Hydrogen sulfide-induced inhibition of L-type Ca 2+ channels and insulin secretion in mouse pancreatic beta cells

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1 Dietologi (213) 56: DOI 1.17/s ARTICLE Hydrogen sulfide-indued inhiition of L-type C 2 hnnels nd insulin seretion in mouse pnreti et ells G. Tng & L. Zhng & G. Yng & L. Wu & R. Wng Reeived: 29 June 212 / Aepted: 29 Novemer 212 / Pulished online: 3 Deemer 212 # Springer-Verlg Berlin Heidelerg 212 Astrt Aims/hypothesis L-type voltge-dependent C 2 hnnels (VDCCs) in pnreti et ells ply ritil role in regulting insulin seretion. The gsotrnsmitter H 2 Sismostlygenerted from L-ysteine in pnreti et ells y ystthionine γ-lyse (CSE) nd hs een reported to inhiit insulin relese y opening ATP-sensitive K hnnels. However, whether nd how H 2 S ffets VDCCs in et ells is unknown. Methods The whole-ell pth-lmp tehnique ws used to reord VDCCs in et ells from Cse (lso known s Cth)- knokout (KO) nd wild-type (WT) mie. Insulin seretion from pnreti islets nd endogenous H 2 S prodution in pnres were mesured. Results The H 2 S donor NHS reversily deresed L-type VDCC urrent density in onentrtion-dependent fshion in WT pnreti et ells, nd the urrent density ws further inhiited y nifedipine. Furthermore, NHS inhiited the hnnel reovery from depolristion-indued intivtion, ut did not shift the urrent voltge (I V) reltionship. ACS67, nother H 2 S donor, lso inhiited L-type VDCCs in G. Tng : L. Zhng : R. Wng () Deprtment of Biology, Lkehed University, 955 Oliver Rod, Thunder By, ON P7B 5E1, Cnd e-mil: rwng@lkehedu. G. Yng Shool of Kinesiology, Lkehed University, Thunder By, ON, Cnd L. Wu Deprtment of Helth Sienes, Lkehed University, Thunder By, ON, Cnd L. Wu Thunder By Regionl Reserh Institute, Thunder By, ON, Cnd et ells. Inhiiting CSE tivity with DL-proprgylglyine inresed the sl L-hnnel tivity of et ells from WT mie, ut not tht of et ells from Cse-KO mie. Bet ells from Cse-KO mie displyed higher L-type VDCC density thn those from WT mie. Insulin seretion from pnreti islets ws elevted in Cse-KO mie ompred with WT mie. NHS dose-dependently inhiited gluose-stimulted insulin seretion, whih ws further inhiited y nifedipine. By K-8644 inresed gluose-stimulted insulin seretion, ut this ws ounterted y NHS nd nifedipine. Conlusions/interprettion Exogenous nd endogenous H 2 S inhiit L-type VDCC tivity nd pnreti insulin seretion, onstituting novel mehnism for the regultion of insulin seretion y the CSE/H 2 S system. Keywords Betells.C 2 hnnels.h 2 S.Insulin seretion. Pnres Arevitions [C 2 ] i Intrellulr lium onentrtion CBS Cystthionine β-synthse CSE Cystthionine γ-lyse HP Holding potentil I C VDCC urrent I C,pek Pek I C I C,SS Stedy-stte I C I V Current voltge K ATP ATP-sensitive K KO Knokout KRB Kres Ringer ironte PPG DL-proprgylglyine STZ Streptozotoin VDCC Voltge-dependent C 2 hnnel WT Wild-type ZDF Zuker dieti ftty

2 534 Dietologi (213) 56: Introdution Cystthionine γ-lyse (CSE) uses L-ysteine s sustrte to generte H 2 S in mny types of mmmlin ells [1, 2]. CSE is mostly responsile for the endogenous prodution of H 2 Sin pnreti tissues nd loned pnreti et ell lines [2 8]. The inhiition of CSE tivity y DL-proprgylglyine (PPG) signifintly deresed prodution of H 2 S nd inresed plsm insulin levels in Zuker dieti ftty (ZDF) rts [6], while inresed Cse expression nd H 2 S formtion were oserved in streptozotoin (STZ)-indued dieti rts [4, 6, 8]. Geneti deletion of Cse (lso known s Cth)inmiemrkedlyredues H 2 S levels in different tissues, leding to inresed lood pressure, umultion of homoysteine nd impirment of vsulr endothelil funtions [3]. When stimulted with high gluose (2 mmol/l), insulin seretion from pnreses of Cse-knokout (KO) mie is signifintly inresed [4]. Compred with wild-type (WT) mie, Cse-KO mie tht reeive STZ injetions hve delyed onset of dieti sttus [4]. STZ signifintly inreses pnreti H 2 SprodutioninWTmie, ut not in Cse-KO mie, nd it uses more poptoti et ell deth in WT thn in Cse-KO mie. Voltge-dependent C 2 hnnels (VDCCs) in pnreti et ells ply ritil role in the regultion of insulin seretion vi ontrolling C 2 influx. It hs een reported tht H 2 S stimultes ATP-sensitive K (K ATP )hnnels, whih hyperpolrises ell memrne nd indiretly intivtes L-type