Expression of estrogen receptor-beta messenger ribonucleic acid in oxytocin and vasopressin neurons of the rat supraoptic and paraventricular nuclei

The regulatory actions of estrogen on magnocellular oxytocin (OT) and vasopressin (VP) neurons of the paraventricular (PVN) and supraoptic (SON) nuclei are well documented. To date it is still debated whether the effect of estrogens is exerted directly or mediated by estrogen-sensitive interneurons. Previous immunocytochemical (ICC) and in situ hybridization (ISH) studies detected either low levels or absence of the classical estrogen receptor (ER-alpha) in the PVN and the SON of the rat. The present experiments using a combined ICC and ISH method were undertaken to examine the expression of the recently cloned beta form of ER (ER-beta) in OT- and VP-immunoreactive (IR) neuronal systems of the rat hypothalamus. The results demonstrate that the highest cellular levels of ER-beta messenger RNA (mRNA) in OT-IR neurons can be visualized in the caudal portion of the PVN and in an area ventro-medial to the central core of VP-IR cells. These neurons were previously shown to project caudally to the brain stem and the spinal cord to regulate autonomic functions. In addition, the whole rostro-caudal extent of the PVN and the SON contained OT-IR neurons that coexpressed variable levels of ER-beta mRNA. Similarly, the presence of ER-beta mRNA was seen in a large population of VP-IR paraventricular and supraoptic neurons. In the SON, somewhat stronger hybridization signal was detected in VP-IR neurons as compared with OT-IR neurons. Together, these findings provide strong support for the concept that the functions of OT- and VP-IR neurons in the PVN and the SON are regulated directly by estrogen and that the genomic effects of estrogens are mediated by ER-beta.     

Isolation and in vitro translation of zein messenger ribonucleic acid

Zein messenger RNA was isolated from membrane-bound polyribosomes of developing maize kernels by oligo(dT)-cellulose chromatography. Translation of the mRNA in vitro yielded protein similar to native zein in amino acid content, ethanol solubility, and mobility on sodium dodecyl sulfate- polyacrylamide gels. The zein mRNA sedimented as a homogeneous peak on sucrose gradients and contained a poly(A)-rich region based upon hybridization to [3H]poly(U). The mRNA had an apparent molecular weight of 540 000 on agarose-acrylamide gels. It synthesized both 21 800 and 19 000 molecular weight zein components in the wheat-germ cell-free protein synthesis system. The possibility of a polycistronic mRNA or two mRNAs of similar molecular weight is considered.     

In vivo regulation of chromogranin A messenger ribonucleic acid in the parathyroid by 1,25-dihydroxyvitamin D: studies in normal rats and in chronic renal insufficiency

Chromogranin-A (CgA) and PTH are the two major secretory products of the parathyroid gland. In vitro, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] increases CgA, but decreases PTH messenger RNA (mRNA) levels. We investigated the physiological significance of the induced changes in CgA expression by examining the effects of 1,25-(OH)2D3 on parathyroid CgA mRNA levels in vivo. Normal rats were injected with 1,25-(OH)2D3 at 48 and 24 h before blood sampling and isolation of both parathyroid glands. Parathyroid total RNA was extracted and CgA and PTH mRNA quantified by Northern blot analysis. CgA mRNA levels increased 1.6-, 3.2- and 5.6-fold, whereas PTH mRNA levels decreased by 37, 63 and 97%, respectively, with 1,25-(OH)2D3 doses of 10, 50, and 250 pmol/100 g BW. Parathyroid gland CgA expression also was examined in rats with mild chronic renal insufficiency, induced by a 5/6 nephrectomy 5 weeks earlier. Chronic renal insufficiency rats, fed normal chow, had elevated serum urea, creatinine, and PTH levels and reduced 1,25-(OH)2D3 but normal serum levels of calcium and phosphate. PTH mRNA levels were elevated 4-fold and CgA mRNA levels were 50% lower in the uremic animals. This indicates that the regulation of CgA expression in normocalcemic rats occurs at physiological 1,25-(OH)2D3 concentrations. In summary, increases and decreases in serum 1,25-(OH)2D3 levels are associated with corresponding increases and decreases in CgA mRNA levels in the parathyroid glands of rats. Therefore, this study is the first to demonstrate the physiological relevance of the earlier in vitro observations.     

