Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms
1 Departments of Physiology Pharmacology, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E5
2 Department of Biology, Lakehead University, Thunder Bay, Ontario, Canada P7B 5E1
Abstract
H2S is an important gasotransmitter, generated in mammalian cells from L-cysteine metabolism. As it stimulates KATP channels in vascular smooth muscle cells, H2S may also function as an endogenous opener of KATP channels in INS-1E cells, an insulin-secreting cell line. In the present study, KATP channel currents in INS-1E cells were recorded using the whole-cell and single-channel recording configurations of the patch-clamp technique. KATP channels in INS-1E cells have a single-channel conductance of 78 pS. These channels were activated by diazoxide and inhibited by gliclazide. ATP (3 mM) in the pipette solution inhibited KATP channels in INS-1E cells. Significant amount of H2S was produced from INS-1E cells in which the expression of cystathinonie gamma-lyase (CSE) was confirmed. After INS-1E cells were transfected with CSE-targeted short interfering RNA (CSE-siRNA) or treated with DL-propargylglycine (PPG; 1–5 mM) to inhibit CSE, endogenous production of H2S was abolished. Increase in extracellular glucose concentration significantly decreased endogenous production of H2S in INS-1E cells, and increased insulin secretion. After transfection of INS-1E cells with adenovirus containing the CSE gene (Ad-CSE) to overexpress CSE, high glucose-stimulated insulin secretion was virtually abolished. Basal KATP channel currents were significantly reduced after incubating INS-1E cells with a high glucose concentration (16 mM) or lowering endogenous H2S level by CSE-siRNA transfection. Under these conditions, exogenously applied H2S significantly increased whole-cell KATP channel currents at concentrations equal to or lower than 100 μM. H2S (100 μM) markedly increased open probability by more than 2-fold of single KATP channels (inside-out recording) in native INS-1E cells (n= 4, P < 0.05). Single-channel conductance and ATP sensitivity of KATP channels were not changed by H2S. In conclusion, endogenous H2S production from INS-1E cells varies with in vivo conditions, which significantly affects insulin secretion from INS-1E cells. H2S stimulates KATP channels in INS-1E cells, independent of activation of cytosolic second messengers, which may underlie H2S-inhibited insulin secretion from these cells. Interaction among H2S, glucose and the KATP channel may constitute an important and novel mechanism for the fine control of insulin secretion from pancreatic -cells.
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Introduction
Amounting evidence in recent years indicate that H2S is an endogenous molecule of gas with important physiological functions (Wang, 2002). In the cardiovascular system, endogenous production of H2S is mainly catalysed by cystathinonie gamma-lyase (CSE). Being a gasotransmitter, H2S causes vasorelaxation at physiologically relevant concentrations (Zhao et al. 2001) and participates in the modulation of neuronal functions (Abe & Kimura, 1996). One of the mechanisms for cardiovascular actions of H2S is activation of KATP channels. H2S increased whole-cell KATP channel currents in rat aortic vascular smooth muscle cells (SMC) (Zhao et al. 2001). Similarly, in rat mesenteric artery SMCs H2S at physiologically relevant concentrations stimulated KATP channels (Cheng et al. 2004). H2S exerted a negative inotropic effect on heart. This effect was partially blocked by glibenclamide, a classical sulphonylurea KATP channel blocker (Geng et al. 2004). Although no electrophysiological recordings were performed on isolated cardiomyocytes, indication of involvement of KATP channels in cardiac effect of H2S is noted.
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KATP channels are sensitive to changes in intracellular ATP concentrations. Elevation of intracellular ATP level leads to closure of KATP channels in many metabolically active cells. In this way, the KATP channel is a coupling factor to link metabolic activity and membrane excitability. This feature is especially important for pancreatic -cells. When circulating glucose level is elevated, glucose influx into pancreatic -cells increases and so does ATP production. Consequential closure of KATP channels on plasma membrane depolarizes membrane and opens voltage-dependent calcium channels. The final eventuality of this chain reaction is increased insulin release due to increased intracellular free calcium. There is no doubt that KATP channels in -cells are critical in regulation of glucose-induced insulin secretion (Cook et al. 1988; Ashcroft et al. 1989; Ashcroft & Gribble, 1998). However, beyond the regulatory role of glucose, via alteration of intracellular ATP level, on KATP channels, little is known about the existence of other endogenous regulators for KATP channels in pancreatic -cells. By analogy to the stimulatory effect of H2S on KATP channels in vascular SMCs, it is reasonable to believe that H2S may be a novel KATP channel opener in pancreatic -cells. To date, endogenous production of H2S in the pancreas, effect of H2S on insulin secretion, and interaction of H2S with pancreatic KATP channels have not been determined.
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In the present study, effects of H2S on KATP channels in an insulin-secreting insulinoma cell line, INS-1E cells, were examined using the whole-cell and single-channel recording configurations of the patch-clamp technique. Endogenous levels of H2S were either decreased by transfecting INS-1E cells with CSE-siRNA vector or dialysing the cells with a specific inhibitor for H2S-generating enzyme, or increased by overexpressing CSE gene in INS-1E cells. Actual production of endogenous H2S, expression level of H2S-generating enzymes, and insulin secretion in INS-1E cells were determined. Our study characterized KATP channels in INS-1E cells, demonstrated the important regulatory role of H2S on insulin secretion and pancreatic KATP channel activation for the first time, and revealed endogenous enzymatic production and metabolism of H2S in insulin-secreting cells.
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Methods
Cell culture
INS-1E cells derived from a rat insulinoma (kindly provided by Dr C. B. Wollheim, Geneva, Switzerland) were grown in a humidified (5% CO2, 95% O2) atmosphere for up to 2 days in Hepes-buffered RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol (Sigma), 100 units ml–1 penicillin, and 100 μg ml–1 streptomycin (Sigma). For patch-clamp study, cultured INS-1E cells were placed in a Petri dish mounted on the stage of an inverted phase contrast microscope (Olympus IX70). For other biochemical and molecular biology assays, INS-1E cells were harvested and centrifuged at 500 g for 10 min after being rinsed twice with PBS solution.
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Electrophysiological recording and analysis
Both whole-cell and single-channel recordings of KATP channel currents were performed as previously described (Cook & Hales, 1984; Zhao et al. 2001). Patch pipettes were pulled from borosilicate glass capillaries using a Flaming/Brown micropipette puller (Model P-87, Sutter Instruments). Pipette resistance was 8–12 M for single-channel recordings and 1–5 M for whole-cell experiments when filled with electrolyte solution. All electrophysiological recordings were performed at room temperature (20–24°C).
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Whole-cell recordings were carried out with an Axopatch 200A patch clamp amplifier via a Digidata 1200 (Axon Instruments Inc.) interface, and analysed off-line using pCLAMP software (version 6.02; Axon Instruments Inc.). Test pulses of 600 ms were made with 10 mV increments from –150 to +50 mV. The holding potential was set at –20 mV. I–V relationships were constructed using stable current amplitude at the end of 600 ms test pulses. Pipettes were filled with a solution containing (mM): KCl 105, MgCl2 1.0, CaCl2 1.0, EGTA 10, and Hepes 10 (pH adjusted to 7.3 with KOH). Unless otherwise specified, ATP concentration of the pipette solution was 0.3 mM. The bath solution contained (mM): NaCl 102, KCl 40, CaCl2 1.0, MgCl2 1.2, glucose 4.5, and Hepes 10 (pH adjusted to 7.4 with NaOH). When glucose concentration was increased in some experiments, equimolar NaCl was removed to maintain osmolality of the bath solution. No leakage subtraction was performed to the original recordings and all cells with visible changes in leakage currents during the course of the study were excluded from further analysis.
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For single-channel recording, inside-out configuration of the patch-clamp technique (Hamill et al. 1981) was used. Seal resistance was 10 G. Single-channel currents were filtered at 1 kHz (eight-pole Bessel, –3 db) and recorded with 100 μs sampling interval in a gap-free model. Single-channel current records were displayed and analysed using pCLAMP 7.0 software (Axon Instruments Inc.). For each concentration of tested agents, at least 30 s of channel activity was directly recorded on computer hard disk. Open probability (NPo), i.e. the fraction of time when the channels stay open within the total observation period with N representing the number of single channels in one patch (Wang & Wu, 1997) and single-channel conductance were determined from an all-point amplitude histogram using Fetchan and Pstat programs (Axon Instruments Inc.). The pipette solution contained (mM): KCl 140, MgCl2 1.2, EGTA 10 and Hepes 5 (pH adjusted to 7.2 with KOH). Inside-out patches were bathed in a solution containing (mM): KCl 140, MgCl2 0.53, glucose 4.5, ATP 0.3, ADP 0.3 and Hepes 5 (pH adjusted to 7.4 with KOH).
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Measurement of endogenous H2S production
H2S production rate was measured as previously described (Stipanuk & Beck, 1982) with modifications, which has been routinely used in our laboratory (Zhao et al. 2001, 2003; Cheng et al. 2004). Briefly, INS-1E cells cultured for 3–7 days were collected and homogenized in 50 mM ice-cold potassium phosphate buffer pH 6.8. The reaction mixture contained (mM): 100 potassium phosphate buffer pH 7.4, 10 L-cysteine, 2 pyridoxal 5'-phosphate, and 10% (w/v) homogenate. Cryovial test tubes (2 ml) were used as the centre wells, each containing 0.5 ml 1% zinc acetate as trapping solution and a filter paper 2 cm x 2.5 cm to increase air: liquid contacting surface. Reaction was performed in a 25 ml Erlenmeyer flask (Pyrex, USA). The flasks containing the reaction mixture and centre wells were flushed with N2 before being sealed with a double layer of Parafilm. Reaction was initiated by transferring the flasks from ice to a 37°C shaking water bath. After incubating at 37°C for 90 min, 0.5 ml of 50% trichloroacetic acid was added to stop the reaction. The flasks were sealed again and incubated at 37°C for another 60 min to ensure a complete trapping of H2S released from the mixture. Contents of the centre wells were then transferred to test tubes, each containing 3.5 ml of water. Subsequently, 0.5 ml of 20 mMN,N-dimethyl-p-phenylenediamine sulphate in 7.2 M HCl was added immediately followed by addition of 0.5 ml 30 mM FeCl3 in 1.2 M HCl. Absorbance of the resulting solution at 670 nm was measured 20 min later with a spectrophotometer (Siegel, 1965). H2S content was calculated against the calibration curve of standard H2S solutions.
