Historical Perspectives

                                    Butler, Hudibra, I.

Throughout the annals of time, men even the prehistoric ones have been fascinated by the ever-existing rhythmic processes in living systems. However, this idea wasn’t completely in the open till 1797 when Hufeland proposed rhythmic events of life in relation to 24 hours or solar day as a prime unit of functional chronology¹. In Florida, nose and throat surgeons found that hemorrhages in throat operation were 82% higher during the second quarter of the cycle of the moon than at other times². Similarly, insulin sensitivity index was found to be lower during winter than summer in Swedish population³. Seasonal variations of HbA1C in diabetic subjects and glycemic variations in healthy subjects have also been   reported^4-6.Thus, it seems that periodic biological events are intimately related to the non-biological cycles, whether terrestrial, astronomical, physical, electrical or others. But certainly it has been realized by the early scientists that the universe is rhythmic and displays incessant movement in the form of periodicity. The capacity to follow them, to oscillate, would enhance the survival potential of a species, including we, the human beings.

In 1843, nearly half a century after Hufeland’s Publication, Chossat presented his report of 20 years of study on the changes in cloacal temperature of pigeons under various experimental conditions as to environmental temperature and nutrition¹. Further analyses on biological rhythms revealed that ‘a close study of these rhythms should yield vital information concerning the construction of various biochemical reactions in the body, especially if cybernetic and thermodynamic principles are applied’. Many enthusiastic scientists and clinicians then devoted themselves for basic understanding of the fundamentals of biological rhythms. In the 30’s of the twentieth century, the periodic behavior of the normal blood glucose was characterized. In this case small meals were given regularly throughout the day and generally each meal produced a variable, transient increase. However, it was found that the glucose concentration often tended to drop somewhat at about 2 to 3 p.m. even if food was given⁷. The essential feature of this periodic behavior is that the blood glucose level is relatively stable, varying between 4.4 and 6.6 mM/L. It was discovered later, that under physiological conditions the blood glucose level is kept at around 5 mM/L in fasting mammals, including humans due to the pulsatile release of the glucostatic hormone insulin.

Insulin is secreted from the pancreatic β-cells in a highly regulated fashion. Among the factors affecting insulin release, glucose is the most important physiological stimulant and is considered as the primary regulatory signal. The exact sequence of biochemical events involved in glucose stimulated insulin secretion (GSIS) has not yet been identified. Nevertheless, it is well documented that GSIS is manifested due to a rise of cytosolic Ca²⁺ ([Ca²⁺]i), which acts as the primary intracellular messenger that couples physiological or pharmacological insulin secretegogues to insulin release from stored granules⁸. A characteristic feature of the [Ca²⁺]i response to glucose is its oscillatory nature observed both in individual pancreatic β-cells and in intact pancreatic islets^9-12. Recent experiments have shown that the [Ca²⁺]i oscillations correspond to pulsatile insulin secretion and electrical activity of β-cells and thus it has been suggested that the [Ca²⁺]i oscillations may have important role in maintaining pulsatile release of insulin^13-18.

Pancreatic β-cell’s response to glucose is oscillatory. When glucose enters the b-cells through high capacity glucose transporters, its metabolism through glycolysis and Kreb’s cycle causes changes in the ATP/ADP ratio in the cytoplasm resulting in closure of the ATP-sensitive K⁺ channels and thereby trigger membrane depolarization^19,20. This leads to the opening of voltage dependent calcium channels (VDCCs), Ca²⁺ influx and to subsequent rise in [Ca²⁺]i that promotes insulin secretion (Fig 1). Apart from the voltage-sensitive  Ca²⁺ influx from extracellular space, another major source of [Ca²⁺]i rise is mobilization of Ca²⁺ from intracellular stores^21-23. Recent studies also suggest the existence of store operated Ca²⁺ entry in pancreatic b-cells^24-26. However, once the [Ca²⁺]i is elevated, to restore it to its basal level, the b-cells drive Ca²⁺ actively either out of the cell across the plasma membrane through calcium pump and Na/Ca exchanger or to various intracellular stores^27-29. We can simply postulate that the upstroke of the oscillation is due to Ca2+ influx and/or the release and the descending phase involves stimulation of outward Ca²⁺ transport and/or intracellular sequestration. And oscillations are generated and maintained via dynamic interplay of discrete signaling cascades which provides complex feedback, as well enhances co-ordination that critically maintains the fine tuning of [Ca²⁺]i fluctuations during different phases of the oscillation.

Figure 1. Schematic representation of ionic events in ‘glucose stimulated insulin secretion’ from pancreatic B-cells. IC = intracellular compartments.

Properties of [Ca²⁺]i Oscillations

Oscillations in [Ca²⁺]i are of different fundamental types, involving different mechanisms. However, in secretory cells two major kinds of [Ca²⁺]i oscillations are seen – baseline transients or spikes and sinusoidal oscillations^30,31. Spikes are characterized by transient increase in [Ca²⁺]i that rise rapidly from a baseline of [Ca²⁺]i. The shape of transients may vary depending on agonist-type: they may be symmetrical or may have a relatively rapid rising phase with a slower falling phase (Fig 2). Sinusoidal oscillations generally appear as symmetrical oscillations superimposed on a sustained level of [Ca²⁺]i usually above the pre-stimulus baseline level. They resemble more closely to true sine waves. These oscillations are generally considered insensitive to variations in agonist concentrations and may simply reflect how certain cells respond to a maintained elevation of calcium30. A less common [Ca²⁺]i oscillatory pattern that seems to be distinct from the spiking and sinusoidal patterns has been described by Rooney and Thomas³². The oscillations are highly asymmetric, consisting of a rapid increase in [Ca²⁺]i followed by a slow decline during which the next asymmetric oscillation is initiated. Such a pattern is extremely prominent in adrenal glomerulosa cells, in neutrophils and in mucosal mast cells³³.

Figure 2. Different patterns of calcium oscillations. (A) Transient oscillations or spikes. Symmetrical transients (left) and transients  with slower recovery phase (right). (B) Sinusoidal oscillations. (C) Asymmetric oscillations.

In pancreatic islets, stimulatory glucose concentrations (>7mM) induce two types of [Ca²⁺]i oscillations – fast and slow. Fast oscillations are transient spikes in which the [Ca²⁺]i level rises sharply and then subsequently decreases along an exponential-like time course¹⁰. They oscillate at a frequency ranging from 2 to 5/min with duration of 3-11s (Fig 3). They are the direct consequence of β-cell bursting electrical activity, their duration depends on glucose concentration, and they are synchronous throughout the islet³⁴. In contrast, slow oscillations are characterized by smooth rising and falling phase with duration of 1-3 min and frequency of 0.2-1/min. A mixed pattern of fast oscillations superimposed on the slow pattern is also a common observation. Both the slow and fast oscillations of [Ca²⁺]i in pancreatic islets depend on periodic entry of Ca²⁺.However, the fast ones somehow depend also on mobilization of Ca²⁺ from intracellular stores35.

Figure 3. Traces showing different oscillatory patterns of (Ca²⁺), in pancreatic islets. Stimulation of pancreatic islets with 11 mM glucose can produce fast (upper trace), slow (middle trace) or mixed (lower trace) patterns of (Ca²), oscillations.

Individual pancreatic b-cells exhibit different types of [Ca²⁺]i oscillations36. Type a or slow [Ca²⁺]i oscillations are sinusoidal which usually appear at glucose concentration of 7-20 mM with different thresholds for the individual cells (Fig 4). These oscillations have typical frequencies of 0.05-0.5/min, starting from the basal level with amplitudes of 300-500 nM37,38. The initial response of individual β-cells to glucose is characterized by a transient initial lowering of [Ca²⁺]i,due to sequestration of Ca²⁺ into intracellular compartments^39-41, followed by a sharp rise of Ca²⁺ (Fig 5). The slow [Ca²⁺]i oscillations are strictly dependent on extracellular Ca²⁺ and disappear in the presence of the voltage-dependent Ca²⁺ channels (VDCC) blockers⁴². The slow [Ca²⁺]i oscillatory response is elicited not only by glucose as well leucine⁴³, isoleucine⁴⁴, ±-ketoisocaproate⁴⁵ and tolbutamide⁴⁶. Various mechanisms have been proposed to explain the generation of this [Ca²⁺]i fluctuations at single cell level including oscillations in glucose metabolism^47-51, fluctuations of inositol 1,4,5 trisphosphate production⁵², oscillations of Ca²⁺ in the endoplasmic reticulum⁵³, periodic Ca²⁺ influx during bursting electrical activity^42,54 and cyclical periods of Ca²⁺ induced Ca²⁺ release⁵⁵. But still it is an ongoing problem and no definitive conclusion has been reached so far.

Figure 4. (Ca²⁺)1 oscillations in individual pancreatic β -cell. Differentt types have been reffered to as a-d. (Reproduced with permission from Elsevier Science Publishers. Hellman B. Gylfe E, Grapengiesser E, Lund PE, Berts A. Cytoplasmic Ca²⁺ oscillations in pancreatic B-cells. Biochem Biophys Acta 1992, 1113:295-305).

Type b or fast [Ca²⁺]i oscillations usually appear as superimposed on the slow oscillations or on a sustained level of elevated [Ca²⁺]i. They occur at a frequency of 2-8/min with a duration of approximately 10s and amplitudes of 70-250 nM (Fig 4). The proportion of b-cells responding to glucose with the type b oscillations is higher in cells analyzed shortly after isolation than in those kept in culture for 1-2 days36. A critical cAMP concentration may be required for the appearance of these type b oscillations^38,56.

Figure 5. Effect of raising glucose concentration from 3 to 11 mM on (Ca²⁺), of a single pancreatic β-cell. The horizontal bar indicates the period with the higher glucose concentration.

Type c oscillations are irregular [Ca²⁺]i transients with a duration of <10s and sometimes observed during glucose stimulation alone but becomes more frequent when cells are exposed to high concentrations of glucagon or when the adenylate cyclase activity has been stimulated with forskolin. The [Ca²⁺]i transients are independent of voltage dependent Ca²⁺ influx and disappear after addition of sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) blocker, thapsigargin, indicating that the mobilization of Ca²⁺ from intracellular stores is responsible for their generation⁵⁷.

Type d oscillations are seen when b-cells, stimulated with glucose, are exposed to extracellular ATP or charbachol, which results in a series of [Ca²⁺]i transients of decreasing amplitude and increasing duration (Fig 4). It reflects mobilization of Ca²⁺ from intracellular stores mediated by activation of inositol 1,4,5-trisphosphate receptors and/or ryanodine receptors. These transients exhibit characteristic patterns, making it possible to identify individual b-cells by their [Ca²⁺]i ‘fingerprints’⁵⁸.

Significance of oscillatory [Ca²⁺]i signals

In pancreatic b-cells, Ca²⁺ is the ‘naturally selected’ second messenger^59,60 that decodes signals from different stimuli and relays messages to the biochemical machinery within the cell. But why does [Ca²⁺]i oscillate? Does it really need to oscillate for proper signal transduction in pancreatic β-cells? Although results of some experiments intriguingly suggest that [Ca²⁺]i oscillations are no more effective in insulin release than a sustained signal in pancreatic β-cell^61-63, certainly [Ca²⁺]i oscillations confer positive functional advantages. In the following sections we will focus on the functional significance of oscillatory [Ca²⁺]I signals in the pancreatic β-cells.

