Effects of hypophysiotropic factors on growth hormone and prolactin secretion from somatotroph adenomas in culture

In an attempt to characterize GH and PRL secretion in acromegaly, the effects of various stimuli on GH and PRL release by cultured pituitary adenoma cells derived from acromegalic patients were studied. In addition, the PRL responses of somatotroph adenoma cells were compared to those of prolactinoma cells. GH-releasing hormone-(1-44) (GHRH) consistently stimulated GH secretion in all 14 somatotroph adenomas studied in a dose-dependent manner. The sensitivity as well as the magnitude of the GH responses to GHRH were highly variable in individual tissues. Somatotroph adenomas that did not respond to dopamine were more sensitive and had greater GH responses to GHRH. In 8 of 9 somatotroph adenomas that concomitantly secreted PRL, the addition of GHRH likewise increased PRL release. Omission of extracellular Ca2+ blocked the stimulatory effect of GHRH on GH and PRL secretion. When cells were coincubated with 0.1 nM somatostatin, GH and PRL secretion induced by 10 nM GHRH were completely blocked in most adenomas. Similarly, coincubation of dopamine resulted in inhibition of GHRH-induced hormone secretion in some adenomas. Addition of TRH to the incubation medium, on the other hand, significantly stimulated GH secretion in 8 of 14 adenomas, while TRH stimulated PRL release in all of the adenomas. Vasoactive intestinal peptide (VIP) and corticotropin-releasing hormone (CRH) produced an increase in GH and PRL secretion in other adenomas. In prolactinoma cells, somatostatin and dopamine unequivocally suppressed PRL secretion; however, other stimuli including GHRH, VIP, and CRF were ineffective. TRH induced a significant increase in PRL secretion in only one prolactinoma. These results suggest that responsiveness to GHRH and somatostatin is preserved in somatotroph adenomas; the responsiveness to GHRH is inversely correlated to that to dopamine; and PRL cells associated with somatotroph adenomas possess characteristics similar to those of GH cells. Further, the GH stimulatory actions of TRH and VIP are different.     

Structure-function correlations of growth hormone or/and prolactin-producing pituitary adenomas: an in vitro study with the reverse hemolytic plaque assay

The purpose of this study was to detect in vitro growth hormone (GH) and prolactin (PRL) secretion from adenomas clinically associated with GH or PRL hypersecretion. The reverse hemolytic plaque assay (RHPA) was applied in order to reveal possible differences among various morphologic adenoma types, and to examine the inhibitory effects of octreotide on GH release as well. The 20 surgically resected pituitary adenomas studied included 15 from acromegalic patients and 5 from patients with hyperprolactinemia. All adenomas were diagnosed by histology, immunocytochemistry and electron microscopy. Among tumors associated with acromegaly, 5 were densely granulated (DG), 5 were sparsely granulated (SG) somatotroph (SM) adenomas, 2 were mammosomatotroph (MSM) and 3 mixed somatotroph-lactotroph cell (mixed SM-LT) adenomas; tumors causing hyperprolactinemia included 4 lactotroph (LT) adenomas and 1 mixed SM-LT adenoma. GH release assessed by the RHPA corresponded to in vivo hormone secretion and to tissue immunoreactivity. Statistical analysis showed significant differences among all morphologic types of SM adenomas, exclusive of SG-SM adenomas compared to mixed SM-LT adenomas. The mean plaque size in DG-SM and MSM adenomas was significantly greater than that of SG-SM and mixed SM-LT adenomas, indicating higher GH secretion by the former two types during the same incubation time. PRL secretion was documented in 2 mixed SM-LT adenomas. Plaques for PRL, but not for GH were formed in all LT adenomas. In all SM and LT adenomas, cells producing large plaques represented a minority of the plaque-forming cell population, however, they accounted for the largest part of the total plaque area, thus the largest part of hormone secretion. Octreotide effects on GH release were studied in 6 adenomas by the RHPA. Octreotide treatment induced a rapid and significant reduction in GH secretion by SM cells in vitro, with a selective effect on high-secreting cells.     