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Measurement of insulin secretion from INS-1E cells
Native or transfected INS-1E cells were plated into 24-well plates at a density of 5 x 104 cells per well and tested 24–48 h later when cells reached about 80% confluence. Cells were maintained at 37°C for 2 h in glucose-free RPMI 1640 medium, washed and pre-incubated in glucose-free (0 mM) Krebs-Ringer-bicarbonate medium (pH 7.4) containing (mM): 135 NaCl, 3.6 KCl, 5 NaHCO3, 0.5 NaH2PO4, 0.5 MgCl2, 1.5 CaCl2, 10 Hepes and 0.1% BSA. After 30 min pre-incubation, cells were incubated for 30 min at 37°C in the presence of different glucose concentrations. At the end of each incubation period, the medium was collected and centrifuged for 10 min at 2500 g to remove cell debris. The supernatant was immediately stored at –20°C until insulin determination using the rat insulin ELISA kit (Mercodia AB, Sylveniusgatan, Uppsala, Sweden).
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Western immunoblotting
Cultured cells were harvested and lysed in a lysis buffer (EDTA 0.5 M; Tris-Cl 1 M, pH 7.4; sucrose 0.3 M; antipain hydrochloride 1 μg ml–1; benzamidine hydrochloride hydrate 1 M; leupeptin hemisulphate 1 μg ml–1; 1,10-phenanthroline monohydrate 1 M; pepstatin A 1 μM; plenylmethylsulphonyl fluoride 0.1 mM, and iodoacetamide 1 mM). Extracts were separated by centrifugation at 14 000 g for 15 min at 4°C. SDS-PAGE and Western blot analysis were performed as previously described (Yang et al. 2004a). Briefly, equal amount of proteins were boiled in 1 x SDS sample buffer (62.5 mM Tris-Cl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, and 0.01% bromophenol blue) and resolved on a 10% SDS-PAGE gel, and transferred onto polyvinylidene chloride (PVDC) nitrocellulose membranes. Dilutions for the primary antibodies were 1: 1000 for CSE, and 1: 5000 for -actin. HRP-conjugated secondary antibody was used at 1: 5000. Immunoreactions were visualized by enhanced chemiluminescence (ECL) and exposed to X-ray film (Kodak Scientific Imaging film). Membranes were stripped by incubating in a buffer containing 100 mM-mercaptoethanol, 2% SDS and 62.5 mM Tris-HCl (pH 6.8).
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CSE-siRNA transfection of INS-1E cells
CSE-targeted 21 nucleotide siRNA was designed using a web based siRNA design program (http://www.ambion.com/techlib/misc/siRNA_finder_html) according to the AA-N19 rule (Brummelkamp et al. 2002; Lake & Castellot, 2003). The targeted sequence was localized at a position 192 bases downstream of the start codon of CSE (GenBank Accession No. NM001902). Forward (ggu uau uua ucc ugg gcu g dtdt) and reverse (cag ccc agg cua aau aac c dtdt) RNA strands with two 5' deoxy-thymidine overhangs were commercially synthesized by Ambion (Austin, TX, USA). GADPH-targeted siRNA was also produced for optimizing transfection conditions. Negative control siRNA, a 21 nucleotide RNA duplex with no sequence homology with all known genes, was also purchased from Ambion. Transfection of siRNA into INS-1E cells was achieved using the siPORT lipid transfection agent from Ambion. Briefly, cells were plated overnight to form 60–70% confluent monolayers. CSE siRNA at 30 nM and the transfection reagent complex were added to cells in serum-free medium for 4 h. Fresh normal growth medium was then added and cells were incubated for another 20 h. As a control, negative siRNA was used to transfect INS-1E cells.
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Construction of recombinant CSE adenovirus vector and infection of INS-1E cells
A PCR was performed to amplify opening read frame (ORF) of CSE (GenBank accession number AB052882) from reverse-transcribed rat vascular tissue using a set of primers: 5'-CGTCCCAGCATGCAGAAGAA-3' and 5'-CAGTTATTCAGAAGGTCTGGCCC-3'. The amplified ORF of CSE was ligated into PCR2.1 vector (Invitrogen), and the KpnI–XhoI restriction fragment of CSE was subcloned into the KpnI–XhoI sites of the shuttle vector pAdShuttle-CMV (Qbiogene, Inc.), which contains cytomegalovirus promoter/enhancer element and simian virus 40 polyadenylation signals. Positive clone containing CSE ORF insert was sequenced to confirm the accuracy of the inserted CSE sequence. The resultant plasmid was linearized with PmeI and cotransformed with the adenovirus backbone vector pAdeasy-1 into E. coli BJ5183 cells by electroporation. Homologous recombinants containing CSE cDNA were detected by restriction endonuclease digestion and agarose gel electrophoresis. Recombinant CSE adenovirus vector (Ad-CSE) was then transformed into E. coli DH5 cells for large-scale amplification. The PacI-digested E1-deleted replication-deficient Ad-CSE vector was then transfected into mammalian HEK-293 cells using calcium phosphate–DNA precipitates. The recombinant Ad-CSE was expanded, purified and titrated (He et al. 1998). The recombinant adenovirus encoding bacterial -galactosidase (Ad-lacZ) derived from the same vector was used as a control. For adenoviral infection, subconfluent INS-1E cells were incubated with Ad-CSE or Ad-lacZ in serum-free media. After 4 h of incubation, media was removed, and cells were incubated in appropriate media for 48 h. The transfection efficiency of adenoviral vector in INS-1E cells was first determined by infecting cells with Ad-lacZ at various multiplicities of infection (MOI). The cells infected with Ad-lacZ were assayed for -galactosidase expression by the in situ X-gal staining method (Hirooka & Sakai, 2004). At MOI 50, > 90% of cells showed nuclear staining for -galactosidase. Subsequent experiments were performed at MOI of 50.
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Real-time RT-PCR determination of the transcriptional level of CSE
Native untransfected cells, negative siRNA- or CSE siRNA-transfected cells were harvested from 100 mm culture dishes 24 h after transfection. Monolayers were rinsed twice with PBS, and total RNA was collected using Tri Reagent (Molecular Research Center, Cincinnati, OH, USA). Contaminating DNA was avoided using the DNA-free kit (Ambion), and total RNA (2 μg) was reverse-transcribed into cDNA with AMV reverse transcriptase using random hexamer primers according to the manufacturer's protocol (Roche Applied Science, IN, USA). Controls containing no reverse transcriptase were used to safeguard for genomic DNA contamination in each sample.
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Real-time PCR was performed in an iCycler iQ apparatus (Bio-Rad, Harcules, CA, USA) associated with the iCycler optical system software (version 3.1) using the SYBR Green PCR Master Mix. All PCRs were performed in a 20 μl volume using 96-well optical grade PCR plates and optical sealing tape. Negative controls for this experiment were samples without a template. Cycling conditions were 95°C for 90 s followed by 38 cycles of 95°C for 10 s and 60°C for 20 s. For quantification, the target gene was normalized to the internal standard gene -actin. A standard curve was constructed with a series of dilutions of total RNA (Ambion) transcribed to cDNA using the protocol outlined above to confirm the same amplifying efficiency in PCR. A standard melting curve analysis was performed using a thermal cycling profile that began at 95°C for 1 min, decreased to 55°C for 1 min, and then ramped to 95°C in 1°C increments to confirm the absence of primer dimers. Product size was determined by running PCR products on a 1.8% agarose gel. Relative mRNA quantification was calculated by using the arithmatic formula ‘2–CT’, where CT is the difference between the threshold cycle of a given target cDNA and an endogenous reference cDNA (Yang et al. 2004a). Thus, this value yields the amount of the target normalized to an endogenous reference.
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Chemicals and statistical analysis
H2S-saturated solution (0.09 M) was freshly made by bubbling pure H2S gas (Praxair; Mississauga, Canada) into Krebs' solution at 30°C for 40 min as previously described (Zhao et al. 2001, 2003; Cheng et al. 2004). At 37°C and pH 7.4, the concentration of H2S in solution was relatively stable (Zhao et al. 2003). Data are expressed as mean ±S.E.M. Multiple comparisons were made with one-way ANOVA followed by a post hoc analysis (Tukey test). Statistical significance was set at P < 0.05.
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Results
Endogenous H2S production and insulin secretion from INS-1E cells
To investigate whether cultured INS-1E cells produced H2S under different in vivo conditions, INS-1E cells were incubated with either 5 or 20 mM glucose for 24 h. Increase in extracellular glucose concentration from 5 to 20 mM significantly decreased endogenous production of H2S in INS-1E cells by about 46% (Fig. 1A). Similar inhibition of H2S production in INS-1E cells was also observed with 16 mM glucose (not shown). To further examine whether this glucose-mediated endogenous H2S production was regulated by specific enzymatic process, DL-propargylglycine (PPG), a selective inhibitor of CSE, was used. Lysed INS-1E cells were mixed with PPG to facilitate interaction of this inhibitor with cytosolically located CSE, and then H2S production was assayed. It was found that PPG significantly inhibited H2S production in INS-1E cells (Fig. 1A).
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A, glucose-mediated H2S production in INS-1E cells. Endogenous production of H2S was significantly decreased by high glucose concentration in the incubation medium. PPG (5 mM) treatment of INS-1E cells significantly reduced endogenous H2S production. *P < 0.05 versus other groups. n= 4 for each group. B, glucose-stimulated insulin secretion from INS-1E cells with Ad-CSE transfection to increase endogenous H2S level. *P < 0.05 versus native or Ad-LacZ-transfected INS-1E cells in the presence of 16 mM glucose. n= 6–8 for each group. C, production of H2S in Ad-CSE-transfected INS-1E cells. Cells were cultured with 16 mM glucose in the medium. *P < 0.05 versus other groups. n= 4–6 for each group.
