Alpha-subunit Secretion Research Articles

Effects of insulin-like growth factor-I on growth hormone and prolactin secretion and cell proliferation of human somatotrophinomas and prolactinomas in vitro.

IGF-I inhibits GH secretion from normal and some tumorous pituitary tissue, and has been shown to be mitogenic for gonadotrophinoma cells in vitro. It is not known whether IGF-I affects somatotrophinoma cellular proliferation or the secretion of other hormones, such as PRL and alpha-subunit, which are often co-secreted by these tumours. We have therefore examined the effects of IGF-I on proliferation and hormonal secretion of human somatotrophinomas and prolactinomas in vitro. Pituitary adenoma tissue was dispersed to single cells in monolayer culture. The effects of 100 nM IGF-I on GH, PRL and alpha-subunit secretion were determined over 4-hour and over 4-day periods, and a 4-day dose-response study using 1-100 nM IGF-I was performed on two tumours. Adenoma cell S-phase proliferation was determined after bromodeoxyuridine incorporation for 1 hour after 4 days, using a double immunostaining method. Over 4 hours, 100 nM IGF-I had no effect on GH, PRL or alpha-subunit secretion in 7 tumours. Over 4 days, 100 nM IGF-I reduced GH secretion in 5/8 somatotrophinomas (range 17-84%, P < 0.05) compared to controls, with tumours responding to IGF-I having lower basal serum and in-vitro GH levels than tumours unaffected by IGF-I (P < 0.05). There was no effect on alpha-subunit secretion in any of the three tumours studied. PRL cosecretion was increased in 3/5 somatotrophinomas compared to control (20, 30 and 37%, P < 0.05), with tumours responding to IGF-I being associated with lower basal serum and in-vitro PRL levels than those tumours unaffected by IGF-I. IGF-I also increased PRL secretion in 2/2 prolactinomas (27 and 32%, P < 0.05) compared with control. GH was inhibited and PRL secretion was stimulated by 1 and 10 nM IGF-I in the two dose-response studies. The proliferative labelling index did not exceed 1.9% in any tumour and no proliferative effect was found with 100 nM IGF-I in any somatotrophinoma. IGF-I inhibited tumorous GH in 62% and stimulated PRL secretion in 71% of tumours over 4 days, without affecting alpha-subunit secretion or being mitogenic for somatotrophinoma cells in vitro. No hormonal effects were observed over short (4-hour) incubations. IGF-I may be a newly recognized factor directly stimulating tumorous PRL secretion.

Effects of pituitary adenylate cyclase-activating polypeptide on gonadotropin secretion and subunit messenger ribonucleic acids in perifused rat pituitary cells.

Pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) is a neuropeptide related to vasoactive intestinal peptide-secretin-glucagon which stimulates adenylate cyclase in cultured rat pituitary cells and stimulates LH and FSH release in vitro and in vivo. Because the cAMP-protein kinase-A pathway regulates the gonadotropin subunit messenger RNAs (mRNAs) and modulates GnRH-stimulated gonadotropin secretion in vitro, we examined the effects of PACAP38 on gonadotropin secretion and subunit mRNA levels. Anterior pituitary cells were prepared from 7-week-old male rats castrated at 5 weeks of age. In monolayer cultures stimulated with GnRH, 0.1-10 nM PACAP38 decreased (P < 0.05) the EC50 for GnRH dose-dependently without affecting the maximum LH secretory response. Cells were next stimulated with 1-min pulses of 2.5 nM GnRH every hour for 9 h in the absence or presence of 10 nM PACAP38, which was perifused continuously. The amplitude of GnRH-induced LH, FSH, and alpha-subunit secretory episodes from PACAP38-treated cells rose (P < 0.01) gradually to 233 +/- 54%, 197 +/- 44%, and 378 +/- 104%, respectively (mean +/- SEM; n = 5 experiments), of the value for control cells lacking PACAP38. This enhancement was sustained for at least 3 h after PACAP38 was removed from the perifusion medium. With PACAP treatment, interpulse secretion of LH and alpha-subunit increased gradually (P < 0.01) to 174 +/- 21% and 212 +/- 64% of the value for chambers stimulated with GnRH alone (control), respectively, whereas interpulse secretion of FSH declined (P < 0.001) to 75 +/- 7% of the control value. In contrast to the gradual effect of PACAP38 to enhance GnRH-induced hormone secretion, PACAP38 alone produced a transient burst of gonadotropin secretion. At the completion of the perifusions, total RNA was extracted and gonadotropin subunit mRNA levels were determined by Northern analysis. GnRH increased (P < 0.01) FSH beta mRNA to 438 +/- 52% of the level in cells stimulated with medium alone (control). Adding PACAP38 to the perifusion medium partially blocked (P < 0.01) the effect of GnRH (178 +/- 20% of the control value), and PACAP38 alone reduced (P < 0.01) FSH beta mRNA levels to 31 +/- 3% of the control value. By contrast, alpha-subunit mRNA levels were increased by both PACAP38 (143 +/- 4% of the control value; P < 0.01) and GnRH (121 +/- 2% of the control value; P < 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Glycoprotein hormone alpha-subunit secretion in prolactinomas and in non-functioning adenomas: relation with the tumour size.

