Radioimmunoassay For Prolactin Research Articles

Studies on the Endocrine and Neuroendocrine Control of Broodiness in the Yuehuang Hen

The hypothesis that serotonin (5-HT) and dopamine stimulate prolactin (PRL) release, either directly from the pituitary gland or by acting through Vasoactive Intestinal Peptide (VIP), was investigated by assessing the role of hypothalamic dopamine and 5-HT in the control of broodiness and PRL release in Cantonese native chicken breed called the Yuehuang hen. A second objective was to assess the involvement of hypothalamic VIP in the control of broodiness and PRL release by using dopamine and serotonin receptor antagonists respectively. In Experiment 1, two hundred laying hens from battery cages were transferred to floor pens with nest boxes to induce broodiness. Starting from the second day after the onset of broodiness, sixty hens were allotted into groups 1, 2 and 3 respectively, corresponding to chlorpromazine-treated (n = 20), cyproheptadine-treated (n = 20) and control (n = 20) groups. Blood samples were collected from the wing veins of hens in each group on days 2, 5, 9, 14 and 19 respectively after the onset of broodiness for radioimmunoassay of PRL and Luteinizing Hormone (LH). Results indicate that in the drug treated hens which terminated broodiness, the concentration of plasma PRL decreased significantly (p 0.05) changes between the chlorpromazine and cyproheptadine treated hens. Within the chlorpromazine and cyproheptadine treated hens, there were significant (p 0.05) changes throughout the blood sampling periods. Sixteen (80%) chlorpromazine treated hens terminated broodiness on an average of 4.6±0.8 days but four hens (20%) did not. However, thirteen (65%) cyproheptadine treated hens terminated broodiness on a n average of 2.3±0.2 days while seven (35%) did not. In Experiment 2, sixty animals were used but their management and drug treatment for group 1 (n = 10) and group 2 (n = 10) were the same as described in Experiment 1. Group 3 (n = 10) served as the control. Eight hens each from groups 1, 2 and 3 were randomly selected for immunohistochemical studies. On day 7 after the onset of broodiness, hypothalamus from anaesthetized chlorpromazine and cyproheptadine treated hens as well as control hens were processed for immunohistochemical localization of VIP neurons in the hypothalamus of Yuehuang hens. Morphological observation showed a higher number of VIP neurons in the hypothalamus of the control hens. A few VIP neurons which were very faint were also found in the hypothalamus of the chlorpromazine and cyproheptadine treated hens. Results of these studies indicate a relationship between the functions of dopamine and 5-HT neurons in the hypothalamus and reproductive activities in domestic hens. They are consistent with the view that hypothalamic dopamine and 5-HT are regulators of PRL release and that using drugs which inhibit the functional activities of these neurotransmitters can inhibit PRL release to disrup t broodiness in hens to maintain egg production. The results also indicate a causal relationship between hypothalamic VIP and changes in PRL secretion associated with reproductive activities in domestic hens. This is consistent with the view that VIP might be an important hypothalamic PRL releasing neuropeptide and also indicate that VIP might be a physiological PRL regulatory hormone in domestic hens.

