Enhancement of insulin secretion by human chorionic somatomammotropin and related hormones.

Hypophysectomized rats were treated for 6 days with 200 mug per day of either human chorionic somatomammotropin, human pituitary growth hormone, plasmin-modified human pituitary growth hormone, or ovine prolactin. All hormone preparations except ovine prolactin enhanced the ability of the pancreases of hypophysectomized rats to secrete insulin in the isolated pancreas perfusion system.     

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

Summary 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.     

Radioimmunoassay for ovine, caprine and bovine prolactin in plasma and tissue extracts

1. A radioimmunoassay for ovine prolactin is described based on the inhibition of the reaction between (131)I-labelled ovine prolactin and guinea-pig or rabbit antiserum to ovine prolactin. The extent of the reaction after a 4-day incubation period is determined by chromatoelectrophoresis or by adsorption of unchanged (131)I-labelled ovine prolactin on charcoal. The sensitivity is equal to 5.9ng. of prolactin/ml. of plasma with chromatoelectrophoresis, or 0.2ng. of prolactin/ml. of tissue extracts with the charcoal separation. 2. A complete cross-reaction demonstrated between ovine prolactin and caprine pituitary extracts allows the assay to be used to measure caprine prolactin. The partial cross-reactions between ovine prolactin and bovine prolactin and between ovine prolactin and bovine pituitary extract differ, and an alteration in the immunological activity of bovine prolactin during its isolation is suggested. Bovine prolactin in plasma may be measured against a bovine pituitary extract as standard. No cross-reactions were demonstrated with pituitary extracts from a number of other species. The extent of the contamination of ovine and bovine growth hormone preparations by their respective prolactins is shown. 3. Dilutions of ovine and caprine plasma inhibit the reaction between (131)I-labelled ovine prolactin and antiserum with the same characteristics as ovine prolactin. 4. The immunoreactive material in plasma fractionates on Sephadex G-200 and in sucrose density gradients as a single peak similar to that shown by freshly dissolved ovine prolactin. There is no evidence that ovine prolactin is bound to a plasma protein. 5. By suppressing prolactin secretion and assaying serial samples of plasma thereafter it is shown that the immunological activity of the surviving hormone becomes progressively altered with time. It is suggested that this alteration is usually not detected but introduces an element of uncertainty into the quantitative but not the qualitative value of the measurements obtained by reference to standard ovine prolactin.     

Glucocorticoid modulation of prolactin receptors on mammary cells of lactating mice

Prolactin receptors were monitored by measuring ¹²⁵I-labeled prolactin binding to collagenase-dissociated mammary epithelial cells of lactating BALB/c mice. Specific receptors for iodine-labeled prolactin with an apparent dissociation constant (K_d) of 0.99 · 10^?9 M were present on the dissociated mammary cells. The binding was inhibited by ovine prolactin, human growth hormone and human placental lactogen but not by follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, bovine growth hormone or insulin. Adrenal ablation of nursing mothers caused a reduction of the number of prolactin receptors and this effect was preventable by hydrocortisone therapy. Hydocortisone injections to mothers 3 days after adrenalectomy also induced a replenishment of the prolactin receptors on the mammary cells. Injections of progesterone failed to sustain the high level of mammary cell prolactin receptors in adrenalectomized animals. Stimultaneous injections of hydrocortisone and progesterone to animals 3 days after adrenalectomy caused a partial suppression of the stimulatory action of hydrocortisone alone. The results suggest that hydrocortisone can exert a modulatory influence on mammary cell prolactin receptors in non-hypophysectomized post-partum mice without altering the dissociation constant (K_d) of the receptors.     

Studies on prolactin 48: Isolation and properties of the hormone from horse pituitary glands

Isolation of prolactin from equine pituitary glands has been described. It has a potency of 42 IU/mg in the pigeon crop-sac test and consists of 199 amino acids. The hormone has only four half-cystine residues in contrast to other mammalian prolactins which have six residues. From NH₂-terminal sequence analysis and amino acid composition of cyanogen bromide fragments, the NH₂-terminal disulfide loop is missing in the equine prolactin molecule. Circular dichroism spectra indicate that the α-helical content of equine prolactin appears to be lower (50%) than that found in the ovine hormone (65%).     

Prolactin binding in rat mammary gland during pregnancy and lactation

Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the mammary gland during pregnancy and lactation. In the present study we describe the optimal conditions for the measurement of ¹²⁵I-labeled ovine prolactin binding to mammary gland slices of pregnant and lactating rats. Prolactin binding is saturable (K_{d approx. 2.36 · 10^?9 M)}, hormone specific and destroyed by proteases. The hormonal environments of pregnancy and lacation dramatically influence the availability and measurement of prolactin binding sites. Whereas binding consistently appears to be low in mammary glands removed from rats during pregnancy, binding levels rise 7–8-fold shortly after birth and remain high during the 22 days of lactation. However, the removal of the ovaries and gravid uteri at specific times during pregnancy results in prompt 3–6-fold increase in prolactin binding. Elevated levels in potential prolactin binding capacity appear in mammary tissue coincident with the reported rise in serum rat placental lactogen between the eight and eleventh days. We suggest that high levels of this lactogenic hormone promote the appearance of prolactin binding sites during pregnancy and mask the sites such that they are not available for measurement in vitro.     

Changes in pituitary hormone levels induced by met-enkephalin in man—The role of dopamine

The interaction of dopamine with the effects of the opiate agonist peptide D-Ala²-MePhe⁴-met-enkephalin-O-o1 (DAMME) on anterior pituitary hormone secretion was investigated in normal male subjects. DAMME produced clear elevations in prolactin, growth hormone and thyroid-stimulating hormone, while inhibiting the release of luteinising hormone and cortisol. There was no change in follicle stimulating hormone. The elevations in prolactin and TSH were enhanced by the dopamine antagonist, domperidone, and blocked by an infusion of dopamine. Neither dopamine nor domperidone modulated the changes in growth hormone, luteinising hormone or cortisol. The data are comptible with the association of the release of prolactin and TSH by opiate peptides with decreased hypothalamic dopaminergic activity; changes in the other anterior pituitary hormones seem to involve different mechanisms.     

Inhibition of growth hormone synthesis by somatostatin in cultured pituitary of rainbow trout

Summary When the pituitary of rainbow trout (Oncorhynchus mykiss) was incubated in a serum-free medium, a high level of growth hormone release as well as an activation of growth hormone synthesis were observed, suggesting the existence of hypothalamic inhibitory factor(s) on growth hormone synthesis. Although an inhibitory effect of somatostatin on growth hormone release is well established in both mammals and teleosts, an effect on growth hormone synthesis has not been demonstrated. In this study, we examined the effect of somatostatin on growth hormone synthesis in organ-cultured trout pituitary using immunoprecipitation and Northern blot analysis. Somatostatin inhibited growth hormone release from the cultured pituitary within 10 min after addition without affecting prolactin release. Incubation of the pituitary with somatostatin also caused a significant reduction in newly-synthesized growth hormone in a dose-related manner, as assessed by incorporation of [³H]leucine into immunoprecipitable growth hormone. There were no changes in the level or molecular length of growth hormone mRNA after somatostatin treatment, as assessed by Northern slot blot and Northern gel blot analyses. Human growth hormone-releasing factor stimulated growth hormone release, although the spontaneous synthesis of growth hormone was not augmented. However, somatostatin-inhibited growth hormone synthesis was restored by growth hormone-releasing factor to the control level. The spontaneous increase in growth hormone synthesis observed in the organ-cultured trout pituitary may be caused, at least in part, by the removal of the inhibitory effect of hypothalamic somatostatin.Abbreviations GH growth hormone - GHRF GH-releasing factor - PRL prolactin - SDS sodium dodecyl sulphate - SRIF somatostatin (somatropin release-inhibiting factor)     

Immunocytochemical localisation of prolactin and growth hormone in the ovine pituitary

Summary Using the peroxidase-antiperoxidase immunocytochemical staining technique, prolactin and growth hormone cells have been identified and described in the ovine pituitary. The image analysing computer, Quantimet 720, was used to assess accurately the size range of the secretory granules in these cell types. The area size distributions of the prolactin and growth hormone granules are similar. An increased proportion of larger granules was observed in the prolactin cells post-partum. Serial sections stained alternately for prolactin or growth hormone confirmed that the cells contain either prolactin or growth hormone but not both.     

