Prolactin Binding Sites Research Articles

Histochemical demonstration of prolactin binding sites.

We have developed a new probe for histochemical demonstration of prolactin binding sites. Ovine prolactin (oPRL) was conjugated with the N-hydroxy-succinimide ester derivative of the fluorochrome 7-amino-4-methylcoumarin-3-acetic acid. Under mild reaction conditions the ester derivative reacted with available NH2 groups of the prolactin molecule to form stable bonds. The coupling reaction yielded products that co-migrated with oPRL but had slightly decreased isoelectric points. The receptor binding and bioactivity of the flurochrome-hormone derivatives were decreased essentially proportional to the extent of conjugation. The derivatives were further tested for their ability to label PRL binding sites, using frozen sections of mammary gland and brain tissue of lactating rats. The results presented in this report describe the validation of this probe with regard to labeling of PRL binding sites at the light microscopic level in known target organs (mammary gland and choroid plexus). In addition, this fluorescent probe was used to demonstrate the presence of PRL binding sites at a novel site, the ependymal lining of the third ventricle.

Further study on the changes in the concentration of prolactin-binding sites in different organs of Xenopus laevis male and female, kept under dry conditions and then returned to water (their natural habitat)

The binding of 125I-labeled ovine prolactin (125I-oPRL) to membranes from the kidney and liver of Xenopus laevis male and female specimens (Experiment I) and from the kidney, epidermis, and liver of female specimens (Experiment II) (1) kept in an aquatic environment, (2) exposed for 2 weeks to dehydrating conditions, and (3) then placed back into their aquaria after exposure to dehydrating conditions (Experiment II) was studied. No significant sex differences in PRL binding to kidney, epidermis, and liver were found. A highly significant drop in PRL specific binding to the membranes from the kidney and epidermis is brought about in the specimens from both sexes exposed to dehydrating conditions. The results obtained by MgCl2 treatment in vitro of the membranes under study for an estimation of total PRL receptor concentrations seem to point to an actual decrease in PRL specific binding sites. The values of PRL specific binding to the membranes from the liver are not affected by dehydration of the animals (Experiment I and II) or their subsequent rehydration (Experiment II). In rehydrated females (Experiment II), PRL binding values were closely related to those recorded in females permanently maintained in water (controls).

Localization of prolactin binding sites in ring dove brain by quantitative autoradiography

Specific binding sites for prolactin have been previously detected and characterized in ring dove brain membranes. In order to map the distribution of these sites, specific binding of 125I-ovine prolactin was examined in slide-mounted sections of 6 male and 6 female ring dove brains by in vitro film autoradiography and densitometry. Analysis of 34 brain regions revealed a sex-specific pattern in specific binding activity. Although no significant sex differences were observed in any individual brain region, a trend in this direction was observed in the preoptic area. Specific binding levels in which the lower limit of the 99% confidence interval was greater than zero were detected in choroid plexus, medial habenula, lateral mesencephalic nucleus, hippocampus, parahippocampal area, preoptic area and 4 hypothalamic sites: paraventricular nucleus, ventromedial nucleus, suprachiasmatic nucleus, and tuberal region. Autoradiographic analysis of specific binding in cerebellum, ventromedial hypothalamus, and hippocampus/parahippocampus yielded relative differences that closely approximated those obtained in binding studies on tissue homogenates from these regions. These results suggest possible sites of prolactin action in altering behavioral state and neuroendocrine function in this species.

Magnesium-dependent non-specific binding of [ 125I] prolactin to myelinated tracts in the rat central nervous system

An in vitro autoradiographic assay was used in identifying a magnesium-dependent, non-specific binding of [ 125I] prolactin to myelinated fiber tracts in the rat brain. Frozen tissue sections were incubated for 18 h at 4°C in media which included [ 125I] prolactin alone or with a 500 fold excess of unlabelled prolactin. Magnesium in the incubation medium caused a non-specific binding of radiolabelled prolactin to the myelinated fiber tracts in the brain. In contrast, calcium did not facilitate prolactin non-specific binding to myelin. Hence, calcium should optimize the detection of specific prolactin binding sites in the brain by in vitro autoradiographic or radioreceptor assays.