In memoriam: H. Allen Tucker, PhD

It is with great sadness that I share news of the passing of Herbert Allen Tucker (“Tuck”; Figure 1), professor emeritus of animal science and physiology at Michigan State University (MSU). Tucker was a leading expert on mammary biology and lactation in dairy cattle who had served on the Michigan State faculty for 38 years. He died Wednesday, July 15, 2009, at Sparrow Hospital. The following remembrance is an attempt to capture in words the remarkable impact Tuck had on his colleagues and field of research. H. Allen Tucker (“Tuck”) was born in 1936 in Milford, Massachusetts. Throughout his adult life Tuck thoroughly enjoyed gardening, fishing, deer hunting, scouting, camping, antiquing, and going to his cabin. At work, he was the consummate old-style professor who believed the purpose of his research program was to train others. He labored tirelessly to train graduate students, fellows, visiting scientists and colleagues like me how to write about, evaluate and conduct research. Tuck often used the word “pithy,” although few of us ever knew what the word meant. He also strongly believed, even preached, that everyone’s research design, lectures, and scientific writings, including his own, could be improved by constructive criticism. Many of us owe much of the success in our scientific careers to Tuck’s pithy, sometimes stinging, yet always welldeserved criticisms. Tucker obtained his BS degree in animal husbandry from the University of Massachusetts in 1958 and his MS and PhD in animal physiology at Rutgers University in 1960 and 1963 under the guidance of Ralph Reece. Tucker joined the Michigan State University Dairy Science Department in 1962 where he rose to the rank of professor in the Departments of Animal Science and Physiology and remained until his retirement in 2000. During his highly productive career, he and his colleagues were responsible for development of radioimmunoassays for prolactin, growth hormone, insulin, glucocorticoids, and thyroid hormones in cattle. His descriptions of variations in these hormones during mammary growth, lactation, and the different seasons were among the first published. He also concentrated his efforts on understanding how temperature and photoperiod might be used to control hormone secretion and, as a consequence, rate of growth and lactation in cattle. Results of these efforts demonstrated clearly that 16 h of light increased growth and lactational performance in cattle. This novel research was highlighted by a publication in Science. Tucker was internationally recognized as a leader in the field of endocrine control of lactation. He authored 355 scientific publications and co-authored the most widely used textbook for college courses in dairy science, Dairy Cattle: Principles, Practices, Problems, Profits. He was the first editor of the Michigan Dairy Review at MSU. Tuck was recipiIn memoriam: H. Allen Tucker, PhD

Production of Recombinant Goldfish Prolactin and Its Applications in Radioreceptor Binding Assay and Radioimmunoassay

Goldfish prolactin cDNA was subcloned into a pRSET A vector and expressed in Escherichia coli. Recombinant goldfish prolactin was expressed mainly as insoluble inclusion bodies in the form of N-terminal 6× His-tagged fusion protein. This fusion protein was purified, refolded, and 125I-labeled to generate a radioligand for receptor binding and validation of a radioimmunoassay for goldfish prolactin. Using goldfish gill membrane as the substrate for prolactin receptor binding, both recombinant and native forms of goldfish prolactin were effective in displacing the specific binding of the radioligand in a similar dose range, suggesting that the fusion protein was refolded properly and could be recognized by goldfish prolactin receptors. To quantify prolactin contents in biological samples from the goldfish, a radioimmunoassay using the 125I-labeled recombinant prolactin as a tracer was established. This assay was shown to be selective for goldfish prolactin without cross-reactivity with mammalian prolactin and pituitary hormones from other fish species (e.g., growth hormone and gonadotropin II). This newly validated assay system was used to investigate neuroendocrine and signal transduction mechanisms regulating prolactin release in the goldfish. In this case, the Ca2+ ionophore A23187 and protein kinase C activator TPA were effective in elevating basal levels of prolactin secretion in perifused goldfish pituitary cells. In parallel studies using a static incubation approach, somatostatin and dopamine, but not vasoactive intestinal polypeptide, were inhibitory to basal prolactin release in goldfish pituitary cells. These results suggest that somatostatin and dopamine may serve as negative regulators of basal prolactin secretion and that extracellular Ca2+ influx and protein kinase C activation may be important signaling events mediating prolactin release in the goldfish.

