Bursting and Calcium Oscillations in Pancreatic Beta-Cells - Specific Pacemakers For Specific Mechanisms

Bursting and calcium oscillations in pancreatic -cells: specific pacemakers for specific mechanisms
L. E. Fridlyand, N. Tamarina and L. H. Philipson

Am J Physiol Endocrinol Metab 299:E517-E532, 2010. First published 13 July 2010; doi:10.1152/ajpendo.00177.2010 You might find this additional info useful... Supplemental material for this article can be found at: http://ajpendo.physiology.org/content/suppl/2010/08/09/ajpendo.00177.2010.DC1.html This article cites 150 articles, 78 of which can be accessed free at: http://ajpendo.physiology.org/content/299/4/E517.full.html#ref-list-1 This article has been cited by 1 other HighWire hosted articles Ionic mechanisms and Ca2+ dynamics underlying the glucose response of pancreatic cells: a simulation study Chae Young Cha, Yasuhiko Nakamura, Yukiko Himeno, JianWu Wang, Shinpei Fujimoto, Nobuya Inagaki, Yung E Earm and Akinori Noma J Gen Physiol, July , 2011; 138 (1): 21-37. [Abstract] [Full Text] [PDF] Updated information and services including high resolution figures, can be found at: http://ajpendo.physiology.org/content/299/4/E517.full.html Additional material and information about AJP - Endocrinology and Metabolism can be found at: http://www.the-aps.org/publications/ajpendo
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Am J Physiol Endocrinol Metab 299: E517E532, 2010. First published July 13, 2010; doi:10.1152/ajpendo.00177.2010.
Review
Bursting and calcium oscillations in pancreatic -cells: specic pacemakers for specic mechanisms
L. E. Fridlyand, N. Tamarina, and L. H. Philipson
Department of Medicine, University of Chicago, Chicago, Illinois
Submitted 18 March 2010; accepted in nal form 9 July 2010
Fridlyand LE, Tamarina N, Philipson LH. Bursting and calcium oscillations in pancreatic -cells: specic pacemakers for specic mechanisms. Am J Physiol Endocrinol Metab 299: E517E532, 2010. First published July 13, 2010; doi:10.1152/ajpendo.00177.2010.Oscillatory phenomenon in electrical activity and cytoplasmic calcium concentration in response to glucose are intimately connected to multiple key aspects of pancreatic -cell physiology. However, there is no single model for oscillatory mechanisms in these cells. We set out to identify possible pacemaker candidates for burst activity and cytoplasmic Ca2 oscillations in these cells by analyzing published hypotheses, their corresponding mathematical models, and relevant experimental data. We found that although no single pacemaker can account for the variety of oscillatory phenomena in -cells, at least several separate mechanisms can underlie specic kinds of oscillations. According to our analysis, slowly activating Ca2 -sensitive K channels can be responsible for very fast Ca2 oscillations; changes in the ATP/ADP ratio and in the endoplasmic reticulum calcium concentration can be pacemakers for both fast bursts and cytoplasmic calcium oscillations, and cyclical cytoplasmic Na changes may underlie patterning of slow calcium oscillations. However, these mechanisms still lack direct conrmation, and their potential interactions raises new issues. Further studies supported by improved mathematical models are necessary to understand oscillatory phenomena in -cell physiology. endoplasmic reticulum; channels; diabetes; mathematical model; metabolism
IN PANCREATIC -CELLS, glucose-stimulated insulin secretion is mediated by an elevated cytosolic free calcium concentration ([Ca2 ]c). This results from calcium inux through voltagedependent Ca2 channels (VDCCs) located in the plasma membrane (PM), which open in response to secretagogues, primarily glucose. Stimulation-secretion coupling in -cells is different from most other cell types, because instead of being mediated by receptor binding, glucose must be transported into the cytoplasm and metabolized. Glucose initiates changes in the PM potential via an increase in the cytoplasmic [ATP]/[ADP] ratio derived from glycolysis and oxidative phosphorylation. This results in closure of ATPsensitive K (KATP) channels. Closure of these channels leads to PM depolarization up to a threshold potential, causing the cell to move from quiescence to initiate electrical activity and VDCC opening. Ca2 inux through VDCCs leads to increased [Ca2 ]c, which is a key signal in the initiation of insulin secretion from the pancreatic -cells. Consensus mechanisms of glucose-induced PM, cytosolic, and mitochondrial processes are summarized in Fig. 1 (for recent review, see Refs. 71, 74, 76, and 140). The -cell membrane is hyperpolarized to a resting potential of about 60 mV at low glucose levels ( 3 6 mM). Dean and Matthews (32, 33) showed that when glucose rises the PM
Address for reprint requests and other correspondence: L. E. Fridlyand, Dept. of Medicine, MC-1027, Univ. of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637 (e-mail: lfridlia@medicine.bsd.uchicago.edu). http://www.ajpendo.org
depolarizes and then generates an electrical activity organized into slow depolarizing waves, called bursting, with a plateau from which action potentials (APs) rapidly re. Bursts (or active phases) are separated by quiescent (resting) periods at potentials below the AP threshold (Fig. 2). This bursting process results from the metabolic processes and electrical activity of ion channels localized on the -cell PM and regenerates as long as the glucose concentration is elevated. Rapid depolarization at the beginning of a burst activates VDCCs, whereas the fast repolarization at the end of a burst results in inactivation of VDCCs. This leads to synchronized cyclical spike-burst activity and corresponding cytoplasmic Ca2 oscillations in response to a rise in extracellular glucose that ultimately determine the oscillatory nature of insulin secretion (71, 76, 140). Increased glucose concentration induces several types of cyclical spike-burst activity and [Ca2 ]c oscillations in -cells with period ranges from several seconds to several minutes (11, 19, 71). We have divided these oscillations into several classes: 1) bursts and [Ca2 ]c oscillations with a period of several seconds we term ultrafast; 2) oscillations with a period from 10 to 60 s, denoted as fast (Fig. 2A); 3) Ca2 oscillations with a period ranging from 1 to several minutes and long bursts (Fig. 2B), designated as slow; and 4) mixed (or compound) [Ca2 ]c oscillations, characterized by ultrafast or fast [Ca2 ]c oscillations superposed on slow oscillations; this is due to periodic bursts, called compound bursting (Fig. 2, C and D) (17).
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BURSTING AND CALCIUM OSCILLATIONS IN -CELLS
Fig. 1. General scheme of the main processes involved in bursting and intracellular Ca2 oscillations in pancreatic -cells. Plasma membrane currents: voltage-dependent Ca2 current (IVCa), calcium pump current (ICap), Na /Ca2 exchange current (INaCa), Ca2 release-activated nonselective cation current (ISOC), inward Na currents (INa), sodium-potassium pump current (INaK), slow Ca2 -sensitive K current (IKCas), delayed rectifying K current (IKDr), small conductance Ca2 -activated K current (IKCa), ATP-sensitive K current (IKATp). ksg is a coefcient of the sequestration rate of Ca2 by the secretory granules. SERCA is a calcium pump in endoplasmic reticulum (ER), and Ca2 leaks from ER throw into inositol 1,4,5-triphosphate (IP3) receptor (IP3R) and ryanodine receptor (RyaR). ATP is the free cytosolic form of ATP, and ADPf is free cytosolic ADP. Signals originating from fuel metabolism increase cytosolic calcium. F6P, fructose 6-phosphate. Synthesis and degradation of cAMP are catalyzed by adenylyl cyclase (AC) and phosphodiesterase (PDE), respectively. AC was activated by stimulatory G protein and Ca2 and deactivated by inhibited G protein, whereas PDE activity was enhanced by Ca2 . Synthesis of IP3 is catalyzed by phospholipase C (PLC). PLC was activated by stimulatory G protein. Solid lines indicate uxes, and dashed lines indicate inhibitory or stimulatory inuences on currents or uxes.
Ultrafast oscillations can be observed in isolated single -cells (91, 150). Fast electrical bursting and cytoplasmic Ca2 oscillations are usually observed in isolated mouse islets (11, 57). Slow and mixed cytoplasmic Ca2 oscillations were found in single cells, -cells clusters, and isolated islets at stimulating glucose concentrations (10, 11, 57, 70, 99). Slow and mixed oscillations most likely constitute a physiological oscillatory pattern in -cells (57, 140). Gap junctions are responsible for tight electrical coupling at least in mouse islets. This leads to the synchronization of bursting and calcium oscillations in an islet (12, 94, 144). For this reason, as a rst approximation we can consider the islet as a community of coupled -cells with similar electrical behavior. Experimental data and theoretical concepts support the idea that the AP bursts and the corresponding [Ca2 ]c oscillations reect a periodic depolarization of the PM (7, 11, 37, 48). Whereas a depolarizing component predominates at the beginning of a burst, a progressive increase in outward and/or decrease in inward cation currents take place during the burst, leading to a repolarization and burst termination. Slow depolarization in a resting (silent) phase leads again to a burst initiation (7, 48). Experimental data and theoretical analysis agree that, after the large depolarizing effect following KATP channel closure, the subsequent generation and termination of the bursts can be maintained by small cyclical changes of any PM current (9, 48, 132, 133). Consequently, initiation and termination of bursts can be connected directly with activation and deactivation of different
