Ram rete testis fluid is shown to elicit clustering of suspensions of Sertoli cells from testes of immature rats, TM-4 cells derived from mouse testis, and erythrocytes from several species. Details of bioassay procedures and characteristics of the phenomenon are reported. Concanavalin A and wheat germ agglutinin prevent aggregation elicited by rete testis fluid, and this inhibition is specifically prevented by alpha-methylmannoside and N-acetyl-glucosamine, respectively. Influences of rete testis fluid on cell aggregation are not dependent on exogenous calcium, but clustering is blocked by various metabolic inhibitors such as dinitrophenol. Rete testis fluid addition to mixed suspensions of erythrocytes and TM-4 cells is followed by separate aggregation of each cell types. Using aggregation of TM-4 cells suspended in simple medium at low density in rotation as a bioassy, we have determined which fractions in rete testis fluid retain activity. We have shown that a heat-stable, trypsin-sensitive protein, having an isoelectric point below pH 4.0, retains the capacity to aggregate cells. We discuss the possible functions of this protein, named clusterin, in cell interactions.
A novel effect of carnitine and O-acylcarnitine derivatives has been described. The presence of these compounds has been shown to inhibit the aggregation of erythrocytes otherwise elicited by the addition of clusterin or fetuin. The specificity of carnitine action has been investigated by comparing influences of chemically related compounds. The concentrations required for inhibition by approximately 50% of aggregation of erythrocytes by clusterin under in vitro conditions defined were determined to be 1.5 mM for L(-) or D(+) enantiomers of carnitine; 0.5 mM for decanoyl(-)- or (+)-carnitine; 0.13 mM for lauroyl(-)- or (+)-carnitine, and 0.05 mM for myristoyl(-)- or (+)-carnitine. In contrast, concentrations up to 12.5 mM of dimethylcarnitine, deoxycarnitine, acetylcholine, acetyl-beta-methylcholine, or inositol had no detectable inhibitory effect on aggregation elicited by clusterin. Clusterin addition also resulted in the aggregation of three other cell types examined (guinea pig spermatozoa, a cell line derived from testes of neonatal mice called TM4 cells, and Sertoli cells from testes of 20 day-old rats). As in the case with erythrocytes, the presence of carnitine inhibited aggregation of spermatozoa, TM4 cells, and Sertoli cells in suspension. We consider possible mechanisms by which carnitine inhibits aggregation of erythrocytes and other populations of dispersed cells incubated in the presence of clusterin.
Carnitine is bound by intact red blood cells, by red blood cell ghosts, and by glutaraldehyde-fixed human erythrocytes in a non-saturable, temperature-dependent manner. Binding of carnitine by these preparations is blocked by sulfhydryl reagents. Incubation or preincubation of red blood cell preparations with carnitine inhibits the aggregation of erythrocytes otherwise elicited by fibrinogen. Identical effects are obtained with red blood cell ghosts. In contrast, choline, even at high concentrations, is inactive in preventing the aggregation of erythrocytes. We discuss possible mechanisms by which carnitine favors the dispersion of red blood cells, and we present data indicating that sulfhydryl groups on erythrocyte membranes are required to permit these carnitine actions to be manifested.
Clusterin, a glycoprotein which elicits the aggregation of a wide variety of cells (Fritz, I. B., and Burdy, K.:J. Cell Physiol., 140:18-28, 1989), has been utilized to investigate some of the factors modulating the competition between cell-substratum interactions and cell-cell interactions. We compared the responses to clusterin by anchorage-independent cells (erythrocytes) with those by anchorage-dependent TM4 cells (a cell line derived from neonatal mouse testis cells). Cells were maintained in culture in the presence of various substrata chosen to enhance cell-substratum interactions (laminin-coated wells), or to diminish cell-substratum interactions (agarose-coated wells). Results obtained showed that the aggregation of erythrocytes elicited by clusterin was independent of the nature of the substratum. In contrast, clusterin addition resulted in aggregation of anchorage-dependent TM4 cells only when TM4 cell-substratum interactions were weak. Thus, clusterin did not aggregate TM4 cells plated upon a laminin substratum, but readily aggregated TM4 cells plated upon an agarose-coated substratum, independent of the sequence of addition of cells and clusterin to the culture dish. We utilized YIGSR, a peptide which competes with laminin for laminin receptors, to determine the possible role of laminin receptors on TM4 cells in the competition between cell-substratum interactions and cell-cell interactions. The presence of YIGSR did not alter responses of erythrocytes to clusterin under all conditions examined. In contrast, the responses of TM4 cells to clusterin were greatly changed. YIGSR addition resulted in the inhibition of aggregation of TM4 cells otherwise elicited by clusterin. YIGSR also prevented attachment of TM4 cells to a laminin-coated surface, but this was reversed by the presence of clusterin. We discuss the possible roles of clusterin and laminin in altering the balance in the competition between cell to cell interactions and cell to substratum interactions.
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