Cytoplasmic Ca2+ oscillations in pancreatic ß-cells

Biochimica et Bioph~,sica Acta, I 113 (1992) 295-305

295

© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4157/92/$05.00

BBAREV 85410

Cytoplasmic Ca z oscillations in pancreatic fl-cells Bo Heliman, Erik Gylfe, Eva Grapengiesser, Per-Eric Lund and Alf Berts Department o f Medical ('ell Biology, lIiomedicum. Uppsula U,~icerxity, Upp.sala t Swcden )

(Received 9 March 1902)

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29.';

II.

Methodological considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

296

Ill.

Types of Ca ~' oscillations in pancreatic fl-cclls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Type.a (J~illat~ons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Type-b oscillations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Type-c oscillations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Type-d o~illations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

297

IV.

The induction of type-a o~illations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

298

V.

Recognition of glucose as reflected by the C a : " oscillatory pattern . . . . . . . . . . . . . . . . . . . .

299

VI.

Cyclic mobilization of intracellular calcium by G-proteins . . . . . . . . . . . . . . . . . . . . . . . . . . .

3f10

297

298 298 298

VII. Propagation of cytoplasmic C a : " oscillations in clusters of 0-cells . . . . . . . . . . . . . . . . . . . .

301

VIII. Cytoplasmic Ca"* oscillations in intact islets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.~)2

IX.

Functional significance of the Ca ~ + oscillations in pancreatic/.:l-cells . . . . . . . . . . . . . . . . . .

303

X.

Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.'~)3

Acknowledgemenl~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

;t)4

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

304

!. Introduction

The cy;.oplasmic concentration of free Ca 2+ ([Ca-"+ ],) is a primary regulator of cellular functions. The eDvation of [Ca 2÷]i mediates various responses of the pancreatic fl-cells, including the release of insulin [34]. The role of Ca 2÷ in the insulin release process has been extensively evaluated using intact pancreatic islets or suspensions of islet cells. In addition to providing the final evidence that stimulation of insulin release is mediated by increased entry of Ca 2+ into the fl-cells,

Correspondence to: B. tlellman. Department of Medical Cell Biology, Biomedieum, U p p ~ l a Universi~. Uppsala. Sweden.

these studies a l ~ indicate that glucose and other nutrients have a dual action on [Ca2÷]i. implying that these secretagogues may under certain conditions even inhibit insulin release. Studies of the role of Ca 2- in insulin release using intact or dispersed pancreatic i~!e'.s arc complicated by the presence of non-/I-cells. Moreover, measurements of average [Ca2+]i in the total cell population may hide variations in the ~esponses of the B-cells. The access to new techniques allowing analyses of individual mammalian B-cells made it possible in 1988 to demonstrate that glucose, the major natural stimulator of insulin release, generated large amplitude oscillations of [Ca2+]i from a basal level to 5-10-fold higher values [12]. In further studies we have been able to demon-

206 strafe lhat glucose also induces faster oscillations supcriml~)sed on an elevated [Ca2"], [18}, and that the cyclic mobilization of intraccllular calcium following inositol phospholipid brcakdown it~ insulin¢~ma cclls [59] al~) occurs in the nt~,mal/3-ccll [14]. The disctwcry of an oscillating [Ca-~+], is fundamcnt::l for the understanding of insulin release, indicating that rhythmicity is an intrinsic property of the/~-cells rather than bcing the result of a neural pacemaker activity. The aim of this report is to review the rapidly, expanding knowledge about the oscillations of [Ca '~ ], in the pancreatic/J-cells. For complementary information on other aspects of the role of Ca"* in the insulin release process reference is made to a number of extensive reviews during the last decade [2.4.2527,30,31,34,35,40,60,61.75]. !1. Methodological considerations Until recently it has been difficult to monitor dynamic changes of [Ca -~*], in isolated small cells like the pancreatic /J-cells. However, the situation improved considerably with the synthesis by Tsicn and colleagucs [20,72] of fluorescent Ca -'+ indicators available as membrane-permeable acetoxymcthyl esters. So f~tr. most recordings of the [Ca-" ~], changes in individu.'tl /J-cells have been performed with thc fluorescent dye fura-2 using a ratiomctric approach with excitation at 341) and 380 rim. At these wavelengths the fluorescence increases and decreases ~espcctivcly with the Ca -'+ binding. The emitted fluorescence at 510 nm has either been recorded with a photomultiplier, estimating aver° age [Ca-'*]i in the entire cell, or with an intensified vide() camera to reveal intracellular hetcn~geneity and how the Ca-"~ signal propagates between the cells. A suitable microscopic set-up allowing continuous recordings of [Ca-'+]i in pancreatic /3-cells based on the 340/380 nm fluorescence excitation ratio of fura-2 is shov, n in Fig. 1. The equipment enables ratio determinations based on measurements of 1 ms light flashes at each o| the excitation wavelengths every 10 ms. After determining separately the 340/380 nm fluorescence excitation ratio and the 381) nm excitation fluorescence tx)th in a Ca-'+-deficient medium and at excessive concentrations of the ion, the [Ca -'+ ], is calculated as described by Grynkiewicz et al. [20]. The rapid time-sharing ratiometric approach for measuring [Ca" ÷]i with fura-2 is associated with a noise reduction compared with single wavelength techniques [7,21]. However, if the 381) nm excitation fluorescence, which is the denominator, is allowed to approach zero as [Ca"+], increases, even small fluctuations in the signal will have profound effects on the ratio. It is therefore important to reduce noise and adjust the signal levels when analyzing oscillatory phenomena. Although high excitation intensities reduce noise, they