Newt prolactin and its involvement in reproduction

The amino acid sequence of newt (Cynops pyrrhogaster) prolactin deduced from the nucleotide sequence of its cDNA showed a relatively high homology with sequences of chicken and sea turtle prolactins as well as with those of anuran prolactins. Cynops prolactin receptor transcripts were detected in various tissues and organs, suggesting that prolactin plays multiple roles in urodeles. Urodele prolactin was purified from the pituitaries of C. pyrrhogaster. Antiserum against this prolactin was used for radioimmunoassay of plasma prolactin and immunoneutralization experiments. Endogenous prolactin was shown to induce migration to water, courtship behavior, and cessation of spermatocytogenesis in the Cynops newt. The hormone was found to be involved in the development of cloacal glands such as the lateral and abdominal glands, growth of the tail and Mauthner neurons, secretion of oviducal jelly, and enhanced synthesis of a female attracting pheromone (sodefrin), and responsiveness of the olfactory epithelium to sodefrin. In most of these cases, prolactin was found to act synergistically or antagonistically with sex steroids. We also discovered that hypersecretion of prolactin in the newts subjected to cold temperature was induced by hypothalamic stimulation rather than release from hypothalamic inhibition.Key words: prolactin, newts, reproduction.

Changes in Serum Progesterone, Estradiol-17.BETA., Luteinizing Hormone and Prolactin in Lactating and Non-lactating Japanese Black Bears (Ursus thibetanus japonicus).

The concentrations of progesterone (P4), estradiol-17 β (E2), luteinizing hormone (LH) and prolactin (PRL) in serum obtained from 9 captive female Japanese black bears, Ursus thibetanus japonicus, (7 lactating and 2 non-lactating) were obtained by a heterologous radioimmunoassay to clarify hormonal profiles during the pre- and post-parturition and lactation periods. The present study provides the first evidence to validate LH and PRL radioimmunoassay in bear sera by using the canine LH and PRL and the antibody to these hormones. In 7 bears which gave birth and lactated, serum P4 concentrations were high during prepartum phases in December and January but fell rapidly after parturition in February (p<0.05; comparison of pre- and post-parturient phases). Serum E2 and LH concentrations did not change significantly throughout pre- and post-parturition. Serum PRL concentrations increased significantly after parturition in February (p<0.05; comparison of pre- and post-parturient phases) and exceeded 10 ng/ml in all lactating bears from Days 41~60 after parturition in March. On the other hand, in 2 bears which did not produce cubs, serum P4 concentrations were high in December and January and decreased after February. Serum E2 concentrations were relatively low between December and February but increased in March. Serum LH concentrations were relatively high in December and remained low throughout January to April, whereas PRL concentrations increased gradually after February, but were below 10 ng/ml. These results suggest that a rapid decline in P4 and a marked increase in PRL occurred in association with events such as parturition and lactation, but there is no clear evidence that changes in serum E2 and LH concentrations are associated with parturition and lactation.

Newt prolactin and its involvement in reproduction

The amino acid sequence of newt (Cynops pyrrhogaster) prolactin deduced from the nucleotide sequence of its cDNA showed a relatively high homology with sequences of chicken and sea turtle prolactins as well as with those of anuran prolactins. Cynops prolactin receptor transcripts were detected in various tissues and organs, suggesting that prolactin plays multiple roles in urodeles. Urodele prolactin was purified from the pituitaries of C. pyrrhogaster. Antiserum against this prolactin was used for radioimmunoassay of plasma prolactin and immunoneutralization experiments. Endogenous prolactin was shown to induce migration to water, courtship behavior, and cessation of spermatocytogenesis in the Cynops newt. The hormone was found to be involved in the development of cloacal glands such as the lateral and abdominal glands, growth of the tail and Mauthner neurons, secretion of oviducal jelly, and enhanced synthesis of a female attracting pheromone (sodefrin), and responsiveness of the olfactory epithelium to sodefrin. In most of these cases, prolactin was found to act synergistically or antagonistically with sex steroids. We also discovered that hypersecretion of prolactin in the newts subjected to cold temperature was induced by hypothalamic stimulation rather than release from hypothalamic inhibition.

