oxadiazoles and Adenoma

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

Topics: Action Potentials; Adenoma; Adrenocorticotropic Hormone; Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Corticotropin-Releasing Hormone; Cytosol; Egtazic Acid; Female; Humans; Isradipine; Membrane Potentials; Oxadiazoles; Pituitary Gland; Pituitary Neoplasms; Rats; Rats, Inbred Strains; Spectrometry, Fluorescence; Tetrodotoxin; Tumor Cells, Cultured

Endocrine cells possess voltage-sensitive Ca2+ channels involved in the modulation of hormonal secretion. Using the dihydropyridine, (+)-PN 200-110, we have investigated the binding characteristics of this ligand to pituitary membrane Ca2+ channels from normal rat, normal and adenomatous human pituitaries. [3H]PN 200-110 binds specifically to rat pituitary membranes to one class of sites (Kd = 0.41 +/- 0.10 mM; Bmax = 39 +/- 1.3 fmol/mg protein). At 37 degrees C, equilibrium is reached in 45 min and half-life of the binding is 13 min. No significant changes were observed for either the Kd or Bmax values between normal rat and human pituitaries or between the different types of adenomas (GH- and PRL-secreting and non-secreting). As the secretory activity of the pituitary adenomas, involving Ca2+ mobilization, varies from one adenoma to another, our results could indicate that, if there is a modified regulation of Ca2+ entry in the adenomas, it may not be related to a varying number of calcium channels, at least the channels labeled by the dihydropyridine (+)-PN 200-110.

Topics: Adenoma; Animals; Binding, Competitive; Brain; Calcium Channel Blockers; Calcium Channels; Female; Humans; In Vitro Techniques; Isradipine; Myocardium; Oxadiazoles; Pituitary Gland; Pituitary Neoplasms; Rats; Rats, Inbred Strains; Receptors, Nicotinic