Prolactin receptor localization to the area postrema.

The area postrema, which lacks a blood-brain barrier, was examined for the presence of prolactin receptors, which would render it a potential site for vascular prolactin to directly interact with neuronal elements. Using an in vitro autoradiographic technique, frozen sections of New Zealand white rabbit medulla were incubated with radiolabelled ovine prolactin alone (total binding) or radiolabelled ovine prolactin in the presence of excess unlabelled ovine prolactin (non-specific binding). The specificity of the binding was also assessed using excess unlabelled human prolactin or ovine LH. While excess unlabelled ovine and human prolactin caused a statistically significant reduction in radio labeled prolactin binding, unlabelled LH was without effect. Results reveal the presence of specific prolactin binding sites within the area postrema, a previously unknown prolactin target area of the CNS.

Placental lactogen binding sites in the pregnant rabbit choroid plexus.

Prolactin has direct effects on the CNS. The highest concentration of prolactin receptors resides within the choroid plexus where they probably function to transport prolactin from blood into CSF. Another member of the lactogen family of hormones, placental lactogen (PL), also affects CNS activity and may similarly employ the cerebroventricular system as an intermediary. In order to determine whether the choroid plexus was a PL target tissue, in vitro autoradiography was used to identify specific PL binding sites in the choroid plexus of pregnant New Zealand White rabbits. Frozen brain sections were incubated in a medium containing 125I human PL (hPL) alone (total binding) or with a 500-fold excess of unlabelled hPL (nonspecific binding). The specificity of the binding was assessed with unlabelled human growth hormone (hGH) and ovine luteinising hormone (oLH). An intense autoradiographic reaction occurred over the choroid plexus of tissue sections incubated with 125I hPL alone. Excess unlabelled hPL and hGH, which is lactogenic in the rabbit, caused a significant reduction (P < 0.001) in the binding of radiolabelled hPL to the choroid plexus. In contrast, unlabelled oLH had no effect on radiolabelled hPL binding to this tissue. The results support a role for the choroid plexus in the interactions between PL and the CNS.

Prolactin enhancement of its own uptake at the choroid plexus.

The choroid plexus contains PRL receptors that function in part to transport PRL from the blood into the cerebrospinal fluid (CSF). The blood PRL concentration of female rats was altered by 1) three daily injections of haloperidol (chronic hyperprolactinemia) with or without bromocriptine administration 4 h before death, 2) bromocriptine alone for 4 h (acute hypoprolactinemia), and 3) a single vascular injection of ovine PRL (acute hyperprolactinemia). Changes in the uptake of PRL by the choroid plexus was assessed by quantitative in vivo autoradiography after the injection of radiolabeled PRL. Correlation of changes in PRL uptake at the choroid plexus with changes in PRL transport from blood to CSF was evaluated by subjecting CSF samples to sodium dodecyl sulfate-polyacrylamide gel electrophoresis after vascular injection of radiolabeled PRL. Autoradiography revealed that both chronic and acute hyperprolactinemia resulted in a significant increase in the uptake of radiolabeled PRL by the choroid plexus compared to that in untreated control animals. In contrast, bromocriptine had no effect on PRL uptake at the choroid plexus relative to that in control (untreated) animals. Chronic hyperprolactinemia, but not acute hyperprolactinemia, resulted in a significant increase in the transport of radiolabeled PRL from the blood to the CSF compared to that in untreated controls. The results are consistent with the up-regulation of PRL receptors in the choroid plexus by circulating PRL and the consequent augmentation of transport of PRL from blood to CSF.

The distribution of lactogen receptors in the mammalian hypothalamus: an in vitro autoradiographic analysis of the rabbit and rat.

The hypothalamus contains a high concentration of lactogen receptors as detected with in vitro radioreceptor assay techniques. In an effort to define the location of the lactogen receptors relative to specific hypothalamic nuclei, an in vitro autoradiography technique was applied to frozen sections of rat and rabbit brains. Three lactogenic hormones, i.e. human growth hormone (hGH), ovine prolactin (oPRL), and rat prolactin (rPRL), were radiolabeled with iodine-125. Competition for observed binding sites was assessed with unlabeled hGH, oPRL, and bovine growth hormone (bGH). Analysis of the autoradiographs with a microcomputer-based densitometry system revealed that the rabbit hypothalamus contains specific lactogen binding sites within the supraoptic, paraventricular, suprachiasmatic, ventromedial, arcuate, and dorsomedial nuclei and the medial preoptic area. Unlabeled bGH was effective in competing for binding sites in all areas when hGH but not oPRL was used as the radiolabeled ligand, suggesting the presence of growth hormone receptors in the rabbit hypothalamus with a distribution similar to that of the lactogen binding sites. In contrast to the rabbit, no lactogen binding sites were detected in the rat hypothalamus regardless of the ligand used in the assay. All of the ligands were successful, however, in detecting lactogen receptors within the rat choroid plexus and liver. The results from the rabbits indicate that the influences of prolactin on hypothalamic activity are mediated via lactogen receptors that are widely distributed throughout the various pertinent hypothalamic nuclei. The broad distribution of lactogen receptors in the rabbit hypothalamus attests to the extensive influence of prolactin on hypothalamic regulatory systems. The results from the rat raise questions as to the nature of rat brain prolactin receptors in comparison to prolactin receptors in rat peripheral tissues.

Prolactin receptors in the primate choroid plexus.

The presence of prolactin receptors in the choroid plexus of primates has until now been assumed to be based on observations made on lower vertebrate animal models. An in vitro autoradiographic technique employing the principles of competitive binding was used to demonstrate the presence of specific prolactin binding sites in the choroid plexus of monkeys. Thus the primate choroid plexus resembles that of lower vertebrate mammals in that it possesses the prerequisite receptor for a prolactin blood-to-CSF transport mechanism.

Development of peroxisomal beta-oxidation activities in brown fat of perinatal rabbits.

The development of peroxisomal activities in brown fat was analyzed in perinatal rabbits, 25- and 30-day fetuses, newborns, 5- and 10-day-old pups. Purified peroxisomal fractions were obtained from brown fat homogenates by sucrose gradient centrifugation at a density of 1.22 g/cm3. Enzymes specifically associated with the isolated peroxisomes, acyl-CoA oxidase, catalase and KCN-insensitive beta-oxidation, were most active in 25-day fetuses and much less active after birth. Mitochondrial-associated activities such as cytochrome c oxidase were greatest at birth. The beta-oxidation enzymes, enoyl-CoA hydratase, beta-hydroxyacyl-CoA dehydrogenase, and thiolase were present in mitochondrial as well as peroxisomal fractions. Nevertheless, the levels of these three enzymes in peroxisomes were higher in fetuses. Thus, peroxisomes, in contrast to mitochondria, were most active prior to birth. This suggests that peroxisomes do not participate in brown fat thermogenesis but that they have some role in priming the development of brown fat in perinatal rabbits.

Magnesium-dependent non-specific binding of [125I]prolactin to myelinated tracts in the rat central nervous system.

An in vitro autoradiographic assay was used in identifying a magnesium-dependent, non-specific binding of [125I] prolactin to myelinated fiber tracts in the rat brain. Frozen tissue sections were incubated for 18 h at 4 degrees C in media which included [125I]prolactin alone or with a 500 fold excess of unlabelled prolactin. Magnesium in the incubation medium caused a non-specific binding of radiolabelled prolactin to the myelinated fiber tracts in the brain. In contrast, calcium did not facilitate prolactin non-specific binding to myelin. Hence, calcium should optimize the detection of specific prolactin binding sites in the brain by in vitro autoradiographic or radioreceptor assays.