Sequential up-regulation of thyroid hormone β receptor,ornithine transcarbamylase,and carbamyl phosphate synthetase mRNAs in the liver of Rana catesbeiana tadpoles during spontaneous and thyroid hormone-induced metamorphosis

During both spontaneous and thyroid hormone (TH)-induced metamorphosis, the Rana catesbeiana tadpole undergoes postembryonic developmental changes in its liver which are necessary for its transition from an ammonotelic larva to a ureotelic adult. Although this transition ultimately results from marked increases in the activities and/or de novo synthesis of the urea cycle enzymes, the precise molecular means by which TH exerts this tissue-specific response are presently unknown. Recent reports, using RNA from whole Xenopus laevis tadpole homogenates and indirect means of measuring TH receptor (TR) mRNAs, suggest a correlation between the up-regulation of TRβ-mRNAs and the general morphological changes occurring during amphibian metamorphosis. To assess whether or not this same relationship exists in a TH-responsive tissue, such as liver, we isolated and characterized a cDNA clone containing the complete nucleotide sequence for a R. catesbeiana urea cycle enzyme, ornithine transcarbamylase (OTC), as well as a genomic clone containing a portion of the hormone-binding domain of a R. catesbeiana TRβ gene. Through use of these homologous sequences and a heterologous cDNA fragment encoding rat carbamyl phosphate synthetase (CPS), we directly determined the relative levels of the TRβ, OTC, and CPS mRNAs in liver from spontaneous and TH-induced tadpoles. Our results establish that TH affects an up-regulation of mRNAs for its own receptor prior to up-regulating CPS and OTC mRNAs. Moreover, results with cultured tadpole liver demonstrate that TH, in the absence of any other hormonal influence, can affect an up-regulation of both the TRβ and OTC mRNAs. © 1992 Wiley-Liss, Inc.     

Molecular cloning of hepatic mRNAs in Rana catesbeiana responsive to thyroid hormone during induced and spontaneous metamorphosis

Amphibian metamorphosis affords a useful experimental system in which to study thyroid hormone regulation of gene expression during postembryonic vertebrate development. In order to isolate gene-specific cDNA probes which correspond to thyroid hormone-responsive mRNAs, we employed differential colony hybridization of a cDNA library constructed from poly(A)+ RNA of thyroxine-treated premetamorphic tadpole liver. From an initial screening of about 6000 transformants, 32 "potentially positive" colonies were obtained. The recombinant cDNA-plasmids from 13 of these colonies plus two "potentially negative" colonies were purified for further study. Southern blot analysis of the plasmid DNA was employed to determine whether different cDNAs encoded for the same mRNA. The effect of thyroid hormone on the relative levels of specific mRNA species was examined by Northern analysis of liver RNA from premetamorphic tadpoles, thyroxine-treated tadpoles, and adult bullfrogs. Three independent cDNA clones were obtained which encoded thyroid hormone-enhanced mRNAs. We also obtained two independent cDNA clones encoding thyroid hormone-inhibited mRNAs and three independent clones encoding thyroid hormone-unresponsive mRNAs. The levels of two thyroid hormone-enhanced mRNAs and one thyroid hormone-inhibited mRNA were essentially the same in the thyroid hormone-treated tadpole liver and adult liver, suggesting that thyroid hormone induces stable changes in liver gene expression during spontaneous metamorphosis. Using selected cDNAs, RNA dot blot analysis of liver mRNA from tadpoles at different stages of metamorphosis showed that the level of one thyroid hormone-enhanced mRNA increased during late prometamorphosis and metamorphic climax. Similarly, a mRNA which was strongly inhibited by thyroid hormone treatment was observed to decline during prometamorphosis and reach undetectable levels during metamorphic climax. One mRNA was detected which was reproducibly inhibited by thyroid hormone treatment but which remained essentially unchanged during spontaneous metamorphosis. These results provide the first direct evidence for the coordinate and selective pretranslational regulation by thyroid hormone of several liver genes during the developmental process of metamorphosis.     

Characterization of Rana catesbeiana HSP30 gene and its expression in the liver of this amphibian during both spontaneous and thyroid hormone-induced metamorphosis

During metamorphosis, the Rana catesbeiana tadpole undergoes developmental changes in almost every tissue/organ. These changes prepare the ammonotelic, swimming larva for its transition to a ureotelic, terrestrial adult, and involve dramatic remodeling. The postembryonic changes in this tadpole are initiated by the thyroid hormones (TH) and result in the extensive degradation of proteins and degeneration of tissues characteristic of the larval phenotype and in the de novo synthesis of proteins characteristic of the adult phenotype. We questioned whether the drastic nature and abruptness of the TH-dependent, postembryonic changes occurring in the tissues of this tadpole might be perceived by the cells in some tissues as stressful and, therefore, cause them to express heat shock and/or stress-like proteins. To address this question, we isolated and characterized a Rana catesbeiana hsp30 gene and used sequences from it to determine if mRNAs encoded from it, or other members of this gene family, are expressed in tissues of tadpoles undergoing metamorphosis. Our results demonstrate that the liver of metamorphosing Rana catesbeiana tadpoles accumulate hsp30 mRNAs and express the heat shock proteins they encode. The fact that the expression of these hsp30s in the liver of these tadpoles is coincidental with the TH-induced expression of genes encoding the liver-specific urea cycle enzymes suggests that TH may influence, directly or indirectly, the expression of these hsp30 genes and, moreover, implies that the presence of one or more of these heat shock proteins may be necessary for the developmental transitions occurring in this organ. © 1996 Wiley-Liss, Inc.     

