Biochem. J. (1985) 228, 529-536Printed in Great Britain

Protein phosphorylation in permeabilized pancreatic islet cells

Jerry R. COLCA,* Bryan A. WOLF, Patricia G. COMENS and Michael L. McDANIELtDepartment of Pathology, Washington University School of Medicine, St. Louis, MO 63110, U.S.A.

(Received 20 August 1984/14 February 1985; accepted 8 March 1985)

A system of digitonin-permeabilized islet cells was developed to characterize Ca2+-and calmodulin-dependent protein phosphorylation further and to determine whetheractivation of this membrane-bound process was sufficient for initiation of Ca2+-stimulated insulin secretion. The efficacy of digitonin in permeabilizing the plasmamembrane was assessed by Trypan Blue exclusion, by extracellular leakage of lactatedehydrogenase, and by permeability to [y-32P]ATP. This treatment did notdetectably alter the ultrastructure of the permeabilized cells. Digitonin was equallyeffective when presented to islet cells that had been previously dispersed or directly tointact isolated islets. The Ca2+- and calmodulin-dependent phosphorylation ofendogenous membrane-bound substrates could be demonstrated in the permeabilizedcells incubated with [y-32P]ATP. This activity displayed characteristics that weresimilar to those described for the protein kinase measured in subcellular fractions andwas dependent on addition of exogenous calmodulin, indicating that calmodulin hadbeen removed from the kinase by permeabilization of the cells. Ca2+-dependentinsulin release by the digitonin-permeabilized islet was demonstrated, with half-maximal release occurring at 0.1 M-free Ca2+ and maximal secretion at 0.2uM-freeCa2 . Under these conditions, calmodulin did not further enhance insulin release,although a stimulatory effect of calmodulin was observed in the absence of free Ca2 .These studies indicate that the permeabilized-islet model will be useful in dissectingout the factors involved in Ca2+-activated insulin secretion.

The mechanism by which glucose stimulates thesecretion of insulin from pancreatic islets remainsunresolved. Various pieces ofevidence suggest thatalterations in cellular levels of Ca2+ are centrallyinvolved in modification of the fl-cell response. Allknown insulin secretagogues affect Ca2+ fluxes inintact islets; insulin secretion is dependent onextracellular Ca2+, and alteration of extracellularCa2+ concentrations can effect the release ofinsulin in the absence of glucose. Thus it appearsthat Ca2+ represents a final effector mechanismwhich initiates the secretory response (Wollheim &Sharp, 1981).Many of the effects of Ca2+ are mediated by

modification of protein kinase activities. We havefairly recently described a membrane-bound Ca2+

Abbreviation used: SDS, sodium dodecyl sulphate.* Present address: The Upjohn Company, Kalama-

zoo, MI 49001, U.S.A.t To whom correspondence and reprint requests

should he addressed at: Box 8118, Washington Univer-sity School of Medicine, 660 S. Euclid Avenue, St. Louis,MO 63110, U.S.A.

and calmodulin-dependent protein kinase activityin subcellular fractions of pancreatic islets (Landtet al., 1982). Various lines of evidence suggest thatthis protein kinase plays a role in insulin secretion.The protein kinase is activated by Ca2+ concentra-tions thought to be obtained in the stimulated ,B-cell (Colca et al., 1983a) and is enriched in a micro-somal fraction that contains a calcium pumpcapable of controlling Ca2+ concentrations in thisrange (Colca et al., 1982). The Ca2+- and calmodu-lin-dependent protein kinase phosphorylates endo-genous islet-cell substrates of 57 and 54kDa and isinactivated by alloxan under conditions wherealloxan inhibits insulin secretion (Colca et al.,1983b). Furthermore, studies performed with in-tact islets preloaded with [32P]P, indicate that thisprotein kinase activity is correlated with glucose-stimulated insulin release (Colca et al., 1983a).