Autoradiographic identification of prolactin binding sites in rat median eminence.

Binding sites for prolactin were localized and quantified in the female rat brain by in vitro autoradiographic analysis with iodine-125-labeled ovine prolactin. Following incubation, labeled prolactin bound preferentially to the median eminence and choroid plexus of the lateral and third ventricles. The addition of excess unlabeled ovine prolactin blocked binding of the labeled hormone in the choroid plexus, and attenuated prolactin binding in the median eminence. These results provide evidence for median eminence. These results provide evidence for prolactin-specific recognition sites in the median eminence, a region intimately involved in the hypothalamic regulation of prolactin secretion.

Association of prolactin and insulin receptors with mammogenesis and lobulo-alveolar formation in pregnant ewes

1. 1. Pregnant, multiparous ewes were sacrificed at d50 ( n = 3), 80 ( n = 4), 115 ( n = 3), or 140 ( n = 4) for biochemical, histological and autoradiographical quantitation of mammary growth. 2. 2. Significant increases in concentration of mammary parenchyma DNA (0.25–24 mg/g tissue), total udder DNA (57–1304 mg), and total udder RNA (36–1504 mg) were observed by d 115. 3. 3. Mammary tissue at d 115 contained the maximal percent of tissue volume occupied by epithelium (41.2%), number of cells per alveolar cross section (36.6) and percent of [methyl- 3 H]-thymidine labeled epithelial cell nuclei (3.5%). 4. 4. Concentration prolactin binding sites were significantly increased at d 115 of gestation, but serum prolactin and growth hormone concentrations remained low until d 140. 5. 5. Mammary insulin binding sites (per unit membrane protein) progressively decreased during gestation. 6. 6. Results suggest that serum growth hormone concentration and quantity of insulin receptors do not limit mammogenesis but greater concentrations of prolactin binding sites coincident with lobulo-alveolar formation, suggest a primary association with mammogenesis in the ewe.

Unoccupied prolactin binding components of the benign and malignant human prostate in a subclinical and clinical procedure

Optimal conditions for the quantitation of free prolactin binding components of human prostatic tissue obtained by TURP were studied by applying γ receptor assay. The radioligand used was 125I-prolactin. Significantly greater heat stability of the prostate membrane prolactin binding sites, when compared to that of androgen cytoplasmic receptors, was confirmed. The saturability and specificity of the prolactin binding components was demonstrated by the results of both Scatchard plot analysis and displacement studies. Free prolactin receptors were found in none of the poorly differentiated (G3) prostatic tumors examined, and only in 62.5% of medium differentiated (G2) prostatic malignancies. The majority of tissue specimens coming from patients with either BPH or well differentiated prostatic tumor (G1) contain measurable amounts of free prolactin membrane binding components. In the present study we report also the case in which the change in tumor differentiation toward a higher grade (G2 to G1, provoked by the successful chemohormonal treatment) is accompanied with the appearance of previously absent free prolactin binding components. In histologically proven BPH tissue specimens free prolactin receptor negative status has been found in most patients with a slight increase in serum PAP values, while receptor rich status was detected in the majority of those with elevated PSA concentrations. We believe therefore that the prolactin receptor values, when used as part of the multivariable analysis, may participate in further delineation of the role of prolactin in the development of prostate cancer, but may also play a role in a subclinical prediction related to the conversion of either an adenoma or a latent adenocarcinoma to the clinically manifest prostatic malignancy.

Characterization of prolactin binding sites in human choroid plexus

Characterization of prolactin binding sites in human choroid plexus

Prolactin receptors on large granular lymphocytes: Dual regulation by cyclosporin A

