chloromercuribenzene-p-sulphonic acid and inhibited by 20 mm glucose. expel Na+ by an active mechanism similar to, or identical with, the Na+/

Transcription

1 J. Phynol. (1974), 242, pp With 3 text-figures Printed in Great Britain TRANSPORT OF RUBIDIUM AND SODIUM IN PANCREATIC ISLETS BY J. SEHLIN AND I.-B. TALJEDAL From the Department of Histology, University of Umea, Umed, Sweden (Received 16 April 1974) SUMMARY 1. Fluxes of 86Rb+ and 22Na+ were measured in pancreatic islets of ob/ob-mice. The islets, which contain more than 90 % fl-cells, were incubated at 370 C in Krebs-Ringer bicarbonate buffer with modifications known to influence insulin release. 2. In the presence of Na+, the islets vigorously accumulated Rb+. The Rb+ uptake was inhibited by depletion of islet Na+ or by 1 mm ouabain or 0-1 mm chloromercuribenzene-p-sulphonic acid. Rb+ uptake was stimulated by 1 mm-5,5'-dithiobis (2-nitrobenzoic acid) or by depletion of islet Ca2+, while 20 mm glucose, 5 mm theophylline, 041 mm iodoacetamide, or 1 mm-6,6'-dithionicotinic acid had no significant effects. 3. The efflux of Rb+ from preloaded islets followed exponential kinetics with a half-life of about 16 min. The rate of efflux was enhanced by 0x 1 mm chloromercuribenzene-p-sulphonic acid and inhibited by 20 mm glucose. Omission of Na+, K+ or Ca2+ from the incubation medium had no significant effects. 4. The efflux of 22Na+ from islets preloaded with this isotope was enhanced by 041 mm chloromercuribenzene-p-sulphonic acid or by Ca2+ deficiency. It was inhibited by 1 mm ouabain, 041 mm-2,4-dinitrophenol, or by omission of Na+ from the incubation medium. Omission of K+ or the addition of 20 mm glucose had no significant effects. 5. It is concluded that the fl-cells are permeable to Na+ and Rb+ and expel Na+ by an active mechanism similar to, or identical with, the Na+/ K+-pump in other cells. The mechanisms of active and passive cation movements are discussed in relation to current hypotheses of stimulussecretion coupling in the fl-cells depending on interactions between Na+ and Ca2+. In particular, the results support the hypotheses of insulin release being stimulated by ouabain through inhibition of the Na+/K+pump and by organic mercurials through enhancement of membrane permeability to cations.

2 506 J. SEHLIN AND 1.-B. TALJEDAL INTRODUCTION The uptake of 22Na+ by pancreatic islets indicated that the fl-cells are permeable to Na+ (Sehlin & T5iljedal, 1974). Assuming that radioisotope equilibrium was reached within 60 min of incubation in medium containing mm labelled Na+, the concentration of Na+ in the f-cells was estimated as about 95 mm (Sehlin & Taljedal, 1974). The existence of an inward Na+ gradient, albeit small, and the observation that ouabain stimulated the net uptake of 22Na+ suggested that the fl-cells contain a Na+ extrusion mechanism with properties similar to those of the seemingly ubiquitous Na+/K+-pump. The mechanisms of Na+ transport in pancreatic fl-cells deserve further characterization because Na+ fluxes may be important in the regulation of insulin release. Insulin release is stimulated by ouabain (Hales & Milner, 1968), is initially stimulated on withdrawal of Na+ from the incubation medium (Hales & Milner, 1968; Henquin, 1973; Hellman, Idahl, Lernmark, Sehlin & Tdljedal, 1974), and is inhibited on equilibration of the islets with a Na+-deficient medium (Hales & Milner, 1968; Henquin, 1973; Hellman et al. 1974). Na+ also influences the electric activity (Dean & Matthews, 1970), the uptake of 45Ca2+ (Hellman, Sehlin & Taljedal, 1971 a), and the rate of glucose metabolism (Ashcroft, Bassett & Randle, 1972; Matschinsky & Ellerman, 1973; Hellman et al. 1974) in the pancreatic islets. Because it has been postulated that the Na+/K+-pump plays an important role in insulin release (Hales & Milner, 1968; Milner & Hales, 1970), we investigated its existence in microdissected islets of ob/ob-mice by studying inward pumping as the concentrative uptake of 86Rb+; Rb+ is an effective substitute for K+ in the function of the Na+/K+-pump in other cells (Bonting, 1970). Outward pumping was studied as 22Na+ efflux from preloaded islets. By comparing the two parameters and by also studying Rb+ efflux, we tried to find out whether glucose, Ca2+, ouabain, and other modifiers of insulin release affect the activity of the pump or the permeability of the fl-cell membrane to passive cation movements. METHODS The sources of chemicals were as follows. The Radiochemical Centre, Amersham, Bucks., U.K.: 86RbCl, 22NaCl and (6,6'-3H)sucrose; Sigma Chemical Co., St Louis, Mo., U.S.A.: chloromercuribenzene-p-sulphonic acid, ouabain and theophylline; British Drug Houses Ltd, Poole, Dorset, U.K.: 5,5'-dithiobis(2-nitrobenzoic acid), ethanedioxybis(ethylamine)tetra-acetic acid, iodoacetamide (less than 0005 % free iodine), and D-glucose; Newcell Biochemicals, Berkeley, Calif., U.S.A.: 6,6'-dithiodinicotinic acid; Leo Pharmaceuticals, Helsingborg, Sweden: diphenylhydantoin; E. Merck A.G., Darmstadt, Germany: 2,4-dinitrophenol. All chemicals were of analytical grade. Double-distilled water was used throughout.

3 Rb+ AND Na+ TRANSPORT IN PANCREATIC ISLETS 507 Adult non-inbred ob/ob-mice of the UmeA colony were starved overnight. Fresh pancreatic islets were isolated by free-hand microdissection (Hellerstr6m, 1964) with the pancreas immersed in Krebs-Ringer bicarbonate buffer (De Luca & Cohen, 1964) equilibrated with 02-CO2 (95:5). Expressed in mm, the buffer had the following composition: Na , K+ 5-9, Ca2+ 2-6, Mg2+ 1-2, Cl , HC , S , and H2P The same buffer supplemented with 3 mm-glucose was used as the basal medium in subsequent incubations at 370 C. TABLE 1. Effects of various agents on the capacity of islets to concentrate Rb+ Modification of medium None (control) 0-01 mm ouabain 0-1 mm ouabain 1-0 mm ouabain DPH 5 jug/ml DPH 10 /zg/ml DPH 40,ug/ml No Ca2+ No Na+ 1-0 mm-dtnb 1-0 mm-cpds 0-1 mm-cmbs 1-0 mm-cmbs 0-1 mr iodoacetamide 5 mm theophylline 20 mm glucose 20 mm glucose plus 0-1 mm iodoacetamide 20 mm glucose plus 5 mm theophylline Islet content of Rb+ (mmol/kg dry wt. of islets) I A Primary data (36) (5) (5) (5) 0-93 ± 0-06 (5) (5) (5) (8) (5) (6) (6) (6) (6) (5) (5) (5) (5) (5) Test minus control (5) (5) (5) (5) (5) (5) (8) (5) (6) (6) (6) (6) (5) (5) (5) (5) (5) P-value < < 005 < 0-02 < < < 0-02 < Islets were incubated for 8 min in basal medium supplemented with 0-07 mm- 86RbCl and 0-1 mm-(6,6'-3h)sucrose. Control incubations were performed without further additives to the medium, while parallel test incubations were performed in media modified as indicated. 'No Ca2+' means that CaCl2 was omitted and 'no Na+' that all Na+ was replaced by choline ions. In all experiments, except those with 20 mm glucose, 0-1 mm iodoacetamide and 5 mi theophylline, the indicated modifications also applied to the preliminary incubation for 30 min in non-radioactive medium. Results are presented as the islet uptake of Rb+ in excess of the sucrose space. The sucrose space was unaffected by all treatments except 1 mm chloromercuribenzene-p-sulphonic acid, which increased it (cf. Bloom, Hellman, Idahl, Lernmark, Sehlin & Taljedal, 1972). In addition to results obtained with each type of medium, the differences between parallel test and control incubations are given as mean values and s.e. for the numbers of experiments (animals) stated in parentheses. Statistical tests were based on t values computed from the mean and S.E. of differences. DPH = diphenylhydantoin, DTNB = 5,5'-dithiobis (2-nitrobenzoic acid), CPDS = 6,6'-dithiodinicotinic acid, CMBS = chloromercuribenzene-p-sulphonic acid.

