Biochimica et Biophysica Acta 929 (1987) 81-87 81 Elsevier

BBA 12039

C a d m i u m - i n d u c e d insu l in r e l e a s e d o e s n o t invo lve c h a n g e s

in i n t r a c e l l u l a r h a n d l i n g o f c a l c i u m

T h o m a s N i l s s o n , P e r - O l o f B e r g g r e n a n d Bo H e l l m a n

Department of Medical Cell Biology, Biomedicum, University of Uppsala, Uppsala (Sweden)

(Received 12 December 1986)

Key words: Cadmium ion; Insulin release; Calcium channel; Inositol trisphosphate: (Pancreatic/3 cell)

A possible interaction between Cd 2+ and Ca 2+ as a component in Cd2+-induced insulin release was investigated in /3 cells isolated from obese hyperglycemic mice. The glucose stimulated Cd ~+ uptake was dependent on the concentration of sugar. This uptake was sigmoidal with a K,, for glucose of about 5 mM and was suppressed by both 50/~M of the voltage-activated Ca 2+ channel blocker D-600 and 12 mM Mg z+. In the presence of 8 mM glucose 5 /~M Cd 2+ evoked a prompt and sustained stimulatory response, corresponding to about 3-fold of the insulin release obtained in the absence of the ion. Whereas 5/~M Cd 2 + was without effect on the glucose-stimulated 45Ca efflux in the presence of extracellular Ca 2+, 40 /~M inhibited it. At a concentration of 5 #M, Cd 2+ had no effect on the resting membrane potential or the depolarization evoked by either glucose or K +. In the absence of extracellular Ca 2 + there was only a modest stimulation of 45Ca efflux by 5 /~M Cd 2+. Studies of the ambient free Ca 2+ concentration maintained by permeabilized cells also indicate that 5 /~M Cd ~+ do not mobilize intracellularly bound Ca 2+ to any great extent. On the contrary, at this concentration, Cd 2+ even suppressed inositol 1,4,5-trisphosphate (IP3) -induced Ca 2+ release. The present study suggests that Cd 2+ stimulates insulin release by a direct mechanism which does not involve an increase in cytoplasmic free Ca 2 + concentration.

Introduction

In a recent study, Cd 2+ fluxes and related effects on secretory activity and metabolism were characterized in /3 cell-rich pancreatic islets iso- lated from obese hyperglycemic mice [1]. It was demonstrated that the /3-cell uptake of Cd 2+ is facilitated by opening of voltage-activated Ca 2+ channels. Furthermore, the accumulation of Cd 2+ is similar to that of Ca 2+ in the involvement of a component of intracellular sequestration promo- ted by glucose. Whereas basal insulin release is

stimulated by low concentrations of Cd 2+, con- centra t ions that drastically suppress glucose oxidation do not affect basal release but signifi- cantly inhibit the secretory response to the sugar.

In the present study, attempts were made to characterize further the extent to which Cd 2+ and Ca 2+ interact, both at the level of the plasma membrane and intracellularly. Such a characteri- zation should also clarify the involvement of a Cd2+-mediated release of Ca 2+ as a component in CdZ+-stimulated insulin release.

Materials and Methods

Correspondence: P.-O. Berggren, Department of Medical Cell Biology, Biomedicum, University of Uppsala, S-751 23 Up- psala, Sweden.

Animals and preparation of islets and cells. Adult obese-hyperglycemic mice (ob /ob ) of both sexes were taken from a local non-inbred colony [2] and

0167-4889/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

82

starved overnight. The animals were killed by decapitation and the islets were isolated by a collagenase technique. When investigating changes in membrane potential and regulation of the am- bient free Ca 2+ concentration maintained by per- meabilized /~ cells, a cell suspension was prepared essentially as described by Lernmark [3]. Briefly, the islets were dissociated into single cells and small clusters by shaking in a Ca 2 +- and Mg 2 +-de- ficient medium supplemented with EGTA. The cells were incubated overnight at 37 o C, pH 7.4, in 12 ml RPMI 1640 medium supplemented with 10% NU-serum tM (Collaborative Resarch Inc.), 100 I U / m l penicillin, 60 /~g/ml gentamycin and 100 /~g/ml streptomycin. To avoid attachment of the cells to the culture flask, the suspension was shaken gently.