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electrogenic channels, pumps, or exchangers, which can be modulated in turn by numerous intracellular components. Therefore, it is not surprising that a variety of different proposals and corresponding mathematical models have been advanced, underlining the nature of these components in the -cells. A list of the compounds to be considered here is shown in Table 1. Several of these modulators can act simultaneously, generating AP bursts and corresponding [Ca2 ]c oscillations. However, the exact physiological variables and their underlying mechanisms that drive bursting or Ca2 oscillations in -cells remain uncertain. Here, we attempt to identify the possible pacemaker candidates for burst activity and cytoplasmic Ca2 oscillations in the pancreatic -cells. We dene a pacemaker as a component of some pacemaker mechanism that serves to establish a rhythmic physiological activity inside a cell. To distinguish likely pacemaker proposals from less likely hypotheses, we will reject a hypothesis that some component is a part of a pacemaker mechanism if Ca2 oscillations (or the AP bursting pattern) persist even though the proposed pacemaker mechanism was blocked in an experiment. We will term this condition the main criterion for the rejection of a proposed pacemaker mechanism. Disappearance or a change of bursting or Ca2 oscillation behavior under the inuence of an agent or process we will term a modulation, and an agent that creates it we will term a modulator. A pacemaker component can also act as a modulator with a certain concentration dependence, depending on its activity or experimental conditions. However, a modulator in this article is a component that is not a part of a proposed pacemaker
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cells or the electrically synchronized islet. A further consideration of the oscillations in insulin secretion is beyond the scope of the present article. However, islet Ca2 oscillations appear to be the framework behind the phenomenon of pulsatile insulin release observed in vivo (71, 112, 117, 140). Interestingly, disruptions in rhythmic function may be an important early biomarker of islet dysfunction leading to diabetes in animal models (72, 79, 140) and man (116). Although pulsatility appears to be a natural function of islets both in vivo and in vitro, this emphasizes the need for careful study of the oscillatory phenomena on the cellular level.
EXPERIMENTAL PROCEDURES AND MODELING
Ca2 imaging measurements of relative cytoplasmic Ca2 concentrations are presented as the 340/380 nm uorescence excitation ratio obtained with the indicator fura-2, as described previously (137). Fura-2 was from Invitrogen (Carlsbad, CA). All other chemicals were from Sigma (St. Louis, MO). The complex model of processes (51) was used for the following simulations, excluding the model for slowly activated Ca2 -sensitive K channels, that are described in the APPENDIX. This model is available for direct simulation on the web site Virtual Cell (www. nrcam.uchc.edu) in the MathModel Database on the math workspace in the library Fridlyand with name Chicago 1.
RESULTS AND DISCUSSION
Fig. 2. Temporal correlation between membrane potential (MP) and cytosolic free calcium concentration ([Ca2 ]c) oscillations (adapted from Ref. 11). Islets from wild mice were perifused with a solution containing 8 (C and D) or 10 mM glucose(A and B) as indicated. The MP was measured in a -cell within an islet, and [Ca2 ]c was measured simultaneously in the same islet.
We can distinguish two main types of mechanisms underlying bursting and [Ca2 ]c oscillations. Mechanisms With Ca2 Feedback Effect In this case, a fast increase of [Ca2 ]c during an active phase should activate (or deactivate) some processes, leading to slow changes in the intermediate pacemaker components. Slow changes of these components should gradually lead to a membrane repolarization down to the threshold potential for spike activity, shutting down the spikes and inactivating the VDCCs. Then Ca2 inux decreases sharply, leading to decreased PM potential and a resting phase. Processes occur in opposite directions during a resting (repolarized) phase. The [Ca2 ]c decreases because cytoplasmic Ca2 is pumped into the extracellular space and into the endoplasmic reticulum (ER) by specic ATP-consuming Ca2 pumps. Decreases in [Ca2 ]c deactivate (or activate) specic processes of the slow changes in the intermediate pacemaker components, leading to PM depolarization up to the threshold potential, reentry of spike activity, Ca2 inux, and an active phase. Table 1. Candidates for pacemakers regulating -cell bursting and [Ca2 ]c oscillations
Compound or Process Reference No(s).
mechanism. Several examples of such pacemakers and modulators will be considered. We performed an analysis of the existing hypothesis, experimental data, and the corresponding mathematical models and compared the simulations with the experimental results. In this way we tested both the validity of the initial hypotheses and the models. Furthermore, predictions for unmeasured quantities could be made, and the model could then be tested. However, many of the mechanisms that could control electrical activity and Ca2 handling have not been adequately experimentally characterized in pancreatic -cells, so we can consider them only briey. Although most of the analyzed experimental work employed mouse islets with some studies in other species, it is difcult to generalize given the different existing animal and insulinoma models of pancreatic -cells. We have emphasized primary tissues where possible. There are several reasons why a clear understanding of internal oscillatory mechanisms in -cells is worth pursuing. First, bursting and [Ca2 ]c oscillations are readily observable phenomena in cell culture and in isolated mouse islets. Numerous hormones and small molecule agonists as well as toxins can modulate oscillatory behavior. The study of the changes in bursting or [Ca2 ]c oscillations can provide a exible approach to understand the action of specic modulators on the pancreatic -cell. However, this approach requires a comprehensive knowledge of the pacemaker mechanisms of these oscillations. We consider here primarily the internal pacemaker mechanisms for individual -cells that represent behavior of isolated
Ca -dependent K current Slowly activated Ca2 -sensitive K current Voltage-gated Ca2 current [ATP]/[ADP] ratio Glycolytic oscillations [Ca2 ] in endoplasmic reticulum Intracellular Na Inositol (1,4,5)-trisphosphate cAMP Mitochondrial metabolic oscillations [Ca2 ]c, cytosolic free calcium concentration. www.ajpendo.org
8, 26 58, 59 28, 88 3, 126, 133 17, 19, 142, 145 20, 25, 51, 125, 148 51, 93 This article 47 14, 101
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Review E520 Ca2 -Independent Mechanisms This type of mechanism was suggested for oscillations such as glycolytic oscillations that can lead to oscillations in the [ATP]/[ADP] ratio changing the conductance of KATP channels (19, 142). In this case, Ca2 -independent mechanisms create the oscillations of compounds that can lead to cyclic changes in PM potential, generating the bursts and corresponding [Ca2 ]c oscillations (101, 142). [Ca2 ]c as the Pacemaker Component and Ca2 -Dependent K Channels Fast-activated Ca2 -dependent K (SK13 and BK) channels. Initially, a mechanism with [Ca2 ]c as the pacemaker component and a Ca2 feedback effect was proposed for burst generation and [Ca2 ]c oscillations via cyclic activation of a Ca2 -dependent K current. According to this hypothesis, Ca2 inux during the active phase would cause a slow rise in [Ca2 ]c, which activates fast Ca2 -dependent K (KCa) channels repolarizing the PM potential down to a threshold potential. This would shut down AP spiking and inactivate VDCCs, leading to a resting phase. A decrease in the [Ca2 ]c level during the resting period would lead to decreased current through KCa channels, PM depolarization, and burst initiation. According to this proposal, a cyclical change in [Ca2 ]c is a candidate islet pacemaker for burst behavior and [Ca2 ]c oscillations per se (8). Corresponding mathematical models have incorporated the cyclical activation and deactivation of KCa channels by bursting-induced changes in [Ca2 ]c (26). The principle difculty with this hypothesis is that [Ca2 ]c should slowly increase during a period of depolarization, leading to a slowly increasing current through KCa channels and then to a repolarization only at the end of a burst period. However, subsequent Ca2 imaging data indicate that the time for the [Ca2 ]c increase is short relative to the oscillation period (129). Another problem with this hypothesis was that apamin, a KCa (SK13) channel blocker, does not change mouse islet bursting or alter -cell K currents in normal mouse islets (4, 58, 59, 96). Slow [Ca2 ]c oscillations were also observed in the presence of 10 20 mM tetraethylammonium (TEA), i.e., in conditions when TEA-sensitive KCa channels should be nearly completely blocked (124, 125). For these reasons, this mechanism for generation of [Ca2 ]c oscillation was ruled out (see Refs. 125 and 132). Despite these issues, mathematical models for -cells continue to appear with slow [Ca2 ]c oscillations on the basis of this hypothesis (36, 108). However, although here we focus on pacemaker mechanisms, some changes in channel conductance may change PM polarization such that oscillation behavior can be modied. For this reason, although KCa (SK13 and BK) channels do not directly take part in the creation of the pacemaker mechanism, their inhibition can contribute to PM depolarization and lead to changes in the oscillatory behavior (139). Slow-activated Ca2 -sensitive K channels. A specic, slowly activated TEA-insensitive Ca2 -sensitive K current (SK-like current) may underlie the mechanisms of oscillations (58, 59). This current activates gradually with a time constant of 2.3 s and inactivates with a time constant of 6.5 s (59). It was assumed that this current regulates the duration of intervals between successive AP bursts observed when -cells are exposed to moderately elevated glucose (59, 151).
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We developed a special mathematical model to simulate this proposal (see APPENDIX). Here, fast KCa channels (SK13 and BK; current IKCa in Fig. 1) as well as slowly activated KCa channels (SK-like current; IKCas in Fig. 1), which according to Dfer et al. (38) can be represented partly by SK4 channels, were modeled, and the time constants for activation gating variables reported by Gopel et al. (59) were used for modeling of IKCas. The behavior of [Ca2 ]c, Vm, and the IKCas is shown in Fig. 3. The reversible slow cyclical changes in IKCas were obtained, leading to bursting behavior and [Ca2 ]c oscillations. The simulated [Ca2 ]c increased rapidly in the beginning of burst period, stabilized by activation of the fast KCa (SK13) channels and kept at a high level during this period. IKCas increased during a burst period, leading to repolarization, termination of the spikes, and transition to a silent phase. Inhibition of IKCas during a resting period led to repolarization and reentry to an active phase. However, the simulated bursts and [Ca2 ]c oscillations had a period of several seconds and corresponded well only to ultrafast bursting and [Ca2 ]c oscillations. Therefore, the experimental data on the dynamics of this slow SK-like current and our simulation support the idea that ultrafast bursts and Ca2 oscillations can be driven by an interaction of these slow-activated KCa channel parameters with [Ca2 ]c changes. Cyclic Changes in KATP Channel Conductance: The Pacemaker Role of [ATP]/[ADP] Ratios KATP channels contribute the predominant -cell K conductance in resting low-glucose conditions. Following glucose entry and metabolism, their conductance is greatly diminished. However, the small remaining KATP conductance in elevated glucose is still comparable with the conductances of other channels, exchangers, and pumps (133). For this reason, cyclical changes in the KATP channel conductance have been