Prolactin (PRL)-releasing peptide stimulates PRL secretion from human fetal pituitary cultures and growth hormone release from cultured pituitary adenomas

The hypothalamic peptide PRL-releasing peptide (PrRP) has recently been cloned and identified as a ligand of an orphan pituitary receptor that stimulates in vitro PRL secretion. PrRP also induces PRL release in rats in vivo, especially in normal cycling females. However, no information on the effects of PrRP in the human is available. To elucidate the role of PrRP in regulating human anterior pituitary hormones, we used human PrRP-31 in primary cultures of human pituitary tissues, including fetal (20--27 weeks gestation) and normal adult pituitaries, as well as PRL- and GH-secreting adenomas. PrRP increased PRL secretion from human fetal pituitary cultures in a dose-dependent manner by up to 35% (maximal effect achieved with 10 nM), whereas TRH was slightly more potent for PRL release. Coincubation with estradiol resulted in enhanced fetal PRL response to PrRP, and GH release was only increased in the presence of estradiol. Although PRL secretion from PRL-cell adenomas was not affected by PrRP, PrRP induced PRL release from cultures of a GH-cell adenoma that cosecreted PRL. PrRP enhanced GH release in several GH-secreting adenomas studied by 25--27%, including GH stimulation in a mixed PRL-GH-cell tumor. These results show for the first time direct in vitro effects of PrRP-31 on human pituitary cells. PrRP is less potent than TRH in releasing PRL from human fetal lactotrophs and is unable to release PRL from PRL-cell adenomas in culture, but stimulated GH from several somatotroph adenomas. Thus, PrRP may participate in regulating GH, in addition to PRL, in the human pituitary.     

The effects of growth hormone-releasing factor (GRF) and dopamine on growth hormone (GH) secretion in acromegaly

GRF (1-40), dopamine (DA), DA plus GRF and placebo were administered to 6 acromegalic patients. The GRF administration induced a highly variable GH release (GH delta % 167.3 +/- 21.4; mean +/- SE). GRF did not provoke any change in PRL serum levels. During simultaneous GRF and DA administration GH release was found to be reduced (GH delta % 80.2 +/- 17.8) compared to that observed for a corresponding period of time after GRF alone (p less than 0.05). Our data underscore that in acromegaly the DA tonus inhibits GH secretion after GRF by acting directly at the pituitary level.     

Phorbol ester pretreatment attenuates the growth hormone (GH) response to GH-releasing factor in cultured rat pituitary cells

The effect of phorbol ester pretreatment on rat (r) GH release induced by GH-releasing factor (GRF) or 8-bromo-cyclic (c)AMP was investigated using rat pituitary cells cultured in monolayers. Pretreatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 3 h significantly suppressed the rGH release induced by GRF, but not that by 8-bromo-cAMP 20 h later; this suppressive effect of TPA was concentration-dependent from 8 to 160 nmol/l, and complete suppression was observed after pretreatment with 80-160 nmol TPA/l. Production of cAMP by pituitary cells stimulated with GRF was similarly attenuated in TPA-pretreated cells. The rGH responsiveness to GRF of these cells was fully recovered on prolonged culture (40 h), suggesting that the inhibitory effect of TPA is reversible. In contrast, pretreatment with GRF (5 nmol/l) resulted in suppression of the rGH response to subsequent exposure to GRF (5 nmol/l) or 8-bromo-cAMP (10 mmol/l), but not to TPA. These observations suggest that pretreatment with TPA modifies the rGH response to GRF at steps before the formation of cAMP.     

Insulin-like growth factor I regulates growth hormone secretion and messenger ribonucleic acid levels in human pituitary tumor cells

GH secretion and mRNA levels were measured in cultured human GH adenoma cells incubated in serum-free medium for up to 48 h. A human recombinant insulin-like growth factor I (IGF-I) analog, Thr-59-IGF-I (6.5 nM), inhibited basal GH secretion by up to 60% in tumor cell cultures. The 30-50% stimulation of GH secretion by GH-releasing hormone (GHRH) was prevented by simultaneous exposure of the cells to IGF-I (6.5 nM). Gel electrophoresis of total RNA derived from GH cell adenoma tissue, followed by transfer and hybridization with 32P-labeled human GH cDNA, revealed a distinct mRNA species of about 1.0 kilobases. Using cytoplasmic dot blot hybridization, IGF-I inhibited the levels of human GH mRNA sequences in these cells and also prevented the GHRH-induced stimulation of GH mRNA. A monoclonal antibody to the type I IGF-I receptor (alpha IR3) prevented the inhibitory effects of IGF-I on basal and GHRH-stimulated GH secretion. This antibody also prevented the IGF-I-induced suppression of GH mRNA sequences. PRL secretion in these cells was not altered by IGF-I. Furthermore, relative levels of beta-actin mRNA were unaltered by IGF-I. Thus, IGF-I suppresses basal and GHRH-stimulated GH secretion and GH mRNA levels in pituitary adenoma cells, indicating that IGF-I acts selectively on the somatotroph to directly regulate GH gene expression.     

Evidence of a circulating growth hormone stimulating factor other than growth hormone releasing hormone in a patient with pituitary tumour and acromegaly

This study is a report on the growth hormone (GH) stimulatory effect of serum and plasma from a patient with notably active acromegaly due to a GH producing pituitary adenoma. Pituitary adenomatous tissue from 7 patients with GH producing adenomas, one with a prolactin (Prl) producing adenoma, one with a TSH producing adenoma, and one with a non-secreting adenoma, were cultured in vitro for 8-10 days. Media were changed every 48-72 h and contained Neumann Tytell buffer with the addition of 1) foetal calf serum, 2) patients' own serum or plasma, 3) serum or plasma from the patient with notably active acromegaly. GH release expressed as microgram GH/1/48-72 h between day 6 and 8 in culture did not differ when adenomatous tissue was cultured in buffer, foetal calf serum or the patients' own serum or plasma. In contrast, GH release was increased in 9/10 patients, when media contained serum or plasma from the patient with notably active acromegaly. This GH stimulatory effect was demonstrated in vitro in human pituitary adenomatous tissue from patients with pathological as well as normal GH secretion in vivo. Furthermore, this GH releasing plasma in a concentration of 10% increased GH release in cultures of dispersed rat anterior pituitary cells. In the same system, synthetic growth hormone-releasing hormone (GRF)-44 stimulated the release of GH in a dose-dependent manner. However, at all dose levels including maximally stimulating doses of GRF, an additive effect on GH release was seen with 10% of the GH releasing plasma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of growth hormone (GH) release by individual pituitary adenoma cells from acromegalic patients, as determined by the reverse hemolytic plaque assay: effects of SMS 201-995, GH-releasing hormone and thyrotropin-releasing hormone

We used the reverse hemolytic plaque assay to study the dynamics of GH secretion by individual pituitary adenoma cells from eight acromegalic patients. There was a considerable variation between the adenomas with respect to the percentages of GH-secreting cells (25-78.5%) and also with respect to the amount of GH released per individual pituitary adenoma cell (mean plaque areas varying from 901-3559 micron 2). The GH plaque area frequency distributions from the adenoma cells were not normally distributed, but revealed a preponderance of small plaques, defined as those with areas smaller than the mean plaque area. The large plaques, that is those with areas larger than the mean plaque area, constituted 24-38% of the total cell population from different tumors and accounted for a large fraction (63-80%) of the total plaque area (the total amount of GH released by the adenoma cells). The somatostatin analog SMS 201-995 caused a shift in the GH plaque area frequency distributions toward smaller plaques, but had no effect on the overall percentages of GH plaque-forming cells in three of the five adenomas in which it was studied. This finding suggests that the adenoma cells from these patients that formed large plaques were preferentially inhibited by SMS 201-995. GHRH (studied in two adenomas) and TRH (studied in one adenoma) had no preferential effect on any subpopulation of adenoma cells. We conclude that GH secretion by individual somatotroph adenoma cells is highly variable both within and between adenomas and that SMS 201-995 has a preferential inhibitory effect on a subpopulation of adenoma cells in some adenomas.     

Cortistatin inhibits growth hormone release from human fetal and adenoma pituitary cells and prolactin secretion from cultured prolactinomas

CONTEXT: Cortistatin (CST) is a neuropeptide that shares high homology with somatostatin and binds with high affinity to all somatostatin receptor (SSTR) subtypes. Many of its endocrine and biological activities overlap with those of somatostatin. OBJECTIVE/DESIGN: The objective of the study was to assess the direct in vitro effects of CST on human pituitary hormone secretion. SETTING: This study was performed in the endocrine laboratory of a tertiary academic medical center. MATERIALS: Primary cell cultures of human fetal (21-25 wk gestation) pituitary tissues and cultured hormone-secreting adenoma cells were used in this study. INTERVENTIONS: Cell cultures were incubated with CST-14 or CST-17, somatostatin, GHRH, SSTR analogs, and ghrelin analogs, and hormone secretion was analyzed. OUTCOME MEASURES: GH and prolactin (PRL) medium concentrations were tested by hormone assay, and SSTR mRNA was tested by RT-PCR. RESULTS: CST-14 (10 nm) inhibited GH secretion by up to 65% in all fetal pituitary specimens after 4-h incubation (P < 0.05). CST-14 or CST-17 (10 nm) inhibited basal GH secretion in six of the 13 GH-cell adenomas and two of the three GH-PRL mixed adenomas. CST-17 (100 nm) suppressed the GH response to GHRH and ghrelin analog (10 nm each) by 30-50% in adenomas (P < 0.05). Three PRL-adenomas treated with CST-17 (10 nm) showed a 20-40% inhibition of PRL release (P < 0.05), whereas in three others no suppression or mild response was achieved at this concentration. A comparable inhibition of PRL secretion was obtained with SSTR5-selective analog but significantly less with SSTR2-preferential compounds. RT-PCR revealed the expression of both SSTR2 and SSTR5 in all GH-cell and mixed adenomas studied and all PRL-secreting adenomas studied, except for two of the CST-resistant prolactinomas, in which SSTR5 was absent. CONCLUSIONS: This is the first report of in vitro CST suppression of human GH and PRL in cultured pituitary tissues. The regulation of PRL release from cultured adenomas appears to be primarily mediated by SSTR5.     

GLYCOPROTEIN HORMONE ALPHA-SUBUNIT AND PROLACTIN RELEASE BY CULTURED PITUITARY ADENOMA CELLS FROM ACROMEGALIC PATIENTS: CORRELATION WITH GH RELEASE

In-vitro data of pituitary adenoma cells from 28 acromegalic patients were evaluated. In addition to GH, PRL was produced by 16 adenomas (57%) and alpha-subunit by 15 adenomas (54%) while there was a significantly higher incidence of tumours producing PRL and alpha-subunit simultaneously. From 26 pituitary adenomas enough cells were obtained in order to perform secretion studies. Percentage basal hormone release (medium: (medium + intracellular hormone)) x 100% of GH and alpha-subunit by 11 adenomas showed a close correlation while such a correlation for GH and PRL was present only in a subgroup of 10 of 13 adenomas. The responses of GH and alpha-subunit release to 10nM SMS201-995, 10nM bromocriptine, 100 nM TRH and 10nM GHRH were closely related in that a response or an absent response of GH release to the four secretagogues was virtually always attended with a response or an absent response respectively of alpha-subunit release. Such a relationship was less evident with respect to the effects of SMS201-995, bromocriptine. TRH and GHRH on GH and PRL release. We conclude that basal and secretagogue-induced alpha-subunit release by cultured pituitary adenoma cells from acromegalic patients closely follows the pattern of GH release while such a relationship for GH and PRL is present only in a subgroup of the adenomas secreting GH and PRL simultaneously.     

In vitro effect of dopamine and L-dopa on prolactin and growth hormone release from human pituitary adenomas.

To determine the site of action of dopaminergic drugs on human PRL and GH release from pituitary adenomas, five PRL-and five GH-secreting adenomas were incubated with and without dopamine and L-dopa. Bromocriptine was also tested in order to compare its effect to that of the other drugs. In all of the experiments except one, a decrease of PRL, which was often statistically significant, was observed. When pooling the results of the PRL-secreting adenomas, the mean levels of PRL with dopamine, L-dopa, and bromocriptine were, respectively, 49%, 55%, and 60% of the control levels. In the GH-secreting adenomas, they were 60%, 67%, and 55% of that of the control. For GH, a decrease of the release was observed in four out of five GH-secreting adenomas. When pooling the results from these tumors, the mean levels of GH with dopamine, L-dopa, and bromocriptine were, respectively, 63%, 76%, and 64% of the control levels. In one case, a significant increase of GH was observed with the three dopaminergic drugs. This study produced the following conclusions. 1) Dopamine acts directly on PRL and GH release from human pituitary adenomas; in vitro, L-dopa effects are similar (its action probably occurs after conversion to catecholamines). These observations strongly suggest the presence of dopaminergic membrane receptors on human lactotroph and somatotroph adenomatous pituitary cells. 2) In vitro hormonal results are in good agreement with in vivo dynamic tests using L-dopa and bromocriptine. 3) The paradoxical effect of dopaminergic drugs on GH secretion in acromegalic patients may be attributed to modified dopamine membrane receptors. However, the paradoxical response is not a constant feature in acromegaly, and its mechanism needs further investigations.     

