Effects of dextran-linked chloromercuribenzoic acid on insulin release from microdissected pancreatic islets

96

Biochimica et Biophysica Acta, 451 (1976) 96--105 © Elsevier]North-Holland Biomedical Press

BBA 28059 EFFECTS OF DEXTRAN-LINKED CHLOROMERCURIBENZOIC ACID ON INSULIN RELEASE FROM MICRODISSECTED PANCREATIC ISLETS

STIG -~KERSTROM, BO HELLMAN *,/~KE LERNMARK, BERNT LINDBEI~G, MONICA SODERBERG and INGE~BERT TALJEDAL Department of Histology, University of Ume~, S-901 87 Ume8 and Pharmacia AB, S-751 25 Uppsala (Sweden)

(Received April 2nd, 1976)

Summary Insulin release in response to dextran-linked p-chloromercuribenzoic acid was studied in microdissected pancreatic islets of non-inbred o b / o b - m i c e . No contamination of the dextran-linked mercurial with free chloromercuribenzoic acid was detected before or after the incubation with islets. In comparison with free mercurial, of the same thiol-blocking activity, the dextran-linked compound had a weak insulin-releasing action with a different dose vs. response relationship. The dextran-linked mercurial had no demonstrable effect on the islet content of cyclic AMP. The results support the hypothesis that free organic mercurials mainly stimulate insulin release by blocking thiol groups t h a t are embedded within the ~-cell plasma membranes beneath their surfaces.

Introduction Chloromercuribenzene-p-sulphonic acid (CMBS) or p-chloromercuribenzoic acid (CMB) promptly stimulates the release of insulin from microdissected pancreatic islets [1]. CMBS also increases the islet cell permeabilities to Na ÷ and Rb * [2--4] and appears to stimulate the formation of cyclic 3',5'--AMP in the islets [5]; these effects may be responsible for the enhancement of insulin release [4]. Because CMBS is a slowly permeating thiol reagent and y e t exerts a fast effect on insulin release, it was suggested that target thiols of importance for secretion are located in the plasma membranes of the ~-cells [1]. This hypothesis was supported by subsequent observations that other thiol reagents could also stimulate insulin release [6--8]. * Present address: Department of Histology, Biomedicum, University of Uppsala, S-751 28 Uppsala (Sweden). A b b r e v i a t i o n s : C M B S , c h l o r o m e r c u r i b e n z e n e - p - s u l f o n i e a c i d ; C M B , p-chloromereuribenzoic acid~ SITS, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid; AE-dextran° aminoethyldextran.

97 The effects of CMBS on insulin release, as well as on monovalent cation fluxes and cyclic 3',5'-AMP, are inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS). SITS is an amino-reactive probe of plasma membranes [9,10] that markedly inhibits the permeation of CMBS in islet cells [11]. It was therefore suggested that the target thiols for CMBS are embedded within the fl-cell plasma membranes beneath their surfaces [11]. To test this hypothesis further, we have investigated the effects of CMB linked to dextran, the idea being that the dextran carrier would prevent the mercurial from reaching all but the most superficial thiol groups in the ~-cells. CMB
Chemicals Reagents of analytical grade were used. p-chloromercuribenzoic acid (CMB) linked to dextran (CMB
Chromatography Ion exchange cellulose was prepared for chromatography by washing with 0.2 M HC1 and 0.1 M NaOH. The cellulose was then rinsed in water and swollen in Krebs-Ringer bicarbonate buffer [16] supplemented with 20 mM Hepes to stabilize pH at 7.4 (buffer I). CMB-dextran or free CMB was applied to a 0.6 cm × 7 cm column in 0.5 ml buffer I and eluted with the same buffer (room temperature). Fractions of 0.5 ml were collected and E23s determined. To obtain chromatographically pure samples of CMB
98 solved in this type of control medium. The thiol-blocking activity of the various chromatographic fractions was also determined. Diluted samples of effluent were incubated for 10 min at room temperature with 0.5 mM L-cysteine. After incubation, 0.1 ml of the medium was mixed with 1 ml of 0.2 mM 6,6'~lithiodinicotinic acid and the formation of 6-mercaptonicotinic acid photometrically recorded at 344 nm.

