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Nitric oxide involvement in pancreatic β cell apoptosis by glibenclamide
Nitric Oxide 14 (2006) 39–44 www.elsevier.com/locate/yniox
Nitric oxide involvement in pancreatic cell apoptosis by glibenclamide Malek Moien Ansar a,b,¤, Mohammad Ansari a a
b
Department of Clinical Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran Department of Biochemistry and Biophysics, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran Received 9 May 2005; revised 4 September 2005 Available online 26 Octoebr 2005
Abstract Glibenclamide as a second-generation compound of sulfonylurea has widely been used in the treatment of type 2 diabetes patients. It has been shown that it induces apoptosis in cells, which is partially mediated by Ca2+ inXux. Here, we investigated the role of nitric oxide (NO) and nitric oxide synthase (NOS) isoforms on glibenclamide-induced apoptosis in rat insulinoma cells. Our results showed that glibenclamide induces NO generation (measured as nitrite) that is accompanied with decrease of cell viability in a deWned concentration of glibenclamide. The eVects of glibenclamide on cell viability were partially inhibited after treatment with NG-nitro-L-arginine methyl ester (L-NAME), inhibitor more selective for constitutive nitric oxide synthase, and in the presence of D600— a blocker of voltage-gated L-type Ca2+ channels inhibited Ca2+ inXux into cells, whereas aminoguanidine (AG), a preferential inhibitor of inducible NOS, was signiWcantly less eVective. Analysis of DNA fragmentation by electrophoresis and staining with Hoechest 33342 and propidium iodide showed that L-NAME, but not AG, prevented DNA fragmentation and decreased the number of cells with condensed and fragmented nuclei. It revealed that the eVects of glibenclamide on apoptosis were partially inhibited by treatment with L-NAME. In conclusion, we have shown that NO production in glibenclamide treated cells may be involved in the induction of apoptotic cell death in pure cell line and it may be due to Ca2+ dependent activation of constitutive NOS isoforms. 2005 Elsevier Inc. All rights reserved. Keywords: RIN-5F; Glibenclamide; Nitric oxide; Nitric oxide synthase; Apoptosis; L-NAME; Aminoguanidine
Recently, there have been many reports about the deterioration of cell functions in type 2 diabetes [1,2]. After 1986, glibenclamide as a second-generation compound of sulfonylurea has entered into the market and up to now has widely been used in the treatment of type 2 diabetes patients [3]. These drugs close the ATP sensitive potassium channels (KATP channels), producing membrane depolarization and activating the Ca2+ inXux, which triggers insulin secretion [4]. Ca2+ accumulation can trigger apoptotic cell death [5,6], and Iwakura et al. [7] showed that glibenclamide induces apoptosis in RINm5F cells through sustained enhancement of Ca2+ inXux. On the other side, there is some evidence to support the role of nitric oxide (NO) in diabetes development [8]. Over*
Corresponding author. Fax: +98 21 8953004. E-mail address: [email protected] (M.M. Ansar).
1089-8603/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2005.09.002
production of NO can be due to the expression of inducible nitric oxide synthase (iNOS)1 gene or exposure of constitutive nitric oxide synthase (cNOS) to high concentration of calcium [9]. Since some cytotoxic eVects were derived from dispersion in dispersed islet cells itself [10] and the eVects of cells other than cells, we have used rat insulinoma cell line (RIN-5F). Also, it has been shown that RIN-5F cells have KATP channels and sulfonylurea receptor, as do primary cells [7].
1 Abbreviations used: AG, aminoguanidine; cNOS, constitutive nitric oxide synthase; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; iNOS, inducible nitric oxide synthase; KRBB, Krebs Ringer bicarbonate buVer; G L-NAME, N -nitro-L-arginine methyl ester; MTT, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium; NO, nitric oxide; PBS, phosphate buVered saline; PI, propidium iodide.
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M.M. Ansar, M. Ansari / Nitric Oxide 14 (2006) 39–44
In this study, we have investigated the role of NO production and NOS isoforms in glibenclamide-induced apoptosis in cells. Experimental procedures Materials RIN-5F was obtained from The European Collection of Cell Cultures (ECACC), glibenclamide, D600, MTT, Propidium iodide (PI) and Hoechest 33342 from Sigma Chemical (St Louis, MO), and NG-nitro-L-arginine methyl ester (LNAME) and aminoguanidine (AG) from Alexis Co.
described [7,12]. After removing the supernatant, the cells were incubated in RPMI for 16 h. Ten microlitres of phosphate-buVered saline (PBS) containing 0.5% (w/v) MTT was added to 100 L of fresh medium in each well, and plate was wrapped in aluminum foil. After 4–8 h incubation at 37 °C, addition of 100 L DMSO dissolved the remaining MTT-formazone crystals. At last, we added Sorensen’s buVer solution (10 L) to all wells containing DMSO and read the absorbance at 570–620 nm by a microplate reader (Anthous 2020) immediately. All experiments were performed in triplicate and repeated at least twice. Morphological study of apoptosis
Preparations of the cells RIN-5F cells were maintained in RPM1 1640 medium (Gibco BRL, Grand Island, NY) containing 11.1 mM glucose supplemented with 10% fetal bovine serum (FBS), gentamycine (5 mg/dL), Hepes (10 mM), and humidiWed in 5% CO2, at 37 °C. The cells were seeded at a density of 4 £ 105/ml in RPMI. After being incubated overnight, they were used for our experiments.