CELLS

~===J'~ ..... l/::~."."o°.Y

k ....... r==l

, "

..........0V

/ r"- "~ 1i

EMISSIONFILTER PMOTOMULTIPLtE RSIonm

LIGHT BEAM • ELECTRICAL CONNECTION

340 rm AND 380 nm SIGNALS 3~O...

R*TIO ""

RECOBOEB

Fig. I. Micn)scopic set-up for continuous recordings of [Ca 2" 1, in individual pancreatic B-cells k)aded with fura-2. The clwer glass with the attached B-cells is used ;~s the I~*ttom of an open perifusion chamber placed on the stage of an inverted microscope equipped ~,ilh an epifluorescence illuminator and a I(H)× UV-fluorite objective (Nikon). A 75 W xenon arc lamp combined with 5 nm half-bandwith interference filters in the rotating air-turbine filter changer of a time-sharing spectrophotofluorometer [5l provides excitation light flashes of 1 ms duration at 3411 and 380 nm every It) ms. The emitted fluoremence light is measured with a photomultiplier at 5111 nm using a 311 nm half-bandwidth filter. The electronically separated ~;ignals excited at 340 and 381) nm are fed into an analog ratio meier. The :alto as wel! as the 38() nm excitation fluorescence are recorded ~n a strip charier recorder. The microscope is placed within a climate b~)x maintained at 37°(` by an air strc:trn incubator.

cause excessive indicator bleaching and cell damage (see below). The lamp intensity should consequently be reduced by neutral density filters to a level allowing observation periods exceeding 61) min without detectable deterioration of the [Ca 2~], responses. It is also important to equalize the fluorescence excitation intensities at 340 and 381) nm either by amplifying the weaker fluorescence signal or damping the stronger one. An imlx~rtant ~ ay of reducing the noise is to limit the bandwidth to the frequencies of interest. The efficiency of our microfluorometric approach for recording dynamic [Ca~'+], changes in individual /J-ceils has been subject to extensive testing. Using the experimental situation of K ÷ depolarization, which opens the Ca 2 * channels without inducing concomitant action potentials [42], the noise was found to increase 2-3-fold merely b~ raising [Ca2+]i (Fig. 2A). Fig. 2B shows another exFeriment with a comparable initial noise level in a ~-cell depolarized by glucose. It is apparent that glucose, after an initial lowering, initi-

207

A

11 mM

l

::l mM

v

+

300

o

u v FLASH

600

i

200

¢

o

100

÷ ol I

o o •o )

!

I

10

20 TIME (MIN)

I

400

I

I

Fig. 3. Sutprcssion of glucose-induced [Ca:" ], oscillations in an individual fl-cell by a slmrt ( < I ms) pulse of UV-light (~I0-41X)nm). rl he irradiated energy in fl)cusgas estimated as 10 mJ/ram:.

I

B

.J a.

0

300

o 200

/

100

t

2

i

I

i

6

I

10

TIME (MIN)

Fig. 2. Alterations of [Ca2+ ]i in individual /J-cells after de~)larization with K + or glucose. The arrows indicate a rise of K from 5.9 to 30.9 mM (Na" being replaced by equimolar K" ) in a gluco~-free medium (A) or an increase of the glucose eoncenllation from 3 to 211 mM in the pre~nce of 5.9 mM K + (B). ates a rise of [Ca 2 +], with noise fluctuations somewhat exceeding those o b t a i n e d after K + depolarization. However, so far, the use of the ratiometric technique with time-sharing has not adequately r e l i v e d the [Ca"+], transients accounted for by the single /3-cell action potentials. P r e p a r a t i o n of B-ceils for analyses is a two-step p r o c e d u r e with collagenase isolation or intact islets and subsequent dispersicm of these islci,, into sing!e cells. It is imperative to perform this isolation with minimal

