Oscillations of secretion driven by oscillations of cytoplasmic Ca2+ as ...

THEJOURNALOF BloLwlcx.CHEMSTRY Vol. 268, No. 30,Issue of October 25, pp. 22265-22268, 1993 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc. Printed i n U.S.A.

Communication

Here wehave used single pancreatic islets from normal mice to monitor simultaneously insulin release and Ca2+jin p-cells. Pancreatic islets are made u p of a few thousand cells, about 80% of which are insulin-secreting p-cells in the mouse. These cells are electrically coupled and display a rhythmic electrical that induce activity when stimulated by glucose concentrations insulin release (9). This activity, which is largely controlled by (Received for publication, August 5, 1993) K+-ATPchannels, consistsof slow oscillations of the membrane potential superimposed by bursts of spikes and reflects Ca2+ Patrick GilonS, Ruth M. Shepherd, and influx through voltage-dependent Ca2+channels (10,111. It has Jean-Claude HenquinO been reported that insulin release by giant islets of oblob mice From the Unite d'Endocrinologie et Me'tabolisme, is sometimes pulsatile, witha frequency similar tothat of the University of Louvain Faculty of Medicine, UCL 55.30, phases of electrical activity in p-cells (12, 13). Recent studies Avenue Hippocrate 55,B-1200 Brussels, Belgium further showed that, during stimulation by glucose, Ca2+josIt is often assumed, but has not been demonstrated, cillates synchronously (frequencyof 2.Umin) inall p-cells of an that the oscillations of cytoplasmic calcium(Ca2+i)that islet and that these oscillations are triggeredby the oscillations occur in various secretory cells induce oscillations in of Ca2+influx (14, 15). Intact islets may thus constitutea suitsecretion. Here, we have used single pancreatic islets able model to investigate whether the oscillations of Ca2+jinfrom normal mice to monitor simultaneouslyinsulin re- duce oscillations of secretion. To ensure adequate temporal lease and Ca2+iin p-cells for periodsup to 25 min. Ca2+j resolution of the insulin assay, the experiments were performed and insulin secretion were found to oscillate in syn- i n the presence of an elevated concentration (10 mM) of extrachrony during stimulation by glucose. This synchrony cellular Ca2+,as this reduces the frequency of membrane popersisted when the frequency of both events changed tential and Ca2+joscillations by about4-fold (15).

Oscillations of Secretion Driven by Oscillations of Cytoplasmic Ca2+ as Evidenced in Single Pancreatic Islets*

spontaneously or upon addition of the hypoglycaemic sulfonylurea tolbutamide, or when their relative amplitudes varied. Repolarizingthe p-cell membrane with diazoxide abolished both Ca2+i and insulin oscillations. In contrast, epinephrine suppressed insulin oscillations in spite of the persistence of Ca2+ioscillations. This study in an intact organ, is the first direct demonstration that, sustained oscillations of Ca2+j inducedby a physiological stimulus entrain synchronous oscillations of the functional response, and that both events can vary simultaneously or be dissociated depending on the experimental conditions.

EXPERIMENTALPROCEDURES

Mouse pancreatic islets were obtained by collagenase digestionof the gland and cultured overnight in RPMI 1640 medium(15).After loading with 1 PM fura-2 for 40 min, one islet was transferred into a temperature-controlled perifusion chamber (Applied Imaging, Sunderland, United Kingdom)with a bottom made of a coverslip and mounted on the stage of an inverted microscope. The original chamber was given an oval to 110 pl by an inner metal ring. The shape, and its volume was reduced islet was held in place by gentle suction with a glass micropipette. Afine needle was placed just downstream of the islet to collect the perifusion medium as close as possible to the tissue. The preparation was perifused at a flow rate of 1.8 ml/min, and the effluent fractions were collected manually every 10 s; each fraction therefore corresponds to2.7 times the chamber volume. Perifusion solutions were preheated in a It has long been known that Ca2+ plays a pivotal role in water bath, and a temperature controller ensured a temperature of exocytosis (11, but the links between changes in the concentra37.2 "C b0.3 "C) close to the islet as monitored by a thermistor placed tion of cytoplasmic calcium (Ca2+j)1and changes in secretion near the tissue. After switching solutions there was a delay of 90 s are only partially understood. Whereasit is clear that a rise in before the new solution amved in the chamber. Thetime between entry Ca2+ican trigger secretion (2-61, the functional roleof the Ca2+i of the liquid into the outflow system and its collection in the tubes was 22 s. These delays have been corrected forin the figures. The measureoscillations that many agonists produce in various secretory ments of CaZtiwere performedwith the Magical system, (from Applied cells (7, 8 ) has not been elucidated. It is often implicitly asImaging), as described in detail recently (15). The interval between sumed that Ca2+j oscillationstrigger oscillations in secretion, successive ratio of recordings at images (340/380 nm) was 3 s. The but direct demonstration has been difficult to obtain because perifusion medium contained 120 mM NaC1,4.8 mM KCl, 10 mM CaC12, 1.2 mM MgCl,, and 24 mM NaHCOa. It was gassed with OJCOz (94:6)to hormone assays cannot resolve fluctuationsof secretion at the single cell level. One way to circumvent the problem is to resort maintain pH 7.4 and was supplemented with bovine serum albumin (1 mg/ml). Insulin was directly measured in the collection tubes containto a microorgan of coupled cells. ing the 0.3-1111 effluent fractions. The radioimmunoassay included the following steps: addition ofEDTA (final concentration 9 mM) t o avoid calcium precipitation at low temperature, addition of guinea pig anti* This work was supported by Grants 3.4607.90 and 9.4511.89 from insulin serum, incubation for 27 h at 4 "C, addition of a trace amount of the Fonds de la Recherche Scientifique Medicale, Brussels, and by freshly labeled '251-insulin, incubation for 18 h at 4 "C, and precipitaGrant SPPS-AC 89/135 from the Ministry of Scientific Policy, Brussels. tion of bound insulin by ethanol. Standard curves were prepared by The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked diluting rat insulin in exactly the same medium as the samples. The "advertisement" in accordance with 18 U.S.C. Section 1734 solely to minimum detectable amount of insulin was 3.5 pg/tube, which corresponds to 21 pgis1et-l. min-l. Fifty percent displacement of '251-insulin indicate this fact. was achieved by 50 pg of insulidtube. Intra-assay variation of the $ Charge de Recherches of the University of Louvain. 6 To whom correspondence should be addressed: Unit6 d'Endocrinolo- standard curves, run in triplicate, was 15% at 6 pg/tube (36 gie et MBtabolisme, UCL55.30,Avenue Hippocrate 55, B-1200Brussels, pg.islet-'.min-') and 8%or less at 12.5 pg/tube(75 pg.islet-'.min") and above, z.e. in the range of most measurements. The insulin content of Belgium. Fax: 32-2-7645322. ' The abbreviations used are: Caz+i,free cytoplasmic calcium; K+-ATP the islets was determined after extraction in acid-ethanol (HCl/ethanoV water: 1.5/75/23.5). channels, ATP-sensitive K+ channels.