Fig. 3. Slow-activated Ca2 -sensitive K channels as pacemaker for ultrafast burst and [Ca2 ]c oscillations. Burst behavior of the membrane potential (VP; A), the oscillation pattern of [Ca2 ]c (B), and slow Ca2 -sensitive K current (IKCas; C). Simulations were made according the model from the APPENDIX. www.ajpendo.org
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proposed as a possible mechanism underlying oscillatory behavior of -cells (3, 68, 110, 126, 133). In this conceptualization, the [ATP]/[ADP] ratio decreases slowly when [Ca2 ]c increases during a burst, leading to a slow opening of KATP channels and subsequent PM repolarization as the K efux increases. Actually, the opposite should occur since the [ATP]/ [ADP] ratio gradually increases during a silent phase in the presence of stimulatory glucose, when [Ca2 ]c decreases, leading to the closing of KATP channels, PM depolarization, and burst initiation (34, 86, 87, 103105, 126). According to our classication, this is a mechanism with a Ca2 feedback effect, whereas the [ATP]/[ADP] ratio is a pacemaker compound. Several mechanisms and corresponding mathematical models for the [ATP]/[ADP] ratio changes and [Ca2 ]c oscillations have been proposed. An empirical equation for [ATP]/[ADP] ratio changes was introduced in an early mathematical model (133) such that [ATP]/[ADP] ratio slowly decreased as [Ca2 ]c increased and increased with decreased [Ca2 ]c. Oscillatory changes in the [ATP]/[ADP] ratio, KATP channel conductance, bursting, and [Ca2 ]c oscillations were simulated using this model (17, 18, 133). Mechanistic hypotheses underlying the possible inuence of [Ca2 ]c changes on the [ATP]/[ADP] ratio have also been proposed. Decreased [ATP]/[ADP] ratio during [Ca2 ]c rise and an increase of this ratio with [Ca2 ]c decline could result from either stimulated ATP hydrolysis or inhibition of ATP production. [Ca2 ]c-activated ATP consumption. Ca2 activates Ca2 pumps on the PM and in the ER as well and certain other intracellular reactions that use ATP. In this case, increased [Ca2 ]c can increase ATP consumption and decrease the [ATP]/[ADP] ratio. An opposite process that increases the [ATP/ADP] ratio due to a decline in ATP consumption can also occur with decreased [Ca2 ]c (34, 51). We illustrated this mechanism using our mathematical model (51) that includes a simulation of ATP consumption due to the work of Ca2 pumps (PM and ER) as well as the Ca2 -activated ATP consumption in some cytoplasmic processes. The simulated phase relations for the conditions when the free ADP (and corresponding ATP) concentration in the cytoplasm is the only slow pacemaker parameter in this complex model are shown in Fig. 4. Other slow parameters such as cytoplasmic concentrations of Na , inositol 1,4,5-triphosphate (IP3), and Ca2 in ER ([Ca2 ]ER) were frozen at constant levels, blocking other possible mechanisms underlying Ca2 oscillations in this model. A Ca2 feedback effect model was responsible for bursting and Ca2 oscillations; a rise in [Ca2 ]c during the active phase leads to increased Ca2 -activated ATP consumption in the cytoplasm and by Ca2 pumps. As this occurs, free [ADP] increases (and the [ATP]/[ADP] ratio decreases), slowly opening KATP channels. This in turn leads to a slowly increasing IKATP and PM repolarization, damping potential spikes and leading to decreased [Ca2 ]c. These processes have the opposite direction after decreased [Ca2 ]c in the resting period. KCa channels serve to damp depolarization and terminate the [Ca2 ]c increase after initial Ca2 increase during the active phase. Negative [Ca2 ]c effects on ATP production. In several mathematical models the uptake of Ca2 by -cell mitochondria has large energy-dissipative effects, leading to decreased
Fig. 4. Slow ATP/ADP ratio changes as a pacemaker. Burst behavior of the MP and the oscillation patterns of [Ca2 ]c, free ADP ([ADP]), and IKATP are illustrated. It was simulated using a model (51) by a step increase of glucose level. Several variables, [Na ]c, [IP3], and [Ca2 ]ER, were frozen at constant levels to eliminate other mechanisms of fast and slow [Ca2 ]c oscillations ([Na ]c 7 mM, [IP3] 3 M, and [Ca2 ]ER 20 M). Several coefcients were set as gmSOC 2 pS 1 mV, gmKATP 200,000 pS, gmKCa 80 pS, gmVCa 710 pS, gNaCa 500 pS, kADP 0.001 ms, kATP 0.000001 ms, kATP,Ca 0.0000218 M/ms, and PNaK 400 fA. All other parameter settings are as in Ref. 51. A: action potential (VP). B: [Ca2 ]c. C: [ADP]. D: IKATP.
oxidative phosphorylation suppressing the production rate of ATP (15, 103105). This proposal also leads to a decreased [ATP]/[ADP] cytoplasmic ratio with increased [Ca2 ]c, as in rst case, and the models can simulate [Ca2 ]c oscillations through the cyclical changes in [ATP]/[ADP] ratio (103105). However, recent experimental data convincingly shows that the primary role of mitochondrial Ca2 is the stimulation of oxidative phosphorylation (24), suggesting that [Ca2 ]c effects on ATP production are stimulatory rather than inhibitory. Glycolytic oscillations as a mechanism for cyclical changes in the [ATP]/[ADP] ratio. A number of hypotheses have been proposed to explain the mechanism of [ATP]/[ADP] ratio oscillations through glycolytic oscillations. The fundamental proposal of this model is that Ca2 -independent glycolytic oscillations that produce [ATP]/[ADP] ratio oscillations are mediated by the positive feedback of fructose 1,6-bisphosphate. This is the product of the glycolytic enzyme phosphofructokinase (PFK), stimulating PFK activity and subsequently depleting substrate (83, 142). Mathematical models for -cell oscillations have been constructed on the basis of this mechanism (1719, 81, 145). However, the direct evidence for glycolytic oscillations in -cells such as measurements of glycolytic metabolite concentrations is lacking. Periodic variations in mitochondrial membrane potential, NADH, and respiration have been offered as evidence of glycolytic oscillations (18). However, these phewww.ajpendo.org
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nomena can also be a consequence of independent Ca2 cytoplasmic oscillations (see below). The available evidence supports the idea that glycolytic processes are not a requirement at least for slow cytoplasmic Ca2 oscillations. Slow [Ca2 ]c oscillations can be induced at low glucose levels using K and KATP channel blockers (such as TEA and sulfonylureas) in islets and -cell lines (see Fig. 5 and Refs. 51 and 125). Slow [Ca2 ]c oscillations can be generated even in the absence of glucose using such mitochondrial substrates as leucine (64), -ketoisocaproic acid (69, 106), and methyl pyruvate (69). Fast [Ca2 ]c oscillations were found in islets incubated solely in the presence of glyceraldehyde or -ketoisocaproic acid (31). PFK itself is not required for these oscillations, since [Ca2 ]c oscillations remain normal after 9598% suppression of PFK-M activity in mouse islets (121). These data demonstrate that an oscillatory capacity is preserved when glycolytic ux is small or absent and that the slow components of [Ca2 ]c oscillations cannot be fully explained by mechanisms incorporating glycolytic oscillations. However, we cannot entirely rule out that this mechanism could contribute to ultrafast (and possibly fast) [Ca2 ]c oscillations, although the metabolic variability is on the order of minutes. Further studies are needed to establish whether there is any role at all for glycolytic-based mechanisms in -cells regulating Ca2 or other oscillations as opposed to periodic changes in mitochondrial Ca2 or membrane potential. On other hand, glycolytic processes can modulate oscillation behavior. For example, bursting and Ca2 oscillations can change frequency and amplitude and even disappear with changes in glucose concentration (54). Limitations of KATP channel [ATP]/[ADP] ratio-dependent mechanisms of oscillations. The [ATP]/[ADP] ratio drops when [Ca2 ]c rises and increases when [Ca2 ]c falls, indirectly supporting the proposal that [Ca2 ]c oscillations can therefore evoke oscillations in [ATP]/[ADP] ratio and IKATP (2, 34). These data are in agreement with the hypothesis that the KATP channel conductance changes can generate and maintain oscillations. We have also considered above the mechanism for ultrafast oscillations determined by changes in the conductance of slow-activated Ca2 -sensitive K channels. Based on the effects of tolbutamide, a specic KATP channel blocker, it was concluded that KATP channels contribute a signicant fraction