However, experimental approaches that havestudied protein phosphorylation in cell-free sys-tems or after labelling of intact cells with [32p]p;suffer from several theoretical and technicallimitations. There is no assurance that an activity

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J. R. Colca, B. A. Wolf, P. G. Comens and M. L. McDaniel

measured in cell homogenates or sonicated cellsrepresents an interaction of an enzyme and sub-strate that occurs in the intact cell as opposed tobeing an artefact of the cell fractionation. On theother hand, studies with intact cells are limited bythe fact that the experimental period must bepreceded by a loading period with [32P]P; to labelendogenous ATP pools; this also produces a largebackground of protein phosphorylation againstwhich experimental changes must be measured. Iflabelling experiments are carried out in thepresence of [32P]p,, experimental treatments mayalter the labelled-nucleotide pool. Alternatively, ifthe [32P]P, is removed, some experimental treat-ments (e.g. elevation of medium glucose) maydecrease the specific radioactivity of the ATP pool.Furthermore, it is not possible to define preciselychanges in the cellular levels of Ca2+ that arepresumed to occur in response to experimentaltreatments.Measurement of protein phosphorylation in

permeabilized cells offers advantages over thepreviously described systems. The permeabilizedcells can be incubated directly with [y-32P]ATPduring a defined experimental time period. Theconceptrations of Ca2+ or other parameters can becontrolled by direct addition to the externalmedium,, since the plasma membrane is madefreely permeable to these additions.

Detergent-permeabilized cells have been used tostudy pools of intracellular Ca2+ uptake (Hirata &Koga, 1982; Wakasugi et al., 1982; Burgess et al.,1983), DNA synthesis (Miller et al., 1978), cate-cholamine secretion (Dunn & Holz, 1983; Wilson& Kirshner, 1983), and hormone action (Mooney etal., 1983). Such studies have taken advantage ofthe ability of detergents such as digitonin toselectively permeabilize the cholesterol-rich plas-ma membrane. This study describes a technique topermeabilize dispersed islet cells as well as intactislets by a short incubation with digitonin. Thedigitonin-permeabilized islet cells are then used tomeasure Ca2+-activated protein phosphorylationsand insulin secretion.

Experimental proceduresMaterialsMale Sprague-Dawley rats (200-300g) were

purchased from Sasco (O'Fallon, MO, U.S.A.)Collagenase (CLS IV) was obtained from Worth-ington, [y-32P]ATP from New England Nuclear,Dispase from Godo Shusei (Tokyo, Japan), andcalmodulin from Calbiochem (La Jolla, CA,U.S.A.). All other chemicals, including digitonin,were from Sigma Chemical Co. (St. Louis, MO,U.S.A.).

Pancreatic isletsPancreatic islets were obtained from fed rats by

collagenase digestion (Lacy& Kostianovsky, 1967;McDaniel et al., 1983), followed by purification onFicoll gradients (Shibata et al., 1976). The isolatedislets were hand-picked under a dissecting micro-scope and then dispersed into single cells withDispase (Ono et al., 1977). Experiments either usedthese dispersed cells [(1-2) x 104 cells/sample] orintact islets (20-40/sample). All incubation mix-tures contained 3OQul unless otherwise noted.

Digitonin studiesDispersed cells or intact islets were incubated

in a modified Krebs buffer (1 l5mM-NaCl/24mM-NaHCO3 /5mM-KCI / 1 mM -MgCl2 /1 mM -EGTA/20mM-Hepes/0.1% bovine serum albumin,pH 6.8). Permeabilization was performed in theindicated buffer at 37°C with 0-40pg of digiton-in/ml for 15min unless otherwise indicated. Dis-persed cells were centrifuged at 5OOg for 5min.Treated intact islets were allowed to sediment (30s)and the medium was rapidly removed with adrawn Pasteur pipette.

