Relationship of prolactin receptors to concanavalin A binding.

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of 125I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar alpha-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 mu/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (Kd) as the controls. The binding of 125I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (Kd approximately equal to 6.6 . 10(-8) M. At high lectin concentrations, low affinity (Kd approximately equal to 6.7 . 10(-5) M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 microgram/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone. Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized 125I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by alpha-methyl-D-mannopyranoside.     

Pulsatile secretion of prolactin and oxytocin during nursing in the lactating rat

The secretory profile of prolactin and oxytocin in response to suckling stimuli by litters was studied in unanesthetized and urethane-anesthetized lactating rats. Serum prolactin levels were determined by radioimmunoassay. Oxytocin released at milk-ejection reflex was monitored by the changes in the intramammary pressure and/or the characteristic pup's reaction associated with the milk ejection. Serum prolactin concentrations began to rise earlier than the first milk ejection in unanesthetized rats, but they were never elevated without the appearance of milk ejections in urethane-anesthetized rats. Pulsatile fluctuation in serum prolactin levels at 6-15 min intervals was observed in the nursing period when 10 pups were suckling continually. The intermittent milk-ejection reflex occurred not always but preponderantly (64-91%) when the serum prolactin levels were at the nadir of the fluctuation. Injection of an estimated dose of oxytocin released at each milk ejection (1 mU) did not change the serum prolactin levels. These results indicate that the mechanism for prolactin release may be more susceptible to the effects of anesthesia than that for oxytocin release in response to the suckling stimuli and that the release of both the hormones is pulsatile in nature and be influenced by a common biological clock during the nursing period.     

Lipoprotein augmentation of human chorionic gonadotropin and prolactin stimulated progesterone synthesis by rat luteal cells

A collagenase dispersed cell suspension from PMSG-hCG primed immature rats responded to exogenously added hCG, cholera enteroxin, prolactin, and 8-Bromocyclic-AMP with increase in progesterone production in a dose dependent manner, and this stimulation was augmented by the plasma lipoprotein fractions hHDL and hLDL. The responsiveness to low doses of prolactin was not apparent when lipoprotein fractions were not included in the assay mixture. When the incubation mixture contained either LDL or HDL, the stimulatory effect of prolactin on progesterone production was evident at 5 and 10 micrograms prolactin/ml of the incubation mixture. Progesterone production, both basal and hormone stimulated, was maximum on day 7 of pseudopregnancy. Although the extent of hCG and prolactin stimulation of progesterone production and its potentiation by lipoprotein fractions was observed to be higher on days 3 and 5 than that seen on day 7, the net amount of progesterone produced was highest on day 7. The basal as well as hormone and lipoprotein stimulated progesterone production started to decline after day 7, reaching a nadir on day 14. These experiments show that prolactin is effective in stimulating progesterone production by rat luteal cells in vitro and that lipoprotein fractions, LDL and HDL further potentiate this response. This study further suggests that it is important to include LDL or HDL as a source of cholesterol for in vitro experiments in which the steroidogenic response of luteal cells to exogenous stimuli is tested.     

Relationship of prolactin receptors to concanavalin A binding

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of ¹²⁵I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (K_d) as the controls. The binding of ¹²⁵I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (K_d ≈ 6.6 · 10^?8 M). At high lectin concentrations, low affinity (K_d ≈ 6.7 · 10^?5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone.Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized ¹²⁵I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl-D-mannopyranoside.     

Effect of repeated stimulation by thyrotropin-releasing hormone (TRH) on thyrotropin and prolactin secretion in perfused euthyroid and hypothyroid rat pituitary fragments

The previously reported refractoriness of pituitary response to thyrotropin-releasing hormone (TRH) stimuli was investigated here in an in vitro perfusion system using pituitary tissue from euthyroid and hypothyroid rats. Thyroid-stimulating hormone (TSH) and prolactin (PRL) responses to TRH (28 pmol) were significantly greater in hypothyroid tissue compared with euthyroid. Hypothyroid tissue showed a reduction in response to two consecutive stimuli in both TSH and PRL, however the TSH decline in response was more marked than PRL. Euthyroid tissue showed no significant decline in response to TRH. An increase in the dose of TRH (112 pmol), administered to euthyroid tissue, resulted in increased TSH and PRL response, but no decline in response to sequential stimuli was observed. Three consecutive stimuli by TRH (28 pmol) of hypothyroid tissue resulted in a consistent decline in TSH response. The decline in PRL response only reached statistical significance by the third stimulation. Euthyroid and hypothyroid pituitary tissue was subjected to sequential depolarising stimulation with KCl (50 mumol). Euthyroid tissue showed no decline in response in either TSH or PRL. In hypothyroid tissue only, the decline in TSH response reached statistical significance. This decline in TSH response was significantly smaller than the decline in response observed in hypothyroid tissue stimulated with TRH. Refractoriness of hypothyroid pituitary tissue to repeated TRH stimuli is reported here. Our data suggest that the decline in hormonal response cannot be explained solely on the basis of tissue depletion.     