Experiments with islets Fresh pancreatic islets were microdissected free-hand [17] from adult noninbred ob/ob-mice of a local colony. Incubations were performed at 37°C in the presence of 3 mM D-glucose and 1 mg of albumin/ml. In experiments with CMB
Z UJ -J <~

z,O 0

LLI ~Z U W

2oo f-

ud i

I t~ ~

L

200

~00

_L

600

BOO

CMB-DEXTRAN [ pg//ml ) Fig'. 1. S u l p h y d r y l - r e a c t i v i t y o f C M B - d e x t r a n e x p r e s s e d as e q u i v a l e n t s o f f r e e CMB (pM). C M B - d e x t r a n or CMB w a s i n c u b a t e d f o r 1 0 r a i n w i t h 0.5 mM L-eysteine at room t e m p e r a t u r e in 1 ml in b u f f e r 1, p i t 7.4. The sulphydryl-reactive L - c y s t e i n e p r e s e n t a f t e r i n c u b a t i o n w a s d e t e r m i n e d b y m i x i n g 100 pl i n c u b a t i o n m e d i u m w i t h 1 ml 0.2 mM 6 , 6 ' - d i t h i o d i n i c o ~ L n i c acid and measuxing the 6-mercaptonicotinic acid formed b y its a b s o r b a n c e at 3 4 4 n m . S e p a r a t e e x p e r i m e n t s showed that AE-dextran influenced neither the reaction between CMB and L-eysteine, nor the reaction of L-cysteine with 6,6'~ditbiodinicotinic acid.

99 Results

Chemical properties of CMB-dextran The thiol reactivity of CMB
Purification of CMB-dextran by chromatography To purify the CMB
50

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v

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t

w

45

L

5

10 FRACTION

~5

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NUMBER

Fig. 2. Separation of CMB-dextran ( o ) and CMB ( e ) on ion e x c h a n g e c e l l u l o s e . S a m p l e s o f C M B - d e x t r a n ( 4 0 0 /~g/ml) o r C M B (0.2 raM) w e r e a p p l i e d in 0.5 m l b u f f e r I a n d e l u t e d w i t h t h e s a m e b u f f e r . F r a c t i o n s o f 0.5 m l w e r e c o l l e c t e d a n d t e s t e d f o r r e a c t i v i t y a g a i n s t L - c y s t e i n e .

100

5O

!

4

0

5

10

15

FRACTION NUMBER Fig. 3. S u l p h y d r y l - r e a c t i v i t y of c h r o m a t o g r a p h e d C M B - d e x t r a n b e f o r e (o) a n d a f t e r (m) i n c u b a t i o n w i t h m i e r o d i s s e c t e d islets. C M B - d e x t r a n in NaCl was p u r i f i e d b y ion e x c h a n g e c h r o m a t o g r a p h y o n W h a t m a n D E - 2 2 cellulose a n d d i l u t e d w i t h b u f f e r I t o a final c o n c e n t r a t i o n of 8 0 0 p g / m l . S a m p l e s o f 5 0 0 pl w e r e r e c h r o m a t o g r a p h e d e i t h e r i m m e d i a t e l y or a f t e r b e i n g i n c u b a t e d w i t h f o u r p a n c r e a t i c islets f o r 30 rain a t 37°C. The various fractions obtained were analyzed for reactivity against L-cysteine.

was performed immediately or after the CMB
Effects o f CMB-dextran on insulin release Fig. 4 shows the release of insulin during 30 min of static incubation with various concentrations of non-chromatographed CMB~textran and free CMB. The secretory response to CMB-dextran was significant although much smaller than that induced by free CMB added in combination with free AE
101

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f DEXTRAN( pg/ml ) CMS(pM)

0

4~

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320

640

Fig. 4. A m o u n t s of insulin r e l e a s e d in t h e p r e s e n c e o f n o n - c h r o m a t o g r a p h e d s a m p l e s o f A E - d e x t r a n ( o ) , C M B - d e x t r a n (o), o r A E - d e x t r a n + CMB ( e ) . A f t e r 4 0 rain o f p r e l i m i n a r y i n c u b a t i o n in K r e b s - R i n g e r b i c a r b o n a t e b u f f e r c o n t a i n i n g 3 m M glucose a n d 1 m g / m l a l b u m i n t h e islets w e r e i n c u b a t e d f o r 30 rain in t h e s a m e t y p e o f m e d i u m s u p p l e m e n t e d w i t h test s u b s t a n c e s as i n d i c a t e d . M e a n v a l u e s -+ S.E. o f 8 experiments.

