Synthesis and cytotoxicities of mannose conjugated S-nitroso-N-acetylpenicillamine (SNAP)

A series of mannose conjugated S-nitroso-N-acetylpenicillamines (SNAPs) has been synthesized, and their cytotoxicities were assessed for DU 145 human prostate cancer cells and Hela R cancer cells.     

BpV (phen) induces apoptosis of RINm5F cells by modulation of MAPKs and MKP-1

We investigated the mechanism of toxicity of peroxovanadium complex bpV (phen) in RINm5F cells. Treatment with bpV (phen) provoked cell death, predominantly by apoptosis. This compound induced strong and sustained JNK and p38 MAPK activation. However, ERK phosphorylation was not affected. The level of expression of MAPK phosphatase MKP-1 was suppressed after bpV (phen) treatment. In addition, this compound did not stimulate proteolytic processing of procaspase-3, suggesting that caspase-3 is not activated during the course of bpV (phen)-induced apoptosis. A correlative inhibition of JNK activation by immunosuppressive drug FK 506 induced ERK activation and MKP-1 expression, and suppressed RINm5F cell death. A specific p38 inhibitor SB 203580 also stimulated ERK activation and cell survival. Furthermore, simultaneous pretreatment with both FK 506 and SB 203580 almost completely abolished cell death. Thus, our results suggest that stress kinases and MKP-1 have a role in bpV (phen)-induced apoptosis of RINm5F cells.     

Hyperglycemia induces apoptosis and p53 mobilization to mitochondria in RINm5F cells

The mechanisms related to hyperglycemia-induced pancreatic β-cell apoptosis are poorly defined. Rat insulin-producing cells (RINm5F) cultured in high glucose concentrations (30 mM) showed increased apoptosis and protein p53 translocation to mitochondria. In addition, hyperglycemia induced both the disruption of mitochondrial membrane potential (Δ < eqid1 > _m), and an increase in reactive oxygen species (ROS), as shown by fluorescence changes of JC-1 and dichlorodihydrofluorescein-diacetate (DCDHF-DA), respectively. The increased intracellular ROS by high glucose exposure was blunted by mitochondrial-function and NADPH-oxidase inhibitors. We postulate that the concomitant mobilization of p53 protein to the mitochondria and the subsequent changes on the Δ < eqid2 > _m, lead to an important pancreatic β-cell apoptosis mechanism induced by oxidative stress caused by hyperglycemia. This work is part of the thesis required for the doctorate degree in Biological Sciences at the Universidad Autónoma Metropolitana, Mexico City, Mexico.     

Sustained enhancement of Ca(2+) influx by glibenclamide induces apoptosis in RINm5F cells

Cytosolic Ca(2+) elevations are known to be involved in triggering apoptosis in many tissues, but the effect of sustained enhancement of Ca(2+) influx on apoptosis in beta cells remains unknown. We have found that the viability of RINm5F cells is decreased dose-dependently by continuous exposure to glibenclamide at concentrations from 10(-7) to 10(-4) M, and that this effect is partially ameliorated by pretreatment with cycloheximide. Electrophoresis of the cells exposed to glibenclamide revealed ladder-like fragmentation characteristic of apoptosis, and which also is suppressed by cycloheximide pretreatment. By using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, we detected increased DNA fragmentation in the nuclei of the cells exposed to glibenclamide, and staining with Hoechst 33342 and propidium iodide showed a dose-dependent increase in the number of cells with the chromatin condensation and fragmentation in their nuclei that is characteristic of apoptosis. The effects of glibenclamide on cell viability and apoptotic cell death were partially inhibited by treatment with Ca(2+) channel blocker, and by reducing the extracellular Ca(2+) concentration during glibenclamide exposure, suggesting that they may be derived from increased Ca(2+) influx. Furthermore, only the percentage of apoptotic cells, and not that of necrotic cells, increased with the increasing intracellular Ca(2+) concentration during glibenclamide exposure. In conclusion, we have demonstrated that the sustained enhancement of Ca(2+) influx caused by glibenclamide exposure can induce apoptotic cell death in a pure beta cell line.     

Regulation of hepcidin in HepG2 and RINm5F cells

Despite the high impact of the antimicrobial peptide hepcidin in iron homeostasis, the regulation of this hormone is still not completely understood. Studies concerning hepcidin regulation are performed at the mRNA level. For the first time we analyzed the regulation of hepcidin not only at mRNA, but also at protein level in a hepatoma and a pancreatic beta cell line using quantitative RT-PCR and immunoblot analysis. Our data show, that hepcidin is present in HepG2 and RINm5F cells. A significant up-regulation of hepcidin was observed in both cell lines by the inflammatory cytokine interleukin-6, lipopolysaccharide, and a slight upregulation by deferoxamine. A down-regulation was detected after stimulation with erythropoietin. Hepcidin was regulated by iron in a dose dependent manner: low doses up to 3 microM increased hepcidin expression, high doses of iron (65 microM) revealed a switch-over to down-regulation of hepcidin expression. Regulation of hepcidin in HepG2 and RINm5F cells at mRNA and protein level by these substances indicates its involvement in inflammation and iron metabolism.     

High glucose induces mitochondrial p53 phosphorylation by p38 MAPK in pancreatic RINm5F cells

Pancreatic β-cell death in type 2 diabetes has been related to p53 subcellular localisation and phosphorylation. However, the mechanisms by which p53 is phosphorylated and its activation in response to oxidative stress remain poorly understood. Therefore, the aim of this study was to investigate mitochondrial p53 phosphorylation, its subcellular localisation and its relationship with apoptotic induction in RINm5F cells cultured under high glucose conditions. Our results show that p53 phosphorylation in the mitochondrial fraction was greater at ser392 than at ser15. This increased phosphorylation correlated with an increase in reactive oxygen species, a decrease in the Bcl-2/Bax ratio, a release of cytochrome c and an increase in the rate of apoptosis. We also observed a decline in ERK 1/2 phosphorylation over time, which is an indicator of cell proliferation. To identify the kinase responsible for phosphorylating p53, p38 mitogen-activated protein kinase (MAPK) activation was analysed. We found that high glucose induced an increase in p38 MAPK phosphorylation in the mitochondria after 24–72 h. Moreover, the phosphorylation of p53 (ser392) by p38 MAPK in mitochondria was confirmed by colocalisation studies with confocal microscopy. The addition of a specific p38 MAPK inhibitor (SB203580) to the culture medium during high glucose treatment blocked p53 mobilisation to the mitochondria and phosphorylation; thus, the release of cytochrome c and the apoptosis rate in RINm5F cells decreased. These results suggest that mitochondrial p53 phosphorylation by p38 MAPK plays an important role in RINm5F cell death under high glucose conditions.     

Ultrastructural evidence that apoptosis is the mechanism by which human amylin evokes death in RINm5F pancreatic islet beta-cells

A view is emerging that human amylin (HA) kills pancreatic islet beta-cells by apoptosis. This study strengthens this view by documenting time-dependent morphological and ultrastructural changes in 10 microm HA-treated cultured RINm5F islet beta-cells. Membrane blebbing and microvilli loss were the earliest detectable apoptosis-related phenomena, already evident 1 h after HA exposure. Following 6-12 h of HA-treatment, chromatin margination became evident, consistent with detecting DNA laddering about the same time. Nuclear shrinkage, nuclear membrane convolution and prominent cytoplasmic vacuolization were clearly recognized at 22 h post-treatment. Together, these cellular changes constitute a strong case for HA-induced apoptosis, and further demonstrates that electron microscopy is a more sensitive tool for early apoptosis detection in cultured cells than classical biochemical assays like visualizing DNA laddering. The ultrastructural changes reported here contribute further evidence to be included in the ongoing dissection of molecular mechanisms underlying HA-induced apoptosis, as may occur in type-2 diabetes mellitus.     

3-nitrofluoranthene (3-NF)-induced apoptosis and programmed necrosis

Polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs are environmental pollutants formed during incomplete combustion of organic material. Many possess mutagenic and carcinogenic properties, but their effects on cell death are less known. We have found that rather similar PAHs cause death by quite different mechanisms including apoptosis, necrosis as well as various mixtures of the two. In this addendum to our recent publication, Toxicology 2009 (255:140–50), we report that 3-nitrofluoranthene (3-NF) induces apoptosis as well as regulated necrosis with necroptotic features. The typical necroptotic cell exhibited partial nuclear chromatin condensation combined with damaged plasma membrane. The cells were characterized by increased size as well as number of lysosomes and myelinosomes/autophagic vesicles, and also in expression of the autophagic marker, LC3B. However, the induced autophagy appears to be a parallel event rather than the cause of cell death.     

Characterization of serine/threonine protein phosphatases in RINm5F insulinoma cells

This study investigates the occurrence and regulation of serine/threonine protein phosphatases (PPases) in insulin-secreting RINm5F insulinoma cells. PPases types 1 and 2A were identified in crude RINm5F cell homogenates by both enzymatic assay and Western blot analysis. We then characterized and compared the inhibitory actions of several compounds isolated from cyanobacteria, marine dinoflagellates and marine sponges, (viz. okadaic acid, microcystin-LR, calyculin-A and nodularin) cation-independent PPase activities in RINm5F cell homogenates. It was found that okadaic acid was the least potent inhibitor (IC₅₀10^–9M, IC₁₀₀10^–6M), while the other compounds exhibited IC₅₀ values of 5·10^–10 M and IC₁₀₀ 5·10^–9 M. The findings indicate that the inhibitory substances employed in this study may be used pharmacologically to investigate the role of serine/threonine PPases in RINm5F cell insulin secretion, a process that is likely to be regulated to a major extent by protein phosphorylation.     

Potentiation of stimulus-induced insulin secretion in protein kinase C-deficient RINm5F cells.

The role of protein kinase C (PKC) in stimulus recognition and insulin secretion was investigated after long-term (24 h) treatment of RINm5F cells with phorbol 12-myristate 13-acetate (PMA). Three methods revealed that PKC was no longer detectable, and PMA-induced insulin secretion was abolished. Such PKC-deficient cells displayed enhanced insulin secretion (2-6-fold) in response to vasopressin and carbachol (activating phospholipase C) as well as to D-glyceraldehyde and alanine (promoting membrane depolarization and voltage-gated Ca2+ influx). Insulin release stimulated by 1-oleoyl-2-acetylglycerol (OAG) was also greater in PKC-deficient cells. OAG caused membrane depolarization and raised the cytosolic Ca2+ concentration ([Ca2+]i), both of which were unaffected by PKC down-regulation. Except for that caused by vasopressin, the secretagogue-induced [Ca2+]i elevations were similar in control and PKC-depleted cells. The [Ca2+]i rise evoked by vasopressin was enhanced during the early phase (observed both in cell suspensions and at the single cell level) and the stimulation of diacylglycerol production was also augmented. These findings suggest more efficient activation of phospholipase C by vasopressin after PKC depletion. Electrically permeabilized cells were used to test whether the release process is facilitated after long-term PMA treatment. PKC deficiency was associated with only slightly increased responsiveness to half-maximally (2 microM) but not to maximally stimulatory Ca2+ concentrations. At 2 microM-Ca2+ vasopressin caused secretion, which was also augmented by PMA pretreatment. The difference between intact and permeabilized cells could indicate the loss in the latter of soluble factors which mediate the enhanced secretory responses. However, changes in cyclic AMP production could not explain the difference. These results demonstrate that PKC not only exerts inhibitory influences on the coupling of receptors to phospholipase C but also interferes with more distal steps implicated in insulin secretion.     

Protein phosphorylation is required for diazoxide to open ATP-sensitive potassium channels in insulin (RINm5F) secreting cells

The patch-clamp open-cell recording configuration has been used to investigate the effects of non-hydrolyzable analogues of ATP on the diazoxide-activation of K_ATP channels in the insulin-secreting cell line RINm5F. K⁺ channels inhibited by 0.1, 0.5 and 1.0 mM ATP were consistently activated by 200 μM diazoxide. During sustained activation of channels, exchange of ATP for either AMP-PNP, AMP-PCP or ATPγS abolished the effects of diazoxide. If diazoxide was added to the membrane in the continued presence of AMP-PNP, AMP-PCP or ATPγS either no effects were observed or alternatively a small transient activation of channels occurred. This study suggests that protein phosphorylation is necessary for diazoxide to activate ATP-sensitive potassium channels in insulin-secreting cells.     