Free glycoprotein hormone alpha-subunit plasma levels have been reported to be increased in glycoprotein hormone-secreting adenomas and in acromegaly, but rarely in prolactinomas and in only two cases of Cushing's disease. The prevalence of elevated plasma alpha-subunit levels in patients with non-functioning adenomas is still unclear. In addition, no previous work has described plasma alpha-subunit levels in a comprehensive series of adenomas characterized by in-vivo secretion and/or immunocytochemistry. Thirty-seven patients with definite prolactinomas and 48 with non-functioning tumours characterized by immunocytochemistry were studied, from a series of 145 consecutive patients including 33 acromegalics, 18 patients with glycoprotein hormone-secreting adenomas and 9 with Cushing's disease. Plasma free alpha-subunit was measured by radioimmunoassay in all patients and in a large sample of normal subjects to establish normal ranges according to sex, age and menstrual status. Tumour volume index was the product in cm3 of length, width and height of the adenoma as assessed by computerized tomography or magnetic resonance imaging. Twelve of the 37 (32%) patients with prolactinomas had increased plasma alpha-subunit levels; their tumours were significantly larger with significantly higher plasma PRL levels than those of patients without increased plasma alpha-subunit levels (P < 0.02). All prolactinomas above 50 cm3 were associated with alpha-subunit secretion, whereas only 6 of 29 smaller tumours were similarly associated. Twelve of the 48 'non-functioning' adenomas actually secreted alpha-subunit in vivo: 8 gonadotrophin-secreting, 2 'pure' alpha-secreting, one with negative immunocytochemistry and one necrotic adenoma. Their volumes were significantly higher than those of adenomas without increased plasma alpha-subunit levels (P < 0.04). Plasma alpha-subunit levels were increased in the 6 patients with TSH-secreting adenomas, 8 of 12 with FSH-secreting adenomas, 11 of 33 acromegalics and none of those with Cushing's disease. Plasma free alpha-subunit levels were increased in 49 of 145 patients (34%). For prolactinomas and 'non-functioning' adenomas, alpha-subunit hypersecretion was seen more often with larger tumours. Half of the cases with increased free alpha-subunit in this series were patients harbouring an adenoma which did not stain for an intact glycoprotein hormone.

Contrasting effects of a gonadotropin-releasing hormone agonist and antagonist on the secretion of free α subunit

Gonadotropin-releasing hormone agonists and antagonists have initial divergent effects on the pituitary secretion of intact biologically active gonadotropins and long-term divergent effects on the secretion of free alpha-subunit. The antagonists appear to function as true competitive inhibitors, blocking the stimulatory effects of endogenous GnRH without evoking any known postreceptor activity. The agonists, in contrast, initially stimulate pituitary secretion and then incompletely desensitize the gonadotrope, resulting in suppression of intact gonadotropin, but not free alpha-subunit, secretion. The mechanisms by which GnRH-a produce this incomplete gonadotrope desensitization and facilitate limited postreceptor activity remain to be elucidated.

Regulation of glycoprotein hormone free alpha-subunit secretion and intracellular alpha-subunit content in primary pituitary cells.

