Imaging of intracellular calcium in rat anterior pituitary cells in response to growth hormone releasing factor.
1. Changes in intracellular ionized calcium [Ca2+]i induced by human growth hormone releasing factor (hGRF) were analysed by quantitative fluorescent microscopy using a dual-wavelength, ratiometric video imaging system and low light level charge-coupled device (CCD) camera visualizing Fura-2 in dispersed male rat anterior pituitary cells. 2. In cells responding to hGRF, spontaneous basal oscillations in [Ca2+]i were frequently observed, and these were usually characterized by a gradient of [Ca2+]i localized in the subplasmalemmal region of the cell. 3. Of the cells which responded to hGRF, the peptide evoked a rise in [Ca2+]i, especially in the region of the subplasmalemma. Continuous application of 10 nM-hGRF produced several different temporal patterns of the [Ca2+]i response which were not attributable to spatial response profiles. A sustained rise in [Ca2+]i was the most common type of response to hGRF (44% of the cells examined). 4. One-third of the cells responding to 10 nM-hGRF showed spontaneous basal [Ca2+]i oscillations ranging from 100 to 500 nM. Mean values of basal and 10 nM-hGRF-induced [Ca2+]i of these cells were 81 +/- 11 nM (mean +/- S.E.M., n = 27) and 560 +/- 47 nM (n = 27) respectively. There was no significant correlation between basal [Ca2+]i and the hGRF-induced [Ca2+]i increase, nor was there any consistent correlation with regard to the spatial response profile. 5. Application of 2 mM-Co2+ abolished the hGRF-induced rise in [Ca2+]i. Quantitative analysis of this effect, performed by comparing the mean [Ca2+]i evoked during the application of hGRF with and without Co2+, respectively, also showed significant inhibition of the hGRF-induced rise in [Ca2+]i by the application of Co2+ (P less than 0.001). 6. The hGRF-induced rise in [Ca2+]i was completely suppressed by replacing extracellular Na+ with impermeant molecules such as mannitol. The onset and offset of suppression was as rapid as that induced by Co2+. Quantitative analysis showed significant inhibition of the hGRF-induced rise in [Ca2+]i by Na+ replacement (P less than 0.01). 7. Tetrodotoxin, a potent blocker of voltage-sensitive Na+ channels (5 and 20 microM), did not affect the hGRF-induced rise in [Ca2+]i. 8. Extracellular application of the membrane permeable dibutyryl cyclic AMP (DBcAMP) to elevate intracellular levels of cyclic AMP caused a large rise in [Ca2+]i, which was dependent on extracellular Na+ and was abolished by 2 mM-Co2+ applied in the bath.(ABSTRACT TRUNCATED AT 400 WORDS)
Kato
M
M
Department of Neuroendocrinology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge.
Hoyland
J
J
Sikdar
S K
SK
Mason
W T
WT
D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D003035:Cobalt / Q000378:metabolism; D013007:Growth Hormone-Releasing Hormone / Q000494:pharmacology*; D007424:Intracellular Fluid / Q000737:chemistry; D008856:Microscopy, Fluorescence; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism; D051381:Rats; D013779:Tetrodotoxin / Q000494:pharmacology
D003035:Cobalt; D013779:Tetrodotoxin; D013007:Growth Hormone-Releasing Hormone; D002118:Calcium