Prolactin: the initial luteotropic stimulus of pseudopregnancy in the rat

The luteotropic stimuli necessary to transform the corpus luteum of the estrous cycle into a corpus luteum of psuedopregnancy on the morning of diestrus-2 (Day 2), as reflected by a dramatic divergence in progesterone secretion, were studied (Day 1 was taken as the first day of diestrus of pseudopregnancy). The requirement of prolactin (PRL) as a luteotropic stimulus was determined by inhibiting the diurnal and nocturnal PRL surges that occur immediately before and during the divergence in progesterone. Following cervical stimulation, 1 mg of 2-Br-alpha-ergocryptine (EC) was injected at 1100 and 2300 h on Day 1 (lights on 0600-1800 h), and the animals were decapitated at 2-4 h intervals from 1100 h on Day 1 to 1700 h on Day 2. In the control animals, the PRL surges on Day 1 and Day 2 were associated with an increase in progesterone secretion on Day 2. However, the regimen of EC treatment resulted in an inhibition of PRL surges, prolactin remaining at baseline values from 1100 h on Day 1 to 1700 h on Day 2. The inhibition of PRL secretion was associated with a fall in progesterone concentration to reach baseline values by 1700h on Day 2. Furthermore, a group of animals similarly treated with EC returned to vaginal estrus 2 days later. LH concentrations did not differ in control and EC-treated animals. The effect of EC on corpus luteum function could be completely reversed by the simultaneous administration of PRL. In addition, if PRL was administered at 1100 h and 2300 h on diestrus-1 of the estrous cycle, in an attempt to mimic the surges os pseudopregnancy, regression of the corpora lutea did not occur. Progesterone levels increased to reach values comparable to those observed in pseudopregnancy on diestrus-2. The role of LH was studied by administering a dose of LH antiserum at 110 and 2300 h on Day 1 of pseudopregnancy. This treatment failed to inhibit the increase in progesterone observed on Day 2. These results demonstrate that the surges of plasma PRL initiated by cervical stimulation are responsible for transforming a corpus luteum of the estrous cycle into a corpus luteum of pseudopregnancy, as reflected by an increase in progesterone secretion of Day 2. LH seems to have a minor role in maintaining corpus luteum function beyond that observed during the estrous cycle.     

Phosphorylation sites in bovine rhodopsin

Bovine rhodopsin has been phosphorylated in rod outer segments by ATP and endogenous rhodopsin kinase. Mono-, di-, and triphosphorylated rhodopsins have been prepared by chromatofocusing. Nearly all of the phosphate is found in peptide 330-348, formed by digestion of phosphorhodopsins with endoproteinase Asp-N. Sequence analysis of the phosphopeptides shows that monophosphorylated rhodopsin consists of a mixture containing rhodopsins phosphorylated at 338Ser and 343Ser. Diphosphorylated rhodopsin is phosphorylated at both 338Ser and 343Ser. When rhodopsin becomes triphosphorylated it does not become phosphorylated on 334Ser but appears to become phosphorylated on one or more of the four threonine residues: 335Thr, 336Thr, 340Thr, and 342Thr.     

Phosphorylation of myosin regulatory light chain eliminates force-dependent changes in relaxation rates in skeletal muscle

The rate of relaxation from steady-state force in rabbit psoas fiber bundles was examined before and after phosphorylation of myosin regulatory light chain (RLC). Relaxation was initiated using diazo-2, a photolabile Ca2+ chelator that has low Ca2+ binding affinity (K(Ca) = 4.5 x 10(5) M(-1)) before photolysis and high affinity (K(Ca) = 1.3 x 10(7) M(-1)) after photolysis. Before phosphorylating RLC, the half-times for relaxation initiated from 0.27 +/- 0.02, 0.51 +/- 0.03, and 0.61 +/- 0.03 Po were 90 +/- 6, 140 +/- 6, and 182 +/- 9 ms, respectively. After phosphorylation of RLC, the half-times for relaxation from 0.36 +/- 0.03 Po, 0.59 +/- 0.03 Po, and 0.65 +/- 0.02 Po were 197 +/- 35 ms, 184 +/- 35 ms, and 179 +/- 22 ms. This slowing of relaxation rates from steady-state forces less than 0.50 Po was also observed when bundles of fibers were bathed with N-ethylmaleimide-modified myosin S-1, a strongly binding cross-bridge derivative of S1. These results suggest that phosphorylation of RLC slows relaxation, most likely by slowing the apparent rate of transition of cross-bridges from strongly bound (force-generating) to weakly bound (non-force-generating) states, and reduces or eliminates Ca2+ and cross-bridge activation-dependent changes in relaxation rates.     

