O-GlcNAc modification, insulin signaling and diabetic complications.

O-GlcNAc glycosylation (O-GlcNAcylation) corresponds to the addition of N-acetylglucosamine on serine and threonine residues of cytosolic and nuclear proteins. O-GlcNAcylation is a dynamic post-translational modification, analogous to phosphorylation, that regulates the stability, the activity or the subcellular localisation of target proteins. This reversible modification depends on the availability of glucose and therefore constitutes a powerful mechanism by which cellular activities are regulated according to the nutritional environment of the cell. O-GlcNAcylation has been implicated in important human pathologies including Alzheimer disease and type-2 diabetes. Only two enzymes, OGT and O-GlcNAcase, control the O-GlcNAc level on proteins. Therefore, O-GlcNAcylations cannot organize in signaling cascades as observed for phosphorylations. O-GlcNAcylations should rather be considered as a "rheostat" that controls the intensity of the signals traveling through different pathways according to the nutritional status of the cell. Thus, OGT attenuates insulin signal by O-GlcNAcylation of proteins involved in proximal and distal steps in the PI-3 kinase signaling pathway. This negative feedback may be exacerbated when cells are chronically exposed to elevated glucose concentrations and could thereby contribute to alterations in insulin signaling observed in diabetic patients. O-GlcNAcylation also appears to contribute to the deleterious effects of hyperglycaemia on excessive glucose production by the liver and deterioration of ß-cell pancreatic function, resulting in worsening of hyperglycaemia (glucotoxicity). Moreover, O-GlcNAcylations directly participate in several diabetic complications. O-GlcNAcylation of eNOS in endothelial cells have been involved in micro- and macrovascular complications. In addition, O-GlcNAcylations activate the expression of profibrotic and antifibrinolytic factors, contributing to vascular and renal dysfunctions.

Structure-function analysis of a double-mutant cystic fibrosis transmembrane conductance regulator protein occurring in disorders related to cystic fibrosis.

A number of disorders related to cystic fibrosis have been described since the cloning of the cystic fibrosis gene, including infertility due to the congenital bilateral absence of the vas deferens. We have identified, in several patients, complex cystic fibrosis transmembrane conductance regulator genotypes like double-mutant alleles. We have now analyzed the structure-function relationships of one of these mutants, R74W-D1270N cystic fibrosis transmembrane conductance regulator, expressed in HeLa cells, to evaluate the contribution of each mutation in the phenotype. We found that R74W cystic fibrosis transmembrane conductance regulator appears to be a polymorphism, while D1270N cystic fibrosis transmembrane conductance regulator could be responsible for the congenital bilateral absence of the vas deferens phenotype. The combination of the two produced a more severe effect on the chloride conductance pathway as well as on the phenotype.

Retinoic acid stimulates growth hormone synthesis in human somatotropic adenoma cells: characterization of its nuclear receptors.

In order to gain a better understanding on the possible role of retinoic acid (RA) on human GH secretion, we have characterized the expression of its nuclear receptors in somatotropic adenoma cell extracts. By immunoblotting with rabbit polyclonal antibodies directed against RAR alpha, beta, and gamma and RXR alpha and beta, we could only detect the presence of RAR alpha and RXR alpha proteins. The predominant expression of RXR alpha was confirmed at the mRNA level by Northern and slot-blot analysis. We then investigated the effect of RA on GH synthesis in cell culture of adenomatous somatotrophs. In cultured cells, RA (1 microM) stimulated GH secretion, increased intracellular GH content and GH mRNA levels within 72 h, suggesting a modulation of GH synthesis by RA.

[Paracrine regulation of anterior pituitary hormones by neuropeptides]. / Régulation paracrine des hormones antéhypophysaires par les neuropeptides.

The hypothalamus is the source of neuropeptides which, being secreted into the portal system, control the synthesis and the secretion of the anterior pituitary hormones. Besides the well characterized hypothalamic central control and the hormonal peripheral control, recent studies have shown, in the anterior pituitary, the expression, among many other regulatory factors, of neuropeptides that are identical to those produced by the hypothalamus and that seem involved in the local control of anterior pituitary functions through autocrine or paracrine mechanisms. The presence of the neuropeptide mRNAs, precursors and mature forms of the peptides in anterior pituitary tissues as well as the secretion of the mature peptides argue in favor of the intrinsic ability of the normal and tumoral anterior pituitary to express neuropeptides. This expression of neuropeptides occurring in tissues bearing functional receptors for these ligands, anterior pituitary control could rely, at least in part, on endogenous neuropeptides acting locally. Correlations between neuropeptide contents in the anterior pituitary and the plasma levels of anterior pituitary hormones suggest that neuropeptides of anterior pituitary origin can play a local regulatory role, complementary of the classical hypothalamic central control. In the normal anterior pituitary which remains under hypothalamic control, it is presently difficult to evaluate the relative importance of the local and central control. However, anterior pituitary hyperplasia and pituitary tumors represent two models in which the specific contribution of the local control is easier to define.