VDCCs, leding to redued insulin seretion [5, 9 11]. Intertion etween H 2 S nd K ATP hnnels in insulin-sereting ells onstitutes n importnt regultory mehnism for insulin seretion [1, 11]. On the other hnd, the role of L-type VDCCs in H 2 S-regulted insulin seretion is unknown. One report desried n inhiitory effet of H 2 S on L-type VDCCs in isolted rt rdiomyoytes [12]. Another study in ereellr grnule neurons reported the ontrditory result tht H 2 S inresed intrellulr C 2 onentrtion ([C 2 ] i ) y stimulting L-type VDCCs [13]. Whether nd how H 2 S interts with L-type VDCCs in et ells is intriguing nd wrrnts in-depth investigtion. In the present study, Cse-KO nd WT mie were used to isolte pnreti et ells. The effets of NHS, donor of H 2 S, were first determined on the eletrophysiologil properties of L-type VDCCs. Chnges in L-type VDCCs were lso tested in the presene of CSE inhiitor (PPG) or in the sene of endogenous H 2 S. The insulin seretion from nd H 2 S prodution in islets or pnres were mesured with or without loking L-type VDCCs. Methods Animl preprtion The Cse-KO mie were generted nd house red s previously desried [3]. The fourth genertion of 1- to 16-week-old mle Cse-KO offspring nd ge-mthed mle WT littermtes on C57BL/6 J 129SvEv kground were used. All niml experiments were pproved y the Animl Use Committee of Lkehed University, ON, Cnd. Isoltion of intt islets nd et ells from mouse pnres Briefly, the mie were nesthetised with single intrperitonel injetion of ketmine(2.2mg/1gody weight) nd xylzine (.44 mg/1 g ody weight) efore the opening of the dominl wll. The pnres ws distended y injetion of 1 ml Kres Ringer ironte (KRB) ontining.5 1 mg/ml ollgense type V (Sigm, USA),.5 1 μl/ml DNse I (Sigm, USA) nd ovine serum lumin (.5 mg/ml) through nnul inserted into the ommon ile dut. KRB omprised: NCl 129 mmol/l; KCl 4.8 mmol/l; KH 2 PO mmol/l; NHCO 3 5 mmol/l; MgCl mmol/l; HEPES 1 mmol/l; nd CCl mmol/l. The pnres ws then removed refully nd trnsferred to 5 ml Flon tue with the ove enzyme solution, nd inuted for 5 1 min in wter th t 37 C. Clened islets of Lngerhns were hnd-piked under disseting mirosope fter 3 entrifuge wshout (t 1,5 g for 3 min t 4 C) with C 2 /Mg 2 -free KRB. The dispersed islets were dissoited into single ells y vigorous shking in C 2 / Mg 2 -free KRB supplemented with 1 enzyme-free elldissoition regent (HiMedi L Pvt. Ltd, VWR, Mississug, ON, Cnd). The dispersed islet ells were ultured on smll glss over slips pre-oted with poly L-lysine, in RPMI 164 medium supplemented with 1% (vol./vol.) fetl ovine serum, 1 U/ml peniillin, nd 1 μg/ml streptomyin in humidified ir t 37 C ontining 5% CO 2. Primry-ultured (1 3 dys) islet ells were used in the pth-lmp experiments. Mesurement of insulin seretion from isolted pnreti islets Freshly isolted islets (ten size-mthed for eh th) were wshed nd pre-inuted with gluose-free RPMI 164 medium in 24 well pltes [6]. After pre-inution for 3 min, islets were treted for 3 min t 37 C with different hemils in the presene of either 2.8 or 2 mmol/l gluose. In other experiments, insulin relese from isolted islets ws deteted in KRB. At the end of eh inution period, the medium ws olleted nd entrifuged for 1 min t 239 g to remove islet deris. The insulin level in the superntnt frtion ws determined using the mouse insulin ELISA kit (Merodi AB, Sylveniusgtn, Uppsl, Sweden). After insulin mesurement, the islets were srped from 24 well pltes on ie into the entrifuge tue; protein onentrtion ws then mesured using Brdford Regent (Sigm, St Louis, MO, USA). Eletrophysiologil reordings The pth-lmp tehnique ws used to reord whole-ell VDCC urrents in the voltgelmp mode [14, 15]. The glss over slip with primry-