Cells were Wxed with 2% formaldehyde for 10 min. After washing with PBS, staining was done with PBS + 0.1% of Triton X-100 + 20 g/ml Hoechst 33342 + 10 g/ml PI for 15 min. After washing the cells with PBS, they were mounted (glycerin 9 vol. + PBS 1 vol.) and visualized using a Xuorescence microscope with UV excitation at 340–380 (Olympus, Tokyo, Japan). Cells with condensed chromatin or fragmented nuclei were considered to be apoptotic.
Treatment by reagents DNA fragmentation assay After RIN-5F cells were incubated in Krebs Ringer bicarbonate buVer (KRBB; 130 mM NaCl, 5.2 mM KCl, 2.8 mM CaCl2, 1.3 mM KH2PO4, 1.3 mM MgSO4, 25 mM NaHCO3, and 11.1 mM glucose) for 30 min, they were incubated in KRBB either without or with the indicated concentrations of glibenclamide and inhibitors for 24 h. All incubations were performed at 37 °C in humidiWed air containing 5% CO2. Glibenclamide was Wrst prepared in dimethylsulfoxide (DMSO), and the Wnal concentration of DMSO did not exceed 0.1% [7]. Nitric oxide assay The NO production was indirectly determined by Griess reaction for simultaneous evaluation of nitrate and nitrite concentration as previously described [11]. BrieXy, equal volumes of sulfanilamide (2%) in 5% HCl and Naphthylethylenediamine (0.1%) in H2O were mixed immediately prior to measurement, and 50 L of resulting solution was mixed with 50 L of medium plate and 50 L VCl3. After 30 min incubation at 37 °C, absorbance was determined at 540 nm. The approximate simultaneous concentration of nitrate and nitrite in samples was determined from a standard curve that was generated by using known concentrations of sodium nitrate.
For detection of DNA fragmentation, genomic DNA was extracted by the described method [13]. In short, cells were washed with PBS and resuspended in lysis buVer (0.5% SDS, 10 mM EDTA, and 10 mM Tris–HCl, pH 8.0) and treated with proteinase K (0.5 mg/ml) at 56 °C for 2 h and then with 0.25 mg/ml RNase A at 37 °C for 1 h. Afterwards, by the method of Duke and Sellins [14] the supernatant containing fragmented (soluble) DNA was transferred to another tube after centrifugation for 15 min at 4 °C (13,000g). 0.1 ml ice-cold 5 M NaCl and 0.7 ml ice-cold isopropanol was added and the samples were incubated overnight at ¡20 °C. After centrifugation and rinsing by 70% ethanol, they were centrifuged again and dried. At last, the pellets were dissolved in TE (1 mM EDTA, 10 mM Tris– HCl, pH 8.0) and analyzed by electrophoresis on 1% agarose gel. Statistical analysis Statistically signiWcant diVerences were measured by one-way analyses of variance (ANOVA) followed by Turkey’s HSD post hoc test and Student’s unpaired t test. P < 0.05 was considered signiWcant. Results
Measurement of cell viability Nitric oxide production by glibenclamide-exposed cells Cell respiration, an indicator of cell viability, was assessed by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium (MTT) to formazan as previously
Glibenclamide increased NO production in rat insulinoma cell line dose-dependently. The amount of nitrite
M.M. Ansar, M. Ansari / Nitric Oxide 14 (2006) 39–44
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EVect of NOS inhibitors on glibenclamide-induced nitrite formation and cell viability For evaluation of the eVects of diVerent kinds of NOS inhibitors with diVerent selectivity to NOS isoforms—on nitrite production and cell viability related to glibenclamide—two inhibitors: aminoguanidine (AG) and NG-nitroL-arginine methyl ester (L-NAME) were studied. L-NAME (0.5 and 1 mM) decreased NO production to (4.49 § 0.5 M) and (4.2 § 0.54 M), respectively, and increased cell viability to 92 § 3.91% and 93 § 3.59%, respectively, while decrease in NO production and increase in cell viability by AG was not signiWcant (Fig. 2). EVect of methoxyverapamil (D600) on nitrite formation and cell viability by glibenclamide Fig. 1. The eVect of 24 h treatment with increasing dose of glibenclamide on nitrite production and cell viability in RIN-5F cells. Values are the means § SE of triplicate experiments. *P < 0.05, **P < 0.01 vs. 0 M glibenclamide.
(4.7 § 0.34 M) increased signiWcantly in comparison with control (1.63 § 0.32 M) after 24 h incubation with 100 M of glibenclamide (Fig. 1).
To study whether the increase in [Ca2+]i induced by glibenclamide was responsible for increasing of NO production and decreasing of cell viability, we used methoxyverapamil (D600) a blocker of voltage-gated L-type Ca2+ channels. NO production due to glibenclamide was signiWcantly reduced by D600 (50 M) to 4.7 § 0.58 M. Also, cell viability increased to 94.1 § 3% of control (Fig. 2).
Viability of glibenclamide-exposed cells The concentration from 1 to 100 M of glibenclamide decreased cell viability in a dose-dependent manner (Fig. 1). Incubation with 100 M of glibenclamide for 24 h has caused a signiWcant decrease in percentage of cell viability up to 74.5 § 2.2% of control cells.