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High glucose affected insulin secretion from INS-1E cells. With an increased glucose concentration from 5 to 16 mM, insulin secretion from INS-1E cells became 3-fold greater (Fig. 1B). After transfection of INS-1E cells with Ad-CSE to overexpress CSE, insulin secretion at basal level (5 mM glucose) was not altered. However, high glucose-stimulated insulin secretion was virtually abolished (Fig. 1B). Significantly increased production of endogenous H2S from Ad-CSE-transfected INS-1E cells is shown in Fig. 1C. Native INS-1E cells were also transfected with Ad-LacZ as a negative control. Neither basal nor high glucose-stimulated insulin secretion from INS-1E cells were altered by Ad-LacZ transfection (Fig. 1B).
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Characterization of KATP channel in INS-1E cells
Gliclazide is a specific blocker of KATP channels in pancreatic -cells (Trube et al. 1986; Ashcroft, 2000). Gliclazide (1 μM) decreased significantly whole-cell KATP currents in INS-1E cells from –1049.9 ± 115 to 522.8 ± 88 pA at –100 mV (n= 5, P < 0.05). Representative results of the effect of gliclazide on whole-cell KATP channels are shown in Fig. 2A. Diazoxide is a potent opener of KATP channels in pancreatic -cells (Sturgess et al. 1988; D'hahan et al. 1999). Whole-cell KATP channels in INS-1E cells were significantly stimulated by diazoxide (Fig. 2B). Whole-cell KATP channels in INS-1E cells were also characterized by their sensitivity to intracellular ATP. KATP channel currents were 82.9 ± 8.6 pA pF–1 (–120 mV) with 0.3 mM ATP in the pipette solution (n= 6). When the ATP concentration was increased to 3 mM, KATP channel currents were reduced to 29.7 ± 5.6 pA pF–1 (–120 mV) (n= 5, P < 0.05 versus 0.3 mM ATP in the pipette solution).
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Holding potential, –20 mV. A, inhibition of the whole-cell KATP channels by gliclazide in INS-1E cells. Representative KATP channel currents (top panel) and the mean I–V relationship of whole-cell KATP channels (bottom panel, n= 5) before and after the application of gliclazide at 1 μM. B, stimulation of whole-cell KATP channels by diazoxide in INS-1E cells. Representative KATP channel currents (top panel) and the mean I–V relationships of KATP channels (bottom panel, n= 4) before and after application of diazoxide at 0.3 mM.
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With symmetrical K+ (140 mM) in the pipette and bath solutions, KATP channels in INS-1E cells had a single-channel conductance of 78 ± 2.3 pS (n= 5) (Fig. 3A). These single-channel KATP currents appeared in rapid open–close transitions and in brief bursts. Open probability of single KATP channels at –60 mV was 0.08 ± 0.01 (n= 6). When gliclazide (1 μM) was added to the bath, activity of the single channel was greatly decreased with open probability reduced to 0.004 ± 0.001 (n= 5, P < 0.05) (Fig. 3B). When diazoxide (100 μM) was applied to inside-out patches, single-channel activity was greatly increased with open probability of single KATP channels changing from 0.09 ± 0.01 to 0.28 ± 0.03 (n= 5, P < 0.05). However, there was no significant change in amplitude of unitary inward current after application of diazoxide. Unitary current amplitudes of single-channel KATP currents were 4.68 ± 0.09 and 4.70 ± 0.06 pA (–60 mV) before and after application of diazoxide, respectively (n= 5, P > 0.05).
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A, representative single-channel KATP channel currents in one inside-out membrane patch (left panel) and the single-channel conductance of KATP channels (right panel, n= 5). B, open probabilities of single KATP channels in the absence (upper panel) and then presence (lower panel) of gliclazide (1 μM). Membrane potential, –60 mV.
Effects of H2S on KATP channels in INS-1E cells
KATP channels in INS-1E cells were also sensitive to glucose stimulation. After glucose concentration of the bath solution was changed from 5 mM to 16 mM, whole-cell KATP currents were significantly reduced (Fig. 4A). With 5 mM glucose in the bath solution, H2S at 100 μM had no effect on whole-cell KATP currents in INS-1E cells (n= 5, P > 0.05). In the presence of 16 mM glucose, application of H2S (100 μM) to INS-1E cells significantly increased KATP currents (Fig. 4B). Application of DL-dithothreitol (DTT) (3 mM) to INS-1E cells for 5 min did not significantly change KATP channel currents in INS-1E cells (79 ± 2.69 versus 86 ± 2.68 pA pF–1 at –100 mV, n= 5, P > 0.05). A lack of effect of DTT on KATP channels has also been reported previously in pancreatic -cells (Islam et al. 1993; Krippeit-Drews et al. 1994). Since DTT is a reducing reagent, our result suggests that the stimulatory effect of H2S on KATP channels in INS-1E cells is unlikely to be mediated by a general reducing effect.
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Holding potential, –20 mV. A, inhibitory effect of glucose on the whole-cell KATP channels in INS-1E cells. Representative KATP channel current traces (top panel) and the mean I–V relationships (bottom panel) of KATP channels with different glucose concentrations in the bath solutions. n= 5 for each group. *P < 0.05. B, interaction of H2S with whole-cell KATP channels in the presence of high glucose (16 mM) in the bath solution. Representative KATP channel current traces were shown in the top panel and the mean I–V relationships in the bottom panel of KATP channels in INS-1E cells before and after H2S application. n= 5 for each group.
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To examine whether KATP channels in INS-1E cells were desensitized at resting conditions by high endogenous H2S levels, we tried to lower endogenous H2S level by pre-treating these cells with PPG. In the presence of PPG (5–10 mM) in the bath solution containing 5 mM glucose, H2S at 100 μM did not alter the whole-cell KATP currents in INS-1E cells (812 ± 63 versus 796 ± 81 pA at –120 mV, n= 5, P > 0.05). In the next group of experiments, PPG (1 mM) was included in the pipette solution to dialyse cells. This manoeuver alone significantly reduced KATP channel currents by 40 ± 9.6% (n= 5, P < 0.05). More interestingly, including PPG in the pipette solution unmasked a dose-dependent stimulatory effect of exogenously applied H2S on KATP channels (Fig. 5A). Even at a concentration of 10 μM, H2S increased KATP channel currents by 69.4 ± 5.7% after PPG treatment (n= 4, P < 0.05, –120 mV) (Fig. 5C). Effects of H2S on KATP channels were not different with either 1 or 5 mM PPG in the pipette solution (Fig. 5A and B), indicating that even at 1 mM PPG might already significantly inhibit CSE. CSE protein expression was confirmed in INS-1E cells with Western blot analysis (Fig. 6A). To further demonstrate that the preconditioning effect of intracellular PPG was related to reduced endogenous CSE activity, cultured INS-1E cells were transfected with CSE-siRNA to knock down endogenous expression of CSE gene. While negative-siRNA transfection did not alter translational or transcriptional expression levels of CSE, CSE-siRNA transfection reduced expression of the CSE gene by about 80% (Fig. 6A and B). In line with suppressed CSE gene expression, endogenous production of H2S from CSE-siRNA-transfected INS-1E cells was significantly reduced in comparison with that of native INS-1E cells (Fig. 6C).
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A, bath application of H2S at 100 μM increased inward KATP channel currents in INS-1E cells, which were dialysed with 5 mM PPG. n= 4. B, bath application of H2S at 100 μM increased inward KATP channel currents in INS-1E cells, which were dialysed with 1 mM PPG. n= 5. C, the concentration-dependent stimulatory effects of H2S on KATP channels with 1 mM PPG in the pipette solution. n= 5–6 for each group. *P < 0.05 versus the recordings in the absence of H2S.
A, Western blot detection of CSE protein expression in INS-1E cells. B, real-time RT-PCR comparison of CSE expression levels in INS-1E cells with and without siRNA transfection. n= 4. *P < 0.01. C, endogenous production of H2S from INS-1E cells with or without transfection with CSE-siRNA. n= 3–5 for each group. *P < 0.05 versus native INS-1E cells.
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Whole-cell KATP channel current density was significantly smaller in CSE knock-down INS-1E cells (26.8 ± 1.9 pA pF–1, n= 4) than in native untransfected cells (46.4 ± 3.9 pA pF–1 at –120 mV, n= 5). Exogenous H2S at 100 μM significantly increased whole-cell KATP channel currents in CSE-siRNA-transfected cells (Fig. 7). For example, an increase of 67.6 ± 5.6% in KATP channel currents was induced by H2S at –120 mV (n= 5). Furthermore, it was found that PPG did not alter the function of single KATP channel in inside-out patches. Open probability of single KATP channels was 0.079 ± 0.004 after the application of PPG (1 mM) to the cytosolic side of inside-out patches, which was not different from that in the absence of PPG (0.083 ± 0.001 at –50 mV, n= 4, P > 0.05). This observation supports the notion that the inhibitory effect of PPG on KATP channels was mediated by CSE in the cytosolic milieu.
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A, H2S (100 μM) had no effect on KATP channels in INS-1E cells transfected with negative siRNA. n= 4. B, H2S (100 μM) significantly increased KATP channel currents in INS-1E cells transfected with CSE-siRNA. n= 5. *P < 0.01.
