Growth hormone-releasing hormone and somatostatin in normal and tumoral human pituitaries.

In order to further understand the role of endogenous pituitary neuropeptides in pituitary hormonal content and secretion, GHRH, SRIH and GH contents were quantified in GH adenomas obtained from acromegalic patients with plasma GH levels either high (greater than 5 micrograms/l, range 11 to 550 micrograms/l, n = 11) or in the normal range (less than 5 micrograms/l, range 1 to 3.3 micrograms/l, n = 4). Values were compared to those found in normal human pituitaries. No relationship was found between GHRH content and plasma GH or between SRIH and GH content when considering together adenomas and normal pituitaries. Results showed that there is a positive relationship between GHRH and GH content: when GHRH content is high, GH content is also high (normal pituitaries and GH adenomas of acromegalic patients with high plasma GH) and when GHRH content is low, GH content is also low (GH adenomas of acromegalic patients with plasma GH in the normal range). Conversely, SRIH content is negatively related to plasma GH levels: when SRIH is present, plasma GH is in the normal range; when SRIH is undetectable, plasma GH is high.

Effect of oestradiol on dopamine receptors and protein kinase C activity in the rat pituitary: binding of oestradiol to pituitary membranes.

Oestradiol exerts an important modulatory influence on the release of prolactin which is accomplished partly through disruption of the inhibitory influence of dopamine. We have focused on the status of the anterior pituitary D2 dopamine receptor in female rats treated chronically with oestradiol or progesterone. A direct membrane effect of these steroids on the dopamine system was also investigated in vitro. Both steroids affected the status of the D2 receptor, oestradiol decreasing the number of sites in vitro and progesterone increasing it both in vitro and in vivo. The in vitro studies demonstrated that these steroids exert a direct membrane effect on the D2 receptor. These results correlated with an in vitro short-term physiological effect of oestradiol and progesterone on the dopaminergic inhibition of prolactin release, oestradiol decreasing it while progesterone had the opposite effect. Binding studies with [3H] oestradiol on pituitary membranes revealed a site for oestradiol of high affinity and low capacity, indicating that oestradiol's membrane effects could be mediated by a specific receptor. In vivo treatment with oestradiol also induces proliferation of prolactin-secreting cells (lactotrophs). We focused on the effect of oestradiol on protein kinase C activity, which is involved in both secretion and proliferation. In female rats treated with oestradiol total protein kinase C activity was increased by 74% (particulate 90%, soluble 71%) in comparison with controls. This effect was reversed by concomitant treatment with a dopamine agonist. Thus in the pituitary oestradiol and progesterone affect the characteristics of membrane components that are implicated in the physiological control of the cell. Whether these effects are post-transcriptional only or are also mediated through direct membrane mechanisms needs further investigation.

Altered balance between thyrotropin-releasing hormone and dopamine in prolactinomas and other pituitary tumors compared to normal pituitaries.

We measured TRH and dopamine (DA) concentrations in prolactinomas and other pituitary tumors in order to further understand the roles of these two factors in the hormone hypersecretion and growth of these tumors. The mean TRH concentration (by RIA) in 16 prolactinomas was 247 +/- 92 (+/- SE) fmol/mg cell protein (range, 10-1297), near that found in normal pituitary tissue. The prolactinoma TRH content did not correlate with the patient's tumor size or plasma PRL level. By contrast, DA assayed by high pressure liquid chromatography was present in normal pituitary tissue (7.3 +/- 3.5 pmol/mg cell protein), but was very low or undetectable in the prolactinomas (23 fmol/mg cell protein or less). 3,4-Dihydroxyphenylacetic acid, also assayed by high pressure liquid chromatography, was undetectable in both normal pituitary tissue and prolactinomas. This imbalance between TRH and DA content also was found in GH-secreting and nonsecreting adenomas. The TRH content in 18 GH-secreting tumors (24 +/- 6 fmol/mg) was considerably lower than that in the prolactinomas (P less than 0.001). In 8 nonsecreting adenomas, the mean TRH concentration was 109 +/- 28 fmol/mg, about half of that in the prolactinomas. In those 2 types of adenomas, DA also was nearly undetectable (less than or equal to 73 fmol/mg cell protein). We conclude that the imbalance between TRH and DA contents in prolactinomas compared to those in normal pituitary tissue might participate in the mechanisms leading to hypersecretion of PRL and the growth of all types of pituitary adenomas.