Expression of somatotropin receptor messenger ribonucleic acid in bovine tissues

The somatotropin receptor mRNA is controlled by at least two different gene promoters that generate two variants with different exon 1 sequences (1A and 1B). The location of 1A and 1B somatotropin receptor mRNA within cattle tissues and, hence, the tissue specificity of the 1A and 1B promoters are unknown. In addition, the cDNA sequence of the 1B somatotropin receptor has not been determined. Our objective, therefore, was to sequence a cDNA for the 1B somatotropin receptor and to analyze bovine tissues for expression of 1A and 1B somatotropin receptor mRNA. Twenty adult tissues and six fetal tissues were collected at slaughter from each of four cows and two fetuses. Messenger RNA was analyzed using ribonuclease protection assays. The adult liver expressed both 1A and 1B mRNA. All other adult tissues expressed 1B mRNA but not 1A mRNA. The greatest amount of 1B mRNA was detected in liver and adipose (abdominal and subcutaneous) tissues. Other tissues had approximately one-half to one-tenth of the amount of 1B mRNA in the liver or adipose tissue. Fetal tissues (including fetal liver) expressed 1B mRNA and not 1A mRNA. Based on cDNA sequencing, the protein encoded by the 1A and 1B mRNA was nearly identical. We concluded that 1A somatotropin receptor mRNA is specific to adult bovine liver. Other adult and fetal bovine tissues expressed 1B somatotropin receptor mRNA with a predicted protein sequence that was similar to the 1A somatotropin receptor.     

Somatostatin decreases somatostatin messenger ribonucleic acid levels in the rat periventricular nucleus

Growth hormone (GH) secretion from the pituitary is known to be under the dual control of GH-releasing factor (GRF) and somatostatin (SRIF). Hypothalamic SRIF, the major inhibitor of pituitary growth hormone secretion, inhibits its own release by a negative ultrashort-loop feedback mechanism. However, it is not known whether this negative regulation is mediated by inhibition of SRIF mRNA production. GRF may also inhibit its own release, thereby modifying pituitary GH secretion, possibly through an ultrashort-loop feedback mechanism. Thus, SRIF production and GRF release are both regulated by SRIF. Periventricular nucleus (PeN) and mediobasal hypothalamus (MBH) from adult male rats were incubated for 6 h in Waymouth's medium with either SRIF or the SRIF agonist analog RC 160 (10(-9) to 10(-6) M). Levels of SRIF mRNA were determined by an S1 nuclease protection assay using a 32[P]-labeled rat SRIF riboprobe. SRIF (10(-7) M) and RC 160 (10(-8), 10(-7) M) significantly (p< or =0.01) decreased SRIF mRNA levels in the PeN. The levels of SRIF mRNA in the MBH were not modified by either SRIF or RC 160. SRIF (10(-7) and 10(-6) M) significantly (p < or = 0.01 and p < or = 0.001, respectively) inhibited the release of GRF at 30 min in the MBH. Likewise, the release of GRF was slightly decreased by 10(-7) M RC 160, and significantly inhibited by 10(-6) M (p < or = 0.001) at 30 min. At 6 h, the levels of GRF were significantly reduced by 10(-7) M SRIF (p < or = 0.05) and by RC 160 (10(-7), 10(-6) M; p < or = 0.001 and p < or = 0.05, respectively). In contrast with these results, the SRIF analog was unable to alter SRIF release at 30 min. At 6 h incubation, RC 160 (10(-7) M) significantly (p < or = 0.001) reduced SRIF release from MBH fragments. These results demonstrate that SRIF and a SRIF analog decrease SRIF mRNA levels in the PeN and inhibit the release of SRIF from the nerve terminals of the MBH. Thus, SRIF appears to regulate its own gene expression by negative ultrashort-loop feedback. Therefore, when SRIF is secreted from these neurons in response to GRF, it down-regulates the preceding stimulatory input as well as its own secretion.     

Ontogeny of estrogen receptor messenger ribonucleic acid expression in the postnatal rat uterus

During the first 2 wk of postnatal life, the rodent uterus undergoes a period of marked growth and differentiation. To further examine the role of the estrogen receptor (ER) in the mediation of uterine development, we analyzed the ontogeny of ER mRNA expression in the postnatal rat uterus using in situ hybridization. ER mRNA was present in the uterine stroma on the day of birth and progressively increased in abundance during the first 2 wk of postnatal life. In contrast, ER mRNA was not detectable in the luminal epithelium at birth and did not become abundant in this region until postnatal day (P) 7. ER mRNA abundance increased in the luminal epithelium and in the invaginating and fully formed glandular epithelium during the second week of life. At P21 ER mRNA was more abundant in the glandular epithelium than in any other uterine cell type. These results are consistent with, and extend the findings of, previous studies using uterine homogenate binding assays and immunohistochemistry to define ER ontogeny in rodents. Delineation of the temporal and cell-type specific pattern of ER mRNA ontogeny in the postnatal rat uterus furthers our understanding of the molecular basis of both endogenous and exogenous estrogen effects on uterine growth and development.     