With the inside-out single-channel recording configuration, exposure of KATP channels in the excised membrane patch to endogenous H2S was minimized. As such, desensitization of KATP channels in insulin-secreting cells by endogenous H2S can be better confirmed. In this recording configuration, application of H2S at low concentration (100 μM) significantly stimulated KATP channels (Fig. 8A). Open probability of single KATP channels was increased from 0.08 ± 0.002 to 0.26 ± 0.05 (n= 4, P < 0.01) and the closed time of single channels decreased from 189 ± 7 to 59 ± 6 ms (n= 4, P < 0.01) by 100 μM H2S. However, H2S did not change the open time of single channels, nor single-channel conductance of KATP channels. Effect of H2S on single-channel activity at various membrane potentials was also examined. The linear current–voltage relation of single-channel currents was not changed by H2S (100 μM) with a single-channel conductance of 78 ± 2.2 pS (n= 6) (Fig. 8B). To examine whether H2S altered ATP sensitivity of KATP channels, ATP concentrations at the cytosolic side of inside-out patches were changed from 300 μM to 30 and 3 μM. Although open probability of single KATP channels was increased as ATP concentration decreased, H2S increased open probability of KATP channels to the same degree with different ATP levels (Fig. 9). It appears that H2S directly acts on KATP channel proteins, rather than altering ATP sensitivity of KATP channels.
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A, changes in open probability of single KATP channels induced by H2S. The close states of the channel are indicated by a bar beside each record. Membrane potential, +60 mV. Changes in opening probability (Po) are shown in the right panel. B, the effect of H2S on the single-channel conductance of KATP channels in INS-1E cells. Representative original records of KATP channels in the presence of H2S (100 μM) are shown in the left panel. The I–V relationship of KATP channels under these conditions is shown in the right panel. n= 6 for each data point.
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Single KATP channel currents in inside-out membrane patches (n= 4) were recorded with symmetrical 140 mM KCl solutions. A, representative single KATP channel currents in inside-out membrane patches before and after H2S (100 μM) of the bath solution at –60 mV membrane potential with different concentrations of ATP. Bar beside each trace indicates the closed state of the single channel. B, increasing concentrations of ATP in the cytosolic side of the membrane patches reduced open probability of single KATP channels, but did not alter the relative stimulatory effect of H2S. *P < 0.05 versus control.
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Discussion
In recent years, the physiological importance of H2S has gained increasingly recognition. Produced in many types of mammalian cells, H2S modulates neuronal activities (Abe & Kimura, 1996), protects the heart from ischaemic damages (Geng et al. 2004), and participates in regulation of cellular apoptosis and proliferation (Yang et al. 2004a,b). One of the recognized mechanisms for cellular effects of H2S is activation of KATP channels by this gasotransmitter, especially in vascular SMCs (Zhao et al. 2001, 2003; Cheng et al. 2004). KATP channels play a central role in regulating the function of insulin-secreting cells by coupling metabolic change with insulin secretion via changes in membrane potential (Cook et al. 1988; Ashcroft & Rorseman, 1989; Ashcroft & Gribble, 1998). However, little information is available on endogenous regulation of pancreatic KATP channels except the known effects of extracellular glucose and intracellular ATP. Interaction of H2S with pancreatic KATP channels has not been investigated to date. It is reasoned that H2S may also stimulate KATP channels in pancreatic -cells as occurs in vascular smooth muscle cells. It should be aware that molecular composition of KATP channels is different among different cell types. For example, in pancreatic -cells the KATP channel complex is composed of the pore-forming inwardly rectifying K+ channel tetramer Kir6.2 and regulatory sulphonylurea receptors SUR1 (Saskura et al. 1995; Ashcroft, 1996; Gribble et al. 1997; Lorenz et al. 1998). In vascular smooth muscle cells, however, KATP channel complex is combination of Kir 6.1 and SUR2B (Yokoshiki et al. 1998; William & Odle, 2003). In this context, interaction of H2S with KATP channels in different cell types needs to be specifically investigated.
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We recorded a 78 pS KATP channel in INS-1E cells. This single-channel conductance is typical of KATP channels reported in pancreatic -cells (Ashcroft & Gribble, 1998). Other characteristics of KATP channels, including burst opening, ATP sensitivity, glucose sensitivity, voltage insensitivity and gliclazide sensitivity are all similar to those described in native pancreatic -cells (Mukai et al. 1998). Moreover, 0.3 mM Mg-ATP was required for sustaining KATP channel currents in our study, a property shared by SUR1/KIR6.2 type of KATP channels (Inagaki et al. 1995; Gribble et al. 1997). Sulphonylureas such as gliclazide stimulate insulin secretion by closing KATP channels (Sturgess et al. 1985; Trube et al. 1986; Ashcroft, 2000). Potassium channel openers such as diazoxide inhibit insulin secretion by opening KATP channels (Trube et al. 1986; Dunne et al. 1987; Sturgess et al. 1988; Minami et al. 2003). In our study, diazoxide significantly increased, and gliclazide inhibited, KATP channel activity in INS-1E cells. KATP channels in INS-1E cells were also inhibited by high glucose concentration in the bath solution (16 mM) or ATP (3 mM) in the pipette solution. These features are hallmarks of KATP channels in insulin-secreting cells (Cook & Hales, 1984; Ashcroft & Rorseman, 1989; Ashcroft & Gribble, 1998). Insulin secretion and KATP channel functionality in this way respond to changes in glucose levels. These pharmacological and biophysical properties indicate that KATP channels in INS-1E cells share the same characteristics with those in pancreatic -cells (Ashcroft & Gribble, 1998; Mukai et al. 1998).
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INS-1E cells are derived from an insulinoma pancreatic -cell line (Janjic et al. 1999; Merglen et al. 2004). These cells exhibit stable differentiated -cell phenotype, and secrete insulin in response to glucose and non-nutrient secretagogues via the stimulation of KATP channels and a minor amplifying pathway (Merglen et al. 2004). Significant amounts of H2S were produced by INS-1E cells. We have achieved a partial knockdown of the CSE gene in INS-1E cells using the CSE-siRNA technique. Since this partial knockdown significantly reduced the production of H2S, it is believed that CSE is the main enzyme for the production of H2S in INS-1E cells. More importantly, our study demonstrated that this endogenous H2S production in INS-1E cells was mediated by a variance in glucose concentrations, thus providing physiological regulatory mechanisms for H2S production. Functional correlation of H2S levels in INS-1E cells is realized by insulin secretion from these cells. By reducing endogenous H2S production in INS-1E cells, high glucose also stimulated insulin secretion. Furthermore, over-expression of the CSE gene in INS-1E cells via Ad-CSE infection significantly increased endogenous H2S production, thus inhibiting high glucose-stimulated insulin secretion.
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In the present study, we demonstrated for the first time that H2S activated KATP channels in INS-1E cells. Without manipulating the endogenous H2S level, exogenous H2S at concentrations equal to or lower than 100 μM had no effect on whole-cell KATP channels in INS-1E cells. This phenomenon might be explained as KATP channels in INS-1E cells were desensitized to endogenous H2S at resting conditions. When INS-1E cells were incubated with a high concentration of glucose (16–20 mM), these cells became sensitive to exogenous H2S that significantly increased KATP channel activity at 100 μM. This sensitizing effect of high glucose concentration on KATP channels could be linked to a high glucose-induced decrease in endogenous H2S production. This hypothesis was further verified by directly inhibiting CSE activity in INS-1E cells. When PPG (1–5 mM) was used to dialyse INS-1E cells to inhibit CSE and endogenous production of H2S, whole-cell KATP channel currents were greatly increased by exogenously applied H2S in a concentration-dependent manner. Another line of evidence supporting conditioning of KATP channels in INS-1E cells by endogenous H2S was derived from single-channel recording studies. Exogenous H2S significantly increased open probability of single KATP channels in inside-out membrane patches of INS-1E cells. With this cell-free recording configuration, substrates and enzymes for endogenous H2S production are eliminated. Therefore, it is highly possible that KATP channels in insulin-secreting cells have been desensitized by a high level of endogenous H2S at resting conditions. Removal or reduction of endogenous H2S would re-sensitize KATP channels so that these channels regain their sensitive response to H2S. We propose that the endogenous H2S level has one switch for turning on or off KATP channels in insulin-secreting cells, whereas the glucose level regulates endogenous H2S production. Under physiological conditions with low extracellular glucose (5 mM), endogenous H2S level is high, which would keep KATP channels mostly in their open state, thus hyperpolarizing the membrane of insulin-secreting cells. This will result in low activity of voltage-dependent calcium channels and low secretion of insulin from insulin-secreting cells. When the glucose concentration of plasma is elevated, endogenous H2S production in insulin-secreting cells is decreased. Consequent closure of KATP channels leads to increased insulin secretion. In our previous studies, it has been shown that the vasorelaxant effect of H2S was not mediated by any known second messengers, including cGMP, cAMP and PKC pathways (Zhao et al. 2001, 2003; Zhao & Wang, 2002). In the present study, we showed that H2S directly activates KATP channels in cell-free inside-out membrane patches. We also showed that ATP sensitivity of KATP channels was not changed by H2S. Taken together, these observations suggest that the interaction of H2S and KATP channels is not mediated by cytosolic second messengers. As a reducing agent, H2S may reduce selective cysteine residues of KATP channel protein, altering its functional status. However, application of a classical reducing agent (DTT) to INS-1E cells did not replicate the excitatory effect of H2S on KATP channels, suggesting that other mechanisms should be sought to explain the interaction of H2S with the KATP channel complex in a tissue-/cell type-specific manner.
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In summary, KATP channels in insulin-secreting INS-1E cells share many common features with their counterparts in native pancreatic -cells. H2S increased the activity of these KATP channels by increasing single-channel open probability, but not single-channel conductance. An endogenous high level of H2S in INS-1E cells sets up the tune for KATP channel activity and thus insulin-secreting level at resting conditions. An increase in extracellular glucose concentration lowers endogenous H2S level. This will have two effects. Firstly, activity of KATP channels in INS-1E cells will be significantly reduced so that insulin secretion will be increased. Secondly, KATP channels in INS-1E cells will be partially closed and re-sensitized to H2S. Subsequent changes in the endogenous H2S level would exert a much greater effect on the functional status of KATP channels under these conditions. Interaction among H2S, glucose, and KATP channels in insulin-secreting cells may constitute an important and novel mechanism for the fine control of insulin secretion from pancreatic -cells, which is initially triggered by changes in glucose concentration, under physiological and different pathophysiological conditions.
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2 Department of Biology, Lakehead University, Thunder Bay, Ontario, Canada P7B 5E1
Abstract
H2S is an important gasotransmitter, generated in mammalian cells from L-cysteine metabolism. As it stimulates KATP channels in vascular smooth muscle cells, H2S may also function as an endogenous opener of KATP channels in INS-1E cells, an insulin-secreting cell line. In the present study, KATP channel currents in INS-1E cells were recorded using the whole-cell and single-channel recording configurations of the patch-clamp technique. KATP channels in INS-1E cells have a single-channel conductance of 78 pS. These channels were activated by diazoxide and inhibited by gliclazide. ATP (3 mM) in the pipette solution inhibited KATP channels in INS-1E cells. Significant amount of H2S was produced from INS-1E cells in which the expression of cystathinonie gamma-lyase (CSE) was confirmed. After INS-1E cells were transfected with CSE-targeted short interfering RNA (CSE-siRNA) or treated with DL-propargylglycine (PPG; 1–5 mM) to inhibit CSE, endogenous production of H2S was abolished. Increase in extracellular glucose concentration significantly decreased endogenous production of H2S in INS-1E cells, and increased insulin secretion. After transfection of INS-1E cells with adenovirus containing the CSE gene (Ad-CSE) to overexpress CSE, high glucose-stimulated insulin secretion was virtually abolished. Basal KATP channel currents were significantly reduced after incubating INS-1E cells with a high glucose concentration (16 mM) or lowering endogenous H2S level by CSE-siRNA transfection. Under these conditions, exogenously applied H2S significantly increased whole-cell KATP channel currents at concentrations equal to or lower than 100 μM. H2S (100 μM) markedly increased open probability by more than 2-fold of single KATP channels (inside-out recording) in native INS-1E cells (n= 4, P < 0.05). Single-channel conductance and ATP sensitivity of KATP channels were not changed by H2S. In conclusion, endogenous H2S production from INS-1E cells varies with in vivo conditions, which significantly affects insulin secretion from INS-1E cells. H2S stimulates KATP channels in INS-1E cells, independent of activation of cytosolic second messengers, which may underlie H2S-inhibited insulin secretion from these cells. Interaction among H2S, glucose and the KATP channel may constitute an important and novel mechanism for the fine control of insulin secretion from pancreatic -cells.
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Introduction
Amounting evidence in recent years indicate that H2S is an endogenous molecule of gas with important physiological functions (Wang, 2002). In the cardiovascular system, endogenous production of H2S is mainly catalysed by cystathinonie gamma-lyase (CSE). Being a gasotransmitter, H2S causes vasorelaxation at physiologically relevant concentrations (Zhao et al. 2001) and participates in the modulation of neuronal functions (Abe & Kimura, 1996). One of the mechanisms for cardiovascular actions of H2S is activation of KATP channels. H2S increased whole-cell KATP channel currents in rat aortic vascular smooth muscle cells (SMC) (Zhao et al. 2001). Similarly, in rat mesenteric artery SMCs H2S at physiologically relevant concentrations stimulated KATP channels (Cheng et al. 2004). H2S exerted a negative inotropic effect on heart. This effect was partially blocked by glibenclamide, a classical sulphonylurea KATP channel blocker (Geng et al. 2004). Although no electrophysiological recordings were performed on isolated cardiomyocytes, indication of involvement of KATP channels in cardiac effect of H2S is noted.
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KATP channels are sensitive to changes in intracellular ATP concentrations. Elevation of intracellular ATP level leads to closure of KATP channels in many metabolically active cells. In this way, the KATP channel is a coupling factor to link metabolic activity and membrane excitability. This feature is especially important for pancreatic -cells. When circulating glucose level is elevated, glucose influx into pancreatic -cells increases and so does ATP production. Consequential closure of KATP channels on plasma membrane depolarizes membrane and opens voltage-dependent calcium channels. The final eventuality of this chain reaction is increased insulin release due to increased intracellular free calcium. There is no doubt that KATP channels in -cells are critical in regulation of glucose-induced insulin secretion (Cook et al. 1988; Ashcroft et al. 1989; Ashcroft & Gribble, 1998). However, beyond the regulatory role of glucose, via alteration of intracellular ATP level, on KATP channels, little is known about the existence of other endogenous regulators for KATP channels in pancreatic -cells. By analogy to the stimulatory effect of H2S on KATP channels in vascular SMCs, it is reasonable to believe that H2S may be a novel KATP channel opener in pancreatic -cells. To date, endogenous production of H2S in the pancreas, effect of H2S on insulin secretion, and interaction of H2S with pancreatic KATP channels have not been determined.
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In the present study, effects of H2S on KATP channels in an insulin-secreting insulinoma cell line, INS-1E cells, were examined using the whole-cell and single-channel recording configurations of the patch-clamp technique. Endogenous levels of H2S were either decreased by transfecting INS-1E cells with CSE-siRNA vector or dialysing the cells with a specific inhibitor for H2S-generating enzyme, or increased by overexpressing CSE gene in INS-1E cells. Actual production of endogenous H2S, expression level of H2S-generating enzymes, and insulin secretion in INS-1E cells were determined. Our study characterized KATP channels in INS-1E cells, demonstrated the important regulatory role of H2S on insulin secretion and pancreatic KATP channel activation for the first time, and revealed endogenous enzymatic production and metabolism of H2S in insulin-secreting cells.
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Methods
Cell culture
INS-1E cells derived from a rat insulinoma (kindly provided by Dr C. B. Wollheim, Geneva, Switzerland) were grown in a humidified (5% CO2, 95% O2) atmosphere for up to 2 days in Hepes-buffered RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol (Sigma), 100 units ml–1 penicillin, and 100 μg ml–1 streptomycin (Sigma). For patch-clamp study, cultured INS-1E cells were placed in a Petri dish mounted on the stage of an inverted phase contrast microscope (Olympus IX70). For other biochemical and molecular biology assays, INS-1E cells were harvested and centrifuged at 500 g for 10 min after being rinsed twice with PBS solution.
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Electrophysiological recording and analysis
Both whole-cell and single-channel recordings of KATP channel currents were performed as previously described (Cook & Hales, 1984; Zhao et al. 2001). Patch pipettes were pulled from borosilicate glass capillaries using a Flaming/Brown micropipette puller (Model P-87, Sutter Instruments). Pipette resistance was 8–12 M for single-channel recordings and 1–5 M for whole-cell experiments when filled with electrolyte solution. All electrophysiological recordings were performed at room temperature (20–24°C).
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Whole-cell recordings were carried out with an Axopatch 200A patch clamp amplifier via a Digidata 1200 (Axon Instruments Inc.) interface, and analysed off-line using pCLAMP software (version 6.02; Axon Instruments Inc.). Test pulses of 600 ms were made with 10 mV increments from –150 to +50 mV. The holding potential was set at –20 mV. I–V relationships were constructed using stable current amplitude at the end of 600 ms test pulses. Pipettes were filled with a solution containing (mM): KCl 105, MgCl2 1.0, CaCl2 1.0, EGTA 10, and Hepes 10 (pH adjusted to 7.3 with KOH). Unless otherwise specified, ATP concentration of the pipette solution was 0.3 mM. The bath solution contained (mM): NaCl 102, KCl 40, CaCl2 1.0, MgCl2 1.2, glucose 4.5, and Hepes 10 (pH adjusted to 7.4 with NaOH). When glucose concentration was increased in some experiments, equimolar NaCl was removed to maintain osmolality of the bath solution. No leakage subtraction was performed to the original recordings and all cells with visible changes in leakage currents during the course of the study were excluded from further analysis.
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For single-channel recording, inside-out configuration of the patch-clamp technique (Hamill et al. 1981) was used. Seal resistance was 10 G. Single-channel currents were filtered at 1 kHz (eight-pole Bessel, –3 db) and recorded with 100 μs sampling interval in a gap-free model. Single-channel current records were displayed and analysed using pCLAMP 7.0 software (Axon Instruments Inc.). For each concentration of tested agents, at least 30 s of channel activity was directly recorded on computer hard disk. Open probability (NPo), i.e. the fraction of time when the channels stay open within the total observation period with N representing the number of single channels in one patch (Wang & Wu, 1997) and single-channel conductance were determined from an all-point amplitude histogram using Fetchan and Pstat programs (Axon Instruments Inc.). The pipette solution contained (mM): KCl 140, MgCl2 1.2, EGTA 10 and Hepes 5 (pH adjusted to 7.2 with KOH). Inside-out patches were bathed in a solution containing (mM): KCl 140, MgCl2 0.53, glucose 4.5, ATP 0.3, ADP 0.3 and Hepes 5 (pH adjusted to 7.4 with KOH).
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Measurement of endogenous H2S production
H2S production rate was measured as previously described (Stipanuk & Beck, 1982) with modifications, which has been routinely used in our laboratory (Zhao et al. 2001, 2003; Cheng et al. 2004). Briefly, INS-1E cells cultured for 3–7 days were collected and homogenized in 50 mM ice-cold potassium phosphate buffer pH 6.8. The reaction mixture contained (mM): 100 potassium phosphate buffer pH 7.4, 10 L-cysteine, 2 pyridoxal 5'-phosphate, and 10% (w/v) homogenate. Cryovial test tubes (2 ml) were used as the centre wells, each containing 0.5 ml 1% zinc acetate as trapping solution and a filter paper 2 cm x 2.5 cm to increase air: liquid contacting surface. Reaction was performed in a 25 ml Erlenmeyer flask (Pyrex, USA). The flasks containing the reaction mixture and centre wells were flushed with N2 before being sealed with a double layer of Parafilm. Reaction was initiated by transferring the flasks from ice to a 37°C shaking water bath. After incubating at 37°C for 90 min, 0.5 ml of 50% trichloroacetic acid was added to stop the reaction. The flasks were sealed again and incubated at 37°C for another 60 min to ensure a complete trapping of H2S released from the mixture. Contents of the centre wells were then transferred to test tubes, each containing 3.5 ml of water. Subsequently, 0.5 ml of 20 mMN,N-dimethyl-p-phenylenediamine sulphate in 7.2 M HCl was added immediately followed by addition of 0.5 ml 30 mM FeCl3 in 1.2 M HCl. Absorbance of the resulting solution at 670 nm was measured 20 min later with a spectrophotometer (Siegel, 1965). H2S content was calculated against the calibration curve of standard H2S solutions.
