Molecular surgery of the basement membrane by the argon laser

Abstract

Although the argon laser is used successfully to weld a number of different tissues, the underlying chemical and cellular mechanisms for this process are not precisely defined. Consequently, a biochemical model has been developed in vitro using the welldefined extracellular matrix from the murine Engelbreth‐Holm‐Swarm (EHS) sarcoma. Control and experimental samples of EHS basement membranes were irradiated with a Trimedyne argon laser at 500–3,000 Joules/cm² at 0°C. The samples were diluted into cold phosphate‐buffered saline and allowed to gel at 35°C. The time course of the gelation reaction was followed in a spectrophotometer at 360 nm. Irradiation reduced the absorbance 7.5–15% compared to controls and was independent of the dilution over a 10‐fold range. Gelation was also measured by determining the amount of protein by the Bradford assay that could be collected by centrifugation at 10,000g for 10 minutes. Argonirradiated samples had 30–40% less protein in the precipitate than the controls. The addition of 5 mM beta‐mercaptoethanol to the EHS extract blocked the effect of the laser on the gelation reaction. In addition, when gelation was carried out in the absence of calcium and magnesium, there were no differences between laser‐treated samples and controls. The basement membrane proteins were separated by electrophoresis through polyacrylamide gels under denaturing plus reducing or denaturing and non‐reducing conditions. No differences in the polypeptide composition were noted between irradiated and control samples using either Coomassie‐ or silver‐staining techniques. The irradiated and control samples were also analyzed on 1.5% agarose gels in 10 mm Tris‐HCl, pH 8 containing 0.1% SDS. Both samples produced a single Coomassie‐positive band, but the control migrated at 0.5 and the irradiated sample at 0.6. In sum, the conformation of extracellular matrix proteins can be altered by the argon laser at welding energies at low temperatures, even though no new covalent interactions were observed.