(48%) of this mechanism (86). Therefore, we suggest that the mechanism underlying ultrafast bursts and [Ca2 ]c oscillations in -cells can be due partly to the changes in the [ATP]/[ADP] ratio as a pacemaker (and corresponding changes in KATP channel conductivity), in accord with the KATP channel-dependent mechanism. However, there are several valid arguments against a KATP channel-dependent mechanism for slow bursting and [Ca2 ]c oscillations. If KATP channels function as a pacemaker, the conductance of the KATP channels should oscillate during bursting electrical activity following changes in the [ATP]/ [ADP] ratio. However, effective KATP channel blockers such as sulfonylureas do not stop slow bursting and Ca2 oscillations in pancreatic islets and can even induce them in single -cells or -cell clusters isolated from mouse islets (see Fig. 5 and Refs. 43, 65, and 109) and in TC3-neo cells (125). Slow [Ca2 ]c oscillations were also found in a mouse lacking functional KATP channels (40, 67, 120, 135). These results suggest that changes in the [ATP]/[ADP] ratio and, consequently, in KATP channel conductivity are unlikely to function as a pacemaker component for coupled slow oscillatory mechanisms. However, studies are needed to carefully examine whether fast Ca2 oscillations persist upon blocking KATP channels at low glucose levels or in KATP channel-null mice. Until such studies are reported, it is possible that ultrafast and fast bursting and the corresponding [Ca2 ]c oscillations are in part due to [ATP]/[ADP] ratio and KATP channel conductance changes acting as a pacemaker. Intracellular [Na ] As a Slow Component in Pacemaker Mechanisms Several components regulate Na dynamics in -cells. The most important is the electrogenic Na ,K -ATPase that extrudes three Na ions in exchange for two K ions for each molecule of ATP hydrolyzed, generating a net outward ow of cations through the PM. This pump is highly active, since it determines the high cytoplasmic K concentration (113). Like most other cells, the -cells are also equipped with a PM Na /Ca2 exchanger, an electrogenic transporter that couples the exchange of 3 Na for 1 Ca2 (52, 73). Consequently, changes in the cytoplasmic Na concentration ([Na ]c) lead to changes of inward and outward currents and the PM potential. Changes in [Na ]c can have a signicant effect on generation of slow cytoplasmic Ca2 oscillations in -cells (56, 93). For example, Kitasato et al. (93) proposed a specic oscillatory mechanism, including Na pumps and transporters. They suggested that increased [Ca2 ]c during an active phase results in an activation of the Na /Ca2 exchanger and a corresponding increase in [Na ]c. An increase in cytosolic Na would lead to an enhanced ATP consumption at Na /K pump sites and to a decreased ATP/ADP ratio. This in turn could lead to activation of KATP channels followed by membrane repolarization. A decrease in [Ca2 ]c results in the opposite order of events during a silent phase. This hypothesis was modeled by Miwa and Imai (110) as the underlying mechanism of [Ca2 ]c oscillations. This mechanism is a variant of KATP channel-dependent mechanism for a generation of bursts and [Ca2 ]c oscillations. It does not explain the experimental data, suggesting that the [ATP]/[ADP] ratio and KATP
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Fig. 5. Effect of tolbutamide and thapsigargin on slow Ca2 oscillations in mouse islets. Mouse islets were loaded with fura-2, and relative [Ca2 ]c changes were recorded as the 340/380 nm uorescence excitation ratio changes obtained with the indicator fura-2 during incubation in 2 mM glucose with 50 M tolbutamide, with subsequent addition of 1 M thapsigargin. AJP-Endocrinol Metab VOL
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channel conductance changes are not the main driving force for slow [Ca2 ]c oscillations (see above). We proposed and modeled a mechanism with a Ca2 feedback effect where the intracellular [Na ]c is a dynamic pacemaker variable that can govern bursts and slow [Ca2 ]c oscillations, although KATP channels do not change their activity (51). Figure 6 illustrates the roles of the Na ,K -ATPase, the Na /Ca2 exchanger, and [Na ]c dynamics in this model. Other slow parameters such as cytoplasmic concentrations of [ADP], [Ca2 ]ER, and [IP3] were frozen at constant levels, blocking other possible mechanisms of bursting and [Ca2 ]c oscillations. The following mechanism was responsible for bursting and Ca2 oscillations; increased [Ca2 ]c during an active period activates Na inux through Na /Ca2 exchangers. The resultant increase in intracellular [Na ]c leads to a slow increase in outward current through electrogenic Na /K pumps (INaK in Fig. 1), with corresponding plasma membrane repolarization, so that the cell reenters a silent phase. Decreased [Ca2 ]c during a silent phase leads to a slow decrease in [Na ]c because Na inux through the Na /Ca2 exchanger decreases. This leads to a decreased outward current through Na /K pumps and then in turn to PM depolarization and a generation of bursts and [Ca2 ]c oscillations. The effect of modulators leads to changes in the shape of simulated [Ca2 ]c oscillations, bringing them closer to experimental results. For example, Fig. 7 [which reproduces the third gure from our earlier article (51)] shows slow-simulated [Ca2 ]c oscillations that are similar to the experimental data represented in Fig. 2B. In this simulation, as opposed to that in Fig. 6, [ADP], [Ca2 ]ER, and [IP3] were not frozen at constant levels and thus played a role as modulators. In this case,
Fig. 7. Simulation of glucose-induced slow oscillations and SERCA blocking. Glucose-induced slow electrical bursting and [Ca2 ]c oscillations were simulated at a step increase of the rate constant of ATP production from a low to an intermediate value at t 0. A: [Ca2 ]c. B: MP. C: Ca2 in ER. D: cytosolic ATP. E: cytosolic IP3. F: intracellular Na concentration. For simulation of thapsigargin action, the maximal rate of ER (SERCA; PCaER) was decreased from 0.105 to 0.013 M/ms at arrow in B (adapted from Ref. 51).
Fig. 6. Slow [Na ]c changes as a pacemaker. Typical computer simulations using the model by Fridlyand et al. (51). Slow bursting and the oscillation patterns of Ca2 and Na are illustrated. The other slow variables were frozen ([ATP] 3,300 M, [IP3] 3 M, and [Ca2 ]ER 50 M) at constant levels to eliminate other mechanism of [Ca2 ]c oscillations. All other parameter settings are as in Ref. 51. A: VP. B: [Ca2 ]c. C: [Na ]c. D: INaK. AJP-Endocrinol Metab VOL
cytoplasmic [Na ] as a slowly variable component functioned as the pacemaker mechanism. We will discuss the possible mechanisms of this kind of inuence of modulators on the shape of Ca2 oscillators below. Some indirect experimental evidence supports the hypothesis that cyclic [Na ]c changes can act as a pacemaker to induce bursting and slow [Ca2 ]c oscillations. For example, in isolated individual -cells or cell clusters, the slow glucose-induced Na oscillations were found under conditions known to induce oscillations of [Ca2 ]c. Partial suppression of the Na /K pump by ouabain resulted in an increased amplitude of slow Na and Ca2 oscillations with decreased frequency; slow Na and Ca2 oscillations were converted to a tonically elevated Na and Ca2 level following ouabain inhibition of the Na,K-ATPase (62, 63). Interestingly, slow Ca2 oscillations were converted to a sustained elevated Ca2 level following ouabain even in sulfonylurea type 1 receptor (SUR1)null mouse islets, where KATP channels were blocked (39). Our computational simulation resulted in a similar picture (see the 7th gure in Ref. 51) showing that these experiments can be modeled exactly by a partial inhibition of the Na /K pumps by oubain. According to this simulation, decreasing the Na /K pump maximal activity reduced the rate of Na efux and corresponding net outward current, leading to increased [Na ]c and PM depolarization. This resulted in increased
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amplitude of slow Na and Ca2 oscillations with decreased frequency. Greater inhibition of the Na /K pump maximal activity led to a collapse of this Na -dependent mechanism, the disappearance of bursting and slow Ca2 oscillations, and tonically elevated [Ca2 ]c. Veratridine causes voltage-dependent Na channels to stay open during a sustained membrane depolarization by decreasing inactivation (143). Partial activation of Na channels by veratridine resulted in a decreased amplitude and increased frequency of slow Ca2 oscillations, and slow Ca2 oscillations were converted to a tonically elevated Ca2 level in isolated individual -cells or cell clusters (44). We were able obtain similar results in isolated pancreatic islets (Fig. 8). We simulated the effects of veratridine by shifting the half-activation potential for Na channels toward a more positive voltage using our general model (51). According to this simulation (Fig. 9), increased Na inux through voltagedependent Na channels leads to an additional PM depolarization as a result of increased Na inward current and an accelerated Na accumulation during an active phase. This resulted in increased amplitude and frequency of slow Na and Ca2 oscillations. The simulations are in reasonably good agreement with experimental data (compare Figs. 8 and 9). Correlating the model simulations with experimental data shows that cyclic [Na ]c changes can act as a pacemaker to induce bursting and slow [Ca2 ]c oscillations as opposed to the proposals on the role of KATP channels. However, this mechanism underlying oscillations remains incompletely investigated. Simultaneous measurements of [Ca2 ]c and [Na ]c, although difcult, can directly test this hypothesis. Role of the ER and [Ca2 ]ER as a Slow Pacemaker The ER is a high-afnity and high-capacity organelle for calcium storage. The -cell ER sequesters Ca2 when the cytoplasmic calcium level is high and releases it when [Ca2 ]c is low or under the inuence of intracellular messengers (55, 122, 123, 141). Ca2 from the cytosol is transported into the ER by P-type ATPases using ATP and exits through two types of ER Ca2 channels, the IP3 receptor (IP3R) channels and the ryanodine receptor channels (Fig. 1) (56, 82, 123, 138). How-
Fig. 9. Simulated inuence of veratridine on slow [Ca2 ]c oscillations in -cells. Addition of veratridine was simulated by decrease of half-activated potential for Na channels in the model by Fridlyand et al. (51). (Halfactivated potential for Na channels was decreased from 104 to 94 mV at arrow 1 and from 94 to 84 mV at arrow 2 in B). A: VP. B: [Ca2 ]c. C: [Na ]c. D: INa.
Fig. 8. Effect of veratridine on slow Ca2 oscillations in mouse islets. The experiments were performed in medium containing 2.5 mM Ca2 and 14 mM glucose. Veratridine ( M) was added as indicated. The concentration of cytoplasmic Ca2 is presented as the 340/380 nm uorescence excitation ratio obtained with the indicator fura-2. AJP-Endocrinol Metab VOL