Partial purification of prolactin-like substance from snake (Ptyas mucosa) pituitaries

Pituitary extract of the common rat snake (Ptyas mucosa) was found to be capable of displacing the binding of 125I-labelled ovine prolactin to female rat liver membranes, suggesting the presence of prolactin-like substance in snake pituitary. The snake prolactin-like substance was unadsorbed on Concanavalin A-Sepharose, but adsorbed on DEAE-cellulose. The partially purified snake prolactin-like substance was also capable of displacing the binding of 125I-labelled ovine prolactin to snake kidney and large intestine membranes. Chromatographic fractions derived from snake pituitary and which possessed potent growth hormone receptor binding activity were devoid of prolactin receptor binding activity, suggesting the existence of distinct prolactin-like and growth hormone-like substances in snake pituitary.     

Effects of colchicine and 2-Br-alpha-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone by functional pituitary tumor cells in culture

The effects of colchicine and 2-Br-α-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone have been studied in a clonal strain of rat pituitary tumor cells (GH₃) in monolayer culture. These cultures produce both prolactin and growth hormone and release both proteins spontaneously into the medium without storing them in large amounts. Immunological methods were used to measure both intracellular and extracellular concentrations of the hormones. Colchicine (5 × 10^?6 M for 3 hours) caused a 2- to 3-fold increase in intracellular concentrations of prolactin and growth hormone but, under basal conditions, had little or no measurable effect on the amounts of hormone accumulated in the medium during the course of the standard three hour treatment period. This latter finding evidently is due to a lag in the onset of drug action. Colchicine had little or no effect on accumulation of extracellular prolactin during the first two hours of treatment whereas such accumulation was depressed by over 60% during the third hour of treatment. Previous studies have shown that treatment of GH₃ cells with thyrotropin releasing hormone (TRH) and hydrocortisone (HC) increases both intra and extracellular levels of prolactin and growth hormone, respectively. In cultures treated with TRH (5 × 10^?8 M), colchicine (5 × 10^?6 M for 3 hours) increased intracellular prolactin by about 70% and decreased extracellular hormone by 10%. In cultures treated with HC (3 × 1O^?6 M), colchicine increased intracellular growth hormone by more than 100% and decreased medium concentrations of the hormone by 15%. Colchicine did not significantly alter total hormone (intracellular + extracellular) accumulation, cellular uptake of ³H-amino acids, or total cell protein synthesis. The synthetic ergot alkaloid, CB 154, (3.3 × 10^?6 M for 3 hours) caused an 80% increase in intracellular, and a nearly 50% decrease in extracellular, prolactin without affecting the accumulation of growth hormone, the uptake of ³H-labeled amino acids, or overall protein synthesis in the cultures. Elevation of medium potassium concentration from a basal value of 5.3 mM to 3–5 × 10^?2 M (by addition of KCl) decreased intracellular levels of prolactin by 85% and growth hormone by 55%. These effects of high potassium were blocked by colchicine and by CB 154. We conclude that colchicine, after a lag period of two hours, acts to inhibit the release of prolactin and growth hormone from GH₃ cells. By the end of three hours of treatment, this inhibition is over 60% complete in the case of prolactin. The qualitatively different effects of colchicine and CB 154 on prolactin and growth hormone release suggest that these two secretory blocking agents probably act on GH₃ cells by different mechanisms.     