4 508 J. SEHLIN AND I.-B. TALJEDAL All experiments were begun by preliminary incubation of the islets for 30 min in non-radioactive basal medium, in some experiments modified as described in the legend to Table 1. To study Rb+ uptake, the islets were then incubated in basal medium supplemented with 0-07 mm-86rbcl (290 mci/mmol) and 0-1 mm-(6,6'-3h)- sucrose (150 mci/mmol) as well as with test compounds as required; in most experiments the incubation time was 8 min. To study Rb+ efflux, the islets were incubated for 120 min in basal medium with 86RbCl and (6,6'-3H )sucrose as above. They were rinsed briefly (approx. 2 sec) in basal medium and then incubated in non-radioactive basal medium containing test compounds as required; in most experiments the incubation time was 10 min. Efflux of Na+ was studied by incubating the islets for 60 min in basal medium labelled with 22NaCl (0-14 mci/mmol) and subsequently in basal medium with test compounds as required; in most experiments the incubation time was 10 min. Details of experimental design are described in the legends to Figures and Tables. After incubation the islets were freeze-dried overnight (-40 C, 0-1 Pa), weighed on a quartz-fibre balance, and analysed for radioactivity in a liquid scintillation spectrometer. Samples of radioactive incubation media were used as external standards. In measurements of Rb+ uptake and efflux, corrections were made for 86Rb residing in the islet space occupied by 3H; sucrose is distributed as an extracellular space marker in the microdissected islets (Hellman et al b, c). During the simultaneous counting of 86Rb and 3H, the discriminators wqre set so that 2 % of the 86Rb-counts were also counted in the 3H-channel, and less than 0 5 % of the 8H-counts in the 86Rb-channel. Appropriate corrections were made for this spill-over Incubation time (min) Fig. 1. Islet uptake of Rb+ with time. After preliminary incubation in basal medium for 30 min, the islets were incubated for different periods of time in basal medium supplemented with 0 07 mmm86rbcl (290 mci/mmol) and 0- I mm-(6,6'-3h)sucrose (150 mci/mmol). Uptake of Rb+ in excess of the sucrose space is presented for each time as the mean value and S.E. of four different experiments (animals).

5 Rb+ AND Na+ TRANSPORT IN PANCREATIC ISLETS 509 RESULTS Rubidium accumulation Fig. 1 shows the islet uptake of Rb+ with time. The islet cells went on concentrating Rb+ for at least 2 hr, at which time their content of Rb+ was 3-7 mmol/kg dry wt. Assuming that the intracellular water of incubated islets amounts to 1-2 1/kg dry wt. (Hellman et al b), the intracellular concentration was about 3 1 mm, i.e. 44 times the concentration in the medium E3 E.0 EL ~~~~~~~min E 0' Efflux time (min) Fig. 2. Time course of Rb+ efflux. After preliminary incubation for 30 min in basal medium followed by loading for 120 min in basal medium supplemented with 0 07 mm-86rbcl (290 mci/mmol) and 0.1 mm-(6,6'-3h)sucrose (150 mci/mmol), the islets were incubated for different periods of time in non-radioactive basal medium. The amounts of Rb+ retained by the islets are presented as mean values and s.e. of three to four different experiments (animals). The inset shows a semi-logarithmic plot of the same data. The effects of various modifications of medium composition are shown in Table 1. Islets which had been preliminarily incubated for 30 min in a Na+-free medium exhibited a marked inhibition of Rb+ uptake. The Rb+ uptake was also inhibited by ouabain, the effect being statistically significant with 1 mm but not with 0-01 or 0-1 mm of the drug. Diphenylhydantoin did not stimulate Rb+ uptake but at a concentration of 40,ug/ml inhibited it. Depletion of islet Ca2+ or the addition of 1 mm-5,5'-dithiobis(2-nitro-