3 Dietologi (213) 56: ultured pnreti et ells tthed ws inserted into the perfusion hmer, whih ws mounted on the stge of n inverted phse-ontrst mirosope (Olympus IX71, Olympus, Tokyo, Jpn). Cells were wshed with th solution in the hmer for 3 5 min efore strting n experiment. Pth pipettes were pulled from orosilite glss pillries with filments (Sutter Instrument, Novto, CA, USA) with tip resistne of 2 5 MΩ when filled with different pipette solutions. Currents were reorded with n Axopth 2-B mplifier (Axon Instruments, Moleulr Devies, LLC, Sunnyvle, CA, USA),ontrolledyDigidt12interfendpCLAMP softwre (Version 6.2, Axon Instruments). Memrne urrents were filtered t 1 khz with four-pole Bessel filter. At the eginning of eh experiment, the pth-lmp mplifier ws djusted to set I=. No lekge sutrtion ws performed for the originl reordings, nd ll ells with visile hnges in lekge urrents during the ourse of study were exluded from further nlysis. The VDCC urrents were reorded y 3 ms step pulse rnging from 8 to 6 mv with 1 mv inrements. The holding potentils (HPs) were set t 8 or 4 mv, t whih the outwrd voltge-dependent K urrents or inwrd T-type VDCC urrents were lrgely intivted, respetively. The pipette solution for reording VDCC urrents ontined: Cs 2 -sprtte 7 mmol/l, EGTA 1 mmol/l, MgATP 4 mmol/l, MgCl 2 5mmol/l,CCl 2 1mmol/l,N-pyruvte 5 mmol/l, K-suinte 5 mmol/l, gluose 25 mmol/l, HEPES 1 mmol/l, N-retine phosphte 5 mmol/l, retine kinse 5 U/ml, N 2 GTP.3 mmol/l (ph=7.2). The th solution for reording VDCC urrents inluded: Trizm-HCl 11 mmol/l, TEA-Cl 5 mmol/l, BCl 2 2 mmol/l, CsCl 5 mmol/l, HEPES 2 mmol/l, gluose 5 mmol/l (ph=7.4). Gig-ohm sel ws mde in norml extrellulr high-n solution: NCl 135 mmol/l, KCl 4.8 mmol/l, MgCl mmol/l, CCl mmol/l, HEPES 1 mmol/l. In some experiments, tetrodotoxin (TTX) t 1 μmol/l ws inluded in the th solution to lok ny N hnnel urrents. The osmollity of reording solutions ws djusted to 29 mosmol/l, nd ph to Cells were ontinuously superfused with the th solution ontining the test hemils t the desired finl onentrtions. All experiments were performed t room temperture. In eletrophysiologil reordings, et ells were distinguished from other islet ells y lrge size (usully pitne 7 pf), no detetle N urrent when holding t 7 mv, nd hrteristi osilltory or ursting eletril tivity when exposedto1mmol/lgluose[16, 17]. The urrent density of L-type VDCCs ws lulted y normlising the pek mplitude of urrent with ell pitne (pa/pf). The hnnel reovery from depolristion-indued intivtion ws lso produed y doule-pulse protool stepped to mv from 7 mv with n HP of 8 mv. An inrese in the intervl etween the onditioning nd test pulses resulted in reovery of VDCC urrent (I C ), whih n e fitted y mono-exponentil eqution: I=I mx ¼ 1 exp ð t=tþ,wherei mx is the urrent mesured during the first pulse, I is the urrent mesured during the seond pulse, t represents the vlue of the intervl, nd τ represents the time onstnt of I C reovery from intivtion. Chemils nd sttistil nlysis Nifedipine, By K-8644, PPG, nd NHS were purhsed from Sigm Chemils, glienlmide from Reserh Biohemils Interntionl (Ntik, MA, USA), nd ACS 67 from Cymn Chemil Compny (Ann Aror, Mihign, USA). Stok solutions of nifedipine, glienlmide nd ACS 67 were mde in DMSO nd diluted to the desired onentrtions immeditely prior to use. DMSO lone ws without effet t the onentrtion used (up to.3%, vol./vol.). Dt re expressed s mens±sem nd were nlysed usingpiredstudent s t test nd nlysis of vrine in onjuntion with the Newmn Keuls test where pplile. Differenes were onsidered sttistilly signifint t the level of p<.5. Results Eletrophysiologil fetures nd phrmologil sensitivity of VDCC urrents To seprte the whole-ell I C omponents pek I C (I C,pek ) with fst intivtion nd stedy-stte I C urrent (I C,SS ) without intivtion different HPs were employed. At the HP of 8 mv, I C urrent tres were reorded y 3 ms pulse stepped from 8 to 6 mv with 1 mv inrements, exhiiting oth I C,pek nd I C,SS omponents (Fig. 1). Chnging the HP from 8 to 4 mv would intivte T-type VDCCs [18, 19]. In our study, this HP hnge redued the I C,pek density from 52.2±5.5 to 36.7± 3.7 pa/pf (t 1 mv, n=8, p<.5) (Fig. 1,). With the HP t 4 mv, the whole-ell inwrd I C showed only one I C,SS omponent (Fig. 1 ). This I C,SS hd the sme urrent density with 8 or 4 mv s the HPs (n=8, p>.5) (Fig. 1). To onfirm whether I C,SS represents mostly the tivity of L-type VDCCs, speifi gonist nd n ntgonist for L-type VDCC were pplied. By K-8644, n gonist of L-type VDCC, enhned the I C,SS y 53.3±4.2% nd 53.8±6.4% (t 2 mv, n=6, p<.5) t HPs of 8 nd 4 mv, respetively (Fig. 1d). The stimultory effet of By K-8644 on I C,SS ws loked y nifedipine y 81.1±7.2% nd 72.3±6.5% t 8 nd 4 mv HPs, respetively (Fig. 1d). However, oth By K-8644ndnifedipinefiledtoffettheI C,pek (Fig. 1d). Thus, I C,SS ws referred to s L-type VDCCs in this study. Inhiition of L-type VDCCs y exogenous H 2 S in pnreti islet et ells With the HP t 8 mv, NHS signifintly inhiited the I C,SS y 31.3±2.4% (n=12,p<.5), ut not the I C,pek (t 1 mv, n=12, p>.5) (Figs 2, nd 3). The inhiitory effet of NHS on L-type VDCCs ws reversile

4 536 Dietologi (213) 56: mv 1 ms -8 mv By K-8644 By K-8644 HP=-8 mv HP=-4 mv ife 644 di pi ne l K8 N tro y on B C C NHS (µmol/l) on Nifedipine HP= -4 mv -2-4 NHS Fig. 1 VDCC urrents in mouse pnreti et ells. () Originl VDCC tres reorded from 8 mv to 6 mv with HPs of 8 mv () nd 4 mv (), respetively, nd the differentil urrents ( ) generted fter sutrting the urrent reorded in () from those in (). () The I V reltionship of IC,pek reorded with different HPs ( 8 nd 4 mv) is summrised (n=8, p<.5). The IC,pek is mesured t the mximum mplitude of IC t eh testing potentil. () The I V reltionship of IC,SS reorded with different HPs of 4 mv (lk squres) nd 8 mv (white squres) is summrised (n=8). The IC,SS is mesured t the end of eh testing pulse. (d) Originl VDCC tres reorded t the testing potentil of 1 mv with different HPs in the sene nd presene of.2 μmol/l By K-8644 nd 1 μmol/l nifedipine. The dotted line indites zero urrent. The summry shows IC,SS efore nd fter the pplition of By K-8644 nd nifedipine (test potentil, 1 mv, n=6, p<.5) fter wshing out NHS (Fig. 2). NHS did not lter the reversl potentil nd the voltge-dependene of L-type VDCCs (Fig. 2). The IC5 of the inhiitory effet of NHS on L-type VDCCs ws 65.4±5.6 μmol/l (Fig. 2). The IC,SS ws inhiited y NHS y 43.5±4.4% nd further inhiited y nifedipine y 32.3±3.5% (n=5, p<.1) (Fig. 2). After hnging the HP to 4 mv from 8 mv, only the IC,SS existed nd NHS signifintly inhiited IC,SS y 45.6±4.4% (Fig. 3,), onfirming tht NHS trgeted only L-type VDCCs. In generl, NHS did not shift the urrent voltge (I V) urve of L-type VDCCs with the HP t 8 mv (Figs 2 nd 3). It ppers tht with HP t 4 mv NHS used mrginl, ut inpprent, shift of the I V urve of L-type VDCCs to the left (Fig. 3). To evlute the reovery from intivtion, urrents were eliited y the doule-pulse protool, with the intervl seprting two pulses inresed progressively from to 2 ms NHS nifedipine 1 pa/pf 1 ms IC,SS (pa/pf) Nifedipine on tr B ol y K N -86 ife 4 di 4 pi ne HP= -8 mv 1 pa/pf d 1 ms Nifedipine ne l -4 4 pi -3 di tro -2 4 Wshout 1 4 ife 3 NHS (µmol/l) IC,SS (pa/pf) -4 1 Inhiition of IC,SS (%) -8 IC,SS (pa/pf) IC,pek (pa/pf) on -4 N Differene -8 C HP=-4 mv tro l H S N ni H fe S di pi ne HP=-8 mv 1 ms N -8 mv IC,SS (pa/pf) -4 mv C 6 mv -8 mv -8 mv 1 pa/pf 6 mv 1 pa/pf -2-4 Fig. 2 The inhiitory effets of NHS on VDCC urrents in mouse pnreti et ells. () Originl VDCC tres sequentilly reorded in the sme ell from 8 mv to 6 mv with n HP of 8 mv in the sene (white squres) nd presene of different onentrtions of NHS (t 1 [lk irles], 3 [lk squres], nd 1 [lk dimonds] μmol/l) nd hemil wshout (white irles). The orresponding I V reltionship of IC,SS is shown in the right pnel. () The onentrtion-dependent inhiition of IC,SS y NHS (t 1 mv, HP= 8 mv, n=12) with IC5 of 65.4±5.6 μmol/l. () Originl VDCC tres in the left pnel showing tht IC,SS ws inhiited y NHS (1 μmol/l), NHS nd nifedipine (1 μmol/l) s well s y nifedipine lone (TP= 8 to 6 mv, HP= 8 mv). The summry of these dt is shown in the right pnel, with urrent intensity mesured t 1 mv (HP= 8 mv, n=5, p<.1) (Fig. 3d). When the intervl ws short, the IC ws smll nd deyed more slowly. An inrese in the intervl etween the onditioning pulse nd test pulse resulted in reovery of IC. The time onstnts (τ) of IC reovery from intivtion in the HP of 7 mv were 89.4±6.3 nd 115.4±8.5 ms in the ontrol nd NHS group, respetively (Fig. 3d). As the time intervl (t) inresed to 8 2 ms, the I/Imx vlues in the NHS-treted group were signifintly deresed in omprison with those of the ontrol group (n = 6, p <.5) (Fig. 3d). It ppers tht L-type VDCCs in INS-1E ells tke more thn 7 ms for reovery from intivtion. The mehnism underlying this delyed reovery is not ler yet. ACS 67 is n nlogue of ltnoprost (n F-series prostglndin nlogue) tht ontins n H2S-relesing omponent onjugted to the ltnoprost roxyl group, nd is therefore lipid-solule, slow-relesing H2S donor [2].

