Neuron, Vol. 17, 513–522, September, 1996, Copyright 1996 by Cell Press

Neurotransmitter-Induced Inhibitionof Exocytosis in Insulin-Secreting b Cellsby Activation of Calcineurin

Erik Renstrom, Wei-Guang Ding, Krister Bokvist, Their action involves suppression of electrical activityand thus Ca21-dependent exocytosis (Drews et al., 1990,and Patrik Rorsman

Department of Islet Cell Physiology 1994; Debuyser et al., 1991). Studies on suspensions ofpermeabilized insulinoma cells have provided evidenceNovo Nordisk A/S

The Symbion Science Park that they also inhibit secretion by direct interferencewith the secretory machinery (Ullrich and Wollheim,Fruebjergvej 3

DK-2100 Copenhagen 1988, 1989; Sharp et al., 1989). Here, we have used highresolution capacitance measurements, as an indicatorDenmarkof exocytosis (Neher and Marty, 1982), to explore theprocesses underlying agonist-induced inhibition of insu-lin secretion in single voltage-clamped b cells. We showSummarythat inhibition of insulin secretion can be accountedfor by activation of the Ca21-dependent phosphataseNeurotransmitters and hormones such as somato-calcineurin.statin, galanin, and adrenalin reduce insulin secretion.

Their inhibitory action involves direct interferencewiththe exocytotic machinery. We have examined the mo- Resultslecular processes underlying this effect using highres-olution measurements of cell capacitance. Suppres- Effects of Somatostatin and Galanin on b

Cell Electrical Activitysion of exocytosis was maximal at concentrations thatdid not cause complete inhibition of glucose-stimu- In the presence of stimulatory concentrations of glucose

(10–15 mM), b cells in intact pancreatic islets generatelated electrical activity. This action was dependent onactivation of G proteins but was not associated with a characteristic pattern of electrical activity consisting

of bursts of action potentials separated by repolarizedinhibition of the voltage-dependent Ca21 currents oradenylate cyclase activity. The molecular processes silent intervals (reviewed by Henquin and Meissner,

1984). As illustrated in Figure 1A, addition of 400 nMinitiated by the agonists culminate in the activation ofthe Ca21-dependent protein phosphatase calcineurin, somatostatin to a b cell exposed to 10 mM glucose

produced a reversible lengthening of the silent intervals,and suppression of the activity of this enzyme abol-ishes their action on exocytosis. We propose that but did not inhibit electrical activitycompletely (three out

of five cells). The response to somatostatin on electricalmechanisms similar to those we report here may con-tribute to adrenergic and peptidergic inhibition of se- activity was very variable, and in some cells, electrical

activity was not affected (two out of five cells; Figurecretion in other neuroendocrine cells and in nerve ter-minals. 1B). However, subsequent addition of 100 nM galanin

to a cell that did not respond to somatostatin promptlyinhibited electrical activity (Figure 1B, right). Thus, theIntroductionlack of response to somatostatin in some b cells isunlikely to reflect the loss of the membrane receptorsHormonal and neuronal modulation of exocytosis repre-during islet preparation.sents an important mechanism by which neuroendo-

The effects of galanin on B cell electrical activity werecrine cells and neurons adjust to changes of the secre-further analyzed in Figure 2. As was the case with so-tory demand (reviewed by Hille, 1994). Such modulationmatostatin, galanin produced either complete (Figureis generally assumed to be secondary to activation of2A; four out of seven cells) or partial (Figure 2B; threeK1 channels and/or inhibition of Ca21 channels (Bean,out of seven cells) inhibition of electrical activity. In the1989; Hille, 1994; Chen and Clarke, 1992; Chen et al.,latter case, inhibition was manifested as an increased1992; Ishibashi and Akaike, 1995) with resultant sup-duration of the silent intervals.pression of Ca21-dependent exocytosis. These effects

are often mediated by activation of protein kinases, pro-tein phosphatases, and G proteins (Swain et al., 1991; G Protein–Dependent Inhibition of Exocytosis

by SomatostatinGalindo et al., 1992; Ammala et al., 1993a). The insulin-secreting pancreatic B cells share many features with The effects of somatostatin on whole-cell Ca21 currents

and exocytosis in a b cell are illustrated in Figure 3A. Theother neuroendocrine cells. They are electrically excit-able, and exocytosis of the insulin-containing granules recording was carried out using the perforated patch

whole-cell configuration, and the cell was stimulated byis initiated by an increase in the cytoplasmic Ca21 con-centration resulting from activation of plasma mem- the application of a voltage-clamp depolarization from

