51 Covenas, R. etaL (1990) NeuroscL Lett. 114, 160-166 52 Day, T. A. (1989) Prog, Brain Res. 81,303-317 53 Raby, W. N. and Renaud, L. P. (1989) Prog. Brain Res. 81, 319-327 54 Gribkoff, V. K. and Dudek, F. E. (1988) Brain Res. 442, 152-156 55 van den Pol, A. N., Wuarin, J-P. and Dudek, F. E. (1990) Science 250, 1276-1278 56 Meeker, R. B., Swanson, D. J. and Hayward, J. N. (1989) Neuroscience 33, 157-167 57 Sawchenko, P. E., Swanson, L. W. and Steinbusch, H. W. M. (1983) Brain Res. 225, 249-269 58 Sawchenko, P. E. etal. (1988) Nature 344, 615-617 59 Sawchenko, P. E., Arias, C. and Bittencourt, J. C. (1990) J. Comp. Neurol. 291,269-280 60 Jhamandas, J. H. and Renaud, L. P. (1987) Can. J. NeuroL Sci. 14, 17-24 61 Norgren, R. (1984) in The Nervous System: Sensory Processes (Mountcastle, V. B., section ed., Bloom, F. E., volume ed.), pp. 1087-1124, American Physiological Society 62 De Groat, W. C. (1986) Prog. Brain Res. 67, 165-187 63 Tasker, J. G., Theodosis, D. T. and Poulain, D. A. (1986) Neuroscience 19, 495-509 64 Poulain, D. A. and Wakerley, J. B. (1986) Neuroscience 19, 511-521 65 Menetrey, D., Roudier, F. and Besson, J. M. (1983) J. Comp. Neurol. 220, 439-452

66 Menetrey, D. and Basbaum, A. I. (1987) J. Comp. Neurol. 255, 439-450 67 Leranth, C., Zaborszky, L., Matron, J. and Palkovits, M. (1975) Exp. Brain Res. 22, 509-524 68 Zaborszky, L., Leranth, C., Makara, G. B. and Palkovits, M. (1975) Exp. Brain Res. 22,525-540 69 Richard, P., Moos, F. and Freund-Mercier, M-J. (1991) Physiol. Rev. 71,331-370 70 Kawata, M., McCabe, J. T., Harrington, C., Chikaraishi, D. and Pfaff, D. W. (1988) J. Comp. NeuroL 270, 528-536 71 Theodosis, D. T. and Poulain, D. A. (1987) Trends Neurosci. 10, 426-430 72 Theodosis, D. T., Poulain, D. A. and Vincent, J. D. (1989) Brain Res. 484, 361-366 73 Brownstein, M. J. and Mezey, E. (1986) Prog. Brain Res. 68, 161-168 74 H6kfelt, T. et al. (1989) Acta Physiol. Scand. (Suppl.) 583, 105-111 75 Meister, B., Villar, M. J., Ceccatelli, S. and H6kfelt, T. (1990) Neuroscience 37, 603-633 76 Meister, B., Cortes, R., Villar, M. J., Schalling, M. and H6kfelt, T. (1990) Cell Tissue Res. 260, 279-297 77 Bicknell, R. J. (1985) J. EndocrinoL 107, 437-446 78 Bondy, C, A., Gainer, H. and Russell, J. T. (1988) Endocrinology 122, 1321-1327 79 Bondy, C. A., Jensen, R. T., Brady, L. S. and Gainer, H. (1989) Proc. Natl Acad. Sci. USA 86, 5198-5201

Acknowl~n~ We thank Ors

D.F.Hanley, 5. L. Lighlman and L. P. Renaud for commenting on an eady versionof the manuscript. We also thank Mr J. Simon for illustrating Figs I and 4, and Mr K. Trulock and Mrs B. Wamsley for photographic and secretarialassistance, respectively. Our work describedhere was supported by NIH grant HL-35137 and the Clayton Foundation for Research.

