Calcium and pancreatic β-cell function I. Stimulatory effects of pentobarbital on insulin release

766

Biochimica et Biophysica Acta, 497 (1977) 766--774 © Elsevier/North-Holland Biomedical Press

BBA 28244 CALCIUM AND PANCREATIC ~-CELL FUNCTION I. S T I M U L A T O R Y E F F E C T S O F P E N T O B A R B I T A L O N I N S U L I N RELEASE

BO HELLMAN Department of Histology, Biomedicum, University of Uppsala, S-751 23 Uppsala and Department of Histology, University of Ume~, S-901 8 7 Ume3 (Sweden)

(Received November 25th, 1976)

Summary Islets m i c r o d i s s e c t e d f r o m o b / o b - m i c e w e r e e x p o s e d to 3 m M p e n t o b a r b i t a l in m e d i a which were n o r m a l or d e f i c i e n t in Ca 2÷. This t r e a t m e n t r e s u l t e d in a m a r k e d decrease o f the islet c o n t e n t o f cyclic AMP r e c o r d e d in t h e p r e s e n c e o f the p h o s p h o d i e s t e r a s e i n h i b i t o r 3 - i s o b u t y l - l - m e t h y l x a n t h i n e . P e n t o b a r b i t a l had a dual e f f e c t o n insulin release. In a d d i t i o n to being a p o t e n t i n h i b i t o r o f g l u c o s e - s t i m u l a t e d insulin release in m e d i a c o n t a i n i n g 2.56 mM Ca 2+ it increased t h e a m o u n t s of insulin released in high glucose m e d i a d e f i c i e n t in Ca :+ . T h e r e was a t r a n s i e n t s t i m u l a t i o n w i t h o r d i n a r y c o n c e n t r a t i o n s o f Ca 2+ and 3 m M glucose w h e n t h e m e d i a also c o n t a i n e d 3 - i s o b u t y l - l - m e t h y l x a n t h i n e . T h e stimulat o r y e f f e c t o f p e n t o b a r b i t a l persisted a f t e r replacing p a r t of t h e Ca 2÷ in t h e ~cell m e m b r a n e w i t h l a n t h a n u m ions and it c o u l d n o t be m i m i c k e d b y lowering t h e o x y g e n t e n s i o n of the i n c u b a t i o n m e d i u m . It is suggested t h a t p e n t o b a r b i tal s t i m u l a t i o n o f insulin release is t h e result o f a specific a c t i o n o f t h e drug on t h e d i s t r i b u t i o n o f Ca 2+ w i t h i n t h e p a n c r e a t i c ~-cells.

Introduction R e c e n t studies in this l a b o r a t o r y i n d i c a t e d t h a t the Ca2÷-transporting ionop h o r e X - 5 3 7 A was a p o t e n t s t i m u l a t o r of insulin release at n o n - s t i m u l a t o r y glucose c o n c e n t r a t i o n s [ 1]. T h e i o n o p h o r e a c t i o n was m a r k e d l y p o t e n t i a t e d b y conc e n t r a t i o n s o f p e n t o b a r b i t a l k n o w n to abolish g l u c o s e - s t i m u l a t e d insulin release. This s o m e w h a t surprising o b s e r v a t i o n raises t h e q u e s t i o n w h e t h e r p e n t o b a r b i t a l a l o n e can s t i m u l a t e t h e s e c r e t o r y process in the p a n c r e a t i c ~-cells in a n a l o g y to w h a t has b e e n r e p o r t e d f o r the release o f g r o w t h h o r m o n e f r o m the a n t e r i o r p i t u i t a r y [2]. T h e p r e s e n t s t u d y indicates t h a t this is to s o m e e x t e n t true since

767 pentobarbital both stimulates and inhibits insulin release from the fl-cell-rich pancreatic islets microdissected from ob/ob-mice. Evidence will be presented that stimulation might be the result of a direct action of the drug on the intracellular distribution of Ca 2.. Materials and Methods