Regulation of insulin secretion. Oscillations in [Ca²⁺]i permit a finer control of secretion than a sustained elevation of [Ca²⁺]i as prolonged stimulation of cellular processes can cause desensitization^64,65. It is anticipated that the various steps involved in exocytosis are also sensitive to distinct aspects of [Ca²⁺]i signals, eg, kinetics, multiple spikes, amplitude, and localization⁶⁶. In pancreatic β-cells, KCl alone induces a sustained [Ca²⁺]i increase but causes transient insulin secretion⁶⁷. In contrast, when glucose concentration is raised from basal to stimulatory, it induces [Ca²⁺]i oscillations and a continuous oscillatory insulin release from intact islets and individual β-cells^16,17,68.

Regulation of gene expression. Kinetics of oscillatory [Ca²⁺]i signaling exhibit significant variation in patterns and mechanisms of recognition^69,70. It has been suggested that calcium spiking behavior permits information to be encoded and detected over a much broader range of signaling levels than with sustained [Ca²⁺]i increases³¹. Thus, oscillations of [Ca²⁺]i might regulate cellular processes other than insulin secretion, eg, gene expression. Glucose increases insulin gene expression both at transcriptional and translational levels⁷¹. The glucose induction of insulin transcription was inhibited by VDCC blocker suggesting that the stimulatory effect observed is mediated by Ca²⁺. Thus, glucose-induced oscillatory [Ca²⁺]i signal acts as a common pathway for effectively stimulating both the synthesis and release of insulin. Experimental results have clearly shown that [Ca²⁺]i oscillations and their frequencies are specific for gene activation, both in terms of efficiency and selectivity⁷². Li et a1.⁷³ and Dolmetsch et a1.⁷⁴ provided ample evidence for oscillatory [Ca²⁺]i signals to be more effective to activate Ca²⁺-dependent transcription factors than a single, prolonged increase.

Regulation of metabolism. Oscillations in [Ca²⁺]i are also integrated at the level of the metabolic response. In hepatocytes, vasopressin-induced [Ca²⁺]i oscillation with the frequency of 0.5/min induces mitochondrial redox responses that were effectively maintained close to the peak response⁷⁵. By contrast, sustained [Ca²⁺]i increases induced by maximal vasopressin doses were associated with only a single transient increase of NADH. Thus, a Ca²⁺ response system, in this case mitochondrial energy metabolism, can be tuned to the oscillatory change of [Ca²⁺]i signaling and actually tuned out by sustained [Ca²⁺]i signal31. In pancreatic b-cells, KCl induced a sustained [Ca²⁺]i increase and transient [Ca²⁺]m increase, while glucose induced [Ca²⁺]i oscillations and an oscillatory [Ca²⁺]m increase, suggesting that repetitive transients of [Ca²⁺]m associated with [Ca²⁺]i oscillations are necessary for continuous stimulation of mitochondrial metabolism and thereby continuous secretion of insulin^76,77. It is also possible that oscillations prevent mitochondrial calcium overload and damage in chronically stimulated cells^60,78. The Na⁺-dependent carriers, which discharge Ca²⁺ from mitochondrial matrix, are inhibited by increasing the extra-mitochondrial Ca²⁺ concentration within the physiological range⁷⁹. Thus, the sustained increase in [Ca²⁺]i induced by KCl may attenuate the movement of Ca²⁺ from the mitochondrial matrix and consequently prolong the time course of the [Ca²⁺]m decline⁷⁷.

Regulation of apoptosis. Long-lasting sustained elevations of [Ca²⁺]i activates Ca2+-dependent degradative enzymes, e.g., protein kinases, endonucleases, proteases, and phospholipases,^80,81 whose prolonged activation can result in extensive catabolism of cellular constituents and lethal injury. Oscillatory [Ca²⁺]i signals prevent these potentially damaging effects of Ca²⁺-dependent enzymes. McCormack et a1.⁸² have shown that induction of thymocytes apoptosis by glucocorticoid hormones are dependent on an early, receptor-mediated, sustained increase in [Ca²⁺]i concentrations. In hepatoma 1c1c7 cells low ATP concentrations (1-10 µM) stimulate a transient, receptor mediated Ca²⁺ response whereas high concentrations of ATP (mM) can also cause a sustained increase in the [Ca²⁺]i^80,83. It appeared that treatment of the hepatoma cells with high levels of ATP could activate Ca²⁺-dependent, enzymatic DNA cleavage and contribute to cell killing⁸⁰, Uncontrolled steady-state rise of [Ca²⁺]i can also induce Ca²⁺-dependent activation of several genes that characterize many types of acute lethal injury. These genes can be induced within 15 min or less, as in the case of c-fos⁶⁶ and c-jun, to trigger additional events, such as the ced 3/ICE protease family members, which appear to be close to the final phase of cell death81. In pancreatic β-cells, increased cell death has been reported at elevated glucose concentrations when intracellular Ca²⁺ is not oscillating⁸⁴.Recently, Iwakura et al.⁸⁵ have shown that sustained enhancement of Ca²⁺ influx induced by continuous exposure to glibenclamide caused apoptotic cell death in rat insulinoma cell line (RINm5F cells). These results are consistent with the concept that oscillatory [Ca²⁺]i signals in pancreatic β-cells prevent cellular damage. In pancreatic β-cells, increased cell death has been observed at elevated glucose concentrations when [Ca²⁺]i is not exhibiting oscillations⁸⁴.Recently, it has been shown that sustained enhancement of Ca²⁺ influx induced by continuous exposure to glibenclamide caused apoptotic cell death in rat insulinoma cell line, RINm5F cells⁸⁵.These results are consistent with the idea that oscillatory Ca²⁺ signals in pancreatic β-cells prevent cellular damage.