EVect of NOS inhibitors on glibenclamide-induced apoptosis by Xuorescence microscopy Few numbers of apoptotic cells, condensed or fragmented chromatin stained with Hoechst 33342 and PI, were observed in control cells (Fig. 3A). In contrast, a large num-
Fig. 2. EVect of NOS inhibitors, L-NAME and AG, with diVerent concentration and D600 a blocker of voltage-gated L-type Ca2+ channels on glibenclamide-induced nitrite production and cell viability by RIN-5F cells for 24 h, (A) nitrite production (B) cell viability. Values are the means § SE of triplicate experiments. *P < 0.05 vs. to 100 M glibenclamide (GB).
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M.M. Ansar, M. Ansari / Nitric Oxide 14 (2006) 39–44
Fig. 4. Percentage of apoptotic cells detected by Xuorescent microscopy (each condition, 250 cells from 3 diVerent experiments were assessed). Cells were incubated for 24 h of glibenclamide (100 M) (GB), glibenclamide (100 M) with 1 mM L-NAME (GB + L-NAME) or glibenclamide (100 M) with 1 mM aminoguanidine (GB + AG). Values are the means § SE of triplicate experiments. *P < 0.05, **P < 0.001 vs. control.
Fig. 3. Detection of apoptosis in RIN-5F cells by Xuorescent microscopy. Cells incubated for 24 h in the absence (A) or in the presence of glibenclamide (100 M) (B). Few apoptotic nuclei (condensed or fragmented) stained in control cells (A) whereas treatment with glibenclamide increased apoptotic cells (B). Arrows indicate apoptotic nuclei.
ber of cells showed chromatin condensation and fragmentation after exposure to 100 M glibenclamide (Fig. 3B). The percentage of apoptotic cells was signiWcantly increased in cells exposed to glibenclamide (15.7 § 0.32%) compared with control (4.07 § 0.13%). Treatment with L-NAME (1 mM) signiWcantly decreased the percentage of apoptotic cells compared with cells induced by glibenclamide alone. Despite some decrease in the number of apoptotic cells (9.9 § 0.95%), percentage of apoptotic cells has increased compared with control in glibenclamide-exposed cells treated by AG (Fig. 4).
Fig. 5. The eVect of the inhibitors of NOS on DNA fragmentation in RIN5F cells. Lane 1, control; lane 2, glibenclamide (100 M); lane 3, glibenclamide (100 M) + L-NAME (1 mM); lane 4, glibenclamide (100 M) + AG (1 mM); lane M is 100 bp ladder marker.
EVect of NOS inhibitors on glibenclamide-induced DNA fragmentation
exposure could not change DNA fragmentation pattern (Fig. 5).
For determining whether the inhibitors of NOS can change glibenclamide-induced DNA fragmentation, we measured the eVect of L-NAME and AG on DNA laddering in RIN-5F cultured cells. The fragmented DNA in 100 M glibenclamide was decreased by treatment with L-NAME, whereas addition of AG during glibenclamide
Discussion There are many reports indicating that cell mass is decreased in type 2 diabetes and the increase of cell apoptosis is thought to be the main mechanism involved [28,29]. It follows that some studies have demonstrated that sulfo-
M.M. Ansar, M. Ansari / Nitric Oxide 14 (2006) 39–44
nylureas such as tolbutamide and glibenclamide induce apoptosis in cell line, rodent islets, and human islets [7,23,30,31]. Butler et al. [28] showed that patients treated with glibenclamide had an increase in rate of cell apoptosis compared with those treated with diet alone. Also, there is an evidence that deterioration of insulin secretion is seen in patients treated with sulfonylurea in the UK Prospective Diabetes Study Group (UKPDS) [32]. This decrease in cell mass and function raises a concern regarding the consumption of sulfonylureas. Our foregoing results indicated that the concentration from 1 to 100 M glibenclamide caused a decrease in cell viability that was comparable to increase of NO production in a deWned concentration of glibenclamide (Fig. 1). Iwakura et al. [7] showed that Ca2+ inXux by glibenclamide induces cell death, and our results showed that NO production should be involved in this manner as well. The eVective doses and plasma levels of the second-generation sulfonylureas are in the range of 1–10 mg and 50– 100 nmol/L, respectively [33]. Although it seems that the submicromolar therapeutical concentrations of glibenclamide maximally achieved in the post-absorptive state, but there are large interindividual variations in the pharmacodynamics and pharmacokinetics of glibenclamide, which are greatly inXuenced by genetic polymorphisms in cytochrome P-450. There are also several drug interactions that may over increase glibenclamide serum levels, especially in elderly diabetic patients who are often on multiple medications and have impaired drug metabolism [34,35]. Also, interindividual diVerences in pharmacokinetics may be attributable to factors such as body weight, plasma protein binding, sex, age, liver function [35] and long elimination half-life of glibenclamide [36]. Therefore, it seems that our Wnding may have potential clinical importance in the treatment of diabetes type 2 patients. Because previous study [7] used 100 M glibenclamide for the contribution of Ca2+ in cell apoptosis, this concentration was chosen to evaluate the role of NO and NOS isoforms in this study, so that direct comparisons could be made most easily by using the same drug concentration. We know that all three NOS isoforms are present in pancreatic cells [15]. Expression of the iNOS enzyme is in response to inXammatory stimuli [16] and the presence of cNOS enzyme in cells [16–18] is substantiated. For demonstration of the fact that the observed NO formation was due to the activity of iNOS or cNOS, the eVects of two NOS inhibitors, one with known selectivity for iNOS and the other with a lesser degree of selectivity and a preference for constitutive isoform of NOS, were studied. L-NAME as an inhibitor more selective for constitutive NOS isoform [19] can reduce NO production signiWcantly, which is accompanied by an increase in cell viability. This increase in cell viability was due to decrease in apoptotic cell death, suggested by staining with Hoechst 33342 and PI. It showed a decrease in the number of cells with chromatin fragmentation and condensation in glibenclamide treated cells by LNAME (Fig. 4).