Bile pigments and bilirubin UDP-glucuronyl transferase during the metamorphosis of Rana catesbeiana tadpoles

1. Cold-acclimated (1 degree C) goldfish (Carassius auratus) branchial Na/K-ATPase activity was elevated 100% while renal Na/K-ATPase activity was not significantly affected compared with warm-acclimated (20 degrees C) goldfish. 2. Cold-acclimated goldfish branchial and renal Mg-ATPase activity was reduced 18 and 30% on a per mg protein basis, respectively. 3. Renal Na/K-ATPase activity was 4.6- and 1.6-fold greater than gill in cold- and warm-acclimated fish, respectively. 4. The elevated branchial Na/K-ATPase activity and the unchanged renal Na/K-ATPase activity are consistent with the maintenance of the reduced blood ion level in cold-acclimated goldfish.     

Hepatic carbamyl phosphate synthetase and ornithine transcarbamylase in mouse influenze A and influenze B infection.

In mice infected with mouse-adapted influenza A/PR/8/34, hepatic carbamyl phosphate synthetase (CPS) activity was reduced to 88%, and ornithine transcarbamylase (OTC) was reduced to 83% of control values. In mice infected with mouse-adapted B/Lee/40, CPS activity was 98% was 94% of control values. These limited reductions in enzyme activity were attributed to a nonspecific debilitating effect of acute influenzal pneumonia. These findings suggest that the pronounced reduction of CPS and OTC activities reported in Reye's syndrome in man are not a general manifestation of the severity of influenza infection.     

Inhibition of thyroxine-induced metamorphosis by prolactin in tadpoles, Rana catesbeiana.

Thyroxine (T4)-prolactin interactions on hepatic arginase and ornithine transcarbamylase (OTC) as well as hind legs, tail, digestive tract and median eminence were investigated in tadpoles, Rana catesbeiana. Prolactin completely blocked T4-induced tail resorption, but failed to suppress hind-leg growth, shortening of digestive tract and promotion by T4 of the median eminence development. Prolactin blocked T4-induced increase in hepatic arginase activity but not in hepatic OTC activity. A possibility that T4 and prolactin are regulating the hepatic arginase indirectly is discussed.     

Prolactin prevents the autoinduction of thyroid hormone receptor mRNAs during amphibian metamorphosis.

We have recently reported that prolactin (PRL) inhibits both morphogenesis and cell death in thyroid hormone (T3)-induced amphibian metamorphosis (Tata et al., 1991), and that the autoinduction of T3 receptor (TR alpha and beta) mRNA is among the most rapid responses of premetamorphic Xenopus tadpoles to T3 (Kawahara et al., 1991). We now demonstrate that PRL prevents the rapid T3-induced upregulation of TR alpha and beta mRNAs in stages 50-54 Xenopus tadpoles and in organ cultures of tadpole tails. This effect is followed by the inhibition of the de novo activation of 63-kDa keratin gene by T3. We present an experimentally testable model whereby PRL exerts its juvenilizing action by preventing the amplification of TR by its autoinduction by T3.     

Albumin synthesis during induced and spontaneous metamorphosis in the bullfrog Rana catesbeiana

Treatment of premetamorphic tadpoles with triiodothyronine (T₃) alters the in vivo distribution of radioactive amino acids among serum protein fractions. The effects on the albumin fraction have been interpreted as reflections of the relative rate of synthesis. About 12 hr after intraperitoneal injection of 2.5 × 10^?10 mole of T₃ per gram, there is an increase in the relative rate of albumin synthesis. The effect peaks on day 3 at 5 × the untreated level and returns to near the untreated level by day 6. Continuous immersion in 1 × 10^?7M T₃ results in a similar stimulation of albumin synthesis, but with no decline after day 3. The timing of the response is independent of dose or route of T₃ administration. The effect of T₃ on the relative rate of albumin synthesis is also observed in froglets. There is a 6-fold increase in the relative rate of albumin synthesis during spontaneous metamorphosis peaking at stage XXI and returning to the premetamorphic level by stage XXV. The following was concluded: (1) The increase in the relative rate of albumin synthesis during metamorphosis results from increased endogenous thyroid levels. (2) Following a peak, the decline in albumin synthesis observed in induced and spontaneously metamorphosing animals is a result of decreasing thyroid hormone levels. (3) The effect of T₃ on albumin synthesis may be the summation of two effects, a direct effect of T₃ and a stimulation by amino acids from the resorbing tail. (4) A decreased relative rate of albumin degradation or a sparing of albumin is probably responsible for the elevated relative concentration of albumin in the serum of postmetamorphic animals.     

Changes in the rate of production of superoxide anion in tails of Rana catesbeiana tadpoles during spontaneous and triiodothyronine-induced metamorphosis

1. The time course of changes in the rate of production of superoxide anion (O2) was compared with that of beta-glucuronidase in tails of Rana catesbeiana tadpoles during spontaneous and triiodothyronine (T3)-induced metamorphosis. 2. Superoxide production increases in tadpole tail tissue undergoing regression in vivo during spontaneous metamorphosis and in response to T3 treatment. 3. The specific activity of beta-glucuronidase rises three-fold relative to control levels in tadpoles treated with T3 in vivo. 4. The time of onset of the rise in beta-glucuronidase activity precedes the onset of tissue regression and the onset of the increase in superoxide production precedes the rise in beta-glucuronidase activity.     

Immunocytochemistry of luteinizing hormone-releasing hormone in brains of bullfrogs (Rana catesbeiana) during spontaneous metamorphosis

The distribution of immunoreactive luteinizing hormone-releasing hormone (ir-LH-RH) in brains of bullfrogs (Rana catesbeiana) during spontaneous metