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 beta-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 alpha-actin antibody and immunostained with F(ab')2 labeled with tetramethyl rhodamine isothiocyanate. Confocal light microscopy of Triton X-100 and beta-escin permeabilized cells showed homogeneous staining of the cytoplasm, whereas in alpha-toxin treated and intact preparations only damaged cells at the edges of the strips were stained. Both the Ca(2+)-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 beta-escin-treated group, but not from the alpha-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 degrees C, rather than 24 degrees C. Ca(2+)-independent myosin light chain kinase (61 kD) contracted the permeabilized portal vein in the absence of free Ca2+ (pCa < 8). In conclusion, permeabilization with beta-escin allows the transmembrane passage of 150 kD proteins under our experimental conditions that also retain receptor-coupled signal transduction.
Iizuka
K
K
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville.
Ikebe
M
M
Somlyo
A V
AV
Somlyo
A P
AP
D000199:Actins / Q000276:immunology; D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects*; D004928:Escin / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006168:Guinea Pigs; D007082:Ileum; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism*; D007537:Isometric Contraction / Q000187:drug effects; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D009124:Muscle Proteins / Q000032:analysis; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D010656:Phenylephrine / Q000494:pharmacology; D011169:Portal Vein; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000378:metabolism; D015398:Signal Transduction; D013873:Thionucleotides / Q000494:pharmacology; D010738:Type C Phospholipases / Q000494:pharmacology*
D000199:Actins; D002147:Calmodulin; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D009124:Muscle Proteins; D011956:Receptors, Cell Surface; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D010656:Phenylephrine; D004928:Escin; C023427:guanosine 5'-O-(2-thiodiphosphate); D002217:Carbachol; D017830:Octoxynol; D007770:L-Lactate Dehydrogenase; D009219:Myosin-Light-Chain Kinase; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins