K. J. Föhr

Calculation and control of free divalent cations in solutions used for membrane fusion studies.

Publisher Summary This chapter discusses a computer program that allows the calculation of multiple equilibria between different ligands and metal ions. Although the media prepared according to the calculation generally give concentrations in good agreement with measured values, the calculation should always be controlled. The computer program developed considers nine different ligands—EDTA, EGTA, HEDTA, NTA, ATP, ADP, GTP, phosphate, and creatine phosphate—and corrections for temperature and ionic strength. The program calculates either the total amount of metals to give the desired free metal concentrations (Ca 2+ , Mg 2+ ) or, in the reversed mode, it calculates the free metal concentration for a given total amount of metals and the selected mixture of ligands. Furthermore, an option exists for calculating the apparent association constants under different conditions (pH, T, I), to choose the appropriate ligands for the experimental purposes. For this purpose, an easy and inexpensive procedure for the preparation of ion-selective electrodes is then described.

Chapter 4 Poration by α-Toxin and Streptolysin O: An Approach to Analyze Intracellular Processes

Publisher Summary This chapter describes the purification and handling of α-toxin and streptolysin O (SLO) for the poration of cells. In contrast to other permeabilizing procedures, the pores inserted into the plasma membrane with the aid of bacterial toxins are stabilized by a proteinaceous ring like structure. Depending on the aim of an experiment, the cells can be made permeable either for large or for small molecules by selection of a suitable bacterial pore-forming protein. This allows permanent access to the cells interior to investigate intracellular processes as diverse as fusion of secretory vesicles with the inner surface of the cell membrane, contraction, metabolism of hormones, fluxes of ions, or glucose metabolism. Besides the different secretory cells, poration by α-toxin or SLO was carried out with hepatocytes, rat basophilic leukemia cells, fibroblasts, and smooth muscle cells. This indicates that the novel approach of permeabilization of cells by channel-forming toxins has already become a widely used tool for the investigation of a variety of intracellular processes in situ .

Decavanadate displaces inositol 1,4,5-trisphosphate (IP3) from its receptor and inhibits IP3 induced Ca2+ release in permeabilized pancreatic acinar cells

Inositol 1,4,5-trisphosphate (IP3) induced Ca2+ release in digitonin permeabilized rat pancreatic acinar cells is specifically inhibited by decavanadate. The Ca2+ release induced with 0.18 microM IP3 is half maximally inhibited with approximately 5 microM decavanadate. Complete inhibition is achieved with around 20 microM decavanadate. Removal of decavanadate from the permeabilized cells fully restores sensitivity towards IP3, indicating the reversibility of the inhibition. Oligovanadate, which inhibits ATP dependent Ca2+ uptake into intracellular stores, does not influence IP3 induced Ca2+ release. In order to reveal the mechanism underlying the effects of the different vanadate species, binding of IP3 to the same cellular preparations was investigated. We found that binding of IP3 to a high affinity receptor site (Kd approx. 1.2 nM) could be abolished by decavanadate but not by oligovanadate. With 0.5 microM decavanadate, IP3 binding was half maximally inhibited. A similar potency of decavanadate was also found with adrenal cortex microsomes which bind IP3 with the same affinity (Kd approx. 1.4 nM) as permeabilized pancreatic acinar cells. Labelled IP3 was displaced from these subcellular membranes with similar kinetics by unlabelled IP3 and decavanadate. The data suggest that the inhibitory action of decavanadate on IP3 induced Ca2+ release is a consequence of its effect on binding of IP3 to its receptor.

Effect of GTP and Ca2+ on inositol 1,4,5-trisphosphate induced Ca2+ release from permeabilized rat exocrine pancreatic acinar cells

The effects of Ca2+ and GTP on the release of Ca2+ from the inositol 1,4,5-trisphosphate (IP3) sensitive Ca2+ compartment were investigated with digitonin permeabilized rat pancreatic acinar cells. The amount of Ca2+ released due to IP3 directly correlated with the amount of stored Ca2+ and was found to be inversely proportional to the medium free Ca2+ concentration. Ca2+ release induced by 0.18 microM IP3 was half maximally inhibited at 0.5 microM free Ca2+, i.e. at concentrations observed in the cytosol of pancreatic acinar cells. GTP did not cause Ca2+ release on its own, but a single addition of GTP (20 microM) abolished the apparent desensitization of the Ca2+ release which was observed during repeated IP3 applications. This effect of GTP was reversible. GTP gamma S could not replace GTP. Desensitization still occurred when GTP gamma S was added prior to GTP. The reported data indicate that GTP, stored Ca2+ and cytosolic free Ca2+ modulate the IP3 induced Ca2+ release.