Modulation of prolactin receptors in the rat hypothalamus in response to changes in serum concentration of endogenous prolactin or to ovine prolactin administration

Specific binding of 125I-labeled rat prolactin (125I-rat PRL) to hypothalamic membranes was studied in Sprague-Dawley rats after ovine PRL administration and in relation to rat PRL serum variations induced by ectopic pituitary implants or by drugs which stimulate (domperidone) or inhibit (bromocriptine) PRL release. Repeated treatments with ovine PRL markedly increased specific binding values of 125I-rat PRL to hypothalamic membranes of female rats. Repeated treatments with domperidone also increased specific PRL binding in the hypothalamus. This effect was associated with an increase in PRL serum levels. Similar results were obtained in male rats after renal pituitary implants which resulted in a state of chronic hyperprolactinaemia. In contrast, a subchronic treatment with bromocriptine decreased specific PRL binding in the hypothalamus and concomitantly caused a sharp reduction in PRL serum levels. Scatchard analysis of data obtained from competition curves showed that the variations in the level of PRL binding to hypothalamic membranes were related to the number of PRL binding sites but not to the dissociation constant (Kd), which was unaffected by different treatments or by pituitary implantation. These results demonstrate a correlation between circulating concentrations of PRL and number of its receptors in the rat hypothalamus and give further support to the hypothesis that these binding sites may have a specific functional role in regulating the homeostasis of pituitary PRL secretion.     

Enhancement by prolactin of carcinogen induced mammary cancerigenesis in the male rat

Mammary tumours were induced in 3 groups of male Long-Evans rats by a series of 6 fortnightly gastric intubations of 7,12-dimethylbenzanthracene. Two weeks before the initial carcinogen treatment one group of rats was grafted with 3 pituitary homografts underneath the kidney capsule of each recipient (hyperprolactinaemia). A second group, 2 weeks before the initial carcinogen treatment and for the duration of the study (35 weeks), were injected 4 X weekly with 2-Br-alpha-ergocryptine (CB-154) (hypoprolactinaemia). A third group of rats served as controls. A significant increase in the incidence of mammary tumours and a reduced latency period of tumour appearance in the hyperprolactinaemia group, when compared with the controls, were observed in this study. Mammary tumour incidence and latency period of tumour appearance in the hypoprolactinaemia group, however, did not differ significantly from controls. Thus, an increased secretion of pituitary prolactin in rats appears to be an important enhancing endocrinic condition in carcinogenesis of the male mammary gland.     

Altered growth hormone and prolactin responsiveness to TRH in the infant rat

12-day-old female and male pups were killed 10 min after the injection of either saline or thyrotropin releasing hormone (TRH), and plasma growth hormone (GH) and prolactin (PRL) levels were measured by radioimmunoassay (RIA). At all doses used (0.15, 0.3, 0.6 and 1.5 mug/100 g b.w.i.p.), TRH induced a significant, although not dose-related, increase in plasma GH levels, but was effective in releasing PRL only at the greatest dose level (1.5 mug/100 g b.w.). The GH-releasing effect of TRH was even more evident in 12-day-old pups subjected to central sympathectomy of 6-hydroxydopamine (6-OHDA, 60 mug/10 mul intraventricular route) 1 week before; in these animals, TRH was ineffective in releasing PRL even at the greatest dose level (1.5 mug/100 g b.w.). In pups pretreated with 6-OHDA, the GH-lowering effect of insulin hypoglycemia or cold exposure was markedly reduced, while the PRL responses were unmodified. Baseline plasma PRL levels were markedly increased following 6-OHDA administration. It is proposed that in the infant rat the greater GH than PRL responsiveness to TRH, which opposed the pattern of response present in the adult animal, may be due to the existence of a 'physiologic' functional disconnection between the central nervous system (CNS) and the anterior pituitary (AP). Results obtained following central sympathectomy by 6-OHDA, which further disrupted CNS-AP links, substantiate this view.     

