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The cytosolic free calcium concentration and cumulative GH release were measured simultaneously in normal pituitary cells. This was made possible by a novel combination of fluorescence microscopy using the calcium indicator fura-2 and a reverse hemolytic plaque assay. GRF (10 nM) rapidly increased the intracellular free calcium concentration ([ Ca2+]i) from a basal level of 234 +/- 17 nM (mean +/- SE) to a peak value of 480 +/- 61 nM 1 min after stimulation. This GRF-induced calcium rise was totally abolished in calcium-free medium or in the presence of calcium channel blockers cobalt chloride (2 mM) and verapamil (100 microM). When somatostatin (SRIF; 1 nM) was added after basal recordings, cytosolic calcium decreased to 96 +/- 23 nM in identified somatotropes. [Ca2+]i returned to baseline upon the removal of SRIF inhibition. This rebound was higher when a sequential treatment of SRIF followed by GRF was applied. Exposing cells to a combination of GRF (10 nM) plus SRIF (1 nM) resulted in a decrease in [Ca2+]i identical to that caused by SRIF treatment alone. Despite the 10-fold excess of GRF, SRIF not only inhibited hormone secretion, but also totally overcame the GRF-induced rise of [Ca2+]i. In summary, stimulation by GRF increases cytosolic calcium in normal somatotropes. This increase is proposed to be due to the influx of calcium through membrane ion channels. In contrast, SRIF decreases [Ca2+]i. This might explain the cAMP-independent effects of this peptide. The effect of SRIF dominates over that of GRF with respect to both changes in [Ca2+]i and hormone release. Changes in the GH secretory rate are, therefore, accompanied by parallel changes in [Ca2+]i, both of which are primarily regulated by SRIF.

To assess the frequency with which acromegaly is caused by ectopic secretion of GRF, we collected plasma samples from 177 unselected acromegalic patients. The samples together with those of three acromegalic patients with previously diagnosed tumors secreting GRF and of normal subjects were assayed in 3 independent GRF RIAs. Plasma immunoreactive GRF (IR-GRF) levels in normal subjects were either undetectable or detectable at levels up to 62.5 pg/ml. In none of the 177 specimens from acromegalic patients were IR-GRF values detectable in all assays, and in the most sensitive assay, the levels were similar to those in normal subjects, with the highest level measuring 82 pg/ml. In contrast, plasma IR-GRF found in the 3 patients with tumors that secreted GRF ranged from 2.0-24.4 ng/ml. These data suggest that extrahypothalamic GRF secretion is a rare cause of acromegaly. However, it is important that this rare cause of acromegaly be diagnosed before the patient has unnecessary surgery and/or irradiation directed at the pituitary. We recommend that plasma IR-GRF be measured in each new acromegalic patient.

Cytosolic free Ca2+ ion concentrations were ([Ca2+]i) measured in single swine granulosa cells using the Ca2(+)-sensitive fluorescent indicator dye fura-2 and digital imaging videomicroscopy with high spatial and temporal resolution. Ovine FSH (oFSH) elicited specific [Ca2+]i increases, which reached their highest value within 5 min of the onset of stimulation and were sustained for 10-15 min. [Ca2+]i usually returned to prestimulated levels and cells regained full responsiveness to a second exposure to oFSH within 20 min. The effect of FSH was not blocked by pretreatment with pertussis toxin. Forskolin and 8-bromo-cAMP were able to mimic similar increases in [Ca2+]i in the presence or absence of extracellular Ca2+. In contrast, the oFSH-induced [Ca2+]i rises were abolished in the absence of extracellular Ca2+. The Ca2+ channel blocker verapamil completely abolished the oFSH-induced [Ca2+]i rise, but not the 8-bromo-cAMP- or the forskolin-induced [Ca2+]i rise. In summary, we have demonstrated that [Ca2+]i is regulated by oFSH in single swine granulosa cells. We propose that the transducing pathway for this FSH effect might not involve cAMP, and that whichever second messenger is responsible for the Ca2+ signal generation acts upon plasma membrane Ca2+ channels.