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Measurement of insulin secretion from INS-1E cells
Native or transfected INS-1E cells were plated into 24-well plates at a density of 5 x 104 cells per well and tested 24–48 h later when cells reached about 80% confluence. Cells were maintained at 37°C for 2 h in glucose-free RPMI 1640 medium, washed and pre-incubated in glucose-free (0 mM) Krebs-Ringer-bicarbonate medium (pH 7.4) containing (mM): 135 NaCl, 3.6 KCl, 5 NaHCO3, 0.5 NaH2PO4, 0.5 MgCl2, 1.5 CaCl2, 10 Hepes and 0.1% BSA. After 30 min pre-incubation, cells were incubated for 30 min at 37°C in the presence of different glucose concentrations. At the end of each incubation period, the medium was collected and centrifuged for 10 min at 2500 g to remove cell debris. The supernatant was immediately stored at –20°C until insulin determination using the rat insulin ELISA kit (Mercodia AB, Sylveniusgatan, Uppsala, Sweden).
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Western immunoblotting
Cultured cells were harvested and lysed in a lysis buffer (EDTA 0.5 M; Tris-Cl 1 M, pH 7.4; sucrose 0.3 M; antipain hydrochloride 1 μg ml–1; benzamidine hydrochloride hydrate 1 M; leupeptin hemisulphate 1 μg ml–1; 1,10-phenanthroline monohydrate 1 M; pepstatin A 1 μM; plenylmethylsulphonyl fluoride 0.1 mM, and iodoacetamide 1 mM). Extracts were separated by centrifugation at 14 000 g for 15 min at 4°C. SDS-PAGE and Western blot analysis were performed as previously described (Yang et al. 2004a). Briefly, equal amount of proteins were boiled in 1 x SDS sample buffer (62.5 mM Tris-Cl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, and 0.01% bromophenol blue) and resolved on a 10% SDS-PAGE gel, and transferred onto polyvinylidene chloride (PVDC) nitrocellulose membranes. Dilutions for the primary antibodies were 1: 1000 for CSE, and 1: 5000 for -actin. HRP-conjugated secondary antibody was used at 1: 5000. Immunoreactions were visualized by enhanced chemiluminescence (ECL) and exposed to X-ray film (Kodak Scientific Imaging film). Membranes were stripped by incubating in a buffer containing 100 mM-mercaptoethanol, 2% SDS and 62.5 mM Tris-HCl (pH 6.8).
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CSE-siRNA transfection of INS-1E cells
CSE-targeted 21 nucleotide siRNA was designed using a web based siRNA design program (http://www.ambion.com/techlib/misc/siRNA_finder_html) according to the AA-N19 rule (Brummelkamp et al. 2002; Lake & Castellot, 2003). The targeted sequence was localized at a position 192 bases downstream of the start codon of CSE (GenBank Accession No. NM001902). Forward (ggu uau uua ucc ugg gcu g dtdt) and reverse (cag ccc agg cua aau aac c dtdt) RNA strands with two 5' deoxy-thymidine overhangs were commercially synthesized by Ambion (Austin, TX, USA). GADPH-targeted siRNA was also produced for optimizing transfection conditions. Negative control siRNA, a 21 nucleotide RNA duplex with no sequence homology with all known genes, was also purchased from Ambion. Transfection of siRNA into INS-1E cells was achieved using the siPORT lipid transfection agent from Ambion. Briefly, cells were plated overnight to form 60–70% confluent monolayers. CSE siRNA at 30 nM and the transfection reagent complex were added to cells in serum-free medium for 4 h. Fresh normal growth medium was then added and cells were incubated for another 20 h. As a control, negative siRNA was used to transfect INS-1E cells.
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Construction of recombinant CSE adenovirus vector and infection of INS-1E cells
A PCR was performed to amplify opening read frame (ORF) of CSE (GenBank accession number AB052882) from reverse-transcribed rat vascular tissue using a set of primers: 5'-CGTCCCAGCATGCAGAAGAA-3' and 5'-CAGTTATTCAGAAGGTCTGGCCC-3'. The amplified ORF of CSE was ligated into PCR2.1 vector (Invitrogen), and the KpnI–XhoI restriction fragment of CSE was subcloned into the KpnI–XhoI sites of the shuttle vector pAdShuttle-CMV (Qbiogene, Inc.), which contains cytomegalovirus promoter/enhancer element and simian virus 40 polyadenylation signals. Positive clone containing CSE ORF insert was sequenced to confirm the accuracy of the inserted CSE sequence. The resultant plasmid was linearized with PmeI and cotransformed with the adenovirus backbone vector pAdeasy-1 into E. coli BJ5183 cells by electroporation. Homologous recombinants containing CSE cDNA were detected by restriction endonuclease digestion and agarose gel electrophoresis. Recombinant CSE adenovirus vector (Ad-CSE) was then transformed into E. coli DH5 cells for large-scale amplification. The PacI-digested E1-deleted replication-deficient Ad-CSE vector was then transfected into mammalian HEK-293 cells using calcium phosphate–DNA precipitates. The recombinant Ad-CSE was expanded, purified and titrated (He et al. 1998). The recombinant adenovirus encoding bacterial -galactosidase (Ad-lacZ) derived from the same vector was used as a control. For adenoviral infection, subconfluent INS-1E cells were incubated with Ad-CSE or Ad-lacZ in serum-free media. After 4 h of incubation, media was removed, and cells were incubated in appropriate media for 48 h. The transfection efficiency of adenoviral vector in INS-1E cells was first determined by infecting cells with Ad-lacZ at various multiplicities of infection (MOI). The cells infected with Ad-lacZ were assayed for -galactosidase expression by the in situ X-gal staining method (Hirooka & Sakai, 2004). At MOI 50, > 90% of cells showed nuclear staining for -galactosidase. Subsequent experiments were performed at MOI of 50.
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Real-time RT-PCR determination of the transcriptional level of CSE
Native untransfected cells, negative siRNA- or CSE siRNA-transfected cells were harvested from 100 mm culture dishes 24 h after transfection. Monolayers were rinsed twice with PBS, and total RNA was collected using Tri Reagent (Molecular Research Center, Cincinnati, OH, USA). Contaminating DNA was avoided using the DNA-free kit (Ambion), and total RNA (2 μg) was reverse-transcribed into cDNA with AMV reverse transcriptase using random hexamer primers according to the manufacturer's protocol (Roche Applied Science, IN, USA). Controls containing no reverse transcriptase were used to safeguard for genomic DNA contamination in each sample.
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Real-time PCR was performed in an iCycler iQ apparatus (Bio-Rad, Harcules, CA, USA) associated with the iCycler optical system software (version 3.1) using the SYBR Green PCR Master Mix. All PCRs were performed in a 20 μl volume using 96-well optical grade PCR plates and optical sealing tape. Negative controls for this experiment were samples without a template. Cycling conditions were 95°C for 90 s followed by 38 cycles of 95°C for 10 s and 60°C for 20 s. For quantification, the target gene was normalized to the internal standard gene -actin. A standard curve was constructed with a series of dilutions of total RNA (Ambion) transcribed to cDNA using the protocol outlined above to confirm the same amplifying efficiency in PCR. A standard melting curve analysis was performed using a thermal cycling profile that began at 95°C for 1 min, decreased to 55°C for 1 min, and then ramped to 95°C in 1°C increments to confirm the absence of primer dimers. Product size was determined by running PCR products on a 1.8% agarose gel. Relative mRNA quantification was calculated by using the arithmatic formula ‘2–CT’, where CT is the difference between the threshold cycle of a given target cDNA and an endogenous reference cDNA (Yang et al. 2004a). Thus, this value yields the amount of the target normalized to an endogenous reference.
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Chemicals and statistical analysis
H2S-saturated solution (0.09 M) was freshly made by bubbling pure H2S gas (Praxair; Mississauga, Canada) into Krebs' solution at 30°C for 40 min as previously described (Zhao et al. 2001, 2003; Cheng et al. 2004). At 37°C and pH 7.4, the concentration of H2S in solution was relatively stable (Zhao et al. 2003). Data are expressed as mean ±S.E.M. Multiple comparisons were made with one-way ANOVA followed by a post hoc analysis (Tukey test). Statistical significance was set at P < 0.05.
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Results
Endogenous H2S production and insulin secretion from INS-1E cells
To investigate whether cultured INS-1E cells produced H2S under different in vivo conditions, INS-1E cells were incubated with either 5 or 20 mM glucose for 24 h. Increase in extracellular glucose concentration from 5 to 20 mM significantly decreased endogenous production of H2S in INS-1E cells by about 46% (Fig. 1A). Similar inhibition of H2S production in INS-1E cells was also observed with 16 mM glucose (not shown). To further examine whether this glucose-mediated endogenous H2S production was regulated by specific enzymatic process, DL-propargylglycine (PPG), a selective inhibitor of CSE, was used. Lysed INS-1E cells were mixed with PPG to facilitate interaction of this inhibitor with cytosolically located CSE, and then H2S production was assayed. It was found that PPG significantly inhibited H2S production in INS-1E cells (Fig. 1A).
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A, glucose-mediated H2S production in INS-1E cells. Endogenous production of H2S was significantly decreased by high glucose concentration in the incubation medium. PPG (5 mM) treatment of INS-1E cells significantly reduced endogenous H2S production. *P < 0.05 versus other groups. n= 4 for each group. B, glucose-stimulated insulin secretion from INS-1E cells with Ad-CSE transfection to increase endogenous H2S level. *P < 0.05 versus native or Ad-LacZ-transfected INS-1E cells in the presence of 16 mM glucose. n= 6–8 for each group. C, production of H2S in Ad-CSE-transfected INS-1E cells. Cells were cultured with 16 mM glucose in the medium. *P < 0.05 versus other groups. n= 4–6 for each group.