Effect of diethylstilbesterol and prolactin on the induction of follicle stimulating hormone receptors in immature and cycling rats

Induction of follicle stimulating hormone receptor in the granulosa cells of intact immature rat ovary by diethylstilbesterol, an estrogen, has been studied. A single injection of 4 mg of diethylstilbesterol produced 72 h later a 3-fold increase in follicle stimulating hormone receptor concentration as monitored by [¹²⁵I]-_oFSH binding to isolated cells. The newly induced receptors were kinetically indistinguishable from the preexisting ones, as determined by Lineweaver-Burk plot of the binding data. The induced receptors were functional as evidenced by increased ability of the granulosa cells to incorporate [³H]-leucine into cellular proteins. Neutralization of endogenous follicle stimulating hormone and luteinizing hormone by administering specific antisera had no effect on the ability of diethylstilbesterol to induce follicle stimulating hormone receptors, whereas blockade of endogenous prolactin secretion by ergobromocryptin administration significantly inhibited (∼ 30 %) the response to diethylstilbesterol; this inhibition could be completely relieved by ovine prolactin treatment. However, ovine prolactin at the dose tried did not by itself enhance follicle stimulating hormone receptor level. Administration of ergobromocryptin to adult cycling rats at noon of proestrus brought about as measured on diestrusII, (a) a reduction of both follicle stimulating hormone (∼ 30 %) and luteinizing hormone (∼ 45 %) receptor concentration in granulosa cells, (b) a drastic reduction in the ovarian tissue estradiol with no change in tissue progesterone and (c) reduction in the ability of isolated granulosa cells to convert testosterone to estradiol in response to follicle stimulating hormone. Ergobromocryptin treatment affected only prolactin and not follicle stimulating hormone or luteinizing hormone surges on the proestrus evening. Treatment of rats with ergobromocryptin at proestrus noon followed by an injection of ovine prolactin (1 mg) at 1700 h of the same day completely reversed the ergobromocryptin induced reduction in ovarian tissue estradiol as well as the aromatase activity of the granulosa cells on diestrus II, thus suggesting a role for proestrus prolactin surge in the follicular maturation process     

Comparative immunochemical studies with antisera to sheep prolactin and bovine growth hormone on anterior pituitaries of ox,sheep, rats and mice

Summary Rabbit antisera to sheep prolactin and bovine growth hormone were used in the indirect fluorescent antibody technique on cryostat sections of anterior pituitaries of sheep, ox, rats and mice. It is demonstrated that in sheep and ox prolactin and growth hormone are manufactured by different acidophilic pituitary cells. Though the antisera do not precipitate the analogous hormones of rats and mice in gel diffusion tests, evidence is given for the specificity of the cross-reaction of the antisera with the analogous murine hormones in situ as found with the fluorescent antibody technique.We are grateful to Dr. J. D. H. Homan of Organon, Oss (The Netherlands) for the supply of bovine growth hormone and for kindly giving us the information concerning this preparation.We should like to thank Dr. F. J. A. Prop for the prolactin assays, Dr. H. G. Kwa for providing the mouse pituitary tumours and Dr. L. M. Boot for the mouse pituitary isograft in the kidney, Mrs. J. J. Geiger-Koedijk for the conventional staining of pituitary sections, and Mr. J. van der Kamp for the photography.     

Episodic Evolution of Protein Hormones: Molecular Evolution of Pituitary Prolactin

Previous studies have shown that pituitary growth hormone displays an episodic pattern of evolution, with a slow underlying evolutionary rate and occasional sustained bursts of rapid change. The present study establishes that pituitary prolactin shows a similar pattern. During much of tetrapod evolution the sequence of prolactin has been strongly conserved, showing a slow basal rate of change (approx 0.27 × 10⁹ substitutions/amino acid site/year). This rate has increased substantially (∼12- to 38-fold) on at least four occasions during eutherian evolution, during the evolution of primates, artiodactyls, rodents, and elephants. That these increases are real and not a consequence of inadvertant comparison of paralogous genes is shown (for at least the first three groups) by the fact that they are confined to mature protein coding sequence and not apparent in sequences coding for signal peptides or when synonymous substitutions are examined. Sequences of teleost prolactins differ markedly from those of tetrapods and lungfish, but during the course of teleost evolution the rate of change of prolactin has been less variable than that of growth hormone. It is concluded that the evolutionary pattern seen for prolactin shows long periods of near-stasis interrupted by occasional bursts of rapid change, resembling the pattern seen for growth hormone in general but not in detail. The most likely basis for these bursts appears to be adaptive evolution though the biological changes involved are relatively small. Received: 31 August 1999 / Accepted: 9 February 2000     

