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Cadmium-induced insulin release does not involve changes in intracellular handling of calcium
Biochimica et Biophysica Acta 929 (1987) 81-87
81
Elsevier BBA 12039
Cadmium-induced insulin release does not involve changes 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 Thomas Nilsson, Per-Olof Berggren and Bo Hellman Department of Medical Cell Biology, Biomedicum, University of Uppsala, Uppsala (Sweden)
(Received 12 December 1986)
Key words: Cadmiumion; 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 isolated 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 promoted by glucose. Whereas basal insulin release is
stimulated by low concentrations of Cd 2+, concentrations that drastically suppress glucose oxidation do not affect basal release but significantly 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 characterization 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 Uppsala, Sweden.
Animals and preparation of islets and cells. Adult obese-hyperglycemic mice ( o b / o b ) 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 ambient free Ca 2+ concentration maintained by permeabilized /~ 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 +-deficient medium supplemented with EGTA. The cells were incubated overnight at 37 o C, p H 7.4, in 12 ml R P M I 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, 4 5 C a 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 radioimmunologically using crystalline mouse insulin as a reference. Measurements of membrane potential Qualitative changes in membrane potential were measured by the fluorescent dye bis-oxonol [8] obtained from Molecular Probes, Junction City, OR, U.S.A. The recordings were made at 3 7 ° 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 solution 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 containing 110 m M KC1, 10 m M NaC1, 2 mM KH2PO4, 1 mM MgC12, 25 m M Hepes and 1 m g / m l albumin was used. The p H 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 m M MgATP and an ATP-regenerating system consisting of 10 m M phosphocreatine and 20 U / m l creatine phosphokinase. The suspension was subjected to constant stirring and the free Ca 2+ concentration in the medium was measured with a Ca2+-selective minielectrode, constructed and calibrated essentially as described by Tsien and Rink [9]. All experiments were performed at room temperature. Test substances were added with 250 nl constant volume pipettes, prepared 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 bottom was cut off, freeze-dried overnight and the pellet was weighed on a quartz-fibre balance. The approximate number 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 m M the uptake levelled off, corresponding to a maximal 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+ uptake 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 m M Mg ~+, a concentration not affecting the basal uptake of the ion (data not shown).
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 stimulatory 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-stimulated 45Ca efflux, there was no effect of 5 ~M Cd 2+. In the presence of 8 mM glucose and 5/.tM
160 f
140
'~
120
Z
100
i
i
~
i
i
0
5
10
15
20
CONCENTRATION OF D-GLUCOSE ( m M O L / 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 glucose 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
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.
Cd 2+ (5 pM)
Cd 2+ (0.5 tiM) hi
g )r~ r~ 20
hi U3 < hi J UJ a~
10
Z
J Z
i
i
6O
~0
i
100 PERIFUSION
i
i
60
80
i
100
TIME(M[N)
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
Cd
A
2+
2+
Ccl
GLUCOSE
(20
GLUCOSE
mM)
(8 mM)
T
uM
200 Z
uJ O n,IJJ n v
150
X _J la... U.. UJ ~(..)
100
50
L____ 60
80
100
40 uM
,
120
,
L
60
80
-
-
_
_
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. 5 /JM Cd
5 ,uM Cd 2.'
2 5 mM K+
2+
I GucoE
Z O I-
25 mM
N
E:
H20
K+
12 mM H20 G L U C O S E
W a
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 depolarization 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 e a s e (see a b o v e ) , t h e r e w a s 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 t h e 45Ca efflux. I n a d d i t i o n , w h e n t h e C d 2+ 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 to 40 ~ M , t h e r e w a s e v e n a s u p p r e s s i o n o f t h e e f f l u x ( F i g . 3, p a n e l B). N o t w i t h s t a n d i n g , t h e f a c t t h a t 5 F M C d 2+ l a c k e d m a j o r e f f e c 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 efflux, it w a s n e c e s s a r y to r u l e o u t a d i r e c t e f f e c t o f t h i s C d 2+ c o n c e n t r a t i o n o n t h e m e m b r a n e p o t e n t i a l ( F i g . 4). A s is e v i d e n t f r o m t r a c e A, r e p e t i t i v e a d d i t i o n s o f C d 2+ w e r e w i t h o u t e f f e c t o n t h e r e s t i n g m e m b r a n e potential and did not interfere with the depolariz 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 o f K +. F u r t h e r m o r e , t r a c e 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 t h e m e m b r a n e p o t e n t i a l in t h e p r e s e n c e of 8 mM glucose nor interfered with the depolariz 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 o f t h e sugar.
