Induction of prolactin receptors in the liver is more closely related to the growth-promoting than to the lactogenic potency of peptides

The sex differentiated binding 125I-human prolactin (PRL) to rat liver membranes was studied and the present results extend our previous studies on induction of hepatic PRL receptors by growth hormone (GH). In prepubertal female rats, PRL receptor levels are low compared with those in mature female rat livers. Infusion of hGH during one week to 17-day-old female rats resulted in a receptor level typical of adult female rats. The time course of receptor disappearance in male rats treated with hGH was also studied. When the receptor-inducing hormone was removed, receptor levels in hGH-treated male rats returned to the normal level characteristic of male rats after approximately 96 h. The specificity of various GH-like and PRL-like hormones in PRL receptor induction was studied in hypophysectomized rats. The PRL-like hormones were identified by measuring their potency to displace 125I-hPRL from a receptor preparation obtained from female rat livers, and the GH-like hormones were identified by their potency to increase body weight in hypophysectomized rats. Using similar doses of hormones it was found that in vivo administration of growth-promoting peptides (rGH, hGH, bGH) induced PRL receptors, whereas lactogenic hormones (rPRL, hPL) had a very small or no effect on PRL receptor induction. This suggests that binding to a type of GH receptor is the first step in PRL receptor induction.     

Heterologous radioimmunoassay for deer prolactin

Antibodies raised in guinea pigs immunized with ovine prolactin (PRL) were used in the development of a heterologous radioimmunoassay to measure PRL levels in sera and pituitary extracts in the white-tailed deer (Odocoileus virginianus borealis). The RIA employs an antiserum with an association constant of 1.8 × 10¹¹M^?1 for ¹²⁵IoPRL, which shows minimal (<0.2%) cross-reactivity with all other hypophyseal hormones tested (human PRL, GH, and CG; rat PRL and GH; bovine GH and TSH; ovine and equine LH; porcine ACTH and FSH), and has an ID₅₀ of 0.34 ± 0.02 ng against oPRL standards. Deer sera and hypophyseal extracts showed parallelism with standards. In adult males and females of a captive, but free-breeding population, baseline and thyrotropin releasing hormone (TRH)-stimulated (200 μg/deer, iv bolus) serum PRL values were measured. Baseline values for both sexes were similar and showed a seasonal variation with a summer peak of ～ 150 ng/ml in June and winter levels of ～ 1 ng/ml. Serum PRL concentrations were elevated by TRH injection in both sexes. Peak postinjection concentrations were attained within 20 min during April through September (N = 49), or within 100 min in October through March (N = 39). In contrast, LHRH (200 μg/deer, iv bolus) produced no significant change in PRL levels, while 2-bromo-α-ergocryptine (CB-154) reduced both baseline serum concentrations and TRH-induced elevations. Pituitary PRL levels in males and nonlactating females were similar and paralleled the serum PRL profile in the months tested, rising from about 1 μ/mg wet weight in winter to about 6μ/mg in summer.     

A radioreceptor assay for purified teleost growth hormone.

Highly purified ¹²⁵I-labelled tilapia (Sarotherodon mossambicus) growth hormone (GH) binds to membrane fractions prepared from tilapia liver. Specific (displaceable) binding occurred with the 600, 15,000, and 90,000g fractions with the greatest binding observed with the 90,000g microsomal membrane fraction. Binding was dependent on time and membrane concentration. Specificity studies showed that up to 60% of the ¹²⁵I-labelled tilapia GH bound to the liver microsomal membrane fraction could be displaced by 500 ng of unlabelled hormone. Scatchard analysis of the binding of ¹²⁵I-labelled tilapia GH revealed a single class of binding site with a binding capacity of 125 ± 7.7 fmol/mg protein and a binding affinity of 1.5 × 10¹⁰ ± 0.4 × 10¹⁰ (SEM) l/mol. GH preparations from several vertebrate classes and tilapia prolactin and ovine prolactin at high concentrations displayed competition for the tilapia GH-binding site.¹²⁵I-Labelled tilapia GH demonstrated specific binding to liver microsomal membrane fractions of the teleosts Gillichthys mirabilis, Salmo gairdneri, and Oncorhynchus tschawytscha but not to those of an amphibian Necturus maculosus or a bird Coturnix japonica. Slight, but significant, specific binding was observed with the microsomal membrane fraction of tilapia kidney and gill and of rat liver. These data suggest that this tilapia GH radioreceptor assay may have an application in the detection of teleost GH-like activity.     