exposure to the mixture of enzymes present in the crude collagenase preparation and to have an adequate oxygen supply. Also the exposure 1o UV-light during the [Ca2-], m e a s u r e m e n t s may disturb the oscillations. As shown in Fig. 3A, a flash of UV-light can break the p a t t e r n of o.~illations with m a i n t e n a n c e of a [Ca -'~ h response to glucose. T h e fact that minor inadequacies in the technique will result in a selective disappearance of the glucose-induced oscillations prevented their detection in our first m e a s u r e m e n t s o n individual ,O-cells loaded with fura-2 [11]. Four months later, hov, ever, improvements in the techniques allowed the d e m o n stration that a glucose-induced rise of [Ca"+], is often manifested as oscillations [12]. IlL Types of Ca z+ oscillations in pancreatic p-cells Four types of cytoplasmic Ca-'* o ~ i l l a t i o n s ha~e been identified in individual p a n c r e a t i c / / - c e l l s (Fig. 4), and some of their characteristics, are summarized in Table I. IlI-A. Type-a oscillations

These sinusoidal [Ca:* ], oscillations are identical to the large amplitudes ones originally observed when -~xposing mouse /3-ceils to 7 - 2 0 m M glucose [12.1417,36]. T h e o ~ i l l a t i o n s start from close to basal [Ca 2" ],

TABLE i Types oJ o'toplasmw Ca: " oscdlations m mdirtduai pam'reatic ~J-cells Type

lnmator

Di~ppcarance on Ca :+ omission

Initial Ca"" level

a

$1ucose

+

ba~l

b c d

glucose with restored or preserved cAMP glucose with excessive cAMP carbachol or ATP

+ + -

elevated elevated basal or elevated

Amplitude (nM) :~N)-5~

70-250 > 2:50 < I 0fl0

Frequency ~(~ill/min) 0.05-0.5

2-8 irregular 1-6

298 with an amplitude of 300-500 nM and a frequer, cy o f 0.05-0.5/min. Several studie~ have demonqrated their presence also in rat B-cells [41,57,74,76].

,?,

iil-B. Type-b oscillations

o;

III-C Type-c oscillations Pronounced [Ca -'~ ]i transients with durations below 10 sec are occasionally seen among the b-oscillations [18]. These oscillations are more frequeat when the B-cells are exposed to high concentrations of glucagon

8

b

1

400

$ vc + (M go O

< -I n

o

I->0

200 _

1000

| 5

I

_._1 10'

d

C

800

600 400 200

;

1 ! mM

20 mM

3 mM

400

These oscillations are about 10-fl~ld faster and superimposed on an elevated [Ca -'~1, in glucose-stimulated B-cells [18]. The proportion of B-cells responding to glucose with the b-type oscillations is higher in cells analyzed shortly after isolation than in those kept in culture for 1-2 days. However, the presence of a low concentration of glucagon (I nM) enables cultured cells to respond regularly to glucose with such oscillations. The latter effect may reflect restoration of normal levels of cyclic AMP in isolated B-cells [18] depleted of the nucleotide due to their separation from the glucagon-producing a,-cells [66]. Also the exposure to membrane-permeable esters of cyclic AMP results in the unmasking of b-type oscillations in cultured B-ceils [18].

600

mM

,o

o

;

TIME (MIN)

Fig. 4. Survey of the [('a2" ], oscillations in individual pancreatic /3-cells expos;ed to 2(1 mM glucose. The different types have been referred to as a-d. The b and c-types of oseillations are suOerim0osed on type-a and shown for a medium supplemented with I nM glucagon or 5 /aM fi~rskolinrespectively. The d-type o.~illations represent the response to 100 V.M earbachol.

o

200

1'0 -

20 TIME (MIN)

30

Fig. 5. Transformation of basal lCa" + ], (state l ) to type-a ~cillations (slate II) with subsequent c:~tablishmcnt o f a sustained elevation (state l i d when exit)sing an individual /J-cell to the indicated concc ntrations of glucose.

(10 or !00 nM) or when stimulating their adenylate

cyclase activity with 5 # M forskolin.

III-D. Type-d oscillations These Ca-'* transients have been observed both in insulinoma cells [59] and mouse /.C-cells [14,22] and reflect mobilization of calcium from intracellular stores mediated by receptor-activated breakdown of inositol phospholipids. In contrast to the a-c oscillations, type-d can be triggered in the absence of extracellular Ca 2+. The d-type oscillations exhibit characteristic patterns, making it possible to identify individual B-cells by their [Ca2 + ]i 'fingerprints' [59]. IV. The induction of type-a oscillations