Glucagon-like peptide-1 improves insulin and proinsulin binding on RINm5F cells and human monocytes

Glucagon-like peptide-1-(7---36) amide (GLP-1) is a potent incretin hormone secreted from distal gut. It stimulates basal and glucose-induced insulin secretion and proinsulin gene expression. The present study tested the hypothesis that GLP-1 may modulate insulin receptor binding. RINm5F rat insulinoma cells were incubated with GLP-1 (0.01-100 nM) for different periods (1 min-24 h). Insulin receptor binding was assessed by competitive ligand binding studies. In addition, we investigated the effect of GLP-1 on insulin receptor binding on monocytes isolated from type 1 and type 2 diabetes patients and healthy volunteers. In RINm5F cells, GLP-1 increased the capacity and affinity of insulin binding in a time- and concentration-dependent manner. The GLP-1 receptor agonist exendin-4 showed similar effects, whereas the receptor antagonist exendin-(9---39) amide inhibited the GLP-1-induced increase in insulin receptor binding. The GLP-1 effect was potentiated by the adenylyl cyclase activator forskolin and the stable cAMP analog Sp-5, 6-dichloro-1-beta-D-ribofuranosyl-benzimidazole-3', 5'-monophosphorothioate but was antagonized by the intracellular Ca(2+) chelator 1,2-bis(0-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. Glucagon, gastric inhibitory peptide (GIP), and GIP-(1---30) did not affect insulin binding. In isolated monocytes, 24 h incubation with 100 nM GLP-1 significantly (P<0.05) increased the diminished number of high-capacity/low-affinity insulin binding sites per cell in type 1 diabetics (9,000+/-3,200 vs. 18,500+/-3,600) and in type 2 diabetics (15,700+/-2,100 vs. 28,900+/-1,800) compared with nondiabetic control subjects (25,100+/-2,700 vs. 26,200+/-4,200). Based on our previous experiments in IEC-6 cells and IM-9 lymphoblasts indicating that the low-affinity/high-capacity insulin binding sites may be more specific for proinsulin (Jehle, PM, Fussgaenger RD, Angelus NK, Jungwirth RJ, Saile B, and Lutz MP. Am J Physiol Endocrinol Metab 276: E262-E268, 1999 and Jehle, PM, Lutz MP, and Fussgaenger RD. Diabetologia 39: 421-432, 1996), we further investigated the effect of GLP-1 on proinsulin binding in RINm5F cells and monocytes. In both cell types, GLP-1 induced a significant increase in proinsulin binding. We conclude that, in RINm5F cells and in isolated human monocytes, GLP-1 specifically increases the number of high-capacity insulin binding sites that may be functional proinsulin receptors.     

Protective effect of (+/-) isoborneol against 6-OHDA-induced apoptosis in SH-SY5Y cells.

Oxidative stress caused by dopamine (DA) may play an important role in the pathogenesis of Parkinson's disease (PD). (+/-) Isoborneol is a monoterpenoid alcohol present in the essential oils of numerous medicinal plants and is a known antioxidant. In this study, we investigated the neuroprotective effect of isoborneol against 6-hydroxydopamine (6-OHDA)-induced cell death in human neuroblastoma SH-SY5Y cells. Pretreatment of SH-SY5Y cells with isoborneol significantly reduced 6-OHDA-induced generation of reactive oxygen species (ROS) and 6-OHDA-induced increases in intracellular calcium. Furthermore, apoptosis induced by 6-OHDA was reversed by isoborneol treatment. Isoborneol protected against 6-OHDA-induced increases in caspase-3 activity and cytochrome C translocation into the cytosol from mitochondria. Isoborneol prevented 6-OHDA from decreasing the Bax/Bcl-2 ratio. We also observed that isoborneol decreased the activation of c-Jun N-terminal kinase and induced activation of protein kinase C (PKC) which had been suppressed by 6-OHDA. Our results indicate that the protective function of isoborneol is dependent upon its antioxidant potential and strongly suggest that isoborneol may be an effective treatment for neurodegenerative diseases associated with oxidative stress.     

Noradrenergic inhibition and voltage-dependent facilitation of omega-conotoxin-sensitive Ca channels in insulin-secreting RINm5F cells.