GH kinase activity in bovine anterior pituitary subcellular fractions

Growth hormone (GH) and prolactin (PRL) share significant structural homology. We have previously characterized the phosphorylation of bovine PRL and wish to determine whether a similar kinase activity phosphorylates bovine GH. Phosphorylation of bovine GH was performed using [α-³²P]ATP labeling of subcellular fractions. Bovine GH phosphorylation was dependent on Zn²⁺ or Cu²⁺ with apparent Km's of 0.9 and 1.0 mM, respectively, and a pH maxima of 7.0. The apparent Km's of bovine GH kinase activity for exogenous bovine GH and ATP were 30 μM and 376 μM, respectively. Exogenous bovine PRL served as a competitive substrate, increasing the apparent Km for bovine GH by threefold compared to the Km determined without exogenous bovine PRL. We conclude: 1) in vitro phosphorylation of bovine GH occurs under conditions that are consistent with those found in anterior pituitary cells, and 2) a similar kinase activity phosphorylates both bovine PRL and GH.     

Somatotropic actions of the homologous growth hormone and prolactins in the euryhaline teleost, the tilapia, Oreochromis mossambicus

It is increasingly clear that growth hormone (GH) has growth-promoting effects in fishes, which are mediated in part by the insulin-like growth factor (IGF)-I. Growth-promoting actions of prolactin (PRL) have been reported in higher vertebrates, but are less well established in teleosts. We examined the effects of injecting homologous GH or the two homologous tilapia PRLs (tPRL₁₇₇ and tPRL₁₈₈) on the in vitro incorporation of [³⁵S]sulfate (extracellular matrix synthesis) and [³H]thymidine (DNA synthesis) by ceratobranchial cartilage explants and on IGF-I mRNA levels in tilapia liver. Tilapia GH (tGH) and tPRL₁₇₇ stimulated sulfate uptake at the highest doses examined. Thymidine incorporation was stimulated by tPRL₁₇₇. tPRL₁₈₈ was without these effects. Consistent with its somatotropic actions, tGH elevated IGF-I mRNA levels in the liver. tPRL₁₇₇ also elevated liver IGF-I levels. Consistent with the previously described osmoregulatory actions of GH and PRL in teleosts, we observed that tGH elevated and tPRL₁₇₇ and tPRL₁₈₈ lowered levels of gill Na⁺,K⁺-ATPase activity. High-affinity, low-capacity binding sites for tGH in the tilapia liver were identified. tPRL₁₇₇ binds with lower affinity than tGH to these sites but can displace ¹²⁵I-labeled tGH from its receptor. The ability of tPRL₁₇₇ to displace tGH was similar to that of ovine GH. tPRL₁₈₈ did not displace ¹²⁵I-labeled tGH binding. Collectively, this work suggests that tPRL₁₇₇ may possess somatotropic actions similar to tGH, but only in freshwater tilapia where tPRL₁₇₇ levels are sufficiently high for it to act as a competitive ligand for GH receptors.     

Isolation and properties of teleost prolactin

Highly purified prolactin (PRL) has been isolated from the pituitary tissue of a euryhaline teleost, Tilapia mossambica. It has a molecular weight of 19,400 daltons, with a single NH₂-terminal residue, valine, and a single COOH-terminal residue, half-cystine. Amino acid composition data revealed the presence of one tryptophan and four half-cystine residues, which is characteristic of all known vertebrate growth hormones (GH) but not of mammalian PRLs. The circular dichroism spectrum of Tilapia PRL was similar to that of porcine PRL and showed an α-helix content of 65%. Tilapia PRL was considerably more potent than ovine PRL in two teleost PRL bioassays: the sodium-retaining assay in Tilapia and the reduction of water permeability in the urinary bladder of Gillichthys mirabilis, but it did not stimulate mammary tissue or the pigeon crop sac. Tilapia PRL had equivocal but suggestive activity in the rat tibia assay, and showed cross reaction in two GH (rat and snapping turtle) radioimmunoassays. A specific Tilapia PRL antiserum was prepared in a rabbit which gave a precipitin reaction against Tilapia PRL in agar diffusion but showed no cross reaction with purified mammalian PRLs or pituitary extracts from other teleosts. The data show that Tilapia PRL has features common to both mammalian PRLs and GHs as well as to Tilapia GH, lending support to the hypothesis that PRL and GH originated from a common ancestral molecule.     

