Calcium and pancreatic β-cell function

534

Biochimica et Biophysica Acta, 540 (1978) 534--542

© Elsevier/North-Holland Biomedical Press

BBA 28523 CALCIUM AND PANCREATIC ~-CELL FUNCTION 3. VALIDITY OF THE La3+-WASH TECHNIQUE FOR DISCRIMINATING BETWEEN SUPERFICIAL AND I N T R A C E L L U L A R 4SCa

BO HELLMAN Department of Histology, Biomedicum, University of Uppsala, S-751 23 Uppsala (Sweden)

(Received October 14th, 1977)

Summary Exposure to La 3÷ has been proposed as a tool for discriminating between superficially and intracellularly located 4SCa. Two different pools of glucosestimulated 4SCa were identified in ~-cell-rich pancreatic islets microdissected from o b / o b mice using a standard procedure of washing with 2 mM La 3+ at 37°C. The results were not critically dependent on the concentration of La 3÷ within a range of 1--20 mM. However, the distribution of 4SCa was markedly influenced by the temperature of the washing medium. Reduction of the temperature to I°C abolished the glucose effect on the La3+-displaceable 4SCa and considerably increased the amounts of 4SCa not displaceable with La 3÷. Separate studies in a perifusion system revealed that La 3+ in itself was less effective than temperature reduction in depressing the efflux of 4SCa from the isolated islets. The data indicate that washing in cold La 3+ solution gives a better estimate of the intracellular islet calcium than the original procedure. It is suggested that the glucose-stimulated part of the 4SCa, which is displaced by washing with La 2+ at 37°C, is derived from the interior of the fl-cells.

Introduction Most of the present ideas of how the fl-cells handle C a 2+ a r e derived from radioisotope measurements of 4SCa uptake and efflux from isolated pancreatic islets. The marked tendency of Ca 2÷ to bind to organic molecules has made it difficult to decide to what extent observations on calcium incorporation reflect the transport characteristics of the plasma membrane or the affinity of Ca 2÷ for the extracellular and intracellular phases. In the attempts to differentiate between the surface binding and transmembrane flows of Ca :+ into the ~-cells, attention has been paid to a m e t h o d originally proposed by van Breemen et al.

535 [1,2] for measuring the intracellular c o n t e n t of radioactive calcium in smooth muscle preparations. In this procedure it is assumed that lanthanum, a trivalent cation which does n o t penetrate the plasma membranes, both displaces extracellular Ca 2÷ and prevents the loss of intracellularly-located Ca 2÷. Preliminary experiments suggested t h a t these demands were essentially satisfied when the technique was applied to the study of calcium metabolism in microdissected pancreatic islets [3,4]. Washing with La 3÷ made it possible to discriminate between two pools of glucose-sensitive calcium in the fl-cell-rich pancreatic islets microdissected from ob/ob mice [3--9]. Only the 4SCa displaceable with La 3÷ was f o u n d to have sufficient mobility to meet the kinetic demands for Ca 2÷ involved in the acute regulation of insulin release. The fact t h a t a short rinsing in a non-radioactive medium proved useful for unmasking the glucose effect suggested that either glucose had increased the affinity of the cell membrane for the superficial 4SCa or that some Laa+-displaceable 4SCa escaped by efflux from interior of the cell [ 5,9]. In the present paper the nature of the glucose-stimulated pools of islet 4SCa has been considered in the light of observations made when modifying the procedure of La a÷ washing. Evidence will be provided to support washing in cold medium giving a better estimate of intracellular 45Ca. It is furthermore suggested that the glucose-stimulated part of the 4SCa, which is displaced when employing the original La3÷-wash procedure, is derived from the interior of the ~3-cells. Material and Methods

