Papers by Eva Grapengiesser
Cell Calcium, 1991
Digital image analysis was employed for resolving the t8mporal and spatial variations of the cyto... more Digital image analysis was employed for resolving the t8mporal and spatial variations of the cytoplasmic Ca2+ concentration ([Ca2+Ii) in pancreatic &cells loaded with the Ca2+-indicator Fura-2. Glucosestimulated individual f&cells exhiblt8d large amplitude oscillations of [Ca2+]i with a mean frequency of 0.33 min'j. When Ca2' diffusion #ms restricted by increasing the Ca2+ buffering capacity, the sugar-induc8d rise of [cS2+Ii preferentially affected the peripheral cytoplasm. When gtucagon was present skrcoge also caused less prominent oscillations with about a X&fold higher frequency on an elevated [Ca2+J. In small clusters of 6-14 &Is the average frequency of the large ampiltude osclilations increased to 0.60 min-'. Th8 clusters were found to contain micro-domains of electrically coupled cells with synchronized osciilatlons. Aft8r incr8aslng the glucorpe concentration, adjacent domains became functionally coupled. The osoiltatlons originated from dlfferent cells in the cluster. Also the fast giucagon-dependent oscklations were synehronlzed betw88n ceils and had different origins. The results indkzrte that coupling of &cells leads to an increased frequency of the large amplitude osclllatlons, and that the osclttatory characteristics are determlned CoHecstively among el8ctrlcalty coupled &cells rather than by particular pacemaker cells. In the light of these data lt is nec88sary to reconsider the previous k&s that glucose-induced osdgatlons of membrane po&ntial and [Ca2']i require coupling between many &c8lis, and that the peak [Ca2% values r8ached durlng oscillations should increase wlth the size of the coupled cluster.
Pancreas, 2017
This study aimed to explore how sulfonylurea blockade of K ATP channels affects the early Ca 2+ s... more This study aimed to explore how sulfonylurea blockade of K ATP channels affects the early Ca 2+ signals for glucose generation of insulin release. Methods: Cytoplasmic Ca 2+ was measured with ratiometric microfluorometry in isolated mouse islets loaded with Fura-PE3. Results: After sulfonylurea blockade of the K ATP channels (50 μM-1 mM tolbutamide or 1 μM-1 mM gliclazide), increase of glucose from 3 to 20 mM resulted in suppression of elevated Ca 2+ during a 3-to 5-minute period. The Ca 2+ decrease was shorter after inhibition of the Na/K pump with ouabain (10 and 100 μM) but prolonged when the α 2A adrenoceptors were activated with clonidine (1 and 10 nM) or epinephrine (10 nM). Inhibition of the sarco/endoplasmic reticulum Ca 2+-ATPase pump with 10 μM cyclopiazonic acid counteracted the action of 10 nM clonidine, making the Ca 2+ decrease shorter than in controls. Extended superfusion of islets with a medium containing 20 mM glucose and 1 mM tolbutamide sometimes resulted in delayed appearance of Ca 2+ oscillations mediated by periodic interruption of elevated Ca 2+. Conclusions: Increase of glucose generates prompt suppression of cytoplasmic Ca 2+ in β-cells lacking functional K ATP channels. Activation of α 2A adrenoceptors markedly prolongs the period of glucose-induced Ca 2+ decrease, an effect counteracted by cyclopiazonic acid.
Lakartidningen, 2007
The concentration of circulating insulin oscillates with periods of 3-6 min due to pulsatile rele... more The concentration of circulating insulin oscillates with periods of 3-6 min due to pulsatile release of the hormone from the pancreas. Pulsatile insulin secretion from the individual * cell is driven by slow cycles of Ca2+ elevation due to periodic depolarisation. The Ca2+ oscillations of individual * cells in the islets of Langerhans are entrained into a common rhythm by gap junctional coupling and diffusible factors. Autonomic ganglia coordinate the oscillatory activity of the million islets in the pancreas. ATP binding to purinoceptors causes pronounced Ca2+ spikes that are important for synchronizing the *-cells within and among islets in the pancreas. Inhibition of purinergic P2Y1 receptors selectively abolishes pulsatile insulin release without reducing the average rate of secretion. The insulin oscillations are particularly important for the liver. This organ is also exposed to oscillating levels of glucagon. The latter oscillations are in opposite phase allowing maximal exposure to insulin when the glucagon concentration is at minimum.