270 mV to 0 mV. Under control conditions, the 200 msbrane voltage-gated L-type Ca21 channels (Ashcroft andRorsman, 1989). Insulin secretion is controlled by a vari- depolarization evoked a peak Ca21 current of 80 pA and

a step capacitance increase of 50 fF; 2 min after theety of neurotransmitters and hormones (reviewed byHowell, 1991; Rasmussen et al., 1990). The molecular addition of somatostatin (400 nM), the depolarization

evoked a peak Ca21 current of 75 pA but failed to elicitprocesses by which somatostatin, adrenalin, and ga-lanin inhibit insulin secretion in the pancreatic b cell exocytosis. The inhibitory action of somatostatin onexo-

cytosis was reversed within 4 min of its withdrawal fromremain unclear (reviewed by Gillison and Sharp, 1994).

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Figure 1. Somatostatin-Induced Inhibition ofGlucose-Induced Electrical Activity

(A) Effects of somatostatin (400 nM) on elec-trical activity evoked by 10 mM glucose.(B) Failure of somatatostatin to affect electri-cal activity evoked by 10 mM glucose andsubsequent inhibition by galanin (100 nM).

the medium, and the depolarization then evoked a Ca21 91% 6 6% (p < 0.001; n 5 6) and 89% 6 7% (p < 0.001;n 5 7), respectively.The action of clonidinewas reversedcurrent of 85 pA and a capacitance increase of 45 fF.

On average, 400 nM somatostatin produced 88% 6 5% by the a2-adrenergic antagonist yohimbine. Inhibition ofexocytosis by galanin and clonidine was not associated(p < 0.001; n 5 7) inhibition of exocytosis and 4% 6 3%

(n 5 7) reduction of the peak Ca21 current (Figure 3B). with a reduction of the Ca21 current, and their actionswere prevented by pretreatment with pertussis toxinThe effect of somatostatin on exocytosis was probably

mediated by activation of inhibitory G proteins, since (data not shown).pretreatment of the b cellswith pertussis toxin abolishedthe inhibitory action (Figures 3C and 3D). Interactions between Guanine Nucleotides

and Inhibitory AgonistsThe effects of somatostatin, galanin, and clonidine onInhibitory Action of Somatostatin on Exocytosis

Is Shared by Adrenalin and Galanin exocytosis remained observable in standard whole-cellrecordings that have the advantage of permitting theThe ability of somatostatin to inhibit exocytosis was

shared by clonidine, an a2-adrenergic agonist, and the cell interior to be dialysed by the pipette solution (Hamillet al., 1981). When the intracellular solution containedneuropeptide galanin. When applied at concentrations

of 100 nM or 5 mM, galanin (Figures 4A and 4B) and 10 mM GTP, application of somatostatin reversibly sup-pressed exocytosis (Figures 5A and 5B). On average,clonidine (Figures 4C and 4D) reversibly suppressed ex-

ocytosis in perforated patch whole-cell recordings by somatostatin produced 86% 6 5% (n 5 6; p < 0.001)

Figure 2. Galanin-Induced Inhibition of Glu-cose-Induced Electrical Activity

Complete (A) and partial (B) inhibition by ga-lanin of glucose-induced electrical activity.The glucose concentration was 15 mM.