Lactate dehydrogenase (EC 1.1.1.27) wasmeasured in a 25 l sample and fluorescence wasread in a 1 ml sample (Lowry et al., 1957). Totallactate dehydrogenase was measured in sonicatedcells or islets (2 x 5 s at 18W with a Bransonsonifier). Total recovery of the lactate dehydrogen-ase by this method was 95-105%.Trypan Blue exclusion was determined after a

5min incubation with 4mg of Trypan Blue/ml atroom temperature and was determined for dis-persed cells only.

Protein phosphorylationDispersed cells or islets pretreated with or with-

out digitonin were washed one to three times withthe modified Krebs buffer and then incubated at37°C in the modified Krebs buffer containing 0-1.2mM-CaCl2, 1 mM-EGTA, 0-1 pM-calmodulinand 5-IOpCi of [y-32P]ATP. For most incubationsthe cells were rapidly centrifuged or the islets wereallowed to sediment and the radioactive mediumwas rapidly removed before addition of the stopsolution [final concns. 3% (w/v) SDS, 2% (v/v) 2-mercaptoethanol and 5% (v/v) glycerol]. Thesamples were then immediately placed in a boiling-wateri bath for 2min. For studies in which thereaction time was varied or for short reactiontimes, i.e. 30s, the assay was terminated by directaddition of the stop solution, followed by placingthe sample in a boiling-water bath for 2min. Thisprotocol totally inactivates protein kinase activity.Phosphoproteins were then separated by electro-phoresis on 10%-(w/v)-polyacrylamide slab gels.

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Protein phosphorylation in islet cells

The gels were dried, exposed to X-ray film, and thephosphoprotein bands of interest were cut from thegels for quantification of 32P incorporation (Landtet al., 1982). Ca2+ and calmodulin-dependentprotein phosphorylation is defined as the amountof phosphorylation occurring in the presence ofCa2+ and calmodulin minus that occurring in theabsence of either component. Quantification wasgenerally of the 57 kDa endogenous substrate,since it is more heavily phosphorylated (Colca etal., 1983a).

Insulin secretionDigitonin-treated islets were washed three times

in Tris buffer (50mM-Tris/100mM-KCI/5mM-MgCl2/0.1% bovine serum albumin, pH6.8)and then incubated at 24°C for 15min in Tris/Pipes buffer (55mM-Tris/Pipes/l 11 mM-KCl/5mM-MgCI2 /1.1 mM-EGTA/ 1 mM-ATP/ l0mM-phosphocreatine/creatine kinase (10 units/ml)/0.1% bovine serum albumin, pH7.5) containing0-1.2mM-CaCl2 and 0167jug of calmodulin/ml.Islets were gassed with O2/CO2 (19: 1). At the endof the incubation period, the medium was with-drawn and the insulin released was quantified byradioimmunoassay. Immunoreactive insulin wasmeasured in triplicate by the method of Wright etal. (1971), with porcine insulin as standard.

Free Ca2+ concentrations were estimated byassuming an apparent stability constant forCa2+ *EGTA of 107-44 (240C, 100mM-KCl, pH 7.5),which was determined from an absolute stabilityconstant of 1010.97 (20°C, I0. 1) by interpolating fortemperature and the H+ activity coefficient assuggested by Martell & Smith (1974). Ionized-Ca2+concentrations were then confirmed by measure-ment in the complete assay medium by usinga Ca2+-specific electrode (Orion 93-20, with adoublejunction reference electrode 90-02) aspreviously described (Kotagal et al., 1983).

ResultsPermeabilization and phosphorylation in dispersedislet cells

Dispersed pancreatic-islet cells were washed inthe modified Krebs buffer and incubated [(4-6)

x 104 cells/300,l sample] with various concentra-tions of digitonin at 37°C. The cells were thensedimented and lactate dehydrogenase activitywas determined in the medium and in sonicated re-suspended pellets. Parallel sets of cells so treatedwere resuspended in medium without addeddigitonin, and incubated with Trypan Blue for5min. Table 1 shows that the percentage of cellsunable to exclude Trypan Blue was directly relatedto the percentage release of the cytosolic marker,lactate dehydrogenase, after pretreatment of cellswith various concentrations of digitonin.