Biochem Biophys Res Commun, 2003
Läkartidningen
The concentration of circulating insulin oscillates with periods of 3-6 min due to pulsatile rele... more The concentration of circulating insulin oscillates with periods of 3-6 min due to pulsatile release of the hormone from the pancreas. Pulsatile insulin secretion from the individual * cell is driven by slow cycles of Ca2+ elevation due to periodic depolarisation. The Ca2+ oscillations of individual * cells in the islets of Langerhans are entrained into a common rhythm by gap junctional coupling and diffusible factors. Autonomic ganglia coordinate the oscillatory activity of the million islets in the pancreas. ATP binding to purinoceptors causes pronounced Ca2+ spikes that are important for synchronizing the *-cells within and among islets in the pancreas. Inhibition of purinergic P2Y1 receptors selectively abolishes pulsatile insulin release without reducing the average rate of secretion. The insulin oscillations are particularly important for the liver. This organ is also exposed to oscillating levels of glucagon. The latter oscillations are in opposite phase allowing maximal exposure to insulin when the glucagon concentration is at minimum.
Endocrine, 2002
Reported species differences in the stimulus-secretion coupling of insulin release made it import... more Reported species differences in the stimulus-secretion coupling of insulin release made it important to compare the Ca2+ handling of rat beta-cells with that previously observed in mice. Single beta-cells and small aggregates were prepared from pancreatic islets of Wistar rats, attached to cover slips and then used for measuring the cytoplasmic Ca2+ concentration ([Ca2+]i) with the ratiometric fura-2 technique. Glucose (11 mM) induced slow oscillations of [Ca2+]i similar to those seen in other species, including humans. Comparison of the oscillations in rat beta-cells with those previously described in mouse revealed that there was a slightly lower frequency and an increased tendency to transformation into sustained [Ca2+]i in response to glucagon or caffeine. Ryanodine (5-20 microM) did not affect existing oscillations but sometimes restored rhythmic activity in the presence of caffeine. Stimulation with glucose resulted not only in oscillations but also in transients of [Ca2+]i so...
Diabète & métabolisme
The role of Ca2+ in initiating exocytosis of granule-bound secretory products was evaluated with ... more The role of Ca2+ in initiating exocytosis of granule-bound secretory products was evaluated with respect to pancreatic islet hormones. Apart from stimulating the transfer of the granules to the plasma membrane and their subsequent extrusion, a rise of the cytoplasmic Ca2+ concentration ([Ca2+]i) may under certain conditions also have depressive effects on insulin release. Glucose has a bidirectional action on [Ca2+]i by stimulating both the entry of the ion and its removal by organelle sequestration and outward transport. The recognition of glucose as a secretory stimulus is based on sudden transitions between oscillatory and steady-state [Ca2+]i at threshold concentrations of the sugar characteristic for the individual beta-cell. The intrinsic ability of each beta-cell to generate oscillations of [Ca2+]i and the subsequent synchronization of these signals result in a pulsatile release of insulin from isolated islets. Glucose regulation of this process is manifested as alterations o...
Virus Research, 1994
Five strains of Coxsackie B4 virus and one of Echo 11 virus were tested with regard to their abil... more Five strains of Coxsackie B4 virus and one of Echo 11 virus were tested with regard to their ability to replicate in pancreatic mouse p-cells and interfere with the alterations of the cytoplasmic Ca* + concentration ([Ca*'],) induced by glucose. All strains except one both multiplied and caused cytopathic effect. In a control group 68% of the &cells responded to 11 mM glucose with large amplitude oscillations of [Ca2+li. After inoculation with the infectious strains these oscillations appeared in only 5% of the p-cells, whereas the non-infectious strain did not modify the glucose effect on [Ca2+li. Despite the virus interference with the glucose response, [Ca*+J was increased after depolarization with excessive extracellular K+ and the oscillations were induced in most p-cells when glucose was combined with the insulin-releasing sulfonylurea tolbutamide.