We found that, besides dihydropyridine-sensitive Ca channels, insulin-secreting RINm5F cells also contain a minority (15-25%) of omega-conotoxin (omega-CgTx)-sensitive channels that show a high-affinity binding to [125I] omega-CgTx (Kd 51 pM). Noradrenaline (NA, 10 microM) slows down Ca-channel activation in these cells and produces a sizeable reduction of Ca currents that is relieved by strong pre-conditioning depolarizations (facilitation). The action of NA is mimicked by intracellular application of GTP-gamma-S and is prevented by pertussis toxin (PTX) or by cell pre-incubation with omega-CgTx. This suggests specific noradrenergic inhibition of omega-CgTx-sensitive Ca channels that is modulated by membrane potentials and PTX-sensitive G-protein activation.     

Discrimination of galanin receptor subtypes in RINm5F cells by structurally different galanin radioligands

Galanin (GAL) is a biologically active peptide that is involved in a variety of physiological functions. The purpose of this study was to evaluate whether porcine and rat galanin radioligands could be used as probes to discriminate GAL receptors (GALR) subtypes using a cell line, RINm5F, that express multiple GALR subtypes. Data from parallel equilibrium binding experiments using the same RINm5F membrane homogenates reveal that [125I]pGAL labels 20% more GALRs with a 2-fold lower affinity than those values identified when using [125I]rGAL. Competition studies using various GAL peptides showed different rank order of potencies depending on the radioligand used. Preincubation of RINm5F membranes with GppNHp, a non-hydrolizable GTP analog, prior to radioligand labeling suggests that a portion of GALRs is precoupled to G proteins. In addition, receptors labeled by [125I]rGAL appear more sensitive to GppNHp-induced uncoupling of G proteins than those labeled by [125I]pGAL. In conclusion, our data suggest that pGAL and rGAL radioligands define different pharmacological profiles of GALRs, and hence, these ligands can be used as pharmacological tools to discriminate GALR subtypes. Additionally, our data suggests that GALRs exist in a precoupled state with their respective G-proteins prior to interaction with the agonist.     

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis is dependent on activation of cysteine and serine proteases

We examined the role of caspases and serine protease(s) in cell death induced by tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). After incubation of adenocarcinoma cells with TRAIL, caspase-3, -8 were activated and the cleavage of Bid induced the release of cytochrome c, from the mitochondria to the cytosol. Tetrapeptide inhibitors of caspase-1, -2, -3, and -8 suppressed DNA fragmentation and attenuated the release of cytochrome c, whereas inhibitors of caspase-5 did not. Interestingly, the general serine protease(s) inhibitor 4-(2-aminoethyl)benzylsulfonyl fluoride (AEBSF) resulted in the arrest of apoptosis. However, the AEBSF did not prevent the release of mitochondrial cytochrome c during TRAIL-induced apoptosis. From these results, we postulate that serine protease(s) may be involved in post-mitochondrial apoptotic events, that lead to the activation of the initiator, caspase-9.     

Isolation of a purified mitochondrial fraction from viable clonal insulin-producing cells (RINm5F) by percoll density gradient centrifugation

Summary A Percoll density gradient was employed for selecting large numbers of viable insulin-producing RINm5F cells. Homogenates of these cells were then subjected to gradient centrifugation and two clearly visible bands were obtained. The light fraction was essentially composed of mitochondria banded at a density of about 1.06 g/ml. The heavier fraction banded at 1.09 to 1.10 g/ml and contained lysosomes and a small number of secretory granules. The distribution of Percoll particles was restricted to the extracellular space and there was no adsorption to any membrane structures. The distribution pattern of marker enzymes for the mitochondria and lysosomes was similar to that of normal pancreatic β-cells. With the use of a Percoll density gradient it was thus possible to isolate a purified mitochondrial fraction from viable RINm5F cells. This work was supported by the Swedish Medical Research Council (03x-4, 12x-562, 12x-6240), the Swedish Diabetes Association, the Nordic Insulin Foundation, Syskonen Svenssons Foundation, and ?ke Wiberg’s Foundation. Per-Olof Berggren is a recipient of a postdoctoral fellowship from the Swedish Medical Research Council.