The effects and interactions of GnRH, TRH, a cAMP analog, a protein kinase-C (PKC) activator, a calcium ionophore, and a calcium channel blocker on pituitary glycoprotein hormone free alpha-subunit secretion and intracellular free alpha-subunit content were investigated. Treatment of dispersed rat pituitary cells with GnRH (100 nM) effected a time-dependent release of alpha-subunit, reaching a 4.5-fold increase (P < 0.05) at 24 h. Smaller effects were observed with TRH (10 nM). A rapid and progressive fall in intracellular alpha-subunit content was observed for 8 h after stimulation with GnRH (61% decrease; P < 0.05) or TRH (55% decrease; P < 0.05), which then remained constant at 24 h. The cAMP analogue 8-bromo-cAMP augmented a late release of alpha-subunit (4.5-fold increase at 24 h; P < 0.05) without affecting levels of alpha-subunit within the cells. Co-addition of 8-bromo-cAMP with GnRH or TRH arrested the marked fall in intracellular alpha-subunit seen with GnRH or TRH alone. These results suggest that although cAMP is capable of stimulating alpha-subunit secretion and maintaining cell content in the face of GnRH- and TRH-stimulated secretion, it does not mediate their effects on alpha-subunit. Like GnRH, the PKC activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) rapidly stimulated alpha-subunit secretion (1.7-fold increase at 4 h; P < 0.05) and progressively lowered cell content over 24h (73% decrease; P < 0.01). This similarity of action and the lack of demonstration of additive effects of TPA with GnRH or TRH imply a role for PKC as a mediator of GnRH and TRH action on alpha-subunit. Using verapamil (50 microM) to block L-type calcium channels had no effect on either basal or GnRH-stimulated alpha-secretion over 24 h. The calcium ionophore A23187 (3 microM) blocked the stimulatory effects of GnRH on alpha-subunit release and alone inhibited free alpha-subunit secretion (28% decrease at 24 h; P < 0.05). Our results suggest that neither cAMP nor an influx of extracellular calcium mediates the effects of GnRH or TRH on free alpha-subunit secretion. Accordingly, we postulate that PKC is involved in the actions of GnRH and TRH on alpha-subunit in rat pituitary cells, although further studies are required in PKC-depleted cells to confirm this hypothesis.

Regulation of glycoprotein hormone free alpha-subunit secretion and intracellular alpha-subunit content in primary pituitary cells

The effects and interactions of GnRH, TRH, a cAMP analog, a protein kinase-C (PKC) activator, a calcium ionophore, and a calcium channel blocker on pituitary glycoprotein hormone free alpha-subunit secretion and intracellular free alpha-subunit content were investigated. Treatment of dispersed rat pituitary cells with GnRH (100 nM) effected a time-dependent release of alpha-subunit, reaching a 4.5-fold increase (P < 0.05) at 24 h. Smaller effects were observed with TRH (10 nM). A rapid and progressive fall in intracellular alpha-subunit content was observed for 8 h after stimulation with GnRH (61% decrease; P < 0.05) or TRH (55% decrease; P < 0.05), which then remained constant at 24 h. The cAMP analogue 8-bromo-cAMP augmented a late release of alpha-subunit (4.5-fold increase at 24 h; P < 0.05) without affecting levels of alpha-subunit within the cells. Co-addition of 8-bromo-cAMP with GnRH or TRH arrested the marked fall in intracellular alpha-subunit seen with GnRH or TRH alone. These results suggest that although cAMP is capable of stimulating alpha-subunit secretion and maintaining cell content in the face of GnRH- and TRH-stimulated secretion, it does not mediate their effects on alpha-subunit. Like GnRH, the PKC activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) rapidly stimulated alpha-subunit secretion (1.7-fold increase at 4 h; P < 0.05) and progressively lowered cell content over 24h (73% decrease; P < 0.01). This similarity of action and the lack of demonstration of additive effects of TPA with GnRH or TRH imply a role for PKC as a mediator of GnRH and TRH action on alpha-subunit. Using verapamil (50 microM) to block L-type calcium channels had no effect on either basal or GnRH-stimulated alpha-secretion over 24 h. The calcium ionophore A23187 (3 microM) blocked the stimulatory effects of GnRH on alpha-subunit release and alone inhibited free alpha-subunit secretion (28% decrease at 24 h; P < 0.05). Our results suggest that neither cAMP nor an influx of extracellular calcium mediates the effects of GnRH or TRH on free alpha-subunit secretion. Accordingly, we postulate that PKC is involved in the actions of GnRH and TRH on alpha-subunit in rat pituitary cells, although further studies are required in PKC-depleted cells to confirm this hypothesis.

Regulation of the ectopically expressed human glycoprotein alpha-subunit gene in the human hepatoma cell line NPLC.

