Permeabilization of rat hepatocytes with Staphylococcus aureus alpha-toxin.

Abstract

Pathogenic staphylococci secrete a number of exotoxins, including a-toxin, aToxin induces lysis of erythrocytes and liposomes when its 3S protein monomers associate with the lipid bilayer and form a hexomeric transmembrane channel 3 nm in diameter. We have used a-toxin to render rat hepatocytes 93-100% permeable to trypan blue with a lactate dehydrogenase leakage <_22%. Treatment conditions included incubation for 5-10 min at 37°C and pH 7.0 with an a-toxin concentration of 4-35 human hemolytic U/ml and a cell concentration of 13-21 mg dry wt/ml. Scanning electron microscopy revealed signs of swelling in the treated hepatocytes, but there were no large lesions or gross damage to the cell surface. Transmission electron microscopy indicated that the nucleus, mitochondria, and cytoplasm were similar in control and treated cells and both had large regions of well-defined lamellar rough endoplasmic reticulum. Comparisons of the mannose-6-phosphatase and glucose-6-phosphatase activities demonstrated that 5-10 U/ml a-toxin rendered cells freely permeable to glucose-6-phosphate, while substantially preserving the selective permeability of the membranes of the endoplasmic reticulum and the functionality of the glucose-6-phosphatase system. Thus, a-toxin appears to have significant potential as a means to induce selective permeability to small ions. It should make possible the study of a variety of cellular functions in situ.The study of the relationship of enzymic activities to internal cell structure requires the development of assay systems that can determine these activities in situ. For most reactions, the development of such assays requires circumventing the permeability barrier presented by the plasma membrane. The use of toluene (1, 2) or detergents to render the plasma membrane permeable to ionic substrates is unsatisfactory since such agents induce permeability changes in intracellular membranes. Toluene treatment also results in large morphological changes in the internal cell structure (2). The ideal reagent would be one that selectively reacts with the plasma membrane, does not induce any alterations of cellular morphology, and does not cause leakage of cytosolic enzymes.Filipin (3-7), digitonin (8-12), and saponin (13-15), all of which complex with membrane-bound cholesterol, have been used to create permeability in cells to a variety of substances. Since the plasma membrane has a higher cholesterol content than most internal membranes (e.g., mitochondria and the 1922 endoplasmic reticulum [ER]'), it has been postulated that these agents would react preferentially with the plasma membrane (6, 7, 1 l, 13). In spite of this selective interaction, the use of filipin or digitonin can cause extensive changes in cell morphology (12,16,17). 2 The rough ER is especially vulnerable, and other studies show that these agents alter the kinetics of ER enzymes (Lange, A. J., B. F. McEwen, and W. J. Arion, unpublished observations; and references 18-20), which suggests the agents interact with the ER membrane or E...

Farnesol Inhibits L-type Ca2+ Channels in Vascular Smooth Muscle Cells

“…The data indicate that farnesol has no direct effect on the Ca 2ϩ -responsive elements implicated in arterial contraction, including those activated by GTP and PKC. The absence of effect of farnesol in ␣-toxin-permeabilized arteries is not due to its inability to penetrate the tissue, because farnesol is a small molecular weight (M r ϭ 222.4) molecule and is likely to diffuse freely inside the arterial smooth muscle cells through the pores created by the toxin (54,55). Therefore, our findings suggest that the vascular action of the isoprenol is solely the consequence of its inhibition of Ca 2ϩ signaling.…”

ERK1/2-mediated phosphorylation of myometrial caldesmon during pregnancy and labor

“…Thin muscle strips were dissected to have a width of 700 m and permeabilized with ␣-toxin by a previously described method (38). ␣-Toxin from Staphylococcus aureus forms pores that allow the passage of molecules Ͻ2-4 kDa across the cell membrane (26). The strips were treated for 30 min at 34°C with 20 g/ml of ␣-toxin (List Biological Lab, Campbell, CA) in pCa 6.4 buffer.…”

Agonist-induced vascular tone.

“…This toxin binds to the cell surface and forms hexamers with other toxin molecules, which insert into the plasma membrane to form pores of 2-3 nm diameter. 58 This limited pore size allows equilibrium of the cytoplasm with inorganic ions and small molecules but prevents the passage of proteins, including a-toxin itself, into or out of the cells. Figure 2 shows a-toxin-mediated permeabilization of the rabbit mesenteric artery smooth muscle to Ca 2+ and ATP.…”