Comparison of potential nuclear precursors for prolactin and growth hormone messenger RNA

Recombinant DNA plasmids containing the coding sequence for rat prolactin or rat growth hormone have been used to investigate the presence of possible precursors for prolactin and growth hormone mRNA. Cytoplasmic and nuclear RNA was prepared from either rat pituitaries or fromthe GC pituitary cell line. RNA was electrophoresed on agarose gels containing methylmercury hydroxide and then transferred to diazobenzyloxymethyl paper. The paper was then hybridized to prolactin or growth hormone recombinant DNA probes labeled in vitro with 32P. The prolactin probe hybridized to RNA species of 7.0, 6.4, 3.8, 1.7, and 1.0 kilobases in nuclear RNA and only to a 1.0-kilobase species in cytoplasmic RNA. Hybridization with a growth hormone probe demonstrated nuclear RNA species of 6.7, 5.6, 2.3, and 1.0 kilobases. These findings demonstrate the presence of multiple species of prolactin and growth hormone RNA which are larger larger than the mature cytoplasmic mRNAs. The large nuclear RNAs are likely precursors for prolactin and growth hormone mRNA.     

Comparison between the time course of changes in nerve growth factor protein levels and those of its messenger RNA in the cultured rat iris

In previous experiments, it has been demonstrated that, in rat irides in culture, a rapid increase in nerve growth factor (NGF) levels occurred (see Barth, E.-M., Korsching, S., and Thoenen, H. (1984) J. Cell Biol. 99, 839-843). We have now determined the levels of mRNANGF in rat irides as a function of time in culture as well. After an initial lag period of 2 h, mRNANGF levels were transiently increased, so that after 12 h, they had increased 35-fold with respect to zero time. In contrast, poly(A)+ RNA levels dropped to 55% of the zero time values within 5 h, recovered to 85% after 24 h, and remained constant until the end of the observation period. Total ribosomal RNA was found to remain constant, indicating that there was no nonspecific decline of overall metabolic function. Actinomycin D prevented the increase in mRNANGF without reducing the basic mRNANGF levels over a 5-h time period, indicating that the enhanced synthesis of NGF in the rat iris in culture is primarily mediated by an augmented production of mRNANGF. The increases of mRNANGF, cellular NGF, and NGF released into the medium were found to be strictly sequential. Monensin selectively abolished the increased production of mature NGF (see Barth et al.) but not of mRNANGF, suggesting that the processing of NGF precursor is prevented.     

The effects of partial opiate agonists on plasma prolactin and growth hormone levels in the rat.

Opiate agonists, partial agonists, and antagonists differed in their effects on release of prolactin and growth hormone. Agonists (morphine, methadone or meperidine) elevated plasma levels of both hormones. An antagonist (naloxone) lowered levels of prolactin but not growth hormone. All partial agonists studied raised growth hormone levels; among these, levallorphan, nalorphine, and ciramadol lowered prolactin levels while pentazocine and meptazinol did not. Naloxone blocked morphine-induced release of prolactin and growth hormone. The partial agonists suppressed morphine-induced prolactin release, and several suppressed the elevated growth hormone levels as well. Data from the opiate radioreceptor assay (displacement of ³H-naloxone) in the presence and absence of sodium agrees with the above placement of agents into three classes. These results suggest that classification of opioid compounds into agonists, partial agonists and antagonists may be made by their effects on prolactin and growth hormone release.     

Age-related changes in renal and hepatic cellular mechanisms associated with variations in rat serum thyroid hormone levels

Aging is associated with changes in thyroid gland physiology. Age-related changes in the contribution of peripheral tissues to thyroid hormone serum levels have yet to be systematically assessed. Here, we investigated age-related alterations in the contributions of the liver and kidney to thyroid hormone homeostasis using 6-, 12-, and 24-mo-old male Wistar rats. A significant and progressive decline in plasma thyroxine occurred with age, but triiodothyronine (T(3)) was decreased only at 24 mo. This was associated with an unchanged protein level of the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) in the kidney and with a decreased MCT8 level in the liver at 24 mo. Hepatic type I deiodinase (D1) protein level and activity declined progressively with age. Renal D1 levels were decreased at both 12 and 24 mo but D1 activity was decreased only at 24 mo. In the liver, no changes occurred in thyroid hormone receptor (TR) TRalpha(1), whereas a progressive increase in TRbeta(1) occurred at both mRNA and total protein levels. In the kidney, both TRalpha(1) and TRbeta(1) mRNA and total protein levels were unchanged between 6 and 12 mo but increased at 24 mo. Interestingly, nuclear TRbeta1 levels were decreased in both liver and kidney at 12 and 24 mo, whereas nuclear TRalpha(1) levels were unchanged. Collectively, our data show differential age-related changes among hepatic and renal MCT8 and D1 and TR expressions, and they suggest that renal D1 activity is maintained with age to compensate for the decrease in hepatic T(3) production.     

Changes in growth hormone and prolactin messenger ribonucleic acid levels during seawater adaptation of amago salmon (Oncorhynchus rhodurus).

To examine the changes in secretion of growth hormone (GH) and prolactin (PRL) with reference to their osmoregulatory roles, changes in pituitary mRNA levels and plasma concentrations of these hormones were examined during seawater adaptation in silvery juveniles (smolts) and precociously mature males (dark parr) of amago salmon (Oncorhynchus rhodurus). Transfer to seawater increased plasma sodium levels in both smolts and dark parr. Smolts adjusted their plasma sodium to the level associated with seawater-adaptation (165 mEq/liter) within 3 days, whereas no adjustment was seen in dark parr; the latter failed to survive in seawater for more than 3 days. In smolts, plasma GH levels increased significantly 1 day after transfer, whereas there was no significant change in dark parr. An increase in GH mRNA levels was observed in smolts in association with increased plasma GH, whereas there was no change in dark parr. In contrast, a reduction in plasma PRL levels was consistently observed in both smolts and dark parr after transfer to seawater. However, there was no significant change in PRL mRNA levels in either smolts or dark parr. These results suggest that both gene expression and release of GH are activated by seawater transfer only in smolts with adequate seawater adaptability, whereas PRL gene expression is decreased after seawater transfer regardless of seawater adaptability.     

Ultrastructural detection of messenger RNA coding for growth hormone in the rat anterior pituitary by in situ hybridization

Ultrastructural detection of the messenger RNA coding for growth hormone in rat pituitary gland could be obtained by association of in situ hybridization and cryoultramicrotomy. Messenger RNAs were localized in the anterior pituitary gland. Silver grain densities observed in autoradiograms after in situ hybridization were dependent to incubation period and to fixation. It was necessary to determine a compromise between ultrastructural aspect and silver grain densities. Messenger RNAs were detected in somatotropic cells, identified by ultrastructural characteristics, in both the nucleus (euchromatin and nuclear membrane) and cytoplasm, in vicinity to endoplasmic reticulum.     

Thyroid hormone regulates type I deiodinase messenger RNA in rat liver

Conversion of the prohormone T4 to the active hormone T3 is catalyzed by 5'-deiodinases, enzymes that have not been purified. Previous studies have shown that modulating thyroid status results in changes in type I deiodinase activity in the rat liver. We have quantitated type I deiodinase mRNA in liver by an expression assay using Xenopus laevis oocytes. We report here that changes in enzyme activity correlate closely with changes in levels of the mRNA for this enzyme, indicating that thyroid hormone regulates type I deiodinase at a pretranslational step. Using the oocyte system to express size-fractionated mRNA, we have also determined that the mRNA coding for this protein is between 1.9-2.4 kilobases in length. It has been proposed that protein disulfide isomerase (PDI) is closely related to the rat type I 5'-deiodinase. Our results indicate that this is not the case, since injection of in vitro transcribed PDI mRNA into oocytes did not result in expression of deiodinase activity, and the deiodinase mRNA could be physically separated from the 2.8-kilobase mRNA species hybridizing to rat PDI cRNA by size fractionation.     

Reduction of T-kininogen messenger RNA levels by dexamethasone in the adjuvant-treated rat

When inflammation is induced in rats following injection of Freund's complete adjuvant, steady state levels of T-I and T-II kininogen mRNAs increase markedly as do plasma levels of T-I and T-II kininogens. When rats are additionally treated with dexamethasone, T-I and T-II steady state mRNA levels and plasma levels of T-kininogens are reduced. The results suggest that dexamethasone may affect the magnitude of T-kininogen gene induction caused by inflammation.     

Developmental,stage-specific,and hormonally regulated expression of growth hormone secretagogue receptor messenger RNA in rat testis

Recent evidence from our research suggested the direct role of ghrelin in the control of testicular function. However, the pattern of expression and hormonal regulation of the gene encoding its cognate receptor (i.e., the growth hormone-secretagogue receptor [GHS-R]) in the male gonad remains to be fully elucidated. In this paper, overall expression of GHS-R mRNA in rat testis was compared with that of the functional receptor form, namely GHS-R type 1a, in different developmental and experimental settings. In addition, cellular distribution of GHS-R within adult testis tissue was assessed. Our analyses demonstrated persistent expression of the GHS-R gene in rat testis throughout postnatal development. In contrast, testicular expression of GHS-R type 1a mRNA remained undetectable before puberty and sharply increased thereafter. In adult testis, GHS-R1a mRNA expression presented a scattered pattern of cellular distribution, including Sertoli and Leydig cells that also showed specific GHS-R1a immunoreactivity. Expression of total GHS-R and specific GHS-R1a mRNAs was detected in isolated seminiferous tubule preparations, with varying levels throughout the defined stages of the spermatogenic cycle. In addition, testicular expression of total GHS-R and GHS-R1a mRNAs was up-regulated by exposure to ghrelin in vitro and after stimulation with FSH in vivo. In conclusion, our data demonstrate that expression of the GHS-R gene in rat testis takes place in a developmental, stage-specific, and hormonally regulated manner. Divergent expression of total GHS-R and type 1a specific mRNAs was detected at certain stages of postnatal development and spermatogenic cycle, thus raising the possibility that, in addition to net changes in GHS-R gene expression, the balance between receptor subtypes may represent a novel mechanism for the tuning of ghrelin sensitivity in rat testis.     