Chemicals Reagents of analytical grade and deionized water were used. Aldrich Chemical Co., Milwaukee, Wisc., U.S.A., supplied 3-isobutyl-l-methylxanthine (IBMX) and British Drug Houses Ltd., Poole, U.K., was the origin of LaC13. N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES) and bovine serum albumin (fraction V) were from Sigma Chemical Co., St. Louis, Mo., U.S.A. Sodium pentobarbital and 12SI-labelled pig insulin were gifts from A b b o t t Laboratories, North Chicago, Ill., U.S.A. and Farbwerke Hoechst A.G., Frankfurt/Main, G.F.R. respectively. Crystalline mouse insulin was prepared by Novo A/S, Copenhagen, Denmark. Schwartz/Mann, Orangeburg, N.Y., U.S.A. supplied 12SI-labelled succinyl cyclic AMP tyrosine methyl ester and cyclic AMP antibodies. Dowex ion exchange resin (AG 50 W-X8, H ÷ form, 100--200 mesh) was purchased from Biorad Laboratories, Richmond, Calif., U.S.A. Animals and isolation of islets Female ob/ob-mice, 7 months old and taken from a non-inbred colony (3), were used as a source of pancreatic islets. These islets contain more than 90% ~cells, known to respond to the c o m m o n l y employed stimulators of insulin release [4]. The animals were starved overnight before being killed by decapitation. Fresh pancreatic islets were isolated by microdissection [ 5]. Design of in vitro techniques All incubations were made at 37 ° C in buffer containing 1 mg/ml albumin. The basal medium consisted of either Krebs-Ringer bicarbonate buffer or a modified version which allowed the introduction of LaC13. In the latter case, phosphate and sulphate were replaced with equimolar amounts of chloride and the buffering capacity provided by 25 mM HEPES instead of bicarbonate. Ca2+-deficient media were prepared by substituting CaC12 with equimolar amounts of NaC1. If not otherwise stated the Krebs-Ringer bicarbonate medium was equilibrated with 02 + CO2 and the HEPES-medium with ambient air. Previous studies have indicated the usefulness of the HEPES-medium for studying insulin release from microdissected islets [6]. The islets were either introduced into closed incubation vials [4] or exposed in a microchamber to non-recirculating perfusion medium [7], the oxygen tension of which was continuously recorded by a Po 2" electrode (type E 5046, Radiometer, Copenhagen, Denmark). With the first alternative, preliminary incubations of batches of t w o islets were followed by two periods of incubation in 315 pl medium, i.e., the first 20 min (early phase ) and subsequent 60 min (late phase) after exposure to pentobarbital. All solutions of pentobarbital were freshly prepared. Experimental details including additions of further com-

768 p o u n d s to media are given in the legends to figures and tables. After incubation the islets were freeze-dried and weighed as previously described [4,7].

Measurements of insulin and cyclic AMP The medium contents of insulin was measured radioimmunologically, using crystalline mouse insulin as a reference. Separation of free and bound insulin was achieved by precipitation with ethanol. It was ascertained that pentobarbital did n o t affect the insulin assay. Perchloric acid extracts of the freeze-dried islets were purified by ion-exchange c h r o m a t o g r a p h y and cyclic AMP was measured by radioimmunoassay [8]. Results The effect of pentabarbital on insulin release in closed vials is shown in Table I. There was a complete blockage of the early and late phases of glucose-stimulated insulin release in the presence of 3 mM pentobarbital or when the media were deficient in Ca 2+. This inhibitory effect of pentobarbital contrasted with what happened when the drug was added to a Ca2+-deficient medium containing 20 mM glucose. In the latter case pentobarbital enhanced the amounts of insulin released both during the initial 20 min and the subsequent 60 min. Table II indicates h o w pentobarbital modified insulin release when the islets were simultaneously exposed to the phosphodiesterase inhibitor isobutylmethylxanthine. In this case the glucose effect was not completely abolished when the medium was deficient in Ca 2+. Again, however, 3 mM pentobarbital proved to have stimulatory effects on insulin release notwithstanding a p o t e n t inhibi-

