Effects of acute sodium omission on insulin release, ionic flux and membrane potential in mouse pancreatic B-cells

198

Biochimica etBiophysicaActa 969 (1988) 198 207 Elsevier

BBA 12236

Effects of acute sodium omission on insulin release, ionic flux and membrane potential in mouse pancreatic B-cells

R. de Miguel a, T. Tamagawa a, W. Schmeer b, M. Nenquin a and J.C. Henquin a,h Unit~ de DiabOtologie et Nutrition, UniversiO~of Louvain, Faculty of Medicine, Brussels (Belgium) and b I Phvsiologisches lnstitut, Universi(v of Saarland, Homburg / Saar (F.R. G.) (Received 22 October 1987)

Key words: Sodium ion; Insulin release; Ion flux; Membrane potential: (Pancreatic B-cell)

The effects of acute omission of extracellular Na + on pancreatic B-cell function were studied in mouse islets, using choline and lithium salts as impermeant and permeant substitutes, respectively. In the absence of glucose, choline substitution for Na ÷ hyperpolarized the B-cell membrane, inhibited S6Rb+ and 45Ca 2+ efflux, but did not affect insulin release. In contrast, Li + substitution for Na + depolarized the B-cell membrane and caused a Ca2+-independent, transient acceleration of 45Ca 2+ efflux and insulin release. Na + replacement by choline in the presence of 10 mM glucose and 2.5 m M Ca 2+ again rapidly hyperpolarized the B-ceU membrane. This hyperpolarization was then followed by a phase of depolarization with continuous spike activity, before long slow waves of the membrane potential resumed. Under these conditions, 86Rb+ efflux first decreased before accelerating, concomitantly with marked and parallel increases in 45Ca 2+ efflux and insulin release. In the absence of Ca 2 +, 45Ca 2 + and 86 Rb + efflux were inhibited and insulin release was unaffected by choline substitution for Na +. Na + replacement by Li + in the presence of 10 m M glucose rapidly depolarized the B-cell membrane, caused an intense continuous spike activity, and accelerated 4 S e a 2 + efflux, 86Rb+ efflux and insulin release. In the absence of extracellular Ca 2+, Li + still caused a rapid but transient increase in 45Ca2+ and 86Rb+ efflux and in insulin release. Although not indispensable for insulin release, Na + plays an important regulatory role in stimulus-secretion coupling by modulating, among others, membrane potential and ionic fluxes in B-cells.

Introduction The first suggestion that sodium ions could play a role in the control of pancreatic B-cell function was prompted by the observation, two decades ago, that omission of extracellular Na increases or decreases insulin release from pieces of rabbit pancreas depending on the concentration of glucose [1]. Very recently, the patch clamp Correspondence: J.C. Henquin, Unit~ de Diabttologie et Nutrition, University of Louvain, Faculty of Medicine, UCL 54.74, B-1200 Brussels, Belgium.

technique demonstrated the existence in neonatal B-cells [2] and in adult mouse B-cells [3] of voltage-dependent Na + channels, which, however, are inactivated at normal resting membrane potential [3], and are thus unlikely to play a role in stimulus-secretion coupling. In the meantime, a number of studies [4-22] have investigated the participation of Na + in different aspects of B-cell function, and have revealed a rather complex picture. This complexity is inherent in the multiplicity of cellular mechanisms under Na + control or influence (electrogenic Na + pump, N a + - C a 2+ and N a + - H + c o u n -

0167-4889/88/$03.50 ~') 1988 Elsevier Science Publishers B.V. (Biomedical Division)

199

tertransports, Ca 2+ sequestration, glucose metabolism). Another difficulty comes from the impossibility of comparing many of these studies because they were performed under very different conditions (distinct preparations, species, Na + substitutes, periods of observations). In the present work, perifused mouse islets were used to characterize the acute effects of extracellular Na + omission on insulin release, ionic fluxes and B-cell membrane potential. Choline was used as an impermeant substitute for Na +, and Li + was used as a permeant substitute, which, however, accumulates in cells because of its slow extrusion by the sodium pump [23]. Materials and Methods

