Morphological effects of osmolarity on purified noradrenergic vesicles

Abstract

Large dense core vesicles (LDV) were purified from bovine splenic nerve homogenates by the sucrose-D2O density gradient method. Vesicles were subjected to a 50% increase and decrease in osmolarity from the control 330 mosmol 1(-1) by adjusting sucrose or potassium phosphate buffer during pre- and/or postfixation. Control vesicles with a mean diameter of 717 A readily swelled to approximately 1050 A and shrunk to approximately 600 A in the hypotonic and hypertonic media, respectively, with either sucrose or phosphate buffer. The dense core responded similarly but to a lesser degree. Prefixation in glutaraldehyde had little effect on vesicle sensitivity to subsequent tonicity change, not did the fixative per se exert an obvious osmotic effect. Thus, final vesicle size was largely determined by the OsO4 postfixation medium and principally by the vehicle rather than the fixative. In controls there was a mixture of spherical to oblate vesicles mostly filled with an electron-dense matrix. Upon swelling, more vesicles became spherical and nearly all had a prominent translucent halo between core and membrane. Upon shrinking, more vesicles became oblate, the halo was obliterated and the electron-density of the matrix increased. Frequency distributions of vesicle diameters at different tonicity clearly indicated that the diameter of LDV could overlap the 400-500 A range characteristic of small dense core vesicles; however, there was no suggestion of a population of the latter in the purified LDV fraction. Implications are discussed concerning the biochemical and morphological identification of 'light' and 'heavy' density peaks of noradrenaline and dopamine beta-hydroxylase from mixed vesicle populations and the possible relevance of changes in vesicle shape to a functional state in situ.

Effects of glutaraldehyde fixative osmolarities on smooth muscle cell volume, and osmotic reactivity of the cells after fixation

“…Results from our Coulter Counter study and tissue strip study both indicated that SMC are still osmotically reactive after GA fixation, which agrees with earlier reports on other cell and tissue types (e.g. Elbers, 1966;Fahimi & Drochmans, 1965;Korneliussen, 1972;Bone & Ryan, 1972;Brunk & Ericsson, 1972;Thureson-Klein et al, 1975;Tisdale & Nakajima, 1976;Boyde et al, 1977). This is in contrast to some reports (Morel et al, 1971;Carstensen et al, 1971 ;Penttila et al, 1974), which stated that cells become osmotically inactive after GA fixation.…”

Catecholamine‐rich cells and varicosities in bovine splenic nerve, vesicle contents and evidence for exocytosis

“…If the LDV are the source, this means that DA could represent up to 7%-8% of the total catecholamines in these vesicles. On the other hand, DA may also be present in the slightly larger dense cored vesicles from the SIF cells, which may occur as a small population in the LDV fraction as indicated by the skew in the vesicle frequency distribution curve (Thureson et al, 1975). Fluorescent varicosities have been described in the splenic nerve (Hamberger e t al., 1971) but are not unique for this nerve (Jacobowitz and Woodward, 1968).…”

Dopamine β‐hydroxylase distribution in density gradients: Physiological and artefactual implications

“…Pellets were postfixed in 2% OsO4 containing 0.2M veronal buffer a t the same pH. E n bloc staining with uranylacetate, dehydration, and embedding in Epon were as previously described (Thureson- Klein et al, 1973Klein et al, , 1975.…”

“…9. There is no indication from histogram analysis of large numbers of vesicles that a minor population of small terminal vesicles occurs in the heavy vesicle peak (Thureson- Klein et al, 1975).…”