Volume 291. number 2. 310-314 0 1991 Federatton of European Ilochemlcdl ADCJNlS 001457939100982L

Measurements

Hendrlk

Bar

Kmdmark’,

October 199 1

FEBS 10273 Socletles 00145793/91/$3 50

sf cytoplasrnic free Ca2+ concentration pancreatic islets an insulinoma cells

in hun-lan

Martin Kohler’, Thomas N~lsson’, Per Arkhammar’, Karl-Ludvlg Patrlk Rorsrnan3, Suad EfendiC’ and Per-Olaf Berggren’

Wlechel’,

’ Tire Rolj L.ufi Center for Dlubetes Research, Depnrment of Endocrmology~ Karolrnska hutltute, Ka, olmka Hospltai, 60 500. S-104 01 Stockhoh, Sweden, ‘Department of Surge, y, Soderyukhuset, Bos 38 100. S-100 64 Stockfrolm, Sweden and ‘Depar ment of Medual Pfysm, Gotflcnburg Unrverslty. Bos 330 31. S-400 33 Gothenhurg, Sweden Received

22 July

1991

In human pdncredtic islets an increase m the glucose concentrntlon from 3 lo 20 mM rdised the free cytoplasnnc Ca“ concentration ([Ca”],), an effect being reversible upon wlthdrdwdl of the sugar Dcpolarlzdtion with a high conccnlration of K’ or the sulphonylurcd tolbutamlde also raised [@*I, Addltlon of extracellular ATP produced a transient rapld rise m [Ca”], Oscdlatlons m [Ca”‘], were observed m the presence of IO mM glucose Insulmoma cells responded to glucose and tolbutamlde with mcrcases m [Cd”], whereas the sulphonamlde dlazoxtde caused a decrease m [Ca”], These findings confirm previous results obtained m rodent p-cells Human pancredtic islet, Human msulinoma.

Stnnulatlon of pancreatic /I-cells with glucose promotes generation of intracellular ATP [l] This leads to closure of ATP-regulated K’ channels m the plasma membrane [2,3], with subsequent depolarlsatlon and mflux of Ca2+ through voltage-activated Ca2’ channels [4,5] The resultmg Increase m the cytoplasmrc free Ca’+ concentration ([Ca”],) mitiatcs secletlon Since the access to viable human Islet tissue IS limited, nearly all mformatlon concerning the /I-cell stimulus-secretion couplmg has, so far, been derlveu from expermlents made on rodent islet pxeparatlons There are only a handful of blochemlcal and blophyslcal studies on human islets describing msulm release, glucose oxid,+ tlon, protem phosphorylatlon and characterization of ATP-regulated K’ channels [6-l I], but none dealing with mtracellular Ca’+ metabohsm In the present study we demonstrate, for the first time, direct measurements of [Ca”], in humdn lslcts and msulmoma cells sttmulated with glucose, hypoglycaemlc sulphonylurea and other secretagogues 2. EXPERIMENTAL i-or isoldtion of islets dnd mcdsurcmcnt5 of [Cd”], WCused d buffer contdmmg (11~mM) N&l 140. KCI 5 9. CXI, I 28, MgCI: I 2, HEPES 25 ,md I mdml ~OVIIIC \crum dlbumm WII~ d pH of 7 4 [I 2) fltlflrc’s\

P -0

Bcrggrcn,

The Roll Lufi Ccnlcr for

B&ctec Rcsc,uch, Dcp,irtmcnt of Cndoct ~nology, Kdro1msk.l Instllute, K;IIO~IIIS~J Hospml, F-&I\ (46) (8) 30 3458

310

free Ca”

concentrdtlon.