Chemicals. Reagents of analytical grade and deionized water were used. Sigma Chemical Company, St. Louis, Mo., U.S.A. supplied N-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES). 4SCa was purchased from the Radiochemical Centre, Amersham, U.K. British Drug House Ltd, Poole, U.K. was the origin of Tris and LaC13. Animals and isolation of islets. Male ob/ob mice, 7--11 months old, were taken from a non-inbred colony and used as a source of pancreatic islets containing more than 90% fl-cells [10]. Unless otherwise stated the animals were starved overnight before being killed by decapitation. After removal of pancreas, islets were microdissected freehand and analysed for their ability to incorporate respective release 4SCa. Efflux studies. Batches of 8--10 islets were exposed to 4SCa in the presence of 20 mM glucose during 120 min of incubation at 37°C in 100 pl salt-balanced Tris buffer [11]. The medium was equilibrated with air and had the following ionic composition in mM: Tris 5; Na ÷ 139; K ÷ 4.7; Ca 2+ 2.6; Mg 2+ 1.2; C1151.2, as well as C1- added as HC1 to give a final pH of 7.4. The specific radioactivity of 4SCa2+ was approx. 390 Ci/mol. After labelling with 4SCa the islets were transferred to non-radioactive medium with the ionic composition as given above (experiments with La 3÷) or somewhat modified (experiments without La 3+) with the buffering capacity provided by 25 mM HEPES [12]. After two washing periods of 5 min in 5 ml medium, the islets were placed in 10-pl chambers of Teflon tubing and perifused with the same type of non-radioactive medium [11].

536

Uptake studies. In the uptake studies the calcium c o n t e n t of the saltbalanced Tris buffer was reduced to approx. 0.04 mM as checked by graphitefurnace atomic absorption spectrophotometry [13]. Preliminary incubation for 45 min of batches of 3 islets in 200 t~l medium containing 3 mM glucose was followed by loading with radioactivity for 20 min at 37°C in the presence of 3 or 20 mM glucose. In the latter type of media part of the stable 4°Ca was replaced with 45Ca to give a specific radioactivity of 440 Ci/mol. In one series of experiments radioactive loading was performed for various periods of time both at 1 and 37°C using identical temperatures in the preincubation media. In some cases, labelling with 45Ca was followed by 2 min of rinsing at 1 or 37°C in non-radioactive medium supplemented with 3 mM glucose. The islet radioactivity was measured with or w i t h o u t subsequent washing for 60 min with LaC13 included in medium kept at 1 or 37°C. When not otherwise stated this washing was performed with 2 mM La 3÷ in glucose-free medium. Evaluation o f results. The islets were freeze-dried overnight, weighed on a quartz fibre balance and dissolved in Hyamine before being analysed for radioactivity by liquid scintillation counting [3]. Comparisons in each animal of the radioactivity in islets washed or not with La 3÷ made it possible to distinguish between La3÷-displaceable and La3÷-nondisplaceable 4SCa. The amounts of 4SCa in both of these pools, as well as in samples of the perifusate collected over successive periods of 2 or 5 min, were expressed in terms of pmol or mmol calcium with the same specific radioactivity as in the loading medium. Results The effect of temperature reduction on the efflux of 4SCa from microdissected islets is shown in Fig. 1. It is evident that lowering of the temperature from 37°C to 1°C resulted in p r o m p t and marked inhibition of the efflux. This effect was readily reversible, as indicated from the rapid reestablishment of the normal efflux rate, when bringing the temperature back to 37°C. Exposure to La 3÷ provided another means of depressing 4SCa efflux; the optimal effect being reached after about 10 min. La 3÷ in itself was less effective than temperature reduction in inhibiting the efflux when introduced into the perifusion medium in a concentration of 2 mM. However, the combination of La 3÷ with temperature reduction practically abolished the efflux of 4SCa, as shown in Fig. 2. In the latter case, the efflux rate at the end of the perifusion period was 1.7 ± 0.4 pmol/kg dry islet per min, which is equivalent to 15% of that noted for controls continously perifused with La 3÷ at 37°C. Although less affected, even the uptake of 4SCa was clearly depressed during hypothermia. The inhibition seen when reducing the temperature from 37 ° to I°C was n o t restricted to initial uptake but was also seen for the total amounts of La3÷-resistant 4SCa incorporated during 180 minutes of incubation (Fig. 3). Table 1 indicates how modifications of the La3÷-wash procedure affect the 45Ca c o n t e n t in islets taken for analyses without previous rinsing in non-radioactive medium. When employing the original technique of La3÷-washing at 37°C the 4SCa, whether displaceable or n o t with La 3÷, increased after raising the glucose concentration in the radioactive loading medium. When 20 mM glucose was present in the washing medium the nondisplaceable 4SCa tended to increase