Development and Application of a Homologous Radioimmunoassay for Xenopus Prolactin

A specific and sensitive homologous radioimmunoassay (RIA) for Xenopus prolactin (xPRL) was developed. PRL isolated from X. laevis pituitary glands was used for generating antiserum in a rabbit, for radioligand and for the standard. Pituitary homogenates and plasma from adult Xenopus produced displacement curves parallel to the xPRL standard. Plasma from hypophysectomized Xenopus showed negligible cross-reactivity. Purified PRLs from other amphibian species such as the bullfrog ( Rana catesbeiana) and toad ( Bufo japonicus) gave inhibition curves which did not parallel the standard. Ovine PRL, mouse PRL, newl ( Cynops pyrrhogaster) PRL, bullfrog GH, and bullfrog LH showed no inhibition of binding even at relatively high doses in this RIA. The sensitivity of the RIA was 0.122 ± 0.005 ng xPRL/100 μl assay buffer. Intraassay and interassay coefficients of variation were 2.46 and 6.65%, respectively. Histological studies of Xenopus adenohypophyses revealed that the cells which reacted immunologically with the antiserum against xPRL corresponded to those positively stained with antiserum against bullfrog PRL. A preliminary application of this homologous RIA for xPRL was performed by evaluating plasma and pituitary PRL levels in subadult and adult Xenopus of both sexes.

Annual changes in serum concentrations of prolactin in captive male black bears (Ursus americanus)

Prolactin may be involved in the regulation of reproduction in black bears (Ursus americanus) as it is a mediator of photoperiodic changes in a number of species. The objectives of this study were to validate a radioimmunoassay to measure prolactin in bear serum and to describe seasonal changes in serum prolactin concentrations in captive male bears. Serum samples were obtained nine times during a year from three captive male black bears that were denning between November and March and active during the other months. The heterologous prolactin radioimmunoassay, using pig 125I-labelled prolactin and goat anti-pig prolactin as a primary antibody, was validated. Injection of thyrotrophin-releasing hormone into the three male bears in June resulted in a rapid increase in serum concentrations of prolactin (t = 0, 11.4-14.8 ng ml-1; t = 15-30 min, 18.4-28.7 ng ml-1). The sensitivity of the assay was 0.08 ng per tube. Intra- and interassay coefficients of variation were 5.5% (n = 6) and 5.7% (n = 6), respectively. Serum concentrations of prolactin changed seasonally, with the lowest concentrations in December (mean +/- SD = 1.1 +/- 0.1 ng ml-1); this was followed by a gradual increase between January (2.6 +/- 0.6 ng ml-1) and April (6.4 +/- 1.2 ng ml-1) and the highest concentrations in May (17.6 +/- 4.7 ng ml-1), preceding peak testosterone concentrations in June. The observation that prolactin secretion increased with increasing daylength suggests that photoperiod may be an external regulator. The presence of high concentrations of prolactin before peak testosterone concentrations suggests that prolactin may play a role in regulating seasonal changes in the testes.

Bioactivity and immunoreactivity of rat pituitary prolactin submitted to various extraction procedures.

Pituitary glands of rats, injected with estrogen to increase the prolactin (PRL) storage in secretory granules, were submitted to various extraction procedures for prolactin. Homogenization and centrifugation of pituitary tissue, in Tris-HCl buffer, pH 7.3, yielded a small amount of radioimmunoassayable prolactin, which increased remarkably after extraction in alkaline pH, disruption of granular membranes with Lubrol and specially after treatment with 2.5 mol/l urea. Nb2 lymphoma cell and pigeon crop sac bioassay (BA) revealed higher levels of bioactivity after extraction in Tris buffer, pH 7.3, in comparison to RIA, with BA/RIA ratios of 1.4 and 2.3, respectively. Bioassays of pituitary PRL extracted with Lubrol and alkaline medium, were less effective in quantifying PRL than RIA. Treatment of pituitary homogenates with urea produced the highest levels of PRL by both BA and RIA, with BA/RIA ratios close to 1. The thorough depolymerization of big PRL effected by urea produced the release of monomeric subunits which allows a complete quantification of the total content in the pituitary gland.

Semicircadian rhythm of dopamine release in the mediobasal hypothalamus in awake rats during pseudopregnancy: evidence that a thyrotropin-releasing hormone analogue stimulates dopamine release and thereby inhibits prolactin secretion.

The release of dopamine (DA) from tuberoinfundibular (TIDA) neurons during prolactin (PRL) surge and nonsurge periods and the effects of the thyrotropin-releasing hormone (TRH) analogue CG 3703 on DA and PRL secretion were studied in awake pseudopregnant (PSP) rats by simultaneous measurement of extracellular DA levels in the mediobasal hypothalamus (MBH) by means of microdialysis and of plasma PRL concentrations by radioimmunoassay of PRL in blood sampled from a permanent heart cannula. Since basal DA levels were low, the experiments were performed under local infusion of the reuptake inhibitor nomifensine (5 mumol/l), which increased extracellular DA levels 5-fold around the probe, but did not affect the occurrence of the nocturnal PRL surge on day 7-8 of PSP. Under nomifensine conditions, during the interphase (18.00-1.00 h) plasma PRL levels were low (< 50 ng/ml), while the DA release from TIDA neurons was elevated (approximately 250%) relative to the DA values measured during the phase when the nocturnal PRL surge occurred (100%; 1.00-12.00 h; plasma PRL levels were elevated to approximately 300 ng/ml). Thus, semicircadian rhythms were detected both for DA and PRL, which were broadly reciprocal in timing. The TRH analogue CG 3703 (100 and 500 micrograms/animal i.v.), when administered at 00.00 h, dose-dependently increased extracellular DA levels (to 300 and 500% of pretreatment values, respectively) and postponed the PRL surge in a corresponding manner (for approximately 2 and 3 h, respectively). Thus, in addition to its PRL-releasing effects, TRH can also suppress the PRL secretion by stimulating the release of DA from TIDA neurons, thereby increasing the inhibitor DAergic tone on lactotrophs. During the interphase, when DA levels were relatively elevated, administration of the DA blocking agent HA 966 (100 mg/kg body weight i.p.) at 20.00 h resulted in a short-lasting decrease in extracellular DA levels (to approximately 60% of pretreatment values for 30 min) accompanied by an immediate, short-lasting increase in PRL levels (to 400 ng/ml for approximately 1 h). Administration of CG 3703 (500 micrograms/animal i.v.) in combination with HA 966 (100 mg/kg body weight i.p.) at 20.00 h prevented the decrease in DA levels and the rise in plasma PRL values. Thus, the TRH analogue and HA 966 counteracted each other regarding the DAergic system, which again illustrates the stimulatory effect of the TRH analogue on the release of DA from TIDA neurons. Moreover, our results suggest that the interphase and the phase during which the nocturnal PRL surge occurs under the condition of PSP differ with regard to the DAergic inhibition of the PRL secretion; during the interphase the DAergic inhibitory tone prevents the rise in PRL secretion, while during the nocturnal PRL surge period another factor in addition to a decrease in DA levels appears necessary to increase plasma PRL levels.

A Radioimmunoassay for Recombinant-Derived Chicken Prolactin Suitable for the Measurement of Prolactin in Other Avian Species

A homologous radioimmunoassay for chicken prolactin is described. The assay is based on recombinant-derived chicken prolactin that has been used to raise an antibody and produce 125I-labeled tracer and assay standards. The radioimmunoassay measures a minimum of 0.03 ± 0.01 ng of the recombinant prolactin with 50% displacement of binding by 0.6 ± 0.1 ng. Chicken plasma and pituitary immunoreactivity dilute in parallel with the standard curve. The mean (± SE) concentrations of plasma prolactin were 12.1 ± 1.5, 433.2 ± 5.5, and 1609.8 ± 18.5 ng/ml in out-of-lay, laying, and incubating bantam hens, respectively. The binding of recombinant prolactin was not displaced by extracts of neural lobe or plasma from a hypophysectomized hen. Plasma prolactin concentrations were increased after intravenous administration of chicken vasoactive intestinal polypeptide or quipazine, a 5-HT agonist. The assay measures immunoreactive prolactin in other birds. Plasma from quail, turkey, great tit, and wryneck displaced the 125 I-labeled prolactin tracer parallel to the standard curve. Starling, ring dove, and penguin plasma displaced the binding in a nonparallel manner.