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Moreover, DNA laddering is a hallmark of apoptosis. Apoptotic cleavage of genomic DNA can be identiWed as DNA fragments or smear on agarose gels [20]. The patterns observed in this study occurred in the treated cell lanes towards the smaller size marker fragments, as opposed to the control cell lane. A decrease in DNA fragmentation in glibenclamide-exposed cells in the presence of L-NAME was observed (Fig. 5). AG as a selective iNOS inhibitor [21] cannot increase cell viability signiWcantly in spite of some decrease in NO production (Fig. 2). In addition, DNA fragmentation assay and morphological study conWrmed no signiWcant decrease in apoptotic cell death in glibenclamide-exposed cells by AG (Figs. 4 and 5). This slight decrease in NO production without any signiWcant diVerence in cell viability showed that to increase cell viability, NO production should be decreased suYciently. Our study shows that activation of the constitutive NOS isoforms that is Ca2+ dependent has more important role than inducible NOS on NO production in cells by glibenclamide. This Wnding consistent with previous study showed that ATP-sensitive K+ channel blocker tolbutamide, which elevates intracellular Ca2+ concentrations, increased DAF-2 Xuorescence through activation of constitutive NOS isoforms in most cells of the isolated islets [22]. On the other side, it has been shown that sustained enhancement of Ca2+ inXux plays a role in the induction of apoptotic cell death in pancreatic cells by glibenclamide [7]. Also, it has been shown that tolbutamide induced cell destruction by apoptosis in a Ca2+ dependent manner [23]. Here, we also showed that glibenclamide-induced NO production and cell death was dependent on [Ca2+]i because inhibition of Ca2+ inXux by D600 abolished NO production and suppressed cell death. Ca2+ as a regulator of the apoptotic process [24,25] can induce apoptosis by several systems [24–26]. Although it has been shown that Ca2+ elevation can induce apoptosis through NF-B activation [25,26] and consequently iNOS expression [27], our results show that Ca2+ enhancement may increase NO production by constitutive NOS activation as well, and this may be involved in cell apoptosis. Also, there was a DNA smear pattern extending to the smaller ladder fragments in glibenclamide-exposed cells in the presence of L-NAME (Fig. 5), it was possibly due to the eVect of glibenclamide on cell apoptosis and this showed that the inhibitor could not prevent cell apoptosis completely. This suggests that some other factors should be involved in cell apoptosis as well. In conclusion, it seems that enhancement of NO production, at least in part, may be involved in the induction of apoptotic cell death in pancreatic cells. Also, NO production was possibly related to activation of constitutive NOS isoforms through Ca2+ elevation in glibenclamide treated cells. Clearly, the present in vitro study needs in vitro study of human islets and especially in vivo conWrmation, because some other factors are involved in glibenclamide-induced apoptosis such as cell proliferation and regeneration. However, if we are able to translate this Wnding into patients with type 2 diabetes, it may be considered as a rea-
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son for the secondary failure which occurred in patients who initially respond to sulfonylureas. These patients may be more exposed to NO production and proapoptotic stimuli during protracted treatment with glibenclamide. Acknowledgments The authors are grateful for the assistance of all biochemistry department staV of Tehran Medical Faculty, especially Dr. Shariftabrizi and Mr. Pazhang for their help. References [1] J. Janson, R.H. Ashley, D. Harrison, S. McIntyre, P.C. Butler, The mechanism of islet amyloid polypeptide toxicity is membrane disruption by intermediate—sized toxic amyloid particles, Diabetes 48 (3) (1999) 491–498. [2] H. Bar-On, R. Ben-Sasson, E. Ziv, N. Arar, E. Shafrir, Irreversibility of nutritionally induced NIDDM in Psammomy obesus is related to beta-cell apoptosis, Pancreas 18 (1999) 259–265. [3] UK Prospective Diabetes Study (UKPDS) Group, Intensive bloodglucose control with sulfonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33), Lancet 352 (1998) 837–853. [4] J.E. Gerich, Oral hypoglycemic agents, N. Engl. J. Med. 321 (1989) 1231–1245. [5] M.J. Berridge, M.D. Bootman, P. Lipp, Calcium: a life and death signal, Nature 395 (1998) 645–648. [6] G.E. Kass, S. Orrenius, Calcium signaling and cytotoxicity, Environ. Health Perspect. 107 (Suppl. 1) (1999) 25–35. [7] O. Iwakura, S. Fujimoto, S. Kagimoto, A. Inada, A. Kubota, Y. Someya, Y. Ihara, Y. Yamada, Y. Seino, Sustained enhancement of Ca2+ inXux by glibenclamide induces apoptosis in RINm5F cells, Biochem. Biophys. Res. Commun. 271 (2000) 422–428. [8] T. Mandrup-Poulsen, -cell apoptosis: stimuli and signaling, Diabetes 50 (Suppl. 