Nitric oxide induces apoptosis via hydrogen peroxide, but necrosis via energy and thiol depletion

We investigated the mechanisms by which two nitric oxide (NO) donors, diethylenetriamine/NO adduct (DETA/NO) and S-nitrosoglutathione (GSNO), induced cell death in a J774 macrophage cell line. Both NO donors induced caspase activation within 6 h, but only DETA/NO-induced caspase activation was sensitive to inhibition of p38 and was completely prevented by antioxidants catalase, ascorbate, dehydroascorbate, or N-acetylcysteine, suggesting that DETA/NO-induced apoptosis may be mediated by H(2)O(2). Consistent with this, DETA/NO acutely stimulated reactive oxygen species (ROS) production by mitochondria and cells, and inhibited catalase-mediated H(2)O(2) breakdown in cells. After prolonged, 24 h exposure of cells to DETA/NO, inactivation of caspases occurred, which was accompanied by an increase in necrosis. DETA/NO-induced necrosis was insensitive to caspase inhibitors, but was partially prevented by catalase or N-acetylcysteine, and was preceded by inhibition of glyceraldehyde-3-phosphate dehydrogenase and a decrease in cellular adenosine triphosphate (ATP). GSNO was even more potent in inhibiting glycolysis and switching apoptosis to necrosis. In cells depleted of glutathione, GSNO and DETA/NO induced rapid necrosis, which resulted from rapid depletion of ATP due to inhibition of glycolysis. Glycolytic intermediate 3-phosphoglycerate decreased DETA/NO-induced necrosis and increased apoptosis. We conclude that: (i). NO-induced apoptosis is mediated by H(2)O(2); (ii). NO-induced necrosis is mediated by energy failure speeded by thiol depletion.     

The giant protein AHNAK is a specific target for the calcium- and zinc-binding S100B protein: potential implications for Ca2+ homeostasis regulation by S100B

Transformation of rat embryo fibroblast clone 6 cells by ras and temperature-sensitive p53val(135) is reverted by ectopic expression of the calcium- and zinc-binding protein S100B. In an attempt to define the molecular basis of the S100B action, we have identified the giant phosphoprotein AHNAK as the major and most specific Ca(2+)-dependent S100B target protein in rat embryo fibroblast cells. We next characterized AHNAK as a major Ca(2+)-dependent S100B target protein in the rat glial C6 and human U-87MG astrocytoma cell lines. AHNAK binds to S100B-Sepharose beads and is also recovered in anti-S100B immunoprecipitates in a strict Ca(2+)- and Zn(2+)-dependent manner. Using truncated AHNAK fragments, we demonstrated that the domains of AHNAK responsible for interaction with S100B correspond to repeated motifs that characterize the AHNAK molecule. These motifs show no binding to calmodulin or to S100A6 and S100A11. We also provide evidence that the binding of 2 Zn(2+) equivalents/mol S100B enhances Ca(2+)-dependent S100B-AHNAK interaction and that the effect of Zn(2+) relies on Zn(2+)-dependent regulation of S100B affinity for Ca(2+). Taking into consideration that AHNAK is a protein implicated in calcium flux regulation, we propose that the S100B-AHNAK interaction may participate in the S100B-mediated regulation of cellular Ca(2+) homeostasis.     