85
To elucidate the effects of C d 2+ o n the intracellular 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+ concentration 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+ directly interfered with the IP3 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+ concentration 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 concentrations exceeding 50 /~M promoted a rapid Ca 2+ efflux.
Cd 2 ~ (5 .uM)
200 Z LU
o
n.LLI
t,, X
150
:D ..-i LI_ I.J_ LU
~0 1 O0
50
6~0
8'0
100
1'20
140
PERIFUSION TIME (MIN)
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 m M 45Ca2 ~ in the presence of 20 m M glucose and perifused with a glucose-free, CaZ+-deficient medium containing 1 m g / m l albumin. 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 experiments during the 10 min preceding the introduction of C d 2+, Mean values_+ S.E. for five experiments are shown.
A CELLS
B
CELLS
1
1
'S cZ*
5.5
0 I
Fig. 6. Effects of Cd 2+ o n the IP3-induced C a 2+ release in permeabilized B-cells. Cells
6.0
6.5 2* Cd
0
5
10
15
i
i
i
i
0
5
10
15
TIME (MIN)
(1.8.10 5 and 2.5.105) were added at 0 min and the additions of Cd 2+, IP 3 and C a 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 demonstrates a control experiment without any additions of Cd 2+. The concentration of Ca 2+ in the medium is given as - l o g [ C a 2+ ] in mol/1. The traces shown are typical for experiments 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 parathyroid 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 stimulatory 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 glucose promoted the sequestration of Cd 2+ in intracellular stores [1]. This assumption was based on the observation that D-600 only partially suppressed 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 concentrations. 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 glucosestimulated insulin release by 160 /~M Cd 2~, it should be recalled that this relatively high concentration 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+, supporting 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 permeabilized 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 mobilization of intracellularly bound Ca 2+ It has been shown that the receptor stimulated phosphodiesteratic hydrolysis of phosphati-
dylinositol 4,5-bisphosphate results in the formation of diacylglycerol and inositol 1,4,5-trisphosphate ( I p 0 [15,16]. Whereas the former compound has been shown to activate protein kinase C, the latter promotes Ca 2+ release from an intracellular 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 stimulatory effect evoked by IP 3. However, in a suspension 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 sulfhydryl 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 modifies 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.
87
Acknowledgements This work was supported by the Swedish Medical Research Council (12x-562), the Swedish Diabetes Foundation, the Nordic Insulin Foundation, the S~itra Brunn Foundation and Ake Wibergs Foundation. P.-O. Berggren is a recipient of a postdoctoral fellowhip from the Sweidsh Medical Research Council.
References 1 Nilsson, T., Rorsman, F., Berggren, P.-O. and Hellman, B. (1986) Biochim. Biophys. Acta 888, 270-277 2 Hellman, B. (1965) Ann. NY Acad. Sci. USA 131,541-558 3 Lernmark, ,~. (1974) Diabetologia 10, 431-438 4 Hellman, B. (1975) Endocrinology 97, 392-398 5 Gutknecht, J. (1983) Biochim. Biophys. Acta 735, 185-188 6 Nilsson, T. and Berggren, P.-O. (1984) Anal. Chim. Acta 159, 381-385 7 Gylfe, E. and Hellman, B. (1978) Biochim. Biophys. Acta 538, 249-257
8 Rink, T.J., Montecucco, C., Hesketh, T.R. and Tsien, R.Y. (1980) Biochim. Biophys. Acta 595, 15-30 9 Tsien, R.Y. and Rink, T.J. (1981) J. Neurosci. Methods 4, 73-86 10 Hellman, B., Ulfendahl, H.R. and Wallin, B.G. (1967) Anal. Biochem. 18, 434-443 11 Byerly, L., Chase, P.B. and Stimers, J.R. (1985) J. Gen. Physiol. 85, 491-518 12 Hagiwara, S. and Byerly, L. (1981) Annu. Rev. Neurosci. 4, 69-125 13 Lrpez-Barneo, J. and Armstrong, C.M. (1983) J. Gen. Physiol. 82, 269-294 14 Fukuda, J. and Kawa, K. (1977) Science 196, 309-311 15 Nishizuka, Y. (1984) Nature 308, 693-698 16 Berridge, M.J. and Irvine, R.F. (1984) Nature 312, 315-312 17 Storey, D.J., Shears, S.B., Kirk, C.J. and Michell, R.H. (1984) Nature 312, 374-396 18 Bruce-Jacobson, K. and Turner, J.E. (1980) Toxicology 16, 1-37
19 Hellman, B., Sehlin, J., S~Sderberg, M. and T~iljedal, I.-B. (1975) J. Physiol. 252, 701-712 20 Thulin, E., Andersson, A., Drakenberg, T., Forsrn, S. and Vogel, H.J. (1984) Biochemistry 23, 1862-1870 21 Mills, J.S. and Johnson, J.D. (1985) J. Biol. Chem. 248, 151-160
81
Elsevier BBA 12039
Cadmium-induced insulin release does not involve changes 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 Thomas Nilsson, Per-Olof Berggren and Bo Hellman Department of Medical Cell Biology, Biomedicum, University of Uppsala, Uppsala (Sweden)
(Received 12 December 1986)
Key words: Cadmiumion; 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 isolated 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 promoted by glucose. Whereas basal insulin release is
stimulated by low concentrations of Cd 2+, concentrations that drastically suppress glucose oxidation do not affect basal release but significantly 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 characterization 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 Uppsala, Sweden.