537

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z ~-

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LO

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20

20

40 PERIFUSION

60

80

100

TIME ( M I N )

F i g . 1. E f f e c t o f t e m p e r a t u r e o n 4 5 C a e f f l u x f r o m i s l e t s l o a d e d w i t h m M g l u c o s e . T h e i s l e t s w e r e m i c r o d i s s e c t e d f r o m oblob m i c e s t a r v e d balanced medium buffered with HEPES and supplemented with 3 mM temperature of the perifuslon medium was reduced from 37 ° to 1°C horisontal black bar. Mean values + S.E. for 4 experiments.

TABLE

radioactivity in the presence of 20 for 3 days and perifused with saltglucose and 1 mg/ml albumin. The during the period indicated by the

I

UPTAKE OF 45Ca ANALYSES AFTER

INTO DIFFERENT CALCIUM POOLS LOADING WITH RADIOACTIVITY

IN

ISLETS

TAKEN

FOR

IMMEDIATE

The islets were incubated for 20 rain at 37°C in 45Ca-labeUed Tris medium containing 3 or 20 mM glucose. The islet radioactivity was measured with or without subsequent washing for 60 rain with 2 mM L a C l 3. W a s h i n g w a s p e r f o r m e d a t 1 o r 3 7 ° C i n m e d i u m c o n t a i n i n g e i t h e r 0 o r 2 0 m M g l u c o s e . T h e r e t e n t i o n o f l a b e l i n e a c h g r o u p is e x p r e s s e d as m m o l C a 2+ w i t h t h e s a m e s p e c i f i c a c t i v i t i e s as t h e m e d i u m u s e d for labelling the islets. The differences between parallel loadings in 20 and 3 mM glucose are also given; the significance between these differences were t-tested: * P < 0.05; ** P < 0.025; *** P < 0.001. Mean values ± S.E. for 8 experiments.

Washing

La3+.displaceable 4 5 Ca

Temperature (°C)

Glucose (mM)

3 mM Glucose (a)

20 mM Glucose (b)

b--a

37 37 1

0 20 0

0.87 + 0.07 0 . 8 3 -+ 0 . 0 7 0 . 7 0 -+ 0 . 0 6

1 . 0 4 -+ 0 . 0 7 1.00 ± 0.08 0.75 ± 0.06

0.17 ± 0.06 ** 0.16 ± 0.07 0.06 ± 0.05

La3+.nondisplaceable 37 37 1

0 20 0

0.14 ± 0.01 0.18 ± 0.03 0.31 ± 0.02

45 Ca 0.21 ± 0.02 0.25 ± 0.02 0.49 ± 0.03

0 . 0 7 -+ 0 . 0 1 * * * 0.07 + 0.03 * 0.18 + 0.02 ***

538

1"C

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o

g o

60

a_ 40 uJ

m

20

20

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60

80

100

120

PERIFUSION TIME (M;N)

F i g . 2. E f f e c t o f 2 m M L a 3+ on 4 5 C a e f f l u x f r o m islets l o a d e d w i t h r a d i o a c t i v i t y in t h e p r e s e n c e of 20 m M g l u c o s e . T h e islets w e r e p e r i f u s e d w i t h s a l t - b a l a n c e d T r i s b u f f e r c o n t a i n i n g 3 m M g l u c o s e . L a 3+ w a s a d d e d a n d t h e t e m p e r a t u r e r e d u c e d f r o m 3 7 ° t o I ° C d u r i n g t h e p e r i o d s i n d i c a t e d by t h e h o r i z o n t a l b l a c k bars. M e a n v a l u e s + S.E. f o r 4 e x p e m m e n t s .

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÷37 °

£3

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60

INCUBATION

120 TIME

(MIN

180 }

Fig. 3. I s l e t u p t a k e o f L a 3 + - n o n d i s p l a c e a b l e 4 S C a w i t h t i m e a t 1 ° o r 3 7 ° C . T h e islets w e r e i n c u b a t e d f o r v a r i o u s p e r i o d s o f t i m e i n 4 S C a - l a b e l l e d T r i s m e d i u m c o n t a i n i n g 20 m M glucose. L a 3+ w a s h i n g w a s perf o r m e d at I ° C w i t h 2 m M L a 3+. M e a n v a l u e s + S.E. f o r 5---6 e x p e r i m e n t s .

