Increase In Prolactin Binding Research Articles

Effect of Feeding Level and Nitrogen Source of the Diet on GH, IGF-T, and Prolactin Receptors in Mammary and Liver Tissue from Pre-pubertal Heifers

Pre-pubertal (86-220 kg BW) Finnish Ayrshire heifers were used to study the effect of feeding level and nitrogen source on GH, IGF-I, and prolactin (PRL) receptors in mammary and liver parenchymal tissue. The heifers were fed a hay-barley diet supplemented with either urea or rapeseed meal. The treatments were: (1) LU, low feeding level+urea; (2) LR, low feeding level+rapeseed meal; (3) HU, high feeding+urea; (4) HR, high feeding level+rapeseed meal. The average daily gains of the heifers on LU, LR, HU and HR diets were 692, 655, 805 and 890 g. At slaughter, mammary and liver parenchymal tissue was collected for preparation of crude membranes, and ligand-binding assays were performed. Specific binding of bGH to mammary membranes could not be detected. Linear Scatchard plots were obtained showing the presence of a single class of high-affinity binding sites for IGF-I in mammary tissue. Specific binding of IGF-I to mammary membranes was unaffected by feeding level and nitrogen source. Furthermore, Scatchard analysis showed unchanged binding capacities and binding affinities of the IGF-I receptors in mammary tissue from low and high fed heifers. Specific binding of prolactin to mammary membranes was increased by high feeding level (P<0.02) and by rapeseed meal (P<0.01). No interaction between feeding level and nitrogen source was identified. Hepatic binding of bGH, IGF-I and prolactin was unaffected by feeding level and nitrogen source. However, specific binding of prolactin tended to be lower (P<0.07) in liver tissue from heifers fed rapeseed meal as diet nitrogen source. The present study shows that the previously reported negative effect of high feeding level on pubertal mammary development (Mäntysaari et al., 1995), cannot be explained by changes in binding to GH and IGF-I receptors. The importance of the increase in prolactin binding to mammary membranes from heifers at high feeding level and with rapeseed meal needs further investigation.

Characterization of plasma and intracellular membrane prolactin receptor in lactating mouse mammary cells.

Mammary cells were prepared from lactating mice by collagenase digestion in order to examine the reaction of prolactin (PRL) with its cellular receptors. Cells were treated with 10% acetic acid following PRL binding. Between 5 and 12.5% acetic acid, the dissociation of PRL remained constant. The ratio of dissociated PRL was comparable to that of tissue slices or disrupted cells with this treatment. In subcellular membranes, about 81% of PRL bound to plasma membrane receptors was dissociated by acid treatment while PRL dissociated from intracellular membrane receptors was about 21%. Based on the nature of their PRL dissociation, PRL receptors were classified as acid-sensitive or acid-insensitive. The reaction of PRL with either species was reversible and saturable, and was dependent on temperature and time. Both species showed high PRL-binding affinity. However, acid-insensitive receptor was unstable at 37 degrees C. The level of acid-sensitive receptor was close to that of acid-insensitive receptor in late pregnancy. During the first 3 days of lactation, the level of acid-sensitive receptor increased more slowly than that corresponding to acid-insensitive receptor. The above criteria suggest that the postpartum increase in PRL binding is characterized especially by an increase in the level of acid-insensitive PRL receptor located mainly on intracellular membrane.

Effects of medroxyprogesterone acetate on serum prolactin levels and liver prolactin binding capacity in the rat

Modifications in liver prolactin (PRL) receptor levels and serum PRL concentration induced by administration of medroxyprogesterone acetate (MPA) were investigated in rats of both sexes. MPA induced a reduction both of the levels of PRL in the serum and of liver PRL receptors in the female rat. The reduction of the number of PRL receptors caused by MPA was rapid and almost complete after 10 days of treatment and appeared earlier than that of serum PRL levels. Furthermore the MPA-induced decrease in PRL receptors was specific, since insulin binding to the same liver membranes was not affected. MPA given simultaneously with oestradiol (which increases both the number of liver PRL receptors and the serum PRL levels in the male rats) was able to counteract the increase in PRL binding induced by oestradiol. On the contrary, the oestrogen-induced increase in serum PRL was not affected by MPA treatment. Similar results were obtained using tamoxifen, a well known antioestrogenic drug. In conclusion, our results show that the reduction of PRL receptor levels induced by MPA in rat liver is specific, not correlated to serum PRL concentration, and seems to depend on the antioestrogenic activity of the drug.