5 Dietologi (213) 56: IC (pa/pf) 1 µmol/l NHS Differene d IC,pek -2 Without PPG dilysis With 2 mmol/l PPG dilysis -2 4 Without PPG dilysis 1. IC,SS I / Imx IC (pa/pf) -4 HP=-4 mv -2 1 ms -4 d -8 mv -8 mv IC,SS ms 1 µmol/l NHS d IC,pek NHS IC,SS (pa/pf) -4 mv -8 mv HP=-8 mv -4 6 mv -8 With 2 mmol/l PPG dilysis Reovery time (ms) Fig. 3 The inhiition of VDCC urrents y NHS t different HPs in mouse pnreti et ells. () Originl VDCC tres reorded from 8 to 6 mv with n HP of 8 mv efore () nd fter () the pplition of NHS (t 1 μmol/l). The orresponding I V reltionship of IC, pek efore (white squres) nd fter (lk squres) the pplition of 1 μmol/l NHS, nd tht of IC,SS efore (white irles) nd fter (lk irles) NHS pplition. () Originl VDCC tres reorded with n HP of 4 mv efore () nd fter (d) NHS pplition. The orresponding I V reltionships of IC, pek nd IC,SS re shown in the right pnel. () Summry of the inhiitory effet of NHS on the pek nd stedy-stte IC urrents t 1 mv t different HPs ( 8 nd 4 mv) (n=1, p<.1). (d) The effet of NHS on the reovery kinetis from VDCC intivtion. A doule-pulse protool is shown with the testing potentils stepped from 7 to mv t n HP of 8 mv. The intervl etween two pulses rnges from ms to 2 ms t 1 ms inrements. A downwrd shift of the reovery urve ws oserved in the presene of NHS (lk squres) in omprison with the urve in the sene of NHS (white squres) (n=6, p<.5) ACS 67 (1 μmol/l) lso inhiits L-type C2 hnnels. It deresed L-type VDCC urrents y 18.3±2.8% t memrne potentil of 1 mv (n=5, p<.5). However, the inhiitory effet of ACS 67 ws weker thn tht of NHS t the sme onentrtion (45.4±4.6% inhiition). Effets of endogenous H2S on L-type VDCCs in pnreti islet et ells Without PPG dilysis, NHS t 1 μmol/l signifintly inhiited IC,SS y 27.5±3.2% (t 2 mv, n=1, p <.5) in WT et ells (Fig. 4,). By inluding 2 mmol/l PPG in the pipette solution, NHS lso inhiited IC,SS y 43.8±5.6% (t 2 mv, n=1) (Fig. 4,). On the other hnd, NHS inhiited IC,SS in Cse-KO mouse et ells with nd without PPG dilysis y 25.2±3.% nd WT 2 mmol/l PPG 1 µmol/l NHS IC,SS (pa/pf) Differene IC (pa/pf) 1 ms 6 mv IC,SS (pa/pf) 1 pa/pf -8 mv 1 pa/pf 6 mv 1 pa/pf Cse-KO Fig. 4 The effet of NHS on VDCC urrents in the sene nd presene of PPG dilysis of pnreti et ells from WT nd Cse-KO mie. () The representtive VDCC tres for inwrd IC,SS urrents reorded in WT mouse pnreti et ells with n HP t 8 mv efore nd fter the pplition of NHS in the sene nd presene of PPG dilysis. The orresponding I V reltionship urves of IC,SS re shown in the right pnel, either without PPG in the sene (white squres) nd presene (lk squres) of 1 μmol/l NHS or with 2 mmol/l PPG dilysis in the sene (white irles) nd presene (lk irles) of NHS. () The representtive IC,SS urrent tres in Cse-KO mouse pnreti et ells with the sme reording onditions s in (). The orresponding I V reltionship urves of IC,SS re shown in the right pnel. () Summry of the effets of NHS on IC,SS, mesured t 1 mv without or with 2 mmol/l PPG dilysis of WT nd Cse-KO pnreti et ells (HP= 8 mv, n = 1, p <.5 for 1 μmol/l NHS vs ontrol, p<.5 for 2 vs mmol/l PPG dilysis) 32.2±4.3%, respetively (t 2 mv, n=1, p<.5), ut PPG dilysis did not lter sl IC,SS level in Cse-KO mouse et ells (Fig. 4,). To test the effet of endogenous H2S on IC,SS, Cse-KO mie were used to isolte pnreti et ells. NHS signifintly suppressed IC,SS y 29.7±3.5% nd 55.6±6.4% in WT nd Cse-KO mouse et ells, respetively (n=1, p<.1) (Fig. 5 ). The sl urrent density of IC,SS in et ells from Cse-KO mie ws greter thn tht from WT mie (p<.5) (Fig. 5). Effets of the CSE/H2S system on insulin relese from pnreti islets Following inution with ulture medium nd norml