The human glycoprotein alpha-subunit is the common subunit of the heterodimeric hormones CG (hCG), TSH, LH, and FSH. Human glycoprotein alpha-subunit is produced eutopically in placenta, pituitary, and choriocarcinoma and ectopically in a large variety of human tumors. We report ectopic glycoprotein alpha-subunit messenger RNA (mRNA) and peptide production in the human hepatoma cell line, NPLC. Neither hCG beta mRNA nor intact hCG peptide was detected. Antimetabolite regulation of glycoprotein alpha-subunit expression in NPLC cells resembled that found in choriocarcinoma cells in that it was stimulated by hydroxyurea. In addition, glycoprotein alpha-subunit mRNA expression and transcription in NPLC were stimulated by activators of the protein kinase A and C second messenger pathways, as well as by glucocorticoid. Glucocorticoid augmented glycoprotein alpha-subunit gene transcription by phorbol ester and forskolin, in contrast to its simultaneous inhibitory effect on phorbol ester activation of the CRH gene, which is also ectopically expressed in these cells. Glucocorticoid thus modulates the activation of these genes by phorbol ester in opposite directions, despite their identical cellular context. The NPLC cell line provides a new model for the study of human glycoprotein alpha-subunit gene regulation and free glycoprotein alpha-subunit secretion. In addition, it should be useful for investigating the role that specific cis-acting DNA sequences play in glucocorticoid modulation of gene induction by second messenger pathways.

Effects of hydrocortisone on pulsatile pituitary glycoprotein secretion.

During states of stress, hypothalamic-pituitary-thyroid and hypothalamic-pituitary-gonadal function can be suppressed. One putative mediator of this stress response may be glucocorticoids, which have widespread effects on thyroid and gonadal function. To characterize dynamic pituitary glycoprotein secretion during glucocorticoid administration, 24-h TSH, LH, FSH, and alpha-subunit pulses were measured in 10 healthy young subjects on 3 occasions: 1) at baseline, 2) during infusions of 100 mg hydrocortisone (HC) over 24 h, and 3) during infusions of 300 mg HC over 24 h. These HC infusions led to serum cortisol levels similar to the endogenous cortisol levels seen in moderate and severe stress. Both HC infusions had profound rapid effects on TSH levels, decreasing TSH pulse amplitude by 60% and abolishing the nocturnal TSH surge. However, TSH pulse frequency was unaltered. In contrast, HC infusions did not change mean or pulsatile LH, FSH, or alpha-subunit secretion. These results suggest that stress levels of cortisol acutely suppress TSH secretion at the pituitary level, with little effect on the TSH pulse generator. On the other hand, the effects of stress and/or hypercortisolism on the gonadal axis may require higher cortisol levels, more prolonged exposure, or other mediators of the stress response.

Increased luteinizing hormone and alpha-subunit secretion in women with hyperandrogenic anovulation

Increased luteinizing hormone and alpha-subunit secretion in women with hyperandrogenic anovulation

Increased luteinizing hormone and alpha-subunit secretion in women with hyperandrogenic anovulation.

Women with hyperandrogenic anovulation (HAA) have increased circulating levels of LH relative to those of FSH. The cause of this disturbance in gonadotropin secretion is uncertain. Previous investigations have sought to determine if increased GnRH drive is responsible for the excessive LH concentrations. Because previous results have conflicted, we addressed this question by comparing the 24-h secretory patterns of alpha-subunit and LH in women with HAA (n = 9) to those in eumenorrheic women in the midfollicular phase (n = 9). The mean (+/- SEM) pulse frequency was increased in women with HAA compared to that in eumenorrheic women of comparable age and percent ideal body weight for both LH (23.0 +/- 0.7 pulses/24 h vs. 3 17.1 +/- 1.7; P = 0.002) and alpha-subunit (23.0 +/- 0.8 vs. 19.1 +/- 1.2; P = 0.02). LH and alpha-subunit, but not FSH, responses to a submaximal dose of exogenous GnRH were increased in HAA, as were basal LH and alpha-subunit levels (P < 0.01). The present observations provide evidence for increased GnRH drive, including pulse frequency, in HAA. Although the results confirm the presence of a disturbance in gonadotropin secretion and suggest that its proximate cause may be of hypothalamic origin, they do not exclude the possibility that other factors, perhaps of ovarian origin, play a role in the establishment and/or maintenance of the altered gonadotropin secretory patterns and the chronic anovulation characteristic of HAA.

Stimulatory effect of oCRH on alpha-subunit secretion during petrosal sinus sampling in patients with Cushing's disease.