Further observations on the autoregulation of the prolactin receptor in rat ventral prostate explants.

In confirmation and extension of an earlier preliminary communication, ovine prolactin was found to elevate prolactin binding by approximately 100% in rat ventral prostate explants incubated for 20 h in vitro. A stimulation was observed with low doses of ovine hormone (150 micrograms/l) which, from available data on the relative biological potencies, could be considered equivalent to the upper limit of the physiological range of endogenous rat prolactin. The response was associated with a lag period of 3-6 h. The effect could be obtained with other lactogenic hormones, including human and rat prolactin and human growth hormone, but not with non-lactogenic hormones such as insulin, hCG, corticosterone, testosterone or oestradiol. The prostaglandin synthesis inhibitor, indomethacin, and the Ca2+-antagonist, verapamil, could not counteract the increase in prolactin binding induced by prolactin treatment, nor could dibutyryl cyclic AMP alone mimic the response. These data suggest that prostaglandins, Ca2+ or cAMP do not mediate the alteration in receptor binding. Furthermore, inhibition of lysosome activity by chloroquine could not alone increase the prolactin binding in the control tissues, suggesting that up-regulation does not simply reflect a protective action of prolactin on receptor degradation.

Modulation of prolactin binding sites in vitro by membrane fluidizers. IV. Differential effects on plasma membrane and Golgi fractions of male prostate and female liver in the rat

In vitro treatment of crude particulate fractions of male rat ventral prostate and female rat liver with membrane fluidizers (aliphatic alcohols) has been previously reported by us to increase prolactin (PRL) receptor levels, presumably by unmasking cryptic prolactin receptors. The objective of this study was to determine if similar in vitro treatment of purified plasma membrane- and Golgi-rich fractions of male rat prostate and female rat liver with ethanol produced differential effects on prolactin binding in these two subcellular fractions. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. 125I-PRL specific binding to Golgi-rich fractions of male ventral prostate and femlale liver was approximately 4-fold higher than that observed in plasma membrane-rich fractions. The microviscosity parameter, inversely related to lipid fluidity, was consistently lower in Golgi-rich fractions than that in plasma membrane-rich fractions in both prostate and liver. In vitro ethanol treatment of prostatic and hepatic plasma membrane fractions produced a dose-related increase and then decline in prolactin binding and a maximal (60–75%) increase in prolactin binding was observed at 4.8% and 2.0% ethanol in prostatic and hepatic membranes, respectively. This in vitro treatment also produced a significant increase in apparent lipid fluidity of plasma membrane-rich fractions of prostate gland and liver. However, similar in vitro ethanol treatment of Golgi fractions of both prostate gland and liver exhibited little increase in prolactin binding without changing microviscosity. Our observations are consistent with the direct relationship between membrane fluidity and prolactin receptor levels. The changes in prostatic and hepatic plasma membrane fractions following in vitro ethanol treatment suggest that prolactin receptors located on the plasma membranes may be modulated (via membrane lipid microviscosity changes) in vitro to a greater extent by various physiological agents than those located within the Golgi fraction.

Modulation of prolactin binding sites in vitro by membrane fluidizers II. Age-dependent effects on rat ventral prostatic membranes

The objectives of this study were to determine (i) if the age-related changes in 125I-labeled ovine prolactin specific binding of rat ventral prostate was correlated with changes in membrane lipid microviscosity and (ii) if membrane fluidizers produced age-dependent effects on prolactin binding of prostatic membranes. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Membrane preparations of ventral prostate glands obtained from immature (24–25 days old), young-adult (80–90 days old) and aged (550–610 days old) male rats were used for prolactin binding and membrane lipid microviscosity measurements. Relative to immature rats, prostatic prolactin binding decreased approximately 50% in young-adult rats and 75% in aged rats. Membrane lipid microviscosity, relative to immature rats, was increased 72% in young-adult rats and 140% in aged rats. Prostatic membranes obtained from immature animals exhibited no significant effects of in vitro alcohol treatment on prolactin binding, whereas, those obtained from aged animals exhibited maximal increase in prolactin binding. The value of the microviscosity parameter, after in vitro alcohol exposure, exhibited no significant changes in immature animals, whereas, this parameter was decreased approximately 15% in young-adults and approximately 30% in aged animals. These data suggest that in vitro fluidization of prostatic membrane exhibits an age-dependent modification of prolactin binding.