6 538 Dietologi (213) 56: mv -8 mv -8 mv 1 µmol/l NHS Cse-KO WT 1 pa/pf 1 ms 1 µmol/l NHS I C (pa/pf) I C (pa/pf) WT 1 µmol/l NHS I C (pa/pf) gluose (t 1 mmol/l) for 3 min, insulin relese from freshly isolted islets from Cse-KO mie ws muh higher thn tht from WT islets (n=5, p<.5) (Fig. 6). Insulin seretion from islets ws inhiited y nifedipine y 45.7± 3.6% nd 41.2±4.5% in WT nd Cse-KO islets, respetively. NHS t 1 μmol/l nd 3 μmol/l inhiited gluosestimulted insulin seretion y 26.7±4.5% nd 49.5±6.5% (n=5, p<.5 vs ontrol), respetively (Fig. 6). NHS or nifedipine lone inhiited insulin relese, nd their opplition hd n inresed effet (n =5, p >.5) (Fig. 6). By K-8644 lone enhned insulin seretion in mouse islets, ut o-pplition of NHS or nifedipine ounterted the stimultory effet of By K-8644 (Fig. 6). These results suggest tht nifedipine nd H 2 S my inhiit the sme L-type VDCCs. Glienlmide lone inresed insulin seretion, whih ws lso inhiited y the ddition of NHS (n=5, p<.5) (Fig. 6). This result indites tht K ATP hnnel tivtion is involved in the Cse-KO Fig. 5 The effet of NHS on VDCC urrents in pnreti et ells from Cse-KO mie. () The representtive inwrd I C,SS urrent tres in WT mouse pnreti et ells (HP= 8 mv) efore nd fter the pplition of NHS. The orresponding I V reltionship urves efore (white squres) nd fter (lk squres) the pplition of 1 μmol/l NHS re shown in the right pnel. () The representtive I C,SS urrent tres in Cse-KO mouse pnreti et ells with the sme reording ondition s in (). The orresponding I V reltionship urves re shown in the right pnel. () Summry of the effets of NHS on I C,SS urrents, mesured t 1 mv, in pnreti et ells from WT nd Cse-KO mie (HP= 8 mv, n=1; p<.1 for 1 μmol/l NHS vs ontrol; p<.5 for Cse-KO vs WT) regultion of insulin relese. Figure 6d further shows the inhiitory effet of endogenous H 2 S on insulin relese from islets of WT or Cse-KO mie, ssyed in KRB. Disussion We nd others hve previously shown tht H 2 S funtions s n endogenous opener of K ATP hnnels in different types of ell, inluding smooth musle ells [2, 21], HEK- 293 ells [22] nd pnreti et ells [5, 11], independent of the tivtion of ytosoli seond messengers. Intertion etween H 2 S nd K ATP hnnels in insulinsereting ells my underlie n importnt mehnism for the regultion of insulin seretion from pnreti et ells. Our present study exmined the effets of exogenous nd endogenous H 2 S on L-type VDCCs in mouse pnreti et ells. We hve found tht: (1) the H 2 S donors NHS nd ACS 67 reversily inhiited L-type VDCCs; (2) PPG dilysis to inhiit CSE tivity nd lower endogenous H 2 S level inresed the sl mplitude of L-type VDCCs in WT mouse et ells; nd (3) the density of L-type VDCCs in et ells from Cse-KO mie, in whih the endogenous H 2 S level is minimised, is greter thn tht from WT mie. These dt represent the first evidene tht H 2 S hs n inhiitory role in the regultion of L-type VDCCs in mouse pnreti et ells. Gluose stimultion of pnreti et ells involves memrne depolristion nd C 2 influx. An upsurge in [C 2 ] i triggers the exoytoti mhinery, with susequent insulin relese. C 2 influx in et ells ours primrily through VDCCs, espeilly L-type VDCCs [23 25]. L-type VDCCs, lrge hetero-oligomeri omplex onsisting of α 1 -, α 2 /δ-, β- ndγ-suunits, is hrterised y highvoltge-tivted persistent C 2 urrents tht re without intivtion or intivte slowly. L-type VDCCs re loked y nifedipine or verpmil nd tivted y By K-8644 or CGP28391 [26]. N- nd R-type VDCCs hve een reported in mouse et ells [27]. These VDCCs elong to the sme group of high-voltge-tivted VDCCs s L-type hnnels. However, N nd R hnnels n e loked y ω-onotoxin nd SNX482, respetively, ut L-hnnels re sensitive to dihydropyridines. In our study, the lokde of L-type VDCCs y nifedipine nd its stimultion y By K-8644 indite tht the L-type VDCCs urrents reorded re unlikely to hve een ontminted y N- or R-type VDCCs. On the other hnd, the effets of H 2 S on N- nd R-type VDCCs in et ells nnot yet e exluded. T-type VDCCs hve een suggested to involve modultion of generl memrne eletril tivity to enhne insulin seretion [28]. They re tivted t more negtive memrne potentils nd re intivted quikly nd ompletely [29]. T-type VDCCs hve een reorded in mouse