539

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20

Fig. 4. A m o u n t s of La3+-nondisplaceable 4 S C a retained after washing with m e d i a containing different concentrations of L a 3+. T h e islets were loaded for 20 rain at 37°C with 4 S C a included in sait-baianced Tris buffer containing 3 (open bars) or 20 (hatched bars) m M glucose. Subsequent washing was performed for 60 rain at 1°C in glucose-fzee m e d i u m containing different concentrations of L a 3+. M e a n values + S.E. for 8 experiments.

at the expense of that displaceable with La 3+. This phenomenon became more pronounced on reducing the temperature of the washing medium to I°C. Moreover, La 3+ washing at this low temperature prevented the increase of La 3+displaceable 4SCa which otherwise occurred after raising the glucose concentration of the radioactive loading medium from 3 to 20 mM. Fig. 4 shows h o w addition of various amounts of La 3+ to the washing medium at I°C affects the La3+-nondisplaceable 4SCa after loading with radioactivity in the presence of 3

TABLE II UPTAKE OF 45Ca INTO DIFFERENT CALCIUM POOLS WHEN THE LOADING OF THE ISLETS WAS FOLLOWED BY 2 min OF RINSING IN NON-RADIOACTIVE MEDIUM CONTAINING 3mM GLUCOSE

T h e experimental design of the radioactive loading and the La3+-washing in glucose-free m e d i u m w a s identical to that described in the legend to Table I. A s indicated, the temperatures of the efflux and washing media were either 1 or 37°C. * P < 0.05; ** P < 0.02; *** P < 0.001. M e a n values -+ S.E. for 8 experiments. Temperature

Rinsing

L a 3 + . d i s p l a c e a b l e 4 5C a

(°C)