PancreaticB cellsare burs#n#,but how? D a n i e l L. C o o k , Leslie S. Satin a n d W i l l i a m F. H o p k i n s

Insulin secretogogues have long been known to stimulate and modulate bursting electrical activity in pancreatic islet B cells and thereby supply extracellular Ca 2+ for the exocytosis of insulin. Recent results have ruled out a long-held hypothesis for the mechanism of burst formation that postulated key roles for intracellular Ca 2+ accumulation and activation of Ca 2+activated K + channels. Here, we present an alternative hypotheses based on a persistent Ca 2+ conductance and, possibly, phasic activation of ATP-sensitive K + channels. These hypotheses are compared with mechanisms of bursting proposed for invertebrate and mammalian neurons. When B cells of the endocrine pancreas are stimulated with glucose to secrete insulin, they display a complex pattern of bursting-membrane electrical activity (for review, see Ref. 1). The bursts correlate with insulin secretion from single islets and appear to be obligatory for providing Ca2+ for the exocytosis of insulin (for review, see Ref. 2). The burst pattern resembles that seen in certain molluscan and mammalian neurons and may, therefore, have a similar electrophysiological origin. Despite years of investigation, the ionic mechanisms underlying B-cell bursting have been a mystery. Recent results, however, have now ruled out a long-standing hypothesis based on accumulation of intracellular Ca2÷ and activation of high conductance Ca2+-activated K + channels [IBK(Ca)channels]. Here, we discuss an alternative explanation for B-cell bursting a that has received support from recent descriptions of a very slowly inactivating component of voltage-dependent Caz+ current. Glucose stimulation of B-cell electrical activity occurs in two stages 1. First, as extracellular glucose TINS, Vol. 14, No. 9, 1991

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increases from zero to a threshold level (about 5 naM; which is roughly a normal plasma level in most species), B cells depolarize from about -75 to - 6 0 mV. At higher glucose levels, bursts of electrical activity are triggered, each beginning with a rapid depolarization and soon reaching a 'plateau' potential (about - 4 0 mV) from which Ca2+ action potentials are triggered (Fig. 1). The initial Caz+ spike frequency is about 10-12 Hz, but decreases to a spike-free interval and a slow repolarization until a hyperpolarized silent phase is reached near - 6 0 mV. During the silent phase there is a slow ramp of depolarization back to a threshold membrane potential, which triggers the next burst. As the glucose level increases, the durations of the plateaux increase while the silent periods become briefer. At a level of glucose (10-15 rnM) where insulin secretory rate is hag-maximal, the plateau and silent phases have nearly equal durations (5-10 s), so that the membrane is depolarized and spiking about half the time. At high levels of glucose (20-25 mM), the silent phases are completely eclipsed and spiking is continuous. Throughout this range of glucose dosage, there is no change in the silent and plateau phase membrane potentials, and no change in the amplitude or shape of Ca2+ spikes. Early studies demonstrated that both depolarization and burst modulation (i.e. changes in patterns of bursting) depended on glucose metabolism and were associated with decreasing membrane K + permeability1. The key link between K ÷ permeability and glucose metabolism was discovered to be the ATP-sensitive K + channel [IK(ATP)channel], which is blocked by ATP (Ref. 4) in cell-free patches and by the metabolism of glucose5,6 in intact cells. These channels are highly selective for K ÷ ions and are

1991, ElsevierSciencePublishersLtd, (UK) 0166- 2236/91/$02.00

Daniel L. Cook, Leslie S. 5ab'nand William F. Hopkins are in the Division of Metabolism (151), Seattle VA Medical Center, 1660South Columbian Way, Seattle, WA 98108, and the Dept of Physiologyand Biophysics, University of WashinEton, Seattle, WA 98195, USA.

