Valinomycin inhibition of insulin release and alteration of the electrical properties of pancreatic B cells

455

Biochimica et Biophysica Acta, 543 (1978) 455--464 © Elsevier/North-Holland Biomedical Press

BBA 28691

VALINOMYCIN INHIBITION OF INSULIN RELEASE AND A L T E R A T I O N OF THE ELECTRICAL PROPERTIES OF PANCREATIC B CELLS J.C. HENQUIN and H.P. MEISSNER Unitd de Diab~te et Croissance, University of Louvain, UCL 54.74, B-1200 Brussels (Belgium) and L Physiologisches Institut der Universit~'t des Saarlandes, Homburg/Saar (F.R.G.) (Received March 22nd, 1978)

Summary The effects of valinomycin on insulin release and rubidium efflux from perifused isolated rat islets were investigated and correlated with its effects on the electrical properties of mouse B cells studied with microelectrode techniques. Valinomycin produced a (1 • 10-9--1 • 10 -6 M) dose- and time-dependent inhibition of (10--15 mM) glucose-stimulated insulin release b u t did n o t affect basal secretion. This inhibitory effect rapidly followed addition of the ionophore and equally affected the two phases of glucose-stimulated secretion. It was n o t reversible by simple washing of the islets, b u t could be reversed transiently by tetraethylammonium or high extracellular potassium ion levels. At low or high glucose, valinomycin rapidly augmented the rate of 86Rb÷ efflux from preloaded islets. Amplitude and rapidity of this effect were dose-dependent and it was antagonized by tetraethylammonium. Glucose metabolism by islet cells was reduced only slightly (15%} by 1 • 10 -7 M valinomycin. During the first 6 to 8 rain of valinomycin addition, the membrane potential of B cells augmented slowly b u t the typical bursts of spikes disappeared rapidly. Later on, B cells hyperpolarized more quickly to a stable value of approx. --70 mV. Increasing extracellular K * immediately depolarized B cells and the linear relationship found between the logarithm of K ÷ concentration and the membrane potential was characterized by a slope of 58 mV for a ten-fold increase in extracellular K ÷. These results suggest that valinomycin interferes with the insulin releasing effect of glucose by increasing the potassium permeability of the B cell membrane.

Introduction

It has been recently demonstrated that physiological concentrations of D-glucose diminish potassium efflux from pancreatic islet cells [1]. The charac-

456 teristics of this effect of glucose support the suggestion [2] that a reduction in the potassium permeability of B cells may be involved in the depolarizing effect of the sugar [3,4]. In addition, tetraethylammonium, a specific blocker of potassium conductance in excitable membranes [5,6], potentiates the effect of glucose on both the stimulation of insulin release and inhibition of S6Rb÷ efflux from isolated islets [7]. In the present study, we have attempted to evaluate further the importance of the secretagogue-induced reduction in potassium permeability of B cells for the stimulus-secretion coupling. Potassium ionophores provide a tool for testing the influence, on the B cell functions, of an artificially increased permeability of the plasma membrane for potassium ions. Valinomycin was selected, because this depsipeptide antibiotic increases the potassium-permeability of artificial and biological membranes with an unsurpassed K*/Na÷ selectivity [8-10]. Materials and Methods