1) (2001) S58–S63. [9] H.T. Chung, H.O. Pae, B.M. Choi, T.R. Billiar, Y.M. Kim, Nitric oxide as a bioregulator of apoptosis, Biochem. Biophys. Res. Commun. 282 (5) (2001) 1059–1075. [10] B.D. Bennett, T.L. Jetton, G. Ying, M.A. Magnuson, D.W. Piston, Quantitative subcellular imaging of glucose metabolism within intact pancreatic islets, J. Biol. Chem. 271 (1996) 3647–3651. [11] K.M. Miranda, M.G. Espey, D.A. Wink, A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite, Nitric Oxide 5 (1) (2001) 62–71. [12] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63. [13] P. Jung-Hyun, N. Shin-Young, L. Yun-Jung, C. Eun-Wee, K. Kil Lyong, Downregulation of MHC class II expression by oxidantinduced apoptosis in EBV-transformed B-cells, Mol. and cells 10 (6) (2000) 654–661. [14] R.C. Duke, C.B. Sellins, in: J.G. Kaplan (Ed.), Cellular basis of immune modulation, Liss, New York, 1989, pp. 311–314. [15] S.R. Smukler, L. Tang, M.B. Wheeler, M.F. Salapatek, Exogenous nitric oxide and endogenous glucose-stimulated cell nitric oxide augment insulin release, Diabetes 51 (2002) 3450–3460. [16] D.L. Eizirik, E. Cagliero, A. Bjorklund, N. Welsh, Interleukin-1 beta induces the expression of an isoform of nitric oxide synthase in insulin-producing cells, which is similar to that observed in activated macrophages, FEBS Lett. 308 (1992) 249–252.
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Nitric oxide involvement in pancreatic cell apoptosis by glibenclamide Malek Moien Ansar a,b,¤, Mohammad Ansari a a
b
Department of Clinical Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran Department of Biochemistry and Biophysics, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran Received 9 May 2005; revised 4 September 2005 Available online 26 Octoebr 2005
Abstract Glibenclamide as a second-generation compound of sulfonylurea has widely been used in the treatment of type 2 diabetes patients. It has been shown that it induces apoptosis in cells, which is partially mediated by Ca2+ inXux. Here, we investigated the role of nitric oxide (NO) and nitric oxide synthase (NOS) isoforms on glibenclamide-induced apoptosis in rat insulinoma cells. Our results showed that glibenclamide induces NO generation (measured as nitrite) that is accompanied with decrease of cell viability in a deWned concentration of glibenclamide. The eVects of glibenclamide on cell viability were partially inhibited after treatment with NG-nitro-L-arginine methyl ester (L-NAME), inhibitor more selective for constitutive nitric oxide synthase, and in the presence of D600— a blocker of voltage-gated L-type Ca2+ channels inhibited Ca2+ inXux into cells, whereas aminoguanidine (AG), a preferential inhibitor of inducible NOS, was signiWcantly less eVective. Analysis of DNA fragmentation by electrophoresis and staining with Hoechest 33342 and propidium iodide showed that L-NAME, but not AG, prevented DNA fragmentation and decreased the number of cells with condensed and fragmented nuclei. It revealed that the eVects of glibenclamide on apoptosis were partially inhibited by treatment with L-NAME. In conclusion, we have shown that NO production in glibenclamide treated cells may be involved in the induction of apoptotic cell death in pure cell line and it may be due to Ca2+ dependent activation of constitutive NOS isoforms. 2005 Elsevier Inc. All rights reserved. Keywords: RIN-5F; Glibenclamide; Nitric oxide; Nitric oxide synthase; Apoptosis; L-NAME; Aminoguanidine
Recently, there have been many reports about the deterioration of cell functions in type 2 diabetes [1,2]. After 1986, glibenclamide as a second-generation compound of sulfonylurea has entered into the market and up to now has widely been used in the treatment of type 2 diabetes patients [3]. These drugs close the ATP sensitive potassium channels (KATP channels), producing membrane depolarization and activating the Ca2+ inXux, which triggers insulin secretion [4]. Ca2+ accumulation can trigger apoptotic cell death [5,6], and Iwakura et al. [7] showed that glibenclamide induces apoptosis in RINm5F cells through sustained enhancement of Ca2+ inXux. On the other side, there is some evidence to support the role of nitric oxide (NO) in diabetes development [8]. Over*
Corresponding author. Fax: +98 21 8953004. E-mail address: [email protected] (M.M. Ansar).
1089-8603/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2005.09.002
production of NO can be due to the expression of inducible nitric oxide synthase (iNOS)1 gene or exposure of constitutive nitric oxide synthase (cNOS) to high concentration of calcium [9]. Since some cytotoxic eVects were derived from dispersion in dispersed islet cells itself [10] and the eVects of cells other than cells, we have used rat insulinoma cell line (RIN-5F). Also, it has been shown that RIN-5F cells have KATP channels and sulfonylurea receptor, as do primary cells [7].