During bilateral and simultaneous venous sampling of the inferior petrosal sinuses for preoperative localization of ACTH secreting microadenomas, alpha-subunit levels, in addition to ACTH, were determined in 9 patients with Cushing's disease. The aim of the study was to evaluate the possible occurrence of unilateral increases of alpha-subunit in basal conditions and the alpha-subunit responsiveness to oCRH. All the patients examined showed a central to peripheral and an intersinus gradient of ACTH concentrations before and/or after oCRH stimulation. Seven patients showed a central to peripheral alpha-subunit gradient in basal conditions. Lateralization of alpha-subunit concentrations was recorded in 4 patients in basal conditions (intersinus gradient > or = 1.55) and paralleled the side with the highest ACTH concentrations. After oCRH stimulation all but one patient showed a unilateral alpha-subunit increase in blood from the inferior petrosal sinus with the highest oCRH stimulated ACTH increase. The present data confirm the occurrence of an increase of alpha-subunit concentration in response to nonspecific stimulation with exogenously administered oCRH, concurrent with an ipsilateral increase of ACTH levels. The mechanism underlying this finding is still unclear, although a paracrine effect from the corticotroph tumour on adjacent pituitary tissue seems so far the most likely explanation.

Differential sorting of lutropin and the free alpha-subunit in cultured bovine pituitary cells.

The glycoprotein hormones lutropin (LH) and follitropin (FSH) are both synthesized by gonadotrophs in the anterior pituitary but are stored in separate secretory granules prior to secretion. Despite having highly homologous beta-subunits and alpha-subunits with the identical amino acid sequence, the Asn-linked oligosaccharides on LH terminate with SO4-GalNAc while those on FSH terminate with sialic acid-Gal. In addition to LH and FSH, gonadotrophs secrete uncombined (free) alpha-subunit which bears the same sulfated oligosaccharides as LH. We have examined the synthesis and secretion of LH and free alpha-subunit in primary cultures of bovine pituitary cells in order to determine if the sulfated oligosaccharides have any impact on sorting. Our results show that newly synthesized free alpha-subunit is secreted exclusively via the constitutive pathway with a t1/2 of 1.8 h and is never found in dense-core secretory granules. In contrast, LH dimer is secreted by both the constitutive and the regulated pathways. Constitutive secretion and arrival in a dense secretory granule both occur with t1/2 values of 1-1.5 h for newly synthesized LH. Sulfation occurs immediately prior to arrival of LH in the secretory granule and is followed by a period of 1-1.5 h before the LH-containing granules become sensitive to release by gonadotropin releasing hormone. As a result the t1/2 for LH secretion in the presence of gonadotropin releasing hormone is 3.5 h. Sulfation of the free alpha-subunit oligosaccharides is not, therefore, sufficient to direct the alpha-subunit to secretory granules, and the information required for directing LH to granules must reside either in the beta-subunit or the alpha beta-complex.

Glycoprotein hormone alpha-subunit production in somatotroph adenomas with and without Gs alpha mutations.

Activating mutations of the Gs alpha subunit have been identified in a subset of somatotroph adenomas. The mutant form of the Gs alpha subunit causes persistent activation of adenylyl cyclase and consequently results in high intracellular levels of cAMP. Because cAMP is known to stimulate the synthesis of the glycoprotein hormone (GPH) alpha-subunit as well as GH, we examined somatotroph tumors with and without Gs alpha mutations for GPH alpha-subunit production. GPH alpha-subunit production was assessed in vivo by measuring serum hormone levels and in vitro by analyzing hormone secretion by cultured pituitary tumor cells. DNA was extracted from the pituitary tumors of 26 acromegalic patients. The Gs alpha gene was amplified by the polymerase chain reaction and screened for mutations at codons 201 and 227 using oligonucleotide specific hybridization. Nine of the 26 tumors (35%) had point mutations at Arg 201. Seven of these tumors contained a CGT to TGT mutation (Arg to Cys) and 2 contained a CGT to CAT mutation (Arg to His). No mutations were detected at codon 227. There were no significant differences in age, sex distribution, tumor size, or serum levels of GH or insulin-like growth factor-1 between the groups of patients with or was Gs alpha mutations. The mean serum level of the free GPH alpha-subunit was 1.9-fold higher in the group with Gs alpha mutations (0.48 +/- 0.37 micrograms/L) than in patients without mutations (0.25 +/- 0.17) (P less than 0.05). In pituitary tumor cell culture, 75% of somatotroph tumors with Gs alpha mutations secreted free GPH alpha-subunit into the media compared with 45% of tumors without Gs alpha mutations. The amount of GPH alpha-subunit secretion was 12-fold greater in the group of tumors containing the Gs alpha mutation (P less than 0.05). Immunocytochemical detection of the free GPH alpha-subunit was similar in the two groups of patients with 75% positive for the GPH alpha-subunit in tumors with Gs alpha mutations and 67% positive in tumors without mutations (P = 0.69). We conclude that GPH alpha-subunit production occurs in somatotroph tumors with and without Gs alpha mutations. The increased levels of GPH alpha-subunit secretion in vivo and in vitro suggest that the Gs alpha mutation may increase the amount of preexisting GPH alpha-subunit biosynthesis in the tumors, perhaps via activation of the cAMP pathway.