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High glucose affected insulin secretion from INS-1E cells. With an increased glucose concentration from 5 to 16 mM, insulin secretion from INS-1E cells became 3-fold greater (Fig. 1B). After transfection of INS-1E cells with Ad-CSE to overexpress CSE, insulin secretion at basal level (5 mM glucose) was not altered. However, high glucose-stimulated insulin secretion was virtually abolished (Fig. 1B). Significantly increased production of endogenous H2S from Ad-CSE-transfected INS-1E cells is shown in Fig. 1C. Native INS-1E cells were also transfected with Ad-LacZ as a negative control. Neither basal nor high glucose-stimulated insulin secretion from INS-1E cells were altered by Ad-LacZ transfection (Fig. 1B).
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Characterization of KATP channel in INS-1E cells
Gliclazide is a specific blocker of KATP channels in pancreatic -cells (Trube et al. 1986; Ashcroft, 2000). Gliclazide (1 μM) decreased significantly whole-cell KATP currents in INS-1E cells from –1049.9 ± 115 to 522.8 ± 88 pA at –100 mV (n= 5, P < 0.05). Representative results of the effect of gliclazide on whole-cell KATP channels are shown in Fig. 2A. Diazoxide is a potent opener of KATP channels in pancreatic -cells (Sturgess et al. 1988; D'hahan et al. 1999). Whole-cell KATP channels in INS-1E cells were significantly stimulated by diazoxide (Fig. 2B). Whole-cell KATP channels in INS-1E cells were also characterized by their sensitivity to intracellular ATP. KATP channel currents were 82.9 ± 8.6 pA pF–1 (–120 mV) with 0.3 mM ATP in the pipette solution (n= 6). When the ATP concentration was increased to 3 mM, KATP channel currents were reduced to 29.7 ± 5.6 pA pF–1 (–120 mV) (n= 5, P < 0.05 versus 0.3 mM ATP in the pipette solution).
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Holding potential, –20 mV. A, inhibition of the whole-cell KATP channels by gliclazide in INS-1E cells. Representative KATP channel currents (top panel) and the mean I–V relationship of whole-cell KATP channels (bottom panel, n= 5) before and after the application of gliclazide at 1 μM. B, stimulation of whole-cell KATP channels by diazoxide in INS-1E cells. Representative KATP channel currents (top panel) and the mean I–V relationships of KATP channels (bottom panel, n= 4) before and after application of diazoxide at 0.3 mM.
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With symmetrical K+ (140 mM) in the pipette and bath solutions, KATP channels in INS-1E cells had a single-channel conductance of 78 ± 2.3 pS (n= 5) (Fig. 3A). These single-channel KATP currents appeared in rapid open–close transitions and in brief bursts. Open probability of single KATP channels at –60 mV was 0.08 ± 0.01 (n= 6). When gliclazide (1 μM) was added to the bath, activity of the single channel was greatly decreased with open probability reduced to 0.004 ± 0.001 (n= 5, P < 0.05) (Fig. 3B). When diazoxide (100 μM) was applied to inside-out patches, single-channel activity was greatly increased with open probability of single KATP channels changing from 0.09 ± 0.01 to 0.28 ± 0.03 (n= 5, P < 0.05). However, there was no significant change in amplitude of unitary inward current after application of diazoxide. Unitary current amplitudes of single-channel KATP currents were 4.68 ± 0.09 and 4.70 ± 0.06 pA (–60 mV) before and after application of diazoxide, respectively (n= 5, P > 0.05).
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A, representative single-channel KATP channel currents in one inside-out membrane patch (left panel) and the single-channel conductance of KATP channels (right panel, n= 5). B, open probabilities of single KATP channels in the absence (upper panel) and then presence (lower panel) of gliclazide (1 μM). Membrane potential, –60 mV.
Effects of H2S on KATP channels in INS-1E cells
KATP channels in INS-1E cells were also sensitive to glucose stimulation. After glucose concentration of the bath solution was changed from 5 mM to 16 mM, whole-cell KATP currents were significantly reduced (Fig. 4A). With 5 mM glucose in the bath solution, H2S at 100 μM had no effect on whole-cell KATP currents in INS-1E cells (n= 5, P > 0.05). In the presence of 16 mM glucose, application of H2S (100 μM) to INS-1E cells significantly increased KATP currents (Fig. 4B). Application of DL-dithothreitol (DTT) (3 mM) to INS-1E cells for 5 min did not significantly change KATP channel currents in INS-1E cells (79 ± 2.69 versus 86 ± 2.68 pA pF–1 at –100 mV, n= 5, P > 0.05). A lack of effect of DTT on KATP channels has also been reported previously in pancreatic -cells (Islam et al. 1993; Krippeit-Drews et al. 1994). Since DTT is a reducing reagent, our result suggests that the stimulatory effect of H2S on KATP channels in INS-1E cells is unlikely to be mediated by a general reducing effect.
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Holding potential, –20 mV. A, inhibitory effect of glucose on the whole-cell KATP channels in INS-1E cells. Representative KATP channel current traces (top panel) and the mean I–V relationships (bottom panel) of KATP channels with different glucose concentrations in the bath solutions. n= 5 for each group. *P < 0.05. B, interaction of H2S with whole-cell KATP channels in the presence of high glucose (16 mM) in the bath solution. Representative KATP channel current traces were shown in the top panel and the mean I–V relationships in the bottom panel of KATP channels in INS-1E cells before and after H2S application. n= 5 for each group.
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To examine whether KATP channels in INS-1E cells were desensitized at resting conditions by high endogenous H2S levels, we tried to lower endogenous H2S level by pre-treating these cells with PPG. In the presence of PPG (5–10 mM) in the bath solution containing 5 mM glucose, H2S at 100 μM did not alter the whole-cell KATP currents in INS-1E cells (812 ± 63 versus 796 ± 81 pA at –120 mV, n= 5, P > 0.05). In the next group of experiments, PPG (1 mM) was included in the pipette solution to dialyse cells. This manoeuver alone significantly reduced KATP channel currents by 40 ± 9.6% (n= 5, P < 0.05). More interestingly, including PPG in the pipette solution unmasked a dose-dependent stimulatory effect of exogenously applied H2S on KATP channels (Fig. 5A). Even at a concentration of 10 μM, H2S increased KATP channel currents by 69.4 ± 5.7% after PPG treatment (n= 4, P < 0.05, –120 mV) (Fig. 5C). Effects of H2S on KATP channels were not different with either 1 or 5 mM PPG in the pipette solution (Fig. 5A and B), indicating that even at 1 mM PPG might already significantly inhibit CSE. CSE protein expression was confirmed in INS-1E cells with Western blot analysis (Fig. 6A). To further demonstrate that the preconditioning effect of intracellular PPG was related to reduced endogenous CSE activity, cultured INS-1E cells were transfected with CSE-siRNA to knock down endogenous expression of CSE gene. While negative-siRNA transfection did not alter translational or transcriptional expression levels of CSE, CSE-siRNA transfection reduced expression of the CSE gene by about 80% (Fig. 6A and B). In line with suppressed CSE gene expression, endogenous production of H2S from CSE-siRNA-transfected INS-1E cells was significantly reduced in comparison with that of native INS-1E cells (Fig. 6C).
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A, bath application of H2S at 100 μM increased inward KATP channel currents in INS-1E cells, which were dialysed with 5 mM PPG. n= 4. B, bath application of H2S at 100 μM increased inward KATP channel currents in INS-1E cells, which were dialysed with 1 mM PPG. n= 5. C, the concentration-dependent stimulatory effects of H2S on KATP channels with 1 mM PPG in the pipette solution. n= 5–6 for each group. *P < 0.05 versus the recordings in the absence of H2S.
A, Western blot detection of CSE protein expression in INS-1E cells. B, real-time RT-PCR comparison of CSE expression levels in INS-1E cells with and without siRNA transfection. n= 4. *P < 0.01. C, endogenous production of H2S from INS-1E cells with or without transfection with CSE-siRNA. n= 3–5 for each group. *P < 0.05 versus native INS-1E cells.
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Whole-cell KATP channel current density was significantly smaller in CSE knock-down INS-1E cells (26.8 ± 1.9 pA pF–1, n= 4) than in native untransfected cells (46.4 ± 3.9 pA pF–1 at –120 mV, n= 5). Exogenous H2S at 100 μM significantly increased whole-cell KATP channel currents in CSE-siRNA-transfected cells (Fig. 7). For example, an increase of 67.6 ± 5.6% in KATP channel currents was induced by H2S at –120 mV (n= 5). Furthermore, it was found that PPG did not alter the function of single KATP channel in inside-out patches. Open probability of single KATP channels was 0.079 ± 0.004 after the application of PPG (1 mM) to the cytosolic side of inside-out patches, which was not different from that in the absence of PPG (0.083 ± 0.001 at –50 mV, n= 4, P > 0.05). This observation supports the notion that the inhibitory effect of PPG on KATP channels was mediated by CSE in the cytosolic milieu.
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A, H2S (100 μM) had no effect on KATP channels in INS-1E cells transfected with negative siRNA. n= 4. B, H2S (100 μM) significantly increased KATP channel currents in INS-1E cells transfected with CSE-siRNA. n= 5. *P < 0.01.