Animals and solutions Electrophysiological recordings were made in partially microdissected islets of fed female NMRI mice (25--30 g). All other experiments were made with islets isolated from fed male Wistar rats (275--325 g) killed 3 h after intraperitoneal injection of 20 mg Pilocarpine/kg [11]. The islets were obtained by combining mild collagenase digestion of the gland (5 mg collagenase per pancreas and shaking for 3.5--4 min) and microdissection of the partially digested pieces. The medium utilized was a Krebs-Ringer bicarbonate buffer, pH 7.4, gassed with O2/CO2 (94 : 6), with the following ionic composition (mM): NaC1, 118; KC1, 4.8; CaCI~, 2.5; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25; and containing 3 mM glucose in basal conditions. Except in electrophysiological experiments, it was supplemented with 0.5% bovine serum albumin. For metabolic studies, 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) was added to the medium to prevent any change of pH in the small volumes of solutions used in these experiments. Measurements o f insulin release, rubidium efflux and glucose metabolism The incubation and perifusion techniques utilized to study insulin release and the method for measurement of immunoreactive insulin have been reported previously [12]. At the highest concentration studied, valinomycin did not interfere with the immunoassay. The technique to monitor the efflux of 86Rb÷ from preloaded islets was almost identical to that recently described in detail [7]. The only difference was that S6Rb* was counted by the Cerenkov radiation after addition to each sample of 10 ml of a 3 mM aqueous solution of the wavelength shifter 7-amino1.3-naphthalenedisulfonic acid [13]. Glucose utilization by islet cells was measured by the production of [all]water from D-[5-3H]glucose [14] and glucose oxidation by the conversion of D-[U-14C]glucose to '4CO~. The technical aspects of these methods have been described elsewhere [ 12 ].

457

Electrophysiological recordings Single B cells were impaled with microelectrodes filled with 2 M potassium citrate (tip resistance between 200 and 300 MFt) and the membrane potential was continuously recorded with an oscilloscope (Tektronix 565) and a fast chart writer (Brush recorder). Details of the m e t h o d have been published previously [ 4].

Chemicals Valinomycin, purchased from Sigma Chemical Co., St. Louis, Mo., U.S.A., was dissolved in dimethylsulfoxide at a concentration of 1 • 10 -3 M. Aliquots of the stock solution, stored at 4°C, were added to the test solutions before each experiment. The final concentration of Me2SO never exceeded 0.1% and was generally 10--100 times lower. 86RbC1 (300 Ci/mol), D-[U-14C]glucose (327 Ci/mol) and D-[5-3H]glucose (1 Ci/mmol) were obtained from the Radiochemical Center, Amersham, U.K.; Me2SO was from Sigma Chemical Co., HEPES from British Drug Houses, Poole, Dorset, U.K., 7-amino-l,3-naphthalenedisulfonic acid from Aldrich, Beerse, Belgium, tetraethylammonium chloride from Merck A.G., Darmstadt, F.R.G. and Collagenase type IV (200 units/mg) from Worthington Biochemical Co., Freehold, N.J., U.S.A. All other reagents were of analytical grade. Results

Effects of valinomycin on insulin release Valinomycin was without significant effect on basal insulin secretion. Islets incubated for 60 min in the presence of 3 mM glucose released 0.50 ± 0.04, 0.54 +- 0.05, 0.51 +- 0.06 and 0.56 +- 0.06 ng immunoreactive insulin per islet (mean + S.E., n = 8) in the absence of or in the presence of 1 • 10 -8, 1 • 10 -7 and 1 • 10 -6 M valinomycin, respectively. Valinomycin produced a dose- and time-dependent inhibition of glucosestimulated insulin release from incubated islets (Fig. 1). In control islets, 15 mM glucose evoked an almost linear immunoreactive insulin release over 90 min. At the lowest concentration studied ( 1 - 1 0 -9 M), valinomycin did not affect immunoreactive insulin release during the first 30 min of presence, b u t reduced it by almost 50% during the subsequent 30 min. At 1 . 1 0 -8 M, valinomycin already clearly inhibited immunoreactive insulin release during the initial 30 min of presence. Yet, the inhibitory effect of all concentrations of the drug was greater during the last 30 min, immunoreactive insulin release being practically abolished b y 1 • 10 -~ or 1 • 10 -6 M valinomycin (Fig. 1). The kinetics of valinomycin inhibition immunoreactive insulin release by perifused islets are shown in Fig. 2. In one series of experiments, the ionophore ( 1 • 10 -8 M) was added to 3 mM glucose 5 min before increasing the concentration of the sugar to 15 mM. Glucose-stimulated immunoreactive insulin secretion was delayed and markedly inhibited (P < 0.001) during the early phase (0.30 + 0.05 vs. 0.89 + 0.10 ng immunoreactive insulin per islet) as well as during the second phase (7.70 + 0.51 vs. 25.60 + 1.60 ng immunoreactive insulin per islet). Valinomycin introduction during the second phase of release produced a rapid, dose-dependent fall in the rate of immunoreactive insulin secre-

