Intracellular Free Calcium Activity Research Articles

Phase transitions in the multi-cellular regulatory behavior of pancreatic islet excitability.

The pancreatic islets of Langerhans are multicellular micro-organs integral to maintaining glucose homeostasis through secretion of the hormone insulin. β-cells within the islet exist as a highly coupled electrical network which coordinates electrical activity and insulin release at high glucose, but leads to global suppression at basal glucose. Despite its importance, how network dynamics generate this emergent binary on/off behavior remains to be elucidated. Previous work has suggested that a small threshold of quiescent cells is able to suppress the entire network. By modeling the islet as a Boolean network, we predicted a phase-transition between globally active and inactive states would emerge near this threshold number of cells, indicative of critical behavior. This was tested using islets with an inducible-expression mutation which renders defined numbers of cells electrically inactive, together with pharmacological modulation of electrical activity. This was combined with real-time imaging of intracellular free-calcium activity [Ca2+]i and measurement of physiological parameters in mice. As the number of inexcitable cells was increased beyond ∼15%, a phase-transition in islet activity occurred, switching from globally active wild-type behavior to global quiescence. This phase-transition was also seen in insulin secretion and blood glucose, indicating physiological impact. This behavior was reproduced in a multicellular dynamical model suggesting critical behavior in the islet may obey general properties of coupled heterogeneous networks. This study represents the first detailed explanation for how the islet facilitates inhibitory activity in spite of a heterogeneous cell population, as well as the role this plays in diabetes and its reversal. We further explain how islets utilize this critical behavior to leverage cellular heterogeneity and coordinate a robust insulin response with high dynamic range. These findings also give new insight into emergent multicellular dynamics in general which are applicable to many coupled physiological systems, specifically where inhibitory dynamics result from coupled networks.

Dimensionality and Calcium Dynamics in Pancreatic β-Cell Clusters

The pancreatic islet is a multicellular organ which provides the sole source of insulin for regulating blood glucose levels. Thus, a functional understanding of the islet is of critical importance to effectively treat diabetes. Beta cells within native islets produce large bursts of insulin to meet physiological needs by mounting a coordinated electrophysiological response. However, when removed from their native three dimensional (3D) environment, they lose much of their coordination and responsiveness. This observation draws parallels with the hindered diabetic response and emphasizes the importance of understanding the electrical dynamics that underlie the coordinated pulsatile secretion of insulin.In this work, we use quantitative microscopy to measure intra-cellular free calcium activity ([Ca2+]i) and coordination of β-cells in 1D, 2D and 3D clusters. using a novel method of aggregating β-cells back into 3D systems, we can provide a better model for understanding the effects of dimensionality and coupling on ([Ca2+]i) dynamics. We have found that by re-aggregating β-cells, they return to a native-like oscillatory behavior, which is lost in 2D cultures. In addition, the overall coordination of 2D clusters was found to decrease with cluster size, showing distinct sub-regions of coordination, while the 3D cultures were better coordinated at comparable sizes and cell numbers.Using a percolation-based theory for modeling cell coordination, we show how higher dimensions of coupling lead to better overall synchrony with larger regions of coordination. This model also predicts the distances over which coupled cells can communicate. This has broader implications in that a loss of cell coupling and a reduction in coordination and responsiveness are often seen in islets from models of type2 diabetes. Also, the application of our mathematical model to predict cellular phenomena yields new insight in understanding the coordinated behavior of electrophysiological signaling in general.

Defects in beta cell Ca2+ signalling, glucose metabolism and insulin secretion in a murine model of KATP channel-induced neonatal diabetes mellitus