Effects of chronic GnRH analogue administration on gonadotrophin and alpha-subunit secretion in post-menopausal women.

To investigate in detail the regulation of LH, FSH, and alpha-subunit secretion by a GnRH agonist analogue under physiological conditions of gonadotrophin elevation. Six normal healthy post-menopausal women. Subjects were given D-Trp6-Pro9-Net-GnRH (GnRHa), 32 micrograms/kg, subcutaneously, daily for 24 days. On days 1, 2, 3, 4, 7, 11, 14, 17, 21, and 24, blood samples before and after GnRHa injection were taken. Sampling was continued off GnRHa twice a week for 4 weeks and then on days 66, 76, and 98. GnRH tests (100 micrograms i.v.) were performed on days 0, 24, and 98. All serum samples were analysed for LH, FSH, and alpha-subunit levels. LH and FSH levels reached a maximum on day 2 after which there was a steady decline to day 24. LH did not begin to rise again until day 44 (20 days off GnRHa), then rose steadily. FSH began to rise earlier, on day 34 (10 days off GnRHa). alpha-Subunit levels also showed maximum elevation on day 2 but remained equally elevated throughout the period of GnRHa administration and then fell rapidly to baseline by day 34. LH, FSH, and alpha-subunit responses to i.v. GnRH were absent on day 24 and were equivalent to baseline on day 98. We conclude that there is a striking dissociation in the regulation of gonadotrophin and alpha-subunit secretion in response to GnRHa in normal post-menopausal women. Gonadotrophin secretion is profoundly suppressed during GnRHa administration and recovers only after a long delay post-treatment, while alpha-subunit is markedly stimulated and recovers rapidly. The difference between this pattern and that seen in patients with pituitary tumours could be useful for diagnosis.

Human pituitary null cell adenomas and oncocytomas in vitro: effects of adenohypophysiotropic hormones and gonadal steroids on hormone secretion and tumor cell morphology.

Human pituitary null cell adenomas and oncocytomas are not associated with evidence of excess hormone secretion in vivo; their cellular derivation has not been clarified by morphologic investigation. In this study we examined 41 null cell adenomas and 58 oncocytomas in vitro to determine hormone release and its response to several adenohypophysiotropic hormones and gonadal steroids. In vitro, 96/99 tumors released LH, FSH, and/or alpha-subunit of glycoprotein hormones. TSH was released by 11 tumors. GH, PRL, and ACTH were found in small quantities in 11, 8, and 5 tumors, respectively. Only 3 tumors released no detectable hormones. Incubations with test substances were examined at 2- and 24-h periods for up to 72 h. All but 3 of 53 tumors showed marked and persistent increases in the release of LH, FSH, and/or alpha-subunit in response to GnRH in short and long duration experiments. Secretion of LH, FSH, or alpha-subunit was stimulated to more than 150% of control by TRH in 37/48 tumors, by CRH in 10/20, by GRH in 7/20. Estradiol, progesterone, and testosterone increased release of FSH, LH, and/or alpha-subunit in 23/32, 3/12, and 3/12 tumors, respectively, and reduced their release in 6/32, 5/12, and 7/12, respectively. In tumors which showed no response to gonadal steroids, GnRH in combination with estradiol, progesterone, or testosterone yielded the same result as GnRH alone; in tumors inhibited by gonadal steroids, GnRH in combination with one of those substances reduced the response to GnRH. No secretion of GH, PRL, ACTH, or TSH was detected after incubation with GRH, estradiol, CRH, or TRH except in the tumors which initially released GH, PRL, or TSH. Ultrastructural examination of cultured cells from 15 cases revealed morphologic alterations that correlated with changes in hormone release and could be quantified by morphometry. This study represents the largest analysis of hormone production and release in vitro and morphologic correlation of clinically nonfunctioning pituitary adenomas. The responsiveness of gonadotropin secretion by null cell adenomas and oncocytomas to GnRH and gonadal steroids resembles that of gonadotroph adenomas. However, the unexpected increases in gonadotropin release attributable to several other adenohypophysiotropic hormones and the release of multiple hormones suggests that null cell adenomas and oncocytomas may represent neoplasms derived from uncommitted or committed precursor cells that can undergo differentiation towards several cell lines.