Modulation of prolactin binding sites in vitro by membrane fluidizers I. Effects on adult rat ventral prostatic membranes

The objective of this study was to determine if aliphatic alcohols, known fluidizers of certain membranes, could increase in vitro the apparent fluidity and prolactin binding capacity of membrane preparations obtained from ventral prostate glands of adult male rats. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Membrane preparations were either incubated with varying concentrations of ethanol, 1-propanol or 1-butanol and 125I-oPRL overnight at room temperature or were exposed to the alcohols for 15 min at room temperature and washed prior to the overnight incubation with ligand. Regardless of the conditions of incubation, alcohol exposure produced the same effects, a dose-dependent elevation and then decline in specific prolactin binding. Butanol produced a maximal 37–42% increase in prolactin binding at a concentration of 1.0%, propanol produced a maximal 40–56% increase in prolactin binding at a concentration of 3.8%, and ethanol produced a maximal 54–77% increase in prolactin binding at concentration of 4.8%. Scatchard analysis of the oPRL binding of ventral prostatic membranes indicated that the in vitro treatment of these membrane fluidizers increased the number of oPRL binding sites rather than the apparent affinity constant. The value of the microviscosity parameter decreased by 10–13%, 13–15% and 21–25%, after a 15 min exposure of prostatic membranes to 1.0% butanol, 3.8% propanol and 4.8% ethanol, respectively. These data suggest that in vitro fluidization of prostatic membrane modifies prolactin binding capacity and are consistent with in vivo prostatic prolactin receptor level-membrane fluidity relationships observed in earlier studies.

Prolactin binding by testes of unilaterally cryptorchid rats: the effect of hCG, testosterone, prolactin and orchiopexy.

The effect of unilateral cryptorchidism on prolactin binding to the testes was studied in the rat. Cryptorchidism was rendered surgically for 3 weeks and 3, 6 and 9 weeks later prolactin binding was measured in testicular homogenates. Prolactin binding to the cryptorchid testes decreased significantly at 3 weeks with a further decrease at 6 and 9 weeks. Binding by the contralateral testes decreased at 3 weeks and increased at 6 and 9 weeks. To examine the possible mechanism of these changes one group of rats was treated for 5 weeks with testosterone and another with hCG. Testosterone treatment resulted in a significant fall in prolactin binding to normal, cryptorchid and contralateral testes. hCG also produced a slight but significant reduction in prolactin binding. To study the effect of surgical relocation of the testes into the scrotum, orchiopexy was performed in another group of rats. Orchiopexy increased prolactin binding only if performed 3 weeks after cryptorchidism. At 6 and 9 weeks after cryptorchidism orchiopexy did not increase prolactin binding. Treatment of cryptorchid rats with prolactin for 5 weeks induced an increase in prolactin binding to control, cryptorchid and contralateral testes. It is concluded that testicular atrophy follows upon placement of a testis within the peritoneal cavity. This atrophy lowers the total amount of prolactin binding and increases binding to the contralateral testes. Intratesticular concentration of testosterone may play a major role in the decrease of prolactin binding. Orchiopexy improves prolactin binding only if performed before 6 weeks of age. Administration of prolactin augments prolactin binding to the testes, irrespective of their location.

Increased detection of prolactin binding of rat ventral prostate after treatment with dextran coated charcoal: evidence for a direct dihydrotestosterone-prolactin interaction.

The initial objective of this study was to determine the effect of pretreatment of membrane fractions with dextran coated charcoal (DCC) on specific prolactin (PRL) binding of rat prostate. Preincubation of 15,000 χ g and 100,000 × g pellets of adult male rat ventral prostate with DCC consistently increased PRL binding. DCC pretreatment failed to increase PRL binding in prostatic membranes from immature male rats (24–25 days old) and from adult male rats castrated 3 days previously. Prostatic PRL binding of immature rats was two-fold greater than that of intact adult rats,while orchidectomy reduced prostatic PRL binding by 40–50%. Scatchard analysis indicated that the increase in PRL binding of intact adult prostate membranes produced by DCC was due to an increase in the number of PRL receptor sites. The postpubertal decrease in prostatic PRL binding and effect of DCC in intact adult prostate suggested that substances of testicular origin inhibited PRL binding of prostatic membranes. Consequently, membran...