Phosphorylation of plant proteins and the identification of protein-tyrosine kinase activity in maize seedlings

Phosphotyrosine was found to be 0.5% of the total phosphoamino acids labelled with [32P]orthophosphate in endogenous maize seedlings proteins. Two peaks of protein kinase activity towards phosphorylation of synthetic peptide poly (Glu80, Tyr20) were obtained after chromatography of protein extract of dark-grown etiolated maize seedlings on phosphocellulose. The phosphorylation of synthetic peptide as well as endogenous proteins was strongly stimulated by Mn2+. At least three endogenous proteins with molecular masses in the range of 40-65 kDa were predominantly phosphorylated. This phosphorylation was resistant to alkali treatment. Chemical, immunological and enzymatic data indicated the presence of tyrosine kinase activity and also phosphotyrosine in proteins of maize seedlings. The plant enzyme(s) is reminiscent known mammalian cytosolic tyrosine kinase(s).     

Circadian rhythms of serum renin activity and serum corticosterone, prolactin, and aldosterone concentrations in the male rat on normal and low-sodium diets

To investigate the control of aldosterone secretion, non-stress levels of serum aldosterone, corticosterone, and prolactin, and renin activity were determined at 4-h intervals during 24-h light-dark cycles in adult male rats on regular and low-sodium diets. Circadian rhythms of plasma aldosterone, prolactin, and corticosterone concentrations and of serum renin activity were demonstrated during a regular sodium diet. When the rats were on a low-sodium diet, a circadian rhythm of serum corticosterone and aldosterone concentration was observed, but there was no circadian variation in serum renin activity or in serum prolactin concentration. Serum aldosterone concentration correlated with serum corticosterone concentration (r = 0.48) and serum renin activity (r = 0.36) during a low-sodium diet. Serum prolactin concentration did not correlate with serum aldosterone concentration or serum osmolality. These data are compatible with a role for renin and ACTH, but not for prolactin, in the modulation of aldosterone secretion in the rat.     

Comparison of secreted and extracted forms of rat pituitary prolactin

Prolactin secreted by rat anterior pituitary explants into organ culture medium was purified by salt fractionation and gel filtration. A yield of 22 mg/g was obtained, which clearly represented de novo synthesis and secretion of the hormone. Comparative characterization studies were performed on the secreted prolactin and pituitary extracted rat prolactin obtained from the National Institute of Arthritis, Metabolism and Digestive Disease. The biological and immunological activity estimates of both forms were comparable, although the specific activities of the secreted prolactin were somewhat lower than those of the pituitary prolactin. The secreted and extracted forms of prolactin appeared to be identical in primary structure as evidenced by similar amino acid compositions and identical NH2-terminal sequences. Circular dichroism spectra suggested that there may be differences in tertiary structure, since the positive tryptophan band at 292 nm, which eas observed with extracted hormone, was absent in the secreted prolactin.     

Estrogen receptor expression in bovine and rat retinas

PURPOSE: To investigate the expression and distribution of estrogen receptor protein and mRNA in bovine and rat retinas. METHODS: Western blot analysis with an antiestrogen receptor monoclonal antibody (mAb) was used to detect estrogen receptor protein in the bovine retina. Immunohistochemistry with an antiestrogen receptor mAb and in situ hybridization with an oligodeoxynucleotide sequence coding for estrogen receptor were applied to study the cellular distribution of estrogen receptor protein and its mRNA in male bovine retina and rat retina of both sexes. RESULTS: Estrogen receptor protein was detected in bovine retina by Western blot analysis. Immunohistochemical staining with the antiestrogen receptor mAb was widespread throughout the neural retina. Specific staining showed extensive distribution localizing in the nerve fiber layer, the ganglion cell layer, the inner nuclear layer, and the outer plexiform layer. Retinal pigment epithelium and choroid were also stained with the antiestrogen receptor mAb. By in situ hybridization, the expression of estrogen receptor mRNA was predominantly observed in ganglion cell layer, the inner nuclear layer, and outer portion of the outer nuclear layer. No significant difference was found between male and female rats in the immunostaining of retinas with the antiestrogen receptor mAb. CONCLUSIONS: This study provides the first evidence for the presence of estrogen receptor in bovine and rat retinas. Expression of estrogen receptor throughout the retina suggests that estrogen may have important functions in the retina.     

