Effect of antimicrotubule agents on secretion of relaxin by large luteal cells derived from pregnant swine.

The role of microtubules in the modulation of secretion of the ovarian protein hormone, relaxin, by porcine large luteal cells (LLCs) was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis, a plaque, developed around relaxin-releasing LLCs. The rate of development of plaques in time-course experiments (1-12 h) and the detection of discontinuities in the pattern of plaque formation were used in this study as an index of the rate of relaxin release and a method to detect differential responsiveness of individual LLCs, respectively. Monolayers were bathed in medium containing three different antimicrotubule agents that bind to tubulin and induce microtubule loss through depolymerization. Exposure of luteal cell-containing monolayers to colchicine (100 microM), vinblastine (1 microM), and nocadazole (30 microM) resulted in a reduction in the rate of relaxin-induced plaque formation by LLCs, unequivocal evidence of an inhibitory effect of all three of these antimicrotubule agents of the rate of basal relaxin release. However, this suppressive effect on relaxin release was strictly time related. None of the antimicrotubule agents significantly affected plaque formation until 3 h of incubation. Thereafter, a plateau of plaque formation was observed in antimicrotubule-treated monolayers over approximately 3-8 h of incubation, indicating substantial slowing or cessation of relaxin release by LLCs during this period. During this middle phase of the incubation, plaque formation in antimicrotubule-treated monolayers was significantly lower (P less than 0.05) than that in controls. By 8-12 h of incubation plaque formation was still significantly suppressed in vinblastine-treated monolayers, but not, however, in colchicine- or nocadozole-treated monolayers. Treatment with beta-lumicolchicine (100 microM; a form of colchicine that fails to bind to tubulin) resulted in no significant change in the rate of plaque formation compared with that in controls. We conclude that microtubule-assisted transport forms an important intracellular mechanism that assists basal release of an ovarian peptide hormone, relaxin. Moreover, the discontinuity of plaque formation in the middle phase of the incubation is consistent with the view that a subpopulation of LLCs responds preferentially to antimicrotubule agents. However, the exact nature and physiological role(s) of microtubules in the control of relaxin secretion and the interrelationships of microtubules with other intracellular regulatory events remain to be clearly defined.