Mutations that render ATP-sensitive potassium (K(ATP)) channels insensitive to ATP inhibition cause neonatal diabetes mellitus. In mice, these mutations cause insulin secretion to be lost initially and, as the disease progresses, beta cell mass and insulin content also disappear. We investigated whether defects in calcium signalling alone are sufficient to explain short-term and long-term islet dysfunction. We examined the metabolic, electrical and insulin secretion response in islets from mice that become diabetic after induction of ATP-insensitive Kir6.2 expression. To separate direct effects of K(ATP) overactivity on beta cell function from indirect effects of prolonged hyperglycaemia, normal glycaemia was maintained by protective exogenous islet transplantation. In endogenous islets from protected animals, glucose-dependent elevations of intracellular free-calcium activity ([Ca(2+)](i)) were severely blunted. Insulin content of these islets was normal, and sulfonylureas and KCl stimulated increased [Ca(2+)](i). In the absence of transplant protection, [Ca(2+)](i) responses were similar, but glucose metabolism and redox state were dramatically altered; sulfonylurea- and KCl-stimulated insulin secretion was also lost, because of systemic effects induced by long-term hyperglycaemia and/or hypoinsulinaemia. In both cases, [Ca(2+)](i) dynamics were synchronous across the islet. After reduction of gap-junction coupling, glucose-dependent [Ca(2+)](i) and insulin secretion was partially restored, indicating that excitability of weakly expressing cells is suppressed by cells expressing mutants, via gap-junctions. The primary defect in K(ATP)-induced neonatal diabetes mellitus is failure of glucose metabolism to elevate [Ca(2+)](i), which suppresses insulin secretion and mildly alters islet glucose metabolism. Loss of insulin content and mitochondrial dysfunction are secondary to the long-term hyperglycaemia and/or hypoinsulinaemia that result from the absence of glucose-dependent insulin secretion.

Quantitative Experimental Measurements And Mathematical Modeling Of Multi-cellular Dynamics In The Islet Of Langerhans

Multi-cellular interactions and dynamics are key to mammalian physiology. Many organs such as the heart or brain have complex functionality that is brought about by the interactions between constituent cells. To discover general rules and principles that govern multi-cellular behavior, we are studying a relatively simple mammalian multi-cellular system- the islet of Langerhans. The islet is located in the pancreas and provides the sole source of the hormone insulin for regulating blood glucose levels. Thus, understanding the function of the islet is of critical importance to effectively treat diabetes. In this work, we have focused on understanding the electrical dynamics in the islet that underlie the coordinated pulsatile secretion of insulin. We have used quantitative microscopy to measure intra-cellular free calcium activity ([Ca2+]i) simultaneously over a large cellular population of the islet. To provide an experimental axis, we introduced graded changes in the electrical coupling between beta cells by applying both chemical inhibitors of gap junction activity as well as a genetic knockout of the gap junction protein. Upon this reduction in electrical coupling, synchronization of pulsatile electrical activity decreased throughout the islet. Furthermore, the propagating [Ca2+]i waves, which serve to synchronize electrical oscillations, slowed and were disrupted as electrical coupling was reduced. Using a mathematical model of islet cell electrical activity and multi-cellular coupling, we can quantitatively reproduce this experimental data. This allows us to make quantitative predictions of the multi-cellular electrical behavior for any given level of electrical coupling, as well as expected behaviors under perturbations of other parameters, such as K+-channel mutations. We can also hypothesize that this behavior is general for electrical coupling in other multi-cellular systems, potentially allowing us to predict the electrical dynamics in other neuro-endocrine cell systems.

Intracellular free calcium activity is increased in migrating gastric epithelial cells

Intracellular free calcium activity is increased in migrating gastric epithelial cells

Regulation of Rabbit Erythrocyte Ca2+-Pump Sensitivity to Calmodulin in Experimental Hyperlipidemia

