ABSTRACT
OBJECTIVES: Clonal kidney cells (Vero cells) are extensively utilized in the manufacture of biological preparations for disease diagnostics and therapeutics and also in preparation of vaccines. In all cells, regulation of volume is an essential function coupled to a variety of physiological processes and is a topic of interest. The objective here was to investigate involvement of ion channels in the process of volume regulation of Vero cells. METHODS: Involvement of ion channels in cell volume regulation was studied using video-microscopy and flow cytometry. Pharmacologically unaltered cells of different sizes, which are presumably at different phases of the cell cycle, were used. RESULTS: Ion transport inhibitors altered all phases of regulatory volume decrease (RVD) of Vero cells, rate of initial cell swelling, V(max) and volume recovery. Effects were dependent on type of inhibitor and on cell size (cell cycle phase). Participation of aquaporins in RVD was suggested. Inhibitors decelerated growth, arresting Vero cells at the G(0) /G(1) phase boundary. Electrophysiological study confirmed presence of volume-activated Cl(-) channels and K(+) channels in plasmatic membranes of the cells. CONCLUSION: Vero cells of all sizes maintained the ability to recover from osmotic swelling. Activity of ion channels was one of the key factors that controlled volume regulation and proliferation of the cells.
Subject(s)
Cell Size , Ion Channels/metabolism , Kidney/cytology , Kidney/metabolism , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Animals , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cell Size/drug effects , Cells, Cultured , Chlorocebus aethiops , Flow Cytometry , Glyburide/pharmacology , Ion Channels/antagonists & inhibitors , Microscopy , Nitrobenzoates/pharmacology , Tetraethylammonium/pharmacology , Vero CellsABSTRACT
Antigen F1 is a protein of 17 kDa produced by Yersinia pestis it is cultured at 37 grade C. When incorporated into planar lipid bilayer membranes this protein induces fluctuations on membrane conductance typical of the formation of ionic channels. These fluctuations reveal two distinct unitary conductance sizes, one in the range of 800 to 1400 pS and the other in the range of 140 to 600 pS. Zero current potential measaurements in the presence of a salt gradient show that the channell is not significantly ion selective. The reversal potential measured in the presence of 0.5 MKCl on the cis side and 0.1 MKCl on the trans side was 3.58 ñ 3.98 mV (N=7). The non-selectivity of the channel, in addition to its large conductance, suggests that it forms large aqueous pores. The present results, taken together with other data showing that nantigen F1 inhibits the activity of phagocytic cells, suggest that antigen F1 acts by forming aqueous pores in the membrane of these target cells
Subject(s)
Bacterial Proteins/metabolism , Lipid Bilayers/metabolism , Yersinia pestis/metabolism , Ion Channels , TemperatureABSTRACT
Porin isolated from bovine skeletal muscle was reconstituted in planar lipid bilayers under voltage clamp conditions. A set of non-electrolytes were used as molecular probes for determining the pore diameter. The maximal diameter of the open channel was estimated to be 3.02 + 0.26 nm. As observed for other porin channels, a large transmembrane potential drove the channel into a "closed" state. The channel transition to the low conductance (closed) state was followed by a decrease in the maximal diameter of the channel to 2.4 +- 0.08 nm.