Reduction in size of a thyrotropin- and gonadotropin-secreting pituitary adenoma treated with octreotide acetate (somatostatin analog).

TSH as well as alpha-subunit, secretion has been shown to decrease after the administration of the somatostatin analog octreotide acetate (SMS 201-995). We have studied a 59-yr-old, male patient with a TSH- and gonadotropin-secreting tumor who, because of severe cardiomyopathy, was treated with long-term somatostatin analog rather than surgical resection of the pituitary tumor. Thirteen weeks of treatment with thrice daily sc injection of 100 micrograms octreotide acetate resulted in decreased TSH and alpha-subunit secretion, normal serum thyroid hormone levels, reduction in LH and testosterone level, and significant tumor size reduction. Long-term treatment for 51 weeks has not been associated with any significant side effects. We have shown that octreotide acetate may be a therapeutically valuable modality for certain patients with neoplastic inappropriate secretion of TSH (NIST). A probable effect of octreotide acetate on neoplastic gonadotropes, as evidenced by the reduction of the LH level with a concomitant decrease in testosterone level, is, likewise, suggested.

Long-term treatment with the dopamine agonist CV 205-502 of patients with a clinically non-functioning, gonadotroph, or alpha-subunit secreting pituitary adenoma.

We aimed to assess the effects of prolonged treatment with the dopamine agonist CV 205-502 on tumour volume, visual field defects, and serum gonadotrophin and alpha-subunit concentrations in patients with gonadotroph, alpha-subunit secreting, or clinically non-functioning pituitary adenomas. The patients were treated with CV 205-502 in a final daily dose of 300 micrograms for at least 1 year. The patients were seen at 2 or 3-week intervals during the first 3 months of treatment, and thereafter every 1 or 2 months. Computerized tomography and Goldmann perimetry were performed before treatment and during follow-up. Blood samples were drawn before treatment and at each out-patient visit. One patient with gonadotroph, two with alpha-subunit secreting, and two with clinically non-functioning pituitary adenomas were studied. Computerized tomography showed tumour shrinkage in one patient. In two other patients an improvement of visual field defects was observed. In four patients, a significant decrease in serum FSH and/or alpha-subunit concentrations occurred within the first 3 months of treatment. In the remaining patient, a significant decrease of serum FSH and alpha-subunit concentrations was found after more than 3 months of treatment. In patients with clinically non-functioning, gonadotroph, or alpha-subunit secreting pituitary tumours, long-term treatment with the dopamine agonist CV 205-502 decreases serum FSH and/or alpha-subunit concentrations. This decreased secretory activity from the pituitary tumour may be accompanied by an improvement of visual field defects, or tumour shrinkage on computerized tomography. Therefore, treatment with CV 205-502 may be useful in patients with clinically non-functioning, gonadotroph, or alpha-subunit secreting pituitary tumours, who cannot be operated upon.

Somatostatin Regulation of Glycoprotein Hormone and Free Subunit Secretion in Clinically Nonfunctioning and Somatotroph Adenomasin Vitro*