Homologous down-regulation of growth hormone-releasing hormone receptor messenger ribonucleic acid levels

Repeated stimulation of pituitary cell cultures with GH-releasing hormone (GHRH) results in diminished responsiveness, a phenomenon referred to as homologous desensitization. One component of GHRH-induced desensitization is a reduction in GHRH-binding sites, which is reflected by the decreased ability of GHRH to stimulate a rise in intracellular cAMP. In the present study, we sought to determine if homologous down-regulation of GHRH receptor number is due to a decrease in GHRH receptor synthesis. To this end, we developed and validated a quantitative RT-PCR assay system that was capable of assessing differences in GHRH-R messenger RNA (mRNA) levels in total RNA samples obtained from rat pituitary cell cultures. Treatment of pituitary cells with GHRH, for as little as 4 h, resulted in a dose-dependent decrease in GHRH-R mRNA levels. The maximum effect was observed with 0.1 and 1 nM GHRH, which reduced GHRH-R mRNA levels to 49 +/- 4% (mean +/- SEM) and 54 +/- 11% of control values, respectively (n = three separate experiments; P < 0.05). Accompanying the decline in GHRH-R mRNA levels was a rise in GH release; reaching 320 +/- 31% of control values (P < 0.01). Because of the possibility that the rise in medium GH level is the primary regulator of GHRH-R mRNA, we pretreated pituitary cultures for 4 h with GH to achieve a concentration comparable with that induced by a maximal stimulation with GHRH (8 micrograms GH/ml medium). Following pretreatment, cultures were stimulated for 15 min with GHRH and intracellular cAMP accumulation was measured by RIA. GH pretreatment did not impair the ability of GHRH to induce a rise in cAMP concentrations. However, as anticipated, GHRH pretreatment (10 nM) significantly reduced subsequent GHRH-stimulated cAMP to 46% of untreated controls. These data suggest that GHRH, but not GH, directly reduces GHRH-R mRNA levels. To determine whether this effect was mediated through cAMP, cultures were treated with forskolin, a direct stimulator of adenylate cyclase. Forskolin (10 microM) significantly reduced GHRH-R mRNA concentrations (37 +/- 6% of control values) indicating that GHRH acts through the cAMP-second messenger system cascade to regulate GHRH-R mRNA. The somatostatin analogue, octreotide (10 nM), which has been previously reported to decrease adenylate cyclase activity, did not affect GHRH-R mRNA levels. Taken together, these results indicate that GHRH inhibits the production of its own receptor by a receptor-mediated, cAMP-dependent reduction of GHRH-R mRNA accumulation.     

Regulation of hypothalamic preprogrowth hormone-releasing factor messenger ribonucleic acid expression in food-deprived rats: a role for histaminergic neurotransmission

To determine the component(s) of dietary protein that regulates GH-releasing factor (GRF) synthesis, we measured hypothalamic prepro-GRF mRNA by solution hybridization/nuclease protection analysis in food-deprived rats refed protein-free diets (PF) supplemented with individual amino acids. Adult male Sprague-Dawley rats were allowed free access to food (Fed), food deprived for 72 h (FD), or FD then refed for 72 h with a normal (NF) diet, a protein-free (PF) diet, or PF diets containing tyrosine, tryptophan (Trp), glutamic acid, or histidine (His). Food-deprived rats displayed the expected 80% reduction in hypothalamic prepro-GRF mRNA. Upon refeeding, levels were normalized in rats refed a normal diet, but not in those refed a PF diet alone or with tyrosine, Trp, or glutamic acid. In contrast, prepro-GRF mRNA was restored to 70% of Fed values by a PF diet with His. Supplementing a PF diet with His was sufficient to maintain hypothalamic prepro-GRF mRNA expression, as 3 days of feeding replete rats with PF diet or PF diet with added Trp resulted in a 50% reduction in prepro-GRF mRNA, whereas levels were reduced 25% by feeding animals a PF diet with His. Groups of rats allowed free access to food were treated for 72 h with two daily injections of 100 mg/kg alpha-fluoremethylhistidine, a specific irreversible inhibitor of histidine decarboxylase, to determine if the effect of His on prepro-GRF mRNA depended on neural conversion to histamine. alpha-Fluoremethylhistidine-treated rats showed a 40% reduction in hypothalamic prepro-GRF mRNA, with no concomitant change in preproneuropeptide-Y or preprosomatostatin. These data indicate that decreased hypothalamic prepro-GRF mRNA in FD rats is due in part to the lack of dietary and provide clear evidence for a role of the histaminergic neural system in the regulation of hypothalamic GRF expression.     