Intracellular free calcium activity is in part determined by a calmodulin-regulated plasma membrane Ca(2+)-pump. Since changes in Ca2+ permeability have been implicated in atherosclerotic plaque formation, we initiated a lipid hyperalimentation protocol during which we measured various erythrocyte calcium flux parameters and early atheroma development. Adolescent New Zealand White rabbits were fed a diet with 0.5% cholesterol and 2.5% lard over a 3-month period. Plasma cholesterol and triacylglycerols increased on average 18.7- and 13.9-fold respectively, while erythrocyte membrane cholesterol content decreased 18% and total phospholipids by 54%. After 3 months of lipid hyperalimentation, 22% of the aortic arch was covered with large, early-stage, raised atheroma. Basal and calmodulin-activated (Ca2+ + Mg2+)-ATPase activities in erythrocyte membranes increased by 31% and 123%, respectively at 2 months, with a concomitant increase in calmodulin affinity (Km) from 15.6 to 4.2 nM. These differences were transient on account of changes in the control animals which exhibited a slowly developing sensitivity to calmodulin during maturation. Basal Ca2+ transport and passive Ca2+ permeability increased about 7-fold during the hyperlipidemic phase. This suggests that overt hyperlipidemia, leading to atherosclerotic plaque development, alters plasma membrane Ca2+ regulatory mechanisms including passive Ca2+ permeability. The changes in enzymatic function, membrane composition, and Ca2+ permeability seen in this red cell model system may be a reflection of early changes in cells that are directly involved in the development of atherosclerotic plaques.

Two-photon-excitation fluorescence imaging of three-dimensional calcium-ion activity

Two-photon excitation of the ultraviolet-absorbing fluorescent calcium indicator Indo-1 in laser scanning microscopy makes possible a quantitative, three-dimensional recording of intracellular free calcium activity ([Ca(2equation)](i)) distributions and dynamics with low background and minimal photobleaching. We have constructed a simple optical system that facilitates collection of the 400-500-nm Indo-1 fluorescence without the use of a confocal spatial filter. Instead of the fluorescence being descanned as is normally required in confocal microscopy, the fluorescence is deflected by a dichroic mirror into a separate detection pathway. Images of [Ca(2+)](i) distributions with three-dimensional submicrometer resolution and 10% precision are obtained at100-µM Indo-1 concentration and 3-s recording time for 384 × 512 pixels. Data on [Ca(2+)](i) in tumor mast cells and cardiac myocytes illustrate the capabilities of this technique.

Effects of histamine on inositol phosphates and intracellular Ca<sup>2+</sup> in human glomerular epithelial cells

The effect of histamine on the phosphoinositide turnover and intracellular free calcium activity [Ca2+]i was examined in human glomerular epithelial cells in culture. Addition of histamine to glomerular epithelial cells resulted in formation of inositol phosphates in a time- and dose-dependent manner. A transient maximum of inositol trisphosphate (InsP3) was observed within 10 s. Stimulation of protein kinase C by short-term pretreatment (15 min) of glomerular epithelial cells with phorbol 12-myristate 13-acetate caused a dose-dependent inhibition of the histamine-induced inositol phosphate accumulation. The baseline of [Ca2+]i in the cells was 115 +/- 2.7 nmol/l (n = 103). Histamine (ED50: approx. 2 x 10(-7) mol/l) caused a rapid and transient increase in [Ca2+]i as detected by fura-2 microfluorimetry studies. In a calcium-free extracellular solution the rapid increase of [Ca2+]i was still present. The H1 receptor antagonist mepyramine (IC50: approx. 8 x 10(-9) mol/l) inhibited the histamine (10(-6) mol/l) response on [Ca2+]i. Cimetidine, a potent H2 receptor antagonist, showed no effect. This data indicates that H1 receptor activation causes hydrolysis of phosphatidylinositol 4, 5-bisphosphate by phospholipase C activation, and consecutive mobilization of intracellular calcium. Since histamine is a mediator of inflammation, antigen response and cellular injury, these findings could be of importance for the understanding of glomerular epithelial cell pathology.

Intracellular free Ca2+ changes during physiological polyspermy in amphibian eggs

