Cytological and immunocytochemical characterization of the insulin secreting insulinoma cell line RINm5F

The rat insulinoma cell line RINm5F, an insulin secreting pancreatic beta cell line, has been used as an attractive model for basic studies of the mechanisms of insulin secretion and, more recently, as a model for the development of alternative methods for the treatment of diabetes. To elucidate the cytological properties and expression patterns of hormones of the gastro-entero-pancreatic system, suspensions of RINm5F cells were investigated by various methods including immunocytochemistry on serial semithin sections, quantitative immunocytochemistry, routine electron microscopy, immuno-electron microscopy, in situ hybridization, and TUNEL technique. At the ultrastructural level, several phenotypes of RIm5F cells were characterized by differences in the number, shape, size, and density of their secretory granules. The most common type contained a mixture of round granules varying in size and electron density. A second type predominantly contained relatively large, moderately dense granules. Moreover, a minority of cells was characterized by the occurrence of polymorphous electron dense granules or the complete absence of any secretory granules. The immunohistochemical data showed that, among the established islet hormones, insulin was present in more than 50% of cells, whereas glucagon and somatostatin occurred only sporadically. Though cells positive for pancreatic polypeptide (PP) were not found, PP-related peptides (NPY and PYY) however could be detected in a minority of cells. The great majority of RINm5F cells were immunoreactive for chromogranin B (CgB), followed by insulin, chromogranin A (CgA), and serotonin (5-HT). In addition to intercellular differences in the density of immunostaining, numerous colocalizations of immunoreactivities were found, suggesting that RINm5F cells represent a mixture of subtypes concerning the individual pattern of hormone expression. The present results reveal a wide range of heterogeneity with respect to the morphology and especially the hormone content between individual RINm5F cells.     

A delayed-type hypersensitivity skin-test system using the insulinoma cell line RINm5F to monitor beta cell-specific cellular autoimmune reactivity in the spontaneously diabetic BB/O rat.

Islet-specific autoimmune reactivity (humoral and cell-mediated) is the basis for the insulitis process of type I diabetes mellitus. In this report a delayed-type hypersensitivity (DTH) skin test was used to monitor the presence of an islet-specific cell-mediated autoimmune component in BB/O rats. The BB/O rat is a strain characterized by the spontaneous development of type I diabetes. Intact RINm5F cells as well as a RINm5F cell membrane preparation were used as DTH skin test antigens. Rats of different ages and disease stages were tested in the ear with the insulinoma cell line and its cell membrane preparation. As control antigens, the fibroblast cell line 3Y1 and a cell membrane preparation made thereof were used. The DTH reaction system showed a positive cell-mediated reactivity in BB/O rats for membrane-bound RINm5F cell antigens, and not for the control fibroblast 3Y1 cell membrane determinants. The true DTH character of the skin test was established by the time-course of the reaction (maximum at 24 h), the histopathology (infiltration by dendritic cells, lymphocytes and macrophages), and the possibility to transfer the reaction with spleen cells and lymph node cells. The DTH test towards RINm5F cells showed the highest prevalence of positivity (100%) in BB/O rats around the onset of diabetes (3 weeks before to 3 weeks after the onset of glucosuria). The prevalence of DTH positivity was 56% in the period of more than 3 weeks before the onset of glucosuria. In BB/O rats with a duration of glucosuria of more than 3 weeks, the prevalence of positivity was around 60-70%.     

NK92细胞的胞吐效应与SNAP-23蛋白转位相关

的研究SNAP-23蛋白在NK细胞系NK92细胞胞吐效应中的作用，探讨NK细胞胞吐的分子机制。方法利用已糖氨酶(β-hexosaminidase)释放实验检测NK细胞的胞吐作用。通过RT-PCR和Western blot显示NK92细胞中SNAP-23a mRNA和蛋白的表达，利用激光共聚焦显微镜扫描术确定靶细胞触发前后SNAP-23a在NK92细胞中的定位。结果NK细胞的胞吐作用在靶细胞刺激后2h明显，4～6h达高峰；NK92细胞组成性表达SNAP-23a mRNA和蛋白，靶细胞刺激前后蛋白表达量虽无明显差异，但其定位发生了显著改变：激光共聚焦显微镜扫描结果显示在静息NK92中SNAP-23a主要表达在胞浆内的颗粒上，经靶细胞刺激后逐渐向细胞膜发生转位，2h明显转位，4～6h几乎完全转位。结论NK细胞的胞吐效应与SNAP-23蛋白转位相关。     