Concentrations of leptin in the serum of pregnant, lactating, and cycling rats and of leptin messenger ribonucleic acid in rat placental tissue

Leptin concentrations were measured in the serum of cycling, pregnant, and lactating Sprague-Dawley rats. Serum leptin concentrations did not vary significantly during the estrous cycle. In contrast, as gestation advanced, serum leptin concentrations increased significantly, p < 0.0001. Following delivery, leptin concentrations declined and remained stable during lactation. Leptin messenger ribonucleic acid (mRNA) was identified in the visceral adipose tissue and placenta of rats sacrificed on days 14 and 21 of pregnancy. The relative abundance of placental leptin mRNA increased approximately 4 to 5 fold from day 14 to 21 of gestation. The pattern of elevated leptin concentrations in the serum of late pregnant rats is similar to that reported in pregnant women, therefore the rat may be a useful model for the study of leptin during pregnancy. The increase in leptin in the serum of late pregnant rats, as well as an increase in placental mRNA, raises the possibility that leptin may serve a physiological role for the late parturient rat and/or its young.     

Messenger ribonucleic acid metabolism in mammalian mitochondria. Discrete poly(adenylic acid) lacking messenger ribonucleic acid species associated with mitochondrial polysomes

The mRNA species released from mitochondrial polysomes prepared by the Mg2+ precipitation technique have been further characterized using various analytical techniques. Mitochondrial polysomes were dissociated by treatment with puromycin and chemically labeled with (3H) dimethyl sulfate. About 51% of steady-state mitochondrial mRNA bind to oligo(dT)-cellulose indicating the presence of poly(adenylic acid)(poly(A)) in this fraction. The poly(A)-containing mRNAs resolve into discrete bands of 9-16 Se, while the RNA fraction unable to bind to oligo(dT)-cellulose representing poly(A)-lacking mRNA contains 8-12 Se species. About 90% of poly(A) lacking RNA hybridizes with mitochondrial DNA and less than 7% hybridizes with nuclear DNA. The extent of hybridization of poly(A)-lacking RNA with mitochondrial DNA was not significantly affected by the presence of excess mitochondrial rRNA, cytoplasmic rRNA, or a tenfold concentration of poly(A)-containing RNA isolated from total mitochondrial RNA. Possible differences in sequence properties between poly(A)-containing and -lacking mitochondrial mRNAs were further verified using a solid phase-bound cDNA procedure. Poly(A)-containing mRNA released from mitochondrial polysomes shows over 85% sequance homology with oligo(dT)-cellulose-bound cDNA prepared against total mitochondrial poly(A)-lacking mitochondrial mRNA hybridizes with the cDNA providing direct evidence for the distinct sequence properties of the two mRNA species.     

Effect of fasting on insulin receptor-related receptor messenger ribonucleic acid in rat kidney

The insulin receptor-related receptor (IRR), a member of the insulin receptor tyrosine kinase family, has structural homology to the insulin receptor (IR) and the IGF-I receptor (IGF-IR). The ligand, gene regulation and biological function of the IRR are not known. Because mRNAs for both the IR and IGF-IR are increased by nutrient restriction, we used RNase protection assays to assess the effects of fasting 48 h on IRR mRNA in kidneys of rats. We compared the changes in IRR with those in IR and IGF-IR mRNAs. We observed a significant increase in steady state levels of IRR (ratio of IRR mRNA to beta-actin in fed P<0.01), suggesting that the ligand for IRR also might be regulated by nutrients.     

Regulation of oxytocin receptor messenger ribonucleic acid in the ventromedial hypothalamus by testosterone and its metabolites

Oxytocin receptor (OR) binding in the ventromedial hypothalamus (VMH) is regulated by testosterone (T) and its metabolites, estrogen (E2) and dihydrotestosterone (DHT). Previous studies have reported that OR binding increases in the VMH in castrated male rats when they are replaced with T or E2 compared to that in vehicle-treated animals. DHT alone had no effect on OR binding, but when given in combination with E2 appeared to have a synergistic effect. This study was designed to determine whether these effects of steroid hormones on OR binding in the VMH are associated with changes in OR messenger RNA (mRNA) expression. Male rats were castrated or sham operated and given T propionate (TP), E2 benzoate (EB), DHT plus EB, or an oil vehicle. OR mRNA was assessed using a rat complementary RNA OR probe and in situ hybridization techniques. OR binding to tissue slices was quantified autoradiographically using an OR antagonist, [125I]d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)] ornithine vasotocin. These experiments showed that TP and EB increased both OR mRNA and OR binding in the VMH significantly above levels in vehicle-treated animals. However, animals given both EB and DHT exhibited significantly lower OR mRNA expression and OR binding in the VMH compared to those in animals treated with TP or EB alone. These data indicate that increases in VMH OR binding in response to gonadal steroids are accompanied by changes at the mRNA level that correspond well in magnitude and direction with those in the OR-binding sites.