7 Dietologi (213) 56: Islet insulin relese (ng/1 islets) 2 1 WT KO WT KO Nifedipine Islet insulinrelese (ng/1 islets) 1 Gluose (mmol/l) NHS (µmol/l) d Islet insulin relese (ng/1 islets) Islet insulin relese (ng/1 islets) 2 1 NHS Nifedipine By K-8644 Glienlmide Gluose (mmol/l) WT Cse-KO Fig. 6 Insulin seretion nd H 2 S prodution of pnreti islets from WT nd Cse-KO mie. () Gluose (1 mmol/l)-stimulted insulin seretion from WT nd Cse-KO mouse islets nd its inhiition y nifedipine (1 μmol/l) (n=5, p<.5 for KO vs WT; p<.5 nifedipine vs ontrol). () The effets of NHS on insulin seretion from WT mouse islets (n=5, p<.5 for 2 mmol/l vs 2.8 mmol/l gluose; p<.5 for 1/3 vs μmol/l NHS). () Effets of NHS (1 μmol/l), nifedipine (1 μmol/l), By K-8644 (.2 μmol/l), nd glienlmide (3 μmol/l) on 2 mmol/l gluose-stimulted insulin seretion from WT mouse islets (n=5, p<.5 for different tretments with NHS, nifedipine, By K-8644, nd glienlmide vs ontrol without tretment; p<.5 for NHSBy K-8644 vs By K-8644, nd NHSglienlmide vs glienlmide). Mouse islets were inuted with RPMI-164 medium in experiments shown in ( ). (d) Insulin seretion from mouse islets inuted in KRB; n=4; p<.5 for 16.8 mmol/l or 6.4 mmol/l vs 2.8 mmol/l; p<.5 for KO vs WT t 6.4 or 16.8 mmol/l gluose et ells y others [18, 3, 31] nd re likely to hve een reorded in our present study. As we foused on the effets of H 2 S on L-type VDCCs, no further effort ws tken to hrterise T-type VDCCs in our study. Both CSE nd ystthionine β-synthse (CBS) re produed in rodent pnreti tissues or loned islet et ell lines, while CSE plys more importnt role in endogenous H 2 S prodution in et ells. The expression level of Cse mrna ws signifintly higher thn tht of CBS in pnreti islets nd PPG olished most of the H 2 S prodution in pnreti islets nd INS-1E ells [5, 6]. Furthermore, H 2 S prodution in pnreti et ells is deresed y inresed lood gluose level [7]. Overexpression of Cse inhiited insulin relese from INS-1E ells, nd lowering endogenous H 2 SprodutionyPPG or Cse-trgeted smll interfering RNA hd the opposite effet [5]. We previously showed tht PPG meliorted high lood gluose of ZDF rts to ner norml levels, while mino-oxyette, speifi loker of CBS, did not signifintly hnge the hyperglyemi sttus of ZDF rts [6]. Furthermore, H 2 S prodution rte ws signifintly deresed in pnreti islets from Cse-KO mie nd PPG tretment drstilly redued H 2 S prodution rte in pnreti islets from WT mie [4]. In ddition, L-ysteine nd H 2 S inhiit gluose-stimulted insulin seretion from insulin-sereting et ell lines (INS- 1E, MIN6 nd HIT-T15) nd isolted rt islets [5, 11]. All these dt suggest ritil role of CSE in produing H 2 Sin et ells, thus regulting insulin seretion under physiologil or pthologil onditions. L-type VDCC ply ruil role in the regultion of insulin seretion in pnreti et ells. The dey of I C,SS during depolristion is lled intivtion, nd is regulted y elevtion of [C 2 ] i s well s y memrne depolristion [32, 33]. Therefore, [C 2 ] i in the viinity of the hnnels ritilly ontrols their tivity during stimulus seretion oupling. The C 2 -dependent proess of intivtion my serve s negtive feedk mehnism for regulting C 2 entry into pnreti et ells nd insulin grnule exoytosis [32]. The reovery from intivtion of I C,SS is ffeted y different ftors, suh s the HP, the voltge nd durtion of the onditioning pulse, the frequeny of the oupled pulses nd the extrellulr ioni milieu. H 2 S inhiits I C,SS nd delys the hnnels reovery from intivtion in et ells, produing negtive feedk regultion on [C 2 ] i.