Different responses of growth hormone secretion to guanfacine, bromocriptine, and thyrotropin-releasing hormone in acromegalic patients with pure growth hormone (GH)-containing and mixed GH/prolactin-containing pituitary adenomas

There is great variability in the GH secretory responses to different stimuli in patients with acromegaly. In the present study, we compared the effects on GH secretion of two compounds (bromocriptine and TRH), which presumably act directly at the pituitary level, with the effect of the centrally acting alpha-adrenergic agonist guanfacine in 14 untreated acromegalic patients. These in vivo responses of GH release were correlated with the results of immunocytochemical studies of the pituitary adenomas. In nine patients with pure GH-containing adenomas, GH secretion was suppressed by bromocriptine by more than 50% in one patient, while TRH stimulated GH release by more than 100% in another patient. Guanfacine (2 mg, orally) did not elicit a change in circulating GH levels in any of these nine patients. In the group of five patients with mixed GH/PRL-containing adenomas, however, bromocriptine suppressed GH levels by more than 50% in all patients, and TRH stimulated GH release by more than 100% in four of them. Guanfacine stimulated GH secretion significantly in four of these five patients. Guanfacine inhibited GH secretion significantly in five other acromegalic patients who had been treated 5-10 yr previously by external pituitary irradiation. We conclude that in acromegaly, the presence of PRL within the GH-secreting pituitary adenoma makes GH secretion more sensitive to bromocriptine and TRH, while normal sensitivity to hypothalamus-mediated stimulation (alpha-adrenergic agonist) is retained to some extent. In contrast, pure GH-secreting tumors responded little or not at all to bromocriptine, TRH, or guanfacine.     

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

OBJECTIVE 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-l affects somatotrophinoma cellular proliferation or the secretion of other hormones, such as PRL and α-subunit, which are often co-secreted by these tumours. We have therefore examined the effects of IGF-l on proliferation and hormonal secretion of human somatotrophinomas and prolactinomas in vitro. DESIGN Pituitary adenoma tissue was dispersed to single cells in monolayer culture. The effects of 100 nw IGF-I on GH, PRL and α-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. RESULTS Over 4 hours, 100 nw IGF-I had no effect on GH, PRL or α-subunit secretion in 7 tumours. Over 4 days, 100 nw IGF-I reduced GH secretion In 518 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 α-subunit secretion in any of the three tumours studied. PRL co-secretion was increased In 315 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 nw 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 nw IGF-I in any somatotrophinoma. CONCLUSION IGF-I inhibited tumorous GH in 62% and stimulated PRL secretion in 71 % of tumours over 4 days, without affecting α-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.     

Cyproheptadine-mediated inhibition of growth hormone and prolactin release from pituitary adenoma cells of acromegaly and gigantism in culture

The effect of cyproheptadine on growth hormone (GH) and prolactin (Prl) secretion from cultured pituitary adenoma cells of acromegaly and pituitary gigantism was studied. When varying doses of cyproheptadine ranging from 0.01 to 1 microM were added to the incubation media, GH secretion was consistently inhibited and a dose-response relationship was observed between the cyproheptadine concentrations and the amounts of GH released into the media. In pituitary adenomas which concurrently produced and secreted Prl, cyproheptadine likewise suppressed Prl release in a dose-related manner. This effect of cyproheptadine was not blocked by coincubation with serotonin. Similarly, coincubation with a dopaminergic antagonist, haloperidol, failed to reverse the inhibitory action produced by cyproheptadine. When coincubated with dopamine, cyproheptadine further inhibited GH and Prl secretion. These results suggest that cyproheptadine possesses a direct action on human somatotroph adenoma cells to inhibit GH and Prl secretion by an unknown mechanism that is different from serotonergic and dopaminergic systems.     