Cyclo (His-Pro): a selective inhibitor of rat prolactin secretion in vitro

Cyclo (His-Pro) (10 ng/ml), inhibits KCl (59 mM) or thyrotropin-releasing hormone (10 ng/ml) stimulated, but not basal, release of prolactin from rat hemipituitaries $\text{in vitro}$. However, cyclo (His-Pro) has no effect on the basal or stimulated release of thyrotropin and growth hormone. Cyclo (His-Pro) does not inhibit the binding of thyrotropin-releasing hormone to pituitary membrane suggesting that cyclo (His-Pro) inhibition of prolactin release is not mediated via the pituitary TRH-receptor.     

Assay of lactogenic hormones using receptors isolated from rabbit liver.

Using 125-I-labelled ovine prolactin and receptors isolated from the livers of rabbits, a sensitive method has been developed suitable for the assay of ovine, bovine, porcine, human and rat prolactins. These hormones showed competitive displacement of 125-I-ovine prolactin which was in general agreement with their respective activities in the pigeon crop sac bioassay. Human and monkey growth hormones and human placental lactogen, which have marked prolactin-like actions on mammary tissue were also effective competitors. Non-primate growth hormones (ovine, bovine, equine and canine) which do not have prolactin-like activity gave little if any displacement as did human FSH, LH, TSH, ACTH and bovine insulin. Preparations of equine and canine prolactin of varying purity gave dose-response curves although their activity as competitors relative to ovine prolactin was poor and not related to their pigeon crop stimulating activity. This indicates species differences between prolactins in hormone-receptor interaction. Experiments with antiserum to human growth hormone have suggested an effective method of making the assay specific in species such as man in which prolactin is not the sole hormone with lactogenic activity.     

Water Soluble Receptors for Human Growth Hormone from Rabbit Liver

Abstract

Soluble receptors that bind human growth hormone have been prepared by incubation of liver membranes from pregnant female rabbits in 1 mM Tris buffer (pH 7.5 or 9.0) at 4°C. Up to 29% of the growth hormone binding sites could be solubilized within 48 hours. The kinetics of binding of human growth hormone to the soluble receptor, the hormonal specificity and the binding parameters calculated by Scatchard analysis (K_a 2.2 × 10⁹ M^-1, capacity 409 fmole/mg) were essentially unchanged compared with those for the parent membrane-associated (particulate) receptor. Gel filtration on Ultrogel AcA22 indicated that the major binding peak eluted at a molecular weight of 300,000 daltons. Specificity studies showed that the soluble binding sites had a moderately high affinity for ovine prolactin (K_a ～1 × 10⁸ M^-1), but negligible affinity for insulin. Although aqueous extraction gives a lower yield of binding sites for human growth hormone than detergent extraction, it nevertheless avoids some of the problems associated with use of detergents and should facilitate the subsequent purification of the receptor in a relatively unaltered state. It may also have applicability for solubilization of other hormone receptor systems.     

Heroin self-administration is under control of vasopressin

Heroin self-administration in rats is attenuated by daily intracerebroventricular treatment with desglycinamide⁹, arginine⁸, vasopressin (50 ng/animal). Removal of vasopressin by injection of antivasopressin serum into the cerebrospinal fluid, leads to facilitation of self-administering behavior. Treatment with oxytocin or human growth hormone antiserum fails to influence this behavior, while prolactin antiserum exhibits similar effects as observed with antivasopressin serum. These results indicate that pituitary hormones are physiologically involved in heroin self-administration.     

Human pituitary growth hormone: solubility in ammonium sulfate solutions

The solubility of human pituitary growth hormone in ammonium sulfate solutions has been investigated and compared under similar conditions with that of three related proteins: ovine pituitary growth hormone, bovine pituitary growth hormone, and human chorionic somatomammotropin.