Media. The basal medium used for isolation of islets, Cd 2+ uptake, 45Ca efflux experiments and studies of the membrane potential was a Hepes buffer (pH 7.4) physiologically balanced in cations with CI as the sole anion [4]. Since Cd 2+ binds to albumin, the concentrations given will refer to the dialyzable concentration of the ion. However, no corrections were made for the possible binding of Cd 2+ to other ions such as C1- [5]. Details of the media composition are given in the legends to figures.

Determination of islet cadmium. Cadmium was measured with electrothermal atomic absorption spectrometry as previously described [6].

Studies of 45Ca eff]ux and insulin release. The dynamics of 45Ca efflux and insulin release were studied using previously described procedures [7]. Samples were analyzed for radioactivity by liquid scintillation counting and insulin was assayed ra- dioimmunologically using crystalline mouse in- sulin as a reference.

Measurements of membrane potential Qualita- tive changes in membrane potential were mea- sured by the fluorescent dye bis-oxonol [8] ob- tained from Molecular Probes, Junction City, OR, U.S.A. The recordings were made at 37°C with constant stirring in a 1-cm cuvette placed in a spectrofluorometer with excitation and emission wavelengths at 540 and 580 nm, respectively. The dye, at a final concentration of 150 nM, was allowed to equilibrate with 1.5 ml of buffer solu- tion before adding the cells. After waiting for the

fluorescence signal to stabilize, test substances were added from stock solutions.

Studies of the ambient free Ca 2 + concentration maintained by permeabilized cells. A buffer con- taining 110 mM KC1, 10 mM NaC1, 2 mM KH2PO4, 1 mM MgC12, 25 mM Hepes and 1 m g / m l albumin was used. The pH was adjusted to 7.0 with concentrated KOH. The cells were permeabiliz~d by exposing the cell suspension (300 /~1) to high-voltage discharges (5 pulses of 2.5 k V / c m ) . After permeabilization, the cells were centrifuged and resuspended in 25 /zl medium supplemented with 1 /~g/ml oligomycin, 0.2 /~M antimycin, 2 mM MgATP and an ATP-regener- ating system consisting of 10 mM phosphocreatine and 20 U / m l creatine phosphokinase. The sus- pension was subjected to constant stirring and the free Ca 2+ concentration in the medium was mea- sured with a Ca2+-selective minielectrode, con- structed and calibrated essentially as described by Tsien and Rink [9]. All experiments were per- formed at room temperature. Test substances were added with 250 nl constant volume pipettes, pre- pared according to a previous description [10]. Inositol 1,4,5-trisphosphate (IP3) was obtained from Amersham, Buckinghamshire, England.

Estimation of cell number. A sample of the cell suspension was taken after each experiment and centrifuged at 8000 × g for 1 min in a 400 ~1 polyethylene test tube. The bot tom was cut off, freeze-dried overnight and the pellet was weighed on a quartz-fibre balance. The approximate num- ber of cells was estimated assuming that 1 /~g dry weight corresponds to 3.6- 103 cells [3].

Results

Glucose stimulated Cd 2÷ uptake sigmoidally, having a K m of about 5 mM (Fig. 1). At 8 mM the uptake levelled off, corresponding to a maxi- mal stimulation 50% higher than the basal uptake. Addition of 50 /~M of the voltage-activated Ca 2+ channel blocker D-600 suppressed the Cd 2+ up- take at glucose concentrations of no less than 8 mM. In this case the maximal stimulation was not greater than 20%. The glucose-stimulated Cd 2+ uptake was also suppressed by 12 mM Mg ~+, a concentration not affecting the basal uptake of the ion (data not shown).

f

140

'~ 120

Z 100

160

i i ~ i i

0 5 10 15 20

CONCENTRATION OF D-GLUCOSE (mMOL/L)

Fig. 1. Effects of different concentrations of glucose on Cd 2+ uptake in the presence or absence of D-600. After 30 rain of preincubation the islets were loaded for 60 min with 2.5 ~M Cd 2+ in the presence of the indicated concentrations of glu- cose in medium containing 1.28 mM Ca 2+ supplemented ( © ) or not (e) with 50 ~M of the voltage-activated Ca 2+ channel blocker D-600. After loading, the islets were washed for 30 min at 2 ° C in a medium lacking Cd 2+, Ca 2+ and glucose and supplemented with 0.5 mM EGTA. The uptake of Cd 2+ is

83

The effects of Cd 2+ on the kinetics of insulin release were investigated under conditions in which the islets were exposed to an intermediate stimula- tory glucose concentration of 8 mM (Fig. 2). At a concentration of 0.5 /~M, Cd 2+ had no marked stimulatory effect on insulin release. However, when the concentration was 5 /~M there was a prompt and sustained stimulatory effect, with a maximum release corresponding to about 3-fold of that obtained in the absence of the ion.

