General Remarks

assay of permeabilization use pre-binding of toxin

In contrast to neuronal cells, a number of toxins, which provide interesting tools for the investigation of cell function, do not cross the cell membrane. Controlled cell permeabilization allows the intracellular application of these molecules in a system which retains the exocy-totic response to stimulating or inhibiting agents. This can be achieved by the use either of detergents or of pore-forming toxins (Holz et a/., 1992, Ahnert-Hilger et a/., 1993). The detergent digitonin solubilizes membranes according to their cholesterol content and is therefore in general suitable for the creation of pores, mainly in the plasma membrane. Although this approach has often been used successfully in chromaffin cells (Holz et a/., 1994, Holz et a/., 1992), we did not find it a reliable method for insulin-secreting cells (Wollheim and Ullrich, unpublished). An alternative approach has been described, i.e., intracellular application of toxins by electrophoresis (Boyd et a/., 1995). This method is feasible for toxins endowed with high biological activity and long half-life, as is the case for botulinum neurotoxins and tetanus toxin.

In our hands, pore-forming toxins offer a far more reliable tool than detergents and are relatively easy to handle (Lang et a/., 1995, Kiraly-Borri et a/., 1996, Regazzi et a/., 1995, Sadoul et a/., 1995). As described in detail elsewhere in this volume, the hemolysin streptolysin O (SLO) from streptococci (commercially available from List, Sigma, Gibco,) binds as a monomer to cell membranes even at 4°C, and induces pores only at higher temperatures by forming oligomers.

The effectiveness of the resulting permeabilization can easily be ascertained by observing dye-uptake by the cells. The major danger of this tool lies in over-permeabilization. Indeed, the streptolysin O monomer can enter the cytosol through the plasma membrane pores created by the oligomer and consequently permeabilize intracellular organelles. Theoretically, permeabilization is therefore achieved in the safest way by prebinding the toxin at 4°C, and washing off the unbound toxin.

Subsequent pore-formation is achieved by shifting the cells to 37°C. However, this approach requires a relatively high amount of toxin, which may not be freely available. In addition, this procedure prolongs the experiment and can lead to the loss of cytosolic proteins essential for various cellular functions. Indeed, insulin-secreting cells demonstrate a run-down of their exocytotic response to Ca2+ over time (Kiraly-Borri et a/., 1996). We therefore prefer to perform the entire experiment at 37°C, thereby limiting the use of toxin. The activity of the toxin can be standardized by measuring the hemolysis of rabbit erythrocytes. In practice this is not required, as the permeabilization of the cell under study constitutes the critical parameter. In addition, the susceptibility of cell lines to the action of SLO may vary according to the passage number.

Cell permeabilization can be performed on cells in suspension or on attached cells. The use of attached cells avoids the need for cell detachment by agents which may alter cellular functions. This is of importance when studying receptor-mediated processes. Furthermore, attached cells do not require a recovery period as is necessary after trypsinization. Unfortunately, only a limited number of cell lines permit cell permeabilization when seeded on normal plastic. As far as the insulin-secreting cell lines are concerned, only the HIT-T15 cells (derived from hamster (3-cells) resist this procedure, whereas others such as RINm5F, INS-1 or primary islet cells tend to detach. Among the various procedures tested to improve cell adherence, only substrata coated with the extracellular matrix from bovine cornea gave good results. These specially coated sterile plastic wells or glass coverslips are commercially available in different forms and sizes (Eldan). Cells should be allowed to reach about 80% confluence, since cell contact generally favours adherence during subsequent manipulations.

Another important issue concerns the intracellular buffers used. We have obtained good results with a potassium glutamate/HEPES buffer (see below). Whenever it might be important, the concentrations of free calcium ions have to be carefully controlled by the use of chelators. As the active concentration of free calcium for exocytosis in insulin-secreting cells ranges from 0.1 [iM (basal) to 10|iM (maximal stimulatory levels) with an EC50 at 2|xM, EGTA is a suitable chelator (Vallar eta/., 1987).

The actual concentration of free Ca2+ can be measured either by the use of an ion-specific Ca2+-electrode, or can be computed using an appropriate programme. The preparation and use of these electrodes have been described in detail (Baudet etal., 1994). For obvious reasons they offer the most direct and reliable method to determine levels of free calcium. Although refined programs are available (Fohr et a/., 1993, Brooks and Storey, 1992), (e.g., WINMAXC) the computed values do not completely coincide with those measured by a calcium-specific electrode. However, they often allow calculation of free magnesium and nucleotides. Fluorescent Ca2+-indicators such as Fura-2 are also commonly used to assess levels of free Ca2+. In practice the variations in computed values of free calcium are of minor importance when using only low (0.1 ¡xM) and high (10 ¡xM) levels of free calcium in insulin-secreting cells, but a considerable error can be introduced at intermediate free calcium concentrations. In addition, certain agents added to the system may in fact behave as calcium chelators and this can obviously only be detected by a calcium-specific electrode. In any case, calcium buffers have to be prepared extremely carefully using standard calcium solutions and paying particular attention to the pH-value of the solution, as the chelating action of EGTA is strongly dependent on the pH.

control of calcium concentration

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