it is possible to demonstrate three states in the glucose generation of the type-a (~sciUations [19,29]. l'hese states are illustrated in Fig. 5. There are no oscillations when cultured mouse B-cells are exposed to glucose conct.'rdrations below 7 mM (state I). The latter state is characterized by a decrease of [Ca2+]i when raising glucose, on effect explained by organelle sequestration and outward transport of the ion l! 1°2527,30,31,34]. The transition to oscillations (state ll) occu~.':~ suddenly with a considera01e variation between cells with regard to threshold concentrations of the sugar. With a further increase of glucose, the type-a oscillations will often be tra,:sformed into sustained elevatton of [Ca2*]i (state !11). Although the latter transition sometimes occurs in response to 11 mM glucose, many B-cells remain in state II even at a glucose concentration of 41) mM. The induction of type-a oscilh::ions is not unique to glucose but has also been obtained with leucine [13] as well as hypoglycemic sulfonylureas [17]. Actually, the type-a oscillations can be seen under the same conditions as those initiating electrical activity in B-ceils of

299 intact islets [38]. Various observations indicate that type-a oscillations ~.fle,-t cyclic v,riations in the Ca 2. permeability of the plasma membrane. Whereas increase of extracellular Ca 2. results in a rise of the amplitude of thc oscillations, they disappear when blocking the voltage-dependent Ca 2+ channels [14]. Indeed, studies of isolated//-cells with the patch clamp technique using either the perforated patch approach at .'~°C [67] or the ceil-attached configuntion at 37°C [36] reveal that the type-a transients correspond to stow bursts of the Ca2+-dependent action potentials, in the search for mechanisms responsible for the cyclic variations in the Ca 2+ permeability different alternatives should be considered, it has been suggested that, apart from controlling the membrane potential, glucose also directly stimulate~ the activity of the Ca 2+ channels [63,68]. Inhibition of Ca 2÷ influx through the voltagedependent channels by a raised [Ca2+]~ may be important. Another feed-back loop involving hyperpolarization by Ca 2+-activated K + channels can also be envisaged [1,21]. Glycolysis is a prototype of an oscillatory metabolic pathway [43], and indirect evidence has been provided that the A T P / A D P ratio is a major determinant for periodic fluctuations in the Ca 2+ entry [10]. When discussing a metabolic origin of the cyclic variations in the Ca 2. permeability, it is notable that the initial rise of [Ca2+]~ is preceded by an increase of reduced pyridine nucleotides, and that the native fluorescence of these nucleotides has been found to fluctuate with a frequency similar to that of the type-a oscillations [57,58]. Arguments for an oscillatory metabolism of glucose can also be taken from measurements of membrane potential in//-cells within the intact islet. During the electrophysiological burst activity the time spent in the depolarized state is strongly dependent on the glucose concentration [38]. it is therefore noteworthy that the time in the depolarized state exhibits spontaneous variations with a type-a-like frequency [8.39]. In the light of the observations of slow bursts of action potentials at 37°C [36] it is no, surprising that individual pancreatic //-cells rcsP~,nd to glucose with type-a oscillations. The max',an! rate of [Ca:+]~ increase during the rising phase of the oscillations does not exceed 60 n M / s [18]. When taking into account that the highest frequency of action potentials in this situation is approx. 4 / s [36], the contribution of each action potential to the increase of [Ca2*]i can be estimated as less than 20 nM. Although this value may be considerably higher due to an underestimate of the a~erage [Ca2+]~ [21], there i~ no doubt that Ca 2+ is effectively buffered in the //-cells. The amounts of Ca 2+ entering during a single action potential has been estimated as 10 -~8 mol, which is sufficient to increase the total calcium concentration of the//.cell by I /aM [~1.

RYANOD~E

1 ~

4OO

,,,

B

;o

-"

3'0

-~

CAFFEIN[ ~

...... IRYANOOINE

4OO

,;

2'0

30

,o

T ~ (MIN) Fig. 6. Effects of ~ancglinc (2(l p . M ) o n lypc-a o'*,cillat,~m', induced by II mM gluco~ (A) or on the sustained elevation of [ C a " ' ] , obtained when combining I I mM gluc(~¢ with I0 mM caffeine (B)

An important question related to the generation of the glucose-indu:ed type-a oscillations is whcthcr the Ca `'+ entering the//-cells induces an additional release of Ca :+ from intraceilular stores. It is evident from reports from several laboratories that Ca"-induced release of Ca 2. is not restricted to the ~rcoplasmic reticulum but is also of physiological relevance for a number of non-muscle cells [50]. The Ca2+-induced Ca 2+ release channel is characterized by its sensitivity to ryanodine, which locks it in an open subconductance state [45,49]. Fig. 6A illustrates experiments intended to reveal whether release from intracellular Ca: + stores contributes to the type-a oscillations. In accordance with the idea that a periodic influx of extracellular Ca:* is sufficient to account h~r the type-a oscillations (sec above), they remained unaffected when the//-cells were exposed to 20/aM r~anodine. The absence of a suppressive actio, of ryanodine on the type-a oscillations cannot be accounted for by a restricted entry of the alkaloid. Actually, ryanodir,c rapidly restores type-a oscillations, when they have been transformed into the sustained increasc of [Ca 2~ ], by caffeine (Fig. 6B). V. Recognition of glucose as reflected by the Ca TM oscillatory pattern The discovery of the cyclic changes of cytoplasmic Ca :+ in the oancreatic //-cells raises the question of how the oscillatory pattern is related to the ability to recognize glucose as a secretory stimulus. Evidcncc has been provided that cellular functions may be regulated by amplitude or frequency modulations of [Ca2*]i oscillations [3,6]. Fig. 7 indicates how a rise of glucose