Evidence of thyrotropin-releasing hormone (TRH) gene expression in rat anterior pituitaries and modulation by estrogens of TRH-like immunoreactivity and TRH-elongated peptide contents.

TRH gene expression in the anterior pituitary has previously been reported in the human in vivo and in the rat in vitro. Until now, modulation of this synthesis with glucocorticoids and thyroid hormones has been observed in rats. The present study demonstrates for the first time that the TRH gene is also expressed, in vivo, in the rat anterior pituitary and that anterior pituitary TRH-like immunoreactivity (TRH-LI) and elongated forms of the immediate TRH progenitor sequence (TRH-elongated peptide) contents are also modulated by estrogens (E2). To investigate the presence of proTRH mRNA in the rat anterior pituitary, total RNA was reverse transcribed (RT) and the RT products were then amplified by PCR. Treatments with E2 were performed on intact and ovariectomized (OVX) rats for 2 months. TRH-LI was measured by RIA with an antibody which did not recognize the TRH-like peptide. pGlu-Glu-Pro-NH2 (< EEP-NH2) (cross-reactivity < 0.1%) and was characterized further as TRH-LI by HPLC. TRH-elongated peptides were measured by EIA and characterized by Sephadex G-50 chromatography and immunoblotting (molecular mass 25-35 kDa). The plasma prolactin levels and the pituitary sizes were increased by E2 treatment in both intact and OVX rats. Anterior pituitary TRH-LI increased in intact E2-treated rats compared with intact rats (82.7 +/- 19.0 versus 39.6 +/- 3.6 fmol/mg protein; means +/- S.E.M.; P < 0.001). This increase was greater when E2 was administered to OVX rats (599.0 +/- 98.4 after E2 treatment versus 58.6 +/- 3.6 fmol/mg protein: P < 0.001). In intact rats, anterior pituitary TRH-elongated peptide contents were not modified by E2 treatment while they were significantly decreased in OVX E2-treated rats (144.6 +/- 8.8 versus 223.7 +/- 9.5 fmol/mg protein; P < 0.001). These results demonstrate TRH gene expression in the rat anterior pituitary in vivo and suggest that E2 treatment is responsible for an increase in anterior pituitary TRH-LI, together with a decrease in TRH-elongated peptide contents.

Vitamin A and retinoic acid stimulate within minutes cAMP release and growth hormone secretion in human pituitary cells.

In order to gain a better understanding on the possible role of vitamin A (VA) and retinoic acid (RA) on human growth hormone (GH) secretion, we used the physiological model of pituitary cells perifusion. In perifused cells from pituitary somatotropic adenomas, RA induced within minutes a peak of GH secretion. This effect was dose dependent, maximal effect being observed with 100 nM. The GH release was associated with a discharge of cAMP delayed by 4 to 8 minutes relative to the GH surge. Similar results were obtained after VA stimulation. Our observations provide the first evidence of an action of VA and RA on cAMP production. They suggest a role of RA and VA in the regulation of human GH secretion via the cAMP dependent pathway.

Characterization and localization of an immunoreactive growth hormone-releasing hormone precursor form in normal and tumoral human anterior pituitaries.

Arguments favor an in situ synthesis of GH-releasing hormone (GHRH) in the normal and tumoral human anterior pituitary. These tissues may express human (h) GHRH messenger RNA, contain hGHRH-(1-44)-NH2, and secrete in vitro an immunoreactive form (ir-form) of the peptide. Here, we characterize and localize the precursor of hGHRH in human anterior pituitary tissues using RIAs specific for the C-terminus or the midportion of hGHRH-(1-44)-NH2, size-exclusion chromatography, HPLC, Western blotting, and immunocytochemistry. The anterior pituitary ir-forms were compared to those found in hypothalamus, posterior pituitary, and GHRH-secreting endocrine pancreatic tumors. Three ir-forms of hGHRH with mol wt of 30-45, and 5 kilodaltons (kDa) were detected. The 30- to 45-kDa ir-form was very likely to consist of hGHRH bound to proteins. The 5-kDa ir-form represented mature forms of hGHRH. It was the major form in tissues actively synthesizing and/or secreting hGHRH. Nontumoral anterior pituitaries contained significant amounts of mature hGHRH. The 10-kDa form was identified as a hGHRH precursor ir-form. In addition to its expected presence in the hypothalamus and GHRH-secreting tumors, normal and tumoral human anterior pituitaries contained an identical ir-form of the hGHRH precursor. Cells immunoreactive for the hGHRH precursor were observed in pituitary adenomas. Evidence for precursor and mature ir-forms of hGHRH in anterior pituitary tissues provides conclusive arguments for the endogenous synthesis of the neuropeptide.