Role in energy homeostasis. Oscillations are less costly for maintenance of cell homeostasis65 considering that elevation of [Ca²⁺]i activates energy-consuming processes for extrusion of the ion^33,60,while shortening of the time with a raised [Ca²⁺]i will conserve energy⁶⁴.As an example of the efficiency of oscillatory system in conserving energy, the calculated results suggest that the dissipation of free energy is reduced by 5-10% in oscillatory glycolysis⁸⁶.

Synchronization of cellular processes. Oscillations can be integrated in single cell or tissue level. [Ca²⁺]i oscillations that result in asynchronous pulsatile responses in individual cells or groups of cells will be integrated into a smooth and continuous response in the total output of the tissue³¹. For example, response of single β-cell to glucose is heterogeneous – some cells display [Ca²⁺]i oscillations, others show a sustained rise, whereas a small proportion appear unresponsive^87-89. However, a consistent oscillatory [Ca²⁺]i response is observed in clusters of 5-8 mouse pancreatic b-cells stimulated with 15 mM Glucose^37,65. The heterogeneous response of isolated cells to glucose is masked when they are organized in the whole islets as a result of a very efficient coupling mechanism, which leads to synchronous glucose-induced oscillations of [Ca²⁺]i^34,35. Analysis of [Ca²⁺]i oscillations in different regions of a single glucose-stimulated islet also showed that they may be of variable amplitude but are always synchronous. Simultaneous measurements of [Ca²⁺]i and insulin secretion in single mouse islets show that each [Ca²⁺]i oscillation is accompanied by an oscillation of secretion⁹⁰. This synchrony persists when the frequency of [Ca²⁺]i oscillations is modified by a change in glucose concentration^9l. Thus, pulsatile insulin secretion, triggered by highly integrated [Ca²⁺]i oscillations, from each islet is ultimately responsible for integrated pulsatile secretion by the whole pancreas and the generation of plasma insulin oscillations which are important for optimal action of the hormone⁶⁵.

Temporal control of cellular activity. In the biological targets the [Ca²⁺]i signal responds to the frequency of [Ca²⁺]i spikes rather than to the amplitude of [Ca²⁺]i change. This has given rise to the concept of frequency-modulated [Ca²⁺]i signaling^78,86. However, it has also been reported that Ca²⁺ oscillations can be modulated both in frequency and amplitude^92-94. Frequency encoding results in enhanced precision of control; it is particularly resistant to distortion by background noise⁸⁶. Frequency dependent control systems also succeed in environments where amplitude dependent controls fail. If the [Ca²⁺]i is to be used as an amplitude-dependent signal, in which case its level would have to be maintained at a higher concentration for prolonged periods, it would cause calcium toxicity⁹⁵. In pancreatic β-cell the frequencies of the [Ca²⁺]i oscillations vary as a function of agonist concentration⁵⁸. For example, 25 µM carbamylcholine produced transients approximately every 15s, while at 200 µM transients occurred at ~10s intervals. Furthermore, Gilon et a1.⁹¹ explicitly demonstrated that when the concentration of glucose is raised, the peak of [Ca²⁺]i oscillations did not change significantly, but the frequency of [Ca²⁺]i oscillations clearly increased. This may result from glucose capacity to increase the efficacy with which frequency-encoded Ca²⁺ signals activates the exocytotic process and increases insulin release.

Spatial control of cellular activity. Oscillations of [Ca²⁺]i show spatial order, which has indeed functional advantage for periodicity in biological control⁸⁶. A distinctive attribute of biological system is to do the right thing at the right time in the right place. In heart, the temporal entrainment sequence (SA-node to Purkinje fibers to the ventricles) ensures the correct spatial sequence. Failures in this entrainment process can result in some of the clinically observed cardiac arrhythmias. Thus, Durham96 has speculated, “it is more plausible that every region (of the organism) can potentially oscillate at some frequency. Those parts with the highest inherent frequency will establish a phase lead and drive other regions. Waves will, therefore, move along membranes down gradients of inherent frequency”. Another crucial biological process in which precise spatial control is of central importance is the development of multicellular organisms from a single fertilized egg cell⁹⁷. The events are particularly ordered in time and in space. However, the spatial organization of Ca²⁺ oscillations in pancreatic β-cell is not yet convincing. With digital imaging of the Ca²⁺-dependent fluorescence signal it has been demonstrated that [Ca²⁺]i varies substantially within the cell^98a. [Ca²⁺]i first increases in a rim close to the plasma membrane. As the duration of the depolarization is increased, the [Ca²⁺]i-transient extends progressively deeper into the cell. However, at least during the first 350ms, [Ca²⁺]i remains highest in the vicinity of the plasma membrane. It is of interest, that the increase in [Ca²⁺]i is particularly rapid and pronounced in the upper right part of the cell whereas other parts of the cell remain relatively unaffected^98a. The observation, that the [Ca²⁺]i-increase is more pronounced in certain parts of the cell may indicate an uneven distribution of Ca²⁺-channels in the β-cell membrane. It is tempting to speculate that regions of the plasma membrane with a high Ca²⁺-channel density correspond to ‘hot spots’ of exocytosis. Thus the spatial organization of oscillatory Ca²⁺ signal could lead to a provision of high Ca²⁺ concentration needed at the exocytotic sites while lower [Ca²⁺]i may be sufficient to activate other essential processes, such as the movement of insulin granules from storage to release sites^98b.

Discrimination between signal and noise. Calcium signal is digitized in the form of oscillations^95,99 and a digitally encoded signal with the all or none property has favorable ‘signal-to-noise’ ratios^70,100. By relying on large, discrete digital events, e.g. calcium oscillations, cells can readily distinguish an “intentional” calcium signal from potentially spurious wanderings of the steady-state, cytoplasmic calcium concentration. Indeed, in the brain, bursts of electrical activity are more readily perceived as signals than are action potentials that arrive singly.