Cervicovaginal prolactin: A marker for spontaneous preterm delivery

Our purpose was to determine whether the presence of prolactin in cervicovaginal washings is associated with preterm birth. A cohort of 80 patients underwent a washing of the ectocervix and vaginal fornices with a normal saline solution. The cohort consisted of two groups: 40 inpatients requiring tocolysis and 40 asymptomatic outpatients. The saline solution aspirates were centrifuged, the supernatant was stored at -70 degrees C, and a radioimmunoassay for prolactin was run in batch fashion. A prolactin concentration greater than the detection limit of the assay was considered a positive test result. Prolactin was identified in significantly more symptomatic patients than asymptomatic controls (50% vs 5%, p < 0.0001). In symptomatic patients cervicovaginal prolactin had an 80% positive predictive value and a 65% negative predictive value for delivery at < or = 34 weeks' gestation. Patients testing positive for prolactin had significantly shorter latency from testing to delivery (16 +/- 17 vs 34 +/- 24 days, p = 0.02) and had significantly lower birth weights (1985 +/- 729 vs 2583 +/- 696 gm, p = 0.01) compared with patients testing negative. Prolactin was also identified in two asymptomatic patients, both of whom were delivered before term. Cervicovaginal prolactin is a biochemical marker for preterm delivery, a shorter latency period to delivery, and lower birth weight in symptomatic patients. This test may also prove to be a valuable marker for preterm birth in asymptomatic women.

Cause of failure of lactation in mouse mammary tumor virus/human transforming growth factor alpha transgenic mice.

Transgenic female mice bearing human transforming growth factor-alpha (TGF alpha) cDNA under the control of the mouse mammary tumor virus enhancer/promoter became pregnant but failed lactation. TGF alpha mRNA was detected in the mammary glands of these mice by the reverse transcriptase-polymerase chain reaction. By the use of collagenase-dissociated mammary epithelial cells, the binding of prolactin to its receptor was determined before and after parturition. At the end of pregnancy, the binding in TGF alpha transgenic (TGF alpha [+]) mice was small and its amount was comparable to that in the TGF alpha negative (TGF alpha [-]) mice. On the day of parturition, prolactin binding in TGF alpha (+) mice increased approximately 1.9-fold (insignificant), while that in TGF alpha (-) mice elevated over 5.3-fold (P < 0.01). The binding sites per cell were also higher in TGF alpha (-)mice. Radioimmunoassay of prolactin suggested that in TGF alpha (+) mice the low level of prolactin binding after parturition was not due to masking effect of serum prolactin. Among six TGF alpha (+) mice assayed, one mother with the highest prolactin binding activity (3.7-fold increase) initiated lactation, but the others did not. As there was little difference between groups in the growth and synthesis in the mammary glands, it was concluded that the failure of lactation in TGF alpha (+) mice is principally due to the lack of elevation of mammary prolactin receptor after parturition. At present, the role of TGF alpha in this process is obscure; however, TGF alpha was revealed not to interfere with the binding of prolactin to the receptor.

Influence of repeated cocaine exposure on the endocrine and behavioral responses to stress in rats.