Differential effects of thyrotropin-releasing hormone, vasoactive intestinal peptide, phorbol ester, and depolarization in GH4C1 rat pituitary cells

The sequence of PRL and GH release from GH4C1 cells was studied in perifusion and static culture systems. The secretory pattern elicited by TRH differed from those caused by depolarizing concentrations of KCl (Ca2+-initiated secretion), vasoactive intestinal peptide (VIP), 8-bromo-cAMP, and forskolin (cAMP-mediated secretion), and 12-O-tetradecanoylphorbol-13-acetate (TPA) (protein kinase C activation). TRH, K+, VIP, and TPA all caused secretion within 1 min in the perifusion system but the peak response to TRH and depolarization occurred earlier than the peak responses to TPA and VIP. PRL and GH release in response to a pulsatile application of TRH (0.4-min pulse every 5 min for 25 min) did not decline with a low dose, indicating that acute desensitization does not occur, but did decrease with a high concentration. When cells in the perifusion system were subjected to continuous stimulation, TRH caused a biphasic response with a 2- to 3-min period of high secretion followed by a second phase in which GH and PRL secretion were 60-70% the rates in the first phase. KCl caused predominantly first-phase secretion, and TPA caused a biphasic secretory pattern with a delay in its peak of action. VIP caused a modest but prolonged response whether administered in a pulsatile or sustained manner. When GH-cells were exposed to 100 nM TRH for 2 days, [3H] [N3-methyl-His2]TRH binding was decreased (down-regulation), intracellular PRL was increased (170% of control), and intracellular GH was decreased (65% of control). In these down-regulated cells, baseline PRL and GH secretion were changed in proportion to the relative intracellular hormone content. The responsiveness to TRH, KCl, and TPA during the initial 10-min period (first phase) was reduced; however, the responsiveness to these substances in the subsequent 50-min period (second phase) was unchanged. The ED50 for TRH stimulation of hormone release was increased 2- to 4-fold in down-regulated cells, but the dose-response curves for other secretagogues were not shifted. These data suggest that the initial burst of hormone release caused by TRH is mediated by Ca2+, and that prolonged exposure to TRH causes homologous desensitization.     

Long-term in-vitro maintenance of growth hormone secretion from human somatotrophinomas with cortisol, and its effects on growth hormone-releasing factor

The effect of cortisol (206 nmol/l) on GH secretion from enzymatically dispersed human somatotrophinoma cells in long-term culture was studied using adenomas from 13 patients with acromegaly. Basal GH secretion from cultures of three out of five tumours measured during periods of 4 hours declined to less than 10 mu u. GH/culture within 21 days. Secretion from two other tumours measured during 24 h also declined but GH concentrations were still readily detectable (greater than 100 mu u./culture) by 28 or 58 days after cell dispersal. The decline in GH secretion was reversed in all seven tumours when cultures were maintained in cortisol-supplemented medium (CM), 4-h secretion rates being increased or retained at greater than or equal to 100 mu u./culture for as long as the studies were continued (range 18-291 days), and 24-h secretion rates at greater than 1000 mu u./culture (range 28-58 days). GH secretion and content from cultures of two additional somatotrophinomas were increased by treatment with cortisol for 5 or 9 days but there was no concomitant effect on cell number. During long-term maintenance of cultures in CM (range 5-40 days), cortisol partially or completely blocked the 4-h GH response to a test dose of GH-releasing factor (GRF) (20 nmol/l) in five out of six tumours, and to a dose range of GRF (0.01-20 nmol/l) in two of them.(ABSTRACT TRUNCATED AT 250 WORDS)     

The relationship between growth hormone (GH) messenger ribonucleic acid levels and hormone release from individual cells derived from human GH-secreting pituitary adenomas