458

G 15ram

o 100

30

10-gM

o 10-gM

2O

75

C o ~J

~o.~M ~

50

10.r~M n~ 10

,

"~ r~ 25

/,

\ \

/ /

\

/

x

/ /

v/

I

I

0

30 INCUBATION

610 TIME (rain)

0

0

90

10;-9

10=8 10]-? VALINOMYCIN (M)

10-6

Fig. 1. E f f e c t o f v a l i n o m y c i n o n i m m u n o r e a c t i v e insulin ( I R I ) release b y r a t islets i n c u b a t e d in t h e prese n c e o f 15 m M glucose (G). A f t e r 30 rain o f p r e i n c u b a t i o n in t h e p r e s e n c e of 3 m M glucose, b a t c h e s of t h r e e islets w e r e t r a n s f e r r e d i n t o 1 m l m e d i u m s u p p l e m e n t e d w i t h 15 m M glucose a n d i n c u b a t e d for 90 rain at 3 7 ° C . L e f t p a n e l . T i m e - d e p e n d e n t i n h i b i t i o n of i m m u n o r e a e t i v e insulin release b y v a l i n o m y c i n . A f t e r 3 0 rain ( a r r o w ) , 1 0 0 W1 o f t h e i n c u b a t i o n m e d i u m w a s t a k e n for i m m u n o r e a c t i v e insulin m e a s u r e m e n t a n d r e p l a c e d b y 1 0 0 pl o f p r e w a r m e d m e d i u m c o n t a i n i n g a p p r o p r i a t e a m o u n t s of v a l i n o m y c i n to give t h e final c o n c e n t r a t i o n s s h o w n in t h e r i g h t p a r t of t h e figure. R i g h t p a n e l . D o s e - d e p e n d e n t i n h i b i t i o n o f i m m u n o r e a c t i v e insulin release b y v a l i n o m y c i n . F o r t h e t w o p e r i o d s o f i n c u b a t i o n w i t h t h e i o n o p h o r e , i m m u n o r e a c t i v e insulin release in t h e p r e s e n c e o f t h e d i f f e r e n t c o n c e n t r a t i o n s o f t h e d r u g w a s e x p r e s s e d as p e r c e n t a g e o f t h e m e a n c o n t r o l release d u r i n g t h e s a m e p e r i o d a n d w i t h i n t h e s a m e e x p e r i m e n t . V a l u e s are m e a n s ±S.E. o f 14 o b s e r v a t i o n s .

/

G3mM

• O15rnM

~

G15mM ",-Valinomycin

',,vo j.L - - ~o G 15ram + Volinornycin 10-8M 110.8M 0.75

-g o.5o

0.25

-

~0_8M

0

T0"~M

20

30

40

50

60 TIME (rnin)

70

80

90

Fig. 2. E f f e c t o f v a l i n o m y c i n o n t h e d y n a m i c s o f i m m u n o r e a e t i v e insulin ( I R I ) release i n d u c e d b y 15 m M glucose ( G ) in p e r i f u s e d r a t islets. I m m e d i a t e l y a f t e r i s o l a t i o n , g r o u p s o f 15 islets w e r e t r a n s f e r r e d i n t o the p e r i f u s i o n c h a m b e r s . G l u c o s e c o n c e n t r a t i o n was i n c r e a s e d f r o m 3 t o 15 m M a t 3 0 rain. In o n e series o f e x p e r i m e n t s (o), v a l i n o m y c i n (1 - 10 -8 M) was p r e s e n t in t h e m e d i u m f r o m m i n u t e s 25 to 90. In the o t h e r series o f e x p e r i m e n t s ( e ) , v a l i n o m y c i n was p r e s e n t only f r o m m i n u t e s 60 t o 90, at the c o n c e n t r a tion i n d i c a t e d in t h e r i g h t P a r t o f t h e figure. Values are m e a n s +S.E. o f five e x p e r i m e n t s .