TABLE I EFFECT OF PENTOBARBITAL ASE INHIBITOR

ON INSULIN

RELEASE

IN T H E A B S E N C E O F P H O S P H O D I E S T E R -

A f t e r 60 m i n of p r e l i m i n a r y i n c u b a t i o n w i t h 3 m M glucose in b i c a r b o n a t e m e d i u m d e f i c i e n t or n o t in C a 2+, t h e i s l e t s w e r e i n c u b a t e d f o r 2 0 m i n ( e a r l y p h a s e ) a n d 6 0 m i n ( l a t e p h a s e ) w i t h 3 o r 2 0 m M g l u c o s e in the same type of basal medium supplemented or not with 3 mM pentobarbital. When not otherwise s t a t e d w i t h i n p a r e n t h e s i s , 8 s e p a r a t e e x p e r i m e n t s w e r e p e r f o r m e d . T h e r e s u l t s are p r e s e n t e d as t h e m e a n v a l u e s -+ S . E . M . f o r t h e a m o u n t s o f i n s u l i n r e l e a s e d i n t h e e a r l y a n d l a t e p h a s e s r e s p e c t i v e l y as w e l l as f o r the differences between parallel incubations after the addition of pentobaxbital. Ca 2+ (mM)

Glucose (raM)

Insulin release (ng/pg dry islet) C o n t r o l (a)

P e n t o b a r b i t a l (b)

b

a

Early phase

0 0 2.56 2.56

3 20 3 20

0.42 0.22 0.34 0.93

± 0.09(7) +- 0 . 0 6 +- 0 . 1 1 + 0.11

0.45 0.51 0.26 0.36

+ 0.11(7) -+ 0 . 1 1 + 0.12 + 0.18

+0.02 +0.29 --0.09 0.56

± 0.16(7) -+ 0 , 0 9 * + 0.18 ± 0 . 0 9 ***

Late phase

0 0 2.56 2.56

3 20 3 20

0.60 0.41 0.52 2.87

-+ 0 . 1 1 + 0.06 + 0.10 +- 0 . 2 4

0.86 0.87 0.47 0.54

± 0.12 -+ 0 . 1 6 + 0.10 -+ 0 . 0 5

+0.26 +0.46 --0.05 --2.33

+ 0.22 ± 0 . 1 3 ** +- 0 . 1 5 + 0 . 2 5 ***

* Levels of significance by f-test for paired observations: P < 0.02; ** P < 0 . 0 1 ;

*** P < 0.001.

769

TABLE

II

EFFECT OF PENTOBARBITAL ASE INHIBITOR

ON INSULIN

RELEASE

IN THE PRESENCE

OF PHOSPHODIESTER-

After 60 min of preliminary incubation with 3 raM glucose in bicarbonate medium deficient or ~ot in C a 2+, t h e i s l e t s w e r e i n c u b a t e d f o r 2 0 m i n ( e a r l y p h a s e ) a n d 6 0 r a i n ( l a t e p h a s e ) w i t h 1 m M i s o b u t y l m e thylxanthine and either 3 or 20 mM glucose in the same type of basal medium supplemented or not with 3 mM pentobarbital, Results are presented as the mean values ± S.E.M. for 7 separate experiments and i n d i c a t e b o t h t h e a m o u n t s o f i n s u l i n r e l e a s e d i n t h e e a r l y a n d l a t e p h a s e s r e s p e c t i v e l y as w e l l as t h e d i f f e r ences between parallel incubations after the addition of pentobarbitai. C a 2+ (raM)

Glucose (mM)

Insulin release (ng/pg dry islet) C o n t r o l (a)

