Introducing specific antibodies into electropermeabilized cells is a valuable tool for eliminating specific cell functions.
A technique is established for the role of intracellular proteins to be eliminated and thereby gives information about their specific role in signal transduction within cells. Rat pancreatic islets as well as INS-1 cells (an insulin secreting cell line) were electrically permeabilized in order to introduce high molecular weight compounds. Optimized conditions were five exposures with 15-s intervals, tau = 200 ms, an electric field of 1.36 kV per 0.4 cm in a specific permeabilization buffer at a calculated Ca++ concentration of 5 x 10(-8) M. In electroporation control experiments the spectrophotometrically measured uptake of the cell membrane-impermeable propidium iodide, FITC-labelled dextran (MW approximately 4000) and FITC-labelled antibodies (MW approximately 150,000) was established as being 81.5 +/- 5.0, 82.7 +/- 3.0 and 81.0 +/- 1.0 per cent of maximum, respectively. These data were corroborated qualitatively by visualizing microscopically the fluorescence of the FITC-labelled compounds in islets as well as in INS-1 cells. The cells appear to reseal since control experiments indicated a short-lived outflow of lactate dehydrogenase (MW of 140,000 which is similar to that of antibodies) and of insulin for the first 15-20 min. After electroporation the cells were functionally intact, i.e. responded to the stimulus carbachol (CCh). Only 18.0 +/- 10.1 per cent of cells had not resealed after 2 h (propidium iodide uptake measured at various time intervals after electroporation). As was shown recently the effect of specific compounds such as CCh and CCK8 on insulin release was eliminated selectively by antibodies against specific G proteins thus proving this method to be a valuable tool. In conclusion, adding antibodies to electrically permeabilized cells is a valuable tool for eliminating a specific cell function in order to elucidate the specific role of intracellular compounds. This method can probably be used for testing the specific role of other proteins in cell functions.
Verspohl
E J
EJ
Department of Pharmacology, Institute of Pharmaceutical Chemistry, University of Muenster, Germany.
Kaiserling-Buddemeier
I
I
Wienecke
A
A
D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000502:physiology*; D003911:Dextrans / Q000493:pharmacokinetics; D018274:Electroporation / Q000379:methods; D005260:Female; D016650:Fluorescein-5-isothiocyanate / Q000493:pharmacokinetics; D005947:Glucose / Q000494:pharmacology; D007074:Immunoglobulin G / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D011419:Propidium / Q000493:pharmacokinetics; D051381:Rats; D017208:Rats, Wistar; D012107:Research Design
D000906:Antibodies; D003911:Dextrans; D007074:Immunoglobulin G; D007328:Insulin; D011419:Propidium; D007770:L-Lactate Dehydrogenase; D016650:Fluorescein-5-isothiocyanate; D005947:Glucose