Hyperprolactinemia-induced modifications in vasoactive intestinal peptide binding site densities in the rat central nervous system and pituitary gland: evidence for an interaction between estradiol-17 beta and prolactin effects.

Hyperprolactinemia was induced in ovariectomized rats by implanting estradiol-17 beta pellets, grafting extrapituitaries, or by a combination of both treatments. Subsequently, the effect of increasing plasma prolactin levels on both central and pituitary receptors for vasoactive intestinal peptide (VIP), a neuropeptide known to stimulate prolactin release was investigated. The results obtained by quantitative autoradiography show that the density of VIP binding sites is modified in restricted areas of the central nervous system (striatum, several cortical, thalamic and limbic structures) and in the pituitary in hyperprolactinemic animals. The present results suggest that changes in plasma prolactin levels may control VIP receptor site density in both brain and pituitary. Moreover, direct effects of estradiol-17 beta and possible interactions between estradiol-17 beta and prolactin are observed on both brain and pituitary VIP binding sites.

Receptors and neurohormones in human pituitary adenomas.

In order to go further into the pathogenesis of human pituitary adenomas, we studied receptors for neurohormones (thyroliberin, TRH; dopamine, DA; somatostatin, SRIH), for estradiol and epidermal growth factor (EGF) thought to influence hormone secretion and/or cell growth. The following results were obtained: (1) the receptors listed above, with the exception of EGF receptors in the adenomas, are present in normal pituitary tissue and in prolactin (PRL)- and growth hormone (GH)-secreting adenomas; (2) they are functional and their affinities are not different in normal or tumoral tissues; (3) their density is variable and depends on the type of secreting adenoma (GH or PRL), the size of the tumor and the plasma level of the hormone which is secreted, and (4) in nonsecreting adenomas, only TRH receptors are found with characteristics identical to those observed in secreting adenomas. We also showed that TRH is contained in normal and tumoral pituitary tissues. TRH and SRIH are released in vitro from adenomatous cells in large amounts, suggesting their possible synthesis by the pituitary. In both cases a local regulation is observed. TRH release is stimulated in the presence of DA while SRIH is inhibited in the presence of TRH. This neuropeptide release may be implicated in the pituitary hormone regulation through a paracrine or an autocrine mechanism. Thus, the neurohormone receptors found in pituitary adenomas should be dependent on a more complex regulation than it has been envisaged till now.

[Release of thyrotropin releasing hormone (TRH) from human prolactin-secreting pituitary adenoma cells. Modulation by dopamine]. / Libération de thyrolibérine (TRH) par les cellules adénomateuses hypophysaires humaines à prolactine. Modulation par la dopamine.

We found, by radioimmunoassay, that thyrotropin-releasing-hormone (TRH) was present in human prolactin (PRL)-secreting adenomas (mean: 89 +/- 45 (SEM) fmol/mg proteins) and was released by perifused adenomatous cells at levels varying from 5 to 60 fmol/10(6) cell/2 min. TRH release was increased in the presence of dopamine (DA) 10(-6) M but was not modified by the presence of somatostatin (SRIH) 10(-6) M.

Evidence of thyrotropin-releasing hormone (TRH) and TRH-binding sites in human nonsecreting pituitary adenomas.