Previous studies have determined that chronic cocaine exposure inhibits the serotonergic stimulation of hormone secretion. The present experiments were conducted to determine whether the endocrine responses to stress could be a useful approach to assess the influence of cocaine exposure on neuronal function. Male rats received twice daily injections of cocaine (1-15 mg/kg, IP) for 7 days. Animals were subsequently exposed to different stressors, i.e. conditioned emotional stress utilizing a low (0.5 mA) or high (1.5 mA) intensity footshock during training, or to immobilization stress. Immediately after the stress procedures, blood samples were collected for radioimmunoassay of plasma corticosterone, prolactin, and renin concentrations. Repeated cocaine exposure attenuated the stress-induced elevations of corticosterone and prolactin secretion, and attenuated some of the behavioral effects of the low intensity conditioned emotional stress. When exposed to the high intensity conditioned emotional stress, cocaine did not alter the endocrine or behavioral effects of stress. Finally, repeated cocaine exposure modified the immobilization stress-induced elevation of renin secretion; low doses of cocaine (1 or 5 mg/kg) attenuated, while higher doses (10 mg/kg) potentiated the renin response to immobilization stress. Thus, the influence of repeated cocaine exposure on the endocrine and behavioral responses to stress appears to depend upon the type and intensity of the stressor. Compared with previous studies which found altered neuroendocrine responses to serotonin releasers and agonists following cocaine exposure, the hormonal responses to stress are less consistently modified by cocaine.

Development of homologous radioimmunoassays for equine growth hormone and equine prolactin and their application to the detection of circulating levels of hormone in horse plasma

Highly purified and well-characterised preparations of equine prolactin and growth hormone from equine pituitary glands were employed to set up highly sensitive and specific homologous radioimmunoassays (RIA) for the measurement of hormone in horse plasma. The limit of sensitivity of the GH RIA was 1.2 ng/ml with mean intra- and inter-assay coefficients of variation (CV) of 6.6 and 10%, respectively. The sensitivity of the equine prolactin (ePRL) RIA was 0.5 ng/ml with mean intra and inter-assay CV of 9.1 and 15.6%, respectively. Dose-response curves of a crude pituitary gland extract and plasma samples collected from a mare and foal were parallel to the standards and the PRL RIA was clinically validated by administration of thyrotropin-releasing hormone (TRH). Plasma samples taken at 15 min intervals over 24 h from lactating mares gave 24 h mean GH values in the range 5.5 to 7.95 ng/ml. Large intermittent elevations of GH activity were detected. The mean 24 h PRL concentrations were between 3.2-10.4 ng/ml in the lactating animals, with higher concentrations earlier in lactation. Long episodic bursts of PRL were detected.

Prolactin bioassay before and after treatment with nortriptyline

Serum prolactin has been measured in patients with depression before and after treatment with antidepressants. Some, but not all, investigators have reported increased prolactin levels; however, these have been measured by radioimmunoassay (RIA) only. We investigated both serum prolactin RIA and the rat lymphoma Nb2 (Noble strain 2) bioassay for prolactin in 13 patients with major depression before and 1 to 2 weeks after treatment with nortriptyline. There were no significant differences with either measure. The relative Nb2 bioactivity of prolactin appears to be unaffected by treatment with nortriptyline.

Purification, partial characterization, and radioimmunoassay of prolactin and growth hormone from the Bennett's wallaby

Bennett's wallaby prolactin (wPRL) and growth hormone (wGH) were purified from an aqueous extract of pituitary glands. The extract from 202 glands (6.5 g wet wt) was processed by gel filtration on Sephadex G-100, gel filtration on Sephadex G-100 SF, and then anion-exchange chromatography on DEAE-Sepharose CL-6B. The yields of wPRL and wGH were 5.2 and 15.7 mg, respectively. Since recovery of wPRL from the anion exchange column was 10%, anion exchange was performed in the presence of 20% acetonitrile in a subsequent purification. Recovery from this column was markedly increased to 42%. The purified hormones each gave a single vand on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight under reducing conditions of 21,000 and 23,000 for GH and PRL, respectively. Each hormone was positively identified by its N-terminal amino acid sequence, which showed high sequence identity with the equivalent eutherian hormone. Semianalytical gel filtration of purified hormone was used to demonstrate that each hormone remained as a monomer in aqueous solution. Each purified hormone was tested in the heterologous PRL radioimmunoassay (RIA) which has been used in many earlier studies to measure marsupial PRL. Highly purified wPRL was less potent than ovine prolactin (5.3 compared with 1.5 ng/ml at 50% displacement) and the cross-reaction of wGH was <0.01%. Antibodies were raised against wPRL and wGH and a homologous RIA was developed for each hormone. The sensitivity of the wPRL assay was 0.8 ng/ml which is similar to that of the heterologous PRL assay. Cross-reaction with a number of eutherian pituitary hormones or wGH was <0.07%. The wGH assay detected 0.8 ng/ml, cross-reacted with GH from several eutherian species, and showed low cross-reaction with wPRL (<0.5). In both the wPRL and wGH assays, pituitary homogenates from several species of marsupial diluted in parallel with the wallaby standard, suggesting that these assays will be of use in studies of a number of marsupial species.