Inhibition of Exocytosis in b Cells515

Figure 3. Somatostatin-Induced Inhibition of Exocytosis

(A) Ca21 currents and changes in membrane capacitance evoked by 200 ms depolarizations before, 2 min after addition of 400 nM somatostatin,and 6 min after its removal.(B) Effects of somatostatin on peak Ca21 current (ICa) and exocytotic responses (DCm) before, during, and after addition of somatostatin (Sst).(C and D) Conditions were as in (A) and (B), but cells had been pretreated for >4 hr with 100 ng/ml pertussis toxin. Data in (B) and (D) aremean values 6 SEM of seven and five experiments, respectively. The double asterisks indicate p < 0.01; the triple asterisks indicate p < 0.001.

inhibition of exocytosis in the standard whole-cell con- a [Ca21]i increase from a basal 0.2 mM to a peak concen-tration of 1.5 mM and a capacitance increase of 50 fF.figuration, similar to that obtained in the intact cells (see

above). Inclusion of 0.5 mM of the stable GDP analog Following the addition of somatostatin, the same depo-larization evoked a [Ca21]i transient that actually wasGDPbS in the intracellular solution prevented the inhibi-

tory effect of somatostatin, and the exocytotic re- larger than that seen under control conditions (from aresting 0.2 mM to a peak >2 mM) and yet exocytosis wassponses in the presence of the agonist amounted to

99% 6 8% (n 5 6) of the control amplitude (Figures 5C almost abolished. The effects of somatostatin on bothexocytosis and [Ca21]i were reversed upon removal ofand 5D). By contrast, when 10 mM GTPgS (a stable GTP

analog that causes irreversible activation of the GTP- the agonist from the medium. In this series of six experi-ments, the average inhibition of exocytosis producedbinding proteins) was added to the pipette solution,

somatostatin produced an irreversible 74% 6 6% inhi- by somatostatin amounted to 71% 6 11% (p < 0.005).The effect was associated with an average 21% 6 15%bition of exocytosis (Figures 5E and 5F). These inter-

actions between somatostatin and the stable guanine increase in the amplitude of the depolarization-evokedpeak [Ca21]i (Figure 6B): an increase being observed innucleotides reinforce the observations made with per-

tussis toxin in intact cells and suggest that the action five out of six experiments.on exocytosis is mediated by activation of GTP-bindingproteins (Neer and Clapham, 1988). Effects of the Inhibitory Agonists Involve

Activation of CalcineurinWe have previously demonstrated that activation of pro-Somatostatin-Induced Inhibition of Exocytosis

Is Not due to Reduction of Cytoplasmic Ca21 tein kinases leads to potentiation of exocytosis via amechanism exerted at a level distal to the elevationSince insulin secretion is a Ca21-dependent process, it

was important to ascertain that somatostatin-induced of [Ca21]i (Ammala et al., 1993b, 1994). Here, we haveexplored the possibility that activation of protein phos-inhibition of exocytosis is not simply attributable to a

reduced amplitude of the depolarization-evoked [Ca21]i phatases has the opposite effect and mediates agonist-induced inhibition of exocytosis. This we tested bytransient. Figure 6A shows parallel recordings of depo-

larization-evoked [Ca21]i and capacitance increases in including phosphatase inhibitors in the cytoplasmicsolution dialyzing the cell interior. Okadaic acid, an in-the absence and presence of somatostatin. Under con-

trol conditions, a 500 ms depolarization to 0 mV evoked hibitor of type 1, type 2A, and type 3 serine/threonine

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Figure 4. Galanin- and Clonidine-Induced Inhibition of Exocytosis

(A) Ca21 currents and changes in membrane capacitance elicited by membrane depolarization before, 2 min after addition of 100 nM galanin,and 6 min after its removal.(B) Effects of galanin on peak Ca21 current (ICa) and exocytotic responses (DCm) before, during, and after addition of galanin (Gal).(C and D) Conditions were as (A) and (B), but cells were exposed to 5 mM clonidine (Clo) and 20 mM yohimbine (Yoh) as indicated. Data in(B) and (D) are mean values 6 SEM of six experiments. The double asterisks indicate p < 0.01; the triple asterisks indicate p < 0.001.