Although evidence has been provided for a modulatory role of prolactin (PRL) on humoral and cell-mediated immune responses and PRL receptors have been found on T and B lymphocytes, no indications exist concerning the influence of PRL on natural killer (NK) activity nor has a structural basis for interaction been found on the NK effector cells (large granular lymphocytes, LGL). We show here that highly purified LGL express binding sites for PRL. The calculated receptor number was 660 per cell and the dissociation constant ( K d ) was 3.0 × 10 −10 M. Since previous studies have reported that cyclosporin (CsA), an immunosuppressive agent used in organ transplant patients, affects the binding of PRL to T and B lymphocytes, but not to rabbit mammary gland cells, we investigated whether this compound could alter the binding of the hormone to LGL. At concentrations from 10 −7 to 10 −6, corresponding to the therapeutical range, CsA induced a complete inhibition of the PRL binding. By contrast, concentrations of CsA ranging from 10 −11 to 10 −9 increased the PRL binding to more than 100% of control levels. In addition to their antitumor role, LGL have been proposed to participate in graft versus host disease and in transplant rejection. The finding that CsA can differently affect PRL-receptor expression on LGL points to an involvement of CsA—PRL interactions in determining the output of these immune responses. In addition, these data strongly support the idea of a close relationship between the neuroendocrine and immune systems.

Regional distribution and species variation of prolactin binding sites in the brain

The binding of 125I-labeled ovine prolactin ( 125I-oPRL) to membranes from different brain regions of pigeon, rabbit, rat, pig, calf, horse, and ewe was studied. The hypothalamus from rabbit, pig, horse, and pigeon showed a low but specific binding for 125I-oPRL clearly different from the other brain regions examined (cortex and cerebellum), whereas in the brain from rat, calf, and ewe the binding was very small and quite uniform in the various regions. Also the membranes from choroid plexus of rabbit, pig, calf, and horse showed an evident specific binding for prolactin. The binding of 125I-oPRL to hypothalamus and choroid plexus membranes from rabbit and horse was inhibited in a dose-dependent manner by unlabeled oPRL and hGH but not by many other polypeptide hormones. Scatchard analysis of the binding revealed the presence of binding sites with low capacity and high affinity for ovine ligard.

Dissociated estradiol (E 2) action on the pituitary-testicular axis in a genetically hypoprolactinemic rat (IPL nude rat)

Prolactin (PRL) has been reported to be a possible mediator in the estradiol (E 2)-induced inhibition of the pituitary-testicular axis. In order to better characterize the role of PRL, we studied the action of chronic hypoprolactinemia on this E 2 inhibitory effect, using a genetically hypoprolactinemic rat (IPL nude). Normal and IPL nude adult male rats were injected either with vehicle or with E 2 valerianate (4 mg/rat) once a week for 2 weeks. Rats were decapitated 7 days after the last injection. Results showed that E 2 increased, similarly in both strains, pituitary weight and serum PRL levels. Serum testosterone values were reduced by 96% in both strains. However, testis weight was significantly reduced by 30% in normal rat, while in IPL nude rat, no significant decrease was observed. PRL binding sites, expressed as fmol/mg protein, were reduced in normal rat by 40%. No decrease was found in IPL nude rat. The dissociated E 2 action observed in IPL nude rat suggested that only testicular growth inhibition could be mediated by PRL and confirm that testosterone level decrease could be due to a direct action of E 2 on Leydig cells.

Prolactin and luteinizing hormone‐releasing hormone receptors in human benign prostatic hyperplasia and prostate cancer

Using a sensitive micromethod for the determination of prolactin (PRL) binding sites based on 125I-human PRL ligand, PRL receptor levels in specimens of benign prostatic hyperplasia (BPH) and human prostate cancer were estimated by the one-point assay system. Ten of 19 BPH specimens (53%), showed significant PRL binding, four being in the 9-12 fmol/mg range. All ten of these cases had an histological diagnosis of nodular glandular hyperplasia. Of ten adenocarcinomas examined, four samples (40%) exhibited positive PRL binding, the highest receptor levels being 10.2 fmol/mg protein. To characterize the receptors from BPH membranes, samples were then separately pooled according to the results obtained in one-point assays. In the PRL-negative pool no displacement could be detected. In the PRL-positive pool, the Scatchard analysis revealed one class of receptors with an average affinity Kd = 1.1 X 10(-9) M and capacity Bmax = 287 fmol/mg protein. In the prostate cancer specimens, luteinizing hormone-releasing hormone receptors with a high affinity and a low capacity were also found. The results indicate the presence of prolactin receptors in prostate cancer and in BPH. The clinical implications of such findings are not clear, but it is possible that a certain proportion of BPH and prostate cancers might be in part PRL dependent. Further studies are necessary to ascertain this hypothesis in an attempt to improve the treatment of BPH and prostate cancer.