With the inside-out single-channel recording configuration, exposure of KATP channels in the excised membrane patch to endogenous H2S was minimized. As such, desensitization of KATP channels in insulin-secreting cells by endogenous H2S can be better confirmed. In this recording configuration, application of H2S at low concentration (100 μM) significantly stimulated KATP channels (Fig. 8A). Open probability of single KATP channels was increased from 0.08 ± 0.002 to 0.26 ± 0.05 (n= 4, P < 0.01) and the closed time of single channels decreased from 189 ± 7 to 59 ± 6 ms (n= 4, P < 0.01) by 100 μM H2S. However, H2S did not change the open time of single channels, nor single-channel conductance of KATP channels. Effect of H2S on single-channel activity at various membrane potentials was also examined. The linear current–voltage relation of single-channel currents was not changed by H2S (100 μM) with a single-channel conductance of 78 ± 2.2 pS (n= 6) (Fig. 8B). To examine whether H2S altered ATP sensitivity of KATP channels, ATP concentrations at the cytosolic side of inside-out patches were changed from 300 μM to 30 and 3 μM. Although open probability of single KATP channels was increased as ATP concentration decreased, H2S increased open probability of KATP channels to the same degree with different ATP levels (Fig. 9). It appears that H2S directly acts on KATP channel proteins, rather than altering ATP sensitivity of KATP channels.
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A, changes in open probability of single KATP channels induced by H2S. The close states of the channel are indicated by a bar beside each record. Membrane potential, +60 mV. Changes in opening probability (Po) are shown in the right panel. B, the effect of H2S on the single-channel conductance of KATP channels in INS-1E cells. Representative original records of KATP channels in the presence of H2S (100 μM) are shown in the left panel. The I–V relationship of KATP channels under these conditions is shown in the right panel. n= 6 for each data point.
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Single KATP channel currents in inside-out membrane patches (n= 4) were recorded with symmetrical 140 mM KCl solutions. A, representative single KATP channel currents in inside-out membrane patches before and after H2S (100 μM) of the bath solution at –60 mV membrane potential with different concentrations of ATP. Bar beside each trace indicates the closed state of the single channel. B, increasing concentrations of ATP in the cytosolic side of the membrane patches reduced open probability of single KATP channels, but did not alter the relative stimulatory effect of H2S. *P < 0.05 versus control.
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Discussion
In recent years, the physiological importance of H2S has gained increasingly recognition. Produced in many types of mammalian cells, H2S modulates neuronal activities (Abe & Kimura, 1996), protects the heart from ischaemic damages (Geng et al. 2004), and participates in regulation of cellular apoptosis and proliferation (Yang et al. 2004a,b). One of the recognized mechanisms for cellular effects of H2S is activation of KATP channels by this gasotransmitter, especially in vascular SMCs (Zhao et al. 2001, 2003; Cheng et al. 2004). KATP channels play a central role in regulating the function of insulin-secreting cells by coupling metabolic change with insulin secretion via changes in membrane potential (Cook et al. 1988; Ashcroft & Rorseman, 1989; Ashcroft & Gribble, 1998). However, little information is available on endogenous regulation of pancreatic KATP channels except the known effects of extracellular glucose and intracellular ATP. Interaction of H2S with pancreatic KATP channels has not been investigated to date. It is reasoned that H2S may also stimulate KATP channels in pancreatic -cells as occurs in vascular smooth muscle cells. It should be aware that molecular composition of KATP channels is different among different cell types. For example, in pancreatic -cells the KATP channel complex is composed of the pore-forming inwardly rectifying K+ channel tetramer Kir6.2 and regulatory sulphonylurea receptors SUR1 (Saskura et al. 1995; Ashcroft, 1996; Gribble et al. 1997; Lorenz et al. 1998). In vascular smooth muscle cells, however, KATP channel complex is combination of Kir 6.1 and SUR2B (Yokoshiki et al. 1998; William & Odle, 2003). In this context, interaction of H2S with KATP channels in different cell types needs to be specifically investigated.
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We recorded a 78 pS KATP channel in INS-1E cells. This single-channel conductance is typical of KATP channels reported in pancreatic -cells (Ashcroft & Gribble, 1998). Other characteristics of KATP channels, including burst opening, ATP sensitivity, glucose sensitivity, voltage insensitivity and gliclazide sensitivity are all similar to those described in native pancreatic -cells (Mukai et al. 1998). Moreover, 0.3 mM Mg-ATP was required for sustaining KATP channel currents in our study, a property shared by SUR1/KIR6.2 type of KATP channels (Inagaki et al. 1995; Gribble et al. 1997). Sulphonylureas such as gliclazide stimulate insulin secretion by closing KATP channels (Sturgess et al. 1985; Trube et al. 1986; Ashcroft, 2000). Potassium channel openers such as diazoxide inhibit insulin secretion by opening KATP channels (Trube et al. 1986; Dunne et al. 1987; Sturgess et al. 1988; Minami et al. 2003). In our study, diazoxide significantly increased, and gliclazide inhibited, KATP channel activity in INS-1E cells. KATP channels in INS-1E cells were also inhibited by high glucose concentration in the bath solution (16 mM) or ATP (3 mM) in the pipette solution. These features are hallmarks of KATP channels in insulin-secreting cells (Cook & Hales, 1984; Ashcroft & Rorseman, 1989; Ashcroft & Gribble, 1998). Insulin secretion and KATP channel functionality in this way respond to changes in glucose levels. These pharmacological and biophysical properties indicate that KATP channels in INS-1E cells share the same characteristics with those in pancreatic -cells (Ashcroft & Gribble, 1998; Mukai et al. 1998).
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INS-1E cells are derived from an insulinoma pancreatic -cell line (Janjic et al. 1999; Merglen et al. 2004). These cells exhibit stable differentiated -cell phenotype, and secrete insulin in response to glucose and non-nutrient secretagogues via the stimulation of KATP channels and a minor amplifying pathway (Merglen et al. 2004). Significant amounts of H2S were produced by INS-1E cells. We have achieved a partial knockdown of the CSE gene in INS-1E cells using the CSE-siRNA technique. Since this partial knockdown significantly reduced the production of H2S, it is believed that CSE is the main enzyme for the production of H2S in INS-1E cells. More importantly, our study demonstrated that this endogenous H2S production in INS-1E cells was mediated by a variance in glucose concentrations, thus providing physiological regulatory mechanisms for H2S production. Functional correlation of H2S levels in INS-1E cells is realized by insulin secretion from these cells. By reducing endogenous H2S production in INS-1E cells, high glucose also stimulated insulin secretion. Furthermore, over-expression of the CSE gene in INS-1E cells via Ad-CSE infection significantly increased endogenous H2S production, thus inhibiting high glucose-stimulated insulin secretion.
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In the present study, we demonstrated for the first time that H2S activated KATP channels in INS-1E cells. Without manipulating the endogenous H2S level, exogenous H2S at concentrations equal to or lower than 100 μM had no effect on whole-cell KATP channels in INS-1E cells. This phenomenon might be explained as KATP channels in INS-1E cells were desensitized to endogenous H2S at resting conditions. When INS-1E cells were incubated with a high concentration of glucose (16–20 mM), these cells became sensitive to exogenous H2S that significantly increased KATP channel activity at 100 μM. This sensitizing effect of high glucose concentration on KATP channels could be linked to a high glucose-induced decrease in endogenous H2S production. This hypothesis was further verified by directly inhibiting CSE activity in INS-1E cells. When PPG (1–5 mM) was used to dialyse INS-1E cells to inhibit CSE and endogenous production of H2S, whole-cell KATP channel currents were greatly increased by exogenously applied H2S in a concentration-dependent manner. Another line of evidence supporting conditioning of KATP channels in INS-1E cells by endogenous H2S was derived from single-channel recording studies. Exogenous H2S significantly increased open probability of single KATP channels in inside-out membrane patches of INS-1E cells. With this cell-free recording configuration, substrates and enzymes for endogenous H2S production are eliminated. Therefore, it is highly possible that KATP channels in insulin-secreting cells have been desensitized by a high level of endogenous H2S at resting conditions. Removal or reduction of endogenous H2S would re-sensitize KATP channels so that these channels regain their sensitive response to H2S. We propose that the endogenous H2S level has one switch for turning on or off KATP channels in insulin-secreting cells, whereas the glucose level regulates endogenous H2S production. Under physiological conditions with low extracellular glucose (5 mM), endogenous H2S level is high, which would keep KATP channels mostly in their open state, thus hyperpolarizing the membrane of insulin-secreting cells. This will result in low activity of voltage-dependent calcium channels and low secretion of insulin from insulin-secreting cells. When the glucose concentration of plasma is elevated, endogenous H2S production in insulin-secreting cells is decreased. Consequent closure of KATP channels leads to increased insulin secretion. In our previous studies, it has been shown that the vasorelaxant effect of H2S was not mediated by any known second messengers, including cGMP, cAMP and PKC pathways (Zhao et al. 2001, 2003; Zhao & Wang, 2002). In the present study, we showed that H2S directly activates KATP channels in cell-free inside-out membrane patches. We also showed that ATP sensitivity of KATP channels was not changed by H2S. Taken together, these observations suggest that the interaction of H2S and KATP channels is not mediated by cytosolic second messengers. As a reducing agent, H2S may reduce selective cysteine residues of KATP channel protein, altering its functional status. However, application of a classical reducing agent (DTT) to INS-1E cells did not replicate the excitatory effect of H2S on KATP channels, suggesting that other mechanisms should be sought to explain the interaction of H2S with the KATP channel complex in a tissue-/cell type-specific manner.
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In summary, KATP channels in insulin-secreting INS-1E cells share many common features with their counterparts in native pancreatic -cells. H2S increased the activity of these KATP channels by increasing single-channel open probability, but not single-channel conductance. An endogenous high level of H2S in INS-1E cells sets up the tune for KATP channel activity and thus insulin-secreting level at resting conditions. An increase in extracellular glucose concentration lowers endogenous H2S level. This will have two effects. Firstly, activity of KATP channels in INS-1E cells will be significantly reduced so that insulin secretion will be increased. Secondly, KATP channels in INS-1E cells will be partially closed and re-sensitized to H2S. Subsequent changes in the endogenous H2S level would exert a much greater effect on the functional status of KATP channels under these conditions. Interaction among H2S, glucose, and KATP channels in insulin-secreting cells may constitute an important and novel mechanism for the fine control of insulin secretion from pancreatic -cells, which is initially triggered by changes in glucose concentration, under physiological and different pathophysiological conditions.
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