Animals and preparation of islets and cells. Adult obese-hyperglycemic mice ( o b / o b ) 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 ambient free Ca 2+ concentration maintained by permeabilized /~ 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 +-deficient medium supplemented with EGTA. The cells were incubated overnight at 37 o C, p H 7.4, in 12 ml R P M I 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, 4 5 C a 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 radioimmunologically using crystalline mouse insulin as a reference. Measurements of membrane potential Qualitative changes in membrane potential were measured by the fluorescent dye bis-oxonol [8] obtained from Molecular Probes, Junction City, OR, U.S.A. The recordings were made at 3 7 ° 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 solution 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 containing 110 m M KC1, 10 m M NaC1, 2 mM KH2PO4, 1 mM MgC12, 25 m M Hepes and 1 m g / m l albumin was used. The p H 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 m M MgATP and an ATP-regenerating system consisting of 10 m M phosphocreatine and 20 U / m l creatine phosphokinase. The suspension was subjected to constant stirring and the free Ca 2+ concentration in the medium was measured with a Ca2+-selective minielectrode, constructed and calibrated essentially as described by Tsien and Rink [9]. All experiments were performed at room temperature. Test substances were added with 250 nl constant volume pipettes, prepared 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 bottom was cut off, freeze-dried overnight and the pellet was weighed on a quartz-fibre balance. The approximate number 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 m M the uptake levelled off, corresponding to a maximal 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+ uptake 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 m M Mg ~+, a concentration not affecting the basal uptake of the ion (data not shown).
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 stimulatory 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-stimulated 45Ca efflux, there was no effect of 5 ~M Cd 2+. In the presence of 8 mM glucose and 5/.tM
160 f
140
'~
120
Z
100
i
i
~
i
i
0
5
10
15
20
CONCENTRATION OF D-GLUCOSE ( m M O L / 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 glucose 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
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.
Cd 2+ (5 pM)
Cd 2+ (0.5 tiM) hi
g )r~ r~ 20
hi U3 < hi J UJ a~
10
Z
J Z
i
i
6O
~0
i
100 PERIFUSION
i
i
60
80
i
100
TIME(M[N)
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
Cd
A
2+
2+
Ccl
GLUCOSE
(20
GLUCOSE
mM)
(8 mM)
T
uM
200 Z
uJ O n,IJJ n v
150
X _J la... U.. UJ ~(..)
100
50
L____ 60
80
100
40 uM
,
120
,
L
60
80
-
-
_
_
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. 5 /JM Cd
5 ,uM Cd 2.'
2 5 mM K+
2+
I GucoE
Z O I-
25 mM
N
E:
H20
K+
12 mM H20 G L U C O S E
W a
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 depolarization 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 e a s e (see a b o v e ) , t h e r e w a s 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 t h e 45Ca efflux. I n a d d i t i o n , w h e n t h e C d 2+ 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 to 40 ~ M , t h e r e w a s e v e n a s u p p r e s s i o n o f t h e e f f l u x ( F i g . 3, p a n e l B). N o t w i t h s t a n d i n g , t h e f a c t t h a t 5 F M C d 2+ l a c k e d m a j o r e f f e c 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 efflux, it w a s n e c e s s a r y to r u l e o u t a d i r e c t e f f e c t o f t h i s C d 2+ c o n c e n t r a t i o n o n t h e m e m b r a n e p o t e n t i a l ( F i g . 4). A s is e v i d e n t f r o m t r a c e A, r e p e t i t i v e a d d i t i o n s o f C d 2+ w e r e w i t h o u t e f f e c t o n t h e r e s t i n g m e m b r a n e potential and did not interfere with the depolariz 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 o f K +. F u r t h e r m o r e , t r a c e 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 t h e m e m b r a n e p o t e n t i a l in t h e p r e s e n c e of 8 mM glucose nor interfered with the depolariz 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 o f t h e sugar.