from I 1 to 20 mM affects the appearancc o! the typc-a oscillations and their transformation to a sustained increase (state liD. in contrast to the pronounced action of glucose on the electrical burst activity in fl-e,:lls from intact islets, theie were no alterations in the frequeneics or durations of the type-a oscillations. Neither were there any changes of their amplitudes. The absence of an effect of glucose on the oscillations did not prevent the sugar from inducing their transformation into a sustained increase, in Ii mM glucose 17% of the//-cells were in state ill as compared with nearly 40% in 20 mM (Fig. 7). It is apparent from these and other observations [28] that glucose sensing of the individual //-cell is based on sudden transitions from the basal into two alternative states of raised cytoplasmic Ca -'~. Even if the peak values of [Ca"'], reached during the oscillation~ exceed the level of the sustained increase, time-average [Ca:+]i will be higher during state 111. Accordingly. it is not modifications of the type-a oscillations but their transformation into a sustai,cd ck:~,atcd level which gencra;cs the maximal rise of cytoplasmic Ca -'+ in the individual //-cell (Fig. 8). The abi!ity of glucose to induce transitions between steady-state and oscillatory cytoplasmic Ca 2~ is critically dependent on a number of factors [19]. Glucose becomes more effective both in inducing type-a oscillations and further transforming them into a sustained elevation when cAMP is raised by the exposure to glucagon or theophylline as well as after addition of the permeable 8-bromo or dibuty,ry.I derivatives of cAMP. Whereas cAMP facilitates the glucose-induced transitions to states 11 and iil, activation of protein

FREQUENCY(OSCILLtMIN)

PERCENTAGETRANSFORMATION TO SUSTAINEDELEVATION

0.4 0.2

40

HALF*WIDTH (MIN)

2.0 1.0 20

AMPLITUDE(riM)

H

400 200

[]

11 mM GLUCOSE

•

20 mM GLUCOSE

Fig. 7. Effects of raising glucose from ! I to ~) m M on the characteristics of the type-a (w,cillations a n d their transformation into a sustained increase (state liD.

...----..

i

BASAL L E V E L

300

T

OSCILLATIONS FROM A B A S A L LEVEL

II

I

SUSTAINED ELEVATED LEVEL

111

I"

o (..i

200

U) .J 0.

100

0

I->0

GLUCOSE CONCENTRATION Fig. X. A ~ h e h , a t i c illm, l r a l h m (ff charhgc,, m Ih¢ average [('a2" ], related l , the lram, ititm,, between ~,lales I. II and III follov.ing an increase of lhe glucose conccnlralion.

kinase C counteracts the occurrence of o~illations. This effect may be due to stimulated outward transport of Ca -~', since state !1 is replaced by a state ili with a lower time-average [Ca-"+]i. The idea that rise of [Ca:* ], to a level initiating feed-back inhibition of further Ca:* entry is involved in the generation of type-a oscillations has support from their disappearance at external Ca:" concentrations below 0.5 raM. Moreover. as with the addition of TPA, moderate reduction of external CaZ* to 0.8 mM sometimes facilitates the transformation of the type-a oscillations into a sustained elevation with a decreased time-average [Ca-~'],. Being superimposed on an elevated [Ca:*t, there will be an increase in the number of the fast type-b oscillations with the glucose induction of transitions into states I! and !11. The question of whether the recognition of glucose as a ~cre;ory stimulus by the individual//-cell also involves altered characteristics of the b-type oscillations was explored by comparing their properties during state 111 when raising the glucose concentration from !1 to 16 mM in the presence of 10 nM glucagon. Such an analysis did not reveal any effects on the amplitudes or half-widths, but di~lo.~d a slight glucose response in terms of increased durations of the peaks [18].