1 Abbreviations used: AG, aminoguanidine; cNOS, constitutive nitric oxide synthase; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; iNOS, inducible nitric oxide synthase; KRBB, Krebs Ringer bicarbonate buVer; G L-NAME, N -nitro-L-arginine methyl ester; MTT, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium; NO, nitric oxide; PBS, phosphate buVered saline; PI, propidium iodide.
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In this study, we have investigated the role of NO production and NOS isoforms in glibenclamide-induced apoptosis in cells. Experimental procedures Materials RIN-5F was obtained from The European Collection of Cell Cultures (ECACC), glibenclamide, D600, MTT, Propidium iodide (PI) and Hoechest 33342 from Sigma Chemical (St Louis, MO), and NG-nitro-L-arginine methyl ester (LNAME) and aminoguanidine (AG) from Alexis Co.
described [7,12]. After removing the supernatant, the cells were incubated in RPMI for 16 h. Ten microlitres of phosphate-buVered saline (PBS) containing 0.5% (w/v) MTT was added to 100 L of fresh medium in each well, and plate was wrapped in aluminum foil. After 4–8 h incubation at 37 °C, addition of 100 L DMSO dissolved the remaining MTT-formazone crystals. At last, we added Sorensen’s buVer solution (10 L) to all wells containing DMSO and read the absorbance at 570–620 nm by a microplate reader (Anthous 2020) immediately. All experiments were performed in triplicate and repeated at least twice. Morphological study of apoptosis
Preparations of the cells RIN-5F cells were maintained in RPM1 1640 medium (Gibco BRL, Grand Island, NY) containing 11.1 mM glucose supplemented with 10% fetal bovine serum (FBS), gentamycine (5 mg/dL), Hepes (10 mM), and humidiWed in 5% CO2, at 37 °C. The cells were seeded at a density of 4 £ 105/ml in RPMI. After being incubated overnight, they were used for our experiments.
Cells were Wxed with 2% formaldehyde for 10 min. After washing with PBS, staining was done with PBS + 0.1% of Triton X-100 + 20 g/ml Hoechst 33342 + 10 g/ml PI for 15 min. After washing the cells with PBS, they were mounted (glycerin 9 vol. + PBS 1 vol.) and visualized using a Xuorescence microscope with UV excitation at 340–380 (Olympus, Tokyo, Japan). Cells with condensed chromatin or fragmented nuclei were considered to be apoptotic.
Treatment by reagents DNA fragmentation assay After RIN-5F cells were incubated in Krebs Ringer bicarbonate buVer (KRBB; 130 mM NaCl, 5.2 mM KCl, 2.8 mM CaCl2, 1.3 mM KH2PO4, 1.3 mM MgSO4, 25 mM NaHCO3, and 11.1 mM glucose) for 30 min, they were incubated in KRBB either without or with the indicated concentrations of glibenclamide and inhibitors for 24 h. All incubations were performed at 37 °C in humidiWed air containing 5% CO2. Glibenclamide was Wrst prepared in dimethylsulfoxide (DMSO), and the Wnal concentration of DMSO did not exceed 0.1% [7]. Nitric oxide assay The NO production was indirectly determined by Griess reaction for simultaneous evaluation of nitrate and nitrite concentration as previously described [11]. BrieXy, equal volumes of sulfanilamide (2%) in 5% HCl and Naphthylethylenediamine (0.1%) in H2O were mixed immediately prior to measurement, and 50 L of resulting solution was mixed with 50 L of medium plate and 50 L VCl3. After 30 min incubation at 37 °C, absorbance was determined at 540 nm. The approximate simultaneous concentration of nitrate and nitrite in samples was determined from a standard curve that was generated by using known concentrations of sodium nitrate.
For detection of DNA fragmentation, genomic DNA was extracted by the described method [13]. In short, cells were washed with PBS and resuspended in lysis buVer (0.5% SDS, 10 mM EDTA, and 10 mM Tris–HCl, pH 8.0) and treated with proteinase K (0.5 mg/ml) at 56 °C for 2 h and then with 0.25 mg/ml RNase A at 37 °C for 1 h. Afterwards, by the method of Duke and Sellins [14] the supernatant containing fragmented (soluble) DNA was transferred to another tube after centrifugation for 15 min at 4 °C (13,000g). 0.1 ml ice-cold 5 M NaCl and 0.7 ml ice-cold isopropanol was added and the samples were incubated overnight at ¡20 °C. After centrifugation and rinsing by 70% ethanol, they were centrifuged again and dried. At last, the pellets were dissolved in TE (1 mM EDTA, 10 mM Tris– HCl, pH 8.0) and analyzed by electrophoresis on 1% agarose gel. Statistical analysis Statistically signiWcant diVerences were measured by one-way analyses of variance (ANOVA) followed by Turkey’s HSD post hoc test and Student’s unpaired t test. P < 0.05 was considered signiWcant. Results
Measurement of cell viability Nitric oxide production by glibenclamide-exposed cells Cell respiration, an indicator of cell viability, was assessed by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium (MTT) to formazan as previously
Glibenclamide increased NO production in rat insulinoma cell line dose-dependently. The amount of nitrite
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EVect of NOS inhibitors on glibenclamide-induced nitrite formation and cell viability For evaluation of the eVects of diVerent kinds of NOS inhibitors with diVerent selectivity to NOS isoforms—on nitrite production and cell viability related to glibenclamide—two inhibitors: aminoguanidine (AG) and NG-nitroL-arginine methyl ester (L-NAME) were studied. L-NAME (0.5 and 1 mM) decreased NO production to (4.49 § 0.5 M) and (4.2 § 0.54 M), respectively, and increased cell viability to 92 § 3.91% and 93 § 3.59%, respectively, while decrease in NO production and increase in cell viability by AG was not signiWcant (Fig. 2). EVect of methoxyverapamil (D600) on nitrite formation and cell viability by glibenclamide Fig. 1. The eVect of 24 h treatment with increasing dose of glibenclamide on nitrite production and cell viability in RIN-5F cells. Values are the means § SE of triplicate experiments. *P < 0.05, **P < 0.01 vs. 0 M glibenclamide.
