Ionophore bromo-A23187 reveals cellular calcium stores in single pituitary somatotropes.
Free cytosolic calcium concentration, [Ca2+]i, in single rat pituitary cells can be measured with the fluorescent, calcium-sensitive probe fura-2 and digital image analysis. A reverse hemolytic plaque assay (RHPA) identifies somatotropes in the mixed population of pituitary cells. Previous studies showed that growth hormone releasing factor (GRF) stimulates growth hormone (GH) release from pituitary somatotropes by increasing the influx of calcium into the cell. Somatostatin reduced [Ca2+]i and inhibits hormone release presumably by closing calcium channels in the membrane. The calcium-ionophore bromo-A23187 rapidly increased [Ca2+]i from a baseline of 226 +/- 38 nM to a peak of 842 +/- 169 nM (mean +/- SEM) which was reached 30 s after exposure to the drug. This spike was followed by a sustained phase of elevated [Ca2+]i approximately 370 nM. When somatostatin (SRIF) (10 nM) was combined with ionophore treatment, the initial rise was preserved. However, the second phase was abolished and SRIF lowered [Ca2+]i to 57 +/- 7 nM. Depolarizing the cellular membrane with high extracellular potassium (60 mM) increased cytosolic calcium as well (797 +/- 178 nM); however, this was not affected by the addition of SRIF (988 +/- 71 nM). KCl depolarization in calcium-free medium (+1.5 mM EGTA) provoked no rise in cytosolic calcium. In contrast, after ionophore, the initial spike was preserved while the sustained phase of elevated [Ca2+]i was abolished. We conclude from these data that (1) membrane depolarization and ionophore treatment lead to an influx of calcium into the cytosol of normal pituitary somatotropes. (2) SRIF inhibits calcium influx induced by ionophore but not influx after depolarization with high potassium concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
Holl
R W
RW
Department of Internal Medicine, University of Virginia Medical School, Charlottesville 22908.
Thorner
M O
MO
Zysk
J R
JR
Leong
D A
DA
D000818:Animals; D000001:Calcimycin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D005453:Fluorescence; D008297:Male; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013004:Somatostatin / Q000502:physiology; D014700:Verapamil / Q000494:pharmacology
D000001:Calcimycin; D013004:Somatostatin; C033170:4-bromo-A-23187; D014700:Verapamil; D011188:Potassium; D002118:Calcium