protein phosphatases (Cohen et al., 1990), failed to Discussioncounteract the inhibitory action of somatostatin (Figure7B), and the agonist reduced the exocytotic responses Using capacitance measurements of exocytosis, we

have explored the molecular mechanisms that underlieevoked by membrane depolarization by 75% 6 10% (p <0.001, n 5 11), similar to that observed in the absence neurotransmitter-induced inhibition of insulin secretion

from pancreatic b cells. Our data reinforce previous ar-of the phosphatase inhibitor (Figures 5A and 7A). Bycontrast, deltamethrin (an inhibitor of the Ca21-depen- guments, based on measurements of insulin secretion

in permeabilized b cells, that agonists such as somato-dent phosphatase calcineurin, type 2B; Enan and Matsu-mura, 1992) abolished the inhibitory action of somato- statin inhibit exocytosis by a G protein–mediated mech-

anism at a site distal to the elevation of Ca21. Here, westatin (Figure 7C). Calcineurin autoinhibitory peptide, ahighly selective inhibitor of calcineurin corresponding to consider a few particularly interesting aspects.the calmodulin-binding domain of calcineurin (residues457–482; Perrino et al., 1995), likewise removed the in-hibitory action of somatostatin (Figure 7D). Collectively, Significance of Membrane Potential Repolarization

Recordings of glucose-stimulated electrical activity inthese observations strongly suggest that the suppressoraction of somatostatin on exocytosis is mediated by mouse pancreatic islets have revealed that galanin and

adrenalin produce a transient membrane repolarizationactivation of calcineurin.Dephosphorylation catalyzed by calcineurin is also and subsequently increase the duration of the silent

(electrically inactive) intervals between two successivelikely to underlie inhibition of exocytosis produced byother neurotransmitters. As illustrated in Figure 8A, clon- bursts of action potentials (Cook and Perara, 1980;

Drews et al., 1990, 1994; Debuyser et al., 1991). Theidine abolished depolarization-evoked exocytosis. Onaverage, clonidine produced 85% 6 5% (p < 0.001; n 5 ability of somatostatin and galanin to inhibit glucose-

stimulated electrical activity was compared with their7) inhibition of exocytosis. This effect was effectivelyprevented by the inclusion of deltamethrin in the pipette capacity to suppress Ca21-dependent exocytosis. We

demonstrate that somatostatin, at the concentration re-solution (Figure 8B), and in the presence of this phos-phatase inhibitor, clonidine lacked inhibitory action in quired to abolish exocytosis, produced (at most) partial

inhibition of electrical activity. The latter effect probablythree out of four cells. Similar observations were madewith galanin (data not shown). resulted from the activation of K1 channels distinct from

Inhibition of Exocytosis in b Cells517

Figure 5. Inhibitory Action of Somatostatin Involves Activation of G Proteins

Exocytotic responses evoked by depolarizations to 0 mV before, 2 min after addition of 400 nM somatostatin (stippled area), and 2 min afterits removal when the cells were dialyzed with 10 mM GTP (A), when the intracellular medium was supplemented with 10 mM GTP and 0.5 mMGDPbS (C), and after replacement of GTP with 10 mM GTPgS (E). The histograms in (B), (D), and (F) show the average exocytotic responsesobtained in the absence and presence of somatostatin (Sst) as indicated. Data are mean values 6 SEM of six experiments in each group.The double asterisks indicate p < 0.01; the triple asterisks indicate p < 0.001.

Figure 6. Somatostatin-Induced Inhibition of Exocytosis Does Not Involve Reduction of Cytoplasmic Ca21 Concentration

(A) Simultaneous measurements of [Ca21]i and membrane capacitance before, 3 min after addition of 400 nM somatostatin (stippled area),and 4 min after its removal.(B) Histograms showing effects of somatostatin onexocytosis (DCm) and depolarization-evoked peak [Ca21]i before, after additionof somatostatin(Sst), and following its removal. Data are presented as mean values 6 SEM of six experiments. The double asterisks indicate p < 0.01.