Conclusion

The periodic changes of [Ca²⁺]i is of great physiological and pathological importance since [Ca²⁺]i oscillates in synchrony with electrical activity and oscillations in [Ca²⁺]i correspond to pulsatile insulin release^13-17. It has also been proposed that oscillatory insulin secretion is important in terms of insulin action on target organs, perhaps because of reducing down-regulation of receptors and thereby enhancing hormone action¹⁰¹. Several studies have demonstrated a greater hypoglycemic effect of insulin infused in a pulsatile manner and an enhancement of glucose disposal^102-104. The greater potency of pulsatile insulin administration has also been demonstrated in perifused liver and in humans with IDDM^105,106. Whether the loss of oscillations during the development of type 2 diabetes contributes to insulin resistance has not yet been established. But it is a widely acknowledged fact that the regular pattern of oscillatory insulin release is altered or lost in both developing type 1 and type 2 diabetes and disappearance of [Ca²⁺]i oscillations is a sensitive indicator of β-cell damage^107-112. Thus, a detailed study of the mechanisms which underlay the presence of regular [Ca²⁺]i oscillations may help to find out the molecular and physiological defects involved in the pathogenesis of diabetes.

References

1.    Visscher MB. Summary of conference on rhythmic functions in the living system. Ann N Y Acad Sci 1962; 98: 1316-1321.

2.    Wolf W. Introductory remarks. Ann N Y Acad Sci 1962; 98: 755-756.

3.    Zethelius B, Berglund L, Lithell H, Berne C. Seasonal variations of insulin sensitivity, plasma glucose and insulin secretion. Ups J Med Sci 2001; 106 Supp12: 87.

4.    Carney TA, Guy SP, Helliwell CD. Seasonal variation in HbA1C in patients with Type 2 diabetes mellitus. Diabet Med 2000; 17: 554-555.

5.    Ishii H, Suzuki H, Baba T, Nakamura K, Watanabe T. Seasonal variation of glycemic control in type 2 diabetic patients. Diabetes Care 2001; 24: 1503.

6.    Maguire GA, Edwards OM. Seasonal variation in glycated haemoglobin in diabetics. Ann Clin Biochem 2001; 38: 59-60.

7.    Mollerstrom J, Sollberger A. Fundamental concepts underlying the metabolic periodicity in diabetes. Ann N Y Acad Sci 1962; 98: 984-994.

8.    Hellman B, Gylfe E, Bergsten P, Grapengiesser E, Lund PE, Berts A, Tengholm A, Pipeleers DG, Ling Z. Glucose induces oscillatory Ca2+ signalling and insulin release in human pancreatic beta cells. Diabetologia 1994; 37 Supp12: S11­S20.

9.    Grapengiesser E, Gylfe E, Hellman B. Glucose-induced oscillations of cytoplasmic Ca2+ in the pancreatic b-cell. Biochem Biophys Res Commun 1988; 151: 1299-1304.

10.   Valdeolmillos M, Santos RM, Contreras D, Soria B, Rosario LM. Glucose­induced oscillations of intracellular Ca2+ concentration resembling bursting electrical activity in single mouse islets of Langerhans. FEBS Lett 1989; 259: 19-23.

11.   Ahmed M, Grapengiesser E, Hellman B. Amino acid transformation of oscillatory Ca2+ signals in mouse pancreatic b-cells. J Endocrinol 1999; 160: 191-195.

12.   Fernandez J, Valdeolmillos M. Synchronous glucose-dependent [Ca2+]i oscillations in mouse pancreatic islets of Langerhans recorded in vivo. FEBS Lett 2000; 477: 33-36.

13.   Rosario LM, Atwater I, Scott AM. Pulsatile insulin release and electrical activity from single ob/ob mouse islets of Langerhans. Adv Exp Med Biol 1986; 211: 413-425.

14.   Santos RM, Rosario LM, Nadal A, Garcia-Sancho J, Soria B, Valdeolmillos M. Widespread synchronous [Ca2+]i oscillations due to bursting electrical activity in single pancreatic islets. Pflugers Arch 1991; 418: 417-422.

15.   Gilon P, Henquin JC. Influence of membrane potential changes on cytoplasmic Ca++ concentration in an electrically excitable cell, the insulin-secreting pancreatic B cell. J Biol Chem 1992; 267: 20713-20720.

16.   Gilon P, Shepherd RM, Henquin JC. Oscillations of secretion driven by oscillations of cytoplasmic Ca2+ as evidences in single pancreatic islets. J Biol Chem 1993; 268:22265-22268.

17.   Bergsten P, Grapengiesser E, Gylfe E, Tengholm A, Hellman B. Synchronous oscillations of cytoplasmic Ca2+ and insulin release in glucose-stimulated pancreatic islets. J Biol Chem 1994; 269: 8749-8753.

18.   Gylfe E, Ahmed M, Bergsten P, Dansk H, Dyachok O, Eberhardson M, Grapengiesser E, Hellman B, Lin JM, Sundsten T, Tengholm A, Vieira E, Westerlund J. Signaling underlying pulsatile insulin secretion. Ups J Med Sci 2000; 105: 35-51.

19.   Ashcroft FM, Rorsman P. Electrophysiology of the pancreatic b-cell. Prog Biophys Mol Biol 1989; 54: 87-143.

20.   Misler S, Barnett DW, Gillis KD, Pressel DM. Electrophysiology of stimulus ­secretion coupling in human b-cells. Diabetes 1992; 41: 1221-1228.

21.   Rana RS, Sekar MC, Mertz RJ, Hokins LE, MacDonald MJ. Potentiation by glucose metabolites of inositol trisphosphate-induced calcium mobilization in permeabilized rat pancreatic islets. J Biol Chem 1987; 262: 13567-13570.