To clarify whether these stimulatory effects of Cd 2+ were secondary to changes in the cellular handling of Ca 2+, studies of 45Ca efflux were performed. In the presence of 1.28 mM Ca 2+, addition of 5 ~M Cd 2+ did not affect the 45Ca

efflux, whereas there was a sustained increase at 4 0 / t M Cd 2+ (Fig. 3, panel A). Although this high Cd 2+ concentration inhibited the glucose-stimu- lated 45Ca efflux, there was no effect of 5 ~M Cd 2+. In the presence of 8 mM glucose and 5/.tM

given as a percentage of that obtained in the absence of glucose, the latter corresponding to 0.14_+0.01 m m o l / k g dry weight. Mean values_+ S.E. for seven experiments.

h i

g ) - r~ r~

2 0

h i U3 < h i

J 10 UJ a~

Z

J Z

Cd 2+ (0.5 tiM)

i i

6O ~0 i i i i

1 0 0 6 0 8 0 1 0 0

P E R I F U S I O N T I M E ( M [ N )

Cd 2+ (5 pM)

Fig. 2. Kinetics of insulin release in response to Cd 2+. Perifusion was performed in an apparatus with three parallel channels. After 60 min of perifusion with medium containing 1.28 mM Ca 2+, 8 mM glucose and 1 m g / m l albumin, the media for two of the channels were supplemented with either 0.5 (panel A) or 5 #M (panel B) Cd 2+ during the periods indicated by the horizontal black bars. The rate of insulin release is given as mean values+_ S.E. for five experiments.

84

2 0 0

Z uJ O n,- IJJ n v

X

_J la... U.. UJ

~( . . )

150

100

50

A 2 +

Cd

GLUCOSE (20 mM)

T

uM

L____ 60 8 0 100 120

2+ Ccl

G L U C O S E (8 m M )

4 0 uM

, , L - - _ _

6 0 8 0

PERIFUSION TIME (MIN)

Fig. 3. Efflux of 45Ca in response to C d 2÷ in a Ca 2 ~ rich medium. The islets were loaded for 90 min with 1.28 mM 45Ca in the presence of 20 mM glucose and then perifused with a medium containing 1.28 mM Ca 2+ and 1 mg/ml albumin. Cd 2~ was introduced at the indicated concentrations during the periods represented by the horizontal black bars. In panel A, 20 mM glucose (open bar) was added to the medium after 100 min of perifusion. In panel B, the glucose concentration was kept at 8 mM throughout the experiment. The efflux of 45Ca is given as a percentage of the average 45Ca efflux in the individual experiments during the 10 min preceding the introduction of Cd 24. Mean values + S.E. for four to five experiments.

Z O I -

N E:

W a

25 mM 5 ,uM Cd 2.' K +

25 mM

H20 K +

2+ 5 /JM Cd

I G u co E 12 mM

H20 GLUCOSE

U,N'

Fig. 4. Effects of Cd 2 + on membrane potential, as indicated by changes in bis-oxonol fluorescence. In trace A, two pulses of Cd 2+, in the absence of glucose, were added prior to depolari- zation with 25 mM K +. In trace C, the experiment was performed in the presence of 8 mM glucose and a pulse of (;d 2~ and additional glucose were added as indicated. Traces B and D served as controls and were obtained under similar conditions as A and C, except no pulses of Cd 2+ were added. Concentrations of cells in A, B, C and D were 1.3.106/ml, 1.5.10~'/ml, 1.2.106/ml and 1.4.106/ml, respectively. The traces shown are typical for experiments repeated three times.

C d ~ +, c o n d i t i o n s i n d u c i n g a s u s t a i n e d s t i m u l a t i o n

o f i n s u l i n r e l ease (see above ) , t h e r e was o n l y a

t r a n s i e n t s t i m u l a t i o n o f the 45Ca eff lux. In ad -

d i t i o n , w h e n the C d 2+ c o n c e n t r a t i o n was in-

c r e a s e d to 40 ~ M , t h e r e was e v e n a s u p p r e s s i o n of

t he e f f lux (Fig . 3, p a n e l B). N o t w i t h s t a n d i n g , t he

fac t t h a t 5 F M C d 2+ l a c k e d m a j o r e f fec t s o n b o t h

b a s a l a n d g l u c o s e - s t i m u l a t e d 45Ca ef f lux , i t was

n e c e s s a r y to ru le o u t a d i r e c t e f fec t of th i s C d 2+

c o n c e n t r a t i o n o n t he m e m b r a n e p o t e n t i a l (F ig . 4).