Pentobarbital

(b)

b -- a

Early phase

0 0 2.56 2.56

3 20 3 20

0.93±0.23 1.96±0.33 0.85±0.16 10.64±0.72

1.12±0.17 3.12±0.69 1.66±0.10 7.06±0.63

+0.19±0.33 +1.16±0.52 +0.82±0.10"** --3.58±0.67**

Late phase

0 0 2.56 2.56

3 20 3 20

0.65±0.17 2.21±0.44 2.14±0.52 ~.5S±1.44

1.19±0.23 0.96±0.10 1.24±0.30 3.62±0.97

+0.54±0.21" --1.24±0.44" --0.90±0.41 --12.96±1.61"**

* L e v e l s o f s i g n i f i c a n c e by t - t e s t f o r p a i r e d o b s e r v a t i o n s : ** P < 0.01; *** P < 0.001.

P < 0.05;

t o r y effect on the glucose-stimulated insulin release at an ordinary Ca 2+ concentration. The stimulatory action of pentobarbital occurred only in the presence of 3 mM glucose and either when the m e d i um was deficient (late phase) or not (early phase) in Ca 2+. Pentobarbital t r e a t m e n t resulted in a marked decrease in the islet c o n t e n t of cyclic AMP irrespective of the concentrations of Ca 2+ or glucose (Table III). Analyses o f the kinetics of insulin release from microperifused islets provided fu r th er evidence t ha t pentobarbital has a dual effect on insulin release in the presence o f i s o b u t y l m e t h y l x a n t h i n e , manifested b o t h as a rapid inhibition of the secretagogic effect of glucose and a stimulation when the medium was deficient

TABLE

III

EFFECT OF PENTOBARBITAL ON PHOSPHODIESTERASE INHIBITOR

ISLET

CONTENT

OF

CYCLIC

A M P 1N T H E

PRESENCE

OF

The islets analyzed are identical to those in Table II. When not otherwise stated within parenthesis mean values ± S.E.M. are given for 7 separate experiments. C a 2+ (raM)

0 0 2.56 2.56

Glucose (raM)

3 20 3 20

Cyclic AMP content

(fmol/pg dry islet)

Control (a)

Pentobarbital

27.1±4.6(5) 40.2±7.6 34.0±5.6(6) 46.6±9.8

5.9±0.6(5) 8.2±1.5 7.0±1.0(6) 13.9±4.0

(b)

* L e v e l s o f s i g n i f i c a n c e b y t-test f o r p a i r e d o b s e r v a t i o n s : P < 0 . 0 2 . ** P < 0.01.

b -- a --21.

--21.2 --31.9 --27.0 --32.7

+- 4.1 ± 7.8 +- 5 . 6 +- 7 . 8

** (5) * ** (6) *

770

PENTOBARBITAL

(3raM)

z - -

=E

300

+w J

123

200

m ~ w

100

z _J z

.~.,_,~.

:

_ ~

!

-

-

~;- - ~ - - -

z

60

90

120

PERFUSION TIME ( M I N ) F i g . 1. I s l e t s w e r e p e r i f u s e d w i t h b i c a r b o n a t e m e d i u m c o n t a i n i n g 1 m M i s o b u t y l m e t h y l x a n t h i n e a n d e q u i l i b r a t e d w i t h O 2 + C O 2 ( 9 5 : 5). A f t e r 6 0 m i n t h e m e d i u m w a s s u d d e n l y c h a n g e d t o o n e a l s o c o n t a i n i n g 3 m M p e n t o b a r b i t a l . T h e m e d i a e m p l o y e d r e p r e s e n t e d t h e f o l l o w i n g 3 a l t e r n a t i v e s w i t h r e g a r d t o t h e C a 2+ a n d g l u c o s e c o n c e n t r a t i o n s p r e s e n t : 2 . 5 6 m M C a 2+ a n d 2 0 m M g l u c o s e ( o ) ; C a 2 + - d e f i c i e n t m e d i u m w i t h 2 0 m M g l u c o s e ( $ ) ; C a 2 + - d e f i c i e n t m e d i u m w i t h 3 m M g l u c o s e (A). M e a n v a l u e s + S . E . M . are g i v e n f o r t h e rate of insulin release in 4 separate experiments with each type of medium.