Immunoreactive TRH (IR-TRH) and TRH-binding sites were sought in nonsecreting pituitary adenomas. [3H]TRH bound specifically to cellular membranes from 11 of 12 such adenomas studied, with a dissociation constant (Kd) of 50 +/- 5 (+/- SEM) nmol/L and a maximum number of binding sites of 76 +/- 16 fmol/mg membrane protein (range, 32-229 fmol/mg protein). IR-TRH was detected in all 8 of the tumors in which it was sought. The identity of the IR-TRH was verified by high pressure liquid chromatography. The tumor IR-TRH concentration varied from 45-248 fmol/mg cell protein (mean, 109 +/- 28 fmol/mg cell protein), about half that in normal human pituitary (229 +/- 55 fmol/mg protein). There was no correlation between the number of binding sites and the IR-TRH content. The role of TRH in nonsecreting pituitary adenomas is unknown at this time.

Epidermal growth factor-binding sites, present in normal human and rat pituitaries, are absent in human pituitary adenomas.

To investigate the involvement of epidermal growth factor (EGF) and its receptor in the pathogenesis of human pituitary adenomas, we examined the presence of EGF-binding sites in normal rat and human pituitaries and in human PRL- and GH-secreting and nonsecreting pituitary adenomas. Using crude membrane preparations, specific binding for [125I] EGF was found in normal rat and human pituitaries. Equilibrium was reached at 25 C in 40 min. There was no change in the Kd values between male rats [Kd, 0.65 +/- 0.35 nM (mean +/- SD)] and female rats (Kd, 0.51 +/- 0.15 nM), while the maximum capacity was significantly higher (P less than 0.05) in male rats (21 +/- 8 fmol/mg protein) than in female rats (10 +/- 2 fmol/mg protein). Scatchard analysis of the data suggested the presence of a single class of binding sites. In the three normal human pituitaries tested, specific [125I]EGF binding was also demonstrated. However, both the Kd and the maximum capacity varied widely. Twenty-two human pituitary adenomas were tested, but no specific binding was detected in any of them. In addition to the binding experiments, a radioreceptor assay using rat liver membranes was developed to detect EGF or EGF-like material in extracts of six human pituitary adenomas (two of each type). No EGF activity was detected in any of the extracts. From these results, we conclude that EGF-binding sites are present in normal pituitary tissue, suggesting a physiological role for EGF in this tissue. Consequently, the reason(s) for the lack of EGF binding in pituitary adenoma membranes is not known.

Binding of (+)-PN 200-110 to rat pituitaries and to normal and adenomatous human pituitaries.

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.

Evidence for a specific estradiol binding site on rat pituitary membranes.

Using adenohypophyses from normal female rats, we demonstrate that estradiol binds pituitary membranes to one homogeneous population of sites with high affinity [dissociation constant (Kd) = 0.041 +/- 0.014 nM; n = 6] and low capacity [maximum binding (Bmax) = 13.6 +/- 5.6 fmol/mg protein]. The binding is thermolabile. Association experiments show that the best experimental conditions are an overnight incubation at 0 C. When the amount of proteins is increased more than 0.3 mg/ml of membrane suspension, binding is rapidly nonlinear. The presence of 0.5 M leupeptin does not improve the binding. Extensive washing of the membranes does not decrease the amount of sites, indicating that the binding is not loosely attached to the membranes. Parenthetically, it should be noted that the membrane fraction was devoid of the cytosolic enzyme marker, lactate dehydrogenase. Binding is specific for estrogenic compounds. When 100% specific binding was determined in the presence of 10(-6) M diethylstilbestrol, 17 beta-estradiol, estrone, and estriol displaced total binding by 110, 80, and 75%, respectively. Neither 4-OH-tamoxifen nor dihydrotestosterone, progesterone, or cortisol displaced the binding. Taken together, these data argue in favor of the presence of specific membrane recognition sites for estradiol in the rat pituitary.

Correlative studies between the presence of thyrotropin-releasing hormone (TRH) receptors and the in vitro stimulation of growth-hormone (GH) secretion in human GH-secreting adenomas.