Calcium and ATP regulate the activity of a non-selective cation channel in a rat insulinoma cell line

A calcium-acivated non-selective cation channel was observed in isolated plasma membrane patches from an insulin-secreting cell line (CRI-Gl). The conductance of the channel was approximately 25 pS with identical (140 mM KCl) solutions on either side of the membrane. However, some rectification was observed (smaller outward current) when sodium ions were present extracellularly. The channel was inactive on exposure to an intracellular calcium concentration of 10^–6 M and required high (greater than 10^–4 M) concentrations for a significant degree of activation. The open-state probability of the channel was voltage dependent, increasing with membrane depolarization. Analysis of single channel kinetics indicated that there were at least two open and two closed states. Application of ATP to the cytoplasmic membrane surface reduced the open state probability in a dose-dependent manner. The channel activity was blocked by quinine and 4-AP but was insensitive to TEA, TTX and amiloride. It is not clear what role this channel might play in the complex electrical activity of -cells.     

In vitro effects of adrenomedullin and calcitonin gene related peptide on the release of serotonin and amino acids from rat dorsal spinal cord

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) are structurally related, interact with each others receptors and show overlapping biological activities. Immunoreactivity (IR) and mRNAs along with binding sites for both CGRP and adrenomedullin have been shown in the rat spinal cord. CGRP mediates the transmission of nociceptive information at the spinal cord level and both peptides has shown to induces c-fos expression and accumulation of cAMP in spinal cells. In this study, HPLC methods were used to investigate the effects of AM and CGRP on the basal and K⁺-evoked release of serotonin, glutamate (Glu), aspartate (Asp), glycine (Gly) and γ amino butyric acid (GABA) from the slices prepared from the rat spinal cord. Neither CGRP (10⁻⁷ and 10⁻⁶ M) nor AM (10⁻⁷ and 10⁻⁶ M) had significant effects on the basal release of serotonin and the amino acids tested in this study. However, CGRP produced statistically significant increases in the K⁺-evoked release of Asp and Glu, whereas AM failed to do so. Neither AM nor CGRP (10⁻⁷ and 10⁻⁶ M) showed any significant effects on the K⁺-evoked release of serotonin, GABA and Gly. Present data suggest that the stimulatory effects of CGRP on the release of Asp and Glu were exerted by distinct types of CGRP receptors.     

Aquaporin expression and cell volume regulation in the SV40 immortalized rat submandibular acinar cell line

The amount of aquaporins present and the cellular ability to perform regulatory volume changes are likely to be important for fluid secretions from exocrine glands. In this work these phenomena were studied in an SV40 immortalized rat submandibular acinar cell line. The regulatory cell volume characteristics have not previously been determined in these cells. Cell volume regulation following hyposmotic exposure and aquaporin induction was examined with Coulter counter methodology, radioactive efflux studies, fura-2 fluorescence, and polymerase chain reaction and Western blot techniques. Cell volume regulation was inhibited by the K⁺ channel antagonists quinine and BaCl₂ and the Cl⁻ channel blocker 5-nitro-2-(3-phenypropylamino)benzoic acid. A concomitant increase in cellular ³H-taurine release and Ca²⁺ concentration was also observed. Chelation of both intra- and extracellular Ca²⁺ with EGTA and the Ca²⁺ ionophore A23187 did not, however, affect cell volume regulation. Aquaporin 5 (AQP5) mRNA and protein levels were upregulated in hyperosmotic conditions and downregulated upon return to isosmotic solutions, but were reduced by the mitogen-activated ERK-activating kinase (MEK) inhibitor U0126. A 24-h MEK inhibition also diminished hyposmotically induced cell swelling and cell volume regulation. In conclusion, it was determined that regulatory volume changes in this immortalized cell line are due to KCl and taurine efflux. In conditions that increased AQP5 levels, the cells showed a faster cell swelling and a more complete volume recovery following hyposmotic exposure. This response could be overturned by MEK inhibition.     