Intracellular zinc fluxes associated with apoptosis in growth plate chondrocytes

Matrix vesicles released by epiphyseal growth plate chondrocytes are known to contain a significant quantity of labile Zn(2+). Zonal analysis of chicken metatarsal bones showed that the resting/proliferative region of the growth plate contained high levels of Zn(2+) with significantly lower levels in the hypertrophic cartilage suggesting a loss of cellular Zn(2+) as the chondrocytes mature. Intracellular labile Zn(2+) was measured in primary cultures of growth plate chondrocytes by assay with the fluorescent Zn-chelator toluenesulfonamidoquinoline (TSQ) and imaged by multi-photon laser scanning microscopy (MPLSM) with the TSQ derivative zinquin. Short-term exposure to Zn(2+), both in the presence and absence of pyrithione resulted in significant increases in cytosolic Zn(2+). Treatment with the membrane-permeant Zn(2+) chelator TPEN rapidly reduced the levels of labile Zn(2+) and triggered apoptosis. Cytosolic Zn(2+) levels were significantly reduced following 24-h incubations with known inducers of chondrocyte apoptosis. The loss of intracellular Zn(2+) was accompanied by a significant reduction in the cytosolic metal-binding protein metallothionein. Examination of Zn(2+)-treated cells with MPLSM showed uniformly higher zinquin fluorescence. Treatment of Zn(2+)-loaded cells with TPEN quenched zinquin fluorescence confirming that the observed fluorescence in chondrocytes is due to the presence of intracellular Zn(2+). A dose-dependent increase in zinquin fluorescence was observed in cells treated with a range of Zn(2+) concentrations. Short-term treatment of cultured chondrocytes with apoptosis-inducing chemicals resulted in transient increases in intracellular labile Zn(2+). These results indicate that Zn(2+) is mobilized from intracellular binding sites in the early stages of chondrocyte apoptosis and is subsequently lost from the cells. The early mobilization of Zn(2+) provides a mechanism for its movement to matrix vesicles and the extracellular matrix.     

Zinc sensing receptor signaling, mediated by GPR39, reduces butyrate-induced cell death in HT29 colonocytes via upregulation of clusterin

Zinc enhances epithelial proliferation, protects the digestive epithelial layer and has profound antiulcerative and antidiarrheal roles in the colon. Despite the clinical significance of this ion, the mechanisms linking zinc to these cellular processes are poorly understood. We have previously identified an extracellular Zn(2+) sensing G-protein coupled receptor (ZnR) that activates Ca(2+) signaling in colonocytes, but its molecular identity as well as its effects on colonocytes' survival remained elusive. Here, we show that Zn(2+), by activation of the ZnR, protects HT29 colonocytes from butyrate induced cell death. Silencing of the G-protein coupled receptor GPR39 expression abolished ZnR-dependent Ca(2+) release and Zn(2+)-dependent survival of butyrate-treated colonocytes. Importantly, GPR39 also mediated ZnR-dependent upregulation of Na(+)/H(+) exchange activity as this activity was found in native colon tissue but not in tissue obtained from GPR39 knock-out mice. Although ZnR-dependent upregulation of Na(+)/H(+) exchange reduced the cellular acid load induced by butyrate, it did not rescue HT29 cells from butyrate induced cell death. ZnR/GPR39 activation however, increased the expression of the anti-apoptotic protein clusterin in butyrate-treated cells. Furthermore, silencing of clusterin abolished the Zn(2+)-dependent survival of HT29 cells. Altogether, our results demonstrate that extracellular Zn(2+), acting through ZnR, regulates intracellular pH and clusterin expression thereby enhancing survival of HT29 colonocytes. Moreover, we identify GPR39 as the molecular moiety of ZnR in HT29 and native colonocytes.     

Zn2+-dependent redox switch in the intracellular T1-T1 interface of a Kv channel

The thiol-based redox regulation of proteins plays a central role in cellular signaling. Here, we investigated the redox regulation at the Zn(2+) binding site (HX(5)CX(20)CC) in the intracellular T1-T1 inter-subunit interface of a Kv4 channel. This site undergoes conformational changes coupled to voltage-dependent gating, which may be sensitive to oxidative stress. The main results show that internally applied nitric oxide (NO) inhibits channel activity profoundly. This inhibition is reversed by reduced glutathione and suppressed by intracellular Zn(2+), and at least two Zn(2+) site cysteines are required to observe the NO-induced inhibition (Cys-110 from one subunit and Cys-132 from the neighboring subunit). Biochemical evidence suggests strongly that NO induces a disulfide bridge between Cys-110 and Cys-132 in intact cells. Finally, further mutational studies suggest that intra-subunit Zn(2+) coordination involving His-104, Cys-131, and Cys-132 protects against the formation of the inhibitory disulfide bond. We propose that the interfacial T1 Zn(2+) site of Kv4 channels acts as a Zn(2+)-dependent redox switch that may regulate the activity of neuronal and cardiac A-type K(+) currents under physiological and pathological conditions.     