ever, Ca2 inux from the extracellular space through VDCCs is the principal regulator of cytoplasmic Ca2 homeostasis in -cells under physiological conditions (6, 51, 127, 146). [Ca2 ]ER and store operating currents. Changes in [Ca2 ]ER may regulate PM potential. Depletion of intracellular Ca2 stores in -cells activates a special Ca2 release-activated current [or store operating current (SOC)] through nonselective PM cation channels and can represent an inward cation current, leading to PM depolarization that potentiates glucose-induced Ca2 inux trough VDCCs (20, 30, 97, 125, 147). This current may be Na inux through nonselective PM cation channels, which have been described in insulin-secreting -cells (30, 97, 125). Transient receptor potential (TRP) ion channels were proposed for this role (74, 78, 125). Although the detailed mechanism for coupling of ER Ca2 store depletion with the nonselective PM channels activation is uncertain, it likely involves translocation of STIM1 and STIM2 proteins from the ER calcium stores to the PM, where they interact with ORAI1 and related proteins associated with cation inux channels (119, 137). Several mechanisms of oscillations with [Ca2 ]ER as a pacemaker employing a Ca2 feedback effect have been proposed (20, 25, 51, 53, 107, 125, 148, 150). These proposals were primarily based on the characteristic properties of SOC that lead to cell depolarization with decreased [Ca2 ]ER. Several mathematical models have also been developed for these proposals using empirical equations for activation of SOC with decreased [Ca2 ]ER (20, 25, 53, 150). SOC increased with decreased [Ca2 ]ER and vice versa in simulations using the empirical equations from these models, yielding periodical changes of [Ca2 ]ER, bursting, and [Ca2 ]c oscillations. We developed a model where SOC includes Na inux through nonselective cation channels that open following dewww.ajpendo.org
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creases in [Ca2 ]ER (SOC was termed CRAN current in our model) (51), following the earlier work of Roe et al. (125). The proposed mechanism is illustrated in Fig. 10. In this case, other possible pacemaker mechanisms of [Ca2 ]c oscillations in the model (51) were blocked by freezing the cytoplasmic concentrations of [ADP], [Na ], and [IP3] at constant levels. In this simulation, a rapid rise in [Ca2 ]c at the beginning of the active phase led to increased Ca2 pumping into the ER and slow [Ca2 ]ER accumulation with corresponding closure of PM nonselective cation channels. This decreased inward Na inux current (ISOC; Fig. 1) through these channels (i.e., SOC) resulted in PM repolarization, termination of the spiking, and the transition to a silent phase. Slowly decreasing [Ca2 ]ER during a silent phase as a result of Ca2 leak through ryanodine or IP3R then led to increased nonselective cation channel conductance, increased inward Na current, and PM depolarization. This resulted in subsequent activation of VDCCs and a new burst. In this case, [Ca2 ]ER is a slow pacemaker component for [Ca2 ]c oscillations with a Ca2 feedback effect. However, an explanation of slow oscillations on the basis of [Ca2 ]ER-dependent mechanisms involves several difculties, since it seems to be at odds with data demonstrating that the slow oscillations can persist in the presence of thapsigargin, a SERCA inhibitor (Fig. 5 and Refs. 6, 51, 55, 98, 109, and 136). Slow [Ca2 ]c oscillations also persist in islets from a SERCA3 knockout mouse, where fast oscillations disappear (11). These results argue against a role of [Ca2 ]ER changes as a pacemaker for slow [Ca2 ]c oscillations. Slow [Ca2 ]c oscillations can also persist even though KATP channels and SERCA activity are blocked simultaneously in mouse islets by tolbutamide and thapsigargin (Fig. 5) or in
Fig. 10. Slow [Ca2 ]ER changes as a pacemaker. Simulations were made as in Fig. 4. [Ca2 ]ER is the only slow parameter at frozen ADP, ATP, Na , and IP3 concentrations that eliminated other possible mechanisms of bursting and Ca2 oscillations in the model by Fridlyand et al. (51). ([Na ]c 6 mM, [IP3] 1 M, [ATP] 3,400 M, and kADP 0.00045 ms 1). All other parameter settings are as in Fridlyand et al. (51). A: VP. B: [Ca2 ]c. C: [Ca2 ]ER. D: ISOC. AJP-Endocrinol Metab VOL
isolated islets from the SUR1-null mouse lacking KATP channels when SERCA is inhibited (39). These complex experimental data show that a mechanism of slow [Ca2 ]c oscillations cannot be explained solely by an interaction of KATP channels and ER-dependent mechanisms. SERCA blockade has led to conicting results for fast bursts and [Ca2 ]c oscillations in -cells converted to a sustained elevated [Ca2 ]c level by thapsigargin (55, 86, 109, 136, 147). However, Tamarina et al. (138) found that an addition of thapsigargin was accompanied by a gradual increase in the amplitude and frequency of fast [Ca2 ]c oscillations in some islets. Bertram et al. (20) had previously found that thapsigargin induced a gradual increase in the amplitude plateau fraction and in electrical burst frequency in the -cell membrane potential. It is possible that in some experiments only partial SERCA blockade was obtained, leading to a partial decrease in [Ca2 ]ER, thereby preserving the ability of the -cells to create an oscillatory mechanism. More profound SERCA block in other experiments could decrease [Ca2 ]ER to a threshold level where oscillatory mechanisms with [Ca2 ]ER as a pacemaker are suppressed. This suggests that the mechanism discussed above in which [Ca2 ]ER is the pacemaker component could be responsible for fast bursting and [Ca2 ]c oscillations. Recently, null mice for one of the TRP ion channels (Trpm5) were found to have islets that had lost the ability to create fast [Ca2 ]c oscillations, whereas slow [Ca2 ]c oscillations were intact (27). The disappearance of fast [Ca2 ]c oscillations in this case is consistent with the hypothesis that Trpm5 channels can be a part of the SOC (or CRAN) mechanism with Na inux (75, 118) that is in part responsible for the fast, not slow, [Ca2 ]c oscillation (see above). [Ca2 ]ER changes can regulate the shape of slow glucoseinduced [Ca2 ]c oscillation in pancreatic -cells (11, 99). Our simulations also support the hypothesis that slow [Ca2 ]c oscillation frequency and the generation of the waveform can be modulated by changes in the rates of Ca2 leak from the ER, although [Ca2 ]ER does not account for the pacemaker mechanism for slow oscillations per se (51, 138). For example, Fig. 7 (reproduced from Ref. 51) shows the changes in the shape of [Ca2 ]c oscillations during simulation of SERCA block that lead to a decrease in [Ca2 ]ER. This is at conditions where the changes in [Na ]c drive the pacemaker mechanism for slow [Ca2 ]c oscillations. In this case, Ca2 is gradually released into the cytosol, and a decrease in [Ca2 ]c can be slow at the beginning of the silent period of slow oscillations (Fig. 7). When [Ca2 ]ER is small after SERCA inhibition, no additional Ca2 release occurs during the silent period of slow oscillations, and the [Ca2 ]c decrease is sharp. A similar result was obtained experimentally (51, 55). Pacemaker due to [Ca2 ]ER regulated by IP3. In pancreatic -cells, as well as in other cell types, external agonists can act on G protein-coupled receptors to stimulate phospholipase C (PLC) activity. Ca2 -activated PLC catalyzes the formation of IP3 from phosphatidylinositol-4,5-bisphosphate (Fig. 1) (23). The resulting rise in IP3 initiates Ca2 discharge from the ER through IP3R channels (13, 56, 138). IP3 can also activate K or Na conductances that can lead to PM repolarization (5, 134). Changes in [IP3]c can play a crucial role in creating Ca2 oscillations through activation of the ER IP3 receptor in nonelectrically excitable types of cells (13, 130). Several mathewww.ajpendo.org
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matical models employed this mechanism for nonelectrically excitable cells. The rst step is that a [Ca2 ]c increase is caused by Ca2 release from the ER. One intracellular signal for this release is IP3, which triggers Ca2 release by binding to the IP3Rs on the surface of the ER. This opens ER Ca2 channels, allowing Ca2 to ow out into the cytoplasm. Subsequent inactivation of the IP3Rs and removal of Ca2 by ER membrane pumps then reduces the Ca2 concentration to lower levels. It can initiate a repeating cycle of release and uptake of Ca2 from the ER, resulting in Ca2 oscillations if the IP3 concentration is in the correct range (13, 130). However, -cells are electrically excitable cells, and as discussed above the changes in PM potential and corresponding Ca2 inux from the extracellular space through VDCCs may be the principal regulator of cytoplasmic Ca2 homeostasis in -cells. For this reason, [IP3]c and IP3R interactions are not likely to function as a pacemaker for [Ca2 ]c oscillations in -cells. We recently investigated the dynamics of IP3 in mouse primary -cells and in MIN6 mouse insulinoma cells upon stimulation with glucose and a muscarinic agonist (carbamoylcholine) (138). We found that IP3 concentrations in the cytoplasm oscillated in parallel with [Ca2 ]c oscillations upon stimulation with glucose. Although this could be interpreted as evidence supporting IP3 oscillations as a pacemaker of Ca2 oscillations, in this case IP3 oscillations can be readily explained by Ca2 activation of PLC leading to [IP3]c changes, as simulated in our model (Fig. 7E). This suggests that slow cyclical changes in IP3 concentration may follow from the activation of PLC by independent cyclical [Ca2 ]c changes; i.e., IP3 may not be a pacemaker for [Ca2 ]c oscillations (51, 138). However, this problem needs further investigation, perhaps by PLC inhibition, to determine the existence of [Ca2 ]c oscillations in the absence of IP3 oscillations. However, the rate of IP3 production can have a dramatic effect on oscillations even though IP3 is not a pacemaker compound; i.e., IP3 can be a strong modulator of oscillatory processes. For example, activation of IP3 synthesis can cause a transformation of slow oscillations into fast ones (20, 138). Although the mechanism is unclear, it may be due to additional PM depolarization following from the opening of SOC or the recently found NALCN channels (134) with decreased [Ca2 ]ER, or this could follow from activation of PM K channels by IP3. cAMP as a Pacemaker Component The cAMP pathway has [Ca2 ]c-dependent components such as some isoforms of adenylyl cyclase and phosphodiesterases (PDEs) (Fig. 1) (29, 60). cAMP may also increase Ca2 -induced Ca2 release from the ER through ryanodine and IP3Rs in mouse pancreatic -cells and may activate some PM channels via phosphorylation or protein binding (66, 84, 85, 131). The suggestion was made for several types of cells that [Ca2 ]c oscillations could result from interactions between cAMP and [Ca2 ]c (61, 102). Therefore, an oscillatory mechanism with Ca2 feedback effect and [cAMP]c as a slow pacemaker component could also occur in -cells. However, recent experimental data does not support the hypothesis that cAMP is a pacemaker component for an oscillatory mechanism, at least for slow [Ca2 ]c oscillations in -cells. For example, in insulinoma -cell lines a PDE inhibitor (IBMX)
