Spontaneous and corticotropin-releasing factor-induced cytosolic calcium transients in corticotrophs.
Spontaneous and CRF-stimulated changes in the cytosolic free calcium concentration ([Ca2+]i) were studied in two types of corticotrophs: 1) cultured human ACTH-secreting pituitary adenoma cells (hACTH cells), and 2) identified small ovoid corticotrophs cultured from normal rat pituitaries. [Ca2+]i was monitored in individual corticotrophs by dual emission microspectrofluorimetry using indo-1 as the intracellular fluorescent Ca2+ probe. In hACTH cells, [Ca2+]i measurements were carried out in combination with electrophysiological recordings obtained using whole cell patch-clamp techniques. It was shown that a single spontaneous Ca(2+)-dependent action potential led to a marked transient increase in [Ca2+]i in human tumoral corticotrophs. Spontaneous fluctuations in [Ca2+]i were also observed in unpatched corticotrophs whether derived from human pituitary tumors or normal rat tissue. Based on their striking kinetic features and their sensitivity to external Ca2+, we suggest that these spontaneous [Ca2+]i transients were the consequence of action potential firing. Under separate voltage-clamp (patch-clamp) conditions, tumor corticotrophs showed two Ca2+ current components: a low threshold, rapidly inactivating (T-type) current, and a higher threshold, slowly inactivating (L-type) current. The dihydropyridine Ca2+ channel blocker PN 200-110 (100 nM) abolished the L-type current without affecting the T-type current, while the inorganic Ca2+ channel blocker Cd2+ (200 microM) suppressed both Ca2+ currents. The Na+ channel blocker tetrodotoxin (5 microM) did not affect inward currents in tumor corticotrophs. Both L- and T-type voltage-gated Ca2+ channels were involved in controlling [Ca2+]i transients in both tumor and normal corticotrophs, inasmuch as Cd2+ (200 microM) abolished [Ca2+]i) transients, while PN 200-110 (100 nM) greatly diminished, but did not completely abolish, [Ca2+]i transients. The latter did not appear to depend on a voltage-dependent Na+ influx, since they were unaffected by tetrodotoxin (5 microM). Corticotrophs generate [Ca2+]i transients in response to the hypothalamic secretagogue CRF by acting on their membrane excitability. Indeed, we demonstrated in combined fluorescent and electrophysiological experiments that CRF (100 nM) had a coordinate action on human tumoral corticotrophs comprised of a modest depolarization and an increase in the frequency of both action potentials and subsequent [Ca2+]i transients. A coincident increase in the peak amplitude of the [Ca2+]i transient and after hyperpolarization was also observed in some CRF-stimulated cells. CRF (100 nM) evoked qualitatively similar [Ca2+]i patterns in human tumoral and normal rat corticotrophs not subjected to patch-clamping.(ABSTRACT TRUNCATED AT 400 WORDS)
Guérineau
N
N
Laboratory of Neurophysiology, CNRS URA 1200, University of Bordeaux II, France.
Corcuff
J B
JB
Tabarin
A
A
Mollard
P
P
D000200:Action Potentials / Q000187:drug effects; D000236:Adenoma; D000324:Adrenocorticotropic Hormone / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D005260:Female; D006801:Humans; D017275:Isradipine; D008564:Membrane Potentials / Q000187:drug effects; D010069:Oxadiazoles / Q000494:pharmacology; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D010911:Pituitary Neoplasms; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence; D013779:Tetrodotoxin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D010069:Oxadiazoles; D013779:Tetrodotoxin; D004533:Egtazic Acid; D000324:Adrenocorticotropic Hormone; D003346:Corticotropin-Releasing Hormone; D002118:Calcium; D017275:Isradipine