Complementary distribution of NADPH-diaphorase and l-arginine in the snail nervous system

Since the interneuronal messenger nitric oxide (NO) can not be stored in neurones, the regulation of the NO-producing enzyme nitric oxide synthase (NOS) is crucial. Neuronal NOS metabolises L-arginine to nitric oxide (NO) and L-citrulline in a Ca(2+)-dependent manner. Thus, availability of L-arginine to NOS may modulate NO production. In this study, we examined the cellular distribution of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, L-arginine and L-citrulline. Using NADPH-diaphorase histochemistry to visualise putative NO-producing cells and immunocytochemistry to localise L-arginine, we showed that the distribution of L-arginine-immunoreactive neurones correlates well with those of NADPH-diaphorase-positive neurones in cerebral ganglia of the pulmonate Helix pomatia. However, substrate and enzyme were visualised in separate but adjacent neurones. We further examined whether NADPH-diaphorase-labelled cells contain the L-citrulline. Following elevation of intracellular Ca(2+) by the Ca(2+) ionophore, ionomycin, or by a high-K(+) solution, the number of L-citrulline-immunoreactive neurones in mesocerebrum and pedal lobe increased up to tenfold. Preincubation of ganglia with the NOS inhibitor N(G)-nitro-L-arginine prevented ionomycin or high-K(+) solution-induced L-citrulline synthesis. Most L-citrulline-immunoreactive neurones contain NADPH-diaphorase activity. In conclusion, these experiments indicate a complementary distribution of NOS and L-arginine and suggest an unknown signalling pathway between neurones to maintain L-arginine and NO homeostasis.     

Nitric oxide donors protect murine myocardium against infarction via modulation of mitochondrial permeability transition

Mitochondrial permeability transition (MPT) pores have recently been implicated as a potential mediator of myocardial ischemic injury. Nitric oxide (NO) donors induce a powerful late phase of cardioprotection against ischemia-reperfusion injury; however, the cellular mechanisms involved are poorly understood. The role of MPT pores as a target of cardioprotective signaling pathways activated by NO has never been explored in detail. Thus mice were administered the NO donor diethylenetriamine (DETA)/NO (4 doses of 0.1 mg/kg i.v. each) 24 h before 30 min of coronary artery occlusion followed by 24 h of reperfusion. Infarct size was significantly reduced in DETA/NO-treated mice (30 +/- 2% of risk region in treated mice vs. 50 +/- 2% in control mice; P < 0.05), which demonstrates powerful cardioprotection. To examine the role of MPT pores, mice were administered atractyloside (Atr; 25 mg/kg i.v.), which induces adenine nucleotide translocase-dependent MPT, 20 min before ischemia. Atr blocked the infarct-sparing effects of DETA/NO (infarct size, 58 +/- 1 vs. 30 +/- 2% of risk region in DETA/NO; P < 0.05), whereas Atr alone had no effect. Mitochondria isolated from DETA/NO-treated mice exhibited increased resistance to Ca(2+)-induced swelling by 20 micromol/l CaCl(2) or by the higher concentration of 200 micromol/l, which suggests that cardioprotection involves decreased propensity for MPT. Preincubation of mitochondria from control hearts with 30 nmol/l of the pore inhibitor cyclosporin A prevented swelling by 200 micromol/l CaCl(2), thereby confirming that Ca(2+) induces mitochondrial swelling via MPT. In accordance with the effects on infarct size, administration of Atr to the mice significantly abrogated DETA/NO-induced protection against Ca(2+)-induced mitochondrial swelling. These phenotypic alterations were associated with an increase in the antiapoptotic protein Bcl-2, which suggests that the underlying mechanisms may involve inhibition of cell death by Bcl-2. These data suggest that a critical process during NO donor-induced cardioprotection is to prevent MPT pore opening potentially via targeting of the adenine nucleotide translocator.     