All experiments were performed with islets of fed female N M R I mice (25-30 g), killed by decapitation. For electrophysiological experiments, a piece of pancreas was fixed in a small perifusion chamber, and the membrane potential of single B-cells was continuously recorded with microelectrodes [24]. B-cells were identified by the typical electrical activity that they display in the presence of 10-15 mM glucose [24,25]. For all other experiments, islets were isolated after collagenase digestion of the pancreas. The technique and the dynamic system of perifusion used to monitor the efflux of 45Ca2+ or 86Rb+ (used as tracer for K +) from preloaded islets have been described in detail [26]. During the experiments of 86Rb+ efflux, a portion of each effluent fraction was drawn for measurement of immunoreactive insulin, with rat insulin as standard (Novo Research Institute, Bagsvaerd, Denmark). The control medium comprised 120 mM NaC1, 4.8 mM KC1, 2.5 mM CaCI 2, 1.2 mM MgC12, and 24 mM N a H C O 3. It was gassed with O j C O 2 (94 : 6) to maintain a pH of 7.4, and supplemented with bovine serum albumin (1 m g / m l ) except for electrophysiological experiments. Ca2+-free solutions were prepared by replacing CaC12 with MgC12. Complete omission of Na + (the residual Na + concentration was less than 1 mM) was achieved either by replacing NaC1 with choline chloride and N a H C O 3 with choline bicarbonate (choline medium), or by replacing NaC1 with LiC1 and N a H C O 3 with choline bicarbonate (lithium

medium). In a few experiments, NaC1 was replaced by 240 mM sucrose or by 120 mM N-methylglucamine chloride. To prevent the muscarinic effects of choline, all solutions (even when they contained Na ÷) were supplemented with 10 ~tM atropine. Radiochemicals were obtained from Amersham International (Amersham, Bucks, U.K.). Choline bicarbonate was from Sigma Chemical Co. (St. Louis, MO, U.S.A.), N-methylglucamine was from Janssen Chimica (Beerse, Belgium), and all other reagents were from Merck A.G. (Darmstadt, F.R.G.). Electrophysiological experiments are illustrated by recordings that are representative of the indicated number of experiments, performed with different mice. All other data are presented as means + S.E. for a certain number of experiments performed with different mice. Results

Effects of Na + omission in the absence of glucose Substitution of Na + by choline in a medium containing Ca z+ hyperpolarized the B-cell membrane (Fig. 1). The resting potential rapidly increased to reach a maximum after about 1 min, and then slowly decreased and stabilized at a level 4 _+ 1 mV more negative than in the presence of Na +. On readmission of Na +, the B-cell membrane transiently depolarized beyond the control

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Fig. 1. Effects of total replacement of extracellular N a ÷ by choline or Li ÷ on the membrane potential of mouse B-cells perifused with a glucose-free medium (GO) containing 2.5 mM Ca 2÷. N a ÷ was withdrawn as indicated by the arrows. The horizontal dotted line is drawn at the level of the steady resting potential in the p_resence of N a +. The two recordings were obtained in different cells and are representative of four similar experiments.

200

level to which it subsequently repolarized (Fig. 1). Replacement of Na + by choline in the presence of Ca 2+ caused an immediate and sustained inhibition of 86Rb+ efflux, but transiently accelerated 4 5 C a 2 + efflux before decreasing it below O0