Raloxifene blocks estradiol induction of the serotonin transporter and 5-hydroxytryptamine2A receptor in female rat brain

Sex steroids have potent effects on mood, mental state and cognition. Our previous findings and those of others suggest that these effects may be due at least in part to estradiol actions on central serotonergic mechanisms. Specifically, estradiol-17beta in its acute positive feedback mode for gonadotropin release in the female rat induces expression of the genes for the 5-hydroxytryptamine(2A) receptor (5-HT(2A)R) and the serotonin transporter (SERT) in the dorsal raphe nucleus (DRN). This is accompanied by an increase in the densities of 5-HT(2A)R and the SERT in forebrain regions which in the human are concerned with the control of mood, mental state, cognition and emotion. Here we report that raloxifene, a benzothiophene and selective estrogen receptor modulator (SERM), completely blocked estradiol stimulation of brain 5-HT(2A)R and SERT expression in acutely ovariectomized rats. Raloxifene also blocked the estrogen-induced surge of luteinizing hormone. Treatment of acutely ovariectomized rats with raloxifene alone increased the density of SERT sites in the mid-frontal cortex and decreased the density of 5-HT(2A)R in the posterior olfactory tubercle. The inhibitory effects of raloxifene on acute estrogen-induction of central serotonergic mechanisms were similar to those of tamoxifen even though there are major differences between the two SERMs in their affinity for the two estrogen receptor subtypes and their actions on the uterus. These findings provide robust evidence that estradiol induction of the 5-HT(2A)R and the SERT in brain is mediated by nuclear estrogen receptors. Our data may provide the basis for obtaining a better understanding of the effects of sex steroids on mood and mental state in the human and the possible rational development of congeners of sex steroids for the treatment of mental disorders.     

Complex action potential waveform recorded from supraoptic and paraventricular neurones of the rat: Evidence for sodium and calcium spike components at different membrane sites

Summary Magnocellular neurones of the supraoptic and paraventricular nuclei display a complex waveform when recorded extracellularly. The present work has examined the possible reasons for the complexity of this waveform by making extracellular recordings from antidromically identified neurones in the anaesthetized rat and extra- and intracellular recordings from similar neurones in the hypothalamic slice preparation, where external solutions can be changed. In extracellularly recorded units, action potentials had two positive going components. The first of these was abolished by tetrodotoxin. The second, slower component was abolished when external Ca⁺⁺ concentration was lowered, but enhanced in magnitude and duration when Ba⁺⁺ was placed in the external medium. The second component was sensitive to electrode movement and was not observed to the same degree when intracellular recordings were made. In light of these observations and the known morphology of these neurones, it is suggested that the magnocellular neurone action potential is comprised of a fast Na⁺-dependent component and a slower component dependent on Ca⁺⁺ entry which may originate from a part of the cell other than the soma. The most likely site for this Ca⁺⁺ component to occur is at the cell dendrite.     

Detection of the release of 5-hydroxyindole compounds in the hypothalamus and the n. raphe dorsalis throughout the sleep-waking cycle and during stressful situations in the rat: a polygraphic and voltammetric approach

Summary In the present work, voltammetric method combined with polygraphic recordings were used in animals under long-term chronic conditions; the extracellular concentrations of 5-hydroxyindole compounds (5-OH^les) and in particular 5-hydroxyindoleacetic acid (5-HIAA) were measured in the hypothalamus and in the nucleus Raphe Dorsalis (n.RD). The hypothesis that extracellular detection of 5-HIAA, in animals under physiological conditions, might reflect serotonin (5-HT) release is suggested by the following observations: — serotoninergic neurons are reported to contain only monoamine oxidase type B (MAO-B); — an inhibitor of such an enzyme, MDL 72145 (1 mg/kg), fails to decrease the extracellular 5-HIAA peak 3 height; — MAO type A is contained in non-5-HT cells or neurons; — only the inhibitor of this last type of enzyme (Clorgyline 2.5 mg/kg) induces a complete disappearance of the voltammetric signal. The 5-HIAA measured in the extracellular space thus comes from the 5-HT released and metabolized outside the 5-HT neurons. Throughout the sleep-waking cycle, 5-OH^les release occurs following two different modes: 1 — during sleep, in the vicinity of the 5-HT cellular bodies in the n.RD; this release might come from dendrites and be responsible for the 5-HT neuronal inhibition occurring during sleep; 2 — during waking, at the level of the axonal nerve endings impinging on the hypothalamus; this release might be related to the synthesis of hypnogenic factors. Finally, we have observed that in the hypothalamus, 30 min. of immobilization-stress (IS) induces a larger increase of the voltammetric signal (+ 80%) than a painful stimulation of the same duration (+ 30%); the possible link between the 5-OH^les release occurring in this area during an IS and the subsequent paradoxical sleep rebound is discussed.     