22.   Roe MW, Lancaster ME, Mertz RJ, Worley JF, III, Dukes ID. Voltage­ dependent intracellular calcium release from mouse islets stimulated by glucose. J Biol Chem 1993; 268: 9953-9956.

23.   Liu YJ, Grapengiesser E, Gylfe E, Hellman B. Crosstalk between the cAMP and inositol trisphosphate-signalling pathways in pancreatic b-cells. Arch Biochem Biophys 1996; 334: 295-302.

24.   Worley JF, III, McIntyre MS, Spencer B, Dukes ID. Depletion of intracellular Ca2+ stores activates a maitotoxin-sensitive nonselective cationic current in b-­cells. J Biol Chem 1994; 269: 32055-32058.

25.   Miura Y, Henquin JC, Gilon P. Emptying of intracellular Ca2+ stores stimulates Ca2+ entry in mouse pancreatic b-cells by both direct and indirect mechanisms. J Physiol 1997; 503 (Pt 2): 387-398.

26.   Liu YJ, Gylfe E. Store-operated Ca2+ entry in insulin-releasing pancreatic b-cells. Cell Calcium 1997; 22:277-286.

27.   Gagliardino JJ, Rossi JP. Ca2+-ATPase in pancreatic islets: its possible role in the regulation of insulin secretion. Diabetes Metab Rev 1994; 10: 1-17.

28.   Varadi A, Molnar E, Ashcroft SJ. A unique combination of plasma membrane Ca2+-ATPase isoforms is expressed in islets of Langerhans and pancreatic b-cell lines. Biochem J 1996; 314 (Pt 2): 663-669.

29.   Van Eylen F, Svoboda M, Herchuelz A. Identification, expression pattern and potential activity of Na/Ca exchanger isoforms in rat pancreatic B-cells. Cell Calcium 1997; 21: 185-193.

30.   Berridge MJ. Cytoplasmic calcium oscillations: a two pool model. Cell Calcium 1991; 12: 63-72.

31.   Thomas AP, Bird GS, Hajnoczky G, Robb-Gaspers LD, Putney JW, Jr. Spatial and temporal aspects of cellular calcium signaling. FASEB J 1996; 10: 1505­1517.

32.   Rooney TA, Thomas AP. Organization of intracellular calcium signals generated by inositol  lipid-dependent hormones. Pharmacol Ther 1991; 49: 223-237.

33.   Fewtrell C. Ca2+ oscillations in non-excitable cells. Annu Rev Physiol 1993; 55: 427-454.

34.   Valdeolmillos M, Nadal A, Soria B, Garcia-Sancho J. Fluorescence digital image analysis of glucose-induced [Ca 2+];oscillations in mouse pancreatic islets of Langerhans. Diabetes 1993; 42: 1210-1214.

35.   Liu YJ, Tengholm A, Grapengiesser E, Hellman B, Gylfe E. Origin of slow and fast oscillations of Ca2+ in mouse pancreatic islets. J Physiol 1998; 508 (Pt 2): 471-481.

36.   Hellman B, Gylfe E, Grapengiesser E, Lund PE, Berts A. Cytoplasmic Ca2+ oscillations ,in pancreatic b-cells. Biochim Biophys Acta 1992; 1113: 295-305.

37.   Gylfe E, Grapengiesser E, Hellman B. Propagation of cytoplasmic Ca2+ oscillations in clusters of pancreatic b-cells exposed to glucose. Cell Calcium 1991; 12: 229-240.

38.   Grapengiesser E, Gylfe E, Hellman B. Cyclic AMP as a determinant for glucose induction of fast Ca2+ oscillations in isolated pancreatic b-cells. J Biol Chem 1991; 266: 12207-12210.

39.   Rorsman P, Abrahamsson H, Gylfe E, Hellman B. Dual effects of glucose on the cytosolic Ca 2+ activity of mouse pancreatic b-cells. FEBS Lett 1984; 170: 196­200.

40.   Gylfe E. Glucose-induced early changes in cytoplasmic calcium of pancreatic b­cells studied with time-sharing dual-wavelength fluorometry. J Biol Chem 1988; 263: 5044-5048.

41.   Roe MW, Mertz RJ, Lancaster ME, Worley JF, III, Dukes ID. Thapsigargin inhibits the glucose-induced decrease of intracellular Ca2+ in mouse islets of Langerhans. Am J Physiol 1994; 266: E852-E862.

42.   Grapengiesser E, Gylfe E, Hellman B. Three types of cytoplasmic Ca2+ oscillations in stimulated pancreatic b-cells. Arch Biochem Biophys 1989; 268: 404-407.

43.   Grapengiesser E, Gylfe E, Hellman B. Ca2+ oscillations in pancreatic b-cells exposed to leucine and arginine. Acta Physiol Scand 1989; 136: 113-119.

44.   Martin F, Soria B. Amino acid-induced [Ca2+]i oscillations in single mouse pancreatic islets of Langerhans. J Physiol 1995; 486 (Pt 2): 361-371.

45.   Martin F, Sanchez-Andres JV, Soria B. Slow [Ca2+]i oscillations induced by ketoisocaproate in single mouse pancreatic islets. Diabetes 1995; 44: 300-305.

46.   Grapengiesser E, Gylfe E, Hellman B. Sulfonylurea mimics the effect of glucose in inducing large amplitude oscillations of cytoplasmic Ca2+ in pancreatic b-­cells. Mol Pharmacol 1990; 37: 461-467.

47.   Corkey BE, Tornheim K, Deeney JT, Glennon MC, Parker JC, Matschinsky FM, Ruderman NB, Prentki M. Linked oscillations of free Ca2+ and the ATP/ADP ratio in permeabilized RINm5F insulinoma cells supplemented with a glycolyzing cell-free muscle extract. J Biol Chem 1988; 263: 4254-4258.