459

G 10ram

.,- Vaiinomycin

....

10"8M

• Ve[

0

o Val

10 - B M ÷ K / * 8 r n M

Val

10-8M + TEA

20ram

!i

0./~

-g tt}

E

& o.2 0

E -

0

I

30

I

l

I

[

I

I

~.0

50

60 TIME (min)

70

80

90

F i g . 3. R e v e r s i b i l i t y o f t h e e f f e c t o f v a l i n o m y e i n on i m m u n o r e a e t i v e i n s u l i n ( [ R I ) release b y r a t i s l e t s p e r i f u s e d i n t h e p r e s e n c e o f 10 rnM glucose (G). T h e i o n o p h o r e (1 • 10 -8 M) w a s a d d e d at rain 40. A t m i n 6 0 , v a l i n o r n y c i n was e i t h e r w i t h d r a w n ( e ) o r r e t a i n e d , w h i l e p o t a s s i u m c o n c e n t r a t i o n w a s i n c r e a s e d f r o m 6 t o 48 rnM ( o ) or w h i l e 20 rnM t e t r a e t h y l a r n r n o n i u m ( T E A ) w a s also a d d e d t o t h e m e d i u m (•). V a l u e s are m e a n s +-S.E. o f 12 e x p e r i m e n t s u n t i l r n i n 6 0 a n d o f f o u r e x p e r i m e n t s t h e r e a f t e r .

tion; the inhibitory effect of the ionophore was already significant (P < 0.02) 2 and 4 min after its addition at concentrations of 1" 10 -7 and 1 . 1 0 -8 M, respectively. The reversibility of valinomycin inhibition of immunoreactive insulin release was studied in experiments illustrated by Fig. 3. Simple removal of the ionophore, after 20 min of presence in the perifusate, failed to restore the normal rate of immunoreactive insulin secretion evoked by 10 mM glucose. In contrast, a rise of extracellular K + from 6 to 48 mM or the addition of 20 mM tetraethyla m m o n i u m resulted in a transient increase in immunoreactive insulin secretion despite the continuous presence of valinomycin.

Effects o f valinomycin on rubidium efflux In a solution containing 3 mM glucose, the fractional efflux of 86Rb + slowly diminished with time (Fig. 4A). Introduction of 1 • 10 -7 M valinomycin caused a p r o m p t increase in the efflux rate, which subsequently declined, even before removal of the ionophore. In the presence of 15 mM glucose, the efflux rate of 86Rb+ was lower and more stable than at low glucose, but was also markedly augmented by addition of valinomycin. Amplitude and rapidity of this effect were clearly dose-dependent (Fig. 4B). No obvious change in the effiux of S6Rb+ was observed when valinomycin was omitted from the perifusate, either at low or high glucose. Fig. 5 shows that, in the presence of 10 mM glucose, 1 • 10 -8 M valinomycin stabilized the rate of 86Rb ÷ efflux at a high level and that its withdrawal was followed only by a slow and partial recovery. In contrast, t e t r a e t h y l a m m o n i u m

460 Vatinomycin 0.030

G 3ram

c_ E

_9o

)

]

I

I

A

i

0,025

.O r/

~6 c .9

0.020 I0 -7 I

M

"'"""'"""

I r

0.015

I

[

I

I

G 15mM c 0.025

t, 10") M

_9 ° 0.020 .,Q oo

"6 ,~ 0.015

I0 "s M I

U.