abolished cAMP oscillations without inhibiting [Ca2 ]c oscillations (see the 5th gure in Ref. 95). Dyachok et al. (42) showed that IBMX can stimulate [Ca2 ]c oscillations even during elevated, nonoscillating cAMP levels, measured using real-time imaging. For this reason, according to our rules for hypothesis rejection (see INTRODUCTION), we believe that cAMP oscillations are not part of a pacemaker mechanism, at least for slow [Ca2 ]c oscillations. We recently analyzed oscillatory behavior of cAMP using our model of cAMP dynamics and found that slow [Ca2 ]c oscillations are a pacemaker for cAMP oscillations in pancreatic -cells and not the other way around (47). However, cAMP (as well as IP3) may modulate the dynamics of the intracellular processes and PM potential, leading to the changes in the oscillation pattern. For example, both IBMX and forskolin, an activator of adenyl cyclase, increase the level of cAMP, transforming [Ca2 ]c oscillations from slow into fast and further increasing their frequency (10). Other agents that increase the concentration of cAMP, such as glucagon, also promote the appearance of fast [Ca2 ]c oscillations (99). The underlying mechanisms of these transformations remain an open question but can be partially explained by altering PM depolarization. The Pacemaker Role of Metabolic Oscillations Metabolic cycles can control glucose-stimulated insulin secretion (80), and a number of hypotheses have implicated metabolic oscillations in determining Ca2 oscillations (101, 142). Oscillations of a metabolic signal in -cells may result from intrinsic properties of various pathways, although glycolytic oscillations are not likely to account for the mechanism of other metabolic oscillations (see above). We cannot rule out that some Ca2 -independent mechanism can create oscillations of compounds that can change the PM potential, leading to [Ca2 ]c oscillations. For example, based on the existence of citrate oscillations in isolated islet mitochondria, it was proposed that there is an independent mitochondrial oscillator and that the exported citrate may feed back and coordinate mitochondrial and cytosolic oscillations (101). Several mathematical models of possible mitochondrial oscillatory mechanisms were discussed recently (14), and these should be testable in -cells. Oscillations of the metabolic signals in -cells may be secondary to oscillations in [Ca2 ]c. As was pointed out by Kennedy et al. (89), an appearance of metabolic oscillations does not prove that metabolic variations are the real pacemakers of oscillations, because these metabolic changes can indeed reect the [Ca2 ]c oscillations. For example, variations in [Ca2 ]c can change ATP consumption because the rate of work of ATP-consuming Ca2 pumps depends on Ca2 concentrations. Using our mathematical model for -cell processes (49, 50), we found that cyclic [ATP] and [ADP] oscillations in the cytoplasm due to the oscillations in [Ca2 ]c can stimulate the oscillations in metabolic processes such as oxygen consumption, the mitochondrial inner membrane potential, the rate of ATP production in mitochondria, and NAD(P)H levels in -cells, i.e., in the oscillating processes that were found in experiments using -cells (89, 92, 100, 111). We can also expect oscillations in metabolism if specic metabolic enzymes can be modulated by cyclic Ca2 concentration changes in cytoplasm or mitochondria. For this reason, the oscillations
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in metabolic variables mentioned above are most likely secondary and likely follow rather than precede bursting and [Ca2 ]c oscillations. Activation or Deactivation of the Voltage-Dependent Channels As a Pacemaker Component As we pointed out in the INTRODUCTION, the cyclic activation and deactivation of several channels can underlie mechanisms that drive bursting and Ca2 oscillations in -cells. Here, we consider other possible suggestions related to regulation of ion channels. For example, it was suggested that PM repolarization and termination of spikes could occur during a burst if some parameters of the voltage-gated Ca2 current could be slowly changed, leading to a decreased current (28). On the other hand, a regenerative recovery from inactivation and reactivation of Ca2 currents in the rest period could lead to a depolarization and a beginning of a new burst. This mechanism for burst generation requires mathematical modeling of timedependent changes in maximal conductance or other coefcients in the equations for VDCC (88). However, this mechanism for the changes in Ca2 current also lacks sufcient experimental support. Similarly, we can suggest that slow activation of any K currents (other than current through KATP and slow KCa channels, which we have considered above) in the active period can lead to repolarization and burst termination. A regenerative deactivation of these currents in the silent interval can lead to depolarization and to a beginning of a burst. However, the available experimental evidence argues against this hypothesis, at least for slow [Ca2 ]c oscillations. For example, stimulation of TC3-neo or MIN6 insulinoma cells with glucose results only in a modest nonoscillatory increase in [Ca2 ]c. However, subsequent addition of TEA (1520 mM, sufcient to substantially suppress most non-KATP K channels) can induce fast and slow [Ca2 ]c oscillations (41, 136, 139) so that inhibiting most K channels activates rather than suppresses [Ca2 ]c oscillations. Slow bursting and [Ca2 ]c oscillations are not lost in mouse islets lacking KV2.1 channels (77). According to our rule for rejection, this indicates that KV2.1 channels are not a part of a pacemaker mechanism, but they do have important modulating effects. Interactions of Mechanisms and Compound Patterns of Bursting and [Ca2 ]c Oscillations Fast and slow modes of oscillation can occur together, as in the mixed pattern, or separately, as in the fast and slow patterns (Fig. 2; see also Refs. 19 and 31). This strongly argues that they stem from distinct mechanisms. These mechanisms can be linked reciprocally and often occur together and can also proceed largely independently of each other. Mixed oscillations also occur in isolated cells, suggesting that this peculiar pattern does not result from the sum of signals produced in distinct -cells in islets (6). Modeling approaches show that compound bursting and corresponding mixed [Ca2 ]c oscillations can be simulated. The results of the modeling favor a scenario where two or more slow variables interact to produce complex oscillations. For example, Bertram and colleagues (17, 18) simulated the ultrafast and fast oscillations by small variations in KATP conductance using the
empirical equation connecting the [ATP]/[ADP] ratio with [Ca2 ]c changes. The slow [Ca2 ]c oscillations were simulated as metabolic glycolytic oscillations such that the two interact to produce compound oscillations consisting of episodes of bursts separated by long periods of silence, or accordion oscillations, that consist of fast bursts with a slowly modulated duty cycle. Similar behavior could be achieved by dening the different slow and fast pacemaker variables (16). For example, slow [Ca2 ]c oscillations in a compound picture may be a result of a [Ca2 ]ERdependent mechanism, and a KATP-dependent mechanism may be responsible for fast [Ca2 ]c oscillations on the top of the slow (35). According to our analysis, it is most likely that [Na ]c can represent a slow-changing compound for slow oscillations and changes in the [ATP/ADP] ratio and/or [Ca2 ]ER for fast oscillations. Modulators of Oscillation Behavior and External Pacemaker Mechanisms
Changes in channel conductance or Ca2 handling can change PM polarization or Ca2 redistribution. This, in turn, may lead to changes in bursting and [Ca2 ]c oscillations even though the cellular components do not participate directly in pacemaker oscillatory mechanisms. For example, activation of anion channels may cause depolarization of the -cell PM (21, 22). Glucose stimulation of -cells is coupled to an increase in Cl permeability, and oscillatory Ca2 signaling may be critically dependent on transmembrane Cl uxes (45). This indicates that agents that regulate anion channels may modulate oscillations. Cyclic nucleotide-gated (HCN) channels may also have a modulating effect because they can inuence -cell PM potential (152). External insulin can also be considered a modulator because it may be able to activate KATP channels (90). Cyclical changes in external modulators can create or synchronize the alterations in PM polarization as well as the corresponding intracellular Ca2 concentration. This property of -cells can be important for synchronization of oscillations of insulin release in vivo in the pancreas. This synchronization is likely achieved through intrapancreatic innervation. This is probably due to cholinergie effects synchronizing islets (1, 149). Interestingly, a single cholinergie pulse per several internal [Ca2 ]c oscillations can synchronize these oscillations (46, 149). Other external pacemaker mechanisms can also contribute to islet synchronization. For example, insulin secreted by islets acts on the liver, resulting in a reduction in plasma glucose concentration that can lead to glucose oscillations in blood, synchronizing an insulin secretion in islets (57, 114, 117). Additional synchronizing factors can be external ATP acting on purinergic receptors (115, 128). Conclusion Our analysis of several different hypotheses for pacemakers of oscillatory processes in -cells shows that the following proposals may be most consistent with the existing experimental data. 1) Changes in the conductivity of slow KCa channels and KATP channels can be the pacemakers for ultrafast [Ca2 ]c oscillations; 2) changes in [ATP]/[ADP] ratio and/or [Ca2 ]ER can be the pacemakers for fast bursts and [Ca2 ]c oscillations; and 3) cyclical [Na ]c changes may be important to explain the pattern of slow bursting and calcium oscillations. However, the exact
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physiological variables and their underlying mechanisms that drive bursting or Ca2 oscillations in -cells remain uncertain. Additional dynamic experiments and mathematical models are necessary to better understand this emerging aspect of -cell physiology.
GRANTS This work has been partially supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DRTC P60-DK-020595, DK-48494, and DK-063493 and a Research Grant from the Keck Foundation. DISCLOSURES The authors have nothing to disclose.
Slow-Activated Ca2 -Sensitive K Channels IKCas dt dKCab where GmKCas 90 pS and [Ca2 ]4 c
dKCas
gmKCas dKCas Vp dKCasb dKCas
EK ,
(11) (12)
d dKCas
dKCas
[Ca2 ]4 c (0.25)4
(13)
2,300 mc.
ATP-Sensitive K Channel Current (IKATP) IKATP where