8 54 Dietologi (213) 56: Gluose-stimulted insulin seretion ws exmined in our study with different mnipultions of H 2 Slevelnd funtion sttus of L-type VDCCs. NHS t different onentrtions hd no sttistilly signifint effet on sl insulin seretion with 2.8 mmol/l gluose (Fig. 6). On the other hnd, signifint portion of insulin relese from pnreti islets of Cse-KO mie remined in the presene of nifedipine (Fig. 6). This result is not in onflit with the notion tht H 2 S inhiits L-type VDCCs to derese insulin relese. H 2 S hs multiple effets on insulin metolism in the pnres. We hve previously demonstrted tht H 2 S indues pnreti et ell poptosis nd dereses et ell mss, thus reduing insulin prodution [4]. The diminished endogenous H 2 SinCse- KO islets would inrese pnreti et ell mss nd more insulin would e produed, so tht with the sme onentrtion of nifedipine tretment more insulin would e ville for relese. Glienlmide lone inresed gluose-stimulted insulin relese, whih ws prtilly suppressed y NHS. This suggests tht H 2 S-inhiited insulin relese relies on K ATP -hnnel-dependent nd -independent mehnisms. The former is medited y the indiret intivtion of L-type VDCCs y H 2 S vi tivtion of K ATP hnnels nd memrne hyperpolristion, wheres the ltter involves diret inhiition of L-type VDCCs y H 2 S. No mtter whether K ATP hnnels or L-type VDCCs or oth re ffeted y H 2 S, the inhiition of L-type VDCCs, leding to lowered [C 2 ] i,is the finl oupling node etween the ellulr eletrophysiologil hnges nd the redued insulin relese induedyh 2 S. In summry, we report n originl oservtion tht H 2 S, pplied exogenously or generted endogenously, inhiits L-type VDCCs nd regultes insulin seretion. This inhiitory effet of H 2 SonL-typeVDCCinpnreti et ells is independent of the effet of H 2 Son K ATP hnnels. As suh, our report dds to urrent understnding of the regultion of pnreti funtion y H 2 S through its effets on different ion hnnels in et ells. This will pve the wy for trgeting different ion hnnels when devising seletive therpeuti strtegies to orret norml H 2 S metolism in the pnres under different pthologil onditions. Funding This work ws supported y n operting grnt from the Cndin Dietes Assoition (to R. Wng) nd post-dotorl fellowship from the Hert nd Stroke Foundtion of Cnd (to G. Tng). Dulity of interest The uthors delre tht there is no dulity of interest ssoited with this mnusript. Contriution sttement All uthors hve: mde sustntil ontriutions to oneption nd design, quisition of dt, nlysis nd interprettion of dt; drfted the rtile or revised it ritilly for importnt intelletul ontent; nd given finl pprovl of the version to e pulished. Referenes 1. Zho W, Ndisng JF, Wng R (23) Modultion of endogenous prodution of H 2 S in rt tissues. Cn J Physiol Phrmol 81: Zho W, Zhng J, Lu Y, Wng R (21) The vsorelxnt effet of H 2 S s novel endogenous gseous K ATP hnnel opener. EMBO J 2: Yng G, Wu L, Jing B et l (28) H 2 S s physiologi vsorelxnt: hypertension in mie with deletion of ystthionine gmm-lyse. Siene 322: Yng G, Tng G, Zhng L, Wu L, Wng R (211) The pthogeni role of ystthionine gmm-lyse/hydrogen sulfide in streptozotoin-indued dietes in mie. Am J Pthol 79: Yng W, Yng G, Ji X, Wu L, Wng R (25) Ativtion of K ATP hnnels y H 2 S in rt insulin-sereting ells nd the underlying mehnisms. J Physiol 569: Wu L, Yng W, Ji X, Yng G, Duridnov D, Co K, Wng R (29) Pnreti islet overprodution of H 2 S nd suppressed insulin relese in Zuker dieti rts. L Invest 89: Zhng L, Yng G, Tng G, Wu L, Wng R (211) Rt pnreti level of ystthionine γ-lyse is regulted y gluose level vi speifiity protein 1 (SP1) phosphoryltion. Dietologi 54: Yusuf M, Kwong Hut BT, Hsu A, Whitemn M, Bhti M, Moore PK (25) Streptozotoin-indued dietes in the rt is ssoited with enhned tissue hydrogen sulfide iosynthesis. 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