Free intracellular calcium ([CA(2+)](i)) in opioid sensitive cells of the porcine anterior pituitary

A new experimental model was utilized to study calcium involvement in the m echanism of opioid influence on cultured porcine pituitary cells. The in vi tro model involved interactive argon laser cytometry of pituitary cells pre -loaded by three dyes (fluo-3AM, fura-red and naloxone-conjugated to fluore scein). We compared: 1) the kinetics of free intracellular calcium ([Ca2+]( i)) in anterior pituitary cells of pregnant pigs (day 25-30) treated in vit ro with naloxone (NAL) or gonadotrophin-releasing hormone (GnRH) and 2) the distribution of the opioid-sensitive cells by image analysis of doubly loa ded cells. In experiment I, the changes in [Ca2+](i) of pituitary cells pre loaded with fluo-3 AM (488(ex) /520(em) nm) in response to NAL (10(-6) M) or to GnRH (10(-8) M) were compared to a control cell group. Repetitive lin e scans across cells were performed and the fluorescence emission from indi vidually selected cells was measured in a time-dependent manner (in 0.5 sec onds intervals during periods of 50 seconds). Analysis of data indicated si gnificant increases of [Ca2+](i) in NAL- (P<0.001) and GnRH-treated cells ( P<0.05) in comparison to the control group. In experiment 2, the fluorescen ce intensity of doubly-loaded pituitary cells (fura-red, 488(ex) /605(em) n m, as principal calcium indicator and NAL-conjugated with fluorescein, 488( ex) /520(em) nm, to distinguish opioid-sensitive cells) were measured using dual detector image analysis. We found that only similar to 8% of the enti re population of anterior pituitary cells exhibited sensitivity to the opio id antagonist treatment. This paper demonstrates calcium involvement in the opioid action on anterio r pituitary cells from pregnant pigs and provides a useful model for studie s at the individual pituitary cell level and in time-dependent manner.