Morphology of rapidly frozen aortic endothelial cells. Glutaraldehyde fixation increases the number of caveolae.

Abstract

SUMMARY. Using a Polaron E7200 quick freeze unit, we investigated the distribution of vesicles (caveolae) bound to the plasma membrane of aortic endothelial cells from adult Sprague-Dawley rats. Counts of caveolae from replicas of rapidly frozen, unfixed samples were made and compared with counts from both aldehyde-fixed, conventionally frozen, and rapidly frozen aortic samples. Aldehyde-fixed samples prepared for freeze fracture by either of the freezing methods revealed a significantly greater number of caveolae than did unfixed, rapidly frozen samples. These findings suggest that the relative number of caveolae is artifactually increased by exposure to aldehyde fixatives. We conclude that previous estimates of the rates of pinocytotic activity in vascular endothelial cells may contain substantial errors based on overestimates of caveolae and underestimates of subplasmalemmal 'free ' vesicles. (Circ Res 53: 424-429, 1983)

Ultrarapid freezing reveals that skeletal muscle caveolae are semipermanent structures

“…Caveolae in cells other than skeletal muscle have been reported to be extremely transient structures, often essentially absent (or present in much lower numbers) in rapidly frozen cells as compared with those in chemically fixed cells (Bretscher and Wytack, 1977;McGuire and Twietmeyer, 1983). In those systems, the observed large number of caveolae in chemically fixed cells is thought to result either from glutaraldehyde-induced fusion of subplasmalemmal vesicles with the plasma membrane, an artifactually increased rate of normal vesicle fusion, or temporal summation during slow fixation of a normally high rate of vesicle fusion.…”

“…In addition, a significant decrease in the diameter of caveolae was reported in rapidly frozen aortic endothelial cells as compared with that in chemically fixed cells (McGuire and Twietmeyer, 1983). Likewise, an initial examination of rapidly frozen rat skeletal muscle sarcolemma by members of this group (Lee et al, 1983) gave preliminary data indicating that rapid freezing drastically reduced the number and size of caveolae in muscle plasma membrane.…”

“…In freeze-fracture replicas of aldehyde-fixed muscle, caveolae are seen either as cup-like or saucer-like P-face indentations having distincitive borders, or as truncated conical E-face projections of the plasma membrane (Bonilla et al, 198 1;Dulhunty and Franzini-Armstrong, 1975;Shotton, 1982). Several models have been proposed concerning the role of caveolae in a variety of tissues (Bonilla et al, 1981;Bundgaard, 1983;Dulhunty and FranziniArmstrong, 1975;Franzini-Armstrong et al, 1975;McGuire and Twietmeyer, 1983;Prescott and Brightmann, 1976;Steinman et al, 1983). Their function in skeletal muscle has been suggested to be a reservoir of membrane that becomes incorporated into the sarcolemma during myofiber stretch (Dulhunty and Franzini-Armstrong, 1975).…”

“…Recent studies of caveolae in rapidly frozen endothelia and fibroblasts (Bretscher and Whytock, 1977;McGuire and Twietmeyer, 1983;Robinson et al, 1984) indicate that there is a significant decrease in the number of caveolae as compared with the number in chemically fixed cells of the same type. In addition, a significant decrease in the diameter of caveolae was reported in rapidly frozen aortic endothelial cells as compared with that in chemically fixed cells (McGuire and Twietmeyer, 1983).…”

Proceedings of the Physiological Society, 12‐14 September 1985, Cambridge Meeting: Communications

“…We conclude that free smooth plasmalemmal vesicles are very rare or absent in rapidly frozen as well as in directly fixed endothelium. Any changes in the numerical density of vesicular profiles in conventional thin sections (McGuire & Twietmeyer, 1983) are therefore likely to reflect a change in the number of invaginations from the cell surface rather than artifactitious fusions between free vesicles and the cell surface.…”