6 510 J. SEHLIN AND I.-B. TALJEDAL benzoic acid) enhanced the rate of Rb+ accumulation, while 0-1 mm or 1 mm chloromercuribenzene-p-sulphonic acid exerted a marked inhibitory effect. The uptake of Rb+ was not significantly affected by 0-1 mm iodoacetamide, 1 mm-6,6'-dithiodinicotinic acid, 5 mm theophylline, or 20 mm glucose. TABLE 2. Effects of various agents on Rb+ efflux Islet content of Rb+ (mnol/kg dry wt. of islets) Modification, _A of medium Primary data Test minus control P-value Experimental 8erie8 I None (control) (8) 20 mm glucose (8) (8) < mm iodoacetamide (7) (7) 20 mm glucose plus (8) (8) 0-1 mm iodoacetamide (7) < 0-01 Experimental 8eres II None (control) (8) No Na (8) (8) No K (8) (8) No Ca (8) (8) 0-1 mm-cmbs 1-66± 0-14 (8) (8) < After loading with 86Rb+ as described in the Methods section, islets were incubated for 10 min in non-radioactive basal medium modified as indicated. 'No Na+' means that all Na+ was replaced by choline ions, 'no K+' that KCI was replaced by choline chloride and KH2PO4 by NaH2PO4, and 'no CO+' that CaCl. was omitted and 0-5 mm ethanedioxybis(ethylamine)tetra-acetic acid added. The islet contents of Rb+ after incubation are given as mean values and se. for the numbers of experiments (animals) stated in parentheses. In addition, the mean values and 8.E. for differences between parallel test and control incubations are given. Statistical testing was performed as in Table 1. Difference versus 0-1 mm iodoacetamide. CMBS = chloromercuribenzene-p-sulphonic acid. Rubidium efflux The process of Rb+ efflux from preloaded islets followed exponential kinetics with a half-life of about 16 min (Fig. 2). Table 2 shows that the rate of Rb+ efflux was increased by 0-1 mm chloromercuribenzene-psulphonic acid, whereas 0-1 mi-m iodoacetamide had no significant effect. Glucose at a concentration of 20 mm inhibited the Rb+ efflux both in the presence and absence of iodoacetamide. Omitting Na+, K+ or Ca2+ from the incubation medium did not significantly affect Rb+ efflux. Sodium efflux The islet uptake of 22Na+ has been described (Sehlin & Taijedal, 1974). Fig. 3 shows the efflux of 22Na+ from islets preloaded with this isotope, as

7 Rb+ AND Na+ TRANSPORT IN PANCREATIC ISLETS 511 well as the inhibition of efflux by 1 mm ouabain. Table 3 summarizes the effects of some other modifications of the medium in which the 22Na-loaded islets were incubated. The efflux of Na+ was enhanced by 0.1 mm chloromercuribenzene-p-sulphonic acid or by omission of Ca2+, and was inhibited by 041 mm-2,4-dinitrophenol or by omission of Na+. The omission of K+ or the addition of 20 mm-glucose had no significant effects. 500 I-.4O0 IV 400.!2 0,; 200 3o ba + z co E 100 E Incubation time (min) Fig. 3. Time course of Na+ efflux. After preliminary incubation for 30 min in basal medium followed by loading for 60 min in basal medium labelled with 22Na+ (0.14 mci/mmol), the islets were incubated for different periods of time in non-radioactive basal medium with (@) or without (0) 1 mm ouabain. The amounts of labelled Na+ retained by the islets are presented as mean values of two to four different experiments (animals). DISCUSSION Studies on other cells have shown that Rb+ can substitute for K+ in the function of the Na+/K+-pump (Bonting, 1970). The concentrative uptake of Rb+ and its inhibition in Na+-depleted islets indicates that the fl-cells contain a mechanism for actively exchanging Na+ for Rb+ that may be identical with the Na+/K+-pump in other cells. In support of this interpretation, ouabain inhibited both the accumulation of Rb+ and the efflux of Na+. As previous evidence for the Na+/K+-pump in pancreatic islets, I8 PHY 242