Toxicology, 1990
Dual wavelength microfluorometry and the indicator fura-2 were employed for measuring cytoplasmic... more Dual wavelength microfluorometry and the indicator fura-2 were employed for measuring cytoplasmic Ca2+ (Ca2+i) in individual pancreatic beta-cells isolated from ob/ob-mice. In most beta-cells, a rise of external glucose from 3 to 20 mM resulted in large amplitude oscillations in Ca2+i, superimposed on a basal level of 60-90 nM. The diabetogenic agents streptozotocin and alloxan (1-4.4 mM) rapidly abolished the glucose-induced oscillations of Ca2+i. The presence of a high glucose concentration during the exposure to the drugs counteracted the action of alloxan but not that of streptozotocin. Perturbation of the cyclic variations of Ca2+i by streptozotocin did not interfere with a glucose-induced increase of the ion in mildly affected beta-cells. The most advanced lesions obtained with the exposure to the diabetogenic agents were manifested as uncontrolled and sustained increases of Ca2+i. Although disrupting the intracellular Ca2+ homeostasis by separate mechanisms, streptozotocin and alloxan may finally kill the beta-cells by activating a common suicidal process due to an excessive rise of Ca2+i.
Pancreas, 2003
Introduction: Nonadrenergic, noncholinergic neurons have been proposed to synchronize pulsatile i... more Introduction: Nonadrenergic, noncholinergic neurons have been proposed to synchronize pulsatile insulin release from the islets in the pancreas by triggering transient increases of the cytoplasmic Ca 2+ concentration ([Ca 2+ ] i) in -cells via an inositol trisphoshatedependent mechanism. Aims: To test whether pancreatic -cells respond to stretch activation with similar types of transients and whether these Ca 2+ signals propagate to other -cells in the presence and absence of cell contacts. Methodology: Single cells and small aggregates were prepared from -cell-rich islets from ob/ob mice. After 2-5 days of culture, [Ca 2+ ] i was measured with digital imaging and the indicator fura-2 during superfusion with a medium containing 20 mmol/L glucose and 50 mol/L methoxyverapamil. Membrane stretch was induced by osmotic swelling or focal touch stimulation. Results: Lowering the medium osmolarity with 100-102 mOSM/L by removal of sucrose or by dilution resulted in a 2-3-fold increase in the number of transients during an initial 5-minute period. Sucrose omission was stimulatory also after isosmolar replacement with readily penetrating urea. The intracellular Ca 2+-ATPase inhibitor thapsigargin suppressed both the spontaneously occurring transients and those initiated by volume expansion. Touch stimuli induced [Ca 2+ ] i transients, which rapidly propagated to cells within the same aggregate or lacking contact. Conclusion: The observations support the idea that -cells both receive and regenerate extracellular signals triggering [Ca 2+ ] i transients. Touch stimulation is a useful tool for investigating the propagation of [Ca 2+ ] i signals between pancreatic -cells lacking physical contact.
Pancreas, 2007
Pancreatic beta cells respond to glucose stimulation with pulses of insulin release generated by ... more Pancreatic beta cells respond to glucose stimulation with pulses of insulin release generated by oscillatory rises of the cytoplasmic Ca2+ concentration ([Ca2+]i). The observation that exposure to external ATP and other activators of cytoplasmic phospholipase A2 (cPLA2) rapidly induces rises of [Ca2+]i similar to ordinary oscillations made it important to analyze whether suppression of the cPLA2 activity affects glucose-induced [Ca2+]i rhythmicity in pancreatic beta cells. Ratiometric fura-2 technique was used for measuring [Ca2+]i in single beta cells and small aggregates prepared from ob/ob mouse islets. Testing the effects of different inhibitors of cPLA2 in the presence of 20 mM glucose, it was found that N-(p-amylcinnamoyl)anthranilic acid (ACA) removed the oscillations at a concentration of 25 microM, arachidonyl trifluoromethyl ketone (AACOCF3) at 10 microM, and bromoenol lactone (BEL) at 10 to 15 microM. Withdrawal of ACA and BEL resulted in reappearance of the oscillations. Suppression of the arachidonic acid production by addition of 5 microM of the diacylglycerol lipase inhibitor 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC 80267) effectively removed the [Ca2+]i oscillations, an effect reversed by removal of the inhibitor or addition of 100 microM tolbutamide. Suppression of the arachidonic acid production had a restrictive influence also on the transients of [Ca2+]i supposed to synchronize the beta-cell rhythmicity. Although less sensitive than the oscillations, most transients disappeared during exposure to 50 microM ACA or 35 microM RHC 80267. The results support the idea that cyclic variations of cPLA2 activity are important for the generation and synchronization of the beta-cell [Ca2+]i oscillations responsible for pulsatile release of insulin.