Prolactin receptors in interstitial cells of testes from rats at different stages of development.

The developmental pattern of prolactin receptors was examined in intact interstitial cells isolated from testes of 19-day old foetuses (F19), 1 to 45-day immature (N1 to N45) and adult animals. The prolactin binding activity in F19 interstitial cells is low, being 27% of that found in cells of the adult. There is an abrupt 60% increase in prolactin binding immediately after birth (N1), followed by a slow, but gradual, increase for the next 34 and adulthood which follows an increment in serum prolactin concentrations after day 24. Equilibrium analysis revealed that the receptors In interstitial cells from N1, N34 and adults have similar affinity (Kd = 2.7 to 3.3 x 10(-10)M) for prolactin. However, cells from adults contain twice as many prolactin receptors per cell as that of immature animals. This increase in prolactin receptors may play a role in the modulation of interstitial (Leydig) cell function during sexual maturation.

Stimulatory effects of prolactin and anti-prolactin receptor serum on prolactin binding sites in rat liver cells in suspension culture

The effects of prolactin and a serum containing anti-prolactin receptor antibodies on prolactin binding sites were investigated in a suspension culture of rat liver cells. In this model, prolactin binding sites decline rapidly with time, with 90% of the sites lost at 24–48 h of culture. The inclusion of 10 to 100 nM ovine prolactin in the incubation medium, results in a 6-fold increase in prolactin binding compared to control cultures. Anti-prolactin receptor serum is capable of preventing this PRL-induced increase in its receptors. However, when incubated alone, these antibodies at lower concentrations (0.5 to 5%) mimic the up-regulatory effect of prolactin on its own binding site. These findings suggest that in rat liver cells, as has been observed for rabbit mammary gland, that the prolactin molecule is not required beyond the initial binding to its receptors for its action to be attained.

Prolactin receptors in the testes of cryptorchid (CRYP) rats

The effect of unilateral CRYP on prolactin(PRL)binding in the CRYP and the con.lat testes was investigated in the rat. CRYP was rendered surgically at 3 weeks, and 3,6 and 9wks later PRL binding was measured in testicular homogenates. Binding decreased at 3 wks from 0.64±0.08ng/testis/100g rat in control to 0.37±0.06 in CRYP and 0.43±0.07 in con.lat testes. At 6 wks control testes bound 0.87±0.01ng/tes/100g rat, CRYP 0.12±0.01 and con.lat increased to 0.82±0.01. At 9 wks: control 0.7±0.01, CRYP 0.12±0.01 and con.lat 1.13±0.04. Orchiopexy was performed in another group of rats at 6, 9 and 12 wks of age. The 6 wks orchp. produced a slight improvement in PRL binding by the CRYP testes. The 9 and 12 wks orchp. did not improve the decreased binding. The con.lat testes bound more than did the nonorchp. con.lat. S.c. PRL in PVP, 0.5mg/kg 3 t.w. for 5 wks induced an increase in PRL binding by control, CRYP and con.lateral testes. To search into the possible mechanism of these changes one group of rats were given Smg/kg s.c. testosterone, and another-500 IU/kg HCG 3 t.w. for 5 wks. Testosterone produced a fall in PRL binding by control, CRYP and con.lat testes. HCG did not alter binding by any of these preparations. In conclusion: 1.CRYP in the rat impair PRL binding and produce a con.lat increase. 2.Orchiopexy restores PRL binding only if done before 6 wks of age. 3.The con.lat increase in PRL binding is not mediated by changes in testosterone or gonadotropine levels.