TABLE I I N S U L I N R E L E A S E IN R E S P O N S E T O A E - D E X T R A N A N D C M B - D E X T R A N B E F O R E A N D A F T E R ION EXCHANGE CHROMATOGRAPHY 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 40 rain in b u f f e r I c o n t a i n i n g 3 m M glucose a n d 1 m g / m l a l b u m i n , islets w e r e i n c u b a t e d f o r 3 0 rain in 3 0 0 #1 o f t h e s a m e t y p e of b u f f e r s u p p l e m e n t e d w i t h d e x t r a n s as i n d i c a t e d . Mean values -+ S.E. o f 8 e x p e r i m e n t s . Dextran tested

None (control) N o n - c h r o m a t o g r a p h e d AEdextran (350 #g/ml) Non-cb-romatographed AEdextran (700/~g/ml) N o n - c h r o m a t o g r a p h e d CMBdextran (350/~g/ml) N o n - c h r o m a t o g r a p h e d CMBdextran (700/~g/ml) C h r o m a t o g r a p h e d CMBdextran (350/~g/ml) C h r o m a t o g r a p h e d CMBdextran (700 #g/ml)

Insulin release ( n g / # g d r y islet) Test

Test minus control

P value for effect

0.16 + 0.03

--

--

0.32 ± 0.05

0.16 ± 0.04

<0.01

0.39 + 0.10

0.23 ± 0.07

<0.02

3.03 ± 0.54

2.87 + 0 . 5 4

<0.005

3.41 + 0.61

3.25 ± 0 . 6 0

<0.005

2.96 ± 0 . 4 8

2.80 ± 0.48

<0.005

3.62 ± 0.85

3.46 + 0.85

<0.01

102

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,~ 2o r~

rY W Q_ Q _LI.I

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60 INCUBATION

120 TIME ( M I N )

Fig. 5. Insulin release w i t h t i m e in the p r e s e n c e o f A E - d e x t r a n (G), n o n - c h r o m a t o g r a p h e d C M B - d e x t r a n (G) or C M B - d e x t r a n purified b y ion e x c h a n g e c h r o m a t o g r a p h y (m). A f t e r 4 0 rain o f p r e l i m i n a r y i n c u b a t i o n in b u f f e r I c o n t a i n i n g 3 m M g l u c o s e and 1 m g / m l a l b u m i n , islets w e r e i n c u b a t e d for various p e r i o d s o f t i m e in 3 0 0 ~ul m e d i u m s u p p l e m e n t e d w i t h 7 0 0 p g / m l o f the d e x t r a n t e s t e d . M e a n values ~ S.E. o f 8 experiments.

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Fig. 6. D y n a m i c s o f i n s u l i n release w h e n p e r i f u s i n g m i e r o d i s s e c t e d islets w i t h K r e b s - R i n g e r b i c a r b o n a t e 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 a n d 1 m g / m i a l b u m i n . Bars i n d i c a t e e x p o s u r e t o A E - d e x t r a n ( u p p e r p a n e l ) or C M B - d e x t r a n ( l o w e r p a n e l ) .

103 T A B L E II C O N T E N T O F C Y C L I C AMP IN I S L E T S E X P O S E D T O A E - D E X T R A N A N D C M B - D E X T R A N P U R I F I E D BY I O N E X C H A N G E C H R O M A T O G R A P H Y 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 for 4 0 rain in b u f f e r I c o n t a i n i n g 3 m M g l u c o s e and 1 m g / m l a l b u m i n , islets w e r e i n c u b a t e d for 3 0 rain in 3 0 0 ~ul o f the s a m e t y p e of m e d i u m s u p p l e m e n t e d as i n d i c a t e d , T h e c o n c e n tration w e r e 7 0 0 ~zg/ml for the d e x t r a n s and 1 m M for IBMX. Mean values -+ S.E. o f 7--8 e x p e r i m e n t s . Compounds tested

C o n t e n t o f c y c l i c AMP ( f m o l / ~ g d r y islet)

I n s u l i n release ( n g / # g d r y islet)

None AE-dextran CMB-dextran IMBX IBMX + AE-dextran IBMX + C M B - d e x t r a n

7.16 5.84 6.30 30.28 30.21 36.69

0.22 0.20 3.97 0.66 0.68 6.98

-+ 1.32 + 1.27 + 1.28 -+ 2.60 -+ 3.85 + 4.66

(7) (7) (S) (8) (8) (8)

+ 0.07 + 0.03 -+ 0 . 7 9 + 0.12 + 0.09 -+ 1.24

(8) (8) (8) (8) (8) (S)

insulin release did not increase with increasing CMB
104 speak against such an interpretation. First, free organic mercurial exerts a p r o m p t action with very high peak rates of insulin secretion occurring within the first 5 rain [1]. Detailed studies of the kinetics of insulin release from microdissected islets have indicated that the initial response is mainly due to secretion from the peripheral fi-cells even when the stimulus is a small-molecular c o m p o u n d such as glucose [23,24]. Yet, CMB
References 1 2 3 4 5

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105

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