Inhibition of pituitary bioactive prolactin secretion in the male rat by the glucocorticoid agonist decadron phosphate.

In the present studies, the soluble glucocorticoid agonist, decadron phosphate (DEC), was administered i.v. to intact adult male rats in order to evaluate the effects of glucocorticoid receptor stimulation on circulating levels of immunoreactive (ir-) and bioactive (bio-) prolactin (PRL). In light of reports that glucocorticoid-specific receptors exist within the rat brain, additional experiments investigated the effects of intracerebroventricular (i.c.v.) administration of graded doses of the same drug on pituitary ir- and bioPRL secretion. Concentrations of ir- and bioPRL in samples obtained before and after drug treatments were determined by standard PRL radioimmunoassay and the Nb2 rat node lymphoma bioassay, respectively. Rats injected i.v. with 0.5 mg DEC/kg body weight, but not those treated with a tenfold lesser dose, exhibited decreased plasma irPRL concentrations. However, both doses promoted a decline in circulating levels of bioPRL compared to vehicle-treated controls, along with an overall reduction in the plasma bio/irPRL ratio. The magnitude and duration of this drug-induced decline in biopotency of secreted hormone was dose-dependent. While the plasma bio/irPRL ratio was diminished only transiently in rats injected with 0.05 mg DEC/kg, treatment with the higher dose led to a sustained decrease in the plasma bio/irPRL ratio for the duration of the experiment. The current studies also show that intracerebral administration of DEC resulted in dose-dependent alterations in pituitary PRL release. Circulating levels of ir- and bioPRL were not altered in rats injected i.c.v. with 10 ng of DEC, the lowest dose tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuroendocrine responses to cocaine do not exhibit sensitization following repeated cocaine exposure

Cocaine-induced enhancement of motor activity and extracellular dopamine concentrations exhibits sensitization upon repeated exposure. In this study, the neuroendocrine responses to cocaine were examined following cocaine pretreatment regimens which have been shown to produce behavioral sensitization. Adult male rats were injected with cocaine (15 mg/kg, IP) once daily for 14 days, followed by a dose-response challenge with cocaine (1–15 mg/kg, IP) either 18 hours or 7 days after the final pretreatment injection. Blood was collected 15 minutes following injections for radioimmunoassay of ACTH, corticosterone, prolactin, and renin. Cocaine increased plasma ACTH and corticosterone, while it decreased prolactin and renin concentrations. Pretreatment with cocaine for 2 weeks did not alter any of these endocrine responses after either the 18 hour or 7 day interval between pretreatment and challenge injections. In contrast, sensitization to the locomotor stimulant effects of cocaine was observed on the final day of pretreatment injections, and 7 days later. These data suggest that these endocrine effects of cocaine do not exhibit sensitization following repeated cocaine exposure.

Fabrication biocompatible de microcapsules par polymérisation ensemencée autour de microsphères d'agarose

AbstractA new process for microencapsulating cells is described. Cells in solution in agarose were extruded from a syringe into a paraffin‐oil containing medium. Solid microspheres were produced when the agarose gelled. A rigid interface around each bead was produced by polymerization of a latex‐seeded aqueous solution of a mixture of 5 wt.‐% acrylamide and 0,25 wt.‐% N,N‐methylenediacrylamide. Cell viability was demonstrated by radioimmunoassay of prolactin liberated from encapsulated pituitary cells.