The effect of some putative neurotransmitters on the release of 5-hydroxytryptamine and gamma-aminobutyric acid from slices of the rat midbrain raphe area.

The effect of various putative transmitters has been studied on the efflux of [³H]5-hydroxytryptamine and [³H]γ-aminobutyric acid from small superfused slices of the midbrain raphe area of the rat. The slices apparently contained functionally intact terminals for these transmitters since a depolarizing stimulus (50 mM KCl) stimulated the rate of efflux of [³H]5-hydroxytryptamine and [³H]γ-aminobutyric acid in a calcium-dependent fashion. Furthermore, slices accumulated radioactivity with apparent high affinity mechanisms. γ-Aminobutyric acid (50 and 100 μ m) and substance P (100 and 500μ m) stimulated the efflux of [³H]5-hydroxytryptamine. The effect of γ-aminobutyrate was blocked by picrotoxin (50 μ m) but not by strychnine (1 μ m). Other inhibitory amino acids (β-alanine, taurine and glycine) were without effect on the release of [³H]5-hydroxytryptamine. l-Noradrenaline (0.2–1 mM) stimulated the efflux of [³H]γ-aminobutyric acid but not that of [3H]5-hydroxytryptamine. Phentolamine (10μ m) but not (±)-propranolol (10 μ m) abolished the effect of 1 mM noradrenaline. Neither dopamine nor 5-hydroxytryptamine influenced the efflux of [3H]γ-aminobutyric acid.It is possible that interactions in the midbrain raphe area proposed from other studies can be mediated through presynaptic influences on transmitter release.     

A new liver metastatic and peritoneal dissemination model established from the same human pancreatic cancer cell line: analysis using cDNA macroarray

To elucidate the mechanisms of metastasis, we established two sublines HPC-1H5 with a highly liver metastatic cell line and HPC-1P5a with a highly peritoneal disseminating cell line, which were sequentially selected from the parental pancreatic cancer cell line HPC-1. Using these three cell lines, we investigated several biological properties and mRNA levels of differentially-expressed genes involved in cancer metastasis by cDNA macroarray. Microscopic findings for the three cell lines were the same. The tumorigenicity, in vitro growth ability, motile activity, adhesive activity and the production of IL-8 of metastatic sublines were higher than those of parental HPC-1 cells. Particularly, HPC-1H5 cells showed clearly higher levels of IL-8 expression and tumors of HPC-1H5 cells grew faster and bigger than those of HPC-1P5a cells. In cDNA macroarray analysis of HPC-1H5 cells, 22 genes were up-regulated and 44 genes were down-regulated compared with parental HPC-1 cells. In HPC-1P5a cells, 9 genes were up-regulated and 28 genes were down-regulated compared with parental HPC-1 cells. This study provides a demonstration of global gene expression analysis of pancreatic cancer cells with liver metastasis and peritoneal dissemination. Furthermore, our results provide a new insight into the study of liver metastasis and peritoneal dissemination of human pancreatic cancer. This revised version was published online in July 2006 with corrections to the Cover Date.     

南海真菌代谢物1386A对胃癌MCG-803细胞增殖、凋亡和膜电位的影响

目的:研究南海真菌代谢物1386A对胃癌MCG-803细胞的作用。方法:采用MTT细胞活性检测法检测1386A对MCG-803细胞活性的影响,用集落形成实验检测1386A对MCG-803细胞增殖能力的影响,用流式细胞仪检测1386A对MCG-803细胞周期分布、凋亡、线粒体膜电位的影响。结果:1386A作用于MCG-803细胞24、48和72h后,MTT检测细胞活性的IC50分别为29.70、14.07和13.47μmol/L,1386A作用48h后集落形成实验检测MCG-803细胞增殖的IC50为3.27μmol/L,1386A作用48h后MCG-803细胞的S期比例由25.6%上升为56.8%,1386A作用24h后MCG-803细胞早期凋亡率由3.34%上升为8.39%,1386A作用24h后MCG-803细胞线粒体膜电位下降率为56.26%。结论:南海真菌代谢物1386A能明显抑制胃癌MCG-803细胞的生长,可能通过线粒体凋亡途径诱导细胞的凋亡。     