85
To elucidate the effects of C d 2+ o n the intracellular 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+ concentration 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+ directly interfered with the IP3 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+ concentration 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 concentrations exceeding 50 /~M promoted a rapid Ca 2+ efflux.
Cd 2 ~ (5 .uM)
200 Z LU
o
n.LLI
t,, X
150
:D ..-i LI_ I.J_ LU
~0 1 O0
50
6~0
8'0
100
1'20
140
PERIFUSION TIME (MIN)
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 m M 45Ca2 ~ in the presence of 20 m M glucose and perifused with a glucose-free, CaZ+-deficient medium containing 1 m g / m l albumin. 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 experiments during the 10 min preceding the introduction of C d 2+, Mean values_+ S.E. for five experiments are shown.
A CELLS
B
CELLS
1
1
'S cZ*
5.5
0 I
Fig. 6. Effects of Cd 2+ o n the IP3-induced C a 2+ release in permeabilized B-cells. Cells
6.0
6.5 2* Cd
0
5
10
15
i
i
i
i
0
5
10
15
TIME (MIN)
(1.8.10 5 and 2.5.105) were added at 0 min and the additions of Cd 2+, IP 3 and C a 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 demonstrates a control experiment without any additions of Cd 2+. The concentration of Ca 2+ in the medium is given as - l o g [ C a 2+ ] in mol/1. The traces shown are typical for experiments 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 parathyroid 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 stimulatory 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 glucose promoted the sequestration of Cd 2+ in intracellular stores [1]. This assumption was based on the observation that D-600 only partially suppressed 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 concentrations. 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 glucosestimulated insulin release by 160 /~M Cd 2~, it should be recalled that this relatively high concentration 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+, supporting 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 permeabilized 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 mobilization of intracellularly bound Ca 2+ It has been shown that the receptor stimulated phosphodiesteratic hydrolysis of phosphati-
dylinositol 4,5-bisphosphate results in the formation of diacylglycerol and inositol 1,4,5-trisphosphate ( I p 0 [15,16]. Whereas the former compound has been shown to activate protein kinase C, the latter promotes Ca 2+ release from an intracellular 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 stimulatory effect evoked by IP 3. However, in a suspension 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 sulfhydryl 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 modifies 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.
87
Acknowledgements This work was supported by the Swedish Medical Research Council (12x-562), the Swedish Diabetes Foundation, the Nordic Insulin Foundation, the S~itra Brunn Foundation and Ake Wibergs Foundation. P.-O. Berggren is a recipient of a postdoctoral fellowhip from the Sweidsh Medical Research Council.
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8 Rink, T.J., Montecucco, C., Hesketh, T.R. and Tsien, R.Y. (1980) Biochim. Biophys. Acta 595, 15-30 9 Tsien, R.Y. and Rink, T.J. (1981) J. Neurosci. Methods 4, 73-86 10 Hellman, B., Ulfendahl, H.R. and Wallin, B.G. (1967) Anal. Biochem. 18, 434-443 11 Byerly, L., Chase, P.B. and Stimers, J.R. (1985) J. Gen. Physiol. 85, 491-518 12 Hagiwara, S. and Byerly, L. (1981) Annu. Rev. Neurosci. 4, 69-125 13 Lrpez-Barneo, J. and Armstrong, C.M. (1983) J. Gen. Physiol. 82, 269-294 14 Fukuda, J. and Kawa, K. (1977) Science 196, 309-311 15 Nishizuka, Y. (1984) Nature 308, 693-698 16 Berridge, M.J. and Irvine, R.F. (1984) Nature 312, 315-312 17 Storey, D.J., Shears, S.B., Kirk, C.J. and Michell, R.H. (1984) Nature 312, 374-396 18 Bruce-Jacobson, K. and Turner, J.E. (1980) Toxicology 16, 1-37
19 Hellman, B., Sehlin, J., S~Sderberg, M. and T~iljedal, I.-B. (1975) J. Physiol. 252, 701-712 20 Thulin, E., Andersson, A., Drakenberg, T., Forsrn, S. and Vogel, H.J. (1984) Biochemistry 23, 1862-1870 21 Mills, J.S. and Johnson, J.D. (1985) J. Biol. Chem. 248, 151-160