Experimental and Clinical Endocrinology & Diabetes, 2009
beta-Cell-rich pancreatic islets from ob/ob-mice were used for evaluating how ATP associated with... more beta-Cell-rich pancreatic islets from ob/ob-mice were used for evaluating how ATP associated with the plasma membrane participates in the regulation of insulin release. Increase of Ca2+ initiates insulin release from permeabilized beta-cells only in the presence of Mg-ATP. When bound to the inner part of the plasma membrane ATP depolarizes the beta-cells by closing a glucose-regulated K+-channel. It is possible that ATP in a plasma membrane compartment modulates insulin release also by stimulating ion pumps and exchange processes. ATP can regulate the secretory activity by binding also to the exterior of the beta-cells. The addition of ATP resulted in stimulation of insulin release related to polyphosphoinositide breakdown. It is suggested that the granule fusion with the plasma membrane is followed by release of sufficient amounts of ATP and ADP for activating a P2-purinoceptor. This receptor may consequently be part of a system for amplifying the secretory response to glucose and other agents facilitating the entry of Ca2+.
Cellular Signalling, 2000
The cytoplasmic concentrations of Cl À ([Cl À ] i) and Ca 2 + ([Ca 2 + ] i) were measured with th... more The cytoplasmic concentrations of Cl À ([Cl À ] i) and Ca 2 + ([Ca 2 + ] i) were measured with the fluorescent indicators N-(ethoxycarbonylmethyl)-6-methoxyquinilinum bromide (MQAE) and fura-2 in pancreatic b-cells isolated from ob/ob mice. Steady-state [Cl À ] i in unstimulated b-cells was 34 mM, which is higher than expected from a passive distribution. Increase of the glucose concentration from 3 to 20 mM resulted in an accelerated entry of Cl À into b-cells depleted of this ion. The exposure to 20 mM glucose did not affect steady-state [Cl À ] i either in the absence or presence of furosemide inhibition of Na + , K + , 2 Cl À co-transport. Glucose-induced oscillations of [Ca 2 + ] i were transformed into sustained elevation in the presence of 4,4 H diisothiocyanato-dihydrostilbene-2,2 H-disulfonic acid (H 2 DIDS). A similar effect was noted when replacing 25% of extracellular Cl À with the more easily permeating anions SCN À , I À , NO 3 À or Br À. It is concluded that glucose stimulation of the b-cells is coupled to an increase in their Cl À permeability and that the oscillatory Ca 2 + signalling is critically dependent on transmembrane Cl À fluxes.
Cell Calcium, 2003
Pancreatic -cells are biological oscillators requiring a coupling force for the synchronization ... more Pancreatic -cells are biological oscillators requiring a coupling force for the synchronization of the cytoplasmic Ca 2+ oscillations responsible for pulsatile insulin release. Testing the idea that transients, superimposed on the oscillations, are important for this synchronization, the concentration of cytoplasmic Ca 2+ ([Ca 2+ ] i) was measured with ratiometric fura-2 technique in single -cells and small aggregates prepared from islets isolated from ob/ob-mice. Image analyses revealed asynchronous [Ca 2+ ] i oscillations in adjacent -cells lacking physical contact. The addition of glucagon stimulated the firing of [Ca 2+ ] i transients, which appeared in synchrony in adjacent -cells. Moreover, the presence of glucagon promoted synchronization of the [Ca 2+ ] i oscillations in -cells separated by a distance <100 m but not in those >200 m apart. The results support the proposal that the repolarizing effect of [Ca 2+ ] i transients provides a coupling force for coordinating the pulses of insulin release generated by pancreatic -cells.