Prolactin-binding sites in tilapia (Sarotherodon mossambicus) kidney.

Highly purified ¹²⁵I-labeled tilapia (Sarotherodon mossambicus) prolactin (PRL) binds to microsomal membrane fractions prepared from tilapia kidney but not liver or gill. Specificity studies revealed that up to 35% of the labeled tilapia PRL bound to the kidney membrane preparation could be displaced by 100 ng of unlabeled hormone. Ovine PRL displaced ¹²⁵I-labeled tilapia PRL from the kidney PRL-binding site. However, ¹²⁵I-labeled ovine PRL demonstrated negligible specific binding to microsomal membrane fractions of tilapia kidney, liver, gill, or muscle. Tilapia PRL did not inhibit the binding of ¹²⁵I-labeled ovine PRL to lactating rat liver membranes. The specific binding of ¹²⁵I-labeled tilapia PRL to tilapia kidney membranes was greater with freshwater-acclimated than with seawater-acclimated fish suggesting that the tilapia kidney PRL-binding site is subject to physiological regulation.     

Androgen inhibition of basal and estrogen-stimulated prolactin binding in rat liver

Estradiol (E₂ 0.25 μg) administered twice a day for 7 days to rats ovariectomized 2 weeks previously caused a 4.5-fold increase in [¹²⁵I]ovine prolactin (PRL) binding to a rat liver crude plasma membrane fraction. Testosterone (T, 500 μug) injected in combination with E₂ caused a 75% reduction in PRL binding. In male rats (both intact and castrated) injected once daily with E₂-benzoate (EB, 10 μg/100 g body wt), T-propionate (TP, 100 μg/100 g body wt.) caused a 62–73% decrease in E₂-induced binding without affecting the high levels of plasma PRL induced by EB. DHT led to a significant inhibition of PRL binding at the relatively low doses of 10 and 2.5 μg/100 g body wt. in control and E₂-treated ovariectomized rats, respectively. T was slightly less effective than DHT in reducing PRL binding. DHT was also found to be effective in reducing PRL binding to rat liver in rats hypophysectomized bearing a pituitary homotransplant, the level of binding decreasing from 10.7 ± 0.8% to 7.4 ± 0.7% (P < 0.01) in animals injected twice a day for 7 days (100 μug/100 g body wt.). A single injection of DHT caused a 24% reduction (P < 0.01) in PRL binding 12 h after its injection in E₂-treated animals, the stimulatory effect of the estrogen being completely inhibited within 3–5 days of DHT treatment. These data indicate that androgens can effectively reduce prolactin binding to rat liver independently of their effect on plasma PRL.     

Prolactin inhibits the induction of its own renal receptors in Rana catesbeiana tadpoles

The effects of ovine prolactin (oPRL) and thyroxine (T₄) on the binding of [¹²⁵I]oPRL to kidney microsomal membrane fractions of Rana catesbeiana tadpoles were studied. T₄ treatment significantly increased receptor binding. Injections of oPRL reduced binding to levels lower than those of controls and antagonized the T₄-induced increase in PRL binding. In vitro and in vivo receptor desaturation methods were employed to minimize the possibility that either injected or endogenous PRL remained bound to receptor sites through the procedures of membrane isolation and assay. These results indicate that the renal PRL receptor is inducible by T₄ and that this induction is inhibited by PRL. These findings illustrate a novel and somewhat unusual form of PRL-T₄ antagonism, but emphasize the possibility of both synergistic and antagonistic interactions between the two hormones.     