_z

150

300

loo

ZOO

I w _J

o

~

cl w

E

w z

N

so

I00 >-

z _J z

60

90

120

PERIFUSION T I M E ( M I N ) F i g . 2. I s l e t s w e r e p e r i f u s e d w i t h C a 2 + - d e f i c i e n t b i c a r b o n a t e m e d i u m c o n t a i n i n g 2 0 m M g l u c o s e a n d 1 m M i s o b u t y l m e t h y l x a n t h i n e a n d e q u i l i b r a t e d w i t h 0 2 + C O 2 ( 9 5 : 5). A f t e r 6 0 r a i n t h e gas p h a s e w a s c h a n g e d t o N 2 + C O 2 ( 9 5 : 5 ) r e s u l t i n g i n a r a p i d d r o p o f t h e o x y g e n t e n s i o n as i n d i c a t e d b y t h e d o t t e d l i n e . M e a n v a l u e s -+ S . E . M . are g i v e n f o r t h e r a t e o f i n s u l i n r e l e a s e i n 4 s e p a r a t e e x p e r i m e n t s .

771 in Ca 2÷ (Fig. 1). The stimulatory action was influenced by the concentrations of glucose in the Ca2+-deficient media. In the presence of 20 mM glucose it appeared promptly as a distinct peak which lasted for about 15 min. At 3 mM glucose the stimulatory action of pentobarbital was less marked, there being a gradual rise of the secretory rate throughout the experiment. Merely lowering the oxygen tension did not result in a stimulation of insulin release. As a matter of fact omission of oxygen slightly diminished the rate of insulin release when the islets were incubated with 1 mM isobutylmethylxanthine iil Ca 2+deficient medium containing 20 mM glucose (Fig. 2). Pentobarbital stimulated insulin release also when the Ca2+-deficient medium was supplemented with 0.2 mM LaC13 (Fig. 3). In the absence of phosphodiesterase inhibitor the addition of pentobarbital now resulted in long-lasting stimulation which was greatest in the presence of high concentrations of glucose. Also in the presence of La3*there was no stimulation of insulin release on lowering the oxygen tension. This is illustrated in Fig. 4, which shows the changes in the secretory rate after lowering the oxygen tension in a Ca2+-defi -

L a C [ 3 ( 0.ZmM )

100

P E N ' I r O B A R B I T A L ( 3raM }

V//Z/Z/1/Z///////////////////////////////////////

Z "~

50

Ltl .-I U3

c~ 60

~

90

120

150

i.n w _1 u.l a:

100

z ..J z 50

60

90 PERIFUSION

120 TIME

( MIN

)

Fig. 3. I s l e t s w e r e p e r i f u s e d w i t h C a 2 + - d e f i c i e n t H E P E S m e d i u m e q u i l i b r a t e d w i t h a m b i e n t air in t h e absence (upper panel) or presence (lower panel) of 1 mM isobutylmethylxanthine. The media contained e i t h e r 3 (A) o r 2 0 ( $ ) m M g l u c o s e . 0 . 2 m M L a C 1 3 w a s a d d e d a f t e r 6 0 m i n o f p e r i f u s i o n a n d a f t e r a n o t h e r 30 rain also 3 mM pentobarbital. T h e rate o f i n s u l i n r e l e a s e w a s r e c o r d e d f o r t h e m e d i a c o n t a i n i n g 3 m M glucose in 2 separate experiments and for the media containing 20 mM glucose in 4--5 separate experim e n t s . I n t h e l a t t e r c a s e t h e m e a n v a l u e s -+ S . E . M . are i n d i c a t e d .

