Introduction of high molecular weight (IgG) proteins into receptor coupled, permeabilized smooth muscle

The permeability to high molecular weight (IgG, 150 kD) proteins of the plasma membrane of receptor-coupled smooth muscles permeabilized with β-escin was determined using confocal microscopy of immunofluorescent tracers and measurement of lactate dehydrogenase (LDH, 135–140 kD) leakage. Permeabilized strips of rabbit portal vein and guinea pig ileum were incubated in a relaxing solution containing mouse anti-smooth muscle α-actin antibody and immunostained with F(ab′)₂ labeled with tetramethyl rhodamine isothiocyanate. Confocal light microscopy of Triton X-100 and β-escin permeabilized cells showed homogeneous staining of the cytoplasm, whereas in α-toxin treated and intact preparations only damaged cells at the edges of the strips were stained. Both the Ca²⁺-sensitizing effect of phenylephrine, in rabbit portal vein, and Ca²⁺ release by carbachol in guinea pig ileum, were retained after permeabilization and the treatment with the primary antibody. During the 30 min permeabilization, 38%, and within the next 75 min an additional approximately 30%, of the total LDH leaked out from the β-escin-treated group, but not from the α-toxin-treated group (3.2%). The responsiveness to agonist and maximum contractility was improved if the preparations were incubated during the introduction of proteins at 4°C, rather than 24°C. Ca²⁺-independent myosin light chain kinase (61 kD) contracted the permeabilized portal vein in the absence of free Ca²⁺ (pCa < 8). In conclusion, permeabilization with β-escin allows the transmembrane passage of 150 kD proteins under our experimental conditions that also retain receptor-coupled signal transduction.