In vivo autoradiographic analysis of prolactin binding in brain and choroid plexus of the domestic ring dove

The binding of intravenously administered prolactin to choroid plexus and brain tissue was determined radioautographically in the ring dove, a species that exhibits prolactin-induced alterations in brain function. An intense autoradiographic reaction was detected over the epithelial cells of the choroid plexus 5 min after the intravenous injection of 125I-ovine prolactin. A significant reaction was also observed over the infundibulum but no significant uptake of prolactin occurred in other brain areas. The binding of radiolabelled prolactin to infundibulum appeared to be non-specific, since excess unlabelled hormone did not reduce silver grain density. In contrast, 125I-ovine prolactin binding in choroid plexus was significantly reduced by excess unlabelled ovine prolactin or human growth hormone, but not by ovine luteinizing hormone. Specific binding to choroid plexus was also detected in vitro. The lack of significant brain uptake of prolactin in vivo is discussed in relation to recent in vitro evidence for specific binding sites for prolactin in several dove brain regions. Similarities between the binding results obtained in this avian species and those reported previously in mammals suggest that the two vertebrate groups exhibit similar patterns of prolactin interaction with neural target tissues.

Prolactin binding sites in human erythrocytes and lymphocytes

Specific binding sites for prolactin (PRL) have been studied in human peripheral lymphocytes and erythrocytes of normal adult volunteers and of term cord bloods. In erythrocytes from healthy adult subjects of both sexes a very low specific binding of 125I-human PRL was found (0.24%), whereas a higher binding was found in term cord blood (1.1 %). The binding was hormone specific, the binding capacity was 2.6 fmol/4x10 9 cells and the Kd was 3.4x10 −10M. In lymphocytes of both adults and term cord bloods an evident specific binding was observed (male adults: 1.6%; female adults: 1.7%; cord blood: 1.8%). The binding was specific for lactogenic hormones and the binding capacity was 3.7 fmol/2x10 6 cells and the Kd was 3.9x10 −10M. The presence of specific binding sites for PRL on human erythrocytes and lymphocytes could be used to study PRL binding on blood cells of patients in different physiological or pathological situations.

Specific binding sites for gonadotrophin-releasing hormone, LH/chorionic gonadotrophin, low-density lipoprotein, prolactin and FSH in homogenates of human corpus luteum. II: Concentrations throughout the luteal phase of the menstrual cycle and early pregnancy.

Corpora lutea were obtained from 52 women undergoing laparotomy during the luteal phase of the menstrual cycle. In addition, stromal, thecal and granulosa cell preparations were obtained from seven women undergoing ovariectomy during the late follicular-preovulatory phase of the cycle. The specific binding of a 125I-labelled gonadotrophin-releasing hormone (GnRH) agonist [D-Ser(But)6] GnRH(1-9)-ethylamide (buserelin) and of human chorionic gonadotrophin (hCG), human FSH (hFSH), human prolactin (hPRL) and human low-density lipoprotein (hLDL) to tissue homogenates was measured under optimal conditions. Bound LH/hCG was estimated by elution with acid-citrate buffer, followed by radioimmunoassay of released hormone. Binding of GnRH agonist, though variable, was highest in mid-luteal corpus luteum and high binding was also present in three out of four corpora lutea of pregnancy. Binding of LH/hCG increased significantly with luteinization, reaching maximal levels in the mid-luteal phase before falling significantly. Occupancy of LH receptors by bound LH was relatively constant throughout the luteal phase (10.7-35.3%), but occupancy increased to greater than 90% in corpora lutea from early pregnancy. Binding of hFSH was variable, with only five out of 50 corpora lutea having binding greater than 10 pg/micrograms DNA. Similarly, hPRL binding varied markedly with only six out of 44 having binding greater than 50 pg/micrograms DNA. Binding of LDL was highest in the early- to mid-luteal phases of the cycle. In corpora lutea from all stages of the menstrual cycle (excluding corpora albicantia), GnRH agonist binding was highly correlated with the levels of unoccupied and occupied LH receptors (P less than 0.001; n = 49 and n = 48 respectively) and with LDL receptors (P less than 0.002; n = 49). Binding of GnRH agonist was also correlated with PRL binding (P less than 0.05; n = 21) but not with FSH receptors (P greater than 0.4; n = 25). In addition, LDL binding was associated with PRL (P less than 0.005; n = 21) and FSH receptors (P less than 0.05; n = 25) and with endogenously bound LH (P less than 0.03; n = 48), but not with unoccupied LH receptors (P = 0.8; n = 49). Moreover, in corpora lutea from the mid-luteal phase, there was a strong association between GnRH agonist binding and LDL receptors (P less than 0.02; n = 23). The correlations between GnRH agonist binding and a number of important indices of luteal function suggest a physiological role for GnRH-like factors in the human corpus luteum.