Analysis of the digitonin-treated islet cells byelectron microscopy showed no significant ultra-structural differences when compared with non-treated islet cells (Fig. 1). In both groups, theultrastructural appearance of the endoplasmicreticulum and the secretory granules was similar,although the mitochondria of permeabilized cellsappeared slightly more condensed.

Studies were undertaken to determine whetherCa2+ and calmodulin-dependent protein kinaseactivity could be detected in the permeabilizedcells. When cells were permeabilized with digi-tonin, washed, and then incubated with [y-32p]-ATP for 10 min at 37°C, the extent of protein phos-phorylation was proportional to the amount ofpermeabilization achieved during the digitoninpretreatment, as shown in Table 1. Whereas theaddition of calcium alone (1 mM buffered withEGTA; 4!LM-ionized Ca2+) did not augmentprotein phosphorylation, the combined presence ofCa2+ and I pM-calmodulin increased the phos-phorylation of 57kDa and 54kDa protein bands(Table 2; see also Fig. 2). This result indicated thatendogenous calmodulin was depleted from or nolonger available to the cellular kinase by thedigitonin treatment.

Permeabilization and phosphorylation in intact isletsWhen intact islets were permeabilized with

various concentrations of digitonin and thenincubated with [y-32P]ATP with or without Ca2+and calmodulin, results similar to those with thedispersed cells were obtained, namely the phos-phorylation of the islet 57 kDa and 54kDa proteins

Table 1. EjIect of digitonin pretreatment on permeability of dispersed islet cellsIsolated pancreatic islets were dispersed into single cells as described in the Experimental procedures section.Samples of the cells were then incubated with the indicated concentrations of digitonin for 15 min at 37°C. The cellswere then sedimented and lactate dehydrogenase activity (LDH) was determined on samples of the supernatant andsonicated pellet as described in the text. A portion of the cell suspension from each sample was also tested for theability of the cells to exclude Trypan Blue. Results are means+ S.E.M. for four experiments.

[Digitonin] (pg/ml) .,..

Percentage of LDH releasedPercentage of cells positive for Trypan

Blue staining

0 4

23+98+2

31+ 1033 + 6

10

63 + 1166+9

20

84+ 787 + 5

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J. R. Colca, B. A. Wolf, P. G. Comens and M. L. McDaniel

-Origin

57 kDa- 57 kDa

Ca2...--- + + - + +-- ++- + +Calmodulin...- - + - - + - -- + - - t

Digitonin pretreatment L.I TJ' p.' J(gig/ml)... 0 5 10 20

LDH release (%.. 5 24 43 73

Fig. 2. Protein phosphorylation in digitonin-permeabilizedislets

Pancreatic islets were permeabilized by 15 minincubations (37°C) with the indicated concentrationof digitonin. Lactate dehydrogenase (LDH) wasthen determined in the supernatants as well as insonicated resuspended pellets. Parallel groups ofislets (25/sample) were then washed twice withalbumin-free medium and incubated for 5min withlO*Ci of[y-32P]ATP with or without 1 pM-calmodu-lin and/or lOuM-Ca2+as described in the text. Theautoradiogram and associated lactate dehydro-genase results shown are representative of threeexperiments of this design. Similar results (shown inTables 1 and 2) were obtained with digitonin treat-ment of dispersed islet cells.

Fig. 1. Ultrastructure ofpermeabilized islet cellsPancreatic islets were incubated in modified KRBmedium with or without 20pg of digitonin/ml for15 min at 37°C as described in the text. The isletswere then fixed in 1.7% (v/v) glutaraldehyde/1.3%(v/v) p-formaldehyde/0. 1 M-cacodylate buffer, post-fixed with 1% OSO4, dehydrated, and embedded inepoxy resin. Ultrathin sections were stained withuranyl acetate and lead nitrate. (a) Control; (b)digitonin-permeabilized. Detailed examination ofthe photomicrographs showed no difference be-tween the groups.