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Figure 7. Protein Phosphatase Activity and Somatostatin-Induced Inhibition of Exocytosis

Whole-cell capacitance responses elicited by depolarizations to 0 mV before and 2 min after addition of somatostatin (stippled area) whenthe cells were dialyzed with control medium (A), with 100 nM okadaic acid (B), with 20 nM deltamethrin (C), or with 0.1 mM of the calcineurinautoinhibitory peptide (D). The horizontal lines above the capacitance traces in the presence of somatostatin indicate the amplitude of therespective control response. The histograms to the right of the capacitance traces show mean exocytotic responses 6 SEM of 5–14 experimentsin the presence and absence of somatostatin (Sst) as indicated. The double asterisks indicate p < 0.01; the triple asterisks indicate p < 0.001.

Figure 8. Deltamethrin Abolishes Inhibitory Action of Clonidine on Exocytosis

Whole-cell capacitance responses elicited by depolarizations to 0 mV before and after addition clonidine (stippled area) when the cells weredialyzed with control medium (A) or with 20 nM deltamethrin (B).

Inhibition of Exocytosis in b Cells519

the K-ATPchannel, as the effects were resistant to appli- involving enhancement of Ca21 influx (Ammala et al.,1993a, 1994). Here, we demonstrate that physiologicalcation tolbutamide and glibenclamide (Rorsman et al.,

1991; Wahlander et al., 1991), pharmacological inhibi- inhibitors of exocytosis also act by a mechanism exertedat a level distal to the elevation of cytoplasmic Ca21. Thetors of K-ATP channels (Ashcroft and Rorsman, 1989).

The failure of somatostatin and galanin to inhibit glu- effect is mediated by G protein–dependent activation ofthe protein phosphatase calcineurin, since the inhibitorycose-induced electrical activity completely and persis-

tently argues that the action on exocytosis is physiologi- effect of somatostatin and adrenalin was abolished bymaneuvers that suppress the activity of this enzyme. Thecally important and contributes significantly to the

overall inhibitory action on insulin secretion. This would ability of protein kinases and phosphatases to modulateCa21-dependent exocytosis suggests that the magni-apply especially to the situation in vivo when electrical

activity represents the combined response to several tude of thesecretory response depends principally uponthe extent of phosphorylation of certain (as yet unidenti-stimuli, many of which depolarize the b cell membrane

potential by mechanisms other than reduction of K1 fied) proteins involved in the distal steps of exocytosisrather than amplitude of the cytoplasmic Ca21 transient.permeability as exemplified by the amino acid arginine

(cf. Drews et al., 1994) that elicits electrical activity byits electrogenic entry via a y1 transporter (Sakura et al.,

Comparison with Neurons and Other1995).Neuroendocrine CellsThe decisive role of an increased cytoplasmic Ca21 con-Neurotransmitters Inhibit Exocytosiscentration for the initiation of exocytosis in neuroendo-without Affecting Ca21 Currentcrine cells and nerve terminals has been extensivelyIt has been proposed that the inhibitory action of so-documented (Thomas et al., 1993; von Ruden and Neher,matostatin, galanin, and adrenalin on electrical activity1993; Artalejo et al., 1994; Heidelberger et al., 1994).and secretion is attributable to inhibition of the voltage-Peptidergic and adrenergic inhibition of exocytosis isgated Ca21 current (Keahey et al., 1990; Hsu et al., 1991;not unique to the pancreatic b cell and is observedHomaidan et al., 1991). Since exocytosis is Ca21 depen-in both other neuroendocrine cells and neurons (Hille,dent in the b cell, such an effect would easily account1994). In general, agonist-induced suppression of hor-for the observed suppression of exocytosis. This maymone and neurotransmitter release is believed to bewell be of importance in the clonal rat and hamster bmediated by a reduction of the rate of Ca21-dependentcells used in these earlier studies. However, our presentsecretion: either by activation of K1 channels, with resul-data indicate that inhibition of the Ca21 current does nottant membrane repolarization and suppression of Ca21