Evidence of gonadotropin-releasing hormone mRNA in the rat anterior pituitary.

We have previously reported the presence and the persistence of immunoreactive GnRH cells in the rat anterior pituitary in the absence of exogenous GnRH. To substantiate the hypothesis of its endogenous production, we have investigated the presence of GnRH mRNA in rat anterior pituitary tissue using a reverse transcription-polymerase chain reaction (RT-PCR) amplification protocol. Total RNA from rat anterior pituitary, hypothalamus (positive control), and muscle (negative control) was reverse transcribed to the first strand of cDNA and amplified by PCR using a set of three exon-specific primers for a target sequence of the GnRH gene, i.e. two external 5' and 3' primers and one internal 3' primer. UV light analysis of the size-fractionated RT-PCR products showed two bands of the predicted sizes [511 and 397 base pairs (bp)] hybridizing with GnRH probes in Southern analysis only in hypothalamus and in anterior pituitary but not in muscle. These two bands were far more intense in the hypothalamus than in the anterior pituitary. Restriction enzyme analysis evidenced that the two RT-PCR products included the HindIII endonuclease site of cleavage present in the GnRH cDNA sequence spanned by the primers, yielding two digested products of the anticipated sizes (86 and 425 bp for the 511-bp fragment, and 86 and 311 bp for the 397-bp fragment). The complementary sequences for the GnRH probes were conserved in the 425-bp and 311-bp fragments. Based on these results, we can conclude that the RT-PCR products generated from RNA tissue were the target GnRH sequences in the anterior pituitary as well as in the hypothalamus.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoreactive thyroliberin (TRH) precursor forms in human hypothalamus and anterior pituitary tissues.

Immunoreactive (IR) proTRH forms were characterized in human hypothalamic tissue with two antisera raised against a hepta- and a decapeptide containing the TRH progenitor sequence (-Gln-His-Pro-Gly-). A similar study was performed in human normal and adenomatous anterior pituitaries, tissues in which TRH synthesis has been previously suggested. IR-proTRH was found in all the samples ranging from 42-775 fmol/mg proteins. Size exclusion chromatography identified a major 25-35 kDa form and a minor 4-8 kDa form. The existence of the major form was confirmed by immunoblotting. The results suggest that both human hypothalamic and normal or adenomatous anterior pituitary tissues synthesize similar IR-proTRH forms.

Detection of thyrotropin-releasing hormone (TRH) mRNA by the reverse transcription-polymerase chain reaction in the human normal and tumoral anterior pituitary.

To investigate the presence of TRH mRNA in the human anterior pituitary tissue, total RNA from human normal and tumoral anterior pituitary, hypothalamus (positive control) and muscle tissues (negative control) was reverse transcribed (RT) to the first strand of cDNA. RT products were then amplified by polymerase chain reaction (PCR) using a set of three exon-specific primers (two external 5' and 3' primers and one internal 3' primer) for a target sequence of the TRH gene including an intronic sequence of about 650 base pairs (bp). Southern analysis of the RT-PCR products specifically hybridizing with a 45-mer TRH probe showed two bands of the predicted sizes (399 and 351 bp) far more intense in hypothalamus than in normal and tumoral anterior pituitary tissue. The 399 and 351 bp RT-PCR products contained the BglII enzyme restriction site included in the TRH cDNA sequences spanned by the primers and the two respective digested fragments which were, as predicted, 337 and 289 bp long, hybridized with the TRH probe. Based on these results, we can conclude that the RT-PCR products generated from RNA tissue were the target TRH sequences in the human normal and tumoral anterior pituitary tissue as well as in the hypothalamus. Our data imply TRH gene expression in the human anterior pituitary.

Somatostatin cells in human somatotropic adenomas.