There is increasing evidence that clinically nonfunctioning pituitary tumors produce and secrete glycoprotein hormone and/or free alpha- and beta-subunits. In addition, hypersecretion of free alpha-subunit occurs in up to 37% of patients with somatotroph adenomas. An understanding of glycoprotein hormone regulation is important in developing effective therapeutic strategies for patients with tumors associated with intact glycoprotein hormone and free subunit hypersecretion. We investigated glycoprotein hormone and free subunit secretion by somatostatin in primary dispersed cultures of pituitary tumor cells from 23 patients with pituitary adenomas. Fifteen tumors from patients with clinically nonfunctioning adenomas (group 1) and 8 tumors from patients with somatotroph adenomas and cosecretion of alpha-subunit (group 2) were studied. Cultures were incubated with control or somatostatin-supplemented media for 24 h. Media samples from group 1 tumors were assayed for intact glycoprotein hormones and free alpha- and beta-subunits secretion levels, while media samples from group 2 cultures were assayed for alpha-subunit and GH secretion levels. Significant (P less than 0.05-0.001) inhibition of secretion of 1 or more intact hormones and/or free subunits was found in 10 of the 15 group 1 tumors. SRIF[10(-7) M] suppressed intact gonadotropin secretion in 60% of FSH-producing tumors and 30% of LH-producing tumors. Media concentrations of FSH beta and LH beta were decreased in 31% and 50% of group 1 tumors, respectively, following somatostatin treatment in those tumors which secreted free beta-subunits. alpha-Subunit was secreted by 12 of the 15 tumors, but significant (P less than 0.02-0.01) inhibition by somatostatin was observed in only 2 tumors. In contrast, significant (P less than 0.05-0.001) inhibition of alpha-subunit in the somatotroph adenomas was found in 6 of the 8 tumors. Significant decreases in alpha-subunit were observed only in those tumors where GH was also significantly inhibited by somatostatin. We conclude that 1) somatostatin inhibits intact glycoprotein or free subunit secretion in the majority of clinically nonfunctioning pituitary tumors in vitro and 2) alpha-subunit secretion is suppressed in 17% and 69% of clinically nonfunctioning and somatotroph adenomas, respectively, consistent with a differential regulation of alpha-subunit by somatostatin in these two tumor types.

Effects of gonadotrophin releasing hormone antagonist and agonist on the pulsatile release of gonadotrophins and α‐subunit in postmenopausal women*

The present study was designed to further assess the mechanism of action of GnRH and GnRH analogues. Both the Nal-Glu GnRH antagonist and the D-Trp6 GnRH agonist were administered sequentially to nine normal, post-menopausal women. A baseline study of pulsatile LH, FSH and free alpha-subunit secretion was performed, with sampling every 10 min for 8 h, and then repeated 8 h after a single subcutaneous injection of Nal-Glu GnRH antagonist (5 mg). Sampling was repeated 21 days after the intramuscular injection of a depot preparation of D-Trp6 GnRH (3.75 mg) in the same women. The baseline sampling period showed synchronous pulses of LH and free alpha-subunit. The antagonist Nal-Glu decreased plasma LH (71%) and free alpha-subunit (43%). However, with the single dose of 5 mg, pulsatile LH and free alpha-subunit release were not completely suppressed and remained temporally correlated. The GnRH agonist had a potent inhibitory action on plasma immunoreactive LH (IRMA) (93%). In contrast, it increased the mean plasma levels of free alpha-subunit from 1.66 +/- 0.01 to 5.06 +/- 0.02 micrograms/l (205%). The pulsatile secretory patterns of both LH and free alpha-subunit were abolished by the agonist. Immunoreactive FSH levels were decreased by the antagonist (24%) and suppressed by the agonist (93%). The pulsatile study confirms the different mechanism of action of GnRH analogues. Following antagonist administration, low amplitude free alpha-subunit pulses persist and are synchronous with residual LH pulses. In contrast, LH and free alpha-subunit are not maintained under agonist treatment. These data provide evidence for the differential regulation of LH and free alpha-subunit by GnRH.

The Effects of Aging on the Secretion of the Common Alpha‐Subunit of the Glycoprotein Hormones in Men

To investigate the effects of aging on the secretion of the common alpha-subunit of the glycoprotein hormones, we measured basal levels of luteinizing hormone (LH), testosterone (T) and alpha-subunit in 176 normal men aged 19 to 89 years. In addition, in two groups of young (less than 65 years; n = 25) and old (greater than 65 years; n = 15) subjects, the effects of luteinizing hormone-releasing hormone (LHRH) on LH and alpha-subunit secretion were determined. Age-related increases in serum alpha-subunit and LH were noted only in the oldest men while T levels decreased progressively with advancing age. LHRH stimulation resulted in significantly greater secretion of alpha-subunit in the old subjects while no difference in LH release between young and old men was observed. Moreover, there was a delay to peak LH and alpha-subunit levels after LHRH in the old subjects. These data suggest that the aging process in males involves deficits in both testicular and gonadotroph functions as demonstrated by (1) the relative hypogonadotropic hypogonadism seen until the ninth decade; (2) the hypergonadotropic hypogonadism apparent in men greater than 80 years; (3) the delay in the timing of peak responses of LH and alpha-subunit after LHRH administration; and (4) the disproportionate increase in the secretion of alpha subunit relative to intact LH.