0.010

I

I I I

20

/.0

60 TIME (rain)

i

I

80

100

Fig. 4. Effect o f v a ] i n o m y e i n on 8 6 R b e f f l u x I-tom rat islet~ peliIIJsed in the presence of 3 or 15 m M ~lucose (G). The i o n o p h o r e was added, at the indicated eoneentrat&on, between minutes 40 and 60. The s t i p p l e d line, in A , r e p r e s e n t s c o n t r o l e x p e r i m e n t s in t h e p r e s e n c e o f 3 m M glucose a l o n e . V a l u e s are m e a n s -+S.E. o f t h r e e e x p e r i m e n t s .

• Va(in0mycin

G10mM l

~

---o

10-eM

• Val 10-aM

val 0 .~ Val 10-8M ÷ TEA 2Dram

c

E 0.020

g~ g

.~ olols g h L

20

I

40

60 TIME (min)

8J0

Fig. 5. R e v e r s i b i l i t y o f t h e e f f e c t of v a l i n o m y c i n o n of 10 m M glucose (G). T h e i o n o p h o r e (1 • 10 -8 M) w i t h d r a w n (o), r e t a i n e d a l o n e (¢) or r e t a i n e d while Values are m e a n s *S.E. o f nine e x p e r i m e n t s u n t i l rain

8 6 R b e f f l u x f r o m r a t islets p e r i f u s e d in t h e p r e s e n c e w a s a d d e d at m i n 40. A t rain 60, v a l i n o m y c i n was 20 m M t e t r a e t h y l a m m o n i u m ( T E A ) w a s a d d e d (~). 60 and of three experiments thereafter.

461 TABLE I EFFECT OF VALINOMYCIN ON GLUCOSE METABOLISM BY ISOLATED ISLETS A f t e r p r e l i m i n a r y i n c u b a t i o n f o r 3 0 r a i n a t 37~C in t h e a b s e n c e o f g l u c o s e a n d v a l i n o m y c i n , b a t c h e s o f islets w e r e i n c u b a t e d at 3 7 ° C as f o l l o w s , a. U t i l i z a t i o n s t u d i e s : t e n i s l e t s in 20 pl o f m e d i u m c o n t a i n i n g 10 m M D - [ 5 - 3 H ] g l u c o s e ( 0 . 5 C i / m o l ) a n d t h e i n d i c a t e d c o n c e n t r a t i o n o f v a l l n o m y c i n ; b. O x i d a t i o n s t u d i e s : t e n islets in 50 pl o f m e d i u m c o n t a i n i n g 1 0 m M D - [ U - 1 4 C ] g l u c o s e ( 1 . 2 C i / m o l ) a n d t h e i n d i c a t e d c o n c e n t r a t i o n o f v a i i n o m y c i n . V a l u e s a r e m e a n s +- S,E. o f s e v e n o b s e r v a t i o n s a f t e r 3 0 m i n a n d 12 o b s e r v a t i o n s a f t e r 6 0 r a i n . S i g n i f i c a n c e levels: a, P < 0 . 0 0 5 a n d b, P < 0 . 0 5 vs. t h e a p p r o p r i a t e c o n t r o l s i n t h e absence of valinomyein. Composition of the medium Glucose (raM)

10 10 10

Glucose utilized (pmol/islet)

Glucose oxidized (pmol/islet)

Vallnomycin (M)

0 1 • 10 -8 1 • 1 0 -7

30 r a i n

60 min

60 r a i n

50.7 +_ 1 . 8 -52.4 _+ 3 . 0

1 0 1 . 5 + 3.1 9 9 , 8 +- 3.1 8 6 . 6 _+ 2.5 a

3 7 . 6 + 1.8 3 7 . 5 _+ 1 . 5 3 1 . 9 _+ 1.6 b

produced a rapid, albeit transient, fall in the fractional S6Rb+ efflux, despite the presence of valinomycin.

Effects of valinomycin on glucose metabolism At a concentration of 1 • 10 -s M, valinomycin did n o t affect glucose metabolism by islet cells. At 1 • 10 -7 M, the ionophore reduced utilization and oxidation of the sugar by 15% after 60 min of incubation {Table I).