Vi. Cyclic mobilization of intracellular calcium mediated by G-proteins GTP-binding proteins play an important role in the signal transduction of the pancreatic //-cells. Repre-

3(11

senting ~n intermediate step in the sequence of events leading to r-ccptor-activated inc;sit0: ph,.'~;pholipid breakdown they ,,-4late the d-type of (~eillations a c counted tot by the moo,;!-ation of calcium from intracellular stores. Although th: oscillatory response to carbachoi and other agonists initiating the phospholipid breakdown is usually manifested as the generation of one or a few [Ca"*], transients [14], it may undcr certain conditions result in the establishment of a regular pattern of small oscillations [22]. Fig. 9 illustrates the induction of such oscillations by carbachol in the presence but not in *.he absence of glucose in /3-cells maintained by diazoxide in a hyperpolarized state. The o b ~ r v e d requirement of glucose for establishing a sustained oscillatory pattern is not unexpected when taking into account that other methodological approaches have demonstrated that the sugar stimulates sequestration of calcium in the pool mobilized by carbachol 124,32,33,371. The important role of the G-proteins for inducing periodic rcleasc of calcium from intracellular stores can be clearly demonstrated by injecting non-hydrolysable GTP-y-S in 0-cells kept in a hyperpolarized state by a patch clnmp pipette [48,55]. Fig. 10 illustrates such an experiment. It is evident that internal perfusion with 100 ttM GTP-y-S induces a regular pattern of oscillations from the basal state and that the [CaZ+], peaks are sufficiently pronounced to activate K + currents. The oscillations induced by direct activation of the G-proteins differ from the glucose-induced type-a oscillations also in other respects than not requiring entry of Ca 2 +. in the original experiments with injection of GTP-y-S a certain concentratton of ATP

A

~E

_(2 3E

600 0

IO0 0

cJ

v

~

0 100 200

o -

1

_ _

J

,

~

t

2

3

4

5

TiME (MtN)

Fig, lt}. Induction of ICa:" ]. (r.,cillations wt~h cnncomitanl K " currents m an individual #-cell kept at a membrane I~tentlal of --70 inV. The cell was mternally pc:rlu~4ed with a patch clamp pipette c(mtaining IIlO bt M GTP-~-S added to a K "-rich medium c~nlaming 3 mM ATP and It~:t tam of the fluorescem indicator In&~- I

was found to bc a prerequisite for establishing the o~illatory pattern [55]. However, when re-evaluating this ATP-dependence it was demonstrated that ATP alone sometimes induces similar oscillations [48]. The latter observation raises the question of whether the ATP produced by glucose metabolism may be involved in the physiological regulation of the G-proteins by promoting conversion of GDP to GTP. The pancreatic islets are known to contain high activities of the nucleotide diphosphate kinase equilibrating the GTP/ GDP .system by the changes in the A T P / A D P ratio [791.

B

Vii. Propagation of cytoplasmic Ca z* oscillations in clusters of ll-ceils ÷ t'q

400

o

c2_ -I n

o

200

I>-

o

t2L 32. I

I

I

I

5

10

5

10

TtME ~ ! N ) Fig. 9. Effects of 100 I~M carbachol on l e a 2" l. in individ,al ~-celL~ exposed to 400 ~aM diaz'.)xide in the absence (A) or pzesencc (B) of 21) mM glucose.

Digital image analysis has enabled studies of how the [Ca"* ], oscillations are affected when several /3cells are electrically coupled [23]. As shown in Fig. I! type-a oscillations become synchronized in clusters. By tollowing the signal propagation between cells it was found that the oscillatory characteristics are dete,mined collectively rather than by particular pacemaker cells [23]. The discovew that the iB-cell co-ordination operates without a particular focus makes it necessary to re-evaluate previous ideas that the electrical activity of the pancreatic islets is governed by glucose-sensitive pacemaker cells located in close vicinity to the blood capillaries [511. The frequency of the type-a oscillations is aim)st do,blec; in 6-14 cell dusters as compared with is,)lated cells [231. Whether this observation means that tl,e frequency will be raised even more with a much larger number of ~.ells remains to be elucidated.

~)2

v 4-

¢q

GLUCOSE

,¢ .J

ft. O F-

0

I

I

I

5

10

15

--

I

20

TIME (MIN)

Fig. I I. induetitm of [('~,"" ], tz..cillati(msin a clusler of t) cells after raising the glucose cont'entration fr(nn 3 Io i I mM. The lit.vet panel sh(~,'s [Ca:" ], for the cells numbered in the 10.,ppanel. The marks or, the ordinate indicate the zero nM level of cyh~pla,,mlc('.,"" ,~r the trace aM)re and/or the 411(InM level lot the trace beh~v,'.