Characteristics of prolactin binding sites in the brain of the ring dove ( Streptopelia risoria)

Radiolabeled ovine prolactin ([ 125I]oPRL) was found to bind specifically to crude membrane fractions prepared from ring dove brain homogenates. Specific binding changed with incubation time and temperature but did not fluctuate systematically with pH. Specific binding levels were highest in hypothalamus and forebrain and were lowest in cerebellum and brain stem. A linear increase in specific binding was observed in pooled midbrain and forebrain membrane fractions as membrane protein concentrations increased from 0.2 to 6.0 mg/ml. Scatchard analysis of binding saturation and competitive binding data revealed that dove midbrain/forebrain membranes bound oPRL with high affinity ( K d = 2.1–2.6 × 10 −10 M) but had low binding capacity ( B max = 2.9–4.8 fmol/mg protein). Unlabeled oPRL and human growth hormone competed most effectively with radiolabeled oPRL for occupation of midbrain/forebrain binding sites. Turkey PRL and human placental lactogen were less effective as competitors but were 8–15 times more effective than ovine or turkey growth hormone, porcine insulin, or ovine luteinizing hormone. Subcutaneous injection of unlabeled oPRL lowered specific binding in liver and midbrain/forebrain by 76 and 39%, respectively, over that observed after saline injection. Prior MgCl 2-induced desaturation of membrane binding sites greatly reduced or eliminated these treatment differences. These results suggest the existence of specific, saturable binding sites for PRL in dove brain which conceivably could mediate the reported effects of PRL on behavior and gonadal function in this species.

Prolactin binding sites in Xenopus laevis tissues: Comparison between normal and dehydrated animals

The binding of 125I-labeled ovine prolactin ( 125I-oPRL) to membranes from the kidney, epidermis, liver, and testis of Xenopus laevis adult speciments either kept in an aquatic environment or exposed for 2 weeks to dehydrating conditions was studied. Prolactin binding specificity was assayed through competition with several unlabeled hormones (oPRL, hGH, rGH, rLH, and porcine insulin). In the animal exposed to dehydrating conditions a statistically highly significant reduction in prolactin binding to the membranes from the kidney and epidermis was recorded. No significant variations were revealed by the membranes from the liver and testis. The reduction detected in the binding of 125I-oPRL is not related to the dissociation constant, but to the number of PRL binding sites. Since PRL ranks among the few peptide hormones whose rise in the bloodstream promotes an increase in the number of their own receptors, the reduction of its binding sites in Xenopus specimens exposed to dehydration might lend some support to our earlier hypothesis that transfer to a dehydrating environment may bring about, in this totally aquatic species, some decrease in the blood PRL levels.