Specific receptors for TRH were characterized on cellular membranes of 6 out of 13 somatotrophic adenomas obtained from acromegalic patients. These receptors had the same dissociation constant (Kd: 62 +/- 10 nM) as those found in human PRL-secreting adenomas, but their maximal number of binding sites (Bmax: 76 +/- 24 fmol/mg of protein) was six fold smaller. A good correlation was found between the presence of TRH receptors and the in vitro TRH-induced stimulation of GH secretion. The increase in GH release varied from 25 to 200%. It was thus concluded that these receptors are functional. However, why only some of the human somatotrophic adenomas possess TRH receptors and respond to TRH in vitro needs further investigations.

In vitro and in vivo antagonistic regulation by estradiol and progesterone of the rat pituitary domperidone binding sites: correlation with ovarian steroid regulation of the dopaminergic inhibition of prolactin secretion in vitro.

The influences of in vivo and in vitro estradiol (E2) and progesterone (P) treatments on the characteristics of [3H]domperidone binding to intact and ovariectomized (OVX) rat pituitary membranes were analyzed and compared to the modulation by these steroids of dopamine (DA) inhibition of PRL secretion in vitro from intact and OVX rat pituitaries. Using intact rat pituitaries, high and low affinity binding sites for domperidone were detected; the dose-dependent DA inhibition curve of PRL secretion was biphasic (range, 10(-13) - 10(-10) M DA, IC50 = 6 X 10(-12) M; range, 10(-10) - 10(-6) M DA, IC50 = 2 X 10(-8) M). Using OVX rat pituitaries, only the high affinity sites for domperidone were detected, and the dose-dependent DA inhibition curve of PRL secretion was monophasic (range, 10(-10) - 10(-6) M DA, IC50 = 10(-8) M). E2 and P did not modify the characteristics of the high affinity sites either after in vivo treatment or when directly added to the in vitro binding assay. However, using in vivo and in vitro tests, a modulation of the low affinity sites by E2 and P was demonstrated. When E2 is in excess and P levels are low or undetectable, these sites are not detectable, and P is able to restore there presence. A parallelism has been established between this antagonistic E2 and P regulation and the modulation of DA inhibition of PRL secretion (range, 10(-13) - 10(-10) M DA). When intact rat pituitaries are perifused in the presence of 10(-8) M E2, the biphasic dose-dependent inhibition curve of the control is changed into the monophasic curve of the OVX rat pituitaries. Conversely, when OVX rat pituitaries are perifused in the presence of 10(-6) M P, the monophasic curve of the control is changed into the biphasic curve of the intact rat pituitaries. Thus, the DA inhibition in the range 10(-13) - 10(-10) M might result from an interaction between DA and the low affinity site for domperidone. In summary, the biological regulation of PRL by DA at the pituitary level may be mediated by two different DA sites, one being submitted to an antagonistic E2 and P regulation directly at the membrane level. The consequence of this regulation is that, whereas E2 decreases the sensitivity of the cell to DA, P is necessary for a normal DA response of the lactotroph.

Interaction between somatostatin and TRH on growth hormone secretion in perifused human growth hormone tumor cells.

The interaction between somatostatin (SRIF) and thyrotropin-releasing hormone (TRH) on growth hormone (GH) release has been studied on dispersed somatotrophic tumor cells obtained from 7 acromegalic patients. TRH increased GH secretion in 4 cases and SRIF decreased GH secretion in 5 cases. When TRH and SRIF were concomitantly perifused, SRIF, when active by itself, prevented and reversed the TRH-induced stimulation of GH release, while TRH never antagonized the inhibitory effect of SRIF. We conclude that, in these adenomatous cells, the physiological inhibitory factor (SRIF) overcomes the nonphysiological stimulatory factor (TRH) in the control of GH secretion.

Paradoxical response of thyrotropin to L-dopa and presence of dopaminergic receptors in a thyrotropin-secreting pituitary adenoma.