Inhibition of binding of ovine placental lactogen to growth hormone and prolactin receptors by monoclonal antibodies

From a single cell fusion, five stable hybridomas secreting antiovine placental lactogen (oPL) antibodies were obtained. Three of these secrete immunoglobulin (Ig)G subclass, and the other two secrete IgM class antibodies. Ascites fluids were raised in mice for each clone and were used as the antibody component for the development of solid phase RIA. Three solid-phase RIAs were successfully established using individual IgG subclass monoclonal antibodies, but the IgM class antibodies were ineffective. In all three individual solid-phase RIAs, the binding of [125I]iodo-oPL to the immobilized antibody was inhibited by unlabeled oPL, but not by ovine pituitary PRL (oPRL), ovine GH (oGH), or ovine pituitary extract. Two of the IgG subclass antibodies were able to inhibit the binding of [125I] iodo-oPL to PRL receptors(s) and to GH receptor(s) in rabbit mammary gland and liver, respectively. One of these two IgG subclass antibodies was more effective at inhibiting the binding of oPL to PRL receptor(s) in rabbit mammary gland, whereas the other one is more effective in inhibiting the binding of oPL to GH receptor(s) in rabbit liver. These antibodies, however, could only weakly inhibit the binding of [125I]iodo-oPRL to rabbit mammary gland and were ineffective in inhibiting the binding of [125I]iodo-oGH to rabbit liver. The addition of monoclonal antibodies in both radioreceptor assay (RRA) for PRL (RRA-PRL) and for GH (RRA-GH) did not affect the parallelism of the displacement curve of oPL standard. Our results suggest that oPL might contain two distinct binding sequence(s): one responsible for the binding of oPL to PRL receptor(s) and the other responsible for the binding of oPL to GH receptor(s). These two binding sequences might overlap or be located adjacent to one another. The interaction of monoclonal antibodies with these binding sequences of oPL may block the binding of oPL with PRL and GH receptor(s). Alternatively, our studies suggest that the monoclonal antibodies do not bind to hormone receptor(s)-binding sequence(s) in oPL, but the interaction between oPL and monoclonal antibody might alter the conformational structure of the oPL which will consequently lead to a lower binding of oPL to PRL and GH receptor(s).     

Rat growth hormone (GH) but not prolactin (PRL) induces both GH and PRL receptors in female rat liver

This study was designed to elucidate which hormone is responsible for the induction of GH and PRL receptors in rat liver. Intact female rats were implanted with osmotic minipumps delivering rat GH (rGH) or ovine GH (oGH) or PRL at various rates from 75 to 800 micrograms/day for 7 days, and binding of radioiodinated bovine GH or ovine PRL (oPRL) tracer was measured on liver microsomal membranes. MgCl2 treatment was used to remove bound hormones from receptors before tracer binding. Infusion of rGH resulted in a significant increase (P less than 0.001) in both GH and PRL binding, the effect being maximal (2.5- to 3-fold for both ligands) at rGH infusion rates from 150 to 400 micrograms/day. Serum rGH levels were elevated 3- to 5-fold in these animals, but somatomedin-C concentrations were not higher than in controls. MgCl2 treatment showed that GH, but not PRL, binding sites in rGH-treated animals were significantly occupied by administered hormone. Analysis of competitive binding curves indicated that receptors for both GH and PRL increased in concentration without changes in binding affinity. In contrast to the rGH effect, oGH infusion from 75 to 400 micrograms/day failed in two experiments to consistently alter either bovine GH or oPRL binding sites. This was not explained by the potency of the preparation at the somatogenic receptor; oGH was in fact more potent than rGH. The effects of rat PRL and oPRL infusion on receptor levels were also assessed. In contrast to previous reports, neither preparation caused induction of either PRL or GH binding sites. oPRL decreased PRL binding by 30-40% when infused between 200 and 400 micrograms/day, whereas rat PRL had a less consistent effect. MgCl2 stripping of membranes suggested that administered PRL preparations did not significantly occupy PRL receptors. GH receptors were unaffected in any PRL-treated group. It is concluded that in intact female rats, rGH regulates the concentration of both GH and PRL receptors. The slight down-regulation of PRL receptors resulting from PRL infusion casts further doubt on the concept that PRL induces its own hepatic receptors.     