Multiple transitions to non-B-DNA structures occur in the distal regulatory region of the rat prolactin gene

The developmentally regulated rat prolactin (rPRL) gene presents a promising model system toward understanding the biological role of non-B-DNA structural elements. Two predominantly alternating purine-pyrimidine (APP) (dA-dC)n.(dG-dT)n repeats of 58 and 178 base-pairs flank the (A + T)-rich distal regulatory region. We have characterized several transitions to non-B-DNA structures within this region in negatively supercoiled plasmids by utilizing high resolution chemical probing. Each repeat undergoes a full-length conversion to a novel left-handed helical structure via the stepwise nucleation and propagation of discrete "segments". These segments are delimited by out-of-alternation bases that are susceptible to attack by potassium permanganate and thus appear to be significantly unstacked within the left-handed helices. Moreover, the spatial order of successive right- to left-handed DNA transitions within each repeat exhibits a clear polarity toward the distal regulatory region of the rPRL gene. An additional transition involving the long-range unpairing of (A + T)-rich sequences establishes a directional propagation toward the regulatory region. These data demonstrate a complex series of quasi-independent transitions to non-B-DNA structures that impinge upon a known regulatory control region.     

Analysis of suckling-induced changes in adenohypophyseal prolactin concentration in the lactating rat by three assay methods

Suckling-induced changes in adenohypophyseal prolactin (PRL) concentrations in lactating rats were measured in two experiments by three assays: a disc electrophoretic assay (DEA), a bioassay (BA) and a radioimmunoassay (RIA). Substantial discrepancies among the three assays were found in the absolute amounts of pituitary PRL measured and in the changes in adenohypophyseal PRL concentration that were recorded between suckling intervals. The results indicate that in dynamic states of secretory activity the correspondence among BA, RIA and DEA estimates of pituitary PRL concentration is poor.     

Phosphorylation of triciribine is necessary for activity against HIV type 1

Triciribine (TCN) is a tricyclic nucleoside with known antineoplastic and antiviral activity. It is a potent and selective inhibitor of HIV-1 and HIV-2, including strains known to be resistant to AZT or TIBO. TCN is phosphorylated to its 5'-monophosphate (TCN-P) by intracellular adenosine kinase (AK), but is not converted to di- or triphosphates. We now report that 5'-phosphorylation is requisite for the activity of TCN against HIV-1. CEM cells incubated with TCN at concentrations ranging from 0.1 to 330 microM gave intracellular TCN-P concentrations from 27 to 775 microM, respectively. There was no difference in the amount of intracellular TCN-P detected in uninfected compared with HIV-1-infected CEM cells. The antiviral effect of TCN against HIV-1 was strongly antagonized by the AK inhibitor 5-iodotubercidin (ITu). In contrast, TCN and ITu only exhibited additive cytotoxicity. The 5'-deoxy analog of TCN, which cannot be phosphorylated, had no antiviral effect against HIV-1 at a concentration more than 100 times higher than the IC50 of TCN. Similarly, TCN was not active against HIV-1 in an AK-deficient cell line (AA-2) at concentrations shown to inhibit the virus by >95% in CEM cells. Consistent with its AK-deficient phenotype, this cell line phosphorylated TCN to only 3% of the extent observed in CEM cells. We conclude that TCN must be phosphorylated to TCN-P for activity against HIV-1.     

Termination at midpregnancy of the two daily surges of plasma prolactin initiated by mating in the rat

The pattern of plasma prolactin following uterine cervical stimulation consists of two surges each day, one nocturnal, occurring between 0100-0900 h (lights on 0600-1800 h), and one diurnal, occurring between 1500-2100 h. This pattern of prolactin continued throughout pseudopregnancy; the last surge was observed on the morning of day 11 (day 1 was taken as the first day of diestrus of pregnancy or pseudopregnancy). Prolactin levels remained low thereafter until the spontaneous proestrous surge on the afternoon of day 12, signalling the onset of a new estrous cycle. In contrast, the two daily prolactin surges did not continue throughout pregnancy, and in fact, were terminated sooner in pregnant animals than in pseudopregnant animals. The last diurnal surge was observed on day 8 while the last nocturnal surge was observed on day 10. The early termination of prolactin surges during pregnancy correlated with the increased secretion of rat placental lactogen. However, placental extracts obtained from day 11 of pregnancy and injected in large doses failed to inhibit prolactin surges in pseudopregnant animals. Prolactin surges also continued for a longer period of time in pseudopregnant rats bearing decidualized uteri than in pregnant animals. Thus, the two major components of pregnancy that differed from pseudopregnancy, that is, the presence of rat placental lactogen or decidual tissue, did not appear to account for the early termination of prolactin surges during pregnancy.