We have made the first measurements of intracellular free calcium activity ([Ca2+]i) in urodele eggs during physiological polyspermic fertilization. Jellied eggs of the urodele amphibian Pleurodeles waltlii were impaled with intracellular Ca(2+)-selective microelectrodes and inseminated under various conditions of sperm:egg ratio to obtain various degrees of polyspermy. In 17 out of 45 cases the egg [Ca2+]i level (0.41 microM) showed no variation following fertilization. In 28 other cases, however, the egg displayed a slow increase in [Ca2+]i of 0.15 microM, starting around 15 minutes after fertilization and reaching a plateau level around 10 minutes later. The amplitude of the fertilization-associated increase in [Ca2+]i was found to be independent of the number of sperm interacting with the egg surface. Measurements with two Ca(2+)-microelectrodes impaled in single eggs showed that the increase in [Ca2+]i did not simultaneously occur at distinct places within the egg cortex and, in some cases, could be detected by only one of the two microelectrodes. This latter observation, as well as the absence of [Ca2+]i change at fertilization in some experiments, strongly suggested that each sperm interacting with the egg might, at various places, trigger a localized, non-propagating change in [Ca2+]i. Experiments in which eggs were locally inseminated, using a micropipette directed towards the site of impalement of one of the two Ca(2+)-microelectrodes, clearly established that [Ca2+]i changes, although incapable of propagating over the entire egg cortex, might nevertheless travel very slowly over short distances, their amplitude vanishing rapidly as they propagate from around the sites of sperm entry.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of bradykinin on the cytosolic free calcium activity and phosphoinositol turnover in human glomerular epithelial cells.

The effect of bradykinin (BK) on the intracellular free calcium activity [Ca2+]i and phosphoinositide (PI) turnover was investigated in human glomerular epithelial cells (GEC) in culture. Human GEC exhibited a baseline [Ca2+]i of 114 +/- 3 nmol (n = 81). BK (ED50 10(-9) mol/l) caused a rapid and transient increase in [Ca2+]i, which could also be observed in the absence of extracellular calcium. The effect of BK (10(-8) mol/l) on the [Ca2+]i was inhibited by the BK2 antagonist Hoe 140 (IC50 10(-8) mol/l). BK also induced PI turnover in a time- and dose-dependent manner. A transient increase in (1,4,5)-inositol-triphosphate (InsP3) formation from 1,445 +/- 119 to 4,629 +/- 323 cpm occurred after 5 s. Stimulation of protein kinase C (PKC) by short-term preincubation (15 min) of human GEC with phorbol-12-myristate-13-acetate (PMA) induced a dose-dependent inhibition of the BK-stimulated (10(-7) mol/l) inositol-phosphate formation. Downregulation of PKC by preincubation of human GEC with PMA (24 h, 10(-6) mol/l) or inhibition of PKC by pretreatment with staurosporin (1 h, 10(-6) mol/l) resulted in a slight but significant augmentation of the BK-induced InsP3 stimulation. The data indicate that BK induces stimulation of [Ca2+]i and PI turnover via a BK2 receptor in human GEC. PKC might exert a negative feedback function for the BK-induced PI turnover.

Intracellular pH and intracellular free calcium responses to protein kinase C activators and inhibitors in Xenopus eggs

Cell activation during fertilization of the egg of Xenopus laevis is accompanied by various metabolic changes, including a permanent increase in intracellular pH (pHi) and a transient increase in intracellular free calcium activity ([Ca2+]i). Recently, it has been proposed that protein kinase C (PKC) is an integral component of the Xenopus fertilization pathway (Bement and Capco, J. Cell Biol. 108, 885-892, 1989). Indeed, activators of PKC trigger cortical granule exocytosis and cortical contraction, two events of egg activation, without, however, releasing the cell cycle arrest (blocked in second metaphase of meiosis). In the egg of Xenopus, exocytosis as well as cell cycle reinitiation are supposed to be triggered by the intracellular Ca2+ transient. We report here that PKC activators do not induce the intracellular Ca2+ transient, or the activation-associated increase in pHi. These results suggest that the ionic responses to egg activation in Xenopus do not appear to depend on the activation of PKC. In addition, in eggs already pretreated with phorbol esters, those artificial activators that act by releasing Ca2+ intracellularly, triggered a diminished increase in pHi. Finally, sphingosine and staurosporine, two potent inhibitors of PKC, were found to trigger egg activation, suggesting that a decrease in PKC activity might be an essential event in the release of the metaphase block, in agreement with recent findings on the release of the prophase block in Xenopus oocytes (Varnold and Smith, Development 109, 597-604, 1990).