Immunohistochemistry and Tinctorial Affinity of Adenohypophysial Cells of the Rat SnakePtyas mucosus(Colubridae)

Immunocharacteristics of the adenohypophysial cells of the rat snakePtyas mucosus(Colubridae) were studied with an unlabeled antibody enzyme technique (PAP method) using rabbit antisera against mammalian/synthetic hypophysial hormones. Adenohypophysial cells were identified on the basis of their specific immunoreactivity with various heterologous antisera. As in other reptiles, there was regional localization of pars distalis (PD) cell types in this snake. The gonadotropic (GTH) cells were identified by their specific immunoreactivity with anti-porcine (p)LHβ serum and were distributed uniformly throughout the PD. The thyrotropic (TSH) cells, recognized by their specific immunoreactivity with anti-human (h)TSHβ serum, were found in the medial PD. The prolactin (PRL) and growth hormone (GH) cells were revealed by their specific immunoreactivity with anti-ovine (o)PRL and anti-hGH sera, respectively; the former were confined to the anterior two-thirds of the PD, and the latter were restricted to the posterior third of the PD. The corticotropic (ACTH) cells, identified by their specific immunoreactivity with the anti-ACTH^1–24serum, were localized in the anterior two-thirds of the PD. Though both ACTH and PRL cells were confined to the same area of the PD, they could be distinguished by their distinctive morphology and distribution pattern. Cells of the pars intermedia were revealed by their immunoreactivity to anti-αMSH (melanophore-stimulating hormone) and anti-ACTH^1–24sera. Among each adenohypophysial cell type, there was variation in the intensity of immunoreactivity and morphological features, which may be due to their heterogeneity, reflecting various stages of cellular activity. Unlike most other snake species, the occurrence of a rudimentary pars tuberalis inP. mucosuscontaining a few immunoreactive GTH and TSH cells appeared to be a novel finding. The occurrence of the PRL-like and TSH-like immunoreactivity seen in certain neuronal perikarya and fibers of the hypothalamus and median eminence conforms with earlier observations in other tetrapods. To resolve certain discrepancies in the literature, the tinctorial affinities of immunohistochemically identified adenohypophysial cell types ofP. mucosuswere studied using various conventional staining methods and were compared with those of other reptilian species studied earlier, including snakes of the family Colubridae.     

Effect of teleost pituitary growth hormone on growth of Tilapia mossambica and on growth and seawater adaptation of sockeye salmon (Oncorhynchus nerka)

Purified Tilapia growth hormone (GH), which has already been shown to have slight but significant activity in the rat tibia assay, was found to promote growth in two intact teleost species, Tilapia mossambica and Oncorhynchus nerka (sockeye salmon). Tilapia GH and bovine GH were approximately equipotent in stimulating an increase in both length and weight of these fish. In salmon, ovine prolactin (PRL) stimulated growth with a much lower potency than Tilapia GH, while Tilapia PRL was inactive. Furthermore, bovine GH, Tilapia GH, and ovine PRL facilitated adaptation of the salmon to sea water, while Tilapia PRL had no effect. These data, along with published results of teleost bioassays specific for Tilapia PRL, demonstrate that teleosts can readily distinguish between Tilapia GH and Tilapia PRL. This separation contrasts with previously reported similarities between the two hormones with respect to amino acid composition, immunochemistry, and activity in the rat tibia assay.     