Millimolar concentrations of free magnesium enhance exocytosis from permeabilized rat pheochromocytoma (PC 12) cells

The role of Mg2+ during the final steps of exocytosis was investigated using rat pheochromocytoma cells (PC12) permeabilized with bacterial pore forming toxins. Concentrations of free Mg2+ between 0.2 and 2 mM slightly lowered the basal but greatly enhanced the [3H]dopamine release elicited by 8 microM free Ca2+. Maximal effects were obtained at approximately 1 mM free Mg2+. At higher concentrations Mg2+ was less potent. Similar effects of Mg2+ were obtained in cells permeabilized either for small molecules (by alpha-toxin) or for large ones (by streptolysin O). It is concluded that millimolar concentrations of cytoplasmic Mg2+ play an important role in Ca2+ triggered exocytosis.

GTP and Ca2+ Modulate the Inositol 1,4,5-Trisphosphate-Dependent Ca2+ Release in Streptolysin O-Permeabilized Bovine Adrenal Chromaffin Cells

Abstract: The inositol 1,4,5‐trisphosphate (IP3)‐induced Ca2+ release was studied using streptolysin O‐permeabilized bovine adrenal chromaffin cells. The IP3‐induced Ca2+ release was followed by Ca2+ reuptake into intracellular compartments. The IP3‐induced Ca2+ release diminished after sequential applications of the same amount of IP3. Addition of 20 μM GTP fully restored the sensitivity to IP3. Guanosine 5′‐O‐(3‐thio)triphosphate (GTPγS) could not replace GTP but prevented the action of GTP. The effects of GTP and GTPγS were reversible. Neither GTP nor GTPγS induced release of Ca2+ in the absence of IP3. The amount of Ca2+ whose release was induced by IP3 depended on the free Ca2+ concentration of the medium. At 0.3 μM free Ca2+, a half‐maximal Ca2+ release was elicited with ∼0.1 μM IP3. At 1 μM free Ca2+, no Ca2+ release was observed with 0.1 μM IP3; at this Ca2+ concentration, higher concentrations of IP3 (0.25 μM) were required to evoke Ca2+ release. At 8 μM free Ca2+, even 0.25 μM IP3 failed to induce release of Ca2+ from the store. The IP3‐induced Ca2+ release at constant low (0.2 μM) free Ca2+ concentrations correlated directly with the amount of stored Ca2+. Depending on the filling state of the intracellular compartment, 1 mol of IP3 induced release of between 5 and 30 mol of Ca2+.

Control of Intracellular Free Calcium in Neurons and Endocrine Cells

Intracellular free calcium concentrations in both neurons and endocrine cells are regulated by transporters and ion channels present either in the cell membrane or in the membrane of intracellular organelles. Low intracellular free Ca2+ levels in resting cells are maintained by active Ca2+ extrusion from the cytoplasm into the extracellular space via the Na+/Ca2+-exchanger and the plasma membrane Ca2+-ATPase (PMCA), as well as Ca2+ uptake from the cytoplasm into various intracellular compartments. Sequestration by intracellular compartments is accomplished by uni- and antiporter systems present in mitochondria or secretory granules and a family of endoplasmic and sarcoplasmic reticulum Ca2+-ATPases termed SERCA. Upon cell stimulation extracellular Ca2+ may enter the cell via voltage operated (VOCS), receptor operated (ROCS) or second messenger operated (SMOCS) calcium channels. An increase of cytosolic free Ca2+ may also be caused by Ca2+ release from intracellular compartments by diffusible second messengers activating IP3 or ryanodine receptor calcium channels.