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.
Masumoto
N
N
Department of Obstetrics and Gynecology, Osaka University Medical School, Japan.
Tasaka
K
K
Kasahara
K
K
Miyake
A
A
Tanizawa
O
O
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D006062:Gonadotropins / Q000302:isolation & purification*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D015850:Interleukin-6 / Q000494:pharmacology*; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000502:physiology; D051381:Rats; D011919:Rats, Inbred Strains
D006062:Gonadotropins; D015850:Interleukin-6; D007987:Gonadotropin-Releasing Hormone; D002118:Calcium