772 LaCI 3 (0.2raM)

z

300

150i

A

uJ

E

o

200

100 . . °

,

. . . . . . .

°

. . . . . .

° . . . .

z o ~D z t~

..

°. o.

121 UA W

100

50

z uJ ¢9

UJ Z

Z

°°'..°°.°°.°°°° i

,

60

i

i

90

J

i

120

PERIFUSION TIME (MIN) Fig. 4. Islets were perifused

with a Ca2+-deficient medium buffered with HEPES and containing 20 mM glucose and 1 mM isobutylmethylxanthine. After 60 rain 0.2 mM LaCl 3 was added to the medium. After a n o t h e r 3 0 r a i n t h e gas p h a s e w a s c h a n g e d f r o m a m b i e n t air t o N 2 , r e s u l t i n g in a r a p i d d r o p o f t h e o x y g e n t e n s i o n as i n d i c a t e d b y t h e d o t t e d line. M e a n v a l u e s ± S . E . M . f o r t h e r a t e o f i n s u l i n r e l e a s e in 3 s e p a r a t e e x p e r i m e n t s are i n d i c a t e d .

cient medium supplemented with 1 mM isobutylmethylxanthine and 20 m M glucose. Discussion Several reports have indicated that anaesthetic doses of pentobarbital cause a prompt secretion of growth hormone in the rat [ 9 - - 1 2 ] . Even if there are reasons to believe that this effect is essentially mediated by neural structures associated with the hypothalamic and extrahypothalamic regions [12] it has been suggested from in vitro studies that pentobarbital may also act directly u p o n the pituitary to stimulate growth hormone release [2]. Pentobarbital effects opposite to those observed for growth hormone have been reported for other pituitary hormones including thyrotropin, adrenocorticotropin, luteinizing hormone and prolactin [12]. It has furthermore been found that pentobarbital decreases the release of catecholamines from the isolated cow adrenal gland or more specifically inhibits the effect of various agents k n o w n to promote the process of exocytosis [13]. A characteristic of this inhibition is that it can be obtained with much lower concentrations of pentobarbital than those usually required for blocking the energy metabolism. Intravenous glucose infusions in dogs have been reported to result in more pronounced hyperglycemia but a less marked rise of the serum insulin level during pentobarbital anaesthesia [ 1 4 ] . Nevertheless there was a normal rate of the in vitro secretion of insulin when pieces of pancreas were taken from rats anaesthetized by intraperitoneal injections of pentobarbital or w h e n such