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autonomous biochemical oscillator. Rather, they demonstrated the importance of voltage-dependent processes in plateau generation and suggested the possibility that plateaux are generated by voltagedependent, persistent inward currents as in molluscan bursting neurons n. This latter possibility was not, however, considered in early mathematical models 12 of B-cell 5s bursting in which bursts of Ca 2+ action potentials were terminated solely by the accumulation of intracellular free Ca 2+ and activation of IBK(Ca)channels. In Fig. 1. Glucose-induced bursting electrical activity recorded with a highresistance glass microelectrode from an isolated mouse islet of Langerhans this model, similar to a model for molluscan perifused with 11.1 mM glucose at 37°C (Cook D.L., unpublished obser- neurons 13, spike-associated Ca 2+ influx accumulates vations). Rhythmic 'plateau' potentials trigger rapid (..-40 ms duration) Ca2+ and activates InK(Ca) channels to produce an outward, hyperpolarizing current that increases until the spikes action potentials ('spikes'). cease and the burst terminates. During the spike-free silent phase, the net efflux, or sequestration of free Ca 2+ reverses the activation of IBm(Ca), allowing the blocked by a variety of cytoplasmic metabolites (for cell to depolarize slowly to trigger another burst of review, see Ref. 7). The specific block of IK(ATP~ spikes. In support of this model, IBK(Ca) channels channels by antidiabetic sulfonylurea compounds was were described in B cells 14 and shown to be sensitive used to show that tolbutamide depolarizes B cells and to K + channel blockers that affect electrical activity triggers electrical activity in the absence of glucose s'9, and insulin secretion (for review, see Ref. 15). while at high glucose levels (>10 raM), it increases electrical activity in a manner that mimics glucose IBK(Ca)channels do not underlie the bursting stimulation 8'9. These findings support a role for oscillation Recent evidence, however, appears to have ruled IK(ATP) channels in both the stimulation and modulation of plateaus, but do not address the ionic out two key features of this Ca2+-accumulation mechanisms underlying the formation of the plateaux IBn(Ca)-activation hypothesis. The first feature - that slow accumulation of intracellular free Ca 2÷ during the themselves. plateau phase activates Inn(ca) channels - appears not to be the case. Recently, Valdeoknillos and coPlateaux are voltage dependent A possible plateau mechanism, suggested by the workers 16 used the Ca2+-sensitive dye Indo-1 to demonstrated link between metabolism and mem- measure intracellular free Ca 2+ concentration, while brane depolarization, is that plateau waves are driven simultaneously recording membrane potentials in entirely by oscillations of glycolytic metabolism. To intact mouse islets. They found that the intracellular test this, we used electrical field stimulation 1° of free Ca 2+ level rose rapidly to reach a steady level single intact islets, while recording from a B cell after the first few spikes in the plateau and there was within the islet (see Fig. 2). Plateaux could be no progressive rise that could serve directly as a prematurely triggered in an all-or-none manner by plateau-pacing signal. depolarizing stimuli above a certain threshold, while The other critical feature - that InK(Ca) channels are suprathreshold hyperpolarizing stimuli triggered active and capable of modulating B-cell bursting - is regenerative plateau repolarization. In both cases, the also questionable. Although B-cell IBn(Ca) channels are plateau rhythm was reset. These findings ruled out activated only at positive membrane potentials ~7, the possibility that plateau waves are driven by an even infrequent, brief openings of these highconductance I~I~(Ca) channels could be important. This possibility appears to be ruled out by three recent studies 18-2° showing that 1 mM tetraethylammonium A (TEA) nearly completely blocks IBK(Ca)but not IK(ATP) or delayed rectifier K + channels (IKv channels) in B cells. This dose of TEA, when applied to intact mouse islets 18'21, increased Ca 2+ spike amplitude, but did not affect the durations of either the plateau or silent phases. Furthermore, higher levels of TEA (4 or 20 B mM) had no effect on the B-cell resting membrane potential in the absence of glucose 21, indicating that IBK(Ca) channels do not contribute to the resting K + 1~ conductance. More recently, Kukuljan et al. 22 have shown that doses of charybdotoxin that specifically Fig. 2. Microelectrode recording from a single mouse islet held on a suction block IBK(Ca), but not IK(ATP) or IKV, channels in B electrode, which was used to pass field-stimulation currents to the entire islet cells, also have no effect on the burst pattern in either (in the presence of 11.1 m/v glucose). (,6,) Endogenous bursting electrical cultured rat islets or microdissected mouse islets. activity is unaffected by a field-stimulation current of 3 #A (at the gray bar). (It) A slightly stronger current (4 I~A) triggers an all-or-none, full-length These studies appear to rule out the possibility that plateau potential and completely resets the plateau rhythm. The inset the formation or modulation of the burst pattern (different cell and scales) shows that a hyperpolarizing current pulse (at arrow) depends on IBK(Ca) channels. While these channels prematurely triggers the regenerative repolarization of a plateau and resets the may contribute to Ca 2+ spike repolarization 23, even plateau rhythm. this is now disputed 22'24.