Furd-2

In the experiments shown m Figs 2 dnd 3. the medium Ca” concentration wds raised lo 2 56 mM

1 INTRODUCTION

Cor mpumhrc*c

Cytoplasmlc

DOT GO500, S-104 01 Stockholm,

Swcdcn

2 2 Preparutron oJ rslers Pdncreatlc tissue wds obtained from patients admitted to hospital because of symptomless Jaundice, weight loss dnd fatigue The dlagnosls was m each case settled by ultrasound mvestlgatlon. percutaneous trdnshepdtlc cholangiogrdphy dnd fine needle aspiration biopsy and later verified by hlstologlc exdmmdtlon of the resected specimen The patients wcrc preoperatwely treated by percutaneously transhepdtically pldc cd endoprosthesls m order to decompress the bile duct ,md rcstttute bile flow lo the gut dnd the cntciohepdtlc clrculatlon Surgery wlls perlormed 4-5 weeks ,ifter placement of the endoprosthesis Betore surgery, the bcrum dspdrtdte ammotransferdse, dldnine dmmotransterdPe. blhrubm and alkdlme phosphdtase were m ,ill pdtlents within the normdl leferencc hmlts Crentmme wdb normdl m dll patlents The parlents hdd noimdl fdstmg glucose lcvcls (less than 6 6 mM) and dbsence of glucose m the urme Oral glucose tolcrdnce test (OGTT) was perfonned where blood glucose wds measured m the fdsted state and dl 2 h dfter mtdkc of 75 g glucose Pdtlent I wds d 73.ycdr-old rndn with Cdnler 01 the pdpilld of Vdter. pdtient 2 wds d 66.year-old womnn with ddenocdrcmomd of the pnncreds dnd patlent 3 wds d 67-yenr-old man with ddenOCdrClnOmd of the drsldl part of the common b&e duct All patients hdd resechon for cure, comprlsmg cephdlic pancredtlc resection cn-bloc with duodenum. dccordmg to Whipple A specimen free from cdnCcr WdS obtalcnd trom the resected part of the pdncrcns dnd put m Lold RPM1 1640 medmm supplcmcntcd with 10% fct.31 bovmc serum (Flow), 100 IUlml pcmclllm dnd 100 @nl strcptomycm ‘md stored on ILC I”lbrotlc pnrls of the pdncrcas and .idLposc tissue were removed dnd the tls\uc wds mmced mto pieces of about 2-4 mm Islcrs wcrc IsoLttcd by colldgcndsc (Bochrmgcr. 3 mdml) and plckcd with d br,lkmg plpcttc under mspcctlon m d drsscchon tntLloscopc TIIC IFICISwcie put m Pctrl d~shcs or In rnlcrowclls 111RPM1 1640 medium supplcmcntcd with 10% fet,d bovine set uni dncl dntlblotlcs ds dcscrlbcd dbovc and LulIuIcd .it 37°C m humlficcl ‘ur with 5% CO: fol l-4 d,lvs,

Volume

291,

FEBS LETTERS

number 2

patient 4, a 44-year-old otherwlse healthy woman whc 4 years earher was admitted to hospital because ot hypoglycenuc attacks due to hypermsulmemla Subsequent operation revealed two pancredtlc tumors stammg ImmunohlstochemIcally posltlve for msulm, promsul.n, C -pcptlde and chromogranm A After 3 5 years hypoglycemic attacks reappeared, bcmg more frequent with exercise and reheved after food Intake Laboratory testsshowed hypoglycemia and hypermsuhnerma Surgery was performed 6 months later from which the tissue used m this study was derlvcd The tumor cells sldmed posltlve for C-peptlde and neurospeclfic enolase Chmcally the tumor was considered a relapse of the msulmoma The msuhnoma tissue was mmced Into mdhmeter-sized pieces and suspended with a Pasteur pipette m 3 ml of Hank’s buffer coatamng 0 I % trypsm and 100 m&ml EDTA until a cell suspension was obtamed After washmg twice with culture medmm, the cells were kept m a culture flask under tissue condmons for 3 h under gentle agltatlon to prevent attachment of the cells to the flasks 2 4 Loadrng w~fltjiircr-2 The Islets were loaded m 2 ml of the HEPES buffer with 2 ,uM fura-Bacctoxymethyl ester (AM) (SlgmtiBoehrmger) for GO mm at 37’C under gentle agltatlon In the experiments shown III Fogs 2 and 3, the Islets were laoded m the presence of 2 56 mM Ca2’ The loadmg procedure resulted m a diffuse even fluorescence throughout the Islet except for a bnghter rim along theextreme periphery The msuhnoma cells were loaded m the culture medmm with 1PM fura-Z/AM for 45 mm at 37°C under gentle agltatton