Cycling of intracellular free calcium and intracellular pH in Xenopus embryos: a possible role in the control of the cell cycle

ABSTRACT The scope of this commentary is to propose the incorporation of recent data, obtained from Xenopus eggs and embryos, into the models of cell cycle regulation by MPF, a universal M-phase Promoting Factor operating in most, if not all, mitotic cells, from yeast to human. These new data are: (1) the cycling activity of MPF in Xenopus eggs is temporally and functionally related to the cycling activity of intracellular pH (pHi) (Grandin and Charbonneau, 1990a); (2) cell division in Xenopus embryos is accompanied by oscillations of the intracellular free calcium activity ([Ca2+]1) (Grandin and Charbonneau, 1991). There is now definitive evidence that one of the molecular components of the cell cycle, the ‘master oscillator’ (or cytoplasmic clock), is represented by MPF (Masui and Markert, 1971) and its correlated cdc2 kinase activity and cyclin level (see, for instance, Draetta and Beach, 1989; Murray, 1989; Murray and Kirschner, 1989). In addition, a variety of different systems have revealed a direct implication of [Ca2+]1 variations in mitotic events (reviewed by Berridge and Irvine, 1989; Hepler, 1989). Although Xenopus embryonic cells have become, in addition to yeast, one of the most important systems for studying the molecular biology of the cell division cycle, almost no attention has been paid to the possible involvement of ionic messengers, particularly Ca2+, in the control of mitosis in Xenopus embryos. This lack of attention to the possible role of [Ca2+]1 variations in the cell division cycle of Xenopus embryos was due to their repeatedly noted absence. However, the recent demonstration of Ca2+ oscillations occurring with a periodicity equal to that of the cell division cycle in Xenopus embryos (Grandin and Charbonneau, 1991) now offers an opportunity of re-evaluating the already proposed models of cell division.

Changes in intracellular free calcium activity in Xenopus eggs following imposed intracellular pH changes using weak acids and weak bases

The aim of this work was to determine the potential relationships between rises in intracellular pH (pH i) and intracellular free calcium activity (Ca i 2+) during cell activation in Xenopus eggs. To do this, we used two weak bases, NH 4Cl and procaine, and a weak acid, CO 2, and measured Ca i 2+ variations in response to these imposed pH i variations. NH 4Cl and procaine increased Ca i 2+ in both unactivated and activated eggs. Procaine was found to alkalinize the egg cytoplasm, whereas the other weak base, NH 4Cl, acidified the egg cytoplasm. On the other hand, CO 2 was found to acidify the cytoplasm and to substantially decrease Ca i 2+, also in unactivated and activated eggs. In addition, CO 2 triggered an increase in the conductance of the plasma membrane to Cl − ions, similarly to what had been found previously with weak bases (Charbonneau, M. (1989) Cell Differ. Develop. 26, 39–52). These Cl − channels, similarly to the sperm-triggered Cl − channels during the fertilization potential, are supposed to be Ca 2+-sensitive. Therefore, the changes in Ca 2+ observed in response to CO 2 do not seem to be responsible for the opening of these Cl − channels, which would rather be triggered by an increase in Ca i[su2+] localized near the plasma membrane. We conclude therefore that weak acids and bases represent appropriate tools for studying cytosolic Ca 2+ homeostasis, but not for dissecting the complex pathways involved in signal transduction.

The relationship of intracellular free calcium activity to amylase secretion in substance P- and isoprenaline-stimulated rat parotid acini

The relationship between intracellular free calcium activity ([Ca 2+] i) and stimulated amylase secretion was investigated in rat parotid acini by measuring the effects of substance P methyl ester and isoprenaline on quin2 fluorescence and amylase release. Although both of these drugs evoked concentration-dependent increases in [Ca 2+] i and amylase release, the tachykinin had a greater effect on [Ca 2+] i while the catecholamine was the more effective secretagogue. Whereas isoprenaline exerted equipotent effects on amylase secretion and [Ca 2+] i, the dose-response relationship for stimulation of secretion by substance P was dissociated by three orders of magnitude to the right of that for elevation of [Ca 2+] i by this peptide. It is concluded that these data do not support the hypothesis that substance P-stimulated amylase secretion is mediated solely through an increase in [Ca 2+] i and that other second-messengers may be involved in mediation of this secretory response.