Inhibitory mechanism of store-operated Ca2+ channels by zinc

Capacitative calcium influx plays an important role in shaping the Ca(2+) response of various tissues and cell types. Inhibition by heavy metals is a hallmark of store-operated calcium channel (SOCC) activity. Paradoxically, although zinc is the only potentially physiological relevant ion, it is the least investigated in terms of inhibitory mechanism. In the present study, we characterize the inhibitory mechanism of the SOCC by Zn(2+) in the human salivary cell line, HSY, and rat salivary submandibular ducts and acini by monitoring SOCC activity using fluorescence imaging. Analysis of Zn(2+) inhibition indicated that Zn(2+) acts as a competitive inhibitor of Ca(2+) influx but does not permeate through the SOCC, suggesting that Zn(2+) interacts with an extracellular site of SOCC. Application of the reducing agents, dithiothreitol (DTT) and beta-mercaptoethanol, totally eliminated Zn(2+) and Cd(2+) inhibition of SOCC, suggesting that cysteines are part of the Zn(2+) and Cd(2+) binding site. Interestingly, reducing conditions failed to eliminate the inhibition of SOCC by La(3+) and Gd(3+), indicating that the Zn(2+) and lanthanides binding sites are distinct. Finally, we show that changes in redox potential and Zn(2+) are regulating, via SOCC activity, the agonist-induced Ca(2+) response in salivary ducts. The presence of a specific Zn(2+) site, responsive to physiological Zn(2+) and redox potential, may not only be instrumental for future structural studies of various SOCC candidates but may also reveal novel physiological aspects of the interaction between zinc, redox potential, and cellular Ca(2+) homeostasis.     

Nitric oxide mediates the survival action of IGF-1 and insulin in pancreatic beta cells

Generation of low levels of nitric oxide (NO) contributes to beta cell survival in vitro. The purpose of this study was to explore the link between NO and the survival pathway triggered by insulin-like growth factor-1 (IGF-1) and insulin in insulin producing RINm5F cells and in pancreatic islets. Results show that exposure of cells to IGF-1/insulin protects against serum deprivation-induced apoptosis. This action is prevented with inhibitors of NO generation, PI3K and Akt. Moreover, transfection with the negative dominant form of the tyrosine kinase c-Src abrogates the effect of IGF-1 and insulin on DNA fragmentation. An increase in the expression level of NOS3 protein and in the enzyme activity is observed following exposure of serum-deprived RINm5F cells to IGF-1 and insulin. Phosphorylation of IRS-1, IRS-2 and to less extent IRS-3 takes place when serum-deprived RINm5F cells and rat pancreatic islets are exposed to either IGF-1, insulin, or diethylenetriamine nitric oxide adduct (DETA/NO). In human islets, IRS-1 and IRS-2 proteins are present and tyrosine phosphorylated upon exposure to IGF-1, insulin and DETA/NO. Both rat and human pancreatic islets undergo DNA fragmentation when cultured in serum-free medium and IGF-1, insulin and DETA/NO protect efficiently from this damage. We then conclude that generation of NO participates in the activation of survival pathways by IGF-1 and insulin in beta cells.     

S-Nitroso compounds interfere with zinc probing by Zinquin

The intracellular homeostasis of zinc is postulated to be controlled by signaling through nitric oxide (NO). Administration of the NO donor S-nitrosocysteine (SNOC) caused a rapid drop in the fluorescence of the zinc-specific fluorescence of the zinc probe zinquin in C6 glioma cells. Tentatively, a strong effect of NO on the level of mobile intracellular zinc ions was concluded. However, zinc analysis with atomic absorption spectrometry demonstrated that the total cellular zinc level was not changed under these conditions. Sodium nitrite or an NO donor devoid of sulfhydryl groups (diethylamine NONOate) exerted no degrading effect on the Zn/zinquin fluorescence, but cysteine alone evoked a similar decline as SNOC. Hence, the sulfhydryl groups of cysteine seem to compete for zinc from the Zn/zinquin complex. Analysis of the reaction products by mass spectrometry demonstrated that cysteine caused a depletion of zinc from the Zn/zinquin complex, whereas an NO donor without sulfhydryl groups (diethylamine NONOate) did not. It is concluded that great caution should be employed when using S-nitroso compounds together with zinquin in investigations of intracellular zinc homeostasis.     