The response of plasma prolactin to suckling during normal and prolonged lactation in the rat

In dams which had been kept isolated from pups for 8-10 h, the magnitude of the suckling-induced prolactin rise in the plasma was studied in relation to intensity of suckling stimulus and lactational age of the mother. At midlactation the response of prolactin evoked by suckling was enhanced as litter size increased. Suckling of 2 pups induced a greater prolactin rise in dams adjusted to 2 pups than in dams adjusted to 8 pups. Suckling of 8 pups caused a greater prolactin rise in dams which had been adjusted to an 8-pup litter, than in rats with a 2-pup litter. At late and prolonged lactation the rise of prolactin in the plasma induced by the suckling stimulus of 8 pups was significantly lower than at midlactation. Injection of perphenazine after a period of suckling induced a moderate increase of plasma prolactin in dams at midlactation, and a similar increase in dams at late lactation and at day 42 of lactation. It is concluded that in the first half of lactation the number of pups, i.e. the intensity of the suckling stimulus, is an important factor in determining the magnitude of the prolactin response to suckling. The lower response of plasma prolactin to suckling in late lactation is neither caused by a decrease in suckling stimulus from the pups nor by an increase in prolactin clearance; it is probably due to a gradual reduction in prolactin synthesizing and releasing capacity of the pituitary, brought on by a desensitization of the neural or neuroendocrine system to suckling stimuli as lactation proceeds.     

Effect of isoproterenol on the liberation of histamine from rat lung mast cells]

Rat lung mast cells were stimulated with drugs with distinct mechanisms of action, namely concanavalin A, compound 48/80 ionophore A23187, in the presence of the beta adrenergic agonist (-)isoproterenol. Cells show a high response when they are stimulated with FNa-calcium. Isoproterenol does not inhibit histamine release induced by any stimuli, but enhances the response to concanavalin A and compound 48/80. Results point to the lack of beta activity on rat lung mast cells.     

Testosterone and prolactin are associated with emotional responses to infant cries in new fathers

To determine the responsiveness of new fathers and non-fathers toward infant cues, we exposed fathers and non-fathers to infant cries and to control stimuli and we measured affective, heart-rate, and endocrine responses, including salivary testosterone and cortisol and plasma prolactin concentrations prior to and after cry presentations. We found that (1) fathers hearing the cry stimuli felt more sympathetic and more alert compared to groups who did not hear the cries or to non-fathers who heard the cries; (2) fathers and non-fathers with lower testosterone levels had higher sympathy and/or need to respond to the infant cries than fathers with higher testosterone levels; (3) fathers with higher, as opposed to lower, prolactin levels were also more alert and more positive in response to the cries; (4) fathers hearing the cry stimuli showed greater percentage increase in testosterone than fathers not hearing the cry stimuli; (5) experienced fathers hearing the cries showed a greater percentage increase in prolactin levels compared to first-time fathers or to any group of fathers hearing control stimuli; finally, (6) partial correlations with parity and experience entered as a covariates indicated that both experience and testosterone contributed to the variance in fathers' affective responses to infant cries. Taken together, these results indicate that, as with a number of other biparental species, human fathers are more responsive to infant cues than are non-fathers and fathers' responses to infant cues are related to both hormones and to caregiving experience.     

The patterns of prolactin release by ECT and TRH compared

The patterns of prolactin release after bilateral ECT and after 0.4 mg TRH i.v. were studied in 11 female melancholic patients in a 5-min sampling protocol. Mean prolactin peaking times were 10.2 min after ECT and 20.5 min after TRH. The elimination rate coefficients were significantly lower--and the corresponding half-lives longer--for prolactin released by TRH than by ECT. A significant positive correlation of the maximal prolactin responses by the two stimuli was also found.     