Glucose

A

70

~

B

Valinomycin 10 -7 M

D

1 m,n

-10

Val,nomyc,n 10 -7 M •

6

E

C

1 rain

~ I

J

I

.~ - z o

10ram

D

2o

I

\

6mMK

+_

£

• -1oI -~--T-'~/J 5s

C" , 5s

!

5s

Fig. 6. Effect of valinomycin on glucose-induced electrical activity and on the m e m b r a n e potential in a single m o u s e B cell. A and B represent a continuous recording over a period of approx. 30 rain, obtained in the s a m e cell and d r a w n with a brush-ink recorder. Glucose concentration was maintained at 10 m M throughout. Valinomycin (1 • 10 -7 M ) w a s added and w i t h d r a w n at times indicated by the d o w n w a r d s directed arrows. W h e n vaiinomycin w a s removed, extracellular K + w a s increased from 6 to 20 raM. T h e delay before appearance of potassium-induced depolarization is due mainly to the dead space of the system. C, D and Z s h o w electrical activity recorded with a faster timescale at the times m a r k e d by bars in A.

462

3

5

10

]-K~o(mM) 20 50

100

J

/I /

200 I

// / t

O. /

~-80

Fig. 7. R e l a t i o n s h i p b e t w e e n t h e c o n c e n t r a t i o n o f e x t r a c e l l u l a r K + a n d t h e m e m b r a n e p o t e n t i a l o f m o u s e B cells. T h e solid line c o r r e s p o n d s to B cells t r e a t e d w i t h v a l i n o m y c i n (1 • 10 -6 a n d 1 • 10 -7 M) in t h e p r e s e n c e o f 10 m M g l u c o s e . V a l u e s are m e a n s +S.D. o f f o u r e x p e r i m e n t s . T h e d o t t e d line w a s o b t a i n e d in c o n t r o l e x p e r i m e n t s p e r f o r m e d at 2.8 m M g l u c o s e w i t h o u t v a l i n o m y c i n t r e a t m e n t a n d c o r r e s p o n d s t o data published elsewhere [ 16].

Effect o f valinomycin on the membrane potential o f B cells As shown in Fig. 6, the normal electrical burst activity [4,15] induced by 10 mM glucose was markedly altered by 1 - 1 0 -7 M valinomycin. During the first minutes following valinomycin addition, the membrane potential of B cells augmented slightly, while the regular bursts (Fig. 6C) were replaced by atypical slow waves (Figs. 6D,E) of progressively decreasing frequency, amplitude and duration. The spikes superimposed on the slow waves also decreased in frequency and amplitude and finally disappeared. After 7--8 min, the cell polarized more markedly and the membrane potential stabilized at a value of --71 mV, which was 9 mV more negative than at 3 mM glucose in the absence of valinomycin. Increasing the extracellular concentration of K ÷ from 6 to 20 mM rapidly produced a sustained and reversible depolarization of B cells similar to that seen without valinomycin treatment [16]. After 60 min of washing with a medium n o t containing the ionophore, the B cells still remained hyperpolarized and no recovery of the normal glucose-induced electrical activity could be observed (not shown). There exists a linear relationship between the stable membrane potential of B cells and the logarithm of the external K + concentration (Fig. 7). In control B cells perifused with 2.8 mM glucose (dotted line), its slope was 47 mV per ten-fold increase in K ÷ concentration [ 16 ]. In B cells treated with valinomycin and studied in the range 4.7--20 mM K ÷, the membrane potential changed with a slope of 58 mV for a ten-fold change in extracellular K ÷. Discussion The results show that valinomycin, in nanomolar concentrations, very effectively inhibits glucose-stimulated insulin release; this finding is in agreement with a previous preliminary report [17]. The data further evidence that the potassium ionophore increases the efflux of Rb ÷ from islet cells, suppresses glucose-induced electrical' activity in B cells and hyperpolarizes them. As also observed in other tissues [18--20], the effects of valinomycin in B cells are not readily reversible by simple washing with a drug-free solution.