Modulation of PGE(2) and TNFalpha by nitric oxide and LPS-activated RAW 264.7 cells

Prostaglandins (PGs), the arachidonic acid (AA) metabolites of the cyclooxygenase (COX) pathway, and the cytokine TNFalpha play major roles in inflammation and they are synthesised mainly by macrophages. Their syntheses have been shown to be regulated by several factors, including nitric oxide, a further important macrophage product. Since both positive and negative regulations of PGs and TNFalpha synthesis by NO have been reported, we sought to understand the mechanisms underlying these opposite NO effects by using a recent class of NO releasing compounds, the NONOates, which have been shown to release NO in a controlled fashion. To this aim, we analysed the effect of NO released from PAPA/NO (t1/2 15 min) and DETA/NO (t1/2 20 h) in RAW 264.7 cells. Both NONOates were used at the same concentrations allowing the cell cultures to be exposed either at high levels of NO for brief time (PAPA/NO) or at low levels of NO for long time (DETA/NO). We found that the two NONOates had opposite effect on basal TNFalpha release, being increased by PAPA/NO and decreased by DETA/NO, while they did not affect the release stimulated by LPS. At variance, both NONOates increased the basal PGE(2) production, while the LPS-stimulated production was slightly increased only by PAPA/NO. The modulation of PGE(2) synthesis was the result of the distinct effects of the two NO-donors on either arachidonic acid (AA) release or cyclooxygense-2 (COX-2) expression, the precursor and synthetic enzyme of PGs, respectively. Indeed, in resting cultures AA release was enhanced only by PAPA/NO whereas COX-2 expression was moderately upregulated by both donors. In LPS activated cells, both NONOates induced AA release, although with different kinetics and potencies, but only DETA/NO significantly increased COX-2 expression. In conclusion, by comparing the activities of these two NONOates, our observations indicate that level and time of exposure to NO are both crucial in determining the molecular target and the final result of the interactions between NO and inflammatory molecules.     

The zinc- and calcium-binding S100B interacts and co-localizes with IQGAP1 during dynamic rearrangement of cell membranes

The Zn(2+)- and Ca(2+)-binding S100B protein is implicated in multiple intracellular and extracellular regulatory events. In glial cells, a relationship exists between cytoplasmic S100B accumulation and cell morphological changes. We have identified the IQGAP1 protein as the major cytoplasmic S100B target protein in different rat and human glial cell lines in the presence of Zn(2+) and Ca(2+). Zn(2+) binding to S100B is sufficient to promote interaction with IQGAP1. IQ motifs on IQGAP1 represent the minimal interaction sites for S100B. We also provide evidence that, in human astrocytoma cell lines, S100B co-localizes with IQGAP1 at the polarized leading edge and areas of membrane ruffling and that both proteins relocate in a Ca(2+)-dependent manner within newly formed vesicle-like structures. Our data identify IQGAP1 as a potential target protein of S100B during processes of dynamic rearrangement of cell membrane morphology. They also reveal an additional cellular function for IQGAP1 associated with Zn(2+)/Ca(2+)-dependent relocation of S100B.     