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measure was about 1.5/min (Fig. 2C). Our inability to detect insulin oscillations with a higher frequency is not surprising In thepresence of only 2 mM glucose, Ca2+j in P-cells was low (average 66 2 4 nM (S.E.); n = 8) and fairly stable. On stimula- with sampling periods of 10 s. Short (-20 s) pulses of insulin tion by 20 mM glucose, Ca2+i first decreased by about 10 nM release by oblob mouse islets have been measured recently by before increasing abruptly (Fig. 1)to a peak value that aver- collecting the perifusion medium at intervals of 3 s (13). This, aged 213 ? 10 nM. After this first rise, Ca2+i oscillated at a therefore, suggests that oscillations of insulin release accomfrequency that varied from islet to islet(Fig. 1, compare A and pany the faster oscillations of Ca2+ithat occur at physiological B). When the concentration of glucose was decreased to 2 mM, concentrations of extracellular Ca2+(14, 15). between Ca2+i Fig. 2 shows in greater detail the relationships the oscillations stopped and Ca2+i remained stable at a level and insulin oscillations in three different islets stimulated by that was slightly higher than at the start of the experiments. Others have sometimes reported that a small decrease in in- 15 mM glucose. Insulin release wastypically found to increase sulin release precedes the rise induced by glucose (16). Such a a few seconds after Ca2+i, and thepeak of the insulin oscilladecrease only inconsistently accompanied the fall in Ca2+i in tions was slightly delayed compared to the peak of the Ca2+i our experiments, perhapsbecause the basal rateof secretion is oscillations. This shift does not necessarily mean that there action of Ca2+ion exocytosis. We ascribe close to the limitof detection with only one single islet. On the exists a time lag in the other hand, the rise of Ca2+ibrought about by 20 mM glucose it to the limitations in the time resolution of our system (difwas consistently followed by a large peak of insulin release. fusion of insulin within the islet, dead spaces, etc.). ExperiThereafter, the rate of secretion oscillated at the same fre- ments based on changes in membranecapacitance have shown quency as the Ca2+ioscillations (Fig. L4). In the experiments that exocytosis occurs without delay whenCa2+i isincreased in shown in Fig. lB, no correlation was found between insulin and various cell types (3-6). Fig. 1 also shows that the amplitude of insulin oscillations Ca2+ioscillations when the frequency of the latter was around progressively but markedly decreased with time, although that 2.5/min, but thesynchrony became evident whenthe frequency of the Ca2+i transients was fairly constant. Wedo not know of Ca2+ioscillations dropped. The highest frequency of insulin oscillations corresponding to Ca2+i oscillations that we could whether this apparent desensitization to Ca2+ireflects a decrease of the efficacy of Ca2+on its targets or the progressive be exhaustion of a pool ofreadily releasable granules. It cannot G20 mM explained by emptying of insulin reserves because the total 1 G2 mM amount of insulin released during theperiod of stimulation did not exceed 1%of theinsulincontent of theislets (167 7 ng/islet; n = 41). Moreover, the phenomenon was consistently seen when the islets were stimulated by a rise in the glucose concentrations from 2 to 20 mM but was not, or was much less, observed during continuous stimulation with 15 mM glucose (Fig. 2). We also note that repetitive pulses ofKC1 (l/min) RESULTS AND DISCUSSION

G15 rnM

2 min

3

,

G20 mM

G2 mM

2 min

0 1 5 mM

FIG.1. Glucose-induced oscillations of insulin release Caz+iin single pancreatic islets. Insulin release and CaZti were measured simultaneously from single mouse islets loaded with fura-2. The concentration of glucose ( G ) in the perifusion medium was increased from 2 20 to mM as indicated top at of panels. These experiments are representative of results obtained with eight islets from six cultures. A, in five islets there was a good synchrony between CaZ+, and insulin oscillations during the whole period of stimulation. B , in the other three islets,no synchrony was found for a few minutes after the initial rise, when the frequency of Ca2+, oscillations wastoo high, but appeared thereafter.

2 min

2 min

-

FIG.2. Comparison of the oscillations of insulin release and Caz+;occurring in single pancreatic islets during steady state M glucose. These exstimulation with a medium containing 15 m periments are representativeof results obtained with33 islets from 14 cultures. Ca2+, oscillations wereobserved in all islets tested, and only one islet failed to exhibit corresponding insulin oscillations.

Oscillations of Insulin Release and Cytoplasmic Tolbutamide 200 pM

G15 mM

I

-

U

2 min

2 rnin

G15 mM

Tolbutamide 200 pM

G15 rnM

Epinephrine 1 pM

I r

-

2 rnin

FIG.3. Effects of tolbutamide on insulin and Ca2+i oscillations in single pancreatic islets. Tolbutamide (200 PM) was added to a medium containing 15 mM glucose ( G ) as indicated a t top of panels. These experiments are representative of results obtained with four islets from four cultures.

induced Ca2+i transients of a similar size but progressively smaller bursts of prolactin in pituitary lactotrophs (17). Tolbutamide increases glucose-induced insulin release by depolarizing the p-cell membrane through a blockade of K+-ATP channels (18, 19). In certain islets, tolbutamide increased the frequency of Caz+i oscillations with little influence on their amplitude (Fig. 3A). This was followed by a corresponding increase in the frequency of insulin oscillations. In other islets, tolbutamide raised Ca2+i, which stopped oscillating regularly for a few min (Fig. 3B). A concomitant increase in the rate of insulin releaseoccurred, but no clear oscillationswere detected until Ca2+i againdisplayed regular oscillations. Diazoxide repolarizes the p-cell membrane through anopening of K+-ATPchannels, and the resultingsuppression of Ca2+ influx underlies the inhibition of glucose-induced insulin release (18,19). On addition of the drug to the medium, both insulin and Ca2+i oscillations stopped, and insulin release and Ca2+i remainedlow (Fig. 4A). However, mobilization of intracellular Ca2+by acetylcholine caused a single peakof Ca2+ithat was accompanied by a peak of insulin. This shows that the rise of Ca2+i, whatever itsorigin, is indeed the triggering signal of insulin release. Afteractivation of a2-adrenoceptors by epinephrine,the p-cell membrane repolarizes for a few minutes before phases of electricalactivity of increased durationand decreasedfrequency reappear (20, 21).As shown in Fig. 4B, Ca2+iin p-cells changed in a strikingly similar way. Ca2+iremained low and stable for about 6 min after addition of epinephrine, and then longer and slower oscillations reappeared. On the other hand,