8 512 J. SEHLIN AND I-B. TALJEDAL TABLE 3. Effects of various agents on Na+ efflux Islet content of labelled Na+ (mmol/kg dry wt. of islets) Modification, A of medium Primary data _ Test minus control P-value Experimental series I None (control) (7) 0-1 mm-2,4-dinitrophenol 82-2 ± 5-0 (7) (7) < 0-05 No Na+ 87-2±+41 (7) (7) < No K (7) (7) Experimental series II None (control) (6) 20 mm glucose (6) (6) 0-1 mm-cmbs (6) (6) < No Ca (6) (6) < After loading with 22Na+ as described in the Methods section, islets were incubated for 10 min in non-radioactive basal medium modified as indicated. 'No Na+' means that all Na+ was replaced by choline ions, 'no K+' that KCl was replaced by choline chloride and KH2PO4 by NaH2PO4, and 'no Ca2+' that CaCl2 was omitted and 0-5 mm ethanedioxybis(ethylamine)tetra-acetic acid added. The islet contents of labelled Na+ (with same specific activity as in loading medium) after incubation are given as mean values and s.e. for the numbers of experiments (animals) stated in parentheses. In addition, the mean values and s.e. for differences between parallel test and control incubations are given. Statistical testing was performed as in Table 1. CMBS _ chloromercuribenzene-p-sulphonic acid. Kizer, Vargas-Cordon, Brendel & Bressler (1970) reported that diphenylhydantoin inhibited the 22Na+ net uptake. However, in the present study diphenylhydantoin did not stimulate the uptake of Rb+, and we are unable to explain this apparent discrepancy in results. The failure to demonstrate a significant effect of K+ deficiency on Na+ efflux may be due to incomplete washout of K+ from the islet extracellular space during the short incubations employed. Na+-depleted islets exhibit decreased formation of [14C]CO2 from ['4C]glucose (Ashcroft et al. 1972; Hellman et al. 1974), of [3H]H20 from [5-3H]glucose (Ashcroft et al. 1972) and of lactate (Matschinsky & Ellerman, 1973), together with increased concentrations of glucose-6-phosphate and fructose-1,6-diphosphate (Hellman et al. 1974). Ouabain also inhibits the production of CO2 (Hellman et al. 1974) and lactate (Matschinsky & Ellerman, 1973) and increases the islet content of early glycolytic metabolites (Hellman et al. 1974). These metabolic effects were tentatively attributed to inhibition of the Na+/K+-pump with diminished hydrolysis of ATP leading to a partial block of glycolysis (Ashcroft et al. 1972; Hellman et al. 1974). To test the dependence of Na+ extrusion on ATP, we

9 Rb+ AND Na+ TRANSPORT IN PANCREATIC ISLETS 513 measured the efflux of 22Na+ from islets poisoned with 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation. As expected, 2,4-dinitrophenol inhibited 22Na+ efflux. Hales & Milner (1968) and Milner & Hales (1970) suggested that the activity of the Na+/K+-pump influences insulin release by determining the intracellular concentration of Na+. As part of the evidence, it was shown that mm oubain stimulated insulin release from pieces of rabbit pancreas. In the present study, significant effects on Rb+ accumulation were observed with 1 mm ouabain only, whereas 0 01 or 041 mm ouabain had no effect. This difference in dose-response relationships does not invalidate the idea that ouabain stimulates insulin release by inhibiting the Na+/K+-pump, as the fl-cells in rabbits may be more sensitive to ouabain than those in the mouse. K. Capito & C. J. Hedeskov (personal communication) observed no effect of 0'01 or 041 mm ouabain on the release of insulin from normal mouse islets. Milner & Hales (1970) speculated that Na+ interacts with Ca2+ in such a way that an increase of intracellular Na+, or a decrease of extracellular Na+, would augment the Ca2+ net uptake and hence insulin release. It is noteworthy therefore that we found the Na+ efflux as well as the Rb+ uptake to be enhanced in Ca2+-deficient islets. If the fl-cells contain a saturable mechanism for passive transfer of Na+ and Ca2+, the omission of Ca2+ should enhance the efflux of 22Na+ from preloaded cells along this route. In view of the inward Na+ gradient, an increased ease of passive Na+ transport may also raise the intracellular Na+ and enhance Na+-Rb+ exchange by the Na+/K+-pump. That the fl-cells contain a Na+ transport system other than the Na+/K+-pump is suggested by our