Free intracellular calcium ([CA2+]i) in opioid sensitive cells of the porcine anterior pituitary.
A new experimental model was utilized to study calcium involvement in the mechanism of opioid influence on cultured porcine pituitary cells. The in vitro model involved interactive argon laser cytometry of pituitary cells pre-loaded by three dyes (fluo-3AM, fura-red and naloxone-conjugated to fluorescein). We compared: 1) the kinetics of free intracellular calcium ([Ca2+]i) in anterior pituitary cells of pregnant pigs (day 25-30) treated in vitro with naloxone (NAL) or gonadotrophin-releasing hormone (GnRH) and 2) the distribution of the opioid-sensitive cells by image analysis of doubly loaded cells. In experiment 1, 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 line scans across cells were performed and the fluorescence emission from individually selected cells was measured in a time-dependent manner (in 0.5 seconds intervals during periods of 50 seconds). Analysis of data indicated significant 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 fluorescence intensity of doubly-loaded pituitary cells (fura-red, 488(ex)/605(em) nm, 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 approximately 8% of the entire population of anterior pituitary cells exhibited sensitivity to the opioid antagonist treatment. This paper demonstrates calcium involvement in the opioid action on anterior pituitary cells from pregnant pigs and provides a useful model for studies at the individual pituitary cell level and in time-dependent manner.
Szafranska
B
B
Department of Animal and Range Sciences, North Dakota State University, Fargo, 58105-5727, USA. szanfran@moskit.uwm.edu.pl
Tilton
J E
JE
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002478:Cells, Cultured; D005260:Female; D005300:Fertility Agents, Female / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D009270:Naloxone / Q000494:pharmacology*; D009292:Narcotic Antagonists / Q000494:pharmacology*; D009294:Narcotics / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000187:drug effects* / Q000378:metabolism; D011247:Pregnancy; D013552:Swine
D005300:Fertility Agents, Female; D005456:Fluorescent Dyes; D009292:Narcotic Antagonists; D009294:Narcotics; D007987:Gonadotropin-Releasing Hormone; D009270:Naloxone; D002118:Calcium