was dependent on the addition of both Ca2+ andcalmodulin and proportional to the percentage oflactate dehydrogenase released during the digi-tonin pretreatment (Fig. 2). Although the digitonintreatment did not disrupt the ultrastructure of thefl-cells (Fig. 1), the extent of phosphorylationobtained after pretreatment of islets with 20 or40ug of digitonin/ml was similar to that occurringafter disruption of the intact islets by sonication,i.e. the cells were made sufficiently permeable withdigitonin (20ug/ml). It is noteworthy that 49 +14% of the total islet Ca2+- and calmodulin-dependent protein kinase was recovered from theintact islets after digitonin treatment. Additionalstudies were undertaken to characterize proteinphosphorylation in these permeable islets. Thetime course for the Ca2+ and calmodulin-depen-dent protein kinase in pre-permeabilized islets isshown in Table 3. Interestingly, a similar timecourse for the activity was obtained after preincu-bation of untreated islets with Ca2+, calmodulin,and ATP and initiation of the intracellular phos-phorylation with digitonin (Table 3). That is, thespeed of permeabilization of the islets by digitonin

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Protein phosphorylation in islet cells

Table 2. Ca2+- and calmodulin-dependent phosphorylation ofpermeabilized islet cellsDispersed pancreatic islet cells were permeabilized with various concentrations of digitonin as described in Table 1.The respective cells were then washed and incubated with [y-32P]ATP (5jiM, lOyCi/tube) with and without Ca2+(I mm total, 4pM free) and calmodulin (1 gM) for 1Omin as described in the Experimental procedures section. Resultsshow the mean phosphorylation ofthe 57 kDa membrane-bound protein obtained in two experiments with islet cells.

Protein phosphorylation (fmol of Pi/57kDa band)

Phosphorylation conditions[Digitonin] (pg/ml) ...

No additionsCa2+Ca2+ + calmodulin

0 4 10 20

38 86 139 15644 94 159 133111 298 440 320

Table 3. Time course of Ca2+- and calmodulin-dependent protein phosphorylation in digitonin-permeabilizedpancreatic islets

Incubation mixtures contained pancreatic islets permeabilized with 20pg of digitonin/ml (15 min, 37°C) and thenwashed free of detergent (protocol A) or untreated islets (protocol B). For protocol A the permeabilized islets werepre-incubated with and without 300Mm-free Ca2+ and 1 pM-calmodulin (2min, 37°C): the reaction was initiated with5-lOpCi of [y-32P]ATP (total volume I00yl) and stopped at the indicated times with stop solution as described in theExperimental procedures section. For protocol B, untreated islets were preincubated with 5-lOpCi of [y-32P]ATPwith and without 300gM-free Ca2+ and 1 pM-calmodulin (2min, 37°C) and the reaction was initiated by the additionof 20 or 40pg of digitonin/ml (total volume I00p1) and then stopped at the indicated times as discussed above. Dataare the means + S.E.M. for Ca2+- and calmodulin-dependent Pi incorporation into the 57 kDa and 54 kDa islet-cellsubstrates (i.e., minus phosphorylation in the absence of Ca2+) for three experiments, each performed in duplicate.

Ca2+- and calmodulin-dependent Pi incorporation (c.p.m.)

ProtocolIncubation

time (min) ...

(A) Pre-permeabilized (20pgof digitonin/ml)

(B) Started with digitonin at:20pg/ml40g/ml

0.5

725+104

944 + 382807 + 232

5

1317+ 88

1210+4071829+271

2158 + 248

2288 + 7682522 + 576

was not such as to rate-limit the measurement ofprotein phosphorylation in this system.To determine if the protein kinase activity in the

permeabilized-islet system was similar to thatdescribed previously for the kinase activity in sub-cellular fractions, the concentration of free Ca2+ orexogenous calmodulin was varied. The calmodulinrequirement for this protein kinase activity isshown in Fig. 3(a). Maximal stimulation of the rateof phosphorylation occurred at approximatelylOpg of calmodulin/ml. The Ca2+ dependence ofthe protein kinase activity (Fig. 3b) showed thatmaximal activation was present at approx. 2-3 pM-Ca2+. The characteristics of this protein kinaseactivity as measured in permeabilized islets weresimilar to those described for the activity measuredin subcellular fractions (Colca et al., 1983a). Addi-tional studies have also been performed to addressthis question. Ca2+ and calmodulin-dependentprotein phosphorylation assays were performed indigitonin permeabilized islets as described in Fig.2. The islets were then homogenized in fractiona-tion buffer at 4°C containing EDTA to chelate

Mg2+ and minimize subsequent dephosphoryla-tion. The particulate membranes and cytosol werethen separated by ultracentrifugation, the phos-phoproteins being by autoradiography. The Ca2+-and calmodulin-dependent phosphoproteins asdescribed in the present study were totally associ-ated with the protein pellet as opposed to thecytosol. These studies are consistent with the othersupportive data indicating that the Ca2+- andcalmodulin-dependent protein kinase system indigitonin-permeabilized islets exists in the mem-brane fraction.

Insulin secretion in permeabilized isletsInsulin release by digitonin-treated islets was

examined at 37°C under conditions similar to thosedescribed for protein phosphorylation. However,the addition of Ca2+ (0.1-40Mm-free Ca2+) with orwithout calmodulin did not augment the release ofinsulin (results not shown). Since it seemed likelythat the failure to demonstrate Ca2+-inducedinsulin secretion may have resulted from technicallimitations of the assay, modifications were made

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J. R. Colca, B. A. Wolf, P. G. Comens and M. L. McDaniel

(b)

10 20 30

1Calmodulin1 (,ug/ml)

,,'

l

l

l

,D' ~~~~~I

II o_I

I

3[Free Ca2'+ (pM)

1

Fig. 3. Ca2+- and calmodulin-dependence of the protein kinase activity measured in digitonin-permeabilized isletsPancreatic islets were permeabilized with 20pg of digitonin/ml as described in the text. The islets were then washedin albumin-free medium and incubated with lOpM-Ca2+ and various concentrations of calmodulin (a) or 1 M

(17pg/ml)-calmodulin and various concentrations of Ca2+ (b). Reactions, initiated by lOyCi of [y-32P]ATP were

terminated after 30s and processed as described in the text.

Table 4. Effect of Ca2+ and calmodulin on insulin release in digitonin-permeabilized pancreatic isletsDigitonin-permeabilized (20pg/ml, 20min, 37°C) pancreatic islets were washed free of detergent in Tris buffer(50mM-Tris/l0OmM-KCI/5mM-MgCl,/0.1% bovine serum albumin, pH6.8) and then incubated at 24°C in a

Tris/Pipes buffer mimicking intracellular conditions [55 mM-Tris/Pipes/ 111 mM-KCI/5mM-MgCl,/1.1 mM-EGTA/lOmM-phosphocreatine/creatine kinase (10 units/ml)/O. bovine serum albumin, pH 7.5] containing no or

0.95mM-CaCl, and 0-167pg of calmodulin/ml. After 15 min incubation, insulin released in the media was measuredby radioimmunoassay. Results are expressed as means +S.E.M. of three to four experiments each performed inquadriplicate.

Insulin release(punits/min per islet)

[Calmodulin](pg/ml) [Free Ca2+] (pM) ...