Nitric oxide protects anterior pituitary cells from cadmium-induced apoptosis

Cadmium (Cd2+) is a potent toxic metal for both plants and animals. Chronic exposure to low doses of Cd2+ results in damage to several organs. We have previously reported that Cd2+ induces apoptosis in anterior pituitary cells by a caspase- and oxidative stress-dependent mechanism. Nitric oxide (NO) synthesis is affected by Cd2+ in several systems. NO has been shown to be either cytoprotective or cytotoxic in many systems. The aim of this study was to evaluate the possible participation of NO in the cytotoxic effect of Cd2+ on rat anterior pituitary cells. Cell viability was evaluated by mitochondrial dehydrogenase activity assay and confirmed by microscopy, studying nuclear morphology. Here we show that DETA NONOate ((Z)-1-[2 (2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), a long-term NO donor, at concentrations below 0.5 mM, reduces nuclear condensation and fragmentation and reverses the decrease in cellular activity induced by Cd2+. Cd2+, by itself, induced NO synthesis, and inhibition of this synthesis enhanced Cd2+ cytotoxicity. NO also prevented caspase-3 activation and lipidic peroxidation induced by Cd2+. The NO/cGMP pathway does not seem to be involved in the cytoprotective effect of NO. These results indicate that NO has a cytoprotective role in Cd2+ -induced apoptosis, suggesting that endogenous NO could have a physiological role in protecting anterior pituitary cells.     

Calcium-dependent release of NO from intracellular S-nitrosothiols

The paper describes a novel cellular mechanism for rapid calcium-dependent nitric oxide (NO) release. This release occurs due to NO liberation from S-nitrosothiols. We have analysed the changes of NO concentration in acutely isolated pancreatic acinar cells. Supramaximal acetylcholine (ACh) stimulation induced a Ca(2+)-dependent increase in the fluorescence in the majority of cells loaded with the NO probe DAF-FM via a patch pipette. The ACh-induced NO signals were insensitive to inhibitors of calmodulin and protein kinase C but were inhibited by calpain antagonists. The initial part of the NO signals induced by 10 muM ACh showed little sensitivity to inhibition of NO synthase (NOS); however, cell pretreatment with NO donors (increasing cellular S-nitrosothiol contents) substantially enhanced the initial component of NO responses. Pancreatic acinar cells were able to generate fast calcium-dependent NO responses when stimulated with physiological or supramaximal doses of secretagogues. Importantly, the source of this NO is the already available S-nitrosothiol store rather than de novo synthesis by NOS. A similar mechanism of NO release was found in dorsal root ganglia neurons.     

Sensor specific imaging of proteomic Zn2+ with zinquin and TSQ after cellular exposure to N-ethylmaleimide

The impact of the thiol binding reagent N-ethylmaleimide (NEM) on proteomic Zn(2+) availability was investigated in rat glioma cells. Zinquin (ZQ) or TSQ, two related fluorescent sensors, were used to observe reactive Zn(2+). Control cells contained proteomic Zn(2+) but no detectable low molecular weight (LMW) Zn(2+). With either sensor, basal cellular fluorescence emission centered near 470 nm, indicative of sensor-Zn-proteins. ZQ sequestered 13% of proteomic Zn(2+) as Zn(ZQ)(2); TSQ reacted only with the Zn-proteome. NEM (100 μM) abolished LMW thiols, including glutathione (GSH) and lowered proteomic sulfhydryl content about 30%. In ZQ-treated cells, NEM exposure enhanced fluorescent intensity and the formation of Zn(ZQ)(2) (λ(MAX), 492 nm). Cells incubated with TSQ and NEM also displayed increased fluorescence without a spectral shift in wavelength maximum, consistent with increased formation of TSQ-Zn-protein adducts but not Zn(TSQ)(2). In neither experiment was Zn(2+) lost from cells. NEM altered Zn(2+) accessibility to sensors in membrane-nuclear and cytosolic fractions, but Zn(ZQ)(2) was only generated in the cytosol. Similar results were obtained when cell supernatant replaced cells. In contrast, when isolated proteome was reacted with ZQ and 100 μM NEM in the absence of GSH, 70% of the proteomic thiols underwent reaction. As a consequence, most of the ZQ-Zn-protein adducts were converted to Zn(ZQ)(2). Substituting TSQ for ZQ, only increased TSQ-Zn-proteins were observed. Evidently, the results of imaging cells with Zn(2+) sensors are dependent upon the specific chemical properties of the sensors and can only be understood after detailed chemical analysis.     