The effect of cell coupling on the faster type-b cycles has also been analyzed [23]. Even these o~illations arc highly synchronized in clusters of #]-cells during the glucose-induced sustained i n c r e a ~ of [Ca-" +], (state I!1). Cell coupling had no apparent effect on the frequency of ,re typc-b o~illations. VIIi. Cytoplasmic Ca z+ oscill~tic ~ in whole islets

prctcd ~t~ indicating a metabolically driven pulsatile ~ccrcto~, activity. A puzzling features is that the [Ca 2 +], ,~cillations occurred also in 3 mM glucose. Moretwcr, the lattc~ t)b~crvation was matte when the fura-2 fluorescence was recorded with a video camera but not with a photomultiplier. When raising the glucose concentration from 3 to 20 mM the average [C,~-'+]l was more than doubled and the amplitudes of the o~illations increased 4-5-fold with little change in their durations. After comparing the [Ca2" 1~ signal in different parts of the islets it was suggested that the increase of amplitude is accounted for b~, recruitment of additional responding cells [9,47]. it is difficult to evaluate the relevance of these observations, since coupling of the /3-cells does not increase the amplitude of the type-a oscillation in clusters [23]. The fast [Ca 2~ ], o.,,cillations have been reported to be synchronized throughout the entire islets, the maximal phase shift between cells being 2 s [65]. Moreover. w h o , combining the fluorescence approach with microelectrode recordings it became evident that the oscillations bad their counterpart in the bursts of electrical activit~ often retcrred to as shr,v waves. Silicc the fast [Ca:" ], oscillati(ms resl~)nd to increase ot the glucose concentration with prolongation of the [Ca =" ], peaks without effects on frequency or amplitudes, it was concluded that it is the time at an enhanced [Ca:+], level which e=codcs for the ~ cell ~ccr,:tory response to gluco:~e [65.69]. Although the t~,pe-b oscillations in individual cells and small clusters have a frequency similar t,~ that of the fast o~illa'ions of [Ca-'+], in

0

~

1.2

Z _0

0.7

q,)

The oscillatory behaviour of [Ca -~"], is not restricted to individual /J-cells or smah clusters but has bccn observed al.~0 among the thousands of cells p r c ~ n t in intact islets from mice [52.65.69,73.78] and rats [9.47]. it was established from the work in Dr. Soria s laboratory that glucose induces both slow and fast oscillations in the mouse islets [73]. The similarities of these ~;:=illations with those designated as types a and b are evident in Fig. 12. Cyt.iic variations of [Ca: ÷ ], with a frequency equivalent to that of the type-a oscillations have been reported to occur also in intact islets from rats [9.47]. The coexistence of such oscillations with similar periods of insulin release and oxygen uptake was inter-

:E UJ uJ

t.) z 1,5

w. -

,.J U.

0.8

T O

,

|

!

I

i

2

4

6

8

10

TIME (k."N)

Fig. 12. Of,callalion~, of [Ca: ~ r ~)rded from Ihe~indo-I flm)rcscencc of ¢mm~ islets expmcd to I I mM glucose. Redrawn flx~n ~,~aldc~milk~ ,~; al 173] v,ilh pcrmi~k,n.

islets, it should bc cmphasizcd that the latter arc much more .~L.nsitive to gluc(~c (scc al'~wc) an¢t that the underlying bursts of action Ix)tcntial~ apparcntl~ ~,.+quire a supraphysiological c(mccntration of Ca:+ [62]. When es'aluating such differences it should bc taken into account that all studies of oscillations ot membrane potential and [Ca+'*], in islets have hecn performed in the absence of an intra-islet capillary circulation. in this situation the exchange of cxtraccllul.r medium within the islet is considerably reduced, which accentuates changes in its ionic comlx)sition as well as paracrine and autocrine effects. Since the cxtraccllular K + concentration has been found to vary with the electrical bursting activity [56], this ion may well bc an important feed-back factor, in view' of the pronounced effects on the [Ca-'*], os,dllations it is also possible that release of glucagon from the a.-cclls and ATP together with insulin from the/~-cclls are major determinants for the fast oscillations observed in intact isl,=ls.

iX. Functmmll significance of file Ca:+ oscimLutlonsin pancreatic ~-cells

I A B I I II

+djac+nt lJ.+~clls '~ l)vfc+II+,c syt~chrm+~it=~+~++~++ ~hc c",~+N.+l~+t~= { . + '

-+Kr,.+t ++'++:1++:,++=

lkm of the Ca -'+ signal. Ht~,v-ever. there i~ m~ doubt that minor da~'~ge of the/~-cclls results in a k~s of the typc-a oscillations with maintenance of a gluco~-ind~ced rise of [Ca:" ]=. As discussed in ,~-c'tton I!+ rnin<,r inadequacies in ceil handling can rcsutt =n ladurc t~+ demonstrate c3'clic variations of [(:'a:+ ], in =,=dated ~l-cells. ,~lectivc di~ppcarance oI the typc-a (~.'dlalions with preservation ol a gluco~: resume.: ~ith rai~d [Ca-+'], is ~ e n a l ~ when the: /~-cclls a~c cxp o n d to toxic agents [ Im??J'(]. X+ Su~.uar~ and co~lu~ioas

