title: Effects of ACTH and angiotensin II on cytosolic calcium in cultured adrenal glomerulosa cells. Role of cAMP production in the ACTH effect.
abstract: We have used microspectrofluorometry and video imaging techniques in order to study and compare the changes in intracellular calcium concentrations [( Ca2+]i) of individual Fura-2 loaded glomerulosa cells cultured for three days and stimulated either with angiotensin II (AT), K+, or adrenocorticotropin (ACTH). As previously demonstrated for freshly isolated cells, K+ ion induces an immediate increase in [Ca2+]i, although AT induces a biphasic response, characterized by an initial transient spike, followed by a sustained plateau. In this study, we demonstrate, for the first time, that ACTH is able to induce a [Ca2+]i increase in cultured glomerulosa cells from rat and bovine sources. Moreover, it is clear that the pattern of [Ca2+]i increase elicited by ACTH is different from that observed with AT. In most cases, addition of ACTH leads to a slow increase in [Ca2+]i after a long latency period ranging from 10-15 min, which could be correlated to cAMP time-production. The present results show that: (a) in the absence of extracellular Ca2+, ACTH does not increase [Ca2+]i; (b) the response develops slowly and cases immediately after [Ca2+]e depletion or addition of calcium channel blockers, such as nifedipine or omega-conotoxin; (c) the addition of the calcium channel agonist Bay K 8644 enhances the ACTH response; (d) the cAMP analog, 8-Br-cAMP, induces an increase in [Ca2+]i similar to that observed with ACTH, which is also dependent of the presence of calcium in the extracellular medium; (e) time-production of ACTH-induced cAMP follows quite well the increase in [Ca2+]i; (f) Bay K 8644 also enhances the 8-Br-cAMP induced increase in [Ca2+]i; and (g) ACTH-induced Cai response is inhibited by the specific protein kinase A blocker, HA1004. These observations, combined with previous results obtained on the effects of ACTH on calcium currents and action potentials, suggest that the [Ca2+]i increase induced by ACTH results from a calcium influx through dihydropyridine and omega-conotoxin sensitive calcium channels, which need to be phosphorylated by cAMP for full activation. The use of video-imaging techniques has allowed us to examine the spatial distribution of changes in [Ca2+]i in single cells. The ability to simultaneously record images of a number of cells confirm the heterogeneity of cellular responses, and corroborate results obtained through photocounting only. Our results indicate that ACTH initially increases [Ca2+]i locally beneath the cell membrane and throughout the cell thereafter, whereas angiotensin II elicits a more prominent effect in certain regions of the cell and eventually extends to the entire cell surface.
authors.affiliation: Department of Medicine, Faculty of Medicine, University of Sherbrooke, Quebec, Canada.
authors.forename: E
authors.initials: E
authors.lastname: Tremblay
authors.forename: M D
authors.initials: MD
authors.lastname: Payet
authors.forename: N
authors.initials: N
authors.lastname: Gallo-Payet
mesh_terms: D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology*; D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000096:biosynthesis*; D003592:Cytophotometry; D016257:Fura-2 / Q000737:chemistry; D007091:Image Processing, Computer-Assisted; D008853:Microscopy; D009543:Nifedipine / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000633:toxicity; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D013050:Spectrometry, Fluorescence; D014741:Video Recording; D015384:Zona Glomerulosa / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020960:omega-Conotoxins
chemical_list: D010456:Peptides, Cyclic; D020960:omega-Conotoxins; C053423:Conus magus toxin; D000804:Angiotensin II; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D000324:Adrenocorticotropic Hormone; D000242:Cyclic AMP; D009543:Nifedipine; D011188:Potassium; D002118:Calcium; D016257:Fura-2