Washing

3 mM

Glucose

(a) 37 1 37 1

37 37 1 1

0.57 0.62 0.46 0.57

20 m M

Glucose

b--a

(b) ± 0.03 ,+ 0 . 0 9 -+ 0 . 0 4 ± 0.04

0.72 0.83 0.36 0.41

0.07 0.12 0.08 0.04

+0.15 +0.21 --0.10 --0.16

,+ 0 . 0 6 * ,+ 0 . 0 8 * -+ 0 . 1 0 ± 0.06 *

+- 0 . 0 1 ,+ 0 . 0 1 ,+ 0 . 0 6 ,+ 0 . 0 4

+0.04 +0.05 +0.31 +0.32

-+ 0 . 0 1 -+ 0 . 0 1 -+ 0 . 0 6 ± 0.03

,+ ± ± 2

La3+.nondisplaceabte 4 SCa 37

1 37 1

37 37 1 1

0.14 0.13 0.32 0.36

± 0.01 ± 0.01 ,+ 0 . 0 2 -+ 0 . 0 2

0.18 0.18 0.63 0.68

** ** ** ***

540 or 20 mM glucose. It can be seen that similar results were obtained when varying the La 3÷ concentration between 1 and 20 mM. The effect of glucose on 4SCa incorporation into islets taken for analyses after 2 min of rinsing in non-radioactive medium is shown in Table II. When employing the original procedure of La 3÷ washing, glucose increased the uptake of 4SCa into the La3÷-displaceable pool irrespective of the temperature of the rinsing medium used. This effect was abolished when La 3÷ washing was performed at 1°C. Reduction of the temperature in the washing medium resulted in a considerable increase in the amounts of La3÷-nondisplaceable 4SCa. Glucose served as an effective stimulator of the 4SCa uptake into the latter pool of calcium either when La3+-washing was performed at 1 ° or 37°C. Discussion In previous analyses of intracellular 4SCa the microdissected islets were washed for 60 min at 37°C with 2 mM La 3÷ [3]. This procedure resulted in a complete suppression of 4SCa uptake in excess of the extracellular space marker sucrose, indicating that 2 mM La 3÷ prevents 4SCa from binding to or entering the islet cells. The inhibitory action of La 3÷ on 4SCa efflux was evident by measuring the total c o n t e n t of radioactivity remaining in islets initially loaded with 4SCa. After 60 min of incubation islet radioactivity approached a stable plateau when the non-radioactive incubation medium was supplemented with 2 mM La 3÷ [3]. In the present study the inhibitory action of La 3÷ on 4SCa efflux was demonstrated by continuous monitoring of the radioactivity appearing in the medium during perifusion of the islets. With this more sensitive technique the maximal effect was found to be reached about 10 min after the islets had been exposed to 2 mM La 3÷. This is equivalent to the equilibration time for sucrose in the extracellular islet space [14]. It should be kept in mind that the washout curve for 4SCa is determined also by factors other than the rate by which the intracellular radioactivity escapes from the fl-cells. Initially, exposure to La 3÷ can be expected to displace from the surface of the H-cells such 4SCa as has n o t yet been mobilised during the preceding period of washing and perifusion in non-radioactive medium. Outward transport of calcium across a cell membrane is known to occur against an electrochemical gradient, i.e., the efflux may be an energy-dependent process. Lowering of the temperature to I°C rapidly reduced the efflux of 4SCa from the islets, suggesting a close coupling between metabolism and calcium extrusion in the pancreatic fi-cells. Cooling has been reported to lead to a rapid accumulation of Ca 2÷ in heart muscle cells because the uptake of the ion was relatively little affected as compared to the marked inhibition of its efflux [15]. In the fi-cell-rich pancreatic islets h y p o t h e r m i a was associated with clear inhibition also of the uptake of 4SCa. In fact, cooling of the islets resulted in a significant depression in the amounts of La3÷-nondisplaceable 4SCa incorporated even after loading with radioactivity for as long as 180 min. In the light of the temperature data obtained it was felt that cooling might help to increase the accuracy of islet 4SCa measurements by minimizing the escape of intracellular radioactivity during La 3÷ washing. This expectation was correct. As indicated from the studies of the 4SCa net uptake with time, it may

541 be anticipated that cooling should reduce the amounts of radioactivity incorporated during the period when La 3+ equilibrates in the extracellular space. Nevertheless, the La3+-nondisplaceable 4SCa was substantially increased when washing was performed with La 3÷ medium kept at 1°C. This observation suggests that cooling made La 3÷ more efficient in preventing the loss of intracellularlylocated 4SCa; an idea for which direct experimental support was obtained when studying the efflux of 4SCa from preloaded islets in the perifusion system. In the original procedure for La 3÷ washing employed for measuring intracellular 4SCa in smooth muscle, the preparation was placed in non-radioactive solution containing 2 mM La 3÷ for 60 min [1]. The procedure was later modified so that 10 mM La 3÷ was first added to the radioactive loading medium followed, 3 min later, by transfer of the specimen to non-radioactive solution containing 10 mM La 3÷ [2]. When introduced in concentrations as high as 10 mM, La 3÷ may have non-specific stabilising effects on the cell membrane in addition to the specific Ca~+-antagonistic action [16]. In the present study similar amounts of La3÷-nondisplaceable 4SCa were f o u n d for a wide range of La 3+ concentrations, indicating that an increase of La 3÷ above the previously used 2 mM level does not result in more accurate measurement of the t-cell content of intracellular 4SCa. An important aspect of how modifications of the La3+-wash procedure affect the calcium distribution in the H-cells is the glucose effect. The previous reports [3--9] of a pronounced glucose stimulation of La3+-nondisplaceable 4SCa in islets washed at 37°C was confirmed and found also when washing was per -~ formed at I°C. In fact, reduction of the temperature of the washing medium resulted in a substantial increase in the amounts of La3+-nondisplaceable 4SCa incorporated in response to glucose. Glucose has been found to stimulate the incorporation of 4SCa also into the pool displaceable by La 3+. A prerequisite for demonstrating this effect was that the radioactive loading of the islets was performed in the presence of trace amounts of calcium [8] or that the loading was followed by short rinsing in non-radioactive medium [ 5]. The present data provide further evidence for the view that glucose promotes the incorporation of La3+-displaceable 4SCa from calcium-deficient medium also when the islets are taken for analyses without previous rinsing in non-radioactive medium. Irrespective of whether the islets were rinsed or not, lowering of the temperature of the washing medium resulted in disappearance of the stimulatory effect of glucose on the incorporation of 4SCa in the La3+-displaceable pool. The observation that the stimulatory effect of glucose on the La3+-displaceable 4SCa is unmasked by short rinsing in non-radioactive medium has drawn attention to the possibility that the glucose-sensitive superficial calcium is derived by efflux from the interior of the H-cells [9]. The present study provides support for this idea by demonstrating that the glucose effect is actually abolished when the 4SCa efflux is minimized by lowering the temperature of the La 3+ washing medium. The effect of glucose on the La3+-displaceable 4SCa disappears if the rinsing period in non-radioactive medium is extended to 5 min [5]. In view of its lability and intimate relations to the secretory activity of the H-cells it is tempting to believe that the superficial pool of glucose-stimulated 4SCa, as demonstrated by the standard procedure of La 3÷ washing, originates from the functionally i m p o r t a n t Ca 2+ supposed to exist in the immediate submembrane space.