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control values (Fig. 2). Insulin release was not stimulated under these conditions, a finding that agrees with some [15] but not all previous reports [6]. Reversal of the inhibition of 86Rb+ efflux was much faster than that of 45Ca 2+ efflux upon return to a control medium. Omission of Na + from a Ca2+-free medium rapidly and reversibly decreased S6Rb+ efflux and 45Ca 2+ efflux, without modifying insulin release (Fig. 2). Substitution of Na + by Li + in a medium containing Ca 2 ~ depolarized the B-cell membrane (Fig. 1). The membrane potential progressively decreased, often showed some small oscillations but no electrical activity, and eventually stabilized at a level 19_+ 2 mV less negative than in the presence of Na +. On readdition of Na +, the B-cell membrane progressively repolarized to the control level (Fig. 1). Replacement of Na + by Li + in the presence of Ca 2~ resulted in a rapid, marked but transient acceleration of 45Ca2~ efflux and insulin release, and by a progressive but sustained increase in 86Rb+ efflux (Fig. 3). On readdition of N a ~ 45Ca2 ~ efflux displayed a biphasic rise above control values, S~Rb + efflux showed a transient further acceleration and did not return to control values, and insulin release increased slightly. The stimulation of 45Ca2 + efflux occurring upon substitution of Li t for Na + persisted in the absence of extracellular Ca 2+ but was now followed by a secondary inhibition. It was accompanied by a short-lived release of insulin that was even larger than in the presence of Ca 2 % Under these conditions ~6Rb+ efflux was slightly and belatedly (after the peak of 45Ca 2+ efflux) inhibited (Fig. 3). Table I summarizes the effects of partial replacement of extracellular Na + by choline on 86Rb+ efflux from islets perifused with a glucosefree medium containing Ca 2~. When the concentration of Na + was lowered to 50 mM, a slight decrease in S6Rb+ efflux was observed. This inhibition was not more pronounced when the Na + concentration was lowered further to 20 or 10 raM, and remained much smaller than the inhibition recorded upon complete omission of Na +. A decrease of the Na + concentration to 20 mM caused a small and transient hyperpolarization of the B-cell membrane followed by a return of the membrane potential to control values within 3 5

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TABLE I EFFECTS OF PARTIAL OR TOTAL OMISSION OF EXTRACELLULAR SODIUM ON 86Rb+ EFFLUX Mouse islets were perifused with a glucose-free medium containing 2.5 mM Ca 2+. The experimental protocol was the same as that described in Fig. 2. At 40 min, Na + was partially or totally replaced by choline, N-methylglucamine or sucrose. Values correspond to the average rates of efflux measured between 66 and 70 min, i.e., 26-30 min after the change in Na + concentration. Values are means_+S.E, for (n) experiments.

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Fig. 3. Effects of total replacement of extracellular Na + by Li + on 45Ca2+ efflux, 86Rb+ efflux and insulin release from mouse islets perifused with a glucose-free medium (GO) containing 2.5 mM Ca 2+ (o) or no Ca 2+ (©). Na + was withdrawn between 40 and 70 min. Values are means _+S.E. for 4-6 experiments.

rain ( n o t s h o w n ) . T a b l e I also s h o w s t h a t total replacement of Na + by N-methylglucamine b r o u g h t a b o u t a d e c r e a s e in 86Rb+ e f f l u x s i m i l a r to t h a t r e c o r d e d w i t h c h o l i n e , b u t t h a t a s m a l l e r i n h i b i t i o n w a s o b s e r v e d w h e n s u c r o s e w a s u s e d as a substitute.

T h e t y p i c a l e l e c t r i c a l a c t i v i t y (slow w a v e s of m e m b r a n e p o t e n t i a l w i t h spikes s u p e r i m p o s e d o n t h e p l a t e a u ) i n d u c e d b y 10 m M g l u c o s e in a c o n t r o l m e d i u m is s h o w n in t h e l e f t - h a n d side of Fig. 4. C o m p l e t e s u b s t i t u t i o n of N a + by c h o l i n e c a u s e d a r a p i d b u t t r a n s i e n t (77-t-_ 7 s) h y p e r p o l a r i z a t i o n o f the B-cell m e m b r a n e w i t h s u p p r e s s i o n o f e l e c t r i c a l activity. S u b s e q u e n t l y , the m e m b r a n e d e p o l a r i z e d to a p l a t e a u level u p o n w h i c h c o n t i n u o u s spike a c t i v i t y started. T h e p l a t e a u p o t e n t i a l w a s m o r e n e g a t i v e a n d the s p i k e a m p l i t u d e l a r g e r t h a n in the p r e s e n c e o f N a +. In c e r t a i n cells, o n e o r t w o slow w a v e s a p p e a r e d a f t e r the initial h y p e r p o l a r i z a t i o n , b e f o r e the m e m b r a n e rem a i n e d p e r s i s t e n t l y d e p o l a r i z e d ( n o t shown). W i t h time, spike f r e q u e n c y d r o p p e d a n d slow w a v e s r e a p p e a r e d (Fig. 4, u p p e r r e c o r d i n g ) . T h e s e slow waves were typically characterized by a much longer duration and more negative plateau and r e p o l a r i z a t i o n p o t e n t i a l s t h a n c o n t r o l slow waves. T h e d u r a t i o n o f the i n t e r v a l s w a s also l o n g e r t h a n in t h e p r e s e n c e o f N a + a n d i n c r e a s e d w i t h time. R e i n t r o d u c t i o n o f N a + r a p i d l y d e p o l a r i z e d the B-cell m e m b r a n e a n d t r i g g e r e d a s h o r t b u r s t of