Prolactin's mediative role in male parenting in parentally experienced marmosets (Callithrix jacchus)

Prolactin has been implicated in promoting paternal care behaviors but little evidence of causality has been found to date except for birds and fish. This study was designed to examine the possible causal relationships between prolactin and male parenting behaviors, reproductive hormones, and physical changes in cooperatively breeding common marmosets, Callithrix jacchus. Fifteen parentally experienced fathers were studied over three consecutive infant care periods during two weeks prior and three weeks following their mates' parturition under three-treatment conditions: normal control pregnancy, decreased prolactin and elevated prolactin. The treatments significantly altered the serum prolactin levels in the fathers. Using three methods of determining a father's level of parental care: infant carrying, family effort and responsiveness to infant stimulus tests, we found that only the male response to infant stimuli was altered by the hormone treatments. Lowering prolactin significantly reduced male responsiveness to infant stimuli but elevating prolactin showed the same effect. Hormonal sampling indicated that testosterone levels showed an inverse relationship to prolactin levels during a normal peripartum period and prolactin treatment reduced this relationship. Prepartum estradiol levels were significantly elevated during the lowered prolactin treatment and estradiol was significantly lowered postpartum with the elevated prolactin treatment. Father's weight decreased significantly by the third week of infant care during the normal treatment. Males in the elevated prolactin treatment lost little or no weight from prepartum while in the lowered prolactin treatment showed the most weight loss. The present findings did not distinguish a direct causal relationship of prolactin on behavior in experienced fathers but did find an interaction with other hormones and weight gain.     

Prolactin release and milk removal induced by suckling and milking in lactating ewes is prevented by L-dopa treatment

The effect of L-DOPA on milk removal and on prolactin release during suckling or milking was studied in lactating ewes. Various doses of L-DOPA (25, 50, 100 and 200 mg per animal) were injected iv 30 min before the suckling or milking period. Control ewes were injected with 0.9% NaCl solution only. Milking induced a significant long-lasting release of prolactin. An inhibition of milk removal was obtained with the dose of 200 mg of L-DOPA. An inhibition of prolactin secretion was observed related to the dose of drug administered. The inhibitory effect of 200 mg of L-DOPA on the secretion of prolactin after milking lasted for about 120 min, and thereafter a significant increase in serum prolactin level occurred. This increase in serum prolactin was not due to a "rebound" effect of L-DOPA, since the milking stimulus had to be present to induce the delayed increase in prolactin. Doses of 25 or 50 mg of L-DOPA prevented the surge of prolactin observed immediately after milking, but a long-lasting release of prolactin was obtained thereafter. The inhibitory effect of L-DOPA on prolactin release could be overridden by the suckling or milking stimuli according to the dose administered. The suckling stimulus was more effective than milking in overriding the inhibitory effect of the low dose of L-DOPA. The results indicate that milk removal and prolactin release induced by milking or suckling in lactating ewes is inhibited by an increase in monoamines at the hypothalamic-hypophyseal level.     

Release of oxytocin and prolactin in response to suckling.

The oxytocin and prolactin responses to suckling were measured in 10 women in early (n = 5) and established lactation (n = 5). Oxytocin was released in a pulsatile manner during suckling in all women, but the response was not related to milk volume, prolactin response, or parity of the mother. In all 10 women plasma oxytocin concentrations increased three to 10 minutes before suckling began. In five women this occurred in response to the baby crying, in three it coincided with the baby becoming restless in expectation of the feed, while in two it corresponded with the mother preparing for the feed. There was no prolactin response to stimuli other than stimulation of the nipple associated with suckling. These results clearly indicate that the milk ejection reflex, with release of oxytocin, occurs in most women before the tactile stimulus of suckling. A second release of oxytocin follows in response to the suckling stimulus itself. Thus it is important that care is taken to protect breast feeding mothers from stress not only during suckling but also immediately before nursing, when conditioned releases of oxytocin will occur.     

Stimulation of prolactin secretion in the rat by alpha-neo-endorphin, beta-neo-endorphin and dynorphin

Intraventricular injections of α-neo-endorphin, β-neo-endorphin and dynorphins (dynorphin[1–13], dynorphin[1–17], dynorphin[1–8]) resulted in an increase in plasma prolactin levels in urethane-anesthetized rats. Dynorphin [1–13] was the most potent to stimulate prolactin release among these opioid peptides. Plasma prolactin responses to these stimuli were blunted by naloxone, an opiate antagonist. In $\text{in}\text{vitro}$ studies, prolactin release from perfused pituitary cells was stimulated by α-neo-endorphin, and the effect was blunted by naloxone, whereas neither β-neo-endorphin nor dynorphin[1–13] affected prolactin release. These results suggest that newly identified “big” Leu-enkephalins in the brain stimulate prolactin secretion in the rat and that α-neo-endorphin has a possible direct action on the pituitary.     