observation that Na+ efflux was faster into Na+-containing than into Na+-free medium. Such trans-stimulation of Na+ transport would presumably not occur by means of the Na+/K+-pump, as the concentration of extracellular K+ was kept constant at a physiological concentration. It is tempting to think that the Na+-Na+ exchange mechanism also transports Ca2+ and is identical with the Na+-dependent Ca2+ uptake mechanism postulated by Milner & Hales (1970). Previous evidence for a Na+-sensitive Ca2+ transport system in islet cells was provided by the fact that the initial uptake of 45Ca2+ (Hellman et at a) and the glucose-induced action potentials (Dean & Matthews, 1970) were increased on incubation in Na+-deficient media. However, there is the obvious possibility that the interactions between Ca2+, Na+ and Rb+ reported here were due to Ca2+ exerting a direct inhibitory influence on the Na+/K+-pump. More detailed measurements of Ca2+ fluxes may be useful to help decide between the two alternatives. Stimulation of Na+ uptake in islets exposed to chloromercuribenzene- I8-2

10 514 J. SEHLIN AND I.-B. TALJEDAL p-sulphonic acid has been reported (Sehlin & Tiljedal, 1974). As shown here, the organic mercurial exerted marked effects on Na+ efflux and Rb+ uptake and efflux as well. These results are best explained as being due to increased membrane permeability to cations. Much smaller, if any, effects were noted in islets exposed to the other sulphydryl reagents tested. In view of the fact that chloromercuribenzene-p-sulphonic acid is by far the most potent initiator of insulin release (Bloom et al. 1972; Hellman, Idahl, Lernmark, Sehlin & T&Ijedal, 1973a, b), the results support our hypothesis that organic mercurials stimulate insulin release through a direct action on the f8-cell plasma membrane leading to increased cation permeability (Bloom et al. 1972). Islets incubated with 20 mm glucose exhibited the same Rb+ uptake and Na+ efflux as islets incubated with 3 mm glucose, indicating that physiological stimulation of insulin release is not associated with great changes of Na+/K+-pump activity. On the other hand, Rb+ efflux was significantly inhibited by 20 mm glucose. Since the time course of Rb+ efflux is compatible with the process being a passive flux down the electrochemical gradient, the effect of glucose may be a decreased membrane permeability to Rb+. Whether the effect is specific for the outward movement of Rb+ cannot be decided from the present data. Small changes of membrane permeability to Rb+ and Na+ may not be detectable in the studies of Rb+ accumulation and Na+ efflux, as these processes are presumably the combined results of Na+/K+-pump activity and passive movements. Malaisse, Brisson & Baird (1973) reported that glucose inhibits the unidirectional efflux of 45Ca2+ from perifused islets. Because the net flux of Ca2+ has not been measured in pancreatic islets, the data on 45Ca2+ efflux are as compatible with glucose inhibiting an active Ca2+ extrusion mechanism as with a less specific decrease of membrane permeability to cations. Further experiments are evidently necessary to elucidate the physiological implications of the glucose-induced decrease of Rb+ efflux. This work was supported by the Swedish Medical Research Council grant 12X REFERENCES ASHCROFT, S. J. H., BASSETT, J. M. & RANDLE, P. J. (1972). Insulin secretion mechanisms and glucose metabolism in isolated islets. Diabete8 21, suppl. 2, BLOOM, G. D., HELLMAN, B., IDAHL, L..A., LERNMARK, A., SEHLIN, J. & TALJEDAL, I.-B. (1972). Effects of organic mercurials on mammalian pancreatic f-cells. Insulin release, glucose transport, glucose oxidation, membrane permeability and ultrastructure. Biochem. J. 129, BONTING, S. L. (1970). Sodium-potassium activated adenosinetriphosphatase and cation transport. In Membranes and Ion Transport, vol. 1, ed. BITTAR, E. E., pp London: Wiley-Interscience.