48.   Corkey BE, Deeney JT, Glennon MC, Matschinsky FM, Prentki M. Regulation of steady-state free Ca2+ levels by the ATP/ADP ratio and orthophosphate in permeabilized RINm5F insulinoma cells. J Biol Chem 1988; 263: 4247-4253.

49.   Longo EA, Tornheim K, Deeney JT, Varnum BA, Tillotson D, Prentki M, Corkey BE. Oscillations in cytosolic free Ca2+, oxygen consumption, and insulin secretion in glucose-stimulated rat pancreatic islets. J Biol Chem 1991; 266: 9314-9319.

50.   Dryselius S, Lund PE, Gylfe E, Hellman B. Variations in ATP-sensitive K+ channel activity provide evidence for inherent metabolic oscillations in pancreatic b-cells. Biochem Biophys Res Commun 1994; 205: 880-885.

51.   Larsson O, Kindmark H, Brandstrom R, Fredholm B, Berggren PO. Oscillations in KATP channel activity promote oscillations in cytoplasmic free Ca2+ concentration in the pancreatic b-cell. Proc Natl Acad Sci USA 1996; 93: 5161-5165.

52.   Ämmälä C, Larsson O, Berggren PO, Bokvist K, Juntti-Berggren L, Kindmark H, Rorsman P. Inositol trisphosphate-dependent periodic activation of a Ca2+-activated K+ conductance in glucose-stimulated pancreatic b-cells. Nature 1991; 353: 849-852.

53.   Gilon P, Arredouani A, Gailly P, Gromada J, Henquin JC. Uptake and release of Ca2+ by the endoplasmic reticulum contribute to the oscillations of the cytosolic Ca2+ concentration triggered by Ca2+ influx in the electrically excitable pancreatic B-cell. J Biol Chem 1999; 274: 20197-20205.

54.   Dryselius S, Grapengiesser E, Hellman B, Gylfe E. Voltage-dependent entry and generation of slow Ca2+ oscillations in glucose-stimulated pancreatic b-cells. Am J Physiol 1999; 276: E512-E518.

55.   Leech CA, Holz GG, Habener JF. Voltage-independent calcium channels mediate slow oscillations of cytosolic calcium that are glucose dependent in pancreatic b-cells. Endocrinology 1994; 135: 365-372.

56.   Ahmed M, Grapengiesser E. Pancreatic b-cells from obese-hyperglycemic mice are characterized by excessive firing of cytoplasmic Ca2+ transients. Endocrine 2001; 15: 73-78.

57.   Ahmed M, Grapengiesser E. Ca2+ handling of rat pancreatic beta-cells exposed to ryanodine, caffeine, and glucagon. Endocrine 2002; 17: 103-108.

58.   Prentki M, Glennon MC, Thomas AP, Morris RL, Matschinsky FM, Corkey BE. Cell-specific patterns of oscillating free Ca2+ in carbamylcholine-stimulated insulinoma cells. J Biol Chem 1988; 263: 11044-11047.

59.   Whitaker M. Ways of looking at calcium. Microsc Res Tech 1999; 46: 342­347.

60.   Carafoli E, Penniston JT. The calcium signal. Sci Am 1985; 253: 50-58.

61.   Gilon P, Jonas JC, Henquin JC. Culture duration and conditions affect the oscillations of cytoplasmic calcium concentration induced by glucose in mouse pancreatic islets. Diabetologia 1994; 37: 1007-1014.

62.   Bergsten P. Glucose-induced pulsatile insulin release from single islets at stable and oscillatory cytoplasmic Ca2+. Am J Physiol 1998; 274: E796-E800.

63.   Jonas JC, Gilon P, Henquin JC. Temporal and quantitative correlations between insulin secretion and stably elevated or oscillatory cytoplasmic Ca2 in mouse pancreatic b-cells. Diabetes 1998; 47: 1266-1273.

64.   Jacob R. Calcium oscillations in electrically non-excitable cells. Biochim Biophys Acta 1990; 1052: 427-438.

65.   Henquin JC, Jonas JC, Gilon P. Functional significance of Ca2+ oscillations in pancreatic b-cells. Diabetes Metab 1998; 24: 30-36.

66.   Schlegel W, Mollard P: Electrical activity and stimulus-secretion coupling in neuroendocrine cells, in Scherubl H, Hescheler J (eds): The electrophysiology of neuroendocrine cells, CRC Press, 1995; pp 23-38.

67.   Sato Y, Aizawa T, Komatsu M, Okada N, Yamada T. Dual functional role of membrane depolarization/Ca2+ influx in rat pancreatic B-cell. Diabetes 1992; 41: 438-443.

68.   Cunningham BA, Deeney JT, Bliss CR, Corkey BE, Tornheim K. Glucose­induced oscillatory insulin secretion in perifused rat pancreatic islets and clonal b-cells (HIT). Am J Physiol 1996; 271: E702-E710.

69.   Brabant G, Prank K, Schöfl C. Pulsatile patterns in hormone secretion. TEM 1992; 3: 183-190.

70.   Putney JW, Jr. Calcium signaling: up, down, up, down ...what’s the point? Science 1998; 279: 191-192.

71.   Melloul D, Cerasi E: Transcriptional regulation of proinsulin synthesis by glucose, in Flatt PR, Lenzen S (eds): Insulin secretion and pancreatic B-cell research, Smith Gordon: London, 1994; pp 7-13

72.   Meldolesi J. Calcium signalling. Oscillation, activation, expression. Nature 1998; 392: 863, 865-863, 866.

73.   Li W, Llopis J, Whitney M, Zlokarnik G, Tsien RY. Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression. Nature 1998; 392: 936-941.

