Format

Send to

Choose Destination
See comment in PubMed Commons below
Cell Calcium. 1994 Dec;16(6):431-45.

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

Author information

1
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville.

Abstract

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.

PMID:
7712537
[Indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Loading ...
    Support Center