APPENDIX
gmKATP OKATP Vp
EK ,
(14)
OKATP 0.08(1 (1 2 MgADPf i 17) 0.89( MgADPf i 17)2 ATPfree i 50) (15) Downloaded from ajpendo.physiology.org on January 31, 2012 MgADPf i 17)2(1 MgADPf
i
Modeling of Slow-Activated Ca2 -Sensitive K Channel-Dependent Bursting and [Ca2 ]c Oscillations Equations and coefcients for channels and pumps are taken from the previous model (51) or represented here. Slow-activated Ca2 sensitive K channels as well as the fast-activated Ca2 -sensitive K channels were modeled, with considerably increased time-dependent parameters for activation reported in Ref. 59. The units are time in milliseconds (ms), voltage in millivolts (mV), concentration in micromoles/liter ( M), current in femtoamperes (fA), conductance in picosiemens (pS), and capacitance in femtofarads (fF). Numerical integration of the model was carried out using standard numerical methods (51). This model is available for direct simulation on the web site Virtual Cell (www.nrcam.uchc.edu) in the MathModel Database on the math workspace in the library Fridlyand with the name SlowCa acted Kchan. Ca2 Current (IVCa) IVCa gmVCa dCa fCa (VP d dCa dt dCab
dCa
0.45 MgADPf i 26 0.55 ADPf i ,
(16) where OKATP is the fraction of open KATP channels, [MgADPf]i is the concentration of free Mg-bound ADP, GmKATP 30,000 pS, and [ADPf]i 20 M. Plasma Membrane Ca2 Pump Current (ICap) ICap where PmCap 4,300 fA. PmCap
2 [Ca2 ]ci
[Ca2 ]2 c
(0.2)2
(17)
Na Background Current (INab) ECa) (1) (2) (3) (4) (5) INab gmNab Vp ENa , (18)
dCab
dCa
dCa
where gmNab 12 pS and ENa 70 mV. The differential equation describing time-dependent changes in the PM potential (Vp) is the current balance equation: Cm dVp dt IVCa IKr IKATP IKCa IKCas ICap INab , (19) where Cm is the whole cell membrance capacitance. The PM currents are listed in Fig. 1. Based on the consideration in our general model (51), and including only IVCA and ICap, the equations for [Ca2 ] dynamics can be written as d Ca2
c
1 1 1.79 1 exp 2 VP /10.5 9.7) 70.2]2 VP /6 , 1 exp 100 mV. 15 exp [(Vp
2.2
fVCa where gmVCa
fVCab
800 pS, ECa
Delayed Rectier K Current (IKr) IKr

2 gmKrdKr fKr
fi
IVCa 2FVi
2ICap
Vp dKr
EK ,
(6) (7)
dt
ksg Ca2
(20)
d dKr dt dKrb where gmKr 30,000 pS, 1

dKr
dKrb
dKr
where fi is the fraction of free Ca2 in cytoplasm, F is Faradays constant, Vi is the effective volumes of the cytosolic compartment, and ksg is a coefcient of the sequestration rate of [Ca2 ]c.
REFERENCES 1. Ahren B. Autonomic regulation of islet hormone secretionimplications for health and disease. Diabetologia 43: 393410, 2000. 2. Ainscow EK, Rutter GA. Glucose-stimulated oscillations in free cytosolic ATP concentration imaged in single islet beta-cells: evidence for a Ca2 -dependent mechanism. Diabetes 51, Suppl 1: S162S170, 2002. 3. Ainscow EK, Rutter GA. Mitochondrial priming modies Ca2 oscillations and insulin secretion in pancreatic islets. Biochem J 353: 175 180, 2001. 4. Amml C, Bokvist K, Larsson O, Berggren PO, Rorsman P. Demonstration of a novel apamin-insensitive calcium-activated K channel in mouse pancreatic B cells. Pugers Archiv 422: 443448, 1993. www.ajpendo.org
1 exp 9 VP /5
, 75 mV.
(8)
23 mc, EK
Fast Ca2 -Activated K Current (IKCa) IKCa dKCa where gmKCa 10 pS.
gmKCa dKCa Vp [Ca [Ca2 ]4 c

2
EK , ,
(9) (10)
]4 c (0.25)4
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Bursting and Calcium Oscillations in Pancreatic Beta-Cells - Specific Pacemakers For Specific Mechanisms

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Bursting and Calcium Oscillations in Pancreatic Beta-Cells - Specific Pacemakers For Specific Mechanisms

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Bursting and calcium oscillations in pancreatic -cells: specific pacemakers for specific mechanisms

L. E. Fridlyand, N. Tamarina and L. H. Philipson

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0193-1849/10 Copyright 2010 the American Physiological Society

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gmKCas dKCas Vp dKCasb dKCas

ATP-Sensitive K Channel Current (IKATP) IKATP where

0.45 MgADPf i 26 0.55 ADPf i ,

fVCa where gmVCa

800 pS, ECa

Delayed Rectier K Current (IKr) IKr

d dKr dt dKrb where gmKr 30,000 pS, 1

gmKCa dKCa Vp [Ca [Ca2 ]4 c

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AJP-Endocrinol Metab VOL

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