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0mV -100 mV

frgl~distinct.,l~kl~ilations of Caz+ channels or from a single type of Ca7:~ channel remains to be determined.

Plateau depolarizations may depend on the persistent Ca 2+ current The finding of a substantial portion of inward ~ current that inactivates on the timescale of a plateau depolarization provides an essential element of an s alternative model of B-cell bursting (see Fig. 4). The . . . . 60 initial depolarization to the plateau and the first spike / _ ~_, , , _ , is suggested to be due to activation of both com~ ~ / p°nents °f Ca2+ current" The spikes are rep°larized by a combination of rapid, Ca2+-dependent Ca2+ current inactivation 28'29 and, possibly, voltagedependent activation of IBK(Ca) channels and IKv current (Ref. 23). During the persistent plateau depolarization, the slow component of the Ca2+ . . ~ .......................................................................................... current inactivates by a voltage-dependent mechanism and reduces the depolarizing drive for spiking until spiking slows and ceases. Following this, the plateau Fig. :3. Whole-cell, voltage-clamped Ca2+ current in an repolarizes regeneratively to a level determined isolated, cultured mouse pancreatic B cell held at -100 largely by background IK¢ATP) and residual Ca2+ mV and then stepped up to 0 mV. The data were recorded in the presence of tetrodotoxin (to block Na + current) and conductances. As the Ca2+ current slowly recovers a variety of K + channel blockers (redrawn from Ref. 24). .from inactivation, it reactivates to produce a depolarThe dotted line indicates the linearly extrapolated leak izing current that slowly increases and produces the current for this cell. The inset shows the leak-subtracted slow silent phase depolarization. This reaches a peak current-voltage relationship for the Ca2+ current regenerative threshold that initiates the next plateau. averaged from six mouse B cells that had their membrane In this formulation, the inactivation of inward Ca2+ potentials stepped up from - 1 0 0 mV to test potentials current plays the role of 'pacemaker' current in the - 6 0 to +60 mY. same way that activation of outward IBK(Ca) is the pacemaker in the previous model. Mathematical models based on such currents 3°'31 reproduce the B cells have a persistent Ca 2+ conductance plateau and spike pattern and so demonstrate the What other voltage-dependent mechanisms could feasibility of these concepts. generate the plateaus? Following the field-stimulation There are two essential features in this new model. experiments 1° shown in Fig. 2, we have pursued the First, the plateau depolarization depends on a perpossibility that B cells possess a voltage-dependent, sistent inward current. Such persistent currents slowly inactivating inward current that could contrib- have been features of bursting neuronal models ever ute to the plateau depolarization 3. To this end, we since Ca2+ currents in molluscan neurons were have characterized Ca2+ currents in three kinds of described 13'3~. More recently, bursting in oscillatory insulin-secreting cells - neonatal rat B cells2~, an mammalian central neurons has been associated with insulin-secreting cell line (HIT cells) 26 and adult persistent Na + conductances 33,34 that operate with mouse B cells27 - and have found substantially the contributions from a variety of Ca2+-dependent and same results. The first studies 28'29 described a Ca2+-independent conductances 34-37. The second rapidly activating (in about 10 ms) Ca2+ current, feature we are proposing is that burst termination is a which then inactivated in a Ca2+-dependent manner slow, voltage-dependent process and does not and left a residual current after 200 ms. To examine depend directly on intracellular Ca2+ accumulation. In the residual current, we used long (10 s; which is this way the model differs from the previous B-cell about the duration of a plateau), depolarizing voltage- model, and from models of molluscan neurons where clamp commands and fitted the timecourse of current intracellular Ca2+ accumulation either activates decay to bi-exponential equations. We found (Fig. 3) IBK(Ca) channels 13 or inactivates Ca2+ channels 32. that most of the residual Ca2+ current decayed very slowly (time . F - ~ Slowing of spiking and slow constant - 2 . 7 5 s at 0 mV) and !epolarization due to voltage-dependent after ten seconds only about 10% reactivation of Ca2+ current ,i,I . ,. ~ Recovery from of peak current was left. Unlike \ [ 4 I inactivation and rapid inactivation, slow inactivation ! reactivation of was seen when Ba 2+ was the charge carrier, and was independent of Ca2+ influx. Significant slow inactivation occurred at depolarizations well below the threshold for activating Ca2+ cur--1 Regenerative activation ~ Regenerative deactivation rent, and at positive potentials of Ca2+current I - Jof residual Ca2+=current (e.g. +100 mV) where Ca2+ influx is expected to be minimal. Whether Fig. 4. Diagram showing four essential events proposed to explain pancreatic B-cell bursting these current components arise electrical activity (see text).