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spikes. Thereafter, the m e m b r a n e r e m a i n e d inactive for several m i n u t e s b e f o r e control electrical activity r e s u m e d (Fig. 4, u p p e r recording). R e p l a c e m e n t of N a + b y choline in the presence of C a 2+ c o n s i s t e n t l y caused a small i n h i b i t i o n of 45Ca2+ efflux followed b y a large increase a n d a progressive r e t u r n to c o n t r o l values (Fig. 5). 86Rb + efflux also initially d e c r e a s e d before increasing a n d stabilizing a b o v e c o n t r o l values. Simultaneously, insulin release was s t i m u l a t e d following a time course which p a r a l l e l e d that of 45Ca 2+ efflux (save for the initial decrease). A similar increase in insulin release was previously o b s e r v e d in ob/ob m o u s e islets [5], b u t was much smaller a n d m o r e transient in rat islets [15,27]. R e a d d i t i o n of N a + t r a n s i e n t l y i n c r e a s e d 45Ca2+ efflux, while inhibiting 86Rb+ efflux a n d insulin release. A f t e r a b o u t 10 min, however, 45Ca2+ efflux, 86Rb+ efflux a n d insulin release started to increase steadily. O m i s sion of N a + from a CaZ+-free m e d i u m was foll o w e d by an i m m e d i a t e , s u s t a i n e d a n d reversible i n h i b i t i o n of 4~Ca2+ a n d S6Rb+ efflux, while the low rate of insulin release was u n a f f e c t e d (Fig. 5).

S u b s t i t u t i o n of N a + b y Li + caused a r a p i d and persistent d e p o l a r i z a t i o n of the B-cell m e m b r a n e with c o n t i n u o u s spike activity (Fig. 4, m i d d l e recording). T h e a m p l i t u d e of the spikes decreased with time because their threshold p o t e n t i a l progressively d e p o l a r i z e d , whereas their peak p o t e n tial d i d not change. The frequency of the spikes was not always as high as in the cell shown in Fig. 4, a n d their a m p l i t u d e sometimes decreased sooner. R e a d d i t i o n of N a + was followed by a m a r k e d a n d long-lasting silent h y p e r p o l a r i z a t i o n that could be i n h i b i t e d b y o u a b a i n . It usually took a b o u t 20 min b e f o r e slow waves r e s u m e d (not shown). R e p l a c e m e n t of N a + by Li + in the presence of C a z+ r a p i d l y s t i m u l a t e d 45Ca2+ efflux a n d insulin release following a similar time-course, a n d steadily increased the rate of 86Rb+ efflux up to ext r e m e l y high values (Fig. 6). On r e a d d i t i o n of Na +, 45Ca2+ efflux, S6Rb+ efflux a n d insulin release fell to values lower than in controls m a i n t a i n e d in a N a +-medium throughout. W h e n the c o n c e n t r a t i o n of N a + was only de-

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creased to 20 mM, with choline as substitute, the B-cell membrane hyperpolarized less markedly and for a shorter time than when Na + was totally omitted (Fig. 4, lower recording). Thereafter, slow waves appeared with high spikes superimposed on their plateau potential. These slow waves were longer than in the presence of N a +, but shorter

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Fig. 6. Effects of total replacement of extracellular N a ÷ by Li + on 45Ca2÷ efflux, 86Rb + efflux and insulin release from mouse islets perifused with a medium containing 10 mM glucose (G 10 raM) and either 2.5 mM Ca 2+ (o) or no Ca 2+ (0). Na + was withdrawn between 40 and 70 min. Values are means_+ S.E. for four experiments.

than the very long slow waves belatedly observed in Na+-free solutions, The intervals between these slow waves were also longer than under control conditions, except at the beginning. Control slow waves rapidly resumed after return to a medium containing a normal concentration of Na + (not shown).