542

Acknowledgements This work was supported by the Swedish Medical Research Council (12x562). Gertrud Andersson and Inger RSnnbergh contributed excellent technical assistance. References 1 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. 3 0 , 4 4 - - 5 4 2 V a n B r e e m e n , C., F a r i n a s , B . R . , C a s t e e l s , R . , G e r b a , P., W u y t a c k , F. a n d D e t h , R. ( 1 9 7 3 ) Phil. T r a n s . R. S o c . L o n d . B. 2 6 5 , 5 7 - - 7 1 3 H e l l m a n , B., S e h l i n , J. a n d T~/ljedal, I.-B. ( 1 9 7 6 ) J. P h y s i o l . 2 5 4 , 6 3 9 - - - 6 5 6 4 H e U m a n , B. ( 1 9 7 7 ) in D i a b e t e s R e s e a r c h T o d a y , S y m p o s i a M e d i c a H o e c h s t 12, p p . 2 0 7 - - 2 2 2 , F . K . Schattauer Verlag, Stuttgart 5 H e l l m a n , B., Sehlin° J. a n d T~/ljedal, I.-B. ( 1 9 7 6 ) S c i e n c e 1 9 4 , 1 4 2 1 - - 1 4 2 3 6 B l o o m , G . D . , H e U m a n , B., S e h l i n , J. a n d T~'ljedal, I.°B. ( 1 9 7 7 ) A m . J. P h y s i o l . 2 3 2 , E l l 4 - - E l l S 7 H e l l m a n , B., L e n z e n , S., S e h l i n , J. a n d T~/ljedal, I.-B. ( 1 9 7 7 ) D i a b e t o l o g i a 1 3 , 4 9 - - 5 3 S H e l l m a n , B. a n d G y l f e , E. ( 1 9 7 7 ) H o r m o n e M e t a b . R e s . , in p r e s s 9 H e l l m a n , B. ( 1 9 7 8 ) in R e g u l a t o r y M e c h a n i s m s o f C a r b o h y d r a t e M e t a b o l i s m , P r o c . l l t h F E B S Meet. ( E s s m a n , V., e d . ) , P e r g a m o n Press, O x f o r d , in p r e s s l 0 H e l l 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 11 G y l f e , E. a n d H e B m a n , B. ( 1 9 7 8 ) B i o c h i m . B i o p h y s . A c t a 5 3 8 , 2 4 9 - - 2 5 7 1 2 H e U 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 3 - - 3 9 8 13 B e r g g r e n , P.-O., B e r g l u n d , O. a n d H e U m a n , B. ( 1 9 7 7 ) A n a l . B i o c h e m . , in p r e s s 1 4 H e l l m a n , B., S e h l i n , J. a n d T~/ljedal, I.-B. ( 1 9 7 1 ) D i a b e t o l o g i a 7, 2 5 6 - - 2 6 5 1 5 L a h r t z , H . G . , L f i U m a n n , H. a n d V a n Z w i e t e n , A. ( 1 9 6 7 ) B i o c h i m . B i o p h y s . A c t a 1 3 5 , 7 0 1 - - 7 0 9 16 Weiss, G.B. ( 1 9 7 4 ) A n n u . Rev. P h a r m a c o l . 1 4 , 3 4 3 - - 3 5 4