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Why have slow plateau depolarizations? It may be useful to think of the B-cell bursting as a cascade of coupled oscillators where high frequency (10-12 Hz) oscillations of all-or-none spikes are driven by a lower-frequency (1--6 per minute), all-ornone plateau oscillator. At lower frequencies, there are also 'slow oscillations' of electrical activity that are nearly sinusoidalwith a period of about five minutes 38'39, and (it is assumed) variations of electrical activity and insulin release that are related to eating-fasting cycles and that are hours long. In this cascade, the bi-stable plateau oscillator may serve to couple 'analogue' metabolic signals to triggering the all-or-none Ca2+ spikes. Two advantages may follow from this. (1) The plateau mechanism can extend the frequency range for Ca2+ spiking. Given the shortlasting currents associated with a single spike (20-50 ms), it is difficult to envision a repetitive spike mechanism that can stably produce interspike intervals of more than a few seconds. By driving the spikes with a slower, bi-stable plateau oscillator having a natural period of 5-10 s, spike frequency can be smoothly modulated from 10 Hz to only a few spikes per minute. (2) A voltage-dependent, bi-stable oscillator can be extremely sensitive to underlying bias current such as that carried by IK(ATP) channels. This has been demonstrated theoretically in a B-cell model (which used IBK(Ca) channels as the plateau pacemaker), where modulating only 7 pS of background IK(ATP) channel activity controlled the gating of 7000 pS of total Ca2+ conductance and, thereby, converted brief and infrequent plateaus to continuous spiking4°. Thus, small changes of a background conductance can be smoothly 'transduced' into changes of membrane electrical activity and Ca 2+ uptake. This exquisite sensitivity to IK(ATP)channel activity suggests the possibility that the five-minute sinusoidal oscillations of electrical activity are due to slow metabolic oscillations seen in B cells41. It further suggests that oscillations of submembrane metabolite levels may be linked to spiking activity and may, therefore, play a direct role in pacing plateaux. For instance, Henquin has suggested that ATP hydrolysis by Caz+- or Na+-K+-dependent ATPases could link cation influx to IK(ATP) channel activation by submembrane ATP depletion and ADP production42. Such oscillations might also occur if elevated intracellular Ca2+ accumulation inhibits rnitochonddal ATP production from ADI~3. Thus, it may be possible that oscillations of IK(ATP) channel activity may contribute additional 'pacemaking' current to control the switching on and off of the slow plateau Ca2+ current. Experimental tests of these ideas may depend on the difficult task of demonstrating changes in submembrane levels of ATP, or other IK(ATe)-regulating metabolites. It may also be that the remaining 10% of the Ca2+ current not inactivated after 10 s has activation and inactivation kinetics that could also contribute to plateau pacing. Recent progress in B-cell electrophysiology can be summarized as follows. (1) B-cell IK(ATP)channels are now accepted as a key link between glucose metabolism and stimulation of electrical activity. (2) Experimental evidence has ruled out IBK(ca) channels and intraceUular Ca2+ accumulation as important for creating or modulating B-cell plateau potentialS. (3) The 414

mechanism of plateau formation is being unraveled in terms of persistent Ca 2+ currents, with a possible contribution from phasic activation of KATe channels. It will be important to determine whether the slow Ca2+ conductance described here is subject to metabolic regulation via glycolytic metabolism or other second messenger systems in B cells.