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Fig. 7. Effects of partial replacement of extracellular Na ÷ by choline on 45Ca2+ efflux, 86Rb+ efflux and insulin release from mouse islets perifused with a medium containing 10 mM glucose (G 10 mM) and either 2,5 mM Ca 2+ (e) or no Ca 2+ (o). Na + concentration was lowered to 20 mM between 40 and 70 min. Values are means + S.E. for 4-5 experiments. Lowering the concentration of N a + to 20 m M in the presence of Ca 2+ caused a small initial decrease in 4SCa2+ efflux, followed by a transient increase and a return to values close to control values (Fig. 7). The rate of 86Rb+ efflux first decreased before increasing transiently and eventually stabilizing below control values. The only significant change in insulin release was a transient stimulation coinciding with the peak of 45Ca2+ efflux. On N a + readdition, 45Ca2+ efflux, 86Rb+ efflux and insulin release increased follow-

Previous reports have shown that glucose-induced insulin release is impaired when the stimulus is applied at the time of or after withdrawal of extracellular N a ÷ [1,5,6,21]. The present study was not designed to investigate the mechanisms [5,15,21] of this long-term inhibition, but to characterize the acute effects of N a ÷ withdrawal. The changes in insulin release, ionic fluxes and B-cell m e m b r a n e potential brought about by abrupt removal of extracellular N a ÷ were markedly influenced by the prevailing concentration of glucose and the nature of the N a ÷ substitute. The differences observed in choline- and lithium solutions raise the question of whether the effects are due to the absence of N a ÷ or to the substitute itself. The muscarinic effects of choline are effectively prevented by the concentration of atropine used throughout [28]. Moreover, control experiments showed that each effect observed with choline could be qualitatively reproduced by Nmethylglucamine, and that substitution of NaCI by sucrose reproduced the effects observed in a choline more closely than those in a lithium medium. However, the use of sucrose entails omission of C I - , which, by itself, affects insulin release, ionic fluxes and m e m b r a n e potential [27,29,30], and thus complicates the interpretation of the results previously obtained with this substitute. It thus seems reasonable to assume that the changes occurring upon choline substitution for N a + are mainly due to the absence of Na + The present study confirms that N a + replacement by choline increases the resting m e m b r a n e potential of B-cells [8,18], and that this increase displays a biphasic pattern [18]. As this hyperpolarization is accompanied by a decrease in 86Rb + efflux, it cannot be explained by an increase in K + permeability, but is more likely to be due to the suppression of a depolarizing current pro-

205 duced by a resting influx of N a + a n d / o r the activity of an electrogenic N a + - C a 2+ countertransport. The decrease in 86Rb+ efflux can, thus, be viewed as the consequence of the hyperpolarization. It is possible, however, that its magnitude is also influenced by suppression of a tonic activation of K + conductance normally exerted by Na + and by inhibition of the Na + pump, two mechanisms evoked to explain the belated attenuation of the hyperpolarization seen in Na+-free solutions [181. The inhibition of 45Ca 2+ effhix observed when Na + was withdrawn from a glucose- and Ca2+-free medium confirms the existence of a N a + - C a 2+ countertransport in islet cells [13-15]. In the presence of extracellular Ca 2+, a transient acceleration of 45Ca2+ efflux preceded the inhibition. It can be explained by an increase of Ca 2+ influx [31] which, however, is insufficient to induce insulin release. This influx occurred while the membrane was hyperpolarized and silent, probably through the inverted operation of the N a + - C a 2+ countertransport expected to occur upon abrupt reversal of the N a + gradient. The inhibition of 45Ca2+ efflux produced by choline substitution in a Ca2+-free medium containing 10 m M glucose indicates that the N a + - C a 2+ exchange mechanism is still operative under these conditions. This supports the conclusion that the inhibitory effect of glucose on 45Ca2+ efflux is due to a mechanism other [32,33] than the inhibition of the N a + - C a 2+ countertransport [15]. Two observations suggest that an inward N a + current contributes to the membrane potential of B-cells in the presence of a stimulatory concentration of glucose. First, a marked silent hyperpolarization, consistently followed choline substitution for Na + and was accompanied by a decrease in the rate of 86Rb + efflux. Second, a rapid depolarization occurred on N a + readdition. The amplitude of this hyperpolarization and depolarization was larger than that in the absence of glucose probably because of the increase in PNa+/PK+ ratio that results from the decrease in K + permeability produced by the sugar. The depolarization caused by N a + readdition was quickly followed by a long period of silent membrane polarization, which is probably due to activation of the electrogenic sodium p u m p [34] by incoming N a + [12,18,20].