Projections from the rat cuneiform nucleus to the A7, A6 (locus coeruleus), and A5 pontine noradrenergic cell groups

Stimulation of neurons in the cuneiform nucleus (CnF) produces antinociception and cardiovascular responses that could be mediated, in part, by noradrenergic neurons that innervate the spinal cord dorsal horn. The present study determined the projections of neurons in the CnF to the pontine noradrenergic neurons in the A5, A6 (locus coeruleus), and A7 cell groups that are known to project to the spinal cord. Injections of the anterograde tracer, biotinylated dextran amine in the CnF of Sasco Sprague-Dawley rats labeled axons located near noradrenergic neurons that were visualized by processing tissue sections for tyrosine hydroxylase-immunoreactivity. Anterogradely labeled axons were more dense on the side ipsilateral to the BDA deposit. Both A7 and A5 cell groups received dense projections from neurons in the CnF, whereas locus coeruleus received only a sparse projection. Highly varicose anterogradely labeled axons from the CnF were found in close apposition to dendrites and somata of tyrosine hydroxylase-immunoreactive neurons in pontine tegmentum. Although definitive evidence for direct pathways from CnF neurons to the pontine noradrenergic cell groups requires ultrastructural analysis, the results of the present studies provide presumptive evidence of direct projections from neurons in the CnF to the pontine noradrenergic neurons of the A7, locus coeruleus, and A5 cell groups. These results support the suggestion that the analgesia and cardiovascular responses produced by stimulation of neurons in the CnF may be mediated, in part, by pontine noradrenergic neurons.     

Mapping of T cell epitopes of the major fraction of rye grass using peripheral blood mononuclear cells from atopics and non-atopics. II. Isoallergen clone 5A of Loliuum perenne group I (Lol p I)

Rye grass is the major cause of hay fever which currently affects 20 % of the population. Lolium perenne group I (Lol p I) is a glycoprotein of 240 amino acid residues, representing the main allergen of rye grass. We have used peripheral blood mononuclear cells (PBMC) from controls and subjects allergic to rye grass and cultured them with L. perenne extract (LPE) and Lol p I and measured lymphocyte activation using thymidine incorporation. Patients were further studied against the 115 overlapping peptides of the iso-allergen clone 5A of Lol p I to see whether the 4 amino acid residue differences between clone 1A and clone 5A affect the T cell epitope and thus, lymphocyte activation. There are 24 peptide differences between isoallergen clone 1A and clone 5A occurring in pools 4, 13, 16 and 19 each one of which could be an immunodominant epitope. The PBMC from all allergic patients studied showed a strong proliferative response to LPE and Lol p I. Five immunogenic peptide pools, pool 6, 15, 16, 17 and 19 of the isoallergen clone 5A were also identified. Most of these pools are in the C-terminal region of Lol p I. Out of 20 pools tested in vitro 1 pool (pool-17) induced PBMC proliferation in five out of six patients who were not restricted to an HLA class II DR gene product. However, three out of the six subjects responded to various other peptide pools in addition to the immunodominant pool. In spite of the amino acid differences between the two clones, pool 17 still remains the immunodominant T cell epitope. Control subjects showed only weak responses to LPE and no detectable response to either Lol p I or peptide pools. From within the most active pool we have defined two peptides of the isoallergen clone 5A (identical in sequence with clone 1A) which stimulate lymphocytes from rye grass-sensitive patients in vitro. Previous studies with the two continuous sequences (¹⁹³WGAVWRIDTPDK²⁰⁴ and ¹⁹⁵AVWRIDTPDKLT²⁰⁶) tested in vivo by intradermal skin testing have shown typical delayed-type hypersensitivity reactions after 24—48 h in one patient. Comparison of amino acid sequences of Lol p I, Lol p II and Lol p III proteins revealed a significant level of structural similarity among them. Interestingly, 50% of the residues of the second peptide sequence are also present in Lol p II and Lol p III. Clinically it would be potentially beneficial if a common immunodominant T cell epitope can be used to induce anergy or specific T cell unresponsiveness to several different rye grass allergens locally or systemically.