773 pieces were exposed to 0.12 mM of the drug [15]. Panten et al. [16] noted that the in vitro exposure of islets from ob/ob-mice to 3 mM pentobarbital, the concentration employed in the present study, resulted in inhibition of the glucose-stimulated insulin release with an increased pyridine nucleotide fluorescence suggestive of impaired mitochondrial respiration. The present data are consistent with these observations in indicating a marked inhibition of the glucose-stimulated insulin release at normal Ca2*-concentrations. Moreover, if 3 mM pentobarbital markedly interferes with the respiration of the pancreatic fi-cells the resulting impairment of the ATP production might well account for the observed decrease of the islet content of cyclic AMP. It was evident that pentobarbital has not only inhibitory but also stimulatory effects on insulin release. We have previously pointed out the possibility that reduction of pyridine nucleotides leads to insulin release by suggesting that the ion permeability of the fi-cells is dependent on the redox equilibrium between the sulphydryl groups and disulphide bridges in the plasma membrane [17]. If such a mechanism accounts for the stimulatory action of pentobarbital on insulin release it should be possible to get a stimulation by other means which promptly influence the redox state of the pyridine nucleotides by interfering with the mitochondrial electron transport system. Lowering of the oxygen tension leads to this experimental situation. However, no effect on insulin release except inhibition was observed after lowering the oxygen tension in media whose composition was varied with regard to Ca 2+, glucose or phosphodiesterase inhibitor. It seems therefore most likely that pentobarbital stimulation of insulin release is accounted for by mechanisms other than alteration of the redox state of the pancreatic ~-cells. As in other tissues the binding of Ca 2÷ to subcellular organelles is stimulated by ATP in the pancreatic islets [18,19]. In the case of pentobarbital there is also the possibility for more specific effects on the intracellular distribution of Ca 2÷ as reported in muscle preparations [20--22]. Previous studies have emphasized the significant role of Ca 2÷ for insulin release from islets of ob/ob-mice suggesting that not only a decrease but also an increase of the functionally important pool(s) might lead to a diminished secretory response to glucose [6]. Whereas the Ca 2÷ concentration significant for glucose stimulation of insulin release was within the optimal range already when the concentration of extracellular Ca 2÷ corresponded to that of the ordinary Krebs-Ringer bicarbonate medium (= 2.56 mM) introduction of more extracellular Ca 2÷ increased the amounts of insulin released in media containing a low glucose concentration and/or isobutylmethylxanthine [6,23]. It may be expected that alterations of the distribution of Ca 2÷ within the pancreatic ~-cells result in stimulation of insulin release only when the concentration of Ca 2÷ in the functionally important pool(s) is shifted towards the optimal range. It was therefore of interest to note that, after withdrawal of extracellular Ca 2+, pentobarbital increased the amounts of insulin released in the presence of 20 mM glucose. It is well established that the ~-cells become more sensitive to Ca2+when they are exposed to a phosphodiesterase inhibitor [6,23]. This might explain why isobutylmethylxanthine modifies the pentobarbital effects on insulin release, making pentobarbital stimulatory in ordinary Krebs-Ringer bicarbonate medium with 3 mM glucose.

774 Lan th an u m ions have been e m p l o y e d to block fluxes of Ca 2÷ across the plasma membrane and displace this ion from its superficially located binding sites in different tissues [24,25] including the pancreatic islets [26]. It was evident fr o m studies of muscle cell preparations that pentobarbital diminishes the amounts of superficially located Ca2÷which can be displaced by La 3÷ [22]. The present study indicates that pentobarbital readily stimulates insulin release also when the islets are exposed to 0.2 mM LaC13, a concentration known to reduce significantly the amounts of insulin released in response to glucose at ordinary Ca 2÷ concentrations in the incubation medium [6]. This suggests that pentobarbital stimulation of insulin release also involves Ca 2÷ storage in locations o th er than the most superficial sites in the H-cell membrane.

Acknowledgements This work was supported by the Swedish Medical Research Council (12x562).