(4.7 § 0.34 M) increased signiWcantly in comparison with control (1.63 § 0.32 M) after 24 h incubation with 100 M of glibenclamide (Fig. 1).
To study whether the increase in [Ca2+]i induced by glibenclamide was responsible for increasing of NO production and decreasing of cell viability, we used methoxyverapamil (D600) a blocker of voltage-gated L-type Ca2+ channels. NO production due to glibenclamide was signiWcantly reduced by D600 (50 M) to 4.7 § 0.58 M. Also, cell viability increased to 94.1 § 3% of control (Fig. 2).
Viability of glibenclamide-exposed cells The concentration from 1 to 100 M of glibenclamide decreased cell viability in a dose-dependent manner (Fig. 1). Incubation with 100 M of glibenclamide for 24 h has caused a signiWcant decrease in percentage of cell viability up to 74.5 § 2.2% of control cells.
EVect of NOS inhibitors on glibenclamide-induced apoptosis by Xuorescence microscopy Few numbers of apoptotic cells, condensed or fragmented chromatin stained with Hoechst 33342 and PI, were observed in control cells (Fig. 3A). In contrast, a large num-
Fig. 2. EVect of NOS inhibitors, L-NAME and AG, with diVerent concentration and D600 a blocker of voltage-gated L-type Ca2+ channels on glibenclamide-induced nitrite production and cell viability by RIN-5F cells for 24 h, (A) nitrite production (B) cell viability. Values are the means § SE of triplicate experiments. *P < 0.05 vs. to 100 M glibenclamide (GB).
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Fig. 4. Percentage of apoptotic cells detected by Xuorescent microscopy (each condition, 250 cells from 3 diVerent experiments were assessed). Cells were incubated for 24 h of glibenclamide (100 M) (GB), glibenclamide (100 M) with 1 mM L-NAME (GB + L-NAME) or glibenclamide (100 M) with 1 mM aminoguanidine (GB + AG). Values are the means § SE of triplicate experiments. *P < 0.05, **P < 0.001 vs. control.
Fig. 3. Detection of apoptosis in RIN-5F cells by Xuorescent microscopy. Cells incubated for 24 h in the absence (A) or in the presence of glibenclamide (100 M) (B). Few apoptotic nuclei (condensed or fragmented) stained in control cells (A) whereas treatment with glibenclamide increased apoptotic cells (B). Arrows indicate apoptotic nuclei.
ber of cells showed chromatin condensation and fragmentation after exposure to 100 M glibenclamide (Fig. 3B). The percentage of apoptotic cells was signiWcantly increased in cells exposed to glibenclamide (15.7 § 0.32%) compared with control (4.07 § 0.13%). Treatment with L-NAME (1 mM) signiWcantly decreased the percentage of apoptotic cells compared with cells induced by glibenclamide alone. Despite some decrease in the number of apoptotic cells (9.9 § 0.95%), percentage of apoptotic cells has increased compared with control in glibenclamide-exposed cells treated by AG (Fig. 4).
Fig. 5. The eVect of the inhibitors of NOS on DNA fragmentation in RIN5F cells. Lane 1, control; lane 2, glibenclamide (100 M); lane 3, glibenclamide (100 M) + L-NAME (1 mM); lane 4, glibenclamide (100 M) + AG (1 mM); lane M is 100 bp ladder marker.
EVect of NOS inhibitors on glibenclamide-induced DNA fragmentation
exposure could not change DNA fragmentation pattern (Fig. 5).