A s is e v i d e n t f r o m t r ace A, r e p e t i t i v e a d d i t i o n s of

C d 2+ were w i t h o u t e f fec t o n the r e s t i n g m e m b r a n e

p o t e n t i a l a n d d i d n o t i n t e r f e r e w i t h t he d e p o l a r i -

z a t i o n i n d u c e d b y h i g h c o n c e n t r a t i o n s of K +.

F u r t h e r m o r e , t r ace C s h o w s t h a t C d 2+ n e i t h e r

i n f l u e n c e d the m e m b r a n e p o t e n t i a l in t he p r e s e n c e

o f 8 m M g lucose n o r i n t e r f e r e d w i t h t he d e p o l a r i -

z a t i o n r e s u l t i n g f r o m a n a d d i t i o n a l 12 m M of the

sugar .

50

5 . 5

Cd 2 ~ (5 .uM)

2 0 0 Z LU o n.- LLI t,,

X 150 :D ..-i LI_ I.J_ LU

~0 1 O0

6~0 8'0 100 1'20

PERIFUSION TIME (MIN)

140

Fig. 5. Effects of Cd 2+ on the efflux of 45Ca in a Ca2+-defi - cient medium. The islets were loaded for 90 min with 1.28 mM 45Ca2 ~ in the presence of 20 m M glucose and perifused with a glucose-free, CaZ+-deficient medium containing 1 m g / m l al- bumin. 5 ~aM Cd 2+ was introduced during the period indicated by the horizontal black bar. The efflux of 45 Ca is given as a percentage of the average 45Ca efflux in the individual experi- ments during the 10 min preceding the introduction of Cd 2+,

Mean values_+ S.E. for five experiments are shown.

85

To elucidate the effects of C d 2+ o n the in- tracellular handling of C a 2+, the efflux studies were performed in a Ca2+-deficient medium. Fig. 5 shows that the introduction of 5 /~M Cd 2+ evoked only a modest stimulation of 4 5 C a efflux, demonstrating that Cd 2+, only to a very minor extent, mobilizes intracellularly bound C a 2+. In an extension of these studies we investigated how Cd 2+ affected the ambient free Ca 2+ concentra- tion maintained by permeabilized cells (Fig. 6). As is evident from panel B, addition of 5 ~M Cd 2+ provoked only a small inflexion in the recording. Most of this effect can be accounted for as direct interference with the electrode, since a similar phenomenon was obtained in the absence of cells (data not shown). More importantly, C d 2+ di- rectly interfered with the IP 3 induced Ca 2+ release under conditions where the cells rapidly buffered a given pulse of Ca 2+ (Fig. 6). It is noteworthy, that we do not know the cytoplasmic Cd 2+ con- centration subsequent to exposing the /~ cells to various extracellular concentrations of the ion. Hence, it was somewhat difficult to choose the appropriate concentration for the experiments with the permeabilized/~ cells. We decided to use 5 #M C d 2+, since lower concentrations had less marked effects on IP3-induced Ca 2 + release and concentra- tions exceeding 50 /~M promoted a rapid Ca 2+ efflux.

A C E L L S

1

0 6 . 0 I

6 . 5

0 5 10 15

B C E L L S

1 'S cZ*

2* Cd

i i i i

0 5 10 15

TIME (MIN)

Fig. 6. Effects of Cd 2+ on the IP3-induced Ca 2+ release in permeabilized B-cells. Cells (1.8.10 5 and 2.5.105) were added at 0 min and the additions of Cd 2+, IP 3 and Ca 2+

(0.12 nmol) were prepared as indicated. The final concentrations of Cd 2+ and IP 3 were 5 and 6 p,M, respectively. Panel A demon- strates a control experiment without any ad- ditions of Cd 2+. The concentration of Ca 2+ in the medium is given as - l og [Ca 2+ ] in mol/1. The traces shown are typical for ex- periments repeated at least five times.