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

18 19 20 21 22 23 24 25 26

H e l l m a n , B. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 3 9 9 , 1 5 7 - - 1 6 9 H o w a r d , N.J. a n d M a r t i n , J.M. ( 1 9 7 2 ) E n d o c r i n o l o g y 9 1 , 1 5 1 3 - - 1 5 1 5 H e U m a n , B. ( 1 9 6 5 ) A n n . N.Y. A c a d . Sci. 1 3 1 , 5 4 1 - - 5 5 8 H e l l m a n , B. ( 1 9 7 0 ) D i a b e t o l o g i a 6, 1 1 0 - - 1 2 0 H e l l e r s t r o m , C. ( 1 9 6 4 ) A c t a E n d o c r i n o l . 4 5 , 1 2 2 - - 1 3 1 H e l l m a n , B. ( 1 9 7 5 ) E n d o c r i n o l o g y 9 7 , 3 9 2 - - 3 9 8 I d a h l , L.-A. ( 1 9 7 2 ) A n a l . B i o c h e m . 50, 3 8 6 - - 3 9 8 H e l l m a n , B., I d a h l , L.-A., L e r n m a r k , A. a n d Taljedal, I.-B. ( 1 9 7 4 ) P r o c . Natl. A c a d . Sci. U.S. 71, 3405--3409 H o w a r d , N.J. a n d M a r t i n , J.M. ( 1 9 7 1 ) E n d o c r i n o l o g y 88, 4 9 7 - - 4 9 9 T a k a h a s h i , K., D a u g h a d a y , W.H. a n d K i p n i s , D.M. ( 1 9 7 1 ) E n d o c r i n o l o g y 88, 9 0 9 - - 9 1 7 K o k k a , N., G a r c i a , J . F . , G e o r g e , R. a n d E l l i o t t , H.W. ( 1 9 7 2 ) E n d o c r i n o l o g y 9 0 , 7 3 5 - - 7 4 3 M a r t i n , J.B. ( 1 9 7 4 ) N e u r o e n d o c r i n o l o g y 13, 3 3 9 - - 3 5 0 H o l m e s , J.C. a n d S c h n e i d e r , F . H . ( 1 9 7 3 ) Brit. J. P h a r m a c o l . 4 9 , 2 0 5 - - 2 1 3 R e n a u l d , A. a n d S v e r d l i k , R.C. ( 1 9 7 4 ) E x p e r i e n t i a 3 1 , 4 7 4 - - 4 7 5 Kizer, J.S., V a r g a s - C o r d o n , M., B r e n d e l , K. a n d Bressler, R. ( 1 9 7 0 ) J. Clin. Invest. 4 9 , 1 9 4 2 - - 1 9 4 8 P a n t e n , U., C h r i s t i a n s , J., P o s e r , W. a n d H a s s e l b l a t t , A. ( 1 9 7 3 ) D i a b e t o l o g i a 9, 4 7 7 - - 4 8 2 H e l l m a n , B., I d a h l , L.-/~, L e r n m a r k , A., S e h l i n , J. a n d T a l j e d a l , I.-B. ( 1 9 7 4 ) P r o c e e d i n g s o f t h e 8 t h C o n g r e s s o f t h e I n t e r n a t i o n a l D i a b e t e s F e d e r a t i o n , Vol. 3 2 , p p . 6 5 - - 7 8 , I n t e r n a t i o n a l C o n g r e s s Series, Excerpta Mediea, Amsterdam H o w e l l , S.L. a n d M o n t a g u e , W. ( 1 9 7 5 ) F E B S L e t t . 52, 4 8 - - 5 2 S e h l i n , J. ( 1 9 7 6 ) B i o c h e m . J. 1 5 6 , 6 3 - - 6 9 L a i n , R . F . , Hess, M.L., G e r t z , E.W. a n d Briggs, F . N . ( 1 9 6 8 ) Circ. Res. 23, 5 9 7 - - 6 0 4 D r a n s f e l d , H., G r e f f , K., S c h o r n , A. a n d Ting, B.T. ( 1 9 6 9 ) B i o c h e m . P h a r m a c o l . 18, 1 3 3 5 - - 1 3 4 5 N a y l e r , W.G. a n d S z e t o , J. ( 1 9 7 2 ) A m . J. P h y s i o l . 2 2 2 , 3 3 9 - - 3 4 4 H e l l m a n , B. ( 1 9 7 6 ) F E B S L e t t . 6 3 , 1 2 5 - - 1 2 8 V a n B r e e m e n , C., F a r i n a s , B . R . , G e r b a , P. a n d M c N a u g h t o n , E.D. ( 1 9 7 2 ) Circ, Res. 30, 4 4 - - 5 4 Weiss, G.B. ( 1 9 7 4 ) A n n u . Rev. P h a r m a c o l . 14, 3 4 3 - - 3 5 4 H e l l m a n , B., Sehlin, J. a n d Taljedal, I.-B. ( 1 9 7 6 ) J. P h y s i o l . 2 5 4 , 6 3 9 - - 6 5 6