Selected references 1 Henquin, J. C. and Meissner, H. P. (1984) Experientia 40, 1043-1052 2 Wollheim, C. B. and Pralong, W. F. (1990) Biochem. Soc. Trans. 18, 111-114 3 Cook, D. L. (1984) Fed. Proc. 43, 2368-2372 4 Cook, D. L. and Hales, C. N. (1984) Nature 311,271-273 5 Ashcroft, F. M., Harrison, D. E. and Ashcroff, S. J. (1984) Nature 312, 446-448 6 Rorsman, P. and Trube, G. (1985) Pfl6gers Arch. 405, 305-309 7 Ashcroff, F. M. (1988) Annu. Rev. Neurosci. 11, 97-118 8 Henquin, J. C. (1988) Biochem. Biophys. Res. Commun. 156, 769-775 9 Cook, D. L. and Ikeuchi, M. (1989) Diabetes 38, 416-421 10 Cook, D. L., Crill, W. E. and Porte, D. J. (1980) Nature 286, 404--406 11 Eckert, R. and Lux, H. D. (1976)J. Physiol. 254, 129-151 12 Chay, T. R. and Keizer, J. (1983) Biophys. J. 42, 181-190 13 Gorman, A. L. F., Hermann, A. and Thomas, M. V. (1982) J. Physiol. 327, 185-217 14 Cook, D. L., Ikeuchi, M. and Fujimoto, W. Y. (1984) Nature 311,269-271 15 Rajan, A. S. et aL (1990) Diabetes Care 13, 340-363 16 Valdeolmillos, M., Santos, R. M., Contreras, D., Soria, B. and Rosario, L. M. (1989) FEB5 Lett. 259, 19-23 17 Tabcharani, J. A. and Misler, S. (1989) Biochim. Biophys. Acta 982, 62-72 18 Henquin, J. C. (1990) PflEIgersArch. 416, 568-572 19 Bokvist, K., Rorsman, P. and Smith, P. A. (1990) J. Physiol. 423, 327-342 20 Fatherazi, S. and Cook, D. L. (1991) J, Membr. BioL 120, 105-114 21 Atwater, I., Ribalet, B. and Rojas, E. (1979) J. Physiol. 288, 561-574 22 Kukuljan, M., Goncalves, A. A. and Atwater, I. (1991) J. Membr. BioL 119, 187-195 23 Satin, L. S., Hopkins, W. F., Fatherazi, S. and Cook, D. L. (1989) J. Membr. Biol. 112,213-222 24 Smith, P. A., Bokvist, K., Arkhammar, P., Berggren, P. O. and Rorsman, P. (1990) J. Gen. PhysioL 95, 1041-1059 25 Satin, L. S. and Cook, D. L. (1988) PfliJgers Arch. 411, 401--409 26 Satin, L. S. and Cook, D. L. (1989) Pfl(JgersArch. 414, 1-10 27 Hopkins, W. F., Satin, L. S. and Cook, D. L. (1991) J. Membr. Biol. 119, 229-239 28 Satin, L. S. and Cook, D. L. (1985) Pfl(Jgers Arch. 404, 385-387 29 Plant, T. D. (1988) J. PhysioL 404, 731-747 30 Chay, T. R. (1990) J. Theor. BioL 142, 305-315 31 Keizer, J. and Smollen, P. (1991) Proc. NatlAcad. Sci. USA 88, 3897-3901 32 Kramer, R. H. and Zucker, R. S. (1985) J. PhysioL 362, 131-160 33 Stafstrom, C. E., Schwindt, P. C., Chubb, M. C. and Crill, W. E. (1985)J. Neurophysiol. 53, 153-170 34 Llinas, R. R. (1988) Science 242, 1654-1664 35 McCormick, D. A. and Pape, H-C. (1990) J. PhysioL 431, 291-318 36 Silva, L. R., Amitai, Y. and Connors, B. W. (1991) Science 251,432-435 37 Kiehn, O. (1991) Trends Neurosci. 14, 68-73 38 Henquin, J. C., Meissner, H. P. and Schmeer, W. (1982) PfliJgers Arch. 393, 322-327 39 Cook, D. L. (1983) Metabolism 32,681-685 40 Himmel, D. M. and Chay, T. R. (1987) Biophys. J. 51,89-107 41 Longo, E. A. etal. (1991)J. Biol. Chem. 266, 9314-9319 42 Henquin, J. C. (1990) Diabetes 39, 1457-1460 43 Keizer, J. and Magnus, G. (1989) Biophys. J. 56, 229-242

TINS, VoL 14, No. 9, 1991

Pancreatic B cells are bursting, but how?

Insulin secretogogues have long been known to stimulate and modulate bursting electrical activity in pancreatic islet B cells and thereby supply extra...
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