Cell Calcium, 1995
Individual pancreatic P-cells were used to study the glucose effects on the handling of Ca2+, SP'... more Individual pancreatic P-cells were used to study the glucose effects on the handling of Ca2+, SP' and Ba2+. In extracellular medium containing one of these ions, sin 8-t le P-cells res S?' or B onded to 11 mM glucose with large amplitude oscillations in cytoplasmic Ca , Ba + with indistinguishable average frequencies (0.30-0.33/min). The oscillations disappeared after hyperpolarization with 400 PM diazoxide. Under such hyperpolarization, glucose stimulated the sequestration of Ca2+ and Sr2+ but not of Ba2+ in the inositol 1,4,5-trisphosphate sensitive pool repetitively mobilized by consecutive exposures to 100 PM carbachol. A 2-3 min exposure to 100 nM of the intracellular Ca2+-ATPase inhibitor thapsigargin also mobilized Ca2+ and Sr2' and irreversibly abolished subsequent release by carbachol. However, thapsigargin did not prevent the large amplitude oscillations in Ca2+, Sr2+ or Ba2+ under non-hyperpolarizing conditions although the frequency of the Ca2+ oscillations was almost doubled. The results indicate that the slow oscillatory behavior of glucose-stimulated individual @ells does not depend on inositol 1,4,5-trisphosphate mediated release of intracellular Ca2+.
Cell Calcium, 1999
Isolated pancreatic β-cells respond to glucose stimulation with increase of the cytoplasmic Ca 2+... more Isolated pancreatic β-cells respond to glucose stimulation with increase of the cytoplasmic Ca 2+ concentration ([Ca 2+ ] i) in terms of membrane-derived slow oscillations (0.2-0.5/min) with superimposed transients of intracellular origin. To evaluate under which conditions transients may result also from entry of extracellular Ca 2+ , the cytoplasmic concentration of the ion was measured with dual wavelength fluorometry and fura-2 in individual mouse β-cells exposed to the K + channel blocker tetraethylammonium (TEA). In the presence of 20 mM TEA, the β-cells responded to closure of the K ATP channels (increase of the glucose concentration to 11 mM or addition of 1 mM tolbutamide) with pronounced transients of [Ca 2+ ] i. However, there were no transients when the β-cells were depolarized by raising extracellular K + to 30 mM in the presence of 20 mM TEA. The glucose-induced [Ca 2+ ] i transients became more pronounced after thapsigargin inhibition of the endoplasmic reticulum Ca 2+-ATPase. The tolbutamideinduced transients were amplified when promoting the entry of Ca 2+ (rise of extracellular Ca 2+ to 10 mM or addition of BAY K 8644), unaffected in the presence of thapsigargin and the Na + channel blocker tetrodotoxin and slightly reduced by glucagon. Blockage of voltage-dependent Ca 2+ channels with methoxyverapamil resulted in a prompt disappearance of the transients induced by glucose or tolbutamide. The observations indicate that closure of the K ATP channels can precipitate pronounced transients of [Ca 2+ ] i when other K + conductances are suppressed.
Cell Calcium, 1992
Glucose stimulation of individual the cytoplasmic Ca*+ ancreatic &cells is associated with a rise... more Glucose stimulation of individual the cytoplasmic Ca*+ ancreatic &cells is associated with a rise of concentration ([Ca* i) 5 manifested either as large amplitude oscillations (0.2-O.Umin) or as a sustained increase. Determinants for the transitions between the basal and the two stimulated states have now been studied using dual-wavelength fluorometric measurements on individual ob/ob mouse f&cells loaded with the Ca*+ indicator Fura-2. The transition from the basal state to large amplitude oscillations was induced by raising the glucose concentration to 7 mM or above. The frequencies and shapes of the [Ca*+]i cycles remained largely unaffected when raising glucose as high as 40 mM. However, in some cells the oscillatory pattern was transformed into a sustained increase of [Ca*+]i at high glucose concentrations. Although the peak values for the oscillations exceeded the steady-state increase, the time average [Ca*+]i was higher during the latter phase. Both types of glucose-induced transitions were facilitated by the presence of l-100 nM glucagon. Protein kinase C activation by IO nfvl of the phorbol ester TPA resulted in a transformation of the glucose-induced oscillations into a sustained increase of [Ca*+]i but the levels reached were considerably lower than obtained with glucose alone. It is concluded that the glucose sensing of the individual &cell is based on sudden transitions between steady-state and oscillating cytoplasmic Ca*+. It is these transitions rather than alterations of the oscillatory characteristics which determine the average [Ca*+]i regulating insulin release. Glucose stimulation of insulin release depends on a rise of the cytoplasmic Ca2+ concentration ([Ca2+]i)
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Papers by Eva Grapengiesser