The techniques available do not permit the dcrrv)nstration of pulsatile insulin rclea~ frtun individual ~6-cells. Nevertheless, we have reason to believe thai the type-a 4r~cillations initiate phasic release of insulin. Since coupling of the /~-celis is associated with ~ n chronization of the [Ca: + ], cycles, it is not unexpected that the periodicity of insulin release from the mouse islets [53] is equivalent to that for t)lae-a oscillations. The ,synchronization of the rhythmic activity of the ,(]-cells is probably also co-ordinated between the islets in the pancrea.~+ O t h e r w i . it is difficult to understand why serum insulin shows pronounced cyclic variations in various species including man [46]. in accordance w,:th ot:,er systems for oscillatory re;ease of biologically active pcptides the cyclic tarialions of circulating insulin can bc supposed to prcwent down regulation of the I~ripheral receptors [46]+ it is therefore interesting that the loss of insulin (~illatkms has been regarded as an early indicator of t.s~-2 diabetes [54]+ With down regulation of the receptors it follows that more insulin must bc released to maintain the blood sugar normal+ Such an increase of the secretory aclivit~ imposes not only a load on the/~-ceqs but may also precipitate :mdesirable effects of exc: s++~e insulin such as hyperlonia and atheroselerosis [70,71.771. The discovery of cyclic variations of [Ca: + 1, in the pancreatic/~-cells raises the question whether it is the impairment of the t)l~-a oscillations or '.,>~s of their synchronization within or among the islets which resuits in the disappearance of pulsatile insulin release+ The differem alternatives arc presented in Table !1. So far, there are no reports regarding a defective propaga-

In tnc la~: I~ years it has Ix*on a greying interest =n the cyclic" varia~kms of circulatin~ insulin [~,! ~.+ter the suggc~icm that this phenomenon mat Iv.: due to os4=illalions of the /l-cell membrane potential [8.39]~ it was demonstrated that [Ca:+], oscillates in the glucoscstimulati;d ~-ccll v,~th a similar frequency to that o( pul~tile insulin reload. The pre~nt review describes four types of [Ca :~ ]+ oscillations in the pancrcatk: /~-ccli. The slow sinusoidal oscillations, refi:rlcd to as ~l>c-a+ are those which most closely corrcsp(vnd ,o pulsatilc insulin rclca.~+ Ahhtmgh not alfecting the properties of the typc-a o,~'illation~ m individual /J+ cells, the concentration of glucose: is a determinant for t h o r generation and turtht:r transformation intt~ a sus+ taincd increa.sc+ Accordingly. cytopla,~mic Ca: ' i~ regulated t~ sudden transition~ tvctween (~ciIlat¢+r) and steady-~at¢ Icwel~ at threshold ct)ncentrations o[ giuco*c. which arc characteristic for the individual /3-cell. This behav=our explains the ob~rxation of a gradual recruitment of previously non.~ccreting cells with mcrca~ o i the cxtraccllular g l u c t ~ o~nccntrat~m ~44] H~'cver. it ~till remains to iv.. cluodatcd ht~A' the sudden transitions between II'acse three ~tatc.~ translate into the oa-ordinated slow oscillatkms of [Ca:" ], in the intact islet. Cycl~ variations of circulating insulin require a s)T~hronizatam of the [Ca ~ +], cycles also among the islets in the pancreas+ It is sill! an open que.~ti,m b) which means dlc millions t~ i~let5 c(wnmumcatc mutually. to c~ablish a pattern ol pul~atilc in~uhn release from the whole pancreas+ The di.~overy that the /~-cell is rK~t only the tunelionel unit folr insulin wnthe~s but also generates ti~

3(¼ [Ca ~+], oscillalions required for pulsatile insuhn re;ca~ ha.,, both physiological and clinical implications. The fact that lninor dz~mage to the fl-cclls prcvcnts the type-a o~illations v, iih maintcnancc of a gluc(:~;c reslx)nse in lerms of rated [('a:'], reinforces previou,, arguments [54] that hP;s of insulin tP,cillations is an early indicator of type-2 diabetes. Furthcr analyses of Ihc tea :+ ], oscillations in the /~.cells should include not only lhe mechanisms flit their generalion and sub~quent propagalion within or among Ihe islets hut al~) how m(~lulation of their frequency affects the insulin ~nsitivity of various target cells. The ialler approach may I~ important in the attempt~ to maintain normoglycemia under conditions minimizing the va~ular offeels of insulin suppo~d to precipitate hypcrUmia and alherosclerosi:, [7~).71.77]. Atknosdt'dti~ments

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