74.   Dolmetsch RE, Xu K, Lewis RS. Calcium oscillations increase the efficiency and specificity of gene expression. Nature 1998; 392: 933-936.

75.   Hajnoczky G, Robb-Gaspers LD, Seitz MB, Thomas AP. Decoding of cytosolic calcium oscillations in the mitochondria. Cell 1995; 82: 415-424.

76.   Pralong WF, Spät A, Wollheim CB. Dynamic pacing of cell metabolism by intracellular Ca2+ transients. J Biol Chem 1994; 269: 27310-27314.

77.   Nakazaki M, Ishihara H, Kakei M, Inukai K, Asano T, Miyazaki JI, Tanaka H, Kikuchi M, Yada T, Oka Y. Repetitive mitochondrial Ca2+ signals synchronize with cytosolic Ca2+ oscillations in the pancreatic beta-cell line, MIN6. Diabetologia 1998; 41: 279-286.

78.   Woods NM, Cuthbertson KS, Cobbold PH. Repetitive transient rises in cytoplasmic free calcium in hormone-stimulated hepatocytes. Nature 1986; 319: 600-602.

79.   McCormack JG, Browne HM, Dawes NJ. Studies on mitochondrial Ca2+-­transport and matrix Ca2+ using fura-2-loaded rat heart mitochondria. Biochim Biophys Acta 1989; 973: 420-427.

80.   Nicotera P, McConkey DJ, Dypbukt JM, Jones DP, Orrenius S. Ca2+-activated mechanisms in cell killing. Drug Metab Rev 1989; 20: 193-201.

81.   Trump BF, Berezesky IK. The role of altered [Ca2+]i regulation in apoptosis, oncosis, and necrosis. Biochim Biophys Acta 1996; 1313: 173-178.

82.   McConkey DJ, Nicotera P, Hartzell P, Bellomo G, Wyllie AH, Orrenius S. Glucocorticoids activate a suicide process in thymocytes through an elevation of cytosolic Ca2+ concentration. Arch Biochem Biophys 1989; 269: 365-370.

83.   Mirabelli F, Bellomo G, Nicotera P, Moore M, Orrenius S. Ca2+ homeostasis and cytotoxicity in isolated hepatocytes: studies with extracellular adenosine 5’-­triphosphate. J Biochem Toxicol 1986; 1: 29-39.

84.   Efanova IB, Zaitsev SV, Zhivotovsky B, Kohler M, Efendic S, Orrenius S, Berggren PO. Glucose and tolbutamide induce apoptosis in pancreatic b-cells. A process dependent on intracellular Ca2+ concentration. J Biol Chem 1998; 273: 33501-33507.

85.    Iwakura T, Fujimoto S, Kagimoto S, Inada A, Kubota A, Someya Y, Ihara Y, Yamada Y, Seino Y. Sustained enhancement of Ca 2+ influx by glibenclamide induces apoptosis in RINm5F cells. Biochem Biophys Res Commun 2000; 271: 422-428.

86.   Rapp PE. Why are so many biological systems periodic? Prog Neurobiol 1987; 29: 261-273.

87.   Herchuelz A, Pochet R, Pastiels C, Van Praet A. Heterogeneous changes in [Ca2+], induced by glucose, tolbutamide and K+ in single rat pancreatic B cells. Cell Calcium 1991; 12: 577-586.

88.   Grapengiesser E, Gylfe E, Hellman B. Glucose sensing of individual pancreatic b-cells involves transitions between steady-state and oscillatory cytoplasmic Ca2+. Cell Calcium 1992; 13: 219-226.

89.   Jonkers FC, Jonas JC, Gilon P, Henquin JC. Influence of cell number on the characteristics and synchrony of Ca2+ oscillations in clusters of mouse pancreatic islet cells. J Physiol 1999; 520 (Pt 3): 839-849.

90.   Bergsten P. Slow and fast oscillations of cytoplasmic Ca2+ in pancreatic islets correspond to pulsatile insulin release. Am J Physiol 1995; 268: E282-E287.

91.   Gilon P, Henquin JC. Distinct effects of glucose on the synchronous oscillations of insulin release and cytoplasmic Ca2+ concentration measured simultaneously in single mouse islets. Endocrinology 1995; 136: 5725-5730.

92.   D’Andrea P, Codazzi F, Zacchetti D, Meldolesi J, Grohovaz F. Oscillations of cytosolic calcium in rat chromaffm cells: dual modulation in frequency and amplitude. Biochem Biophys Res Commun 1994; 205: 1264-1269.

93.   Eberhardson M, Tengholm A, Grapengiesser E. The role of plasma membrane K+ and Ca2+ permeabilities for glucose induction of slow Ca2+ oscillations in pancreatic b-cells. Biochim Biophys Acta 1996; 1283: 67-72.

94.   Pabelick CM, Prakash YS, Kannan MS, Jones KA, Warner DO, Sieck GC. Effect of halothane on intracellular calcium oscillations in porcine tracheal smooth muscle cells. Am J Physiol 1999; 276: L81-L89.

95.   Berridge MJ, Galione A. Cytosolic calcium oscillators. FASEB J 1988; 2: 3074-3082.

96.   Durham AC. A unified theory of the control of actin and myosin in nonmuscle movements. Cell 1974; 2: 123-135.

97.   Miyazaki S. Repetitive calcium transients in hamster oocytes. Cell Calcium 1991; 12: 205-216.

98a.  Rorsman P, Bokvist K, Ammala C, Eliasson L: Ca2+-channels, cytoplasmic Ca2+-concentration and exocytosis in pancreatic B-cell, in Flatt PR, Lenzen S (eds): Insulin secretion and pancreatic B-cell research, Smith Gordon: London, 1994; pp 187­-194.

98b.   Barg S, Ma X, Eliasson L, Galvanovskis J, Gopel SO, Obermuller S, P