0

1.78.4

16.7167.2

0

0.48 + 0.051.04+0.140.93 +0.150.82 +0.101.11 +0.30

0.2

0.96+0.131.02 +0.180.89 +0.150.77 +0.090.94+0.27

to optimize the possibility of demonstrating Ca2+-dependent secretion. Assays were performed atroom temperature to minimize background insulinrelease and at pH7.5 in the presence of EGTA inorder to obtain submicromolar concentrations offree Ca2 . In the presence of an ATP-regeneratingsystem and in a media mimicking intracellularconditions, Ca2+-induced secretion by digitonin-treated islets was successfully demonstrated (Fig.4). Under these conditions, a basal rate of 0.64 +0.06 punit of insulin/min per islet was obtained inthe absence of Ca2+ and maximal insulin release(1.08 + 0.09tunit/min per islet, n = 20) was ob-served at 0.2 pM-free Ca2+. The effect of calmodu-

lin (0-167 pg/ml) on insulin release in the absenceor presence of 0.2 pM-free Ca2+ is detailed in Table4. In the absence of Ca2+, exogenous calmodulinsignificantly enhanced insulin secretion, whereas,in the presence of 0.2 pM-free Ca2+, no furthereffect on insulin release could be demonstrated.Ca2+- and calmodulin-dependent protein kinaseactivity similar to that demonstrated in theexperiments defining this permeabilized-islet sys-tem was also observed under these identical assayconditions, i.e. pH 7.5 and room temperature (e.g.basal activity 0.250+0.019fmol of Pi/lOmin perislet, 0.471 +0.06fmol of P0/lOmin per islet at1.1 M-Ca2+ and 1 pM-calmodulin.

1985

C 34la

EC:

2a't.~_

-o2-ea0r _0+, v 11co

534

.0.0---.P ---/.4--o

Protein phosphorylation in islet cells

.E'a

,V.-

0.5_O.~:

8 7

PC

Fig. 4. Ca2+-induced insulinpermeabilizec

Pancreatic islets were permedigitonin/ml as described irwashed three times inTris/100mM-KCI/5 mM-MgCalbumin, pH6.8) and then ii24°C in a Tris/Pipes buffer nconditions [55mmM-Tris/PipeMgCl2/ 1.1 rM-EGTA/lOnmatine kinase (10 units/ml)/Obumin, pH7.5]. Free-Ca2+obtained by addition of 0-scribed in the Experimental ]then confirmed by measurspecific electrode. Results;five experiments each perfor

Discussion

A system has been despermeabilizing the plasmacreatic-islet cells by usingdigitonin. The digitonin treffective in permeabilizingcells or intact islets. Studiesby using the intact islets, vlated under a microscope wilshould be noted that incuta modified Krebs-Ringeisupplemented with EGTAtonin increased the intercellas determined by microscopaugment the effectiveness odispersed islets. Although di,of the cytosolic content fromrelease of lactate dehydrogture of the cells remained iithe digitonin concentraticpresent study, cholesterol-pcare found in the endoplas1966) are not affected by thi& Huby, 1984). The systestudying protein phosphoryally undisturbed intracellul;determine the requirementsThe demonstration of C

dependent protein kinase

meabilized cells with kinetic characteristics simi-lar to those described in subcellular membranefractions (Landt et al., 1982; Colca et al., 1983a)supports the hypothesis that this enzymic systemfunctions in the intact cell. As the cell membraneswere permeable to Ca2+ and calmodulin, it waspossible to define the conditions for measurement

, , of the kinase activity by controlled additions to the6 5 4 extracellular medium and to initiate the reactionsta2+ with [y-32P]ATP. In contrast with studies withsecretion by digitonin impermeable cells, where these parameters cannot

I islets be controlled directly, the experiments support theiabilized with 20ig of existence of this protein kinase system in then the text. Islets were structurally intact membranes. The need forTris buffer (50mM- addition of exogenous calmodulin in this system'12/0.1% bovine serum implies that the source of calmodulin for thencubated for almin at protein kinase activity is depleted by the loss of

ns/ 11kmM-KCl/5lmM- cytosolic calmodulin. It is possible, therefore, thatl-phosphocreatine/cre- alteration of soluble-calmodulin concentrations4.1% bovine serum al- may play a role in this protein kinase activity inconcentrations were vivo.