463 Compartson of the 42K+ and 86Rb+ effluxes from pancreatic islets justifies the use of the more convenient isotope S6Rb+ to trace the efflux of potassium ions, particularly when qualitative changes in response to physiological conditions are looked upon {refs. I and 7 and unpublished observations}. Although a slight difference in valinomycin affinity for Rb* and K ÷ ions has been demonstrated [8--10], it does not seem sufficient to invalidate the extrapolation of the observed changes in R b + efflux to qualitative K ÷ movements. In intact cells, valinomycin may exert its effects at two sites at least: it may incorporate in the plasma membrane and directly augment the potassium permeability [18,20--23] or interfere with the oxidative phosphorylations in the mitochondria [9,24,25]. It is likely that, thanks to the solvent, valinomycin can penetrate B cells and thus directly affect mitochondria. By increasing the mitochondrial membrane permeability for K + and stimulating cyclic transport of the cation, valinomycin is able to shortcircuit energy production [24,25]. In certain cells this uncoupling effect may even stimulate respiration or glycolysis [26]. In islet cells, the ionophore had only minor effects on the overall glucose metabolism, b u t that does n o t preclude shortage of ATP. If valinomycin proved to depress sufficiently ATP levels in appropriate pools, it might ultimately cause an increase in K ÷ efflux by liberation of cellular Ca 2÷ and subsequent activation of calcium-sensitive potassium channels [27]. Thus, the possibility that some of the observed effects of valinomycin in B cells result from a deficient supply of ATP should n o t be overlooked. Several lines of evidence suggest, however, that the major effect of valinomycin is at the level of the plasma membrane. Soon after addition of the ionophore, the efflux of S6Rb+ augments markedly, the electrical activity disappears and the B cells hyperpolarize considerably. After valinomycin treatment, the B cell membrane behaves as an almost perfect potassium-selective membrane; thus, a change in membrane potential of 58 mV per ten-fold increase in extracellular potassium was found, which is very close to the theoretical value of 61 mV predicted by the Nernst equation for experiments carried out at 37°C. The slope of 47 mV found in control islets [16] suggests that the resting membrane potential at substimulatory glucose concentrations is n o t determined only by the potassium permeability. In addition, two conditions n o t expected to bring about an increase in cellular ATP, b u t to antagonize membrane effects of valinomycin, were found to counteract partially the inhibition of insulin release produced b y the ionophore. Firstly, addition of tetraethylammonium reduced the rate of S6Rb+ efflux augmented by valinomycin and increased the depressed rate of secretion. Both effects were, however, transient in contrast to the effects of tetraethylammonium in the absence of ionophore [7]. Secondly, an increase in extracellular K ÷ was able to reverse steadily the hyperpolarizing effect of valinomycin and to restore transiently insulin secretion. The brevity of this latter effect is n o t surprising since, in normal islets, K ÷ depolarization produces only a monophasic immunoreactive insulin release both at low [28] and stimulatory (J.C. Henquin, unpublished observations) glucose concentrations. Keeping in mind the possibility that the effects of valinomycin on insulin release may be due partially to its uncoupling effect, it is reasonable to pro-

464 pose that the drug mainly acts as K ÷ carrier in the B cell membrane. The resulting increase in K ÷ permeability antagonizes the effects of glucose [ 1 ] and leads to suppression of electrical activity, hyperpolarization and cessation of insulin release. In conclusion, the convergent observations that a decrease in the K ÷ permeability of B cells by tetraethylammonium potentiates glucose-induced insulin release [7], whereas an increase in their K ÷ permeability by valinomycin inhibits the releasing effect of the sugar, strengthen the suggestion that the reduction by D-glucose of the K ÷ permeability of B cells [1,2] is a step or param o u n t importance in the stimulus-secretion coupling.