Effect of prolactin and prostaglandins on the stimulation of prolactin binding sites in the male rat liver

Induction of prolactin hepatic receptors in the male rat by exogenous prolactin and the possible involvement of prostaglandins in this process were studied. When ovine prolactin 500 μg/kg was administered sc in saline containing 10% PVP (polyvinylpyrrolidone), twice daily for 6.5 days and the rats killed 48 h after the last injection, the specific binding of 125I-labelled ovine prolactin to prolactin hepatic receptors was raised 26-fold, while administration of prolactin in saline only caused a 7-fold increase. A well correlated log dose-response relationship was demonstrated between 12.5 and 500 μg of prolactin in saline-PVP, with the lowest dose causing an 18-fold increase in binding. A shorter 4.5 day treatment of prolactin in saline—PVP caused only a small 3-fold increase in prolactin binding. Scatchard analysis showed that these increases resulted from increases in receptor concentration. The effect of prolactin on the induction of the hepatic receptors could not be mimicked by PGE 1, PGE 2 or PGF 2α, nor could PGF 2α synergize with a short treatment with prolactin. Further, indomethacin caused no significant effect on this action of prolactin. It seems that prolactin does not induce its own receptors in the rat liver by stimulation of prostaglandins in this tissue.

Regulation of Lactogenic Hormone Binding in Rat Liver by Steroid Hormones

We have measured the effects of testosterone propionate, medroxy-progesterone acetate and cortisol on the binding of ovine [125I]iodoprolactin to 100,000 X g particulate fractions from liver of normal and estrogen-treated female rats. In untreated animals 6.7 +/- 1.1% (SD) of the radioactivity added to 0.5 mg of membrane protein was specifically bound to the hormone receptor. Specific binding was significantly (P less than .05) decreased after 7 daily doses of testosterone (1.0 mg) to 2.8 +/- 1.4%, medroxyprogesterone (0.25 mg) to 2.7 +/- 0.2% and cortisol (5.0 mg) to 3.1 +/- 1.3%. The serum prolactin concentration, 4.2 +/- 3.4 ng/ml in normal animals, was not affected by the hormone treatment. Ethinyl estradiol, 10 mug/day for 7 days, increasing the binding of [125I]iodo-prolactin to 16.6 +/- 6.0% and increased serum prolactin to 50.6 +/- 11.5 ng/ml. Simultaneous administration of testosterone, medroxyprogesterone or cortisol with estradiol did not diminish the estradiol-induced increase in serum prolactin, but completely prevented the increase in prolactin binding. Testosterone or cortisol given to animals pretreated with estradiol suppressed prolactin binding from 16.4 +/- 4.2% to less than 2.5% after 48-72 h. Parellel results were obtained with 125I labeled human growth hormone whereas 125I labeled-insulin binding was not affected by these treatments. Scatchard analysis showed that the decrease in lactogenic hormone binding was due to a reduced concentration of receptors with no significant change in affinity. Since serum levels of prolactin were not changed, we conclude that treatment with testosterone, medroxyprogesterone, and cortisol decreased lactogenic hormone binding by a direct action on the liver.

Prolactin Binding Sites in the Male Rat Liver Following Castration

Specific binding sites for prolactin (PRL) have been detected in membrane preparations from the liver of the male rat following castration. The magnitude of the increased binding following castration varied with the age of the animals and with the time after castration. The effect of castration did not appear to be PRL mediated, since increases or decreases of serum PRL levels after pharmacological agents had no effect on PRL binding. The pituitary, however, seems to have a critical role in mediating the increase in PRL binding. Adrenalectomy did not influence the extent of binding of PRL after castration. Testosterone administration, however, completely prevented the increased PRL binding which followed castration. These studies suggest that testosterone has a modulating effect on hepatic PRL binding sites. The maintenance of such binding activity requires not only PRL, but also a functioning pituitary.

Effects of castration, testosterone, estradiol, and prolactin on specific prolactin-binding activity in ventral prostate of male rats.

Lactoperoxidase-catalyzed 125I-labeled ovine prolactin (PRL) was found to bind specifically to particulate membrane fractions of rat ventral prostate. Unlabeled PRL readily displaced the labeled PRL, whereas ovine GH, LH, FSH, or TSH showed no such competition. Castration reduced the binding of 125I-labeled PRL to about 1/6 of that in intact rats, and injections of testosterone propionate (TP) increased PRL binding to values as great or greater than those in intact controls. Injections of TP into intact immature and mature rats also increased PRL binding. In vitro binding of labeled PRL was inhibited in prostatic tissue removed from intact immature rats 2 h after injecting unlabeled PRL, but not in ventral prostates from rats killed 26 or 74 h after injecting unlabeled prolactin. PRL injected together with TP in castrated rats produced no greater increase in prolactin binding than TP alone, while estrogen appeared to decrease PRL binding beyond that produced by castration alone.