Two different types of lysis of chromaffin granules characterised by freeze-fracture electron microscopy and photon correlation spectroscopy

Abstract

When bovine chromaffin granules are incubated in hyperosmolar sucrose solutions and subsequently transferred back towards isoosmolarity they undergo lysis (‘hyperosmotic relaxation lysis’). This type of lysis was compared with the common effect of hypotonic lysis by means of photon correlation spectroscopy (PCS) and freeze-fracture electron microscopy. Both methods revealed differences regarding mean sizes and size distributions of granules lysing under either hypotonic or hypertonic conditions. However, the results obtained byy these two methods were not consistent. In the case of hypotonic lysis, a nonmonotonic behaviour of the mean diameter as a function of the sucrose concentration was observed by PCS, but not in the micrographs. From EM size determinations we obtained a decrease in the mean diameter and an increase of the width of the distribution due to the appearance of small (50–200 nm) vesicles. Probably these vesicles are intragranular vesicles released during lysis. The maximum in photon correlation spectroscopy (PCS) diameter being 140% of the isotonic diameter is shown to be caused by the changing size distribution and geometry of the lysing granules. In the case of hyperosmotic relaxation, micrographs revealed that originally shrunken, nonspherical granules regained their spherical shape and formed small (60 nm) vesicles upon lysis. In contrast, no difference was observed between the sizes of granules prior to and after hyperosmotic relaxation by means of PCS. The paper discusses the validity of intensity-weighted light scattering data of polydisperse particle suspensions with changing size distributions. The mechanism of hyperosmotic relaxation lysis is considered.