2 5 1 lslers Smgle islets were transferred to a small open penfusIon chamber (volume 150 .~l) with a covershp bottom The medmm flow rate was 300 PI/mm and controlled by a penstaltlc pump The dead space of the penfusIon system corresponded to a lag pccnod of approximately 20 s, which has been corrected for m all figures Fura- fluorescence was measured at 37”C w11h a SPEX Fluorolog-2 system coupled to an Inverted eplfluorescencc mIcroscope (Zeiss Axlovert 35M) equipped with a 40x Plan-Neofluar objective The measurmg

0

5

1991

diaphragm was closed enough to exclude the brighter penpheral Tone of the Islet, thus also mmlmllmg the contrlbutlon of non-/&cell fluorescence Fura-f was alternatmgly excited at 340 and 380 nm by d beam chopper and emitted hght was collected at 515 nm by a photoncountmg photometer A 3401380 nm ratlo WASobtamed at I Hz and data were stored m the system computer Data from all measurements ale expressed ds relative fluorescence of the 340/380 ratms The trdces shown are selected from measurements on islets from three pntlcnts as mchcated m the text Only expenments performed on islets respondmg to glucose with a ruse m [Ca2+],were consldered successful (success rate 250%) Fig I represents 4 Islets sensllive to glucose whereas Fogs 2 and 3 were the only successful experiments performed on the two patients Measurements could be conducted for more than 100 mm, lmplymg that the experImental condltlons were gentle to the Islets 2 5 2 Insulmo~u cells After washmg twice m the HEPES buffer, the cells were resuspended In I 3 ml of the buffer ma 1cm polystyrene cuvette The measurement was made at 37OC m an Ammco-Bowman spsctrofluoromcter, slightly modified to allow constant stlrrmg The excltatlon and emlsslon wavelengths were set at 340 and 500 nm, respectively Test substances were added from concentrated stock solutions ot the cuvette Changes m [Ca2’], are expressed as reldtlve fluorescence Only one experiment could be performed because of the hmlted amount of cell\

3 RESULTS

2 5 Mesuretnents of /Ca2+ J,

04-t-

October

1

,

I

I

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15

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25

Ttme (minutes) Ftg I Effects of glucose and high K’ on [Ca”*], m a smgle human lslct Ch~ngcs In mcdlum conccntmtlon of glucorc and K’ JR mdlcdlcd by the black bm

Fig. 1 demonstrates a [Ca2+], recording of a furaloaded human islet obtained from patient 1. Ra~smg the glucose concentration from 3 to 11 mM produced a blphaslc response, with an mltlal decrease followed by an Increase m [Ca”‘], which reached a plateau after a few minutes. The increase ln [Ca”], IS due to depolanzatloninduced opening of the voltage-activated Cat+ channels promoted by metabolism of the sugar [l-5] and was rapidly leversed upon decreasing the glucose concentration to 3 mM Addltlon of 25 mM K’, to directly open the voltage-activated Ca*+ channels, promoted Ca2+ mflux with a subsequent fast rise m [Ca”], Irnmedlately after hdving reached the peak, [Ca”], started to decrease, possibly due to Ca2’-dependent mactlvation of the voltage-activated Ca*+ channels [I 31 In Fig. 2A, the islet ollgmated from patient 2 and the response to glucose was similar to that shown m Fig 1, with an imtlal decrease m [Ca”+], followed by an increase. It can be observed that the raised level of[Ca2+], was not constant but fluctuated. Subsequent stlmulatlon wrth 25 mM K’, m the presence of high glucose, led to a rapid increase m [Ca”], comparable to that observed m the previous experiment After returning to the basal glucose concentration, the islet responded to the sulphonylurea tolbutamlde with a marked rise in [Ca2+], A measurement from the same islet, performed after a IO min perrod of stlmulatlon with 1 I mM glucose, 1s shown in Fig 2l3 After penfusmg in low glucose for another 3 mm we stimulated with 200 AM ATP to activate P2-purrgenc receptors [14,15]. Thcrc was a rapid mcreasc m [Ca”], in response to the nuclcqtrde, followed by a gradual dccreasc towards the baselmc. 311

Volume

291. number 2

FEBS LETTERS

October

1991

100 pM iolbulom~de 1OmM glucose

0 Y

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10 Time

(minutes)

ZOOUM

ATP

30

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(minutes)

0 56 0

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056

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Fig 2 Effects of glucose. high K’, tolbutamlde and ATP on [Cd2’], in d smglc human ~slet (A) Addlt1ons of glucose, K’ and tolbutamlde are Indicated by the bldck bars (B) The trace shows the same islet ds m (A) dflcr havmg been penfused with 1I mM glucose for IO m1n dnd then exposed to 3 mM glucose ds mdlcdtcd III the figure Add1tlon of ATP IS indicated by the bldck bar Note the different time dnd fluoresccnvz scales compdrcd lo (A)

The trace deplcted m Fig. 3A 1sa recordmg made on an Islet obtamed from patlent 3 In this experiment the glucose concentration was increased from 0 mM to IO mM. This lslct did not respond with an mltlal decrease in [CZL”]~ but rathel with c~rise Followed by oscillations in [Ca”+],,In Fig 3B &Ipart of the same tract 1s shown at an expanded tlmc scale. The oscillations were somcwhat irregular in shape and had a period of about 70-80 S In

312

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4 we demonstrate an cxperlmcnt performed

0 52 10

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Fig 3 Oscllidtlons in [Ca’*], in d smgle human islet 1n iesponse to glucose stlmuldt1on (A) Addmon of IO mM glucose IS 1ndlcdted by the black bar (B) Expanded pdrt of the trace shown m (A) Note :hc chdngc of tnnc ,md fluorescence scales

on human msuhnoma cells derlvcd From patlent 4 In contrast to the previous experiments, the measurement of [Ca”], was performed m a cell suspension m a cuvette, which prevented test substances to be removed The cells were incubated m the absence of glucose and stimulation with 20 mM of the sugar produced a slow increase m [CL”], after a 2 min lag pcrlod. Addmon of dldzoxldc, a sulphonamlde inhlbltmg msulin rclcase, Icd to a decleasc In [Ca’+],.Subsequent additions of tolbutamldc countclactcd the effect of diazoxldc and consequcntly ralscd [Ca*+],,

Volume

291, number

PEBS

2

-TIME (MINUTES) Fig 4 Effects of glucose, duuoxldc and tolbutamlde on [Ca?+], m a suspension of human msulmoma cells Addttlon of glucose, dlazoxldc and tolbutamlde are rndrcdted by the arrows

4 DISCUSSION The present results represent direct measurements of [Ca”‘], m isolated human islets of Langerhans and are olmllar to those obtained m rodent ,O-cells [!6-181, vaMating the use of ammal models m the study of /I-cell The blphaslc [Ca2’], stimulus-secretion coupling response to glucose has been attributed LO mltlal stlmulatlon of sequestration and/or outward transport of Ca” then fo’ollowcdby depolanzatlon-mediated mflux of the ion [19] The mltlal lowermg m [Ca**], m response to glucose 1s most readily seen when /P-cells have been Incubated at subnormal glucose concentrations, probably reflecting restoration of cytoplasmlc ATP to normal levels with subsequent actlvatlon of energy-deprived Ca*’ pumps [20,2!] It IS of interest to note that a similar pattern of msuhn release parallels the changes m [Ca2’], in mouse /I-cells [20] The inma! mhlbltlon of msuhn release by glucose, likely to reflect lowering m [Ca”],. has also been observed m patients with NlDDM and suggested to Indicate lmpalrment offi-cell function [22]. In our study the blphaslc response was only seen m about half of the expcrlments and m those cases it mdy bc attributed to the low prestlmulatory glucose concentratIon [20,2!] More mterestmgly, we found that human islets demonstrate oscillations In [Ca’*], In response to glucose stlmulatlon, m accordance with what has been reported ft)r singlep-cells ds well as Intact Islets from mice [ 16,181. Although the osclllatlons could not be fully charactenzed, they appeared longer than the ‘fast’ osclllatlons obtained m Intact mouse Islets [!8], but shorter than those observed m single /?-cells [16]. The nature of the oscillatory bchavlour 1s unknown but In view of the importance of the ATP-regulated K* channels m the rcgulatlon of /$-cell membrane potential and thereby Ca” influx, osclllatlons m the ATP levc! and/or ATP/ ADP ratlo may bc mvolvcd Furthcrmorc, muscle extracts demonstratmg spontaneous oscillations itI the ATP/ADP ratio, produce oscillattons m the amblent

