Oscillations In Cytosolic Free Ca2 Research Articles

Oscillations in cytosolic free Ca2+, oxygen consumption, and insulin secretion in glucose-stimulated rat pancreatic islets

Insulin secretion in the intact organism, and by the perfused pancreas and groups of isolated perifused islets, is pulsatile. We have proposed a metabolic model of glucose-induced insulin secretion in which oscillations in the ATP/ADP ratio drive alterations in metabolic and electrical events that lead to insulin release. A key prediction of our model is that metabolically driven Ca2+ oscillations will also occur. Using the fluorescent Ca2+ probe, fura 2, digital image analysis, and sensitive O2 electrodes, we investigated cytosolic free Ca2+ responses and O2 consumption in perifused rat islets that had been maintained in culture for 1-4 days. We found that elevated ambient glucose increased the average cytosolic free Ca2+ level, the ATP/ADP ratio, and oxygen consumption, as previously found in freshly isolated islets. Oscillatory patterns were obtained for Ca2+, O2 consumption, and insulin secretion in the presence of 10 and 20 mM glucose. Very low amplitude oscillations in cytosolic free Ca2+ were observed at 3 mM nonstimulatory glucose levels. Evaluation of the Ca2+ responses of a large series of individual islets, monitored by digital image analysis and perifused at both 3 and 10 mM glucose, indicated that the rise in glucose concentration caused more than a doubling of the average cytosolic free Ca2+ value and a 4-fold increase in the amplitude of the oscillations with little change in period. The pattern of Ca2+ change within the islets was consistent with recruitment of responding cells. The coexistence of oscillations with similar periods in insulin secretion, oxygen consumption, and cytosolic free Ca2+ is consistent with the model of metabolically driven pulsatile insulin secretion.

Tumor necrosis factor-induced degranulation in adherent human neutrophils is dependent on CD11b/CD18-integrin-triggered oscillations of cytosolic free Ca2+.

We have recently been able to correlate closely the "spontaneous" oscillatory activity of cytosolic free Ca2+ in adherent human neutrophils with the ability of tumor necrosis factor (TNF) to induce secretion of granule proteins from these cells. In the present work we show with a single-cell technique that preincubation of human neutrophils with antibodies to CD18, the common beta chain of leukocyte adhesion proteins, inhibits TNF-induced secretion of lactoferrin in a time- and concentration-dependent manner. Similar effects of CD18 antibodies were found on chemotactic factor (fMet-Leu-Phe)- but not on phorbol 12-myristate 13-acetate-induced secretion, suggesting that cell-surface-receptor-mediated secretion is dependent on integrin-associated signals. Similarly, antibodies to CD11b (alpha chain of macrophage 1) also inhibited TNF- and fMet-Leu-Phe- but not phorbol 12-myristate 13-acetate-stimulated release of lactoferrin. Antibodies to CD11a (alpha chain of lymphocyte function-associated antigen 1) or CD11c (alpha chain of p150,95) had only a minimal effect on agonist-induced secretion. Data obtained in several laboratories, including our own, made us suspect that integrin interaction with the surface is responsible for the oscillatory activity of cytosolic free Ca2+ in adherent cells. Indeed, preincubation with antibodies to either CD18 or CD11b, but not to CD11c, inhibited the oscillations of cytosolic free Ca2+ in adherent neutrophils. This inhibitory effect was evident both as a reduction of the number of responding cells and as a reduction of the oscillatory activity in the cells. In conclusion, the oscillatory activity of cytosolic free Ca2+ in adherent neutrophils is mediated through the CD18/CD11b integrins. The generation of this Ca2+ signal may explain how adherence, by way of the integrins, changes the functional properties of the cell and enables TNF to induce secretion.

Correlation between spontaneous oscillations of cytosolic free Ca2+ and tumor necrosis factor-induced degranulation in adherent human neutrophils.

By using a hemolytic plaque assay to detect release of lactoferrin and myeloperoxidase, tumor necrosis factor (TNF) was shown previously to induce secretion of these granule proteins from single adherent neutrophils. Secretion was inhibited by loading neutrophils with calcium chelators, indicating a crucial role of cytosolic free [Ca2+] in the signal transduction mechanism of TNF. In the present study, using a microfluorometer technique to follow changes in the cytosolic free [Ca2+] in single adherent neutrophils, we were not able to detect any TNF-induced [Ca2+] transients. However, these adherent cells exhibited spontaneous oscillations of their cytosolic free [Ca2+], as previously reported (Jaconi, M.E.E., Rivest, R.W., Schlegel, W., Wollheim, C.B., Pittet, D., and Lew, P.D. (1988) J. Biol. Chem. 263, 10557-10560). A close correlation was found between a reduced oscillatory activity of cytosolic free [Ca2+] and a reduced ability of TNF to induce degranulation, by reducing the extracellular [Ca2+] or loading the cells with a calcium chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). In addition, when the cells were incubated at 37 degrees C for 3 h there was a parallel decline in the spontaneous oscillatory activity of cytosolic free [Ca2+] and TNF-induced secretion of lactoferrin. Control experiments showed that phorbol 12-myristate 13-acetate-induced secretion was not affected under the same conditions, indicating that the secretory process per se was not disturbed. We conclude that TNF by itself does not give rise to any changes of the cytosolic free [Ca2+] but that the spontaneous oscillatory activity of cytosolic free [Ca2+] in adherent neutrophils is necessary for TNF-induced degranulation.

Fluoroaluminate mimics agonist application in single rat hepatocytes

Single rat hepatocytes microinjected with the photoprotein aequorin were stimulated with glycogenolytic agonists or low concentrations of fluoroaluminate. Both protocols resulted in the generation of oscillations in cytosolic free Ca2+ levels from a resting value of approx. 200 nM and peaking at over 600 nM. However, oscillations induced by receptor-dependent agonists were more regular in both frequency and time course than those induced by direct activation of G-proteins. The role of G-proteins in the generation of repetitive free Ca2+ oscillations is discussed.

Linked oscillations of free Ca2+ and the ATP/ADP ratio in permeabilized RINm5F insulinoma cells supplemented with a glycolyzing cell-free muscle extract.

We show in the accompanying paper that the steady-state level of free Ca2+ maintained by the organelles of permeabilized RINm5F insulinoma cells varies inversely with the ATP/ADP ratio when this ratio is set by addition of creatine phosphokinase and fixed ratios of creatine to creatine phosphate. We, therefore, asked whether acute cyclic alterations in the cytosolic ATP/ADP ratio in the range known to modulate O2 consumption might be involved in regulating the physiological activity of Ca2+ -ATPases and the cytosolic free Ca2+ level. To explore this hypothesis we combined two experimental systems: 1) permeabilized RINm5F insulinoma cells that can maintain a low medium Ca2+ concentration and 2) a cell-free extract of rat skeletal muscle that spontaneously exhibits oscillatory behavior of glycolysis and linked oscillations in the ATP/ADP ratio, when provided with glucose. The free Ca2+ level maintained by the permeabilized cells oscillated in phase with the glycolytic oscillations and correlated closely with the ATP/ADP ratio but not with glucose 6-phosphate, fructose 6-phosphate, orthophosphate, or pH. When glucokinase replaced hexokinase as the glucose phosphorylating enzyme, Ca2+ oscillations were induced by increasing the glucose concentration from 2 to 8 mM. The results demonstrate a link between metabolite changes and free Ca2+ levels in a reconstituted physiological system. They support a model in which oscillations in glycolysis and the ATP/ADP ratio may cause oscillations in cytosolic free Ca2+, beta-cell electrical activity, and insulin release.