Radioreceptor and autoradiographic analysis of FSH, hCG and prolactin binding sites in primary to antral hamster follicles during the periovulatory period

As measured by radioreceptor assays, binding sites for FSH and prolactin were present at 09:00 h on the day of pro-oestrus in Stage 1-10 follicles (primary to antral) with prolactin receptors 3-6 times higher than FSH sites in Stages 1-3 (3 layers of granulosa cells). Specific binding sites for hCG were present in Stage 1 and 2 follicles (2 layers of granulosa cells) but thereafter their distribution was erratic and they were not consistently detectable until Stage 5, when thecal cells first appeared. Using topical autoradiography, specific binding for FSH was evident in Stage 1-4 follicles (4 layers granulosa cells) whereas specific hCG-binding was not. After the preovulatory gonadotrophin surges, by 21:00 h on pro-oestrus, FSH receptors declined in Stages 5-10, prolactin receptors fell in Stages 8 and 10 (small and large antral follicles) and hCG receptors were reduced in Stages 7 (start of antral cavity) to 10. On the morning of oestrus, for follicles from Stage 4 onwards, receptor numbers usually returned to levels found at 09:00 h on pro-oestrus. At oestrus, the few remaining Stage 10 follicles were all atretic and contained significantly reduced FSH and prolactin receptors but numbers of hCG binding sites comparable to those at 09:00 h of pro-oestrus. These results provide evidence of gonadotrophin receptors in small primary and secondary follicles which is consistent with increased DNA synthesis in small hamster follicles on the afternoon of pro-oestrus and on the morning and afternoon of oestrus. Periovulatory changes in gonadotrophin concentrations may therefore affect early stages of folliculogenesis.

Specific binding sites for prolactin and growth hormone in the adult rabbit lung

Specific prolactin (PRL) and growth hormone (GH) binding sites were identified and characterized in lung membranes from male and female adult rabbits. The binding of iodinated human GH ([ 125I]iodo-hGH) and iodinated ovine PRL ([ 125I]iodo-oPRL) was time, temperature and protein dependent and was found to conform to the requirements defining a physiological receptor, in terms of hormonal and immunological specificities as well as kinetic properties. [ 125I]Iodo-hGH was displaced from lung membranes by hGH, oPRL, ovine GH and rat GH, while [ 125I]iodo-oPRL was effectively displaced only by oPRL and hGH. Scatchard plots of the competition curves of [ 125I]iodo-hGH and [ 125I]iodo-oPRL were both linear, suggesting, in each case, a single class of binding sites with affinity constants ( K a) of 1.74 ± 0.64 × 10 9 M −1 and 0.78 ± 0.28 × 10 9 M −1 and binding capacities of 6.43 ± 0.53 and 4.16 ± 0.69 fmol/mg protein, respectively. Anti-PRL-receptor antiserum significantly inhibited the binding of the [ 125I]iodo-oPRL to rabbit lung membranes, while it was less potent in preventing the binding of [ 125I]iodo-hGH, which has both lactogenic and somatogenic activity. Removal of endogenous ligand by treating lung membranes with 4 M MgCl 2 increased specific binding of hGH about 2.5-fold, exposing additional specific binding sites without significantly changing the binding affinity. The level of binding of hGH and oPRL to rabbit lung did not show a pronounced sex differentiation. In summary, PRL and GH binding sites have been demonstrated for the first time in adult rabbit lung membranes, and they support the possibility of a physiological role for PRL and GH in the lung.

Ontogeny of prolactin receptors in rat decidual tissue: binding by a locally produced prolactin-like hormone.

The objectives of this investigation were to determine whether decidual tissue possesses specific binding sites for prolactin, and to examine whether the locally produced prolactin-like hormone binds to these receptors. Characterization of the binding of prolactin to decidual tissue from rats at day 9 of pseudopregnancy revealed specific, high-affinity sites. Binding approached saturation with increasing concentrations of either the ligand or the protein. Cytosolic extracts of day-9 decidual tissue, containing various amounts of decidual luteotrophin which possesses several of the physiological and biochemical characteristics of prolactin, displaced the binding of 125I-labelled prolactin to decidual membranes in a linear fashion. Prolactin-binding sites were detectable 72 h after induction of decidualization and 48 h after the appearance of decidual luteotrophin in the decidua. Prolactin receptor concentrations increased significantly between days 8 and 9, reached a plateau between days 9 and 12 and declined abruptly on days 14 and 15, accompanied by a similar decline in decidual luteotrophin concentration in the tissue. Thus rat decidual tissue possesses specific receptors for prolactin to which decidual luteotrophin locally produced can bind, thereby suggesting an auto/paracrine role for this substance.