GH mRNA expression and GH release by individual cells derived from four GH-secreting pituitary adenomas were studied by in-situ hybridization and the reverse haemolytic plaque assay, respectively. In addition the percentage of PRL mRNA-containing cells was determined in these cell suspensions. The percentages of GH mRNA-containing cells varied between 52 and 89 while the percentages of GH plaque forming cells varied between 25 and 77. Frequency distributions of GH mRNA levels in individual cells and of individual GH plaque areas showed a majority of the cells having low GH mRNA levels and secreting low amounts of GH respectively, while there is a low proportion of cells expressing high GH mRNA levels and forming large GH plaques. There was a significant correlation between the GH mRNA levels and the GH plaque areas of individual cells from the four adenomas (P less than 0.001). The percentages of PRL mRNA-containing cells in the four different adenomas amounted to less than 1, 5, 2 and 18. Cultured cells from the adenomas consisting of 5 and 18% PRL mRNA-containing cells also contained and released measurable amounts of PRL. Our data show that individual cells from GH-secreting pituitary adenomas are heterogeneous with respect to GH mRNA expression, a small proportion of the cells expressing a high amount of GH mRNA. The heterogeneity in GH mRNA expression is correlated with the heterogeneity in GH release. These observations suggest that a considerable part of GH secreted from a GH-secreting pituitary adenoma is produced by a minority of the GH-secreting tumour cell population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory influence of thyrotropin releasing hormone administration on growth hormone response to low doses of growth hormone-releasing hormone in normal man

Literature data show that TRH may have either stimulatory or inhibitory actions on GH release according to pathophysiological conditions of the subject. In view of this dual effect of TRH, we studied the possible interaction of TRH and GRF on GH secretion. Six healthy male volunteers received iv in different occasions and in random order: 1) GRF 0.05 micrograms/Kg; 2) GRF 0.1 micrograms/Kg; 3) GRF 1 microgram/Kg; 4) GRF 0.05 micrograms/Kg + TRH 400 micrograms, simultaneously; 5) GRF 0.05 micrograms/Kg + TRH 20 micrograms, simultaneously; 6) GRF 1 microgram/Kg + TRH 400 micrograms, simultaneously, 7) the vehicle as control treatment. Blood samples were obtained at several time intervals and plasma GH, PRL and TSH were measured by RIA methods. Plasma GH significantly increased in all subjects after all the tested doses of GRF and after the combination of the highest and of the lowest doses of GRF + TRH (treatments 6 and 5). GH responses increased progressively with the dose of GRF administered, even if a clear dose-response relationship could not be demonstrated, owing to the considerable interindividual variability in the responsiveness. The administration of GRF 0.05 micrograms/Kg increased significantly plasma GH levels vs control treatment. The simultaneous administration of a low effective dose of GRF (0.05 micrograms/kg) plus a high dose of TRH (400 micrograms) was able to significantly inhibit the GH secretion elicited by GRF 0.05 micrograms/Kg alone. The other GRF + TRH combinations tested (treatments 5 and 6) did not modify the GH response to the same doses of GRF given alone. Plasma PRL and TSH did not change either after GRF at any dose or after the vehicle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of large numbers of dispersed human pituitary adenoma cells obtained by aspiration

Since the extent of in vitro experiments with human pituitary adenoma cells is often limited by the small number of cells isolated from surgical specimens, ways to obtain more cells are of interest. In this study we made use of tumor tissue removed by aspiration through a suction tube during transsphenoidal surgery. Fifteen out of 28 adenomas could be dispersed without enzymatic treatment. The other 13 adenomas were mildly treated with Dispase. The large number of red blood cells present in the suction tube were removed by Ficoll-Isopaque density centrifugation. More than 90% of the adenoma cells were recovered from the interphase fraction. This material, in contrast with fragments obtained with a forceps, yielded 35.9 (+/-9.0) X 10(6) cells (mean +/- SE, n = 28). Usually less than 10% of these cells were mononuclear leucocytes. Pituitary hormones other than those hypersecreted in vivo were not found in the incubation media; only small amounts of LH were found in 5 out of 25 adenomas. A linear relationship was found between cell concentration and hormone secretion, both for PRL and GH. In vitro hormone secretion by the adenoma cells was in good agreement with in vivo dynamic tests. In longterm culture, GH and ACTH secretion by the adenoma cells declined, while PRL secretion was fairly constant. It is concluded that large numbers of dispersed pituitary adenoma cells can be obtained from the material removed by suction during transsphenoidal operation.(ABSTRACT TRUNCATED AT 250 WORDS)