contribute to the inhibitory action in mouse b cells, asinflux through the voltage-gated Ca21 channels, or byneither the peak Ca21 current nor the amplitude of thedirect inhibition of the Ca21 channels (Bean, 1989; Hille,depolarization-evoked [Ca21]i transient was affected by1994; Chen and Clarke, 1992; Chen et al., 1992; Ishibashiany of the agonists. This is in keeping with the failureand Akaike, 1995). Our data imply that this is not theof the agonists to reduce depolarization-evoked in-only level of control and that exocytosis may also becreases in [Ca21]i (Nilsson et al., 1988, 1989). We con-regulated distally to the elevation of cytoplasmic Ca21.clude that Ca21 channel inhibition isnot part of the mech-It follows from this that an increase in [Ca21]i is notanisms by which somatostatin, adrenalin, and galaninobligatorily associated with acceleration of secretion.suppress insulin secretion. We further suggest that inhi-Indeed, as illustrated by the present study, somato-bition of exocytosis results from a mechanism exertedstatin-induced inhibition of exocytosis in the b cell wasat a level distal to the elevation of [Ca21]i.often associated with an increased amplitude of thedepolarization-evoked [Ca21]i transient.

Inhibition of Exocytosis Does Not Result It has previously been demonstrated that inhibition offrom Inhibition of Adenylate Cyclase catecholamine release from chromaffin cells by chro-Galanin, adrenalin, and somatostatin all suppress ade- mostatin involves activation of an okadaic acid–sensi-nylate cyclase activity (Ullrich and Wollheim, 1988; Sea- tive protein phosphatase (Galindo et al., 1992). The latterquist et al., 1992). It could therefore be argued that the effect was interpreted to be exerted at the level of theobserved inhibition of exocytosis is simply the result of Ca21 channels; the possible contribution of distal effectsreduced cytoplasmic levels of cyclic AMP and suppres- was not investigated. Phosphatases also appear to havesion of protein kinase A–stimulated exocytosis (Ammala the capacity to modulate synapatic transmission, andet al., 1993a). This explanation can be discarded, how- okadaic acid and inhibitors of calcineurin have beenever, as the agents remained effective even when the reported to stimulate evoked transmitter release in neu-cytoplasmic cyclic AMP concentration was clamped at rons (Swain et al., 1991; Nichols et al., 1994; Victor et100 mM by inclusion of the nucleotide in the pipette al., 1995). The present study demonstrates that physio-solution that dialyses into the interior of the cell. logical agonists control exocytosis directly at a site dis-

tal to Ca21 influxby activation of the protein phosphatasecalcineurin. Regulation of exocytosis by both kinasesRole of Ca21 versus Protein Phosphorylation

in the Control of Exocytosis and phosphatases clearly adds flexibility to the controlof the release process. Given the similarities of exo-We have previously reported that pharmacological acti-

vation of protein kinases A and C and inhibition of oka- cytosis in various cell types, it finally seems possiblethat the direct modulation of exocytosis mediated bydaic acid–sensitive protein phosphatases lead to stimu-

lation of Ca21-dependent exocytosis by mechanisms not activation of protein phosphatases we now describe in

Neuron520

K2SO4, 10 mM NaCl, 10 mM KCl, 1 mM MgCl2, and 5 mM HEPESthe pancreatic b cell also underlies inhibition of hormone(pH 7.35, adjusted with KOH). In all perforated patch whole-celland neurotransmitter release in other cell types.recordings, electrical contact with the cell interior was establishedby addition of the pore-forming antibiotic amphotericin B (Sigma,

Experimental Procedures St. Louis, MO) (Rae et al., 1991). Perforation required a few minutes,and the voltage-clamp was considered satisfactory when Gseries ex-

Preparation and Culture of Cells ceeded 40 nS.All experiments were carried out on isolated mouse pancreatic b For the standard whole-cell experiments (Figures 5–8), the intra-cells. NMRI mice were purchased from commercial breeders. The cellular solution dialyzing the cell interior consisted of 125 mM Cs-

glutamate, 10 mM CsCl, 10 mM NaCl, 1 mM MgCl2, 3 mM Mg-ATP,mice were stunned by a blow against the head and killed by cervical5 mM HEPES, 0.05 mM EGTA, 0.1 mM cyclicAMP, and 0.01 mM GTP.dislocation. The pancreas was quickly removed, and pancreaticGuanine nucleotides other than GTP were included as described inislets were isolated by collagenase digestion. Single b cells werethe text. Recordings started >1 min after the establishment of theprepared by shaking in a Ca21-free medium as previously describedwhole-cell configuration to permit wash-in of the pipette solution.(Rorsman and Trube, 1986). Cells were placed on Corning petriAccess resistance ranged between 10–20 MV.dishes and maintained in tissue culture for up to 2 days in RPMI