For determining whether the inhibitors of NOS can change glibenclamide-induced DNA fragmentation, we measured the eVect of L-NAME and AG on DNA laddering in RIN-5F cultured cells. The fragmented DNA in 100 M glibenclamide was decreased by treatment with L-NAME, whereas addition of AG during glibenclamide
Discussion There are many reports indicating that cell mass is decreased in type 2 diabetes and the increase of cell apoptosis is thought to be the main mechanism involved [28,29]. It follows that some studies have demonstrated that sulfo-
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nylureas such as tolbutamide and glibenclamide induce apoptosis in cell line, rodent islets, and human islets [7,23,30,31]. Butler et al. [28] showed that patients treated with glibenclamide had an increase in rate of cell apoptosis compared with those treated with diet alone. Also, there is an evidence that deterioration of insulin secretion is seen in patients treated with sulfonylurea in the UK Prospective Diabetes Study Group (UKPDS) [32]. This decrease in cell mass and function raises a concern regarding the consumption of sulfonylureas. Our foregoing results indicated that the concentration from 1 to 100 M glibenclamide caused a decrease in cell viability that was comparable to increase of NO production in a deWned concentration of glibenclamide (Fig. 1). Iwakura et al. [7] showed that Ca2+ inXux by glibenclamide induces cell death, and our results showed that NO production should be involved in this manner as well. The eVective doses and plasma levels of the second-generation sulfonylureas are in the range of 1–10 mg and 50– 100 nmol/L, respectively [33]. Although it seems that the submicromolar therapeutical concentrations of glibenclamide maximally achieved in the post-absorptive state, but there are large interindividual variations in the pharmacodynamics and pharmacokinetics of glibenclamide, which are greatly inXuenced by genetic polymorphisms in cytochrome P-450. There are also several drug interactions that may over increase glibenclamide serum levels, especially in elderly diabetic patients who are often on multiple medications and have impaired drug metabolism [34,35]. Also, interindividual diVerences in pharmacokinetics may be attributable to factors such as body weight, plasma protein binding, sex, age, liver function [35] and long elimination half-life of glibenclamide [36]. Therefore, it seems that our Wnding may have potential clinical importance in the treatment of diabetes type 2 patients. Because previous study [7] used 100 M glibenclamide for the contribution of Ca2+ in cell apoptosis, this concentration was chosen to evaluate the role of NO and NOS isoforms in this study, so that direct comparisons could be made most easily by using the same drug concentration. We know that all three NOS isoforms are present in pancreatic cells [15]. Expression of the iNOS enzyme is in response to inXammatory stimuli [16] and the presence of cNOS enzyme in cells [16–18] is substantiated. For demonstration of the fact that the observed NO formation was due to the activity of iNOS or cNOS, the eVects of two NOS inhibitors, one with known selectivity for iNOS and the other with a lesser degree of selectivity and a preference for constitutive isoform of NOS, were studied. L-NAME as an inhibitor more selective for constitutive NOS isoform [19] can reduce NO production signiWcantly, which is accompanied by an increase in cell viability. This increase in cell viability was due to decrease in apoptotic cell death, suggested by staining with Hoechst 33342 and PI. It showed a decrease in the number of cells with chromatin fragmentation and condensation in glibenclamide treated cells by LNAME (Fig. 4).
43
Moreover, DNA laddering is a hallmark of apoptosis. Apoptotic cleavage of genomic DNA can be identiWed as DNA fragments or smear on agarose gels [20]. The patterns observed in this study occurred in the treated cell lanes towards the smaller size marker fragments, as opposed to the control cell lane. A decrease in DNA fragmentation in glibenclamide-exposed cells in the presence of L-NAME was observed (Fig. 5). AG as a selective iNOS inhibitor [21] cannot increase cell viability signiWcantly in spite of some decrease in NO production (Fig. 2). In addition, DNA fragmentation assay and morphological study conWrmed no signiWcant decrease in apoptotic cell death in glibenclamide-exposed cells by AG (Figs. 4 and 5). This slight decrease in NO production without any signiWcant diVerence in cell viability showed that to increase cell viability, NO production should be decreased suYciently. Our study shows that activation of the constitutive NOS isoforms that is Ca2+ dependent has more important role than inducible NOS on NO production in cells by glibenclamide. This Wnding consistent with previous study showed that ATP-sensitive K+ channel blocker tolbutamide, which elevates intracellular Ca2+ concentrations, increased DAF-2 Xuorescence through activation of constitutive NOS isoforms in most cells of the isolated islets [22]. On the other side, it has been shown that sustained enhancement of Ca2+ inXux plays a role in the induction of apoptotic cell death in pancreatic cells by glibenclamide [7]. Also, it has been shown that tolbutamide induced cell destruction by apoptosis in a Ca2+ dependent manner [23]. Here, we also showed that glibenclamide-induced NO production and cell death was dependent on [Ca2+]i because inhibition of Ca2+ inXux by D600 abolished NO production and suppressed cell death. Ca2+ as a regulator of the apoptotic process [24,25] can induce apoptosis by several systems [24–26]. Although it has been shown that Ca2+ elevation can induce apoptosis through NF-B activation [25,26] and consequently iNOS expression [27], our results show that Ca2+ enhancement may increase NO production by constitutive NOS activation as well, and this may be involved in cell apoptosis. Also, there was a DNA smear pattern extending to the smaller ladder fragments in glibenclamide-exposed cells in the presence of L-NAME (Fig. 5), it was possibly due to the eVect of glibenclamide on cell apoptosis and this showed that the inhibitor could not prevent cell apoptosis completely. This suggests that some other factors should be involved in cell apoptosis as well. In conclusion, it seems that enhancement of NO production, at least in part, may be involved in the induction of apoptotic cell death in pancreatic cells. Also, NO production was possibly related to activation of constitutive NOS isoforms through Ca2+ elevation in glibenclamide treated cells. Clearly, the present in vitro study needs in vitro study of human islets and especially in vivo conWrmation, because some other factors are involved in glibenclamide-induced apoptosis such as cell proliferation and regeneration. However, if we are able to translate this Wnding into patients with type 2 diabetes, it may be considered as a rea-
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