Data from our group have shown that the human adenomatous and normal anterior pituitary may be the source of somatostatin (SRIH). SRIH-producing cells were identified in two somatotropic adenomas. Immunoreactive SRIH cells were present in both cases. In case 2, material was available for RNA studies, in situ hybridization and electron microscopy. The size of the transcript identified by Northern blot analysis was identical to that of hypothalamic SRIH mRNA. In situ hybridization showed that the SRIH gene was expressed in a cell subset superimposable to that identified by immunocytochemistry. Co-localization studies revealed that SRIH and growth hormone (GH) immunoreactivities were not present in the same cells. Ultrastructural immunogold labelling showed that SRIH cells had features distinct from those of the somatotropes. The results confirm that the somatotropic adenomas have the ability to synthesize SRIH, indicate that SRIH expression is restricted to a subset of adenoma cells different from GH-producing cells, and imply that SRIH cells are involved in paracrine regulation of neighbouring somatotropes.

Apparently silent somatotroph adenomas.

We describe here 9 patients with somatotroph adenomas associated with mild features of acromegaly and basal plasma GH levels in the normal range. In 5 women and 4 men, 26 to 61 yrs old, the diagnosis of prolactinoma or non-secreting pituitary adenoma had been previously made on the basis of amenorrhea-galactorrhea or tumoral symptoms. However, they had discrete signs of coarsening of the facial features and moderate but evolutive changes of hand and foot sizes. Basal GH levels were in the normal range (0.4 to 4.5 micrograms/l, N less than 5 micrograms/l) but unaffected by oral glucose and insulin tolerance tests while IGF-I concentrations were elevated in all the cases (range 1.7 to 5.8 U/ml, N: 0.37-1.41 U/ml). Plasma PRL concentrations were elevated in 5 patients (range 16 to 80 micrograms/l, N less than 13 micrograms/l in men and N less than 19 micrograms/l in women). The 9 patients had a macroadenoma with an extrasellar extension in 8 of them and all were operated on by the transsphenoidal route. Immunocytochemical studies demonstrated IRGH-cells in all the adenomas and IRPRL-cells in 5 of them. Electron microscopic analysis of 3 tumors showed that the secretory granules were sparse and the Golgi apparatus poorly developed. Molecular biology of 7 tumors showed the presence of small amounts of GH mRNA. This result was in agreement with the morphological aspect, suggesting a low rate of GH synthesis. Thanks to these different approaches the diagnosis of silent somatotroph adenoma should sometimes be reconsidered.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Neuropeptides of anterior pituitary origin. Autocrine or paracrine functions?

Several neuropeptides classically associated with the hypothalamus have been found in the anterior pituitary. The question arises whether they are locally synthesized and if they play a paracrine or autocrine role on pituitary cell functions. Using normal and tumoral human pituitaries we found neuropeptides (TRH, SRIH, GHRH) and dopamine in variable quantities according to the nature of the tissue. They were all present in normal pituitaries, while stimulatory hormones (TRH and GHRH) were predominantly found in tumoral tissue, implying an imbalance of pathophysiological importance between the stimulatory and inhibitory control of hypophyseal hormones (PRL and GH) in pituitary adenomas. Both normal and tumoral pituitaries released TRH, SRIH and GHRH in large amounts suggesting their local synthesis. The in situ synthesis was demonstrated for SRIH by the evidence of SRIH mRNA, the detection of SRIH immunoreactivity in peculiar cells and the presence of SRIH precursor. The possible role of these pituitary neuropeptides was suggested for instance by the negative correlation found in vitro between SRIH and GH secretions. Moreover, neuropeptides could interact with each other. Indeed DA stimulated TRH release while PRL secretion decrease at the same time. Pulses of TRH had differential effects on SRIH release according to the nature of the tissue as TRH inhibited SRIH release from adenoma while it stimulated SRIH release from normal pituitary. Concerning the effects of SRIH and GHRH on GH secretion, there was an endogenous regulatory pattern comparable to that observed in rat portal blood vessels. Pulses of GHRH induced GH secretion only when endogenous SRIH release was not stimulated.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth hormone and somatostatin gene expression in pituitary adenomas with active acromegaly and minimal plasma growth hormone elevation.

Some patients with active acromegaly have elevated plasma IGF-I concentrations with only minimal elevation of plasma GH. We compared adenomatous GH and SRIH expression in 3 such patients (patients No. 1, 2 and 3; basal plasma GH level less than 4 micrograms/l) and in 3 acromegalic patients with high basal plasma GH level (patients No. 4, 5 and 6; 51.7 +/- 16.1 micrograms/l, mean +/- SEM). By immunocytochemistry, all the tumours proved to be somatotropic adenomas. At the ultrastructural level, signs of low secretory activity were observed in adenomas from patients No. 2 and 3. Perifused adenoma cells of patients No. 1, 2 and 3 released very little GH compared with those of patients No. 4, 5 and 6 (1 +/- 0.37 vs 51.5 +/- 34.1 micrograms x (10(-6) cells) x min-1, p less than 0.001). Adenoma SRIH content was 65.7 and 30.6 pg/mg proteins in patients No. 1 and 2, whereas it was undetectable in the others (patients No. 4, 5 and 6). Northern blot analysis showed that the GH gene was poorly expressed in the adenomas from patients No. 1, 2 and 3 compared with the adenomas from patients No. 4, 5 and 6. SRIH mRNA was detected in all 6 adenomas. However, the signal was more intense in the adenomas from patients No. 1, 2 and 3 than in those from patients No. 4, 5 and 6.(ABSTRACT TRUNCATED AT 250 WORDS)

[Neuropeptides of anterior pituitary origin]. / Neuropeptides d'origine antéhypophysaire.