1

800

I !

.u 2 min

FIG.4. Effects of diazoxide, acetylcholine, and epinephrine on insulin release and Ca2+iin single pancreatic islets. Diazoxide (DZ, 400 p ~ and ) acetylcholine(ACh, 100 PM) (panel A ) or epinephrine (1 PM)(panel B ) were added to a medium containing 15 mM glucose (GI a s indicated at top of panels. For the experiments with epinephrine, the medium was supplemented with 0.5 mM ascorbic acid. These experiments are representative of results obtained with five islets from at least four culturesfor each protocol.

epinephrine abolished insulin oscillations not only during the first minutes following its addition, but also when Ca2+ioscillations had reappeared (Fig. 4B). Only a minor increase in release accompanied these oscillations. This dissociation first excludes the remote possibility that an artifact accounts for our finding of a good temporal correlation between Ca2+i and insulin oscillations. It also directlyestablishes that the inhibition of secretion by a2-adrenoceptors involves an action distal to the Ca2+i rise. The same conclusion was previously drawn from experiments showing that epinephrine can inhibit insulin release inducedby a fixed concentration of Ca2+in permeabilized islet cells (22, 23). Direct studies of the functional role of Ca2+ioscillations in secretory cells have generally been hampered by insufficient temporal resolution of the hormone assays. In populations of pituitary cells, repetitive stimulations with KC1 or gonadotropin-releasinghormonetriggeredparallel increasesinCa2+i and hormone release (17, 24), but this was not the case with thyrotropin-releasing hormone (25). Using the hemolytic plaque assay to measure hormone release from single pituitary or chromaffin cells made it possible to show that spontaneous release occurred in those cells that exhibited Ca2+ioscillations (26, 27), but no temporal relationship can be established by this approach. In single chromaffin cells, repetitive depolarizations produced transient rises in Ca2+i thatled to transientincreasesin secretion, as evidenced by anincrease in membrane capacitance (3). It is, however, only very

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recently that combined measurements of membrane capacitance and Ca2+iin single gonadotropes demonstrated that the oscillations of Ca2+iinduced by a single application of gonadotropin-releasing hormone are each accompanied by a burst of secretion for about 1 min (28). This technique has an unsurpassed time resolution, but is difficult to use for recordings longer than a few minutes or a t 37 "C, two conditions that must be met to study glucose-induced insulin release.This may be the reason (29)why capacitance measurements failed to disclose pulsatileinsulinrelease evenwhen Ca2+i was made to oscillate in single p-cells (30). In conclusion, our study demonstrates that, in an intact organ studied underalmost physiological conditions (high extracellular Ca"), oscillations of Ca2+i induced by the sustained application of a physiological stimulus entrain synchronous oscillations of the functional response. In addition to providing a first answer to the much debated question of the biological significance of Ca2+, oscillations,the approach used here could help elucidate the consequences of their disorganization. Oscillations inplasma insulin levels have been described in man and animals, but their origin is still unclear(reviewed in Refs. 31 and 32). However, alterations of these oscillations are considered to be involved in the development of non-insulin-dependent diabetes(33).An important challenge will be to determine whether the oscillatory behavior of p-cells is altered in the disease. Acknowledgments-We thank V. Beaujean for efficient help and M. Nenquin for editorial assistance. REFERENCES 1. Douglas, W.W. (1968) Br. J . Pharmacol. 34, 451474 2. Cheek, T. R., Jackson, T. R., OSullivan, A. J., Moreton, R. B., Berridge, M. J.,

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

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