The intense electrical activity that appeared secondarily in the Na+-free medium containing glucose was attended by a CaZ+-dependent acceleration of both 45Ca2+ and 86Rb + efflux, indicating that Ca 2+ influx was stimulated and Ca2+-sen sitive K + channels were activated. The simultaneous increase in insulin release was totally dependent on extracellular Ca 2+, and displayed a time course parallel to that of the Ca2+-dependent 45Ca2+ efflux and electrical activity. This clearly indicates that the increase in release results from an accelerated influx of Ca 2+. Appearance of this Ca2+-dependent electrical activity from a more negative membrane potential than under control conditions is compatible with a shift in the activation potential of Ca 2+ channels in the absence of N a +. Previous experiments [8,12,18] have already established that the spike amplitude is increased in Na+-free solutions, but it has been incorrectly concluded that sodium ions are necessary for slow waves to occur. Nonetheless, the abnormally long duration of the slow waves which belatedly appear in the absence of Na + indicates that sodium ions are involved in the control of glucose-induced electrical activity. In contrast to choline substitution, Li + substitution for N a + did not hyperpolarize, but rapidly and steadily depolarized the B-cell membrane, and accelerated 86Rb+ efflux in the absence and presence of glucose. Omission of extracellular Ca 2+ largely prevented the acceleration of 86Rb + efflux by lithium solutions, but did not disclose any substantial inhibition. The depolarization, therefore, is not the consequence of a decrease in K + permeability, but is more likely to be due to the influx of Li + into the B-cells, and to arrest of the electrogenic Na + p u m p [34] which Li + does not activate [23]. In spite of the large depolarization produced by lithium in the glucose-free medium, no slow waves or spikes were triggered. This is not surprising, since such electrical activity appears only when a decrease in membrane K ÷ permeability contributes to the depolarization [25]. Thus, in the presence of glucose, which decreases K + permeability, Li + substitution for Na + induced a very marked and continuous spike activity. This activity was accompanied by a strong stimulation of 45Ca2+ efflux and insulin release both of which were largely inhibited in the absence of extracellu-

206

lar Ca 2+. Taken together, these experiments and uptake measurements [14] indicate that Ca 2+ influx is stimulated under these conditions. Yet, neither in the absence nor in the presence of glucose is the acceleration of 45 C a 2 + efflux induced by lithium solutions totally suppressed by omission of extracellular Ca 2+. An early transient peak of 45Ca2+ efflux persists, which thus reflects mobilization of cellular Ca 2+ by Li +, and is sufficient to cause a transient peak of insulin release. The early mobilization of cellular Ca 2 ~ and stimulation of insulin release occurring in lithium solutions are reminiscent of the effects of high concentrations of muscarinic agonists (Refs. 35 and 36 and unpublished observations). It is unlikely, however, that these changes result from the alterations in phosphatidylinositol turnover produced by lithium [37]. Thus, lithium mainly increases the concentration of the inactive isomer inositol 1,3,4-trisphosphate [38]. Moreover, the effect of Li + o n 45Ca 2+ mobilization and insulin release was similar in the absence and presence of glucose, whereas the glucose dependence of the muscarinic effect is well established [3536].

45Ca2+

Acknowledgements R.d.M. and T.T. are Research Fellows on leave from the University Complutense of Madrid, Spain, and the University of Nagoya, Japan, respectively. J.C.H. is 'Maitre de Recherches' of the FNRS, Brussels. This work was supported by Grants 3.4552.81 and 3.4546.86 from the FRSM, Brussels, and by the Deutsche Forschungsgemeinschaft, SFB 246. We thank Dr. T.D. Plant for reading the manuscript,

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