Acknowledgements We are grateful to Mrs. B. Debie, Miss M. Nenquin and Mr. W. Schmeer for skilled technical assistance. We also thank Profs. J. Crabb~, A.E. Lambert and H. Schmidt for reading the manuscript. J.C.H. is "Charg~ de Recherches" from the Fonds National de la Recherche Scientifique, Brussels. These studies were supported by grant 3.4509.75 from the Fonds de la Recherche Scientifique M~dicale, Brussels, by the Deutsche Forschungsgemeinschaft SFB 38, Bonn Bad-Godesberg and by a grant-in-aid from Hoechst-Belgium S.A. References 1 2 3 4 5 6 7 6 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Henquin, J.C. (1978) Nature 2 7 1 , 2 7 1 - - 2 7 3 Sehlin, J. and T~/ljedal, I.-B. (1975) Nature 253, 635---636 Dean, P.M. and Matthews, E.K. (1970) J. Physiol, Lond. 210, 255--264 Meissner, H.P. and Schmelz, H. (1974) Pfl~igers Arch. 3 5 1 , 1 9 5 - - 2 0 6 Schmidt, H. and St~/mpfli, R. (1966) Pfliigers Arch. 2 8 7 , 3 1 1 - - 3 2 5 Hille, B. (1970) Prog. Biophys. Biol. 21, 3--32 Henquin, J.C. (1977) Biochem. Biophys. Res. Commun. 7 7 , 5 5 1 - - 5 5 6 Pressman, B.C. (1968) Fed. Proc. 27, 1283--1288 Mueller, P. and Ru din, D.O. (1967) Biochem. Biophys. Res. C ommun. 2 6 , 3 9 8 - - 4 0 4 L~'ugar, P. (1972) Science 178, 24--30 Kuo, W.N., Hodgins, D.S, and Kuo, J.F. (1973) J. Biol. Chem. 248, 2705--2711 Henquin, J.C. and Lambert, A.E. (1976) Am. J. Physiol. 2 3 1 , 7 1 3 - - 7 2 1 Lliuchli, A. (1969) Int. J. Appl. Radiat. Isot. 20, 265--27 0 Ashcroft, S.J.H., Weerasinghe, L.C.C., Bassett, J.M. and Randle, P.J. (1972) Biochem. J. 126, 525-532 Meissner, H.P. (1976) J. Physiol. (Paris) 72, 757--767 Meissner, H.P., Henquin, J.C. and Preissler, M. (1977) Proc. Int. Union Physiol. Sci. 1 3 , 4 9 8 Corkey, B.E. and Mayhew, D.A. (1974) Diabetes 23, Suppl. I, 337 (Abstract) Hinkle, M. and Van der Kloot, W. (1973) Comp. Biochem. Physiol. 46A, 269--278 Schneider, J.A. and Sperelakis, N. (1974) Eur. J. Pharmacol. 2 7 , 3 4 9 - - 3 5 4 Carmeliet, E.E. and Lieberman, M. (1975) Pflllgers Arch. 3 5 8 , 2 4 3 - - 2 5 7 Harris, E.J. and Pressman, B.C. (1967) Nature 2 1 6 , 9 1 8 - - 9 2 0 Spector, I., Palfrey, C. and Littaiier, U.Z. (1975) Nature 2 5 4 , 1 2 1 - - 1 2 4 Daniele, R.P. and HoUan, S.K. (1976) Proc. Natl. Acad. Sci. U.S. 73, 3599--3602 McMurray, W.C. and Begg, R.W. (1959) Arch. Biochem. Biophys. 84, 546--548 Pressman, B.C., Harris, E.J., Jagger, W.S. and Johnson, J.M. (1967) Proc. Natl. Acad. Sci. U.S. 58, 1949--1956 Poole, D.T., Butler, T.C. and Williams, M.E. (1972) Biochim. Biophys. A c t a 2 6 6 , 4 6 3 - - 4 7 0 Meech, R,W. (1972) Comp. Biochem. Physiol. 42A, 493--499 Henquin, J.C. and Lambert, A.E. (1974) Diabetes 2 3 , 9 3 3 - - 9 4 2