We studied the dopaminergic control of TSH secretion in a patient with hyperthyroidism due to a TSH-secreting pituitary adenoma. A 34-yr-old previously thyroidectomized woman had mild clinical hyperthyroidism and a diffuse goiter without exophthalmos. She complained of headaches and had bitemporal hemianopsia. Serum T4 and T3 by RIA were elevated, and TSH was 112 microU/ml (normal range, 1.1-7.2). The alpha-subunit to TSH molar ratio was 1.7 (normal, less than 1), as reported in other patients with tumoral TSH hypersecretion. After TRH, a marked increase in TSH occurred. There was no evidence of pituitary deficiency. Skull x-rays and computerized axial tomography revealed an intrasellar tumor with suprasellar extension. Selective transsphenoidal adenomectomy was performed, and a pituitary tumor was removed. The tumor was almost entirely composed of cells reactive with antihuman beta TSH serum by indirect immunofluorescence. A unique feature of this patient was the marked increase in TSH levels after L-dopa administration. To our knowledge, this paradoxical response has never been reported previously in such patients. Using [3H]domperidone as ligand, dopaminergic receptors were demonstrated in the membranes of the adenomatous thyrotroph cells. The reason for the paradoxical response of TSH to dopaminergic agents is not known.

Modifications of the high and low affinity pituitary domperidone-binding sites in chronic estrogenized rats.

The effect of chronic estrogen treatment on the anterior pituitary domperidone-binding sites was studied in female rats. The rats were implanted from 1-6 months with a Silastic capsule containing 17 beta-estradiol. The Feldman analysis of [3H]domperidone binding to anterior pituitary membranes of control or estrogenized rats revealed the presence of two sites. The binding characteristics of the higher affinity site were identical for both groups (Kd of the high affinity site, 0.30-0.45 nM; maximum number of binding sites of the high affinity site, 74-95 fmol/mg protein); however, those of the lower affinity site were affected by the estrogen treatment: the Kd of the low affinity site increased from 17.4 +/- 3.2 to 41.5 +/- 9 (+/- SE) nM, and the maximum number of binding sites of the low affinity site increased from 214 +/- 22 to 343 +/- 35 fmol/mg protein. Thus, in chronic estrogenized rats, the total number of binding sites was increased by 54%. These changes, induced by chronic estrogenization, were reversible, since 2 weeks after removal of the 17 beta-estradiol pellet, the binding characteristics were no longer different from those observed in control rats. In contrast to chronic estrogen treatment, ovariectomy reduced markedly the total number of [3H]domperidone-binding sites in anterior pituitary membranes (-70%). Feldman analysis revealed that this reduction resulted from the complete disappearance of the low affinity sites in those membranes. No significant change in the binding characteristics of the high affinity site was detected in ovariectomized rats. Since estradiol induces a decrease in the anterior pituitary content of dopamine, a denervation supersensitivity-like mechanism might be responsible for the increase in pituitary domperidone-binding sites in estrogenized rats. Conversely, a hyposensitivity mechanism could be implicated in the decrease in the total number of the pituitary domperidone-binding sites in ovariectomized rats, since pituitary dopamine levels are increased in those animals. Whether the antidopaminergic properties of estrogen are also involved in this modulation after chronic estradiol treatment requires further investigation.

Evidence of receptors for thyrotropin-releasing hormone in human prolactin-secreting adenomas.

Specific receptors for TRH were demonstrated on cellular membranes from 8 out of 10 human PRL-secreting adenomas. One binding site was characterized. The mean (+/- SD) value for the dissociation constant (Kd) from 8 adenomas was 68 +/- 7 nM. The maximal number of binding sites varied from 1 adenoma to another (from 50-1200 fmol/mg protein). In 7 of the 8 patients, there was little or no in vivo plasma PRL response to TRH although receptors for TRH were present on their tumors. It is thus suggested that in vivo absence of stimulatory effect of TRH in human PRL-secreting adenomas is not due to the absence of TRH receptors.