Several neuropeptides classically associated with the hypothalamus have been found in the anterior pituitary. The question arises whether they are locally synthesized and if they play a paracrine or autocrine role on pituitary hormone secretion. Using normal and tumoral human pituitaries we found neuropeptides (TRH, SRIH, GHRH) and dopamine in variable quantities according to the nature of the tissue. They were all present in normal pituitaries, while stimulatory hormones (TRH and GHRH) were predominantly found in tumoral tissue, implying an imbalance of pathophysiological importance between the stimulatory and inhibitory control of hypophyseal hormones (PRL and GH) in pituitary adenomas. Both normal and tumoral pituitaries released TRH, SRIH and GHRH in large amounts suggesting their local synthesis. The in situ synthesis was demonstrated for SRIH by the evidence of SRIH mRNA, the detection of SRIH immunoreactivity in peculiar cells and the presence of SRIH precursor. The possible role of these pituitary neuropeptides was suggested for instance by the negative correlation found in vitro between SRIH and GH secretions. Moreover neuropeptides could interact on each other. Indeed DA stimulated TRH release while PRL secretion decreased at the same time. Pulses of TRH had differential effects on SRIH release according to the nature of the tissue as TRH inhibited SRIH release from adenoma while it stimulated SRIH release from normal pituitary. Concerning the effects of SRIH and GHRH on GH secretion, there was an endogenous regulatory pattern comparable to that described in rat portal blood vessels. Pulses of GHRH induced GH secretion only when endogenous SRIH release was not stimulated.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence of pre-prosomatostatin mRNA in human normal and tumoral anterior pituitary gland.

Expression of the SRIH gene was investigated in six human normal anterior pituitaries, six GH-, three PRL-, three mixed GH/PRL-secreting and four nonsecreting adenomas. Total cellular RNA and poly(A+) mRNAs were analyzed by dot and Northern blot hybridization to a 3'-end labeled oligonucleotide probe specific for the human pre-proSRIH mRNA. A weak but detectable pre-proSRIH hybridization signal was present in human normal anterior pituitaries and in the four groups of adenomas. The size of this pre-proSRIH mRNA was indistinguishable from that found in our hypothalamic samples and close to that described in the literature. The wide variation of the signal intensity from one case to the other in each group of the different types of normal and tumoral antehypophyseal samples prevented establishment of any correlation between the level of pre-proSRIH mRNA and the nature of the pituitary tissue. The presence of SRIH mRNA in human normal and tumoral anterior pituitary tissues provides a sound basis to substantiate the hypothesis of a SRIH biosynthesis in the human anterior pituitary gland.

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.

Somatostatin receptors in human growth hormone and prolactin-secreting pituitary adenomas.

[125I-Tyr]Somatostatin [( 125I-Tyr]SRIH) binding was found in 11 GH-secreting pituitary adenomas [Kd = 0.46 +/- 0.15 (+/- SE) nM; maximum binding, 165 +/- 35 fmol/mg protein). This binding was specific, since it was displaced by somatostatin-14 (SRIH-14), N-Tyr-SRIH-14, and SRIH-28. In contrast, a number of peptides and drugs not structurally related to SRIH, such as bombesin, dopamine, LHRH, met-enkephalin, naloxone, neurotensin, secretin, substance P, TRH, or vasoactive intestinal peptide, did not affect [125I-Tyr]SRIH binding. [125I-Tyr]SRIH specific binding also was found in PRL-secreting pituitary adenomas. The kinetic characteristics of the specific binding were similar to those of GH-secreting adenomas. However, maximal binding was one quarter that of GH-secreting adenomas (37 +/- 9 fmol/mg protein). In contrast, nonsecreting (chromophobe) tumors were devoid of any specific binding. Finally, in acromegaly, the density of [125I-Tyr]SRIH-binding sites in the adenomas was negatively correlated with plasma GH levels before surgery (r = -0.80). This suggests that somatostatinergic control is involved in GH secretion in acromegalic patients.

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.