Age-related differences in the pituitary prolactin response to thyrotropin-releasing hormone

We have previously reported that human subjects undergoing surgery for inguinal hernias exhibit an age-related attenuation in the plasma prolactin response, with no differences during resting conditions. We suggested that these differences were due to age-related neuroendocrine changes, but that peripheral factors may play a role as well. In the present study, we have assessed the pituitary response to 500 micrograms of thyrotropin-releasing hormone (TRH) in the very same subjects previously studied during surgery. Blood samples were drawn immediately prior to, as well as 10, 20, 40 and 60 minutes following the intravenous administration of TRH. There was a clear-cut age-related attenuation in the pituitary prolactin response with no difference in the thyrotropin (TSH) response. Maximum prolactin response in the young subjects was 31.7 micrograms/l and 19.2 micrograms/l in old subjects (F(4) = 3.5, p less than .01, two-way ANOVA). These results indicate that the age-related differences in the prolactin response to stress are mainly due to pituitary changes. However, prolactin-secreting cells are under the control of the hypothalamus. Therefore, the possibility must be considered that aging or other concurrent factors could be exerting their influence via the hypothalamus and not necessarily directly at the pituitary level.     

Developmental expression of corticotropin releasing hormone messenger RNA and peptide in rat hypothalamus

We have examined corticotropin releasing hormone (CRH), arginine vasopressin (AVP) and somatostatin (SOM) mRNA expression and peptide content in the rat hypothalamus from day 20 of fetal life (F20) to the fifteenth day of postnatal life (P15). During this time, hypothalamic CRH mRNA levels did not change significantly, whereas there was a gradual six-fold rise in CRH peptide levels. AVP mRNA levels fell three-fold between F20 and P1 and increased six-fold between P1 and P15. AVP peptide levels increased three-fold, with most of the rise occurring between P1 and P15. From F20 to P15, SOM mRNA and peptide levels rose four- and eight-fold, respectively. The changes in the levels of these three hypothalamic gene products correlate with the previously described alterations in the responsiveness of the HPA axis observed in fetal and early postnatal rats, suggesting a role for these neuropeptides in the modulation of the HPA axis during this developmental period.     

Retinol-binding protein messenger RNA levels in the liver and in extrahepatic tissues of the rat

A retinol-binding protein (RBP) cDNA clone was used to examine the effect of retinol status on the level of RBP mRNA in the liver, and to explore whether extrahepatic tissues contain RBP mRNA. In the first series of experiments, poly(A+) RNA was isolated from the livers of normal, retinol-depleted, and retinol-repleted rats and the levels of RBP mRNA in these samples were determined by both Northern blot and RNA Dot blot analyses. The levels of RBP mRNA in liver were similar in all three groups of rats. These findings confirm and extend previous studies which showed that retinol did not alter the in vivo rate of RBP synthesis or the translatable levels of RBP mRNA. In a second series of experiments, the RBP cDNA clone was used to survey poly (A+) RNA isolated from 12 different rat tissues for RBP mRNA by Northern blot analysis. We found that, along with the liver, many extrahepatic tissues contained RBP mRNA. Kidney contained RBP mRNA at a level of 5-10% of that of the liver, and the lungs, spleen, brain, stomach, heart, and skeletal muscle contained 1-3% of that of the liver. Translation of kidney poly (A+) RNA in rabbit reticulocyte lysates and immunoprecipitation of the translation products with anti-RBP antiserum resulted in a protein band of the same size as liver preRBP. These data suggest that RBP is synthesized in many extrahepatic tissues.It is possible that this extra-hepatically synthesized RBP may function in the recycling of retinol from these tissues back to the liver or to other target organs.     

TSH regulation of ferritin H chain messenger RNA levels in the rat thyroids

Ferritin heavy chain mRNA steady state levels are increased by thyrotropin both in vivo and in two independent thyroid derived permanent cell lines. Maximum induction was achieved 48 hours after thyrotropin addition in the same conditions in which all the thyroid differentiated functions were stimulated. Thyrotropin stimulation of the levels of ferritin heavy chain mRNA seems to be mediated by cyclic AMP since it mimics the hormone induction.