Proteolytic modification of rat prolactin by subcellular fractions of the lactating rat mammary gland

The current study explored prolactin proteolysis by rat lactating mammary gland. 125I-labelled rat prolactin was incubated with tissue fractions of lactating mammary gland and the extent of prolactin degradation and fragment formation was visualized and densitometrically quantitated from autoradiographs derived from SDS-polyacrylamide gel electrophoresis under reducing conditions. At pH 4.5, the 25 000 X g pellet of mammary gland converted intact prolactin (23 kDa band) to proteolytic fragments (8-16 kDa bands) in a time- and tissue concentration-dependent fashion similar to that reported previously for rat ventral prostate. The prolactin-degrading and -fragmenting activity in lactating mammary gland was 5-10-times that observed for ventral prostate, the most active male tissue. This activity at acid pH was also demonstrable in other fractions of mammary gland but appeared to predominate in the cytosol. The above activities in mammary gland virtually disappeared at pH 7.4, appeared sensitive to aspartate and sulfhydryl proteinase inhibitors, and insensitive to serine and metalloenzyme proteinase inhibitors. The distribution of this activity could not be correlated with a particular enzyme marker. These characteristics of mammary gland activity differed significantly from those reported previously for prostate. When electrophoresis was conducted under non-reducing conditions, prolactin proteolysis in prostate and mammary gland was primarily associated with the formation of a more slowly migrating product (24 kDa band) with little spontaneous 8-16 kDa fragment formation. Re-electrophoresis of the 24 kDa band under reducing conditions resulted in the appearance of the 8 and 16 kDa fragments. In conclusion, prolactin is proteolytically modified by prostate and lactating mammary gland to a variant of intact hormone (24 kDa band) with a cleavage site in its large loop, by two or more widely distributed, acid-dependent proteinases. Lactating mammary gland, the principal target for prolactin, has the capacity to cleave the hormone in its loop at rates higher than any other tissue examined to date.     

Milking- and suckling-induced secretion of oxytocin and prolactin in parturient dairy cows

Serum concentrations of prolactin were unaffected by either suckling or milking on Day 2 or 3 postpartum in cows housed with their calves following parturition. In contrast, among cows housed without their calves milking elicited a four- to sixfold increase in serum prolactin concentrations. Serum oxytocin levels increased in response to both suckling and milking among cows housed with their calves with suckling being a more potent stimulus (257 ± 32 vs 189 ± 23 pg/ml at peak). However, the greatest increase in oxytocin levels accompanied milking in cows housed without their calves (375 ± 36 pg/ml at peak). These results suggest that stimuli associated with the presence or the absence of the calf can alter maternal secretion of oxytocin and prolactin. Greater understanding of factors which regulate secretion of these hormones may result in techniques to modify milk synthesis and milk ejection in dairy cows.     

Mechanisms of lysosomal enzyme secretion by human monocytes

Secretion of lysosomal enzymes by human monocytes in response to various stimuli and the effect of conditioned media from lymphocytes and neutrophils was studied. Monocytes were found to release β-glucosaminidase in response to NH₄Cl and to particles (zymosan, opsonised zymosan, asbestos and latex), but do not respond to some soluble stimuli like formyl-methionyl-leucyl-phenylalanine, phorbol myristate acetate, cytochalasin B, concanavalin A and $\text{N-}\text{acetylmuramyl-}\text{l}\text{-alanyl-}\text{d}\text{-isoglutamine}$. Neutrophil conditioned medium or neutrophil components did not have any effect on secretion. When treated with lymphokines the cells are more responsive, especially to zymosan. Even through there are similarities in the secretory activities of mouse macrophages and human monocytes, there are several differences both in the quantity of the response and in the mechanisms involved.     

Experimental allergic encephalomyelitis in male Lewis rats subjected to calorie restriction