Nitric oxide-induced carbonylation of Bcl-2, GAPDH and ANT precedes apoptotic events in insulin-secreting RINm5F cells

Generation of high levels of nitric oxide (NO) following induction of NOS2 by interleukin-1 beta (IL-1beta) triggers beta cell apoptosis in insulin-secreting RINm5F cells. Mitochondrial and nuclear events such as downregulation of the antiapoptotic protein Bcl-2, activation of the pore responsible for the permeability transition (PT) and DNA fragmentation are involved in the process. We report in the present paper that exposure of insulin-producing RINm5F cells to NO donors and to IL-1beta leads to oxidative carbonylation of both Bcl-2 and the adenine nucleotide translocator (ANT) component of the mitochondrial PT pore. When the effect of endogenous generation of high concentrations of NO following exposure of cells to IL-1beta was studied, carbonylation of Bcl-2 preceded downregulation of the protein. Overexpression of Mn-SOD decreases substantially the extent of Bcl-2 carbonylation in SIN-1-exposed cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibition, carbonylation and translocation from cytoplasm to nucleus and DNA fragmentation were also induced by DETA/NO exposure. DETA/NO-induced carbonylation of Bcl-2 and ANT proteins takes place 6 h before apoptotic release of histone-associated DNA to cytoplasm. Time course studies also reveal a close parallel between GAPDH translocation to nucleus and carbonylation. Inhibitors of lipooxidation end products formation such as piridoxamine (PM) and aminoguanidine (AG) block NO-triggered carbonylation of Bcl-2, ANT and GAPDH, prevent NO-induced GAPDH enzyme inhibition and nuclear translocation and DNA fragmentation. Our results support the notion that the oxidative carbonylation of proteins plays a role in the control of NO-induced apoptosis.     

Uvomorulin mediates calcium-dependent aggregation of islet cells, whereas calcium-independent cell adhesion molecules distinguish between islet cell types

Rat islets of Langerhans are organized as a core of B-cells surrounded by non-B-cells. It is believed that cell type segregation during histogenesis is the result of the differential expression of cell adhesion molecules (CAMs). Since we have previously shown that in contrast to non-B-cells, homotypic adhesion of pancreatic B-cells is dependent on the presence of Ca2+, the possibility exists that Ca(2+)-dependent CAMs (cadherins) might be in part responsible for islet topography. We now demonstrate that after selective removal of Ca(2+)-independent CAMs from the surface of islet cells by mild trypsin/Ca2+ digestion (TC-treatment), there is no significant difference in homotypic adhesion between sorted B- and non-B-cells in the presence of calcium, suggesting an identical deployment of cadherins. Flow cytometric analysis reveals high levels of uvomorulin on both B- and non-B-cells, without any difference between the two populations. On a "1 to 100" scale, B-cell aggregation in the presence of Ca2+ was decreased by anti-uvomorulin Fab fragments from 67 +/- 4 to 25 +/- 3 (mean +/- SEM, n = 4, P less than 0.01). This level is not different from the degree of B-cell aggregation seen in the presence of 0.5 mM EDTA (22 +/- 2). Aggregation of non-B-cells was only slightly decreased by anti-uvomorulin Fab fragments (from 69 +/- 3 to 52 +/- 4). However, after TC-treatment, homotypic cell aggregation of both B- and non-B-cells was completely inhibited by anti-uvomorulin Fab fragments. Thus, uvomorulin appears to be the only functional cadherin on islet cells, and cell type aggregation properties diverge only by virtue of higher levels of Ca(2+)-independent CAMs on non-B-cells. Fab fragments with the property of perturbing islet cell aggregation in the absence but not in the presence of calcium also prevented pseudoislet organization in vitro, suggesting that Ca(2+)-independent CAMs play the major role in islet cell type segregation. In conclusion, the results show that uvomorulin is responsible for the Ca(2+)-dependent aggregation of islet cells and suggest that the cellular organization within islets or pseudoislets results from the different level of Ca(2+)-independent CAMs on islet cell types.