Permeabilization Insulin-secreting cells

In electrically permeabilized insulin-secreting cells, the influence of soluble second messengers on exocytosis can be studied directly, since it is possible to dialyze such cells with respect to nucleotides and ions while cytosolic proteins are retained. In the presence of ATP, Ca " stimulates insulin exocytosis with an EC50 of approximately 1.6 l/Vl (Vallar efal., 1987, Ullrich efal., 1990). This is in close agreement with the value for Ca -stimulated exocytosis in patch-clamped mouse P-cells obtained using the capacitance method (Bokvist ef al.,... 

Insulin-secreting cells do not express the receptors required for entry of the neurotoxins. Consequently, the cells need to be permeabilized before the addition of the neurotoxins. SLO has been shown to produce holes in the plasma membrane of several cell types that allow the passage of molecules up to 150 kDa. The permeabilization procedure with SLO described above yields reproducible results in insulin-secreting cells and is, therefore, the method of choice to test the effect of neurotoxins in these cells. 

Ullrich S, Prentki M, Wollheim CB (1990) Somatostatin inhibition of Ca -induced insulin secretion in permeabilized HIT-T15 cells. In Biochem. J. 270 273-76. 

Vallar L, Biden TJ, Wollheim CB (1987) Guanine nucleotides induce Ca -independent insulin secretion from permeabilized RINm5F cells. In J. Biol. Chem. 262 5049-56. 

The use of permeabilized cells in the study of exocytosis is not restricted to the investigation of agents which do not cross normally the plasma membrane, such as the clostridial neurotoxins, peptides or recombinant proteins. This approach can be combined with the transient overexpression of proteins of interest, e.g., components of the fusion machinery. The method described below applies to insulin-secreting HIT-T15 cells (Lang efal., 1995). 

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