This work analyzes the effect of calorie restriction on the development of experimental allergic encephalomyelitis (EAE) in Lewis rats. Plasma levels of ACTH, corticosterone, prolactin and growth hormone (GH) and mitogenic responses in submaxillary lymph nodes were measured. Male Lewis rats (6 weeks-old) were submitted to a calorie restriction equivalent to 66% of food restriction or to a normal diet. Fifteen days later, rats were injected with complete Freund's adjuvant plus spinal chord homogenate (SCH) or with complete Freund's adjuvant alone. Rats were monitored daily for clinical signs of EAE and were killed on day 15 after immunization. Only rats subjected to normal diet exhibited clinical signs of the disease. The increase in plasma ACTH and corticosterone found after SCH immunization in controls was not detectable in calorie restricted rats. Likewise, the correlation between circulating ACTH and corticosterone was no longer found after calorie restriction. Generally, calorie restriction by itself augmented plasma ACTH or corticosterone and this increase was not further amplified by SCH immunization. Only calorie restricted rats exhibited augmented plasma prolactin levels after SCH immunization, and decreased plasma GH levels regardless of immunization. Calorie restriction depressed the mitogenic response of lymphoid cells to concanavalin A but not to lipopolysaccharide. Calorie restricted rats did not exhibit augmented mitogenic response to concanavalin A following SCH immunization as those found in controls. The results are compatible with the view that the course of EAE can be significantly modified by caloric restriction.     

Identification of a nonimmunoreactive but highly bioactive form of prolactin in the mouse pituitary by gel electrophoresis.

Polyacrylamide gel electrophoresis of fresh mouse pituitary extracts in 10% acrylamide reveals three closely situated bands of protein in the area where prolactin usually migrates. The fastest migrating band constitutes only 10–30% of the total, but it is twice as active in the pigeon crop-sac bioassay as the major band and has little or no immunologic cross-reactivity against an antiserum to the major constituent. This newly recognized band may be a hitherto unrecognized molecular variant of prolactin.     

Hormones associated with non-maternal infant care: a review of mammalian and avian studies

Hormonal changes during non-maternal infant care have been demonstrated in many cooperatively breeding bird species, some monogamous rodents and two species of New World primates. Coevolution of hormones and social traits may have provided for the different breeding systems that occur today. Several hormones have been shown to covary with the breeding systems of vertebrates. Elevated levels of the hormone prolactin with male parenting behaviours are common to many birds, rodents and the callitrichid monkeys Callithrix jacchus and Saguinus oedipus. In birds, prolactin may be elevated in both male and female breeders during various stages of nest building, egg laying, incubating and feeding of young. Testosterone levels appear to have an inverse relationship to prolactin levels during infant care in birds and rodents, but this relationship has not been examined for primates. In cooperatively breeding birds, helpers who remain at the nest also have elevated levels of prolactin when displaying parental care behaviours. Prolactin levels are elevated in helper callitrichid monkeys during the postpartum period. Monogamous male rodents demonstrate elevated prolactin levels with parental care behaviour but, in contrast to the birds, the mechanisms mediating prolactin increase appear to differ for male and female rodents. Two factors may influence male parental behaviours and hormonal changes: stimuli from the pregnant female and stimuli from the newborn pups; whereas maternal behaviours are influenced by the maternal hormones of the female and the pup stimuli. An experiential factor may also influence male parental behaviours. Neuropeptides such as oxytocin and vasopressin appear to be involved in male rodent parental care and there may be an interaction between a series of hormones and neurosecretions and stimuli from mates and pups. Studies of Saguinus oedipus, the cotton-top tamarin, suggest that prolactin levels are responsive to stimuli from contact with infants and the level of infant care experience influences the levels of prolactin with male infant care. Father tamarins also have elevated levels of prolactin before the birth of infants suggesting that cues from the pregnant female are important. Prolactin's role in parental care may have evolved from prolactin's role in other reproductive functions. Hormonal regulation of non-maternal care may occur due to a complex interaction of many hormones and neurotransmitters. Studies described here should provide the impetus for further work on parental care hormones in a wide variety of primates.     

The ability of nonspecific T-cell stimulators to induce helper-cell-dependent increases in either polyclonal or isotype-restricted Ig production in vivo

In order to delineate the various roles of T cells in B-cell activation, mice were exposed to a variety of specific or nonspecific T-cell stimuli including mitogens, e.g., concanavalin A, adjuvants, e.g., complete Freund's adjuvant, and colchicine plus nonimmunogenic doses of antigen, anti-lymphocyte serum, and pathogens and their spleens analyzed for total class-specific immunoglobulin-secreting cells as indicators of helper cell generation. The results demonstrate that, depending on the mode of stimulation, markedly different Ig-secreting cell response patterns were induced, differing with respect to their kinetics and the isotype induced. In contrast to polyclonal T-cell stimuli such as concanavalin A and 17X lethal malaria which induced increases in all classes of Ig-recruiting cells, injection of many T-cell-activating agents resulted in the selective production of IgG clones in particular IgG 1. Such findings are discussed in terms of the different mechanisms of T-cell help and provide further evidence for functional heterogeneity in the T-helper-cell pool.