as large, arboreous cells, with appendages extending peripherally. They were extensively immunolabelled with both anti-vimentin and anti-GFAP monoclonal antibody conjugates. Outstanding correspondence in cell configuration was found when Golgi-impregnated specimens were compared to the distribution of immunolabels. Electron micrographs showed cytoplasmic bundles of anti-vimentin decorated intermediate filaments occupying the radial projections. The presence of GFAP confirms the astroglial character of the radial glia in urodeles; the existence of vimentin suggests that the spinal cord of the adult animal retains immature astroglia, which should express enlarged capabilities of adaptation.

Tendon and myo-tendinous junction in an overloaded skeletal muscle of the rat.

Overloading of rat plantaris muscles was produced by aseptic ablation of the synergists. The morphological changes occurring after 1 or 2 weeks were investigated at the light and electron microscopical level in the distal tendon of the plantaris and at the myotendinous junction. Sham-operated rats were prepared as controls. In the tendon, quiescent fibrocytes were replaced by activated fibroblasts displaying a vesicular nucleus with prominent nucleoli and an outstanding increase in cytomembranes, particularly the rough endoplasmic reticulum and the Golgi complex. The plasmalemma of the fibroblasts was modified by the presence of caveolae and the surbsurface cytoplasm contained many membrane-bound vacuoles. In the tendon, the collagen bundles were disrupted, resulting in the formation of empty longitudinally oriented spaces; in these spaces, as in the pericapillary areas, no inflammatory cells were observed. At the myotendinous junction, fibroblast activation was consistently observed in both control and overloaded specimens. At this level, the sarcolemma of the finger-like projections of muscle fibres presented many caveolae close to clusters of large subsurface vacuoles. These observations indicate that, at the beginning of the compensatory hypertrophy, the adaptative changes to overloading include a non-inflammatory reaction of the tendon characterized by enhanced collagen synthesis and intensive membrane renewal and recycling. From the mechanical point of view this reaction can impair the tendon resistance to stretch. At the myotendinous junction the increased membrane turnover of the sarcolemma and the fibroblast activation can be considered permanent phenomena consequent to the increased stress exerted upon the interface connecting the contractile apparatus to the stroma.(ABSTRACT TRUNCATED AT 250 WORDS)

[Median eminence in vitro superfusion membrane dynamics in a presynaptic compartment]. / L'éminence médiane en superfusion in vitro dynamique membranaire dans un compartiment présynaptique.

Gallardo and Ramirez developed in 1977 an in vitro superfusion procedure for rat hypothalamus; this procedure has been adapted for preserving tissues in order to make ultrastructural analysis. The median eminences were superfused with both, standard media and with media rich in potassium. After superfusion, the specimens were fixed either by immersion in chemical fixative, or quick-frozen utilizing the Cryoblock designed by Escaig, at 6 degrees K. A better preservation of granular vesicles and cytoskeleton is obtained by freezing. Both procedures support the existence of microvesicular depletion after potassium stimulation as well as the existence of two types of vacuoles (smooth and coated vacuoles) taking their origins in the plasmalemma. A double mechanism of retrieval of exocytosed membranes can be postulated in the median eminence axonal endings. Smooth vacuoles could be the way to retrieve microvesicles and complete a short exoendocytic cycle, while coated vacuoles could be intermediate elements to retrieve membranes belonging to secretory granules.

Poststimulatory endocytosis, microvesicle repopulation and changes in smooth endoplasmic reticulum in nerve endings of the median eminence superfused in vitro.

Mediobasal hypothalami of adult rats were superfused in vitro. A single 5 min pulse of 60 mM KCl-containing medium was infused, followed by either 15, 30, 45, 60 or 75 min superfusions with standard medium. In some experiments, 5 or 10% dextran was added followed by a 15 min recovery. Morphologically, two recovery phases were recognized. The early phase (15-30 min) was characterized by two features: (1) A clear-cut increase in the quantity of large, pleomorphic vacuoles occupying the axoplasm of nerve endings; these vacuoles were observed to be connected to caveolae of the same diameter in the axolemma and they were either coated or uncoated. (2) Progressive increase in the quantity of microvesicles (synaptic vesicles) from an initial depleted state. The vacuoles were found to contain dextran aggregates. Microvesicle-like protrusions bulged from the membrane of vacuoles. The late phase, from 45 min poststimulation onward, was typically identified after the appearance of tubules of smooth endoplasmic reticulum at the most distal segments of the nerve terminals. During this period, large vacuoles tended to decrease in quantity. Granular vesicles remained scant during the entire observation period. Images suggesting formation of microvesicles from tubules of smooth endoplasmic reticulum were observed. These results open the possibility that endocytosis of patches of membranes forming large vacuoles be an important mechanism for retrieving the membranes belonging to microvesicles and granular vesicles. Some of these large vacuoles may contribute to the early regeneration of microvesicles. More microvesicles could later be produced from the smooth endoplasmic reticulum.

Structural changes in nerve endings of rat median eminence superfused with media rich in potassium ions.

In vitro fragments of male rat mediobasal hypothalami were superfused with Krebs--Ringer solution in the presence or absence of CaCl2. Infusions containing up to 60 mM potassium chloride were applied, at the end of which tissues were fixed in osmium tetroxide and prepared for transmission electron microscopy. Control superfusions were run in parallel. Quantitative measurements performed on electron micrographs of the outermost palisade region showed significant (20-30%) increase in caliber of axon endings after intensive potassium ion stimulation. Ultrastructurally, widespread depletion of granular vesicles and microvesicles was found. Vesicle shift to the outer zone of the terminals, formation of membrane-bound tubules of the same diameter as microvesicles, and images of attachment and collapse of vesicles into the axolemma were found, particularly after 1 min stimulation. These findings were interpreted as consistent with exocytosis. Longer stimulations were followed by the appearance of large pleomorphic vacuoles that are probably the result of post-exocytotic membrane retrieval. Axon enlargement and vesicle depletion were absent in specimens superfused with calcium-free medium containing high potassium. The functional significance of these ultrastructural changes are interpreted as supporting the hypothesis that exocytosis of calcium-loaded microvesicles can contribute to extrude this ion from median eminence nerve endings during secretion.

Ultrastructure of the rat median eminence after superfusion.

Isolated medio-basal hypothalami of adult rats were continuously superfused in a chamber with controllable inputs and outputs, for periods from 30 to 240 min. The median eminence was prepared for transmission electron microscopy under carefully controlled conditions by immersion fixation with osmium tetroxide. The ultrastructure of superfused median eminence was compared with that of directly fixed, non-superfused median eminence. Even after 4h of superfusion, the median eminence displays remarkably well preserved histological and cytological patterns; cytomembranes, cell organelles, intercellular relationships, and extracellular spaces were remarkably similar in superfused and non-superfused tissues. As a consequence of osmium tetroxide fixation, microtubules were not observable. The ultrastructural information obtained from unstimulated rat median eminence superfused in vitro provides a basis for future morphofunctional correlations in the study of neurosecretion.