Gonadal steroids as modulators of the function of the pineal gland

Sex steroids affect pineal function. Castration or the administration of estradiol, testosterone, or progesterone bring about changes in pineal melatonin synthesis, as well as in other aspects of pineal activity in rats. This paper deals with some of the mechanisms underlying steroid action on pineal metabolism. Estradiol is avidly taken up in vivo and in vitro by the pineal gland. A cytosol binding protein having a K_d of, (0.27 ± 0.08) × 10^?9M for estradiol and requiring SH groups for full expression of binding activity was detected in pineal homogenates. Estradiol uptake by the pineal and the uterus of mature rats attained their minima on the day of proestrus. A priming dose of estradiol increased high affinity binding sites for estradiol in pineal and uterine cytosol by about 150%. Testosterone is also readily taken up and retained in vivo and in vitro by the pineal of orchiectomized rats. Saturation kinetics analysis of androgen binding to bovine pineal cytosol indicated a K_d of 1.57 × 10^?9M for testosterone and 0.36 × 10^?9M for 5α-dihydrotestosterone. The cytoplasmic binder was shown to be a protein and to require SH groups for binding activity. Testosterone was metabolized by the pinealocytes into 5α-dihydrotestosterone, 5α-androstanediol, androstenedione and 5α-androstanedione; 5α-dihydrotestosterone was the major metabolite in the nuclei of pineal cells. Superior cervical ganglionectomy or exposure of rats to light increased androstenedione synthesis, whereas ganglionectomy or exposure to darkness decrease 5α-androstanedione synthesis. Progesterone is taken up by the pinealocytes in vitro up to a tissue concentration 18-fold that of the media. [³H]Progesterone is converted in the pinealocytes into 5α-pregnanedione and 3α-hydroxy-5α-pregnan-20-one. These data suggest that the early steps of sex steroid action on pineal cells resemble those of steroid target tissues.     

Avian growth hormone receptor assay: use of chicken and turkey liver membranes

A sensitive avian growth hormone (GH) radioreceptor assay (RRA) was developed using recombinant chicken growth hormone (rcGH) and a membrane receptor preparation of chicken or turkey livers. The specific binding of 125I-labeled rcGH to a 47,800 X g pellet was 33-36% in a 16-20 h incubation period at 4 degrees C. Binding was time, temperature and pH dependent. Scatchard analysis indicated a single class of high affinity GH binding sites in chicken and turkey livers, with binding affinities of 1.03 X 10(10) liter/M and 1.11 X 10(10) liter/M, respectively, and corresponding binding capacities of 10.7 fmol and 21 fmol per mg protein. The sensitivity of the assay was 0.41 ng of rcGH. Intra- and inter-assay coefficients of variation were 5.3% and 9.7%, respectively. Bovine GH, ovine GH, and porcine GH competed effectively for the GH binding sites in chicken and turkey livers. Turkey prolactin (PRL) and porcine PRL showed little cross-reaction (less than 0.07%), while cross-reaction of ovine and bovine PRL was greater (less than 10%). Standard rcGH (0.5-30 ng) was added to sera from hypophysectomized chickens and turkeys (hypox sera) and to tissue culture medium and was measured quantitatively. Untreated medium (10-100 microliters) and hypox sera (5-40 microliters) did not inhibit rcGH binding. These studies report the existence of specific binding sites for avian GH in chicken and turkey liver and validate a sensitive RRA for measurement of bioactive GH in sera and tissue culture medium.     

Identification and Characterization of Growth Hormone Receptors in Snakehead Fish (Ophiocephalus argusCantor) Liver

The specific binding of¹²⁵I-labeled fish growth hormone (GH) to hepatic membranes prepared from several freshwater fish was assessed. A high level of growth hormone receptor (GHR) was detected on the hepatic membranes of the snakehead fish (Ophiocephalus argusCantor). Scatchard analysis of the binding data showed a single class of high affinity binding site with a binding affinity (K_a) of 1.45 ± 0.23 × 10⁹M⁻¹and a binding capacity (B_max) of 198 ± 57 fmol/mg protein. The binding was specific for fish GH and was saturable. In addition, the specific binding was temperature- and time-dependent, reaching a steady state after 16 hr of incubation at 25°. The molecular weight of GHR as measured by Sephadex G-200 column chromatography and Western blot analysis using a monoclonal antibody (Mab263) against GHR was found to be 200–400 and 90–93 kDa, respectively. Two bands at 65 and 89 kDa were identified in ligand crosslinking studies of membrane receptors. A sensitive teleost GH radioreceptor assay (RRA) was developed, using recombinant fish GH and a membrane preparation from snakehead fish liver, capable of measuring bioactive GH in fish sera or other samples.     

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 (Ka) of 1.74 +/- 0.64 X 10(9) M-1 and 0.78 +/- 0.28 X 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 MgCl2 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.     

