Purification of gonadotropes and intracellular free calcium oscillation. Effects of gonadotropin-releasing hormone and interleukin 6

The intracellular free calcium concentration (Ca2+]i) in single gonadotropes was measured with a calcium-sensitive fluorescent dye indo-1 or fura-2 and a digital imaging fluorescence microscopic system to determine how interleukin-6 (IL-6) increases release of gonadotropins. IL-6 induced an increase in the basal Ca2+]i or the amplitude of spontaneous oscillation of Ca2+]i in gonadotropes in a mixed population. Gonadotropin-releasing hormone (Gn-RH) induced a biphasic increase in Ca2+]i, a transient increase, and then a prolonged increase. These effects were inhibited by the absence of extracellular calcium or pretreatment with calcium channel blockers, cobalt or nifedipine. Next, purified gonadotropes were prepared by fluorescence-activated cell sorting and argon laser treatment of the cells. Gonadotropes labeled with anti-luteinizing hormone antibody were sorted by fluorescence-activated cell sorting and then cultured as monolayers for 24-48 h. In this way, gonadotropes were concentrated from 5-10% to 70-85% from whole pituitary cells. After relabeling with anti-luteinizing hormone antibody, 100% purified gonadotropes were obtained by killing other types of cells with an argon laser. Gonadotropin-releasing hormone induced almost the same responses of Ca2+]i in the purified cell population as in the mixed cell population, but IL-6 did not affect Ca2+]i in the purified gonadotropes. These results suggest that IL-6 affects calcium mobilization in gonadotropes indirectly via paracrine pathways.