LETTERS

October

199

1

Ca2+ concentratlon mamtamed by permeablhzed tumor @-cells [23]. It is of Interest to note that osclllationro m WAD(P)H fluorescence have recently been demonstrated m smgle P-cells subsequent to glucose stlmulatlon [17]. For years it has been known that cell extracts, contammg enzymes required for glycolysis, can produce cychc fluctuations m the levels of mtermedlates such as NADH [%4] These osclllatmns are beheved to arise from fcedbaclc control of certam allosterlc glycolytlc enzymes I.241 Whether fluctuations m the NAD(P)H level, K.+ channel activity, glycolysis and Ca2+ cychng of mtracellular organelles are all mvolvad m the control of [Ca2+], oscillations 1s a matter for future mvestlgations. The demonstration that extracellular ATP, used to activate P,-purmerglc receptors, resulted m an mcrease m [Ca’*], most likely reflects activation of phosphohpase C and the subsequent formation of mositol 1,4,Stnsphosphate (IP,) [!4,!5]. The fact that IP, releases Ca” from Internal stores explams the maJor part of the rise m [Ca”‘], promoted by the nucleotlde Human msulinoma cells have previously been used for patch-clamp studies and successfully kept m tissue culture for long periods of time [25]. In contrast to animal msuhnoma cells, a stable human tumor fl-cell line has not yet been possible to establish Cells from the msulmoma used in this study did not proliferate m tlssue culture and the results thus only reflect regulation of [Ca2’], m one /S-cell tumor. The clear-cut response to glucose stlmulatlon m terms of increased [Cat’],, suggested a functional metabohsm of the sugar Thls IS m agreement with previous studies demonstratmg that glucose, m many patients with msulmomas, evokes a prompt stimulation of msuhn release [26] Pnterestmgly, most ammal-derived p-cell lmes are insensitive to glucose [27,28] although there are recent reports of clones which show sensitlvlty to the sugar [29,30] The existence of functlonal ATP-regulated K’ channels m the msulmoma was further mdlcated since the cells responded to diazoxlde and tolbutamlde Tolbutamlde and other sulphonylurea compounds are known to stimulate insulin release by directly closmg the ATPregulated K’ channels with subsequent depolarization and Influx of Ca” [4,311 Dlazoxide, on the other hand, opens these K’ channcla promotmg repolallzatlon and closure of the voltage-activated Ca” channels with a resultmg decrease m [Ca”], 14,311 The usefulness of dlazoxldc m the treatment of hypermsulmlsm m man [32] most likely reflects Its lowermg effect on [Ca2’], m the p-cells The present study provides addmona! support for a close coupling between actlvlty of ATPregulated K’ channels and changes in [Ca’*], in the Icgulatlon of msuhn release m man. ~c~~r~~~~krt~~e~~rcrrr~~ fh~r study

w.ts supported by grants from the %rcd~rh Mcd~~dl RcrcJrch Council (1%00034, 12x-086471, the Bdnk of Swcdcn Tcrccntcndry Foundaclon, the SHcdIsch IXrbctcs A$soci+ t~on. Funds of the Karo1msl.d tnstltulc. Tort N~lsorlsrounddtlnn for Mcd~cdRescrrch. the Swedish Soe~ctyof Mcdlcmc, Navo Industry.

313

Volume 29 I, number 2

FEES LETTERS

Farmltaha Carlo Erba, the Nordic Insulm, Aage LOUISHansens Memonal, Fredrlk and Ingrtd Thunngs, Torsten and Ragnar Soderbergs, Magnus Bergvalls, Amundssons, Anders Otto Swards and the Swedish Hoechst Research Foundations

REFERENCES [I] Kakel, M , Kelly, R P , Ashcroft, S J H and Ashcroft, F M (1986) FEBS Lett 208, 63-66 [2] Ashcroft, F M , HarrIson, D E and Ashcroft, S J H (1984) Nature 3 12,446-448 [3] Cook, D L and Hales, C N (1984) Nature 31 I, 271-273 [4] Arkhammar, P , Ndsson, T , Rorsman, R and Berggren, P -0 (1987) J Blot Chem 262, 5448-5454 [5] Rorsman, P and Trube, G (1986) J Physlol 374. 531-550 [6] Lohmann, D , Jahr, H , Ver!ohren, H -J , Schmidt, S , Hellmann, W , Zdhlke, H , Hartlg, W and Milttlg, H (1980) Norm Metab Res 12, 349-353 [7] Grant, A M , ChrIstIe, M R and Ashcroft, S J I-I (1980) Dlabctologta 19, 114-l I7 [S] Harrison, D E , Chrlstlc, M R and Gray. D W R (1985) Dlabetologta 28, 99-103 [9] Ashcroft, F M , Kakel, M , Kelly, R P and Sutton, R (19X7) FEllS Lett flS, 9-12 [IO] Ashcroft, F M , Kakel, M., Gibson, J S , Gray, D W and Sutton, W (1989) Dztbetologla 32, 591-598 [I I] Mlsler, S, Gee, W M , GIIIIS, K D 1 &harp, D W and Falke, L C (1989) Diabetes 38. 422427 1121 Hellman, B (1975) Endocnnology 97, 392-398 [I31 Plant, T D (1988) J Phys~ol 404, 731-747 1143 Arkhammar, P, Hallberg. A, Kmdmark, N , Nllsson, T, Rorsman, P and Berggren, P -0 (1990) Blochem J 265, 203211 [I 51 Hellman, B , Gylfe, E , Wesslen, N , Hallbcrg, A , Grapenglesser, E and Marcstrdm. A (1989) Exp Clm Endocrmol 93, 125-135

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[I61 Grauenmesser. E , Gvlfe. E and Hellman, B (1988) Blochem Blophys Res Comm& .I 5 I, 1299-l 304 u 71 Pmlong, W-F , Bartley, C and Wolrhzlm, C I3 (1990) EMBO J 9, 53-60 iI81 Valdeolmlllos, M 1 Santos. R M , Contreras D, Sona, B and Rosarlo, L M (1989) FEES Lett 259, 19-23 1191 Hellman, B dnd Gylfe. E (1986) In Calcmm and Cell Function, vol VI (Cheung, WY ed ) pp 253-326, Acadcmlc Press, New York PO1 Nllsson, T , Arkhammar, P and Berggren, P -0 (1988) Blochem B~ophys Res Commun 153, 984-991 Pll Wollhelm. C B and Blden. T J (1987)_Ann NY Acad SCI 488, 317-333 WI Hellman, I3 , Berne, C , Grapenglesser, E , Grill, V , Gylfe, E and Lund, P-E (1990) Eur J Chn Invest 20, SIO-S17 WI Corkey, B E , Tornhelm, K , Deeney, J T , Glennon, M C , Matschmsky, F M and Prentkl, M (1988) J BIOI Chem 263, 4247-4253 Hess, B (1977) Trends J&o&hem SCI 9, 193-195 t::; Sturgess, NC, Carrmgton, CA, Hales, C N and Ashford, M L J (1987) Pfdgcrs Arch 410, 169-172 WI Lms, P and Efcndle, S (1979) Diabetes 28, 190-195 1271 Rorsman, P , Berggren, P -0, Gylfe, E and Hellman, B (1983) BIOSCI Rep 3, 939-946 WI Boyd III, A E , HI]], R S , Oberwetter. J M and Berg, M (1986) J Clm Invest 77, 774-781 PI Dunne, M J , Yule, D I, Gallacher, D V and Petersen, 0 I-I (1990) J Membr BIOI 114, 53-60 1301 Regazzl, R , Guodong, L , Deshusses, J and Wollhelm, C B (1990) J I301 Chem 255, 15003-15009 t3 1I Trube, G , Rorsman, P and Ohno-Shosaku, T (1986) Pflugcrs Arch 407.493-499 1321 Scdndelarl, C , Zaccdrla, M , S~colo, N , Casara, D , Erie, G and Federspd, G (1976) Horm Metab Res (Suppl Senes 6) 46-54