Somatostatin, galanin, yohimbine, deltamethrin, and okadaic acid1640 medium containing 5 mM glucose and 10% (v/v) fetal calfwere dissolved in DMSO (final concentration of DMSO: 0.01%–serum (Flow Laboratories, Irvine, United Kingdom) and supple-0.1%). All other compounds were dissolved in water. The inhibitorymented with 100 mg/ml streptomycin and 100 IU/ml penicillinagonists and antagonists were purchased from Sigma, whereas(both from Northumbria Biologicals, Limited, Cramlington, Unitedthe phosphatase inhibitors okadaic acid and deltamethtrin wereKingdom).supplied by Molecular Probes (Eugene, OR) and Alomone Labs (Je-rusalem, Israel), respectively.Calcineurin autoinhibitory peptide was

Electrophysiology obtained from Calbiochem (La Jolla, CA) and was allowed to washWhole-cell membrane currents were recorded using an EPC-7 into the cells for at least 2 min before the experiments commenced.patch-clamp amplifier (List Electronic, Darmstadt, Federal Republic Except for the experiments involving fluorimetric recordings of theof Germany). The recordings were performed using both the stan- cytoplasmic Ca21 concentration, the experimental chamber con-dard (Hamill et al., 1981) and perforated patch whole-cell configura- sisted of the plastic petri dish with a plastic insert to reduce thetions (Horn and Marty, 1988). To detect changes in membrane ca- volume to approximately 0.5 ml, which was continuously perfusedpacitance, which reflects exocytosis, a 20 mV root-mean-square at a rate of 2 ml/min. All experiments were performed at 348C.(rms) 800 Hz sine wave was applied to the holding potential (270 mV)as previously described (Ammala et al., 1993b; Joshi andFernandez, Measurements of [Ca21]i1988). The resulting current was analyzed at two orthogonal phase The [Ca21]i measurements were made using an Axiovert 135 invertedangles and ten cycles averaged for each data point, giving a resolu- microscope equipped with a Plan-Neofluar 1003/1.30 objectivetion of 100 ms per point. The voltage-clamp depolarizations applied (Carl Zeiss, Oberkochen, Federal Republic of Germany) and an Io-to evoke exocytosis went to 0 mV and were (unless otherwise indi- noptix (Milton, MA) fluorescence imaging system as described else-cated) applied at 2 min intervals. The phase angle was adjusted where (Bokvist et al., 1995). Excitation was effected at 340 andprior to each stimulation to ensure that a change in cell capacitance 380 nm and emitted light recorded at 510 nm with a video cameradoes not arise as a consequence of variations of cell conductance. synchronized to theexcitation light source and a computer interface.Indeed, the step increases in cell capacitance were not associated The experiments were conducted in the standard whole-cell config-with any change of the cell conductance, which usually remained uration using the pipette-filling solution specified above supple-stable over several minutes. mented with 0.1 mM fura-2 (pentapotassium salt; Molecular Probes,

The effects of somatostatin and galanin on the glucose-induced Eugene, OR) and 10 mM GTP.electrical activity membrane potential were measured from superfi-cial b cells in intact collagenase-isolated mouse pancreatic islets Data Analysis