-1.2mM-CaCl2 as de- Several groups of investigators have shown thatprocedures section and the addition of Ca2+ to digitonin-permeabilizedrement with a Ca2+- adrenal chromaffin cells directly augments theare means+S.E.M. for release of catecholamine (Dunn & Holz, 1983;.med in quadruplicate. Wilson & Kirshner, 1983). Pace et al. (1980) and

Yaseen et al. (1982) have shown that intact isletspermeabilized by electric discharge release insulinin proportion to the concentration of Ca2+ presentin the incubation medium. In those studies there

-cribed for effectively was not a depletion of cytosolic contents, althoughmembrane of pan- after a period of time these cells permeabilized by

low concentrations of electric discharge may become depleted of ATPeatment is equally as (Pace et al., 1980). These results obtained witheither dispersed islet islets made permeable by electric discharge areare greatly expedited essentially similar to those described initially by

vhich can be manipu- Baker & Knight (1978, 1981) for adrenal chromaf-thout centrifugation. It fin cells made permeable by electric discharge.bation of the islets in Thus the basic features of Ca2+-stimulated secre-r bicarbonate buffer tion from the pancreatic islets and adrenal chro-with or without digi- maffin cells may be similar.Iular space in the islets We have demonstrated that Ca2+-dependentmy and this may in fact insulin release also can occur in digitonin-permea-f digitonin in the non- bilized islets. The failure to show Ca2+-stimulatedgitonin caused leakage insulin release at 37°C is due to the large non-i the cells (as judged by specific background release of insulin (10 times theenase), the ultrastruc- basal rate observed at 24°C). However, at roomntact. Furthermore, at temperature and under conditions mimicking:n employed in the intracellular requirements, Ca2+-dependent in-)or membranes such as sulin release was present as well as the Ca2+-mic reticulum (Korn, and calmodulin-dependent phosphorylation ofis treatment (Gogelein the 57kDa endogenous substrate. The effect ofXm is thus useful for calmodulin on insulin secretion in this system wasrlation in the structur- unexpected. The lack of further enhancement ofar membranes, and to Ca2+-dependent insulin release with calmodulins for insulin secretion. could be due to the experimental conditions, such'a2+- and calmodulin- as free Ca2+ concentration, resulting in already-activity in the per- maximal insulin release. However, calmodulin in

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536 J. R. Colca, B. A. Wolf, P. G. Comens and M. L. McDaniel

the absence of added Ca2+, over a wide range ofconcentrations, stimulated insulin release. Thisphenomenon could not be accounted for by Ca2+contamination of the calmodulin preparation, asconfirmed by theoretical and Ca2+-electrodedeterminations (see the Experimental proceduressection). The possibility that calmodulin per se actson an unknown link of the exocytosis processcannot be excluded. In any event, the developmentof the digitonin-permeabilized islet system willallow detailed testing of these hypotheses undervarious conditions with inhibitors of the proteinkinase activity such as calmodulin antagonists, di-phenylhydantoin and alloxan (Landt et al., 1982;Norling et al., 1984; Colca et al., 1983b).The present studies demonstrate the usefulness

of the digitonin-permeabilized cells in the study ofprotein phosphorylation and insulin release inintact cellular membranes. This system cancomplement studies with subcellular fractions orcells loaded with [32P]Pi and offers certain advan-tages over both of these techniques. It is alsoapplicable to the direct study of other proteinkinases such as cyclic AMP-dependent proteinkinase and protein kinase C and the impact ofthese activities on insulin secretion.

This work was supported in part by a grant to M. L. M.from the U.S. National Institutes of Health (AM06181)and by grant to J. R. C. from the Juvenile DiabetesFoundation. The excellent technical assistance of Ms.D;eidre Buscetto and Mrs. C. Joan Fink is greatlyappreciated. Electron microscopy was kindly performedby Dr. M. H. Greider.

ReferencesBaker, P. F. & Knight, D. E. (1978) Nature (London) 276,620-622

Baker, P. F. & Knight, D. E. (1981) Philos. Trans. R. Soc.London Ser. B 2%, 83-103

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