Prolactin directly stimulates the liver in vivo to secrete a factor (synlactin) which acts synergistically with the hormone

Recent studies in our laboratory indicated that the liver of rats, mice, and pigeons secretes a PRL-synergizing activity (synlactin) in vitro. Accordingly, we investigated whether PRL and/or GH could stimulate the secretion of synlactin by the pigeon liver or kidney. Young birds received twice daily injections of PBS, ovine (o) PRL, or oGH for 5 days. On the sixth day, their livers and kidneys were removed, and slices of these organs were incubated in medium 199 for 4 h. The medium samples were filtered and diluted, then tested for PRL-like activity and synlactin activity in the local pigeon crop-sac bioassay. The latter test involved adding a dose of 1.0 microgram oPRL to the medium samples. None of the liver or kidney medium samples had PRL-like activity when tested alone. Only the liver incubation medium from the PRL-injected pigeons contained significant amounts of synlactin activity. Our next experiment was designed to determine whether PRL stimulation of hepatic secretion of synlactin involved a direct action of the hormone on the liver in vivo. A catheter attached to a coil of tubing and an osmotic minipump was inserted into an intestinal vein of pigeons, and the pump and coil were left in the abdomen. By this means, solvent, or GH or PRL in solvent, was pulse infused (four pulses of 2 h each per day) into the intestinal venous drainage. Thus, the hormones were delivered directly to the liver via the hepatic portal vein. During the last 3 of the 7 days of infusion, the pigeons received two daily injections of PBS or oPRL over the contralateral lobes of the crop-sac. Intrahepatic infusion of PRL, but not GH, caused a marked augmentation of the response of the crop to local injections of PRL. Pulse infusion of the same dose of oPRL into the external jugular vein of pigeons did not have this effect; hence, it appears to be mediated by the liver. These results indicate that one of the actions of PRL on the liver is to stimulate the secretion of a factor (synlactin) which acts synergistically with the hormone to promote growth of its target organs.     

Somatotropic effects of prolactin and growth hormone in juvenile snapping turtles (Chelydra serpentina)

Juvenile snapping turtles (Chelydra serpentina) were treated with ovine prolactin (PRL) or ovine growth hormone (GH). Organ growth responses were greatest in the GH-treated animals, except for the spleen, which was greatest in the PRL-injected group. A significant increase in body weight occurred in the group injected daily with 10 μg PRL, but less than that observed in the group treated with 10 μg of GH. Increases in carapace length of the turtles injected with PRL or 10 μg of GH were about equal, but less than those treated with 50 μg of GH.     

Distribution of immunoreactive adenohypophysial cell types in the pituitaries of the Atlantic and the Pacific hagfish, Myxine glutinosa and Eptatretus burgeri

The hagfish is considered the most primitive vertebrate known, living or extinct. It remains an enigma whether adenohypophysial hormones similar to those of more advanced vertebrates are present in the hagfish pituitary gland or not. The present study aimed to detect immunoreactive adenohypophysial hormones in the hagfish pituitary gland, using antisera to tetrapod and fish adenohypophysial hormones as immunohistochemical probes. For this purpose, two species of hagfish, the Atlantic hagfish, Myxine glutinosa, and the Pacific hagfish, Eptatretus burgeri, were used. In both species, three different types of immunoreactive cells were detected in the adenohypophysis. (1) The first type of cells was gonadotropin (GTH)-like cells which were stained by antisera to LH-related GTHs, such as ovine LHbeta, human LHbeta, bullfrog LH, salmon LHbeta and sturgeon LHbeta in both species of hagfish. (2) The second type of cells that were detected was growth hormone (GH)/prolactin (PRL)-like cells. In M. glutinosa the cells were stained by antisera to salmon GH, salmon PRL, sturgeon GH, sturgeon PRL, blue shark GH, and lamprey GH. In E. burgeri the cells were only stained by anti-human GH and anti-sturgeon PRL. (3) The last type of cells was adrenocorticotropin (ACTH)-like cells. These cells were stained by antisera to lamprey ACTH and human beta-endorphin. In both species of hagfish, GTH-like cells were relatively abundant, and were distributed throughout the adenohypophysis, whereas GH/PRL-like and ACTH-like cells were few in number in the adenohypophysis. Based on these findings, we suggest that hagfish may have retained ancestral characteristics of key anterior pituitary hormones.