Data are presented as mean values 6 SEM of n cells. Statistical(Figures 1 and 2) using the peforated patch whole-cell configurationsignificances were evaluated using Student’s t test. Experimentsand an EPC-9 patch-clamp amplifier (Heka Electronics, Lambrecht/using the perforated patch whole-cell configuration commencedPfalz, Federal Republic of Germany). Pancreatic b cells were func-when the exocytotic responses evoked by two subsequent depolar-tionally identified by the characteristic electrical activity seen at 10izations (2 min interval) were of the same amplitude to ascertainmM glucose (cf. Figure 1 and Henquin and Meissner, 1984). Thesethat the observed effects do not simply reflect spontaneous changescells had voltage-gated membrane currents similar to those pre-of the exocytotic capacity. For the same reason, only the experi-viously described in isolated cultured b cells (Rorsman and Trube,ments in which exocytosis recovered from inhibition were included1986). We regularly encountered another type of cell, tentativelyin the statistical analysis.identified as glucagon-secreting a cells because they were not elec-

trically active in the presence of 10 mM glucose, and contained aAcknowledgmentslarge voltage-dependent Na1 current (cf. Rorsman and Hellman,

1988). Such cells were not used in the present study.We thank Drs. Lena Eliasson and Jesper Gromada for critically read-ing the manuscript. Dr. W.-G. Ding is on leave from the Department

Solutions of Physiology, Shiga University, Japan. The initial stages of thisThe standard extracellular medium consisted of 118 mM NaCl, 20 project were supported by grants from the Swedish Medical Re-mM TEA–HCl (to block voltage-gated K1 currents), 5.6 mM KCl, 1.2 search Council, The Juvenile Diabetes Foundation International, ThemM MgCl2, 2.6 mM CaCl2, 5 mM D-glucose, 5 mM HEPES (pH 7.4, Novo-Nordisk Foundation, The Swedish Diabetes Association, andadjusted with NaOH). In the perforated patch whole-cell recordings The Magn. Bergvalls Stiftelse.of exocytosis (Figures 3 and 4), the pipette solution contained 76 The costs of publication of this article were defrayed in part bymM Cs2SO4, 10 mM NaCl,10 mM KCl, 1 mM MgCl2, and 5 mM HEPES the payment of page charges. This article must therefore be hereby(pH 7.35, adjusted with CsOH). In these experiments, forskolin (2 marked “advertisement” in accordance with 18 USC Section 1734mM) was included in the extracellular medium to increase the ampli- solely to indicate this fact.tude of the exocytotic responses (Ammala et al., 1993a). The re-cordings of the membrane potential from b cells in intact pancreatic Received March 26, 1996; revised July 1, 1996.islets (Figures 1 and 2) were performed using the perforated patchwhole-cell congfiguration and an extracellular medium consisting Referencesof 140 mM NaCl, 3.6 mM KCl, 2 mM NaHCO3, 0.5 mM NaH2PO4, 0.5mM MgSO4, 5 mM HEPES (pH 7.4, adjusted with NaOH), 2.6 mM Ammala, C., Ashcroft, F.M., and Rorsman, P. (1993a). Calcium-inde-CaCl2, and 10 mM or 15 mM D-glucose (as indicated). In the latter pendent potentiation of insulin release by cyclic AMP in single

b-cells. Nature 363, 356–358.type of recordings, the pipette solution was composed of 76 mM

Inhibition of Exocytosis in b Cells521

Ammala, C., Eliasson, L., Bokvist, K., Larsson, O., Ashcroft, F.M., inhibits a dihydropyridine-sensitive Ca21-current in the RINm5F cellline. Proc. Natl. Acad. Sci. USA 88, 8744–8748.and Rorsman, P. (1993b). Exocytosis elicited by action potentials

and voltage-clamp calcium currents in individual pancreatic B-cells. Horn, R., and Marty, A. (1988). Muscarinic activation of ionic currentsJ. Physiol. 472, 665–688. measured by a new whole-cell recording method. J. Gen. Physiol.

92, 145–159.Ammala, C., Eliasson, L., Bokvist, K., Berggren, P.-O., Honkanen,R.E., Sjoholm, A., and Rorsman, P. (1994). Activation of protein Howell, S.L. (1991). Insulin biosynthesis and secretion. In Textbookkinases and inhibition of protein phosphatases play a central role of Diabetes, J. Pickup and G. Williams, eds. (Oxford: Blackwell Sci-in the regulation of exocytosis in mouse pancreatic b-cells. Proc. entific Publications), pp. 72–83.Natl. Acad. Sci. USA 91, 4343–4347.

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