Failure of 2 Hydroxyestradiol to interact with dopamine inhibition of human prolactin secretion in vitro and with dopamine receptors of prolactin-secreting adenomas.

The ability of 2-Hydroxyestradiol, a catecholestrogen, and 17 beta Estradiol to interact with the dopamine inhibition of prolactin and with dopamine receptors has been tested on dispersed human prolactin-secreting cells obtained from ten pituitary adenomas. There is a 80% inhibition of prolactin secretion obtained by addition of dopamine in a superfusion system. This inhibition is not affected by preexposure to the steroids, or by their introduction into the perifusion medium. Moreover 2 Hydroxyestradiol and 17 beta Estradiol do not interact with the binding of 3H Domperidone to DA receptors.

[Presence of high-affinity binding sites for thyroliberin in human pituitary adenomas secreting prolactin]. / Présence de sites de liaison de haute affinité pour la thyrolibérine dans les adénomes hypophysaires humains sécrétant de la prolactine.

Specific high affinity binding sites for thyrotropin-releasing hormone (TRH) were demonstrated on cellular membranes from human prolactin-secreting adenomas in spite of the lack of in vivo prolactin response to TRH. One binding site was characterized. The mean value for the Kd from four adenomas was 64.8 +/- 12.5 nM. The maximal number of binding sites varied from one adenoma to another (from 80 to 450 fmoles/mg membrane proteins).

Alterations in central neurotransmitter receptor binding sites following estradiol implantation in female rats.

The subcutaneous implantation of an estradiol pellet (10 mg) into female rats induced a hypophyseal hyperplasia with hyperprolactinaemia. Examination of neurotransmitter receptors in the hippocampus, striatum and cerebral cortex one month after the implantation revealed that estrogenization was associated with: an increased density of (3)H-domperidone binding sites (D(2) receptors) in the striatum and reduced numbers of (3)H-serotonin high affinity sites (5-HT(1) receptors) in the hippocampus and of (3)H-muscimol binding sites (GABA receptors) in the hippocampus, striatum and cerebral cortex. In contrast, the characteristics of (3)H-spiperone binding to 5-HT(2) receptors (in the cerebral cortex) and those of (3)H-flunitrazepam binding to benzodiazepine sites (in the three brain regions examined) were not significantly different in estrogenized and in control female rats. However, the enhancing effect of GABA on (3)H-flunitrazepam binding was markedly reduced in brain membranes from estrogenized animals. The respective roles of estradiol and prolactin in mediating these changes in neurotransmitter receptors are discussed notably with regard to the regional heterogeneity of estradiol binding capacity in the rat brain.

Evidence of dopamine receptors in human growth hormone (GH)-secreting adenomas with concomitant study of dopamine inhibition of GH secretion in a perifusion system.

Dopamine (DA) is known to influence human GH release both in vivo and in vitro. The direct control of GH secretion at the pituitary level has been observed using human GH-secreting pituitary adenomas in organ culture, but no dose-response relationship between DA and GH inhibition was demonstrated. Such a dose-response relationship was obtained using GH-secreting adenomatous cells in a perifusion column. The IC50 of DA was 5 X 10(-8) M. Binding studies with [3H]apomorphine on human GH-secreting pituitary adenoma membranes demonstrated the presence of a dopaminergic receptor (Kd = 0.73 +/- 0.24 nM; maximum binding, 18.5 +/- 3.6 fmol/mg protein), which presumably mediates the effect of DA on GH release. In comparison to prior studies with PRL-secreting adenomas, several differences were apparent: 1) the maximum inhibition of GH release by DA is lower than that of PRL (50% vs. 80%), 2) the maximum number of binding sites is much lower (5-20 times) in GH-secreting adenomas than in PRL-secreting adenomas, and 3) [3H]domperidone does not show specific binding on GH-secreting adenomas but does on PRL-secreting adenomas. These experimental results indicate that the dopaminergic control of GH secretion is different from that of PRL secretion, as DA is less effective on GH release than on PRL release from human pituitary adenomas.