11 Rb+ AND Na+ TRANSPORT IN PANCREATIC ISLETS 515 DEAN, P. M. & MATTHEWS, E. K. (1970). Electrical activity in pancreatic islet cells: effect of ions. J. Phy8iol. 210, DE LUCA, H. F. & COHEN, P. P. (1964). Suspending media for animal tissues. In Manometric Technique8, 4th edn., ed. UXBREIT, W. W., BuRis, R. H. & STAUFFER, J. F., pp Minneapolis: Burgess Publishing Co. HALES, C. N. & MILNER, R. D. G. (1968). The role of sodium and potassium in insulin secretion from rabbit pancreas. J. Physiol. 194, HELLERSTROM, C. (1964). A method for the microdissection of intact pancreatic islets of mammals. Acta endocr., Copenh. 45, HELLMAN, B., SEHLIN, J. & TALJEDAL, I.-B. (1971a). Calcium uptake by pancreatic fl-cells as me&,tured with the aid of 45Ca and mannitol-3h. Am. J. Physiol. 221, HELLMAN, B., SEHLIN, J. & TALJEDAL, 1.-B. (1971 b). Transport of z-aminoisobutyric acid in mammalian pancreatic fl-cells. Diabetologia 7, HELLMAN, B., SEHLIN, J. & TALJEDAL, I.-B. (1971 c). Evidence for mediated transport of glucose in mammalian pancreatic fl-cells. Biochim. biophys. Acta 241, HELLMAN, B., IDAHL, L.-A., LERNMARK, A., SEHLIN, J. & TXLJEDAL, 1.-B. (1973a). lodoacetamide-induced sensitization of the pancreatic fl-cells to glucose stimulation. Biochem. J. 132, HELLMAN, B., IDAHL, L.-A., LERNMARK, A., SEHLIN, J. & TALJEDAL, I.-B. (1973b). Role of thiol groups in insulin release: studies with poorly permeating disulphides. Molec. Pharmacol. 9, HELLMAN, B., IDAHL, L.-A.. LERNMARK, A., SEHLIN, J. & TALJEDAI, I.-B. (1974). The pancreatic f-cell recognition of insulin secretagogues. Effects of calcium and sodium on glucose metabolism and insulin release. Biochem. J. 138, HENQUIN, J. C. (1973). Etude de l'influence de certains cations sur la cin6tique de la secretion d'insuline in vitro, pp Thesis, Universite Catholique de Louvain. KIZER, J. S., VARGAS-CORDON, M., BRENDEL, K. & BRESSLER, R. (1970). The in vitro inhibition of insulin secretion by diphenylhydantoin. J. clin. Invest. 49, MALAISSE, W. J., BRISSON, G. R. & BAIRD, L. E. (1973). Stimulus-secretion coupling of glucose-induced insulin release. X. Effect of glucose on 45Ca efflux from perifused islets. Am. J. Physiol. 224, MATSCHINSKY, F. M. & ELLERMAN, J. (1973). Dissociation of the insulin-releasing and the metabolic functions of hexoses in islets of Langerhans. Biochem. biophys. Rles. Common. 50, MILNER, R. D. G. & HALES, C. N. (1970). Ionic mechanisms in the regulation of insulin secretion. In The Structure and Metabolism of the Pancreatic Islets, ed. HELLMAN, B., FALKMER, S. & TALJEDAL, I.-B., pp Oxford: Pergamon Press. SEHLIN, J. & TALJEDAL, I.-B. (1974). Sodium uptake by microdissected pancreatic islets: effects of ouabain and chloromercuribenzene-p-sulphonic acid. FEBS Lett. 39,