86

Discussion

Studies of the permeation mechanism of Ca 2 + conductance have revealed that Cd 2 + can function as an efficient blocker of Ca 2+ channels [11]. In addition, Cd 2+ can also carry current through Ca 2+ channels [12] and both depolarize parathy- roid cells [13] and replace Ca 2+ in the generation of action potentials in muscle fibres [14]. In the present study, glucose was found to stimulate Cd 2 + uptake. With the demonstration that the stimula- tory effect of glucose was suppressed by both D-600 and high concentrations of Mg 2+ it can be postulated that voltage-activated Ca 2+ channels mediate the influx of C d 2+ also into pancreatic cells. In a previous study, we proposed that glu- cose promoted the sequestration of Cd 2+ in in- tracellular stores [1]. This assumption was based on the observation that D-600 only partially sup- pressed the stimulatory effect evoked by 20 mM of the sugar, under conditions where K+-stimu - lated Cd 2+ uptake was abolished. The present study provides further support for such a sequestration in demonstrating that D-600 had similar effects also at lower glucose concentra- tions. The fact that 40 /~M Cd 2+ inhibited a glucose stimulated 45Ca efflux in the presence of extracellular Ca 2+ supports the concept that high concentrations of Cd 2+ also prevent the entrance of Ca 2+ through voltage-activated Ca 2+ channels. Although such an interference is consistent with the previously observed inhibition of a glucose- stimulated insulin release by 160 /~M Cd 2~, it should be recalled that this relatively high con- centration of the ion also suppresses the oxidation of glucose [1].

Under the present experimental conditions, 5 ~M Cd 2+ evoked a transient stimulation of 45Ca efflux in the absence of extracellular Ca 2+, sup- porting the notion that Cd 2+ only promotes a modest release of Ca 2 + from intracellular binding sites. Also, the measurements of the ambient free C a 2 + concentration in a suspension of permeabi- lized cells demonstrate that C d 2+ has no major effect on Ca 2+ release. It is, therefore, unlikely that CdZ+-induced insulin release is due to mobili- zation of intracellularly bound Ca 2+

It has been shown that the receptor stimulated phosphodies tera t ic hydrolysis of phosphat i -

dylinositol 4,5-bisphosphate results in the forma- tion of diacylglycerol and inositol 1,4,5-tris- phosphate ( Ip0 [15,16]. Whereas the former com- pound has been shown to activate protein kinase C, the latter promotes Ca 2+ release from an in- tracellular pool, most likely represented by the endoplasmic reticulum. Since low concentrations of C d 2+ have been reported to inhibit inositol 1,4,5-trisphosphate phosphatase [17], the enzyme responsible for IP~ degradation, the presence of Cd 2+ should be expected to potentiate the stimu- latory effect evoked by IP 3. However, in a suspen- sion of permeabilized/~ cells, 5/~M Cd 2 + markedly reduced the amount of Ca 2+ released by I ~ . Although the mechanism still needs to be defined it is of interest to note that Cd 2+ is the only reagent tested so far, including various other types of Ca 2+ channel blockers, that directly interferes with the mechanism whereby I ~ induces Ca 2+ release. Hence, it is not plausible that interaction with the IP 3 pathway is part of the mechanism whereby Cd 2+ stimulates insulin release.

Considering alternative ways by which Cd 2+ might elicit insulin release, it should be noted that this divalent cation readily binds to protein sulf- hydryl groups [18]. Other sulfhydryl reagents like chloromercuribenzene-p-sulphonic acid stimulate insulin release, an effect that may be related to the capability of this compound to reduce markedly the/~ cell membrane potential [19]. However, when bis-oxonol fluorescence was used to estimate changes in membrane potential, there were no indications that this Cd 2+ concentration induced depolarization of the/~ cells.

It is well established that Cd 2~ binds to calmodulin, inducing the same conformational changes in this regulator protein as Ca 2+ [20]. Moreover, recent studies have demonstrated that Cd 2+ is capable of forming an allosterically active species of calmodulin which cannot be maintained by physiological concentrations of Ca 2 + alone [21]. With the observation that C d 2+ only slightly mod- ifies the intracellular handling of Ca 2+, it seems likely that Cd 2~ promotes insulin release by a direct mechanism not involving an increase in cytoplasmic free Ca ~ + concentration.

Acknowledgements

This work was supported by the Swedish Medi- cal Research Council (12x-562), the Swedish Di- abetes Foundation, the Nordic Insulin Founda- tion, the S~itra Brunn Foundation and Ake Wi- bergs Foundation. P.-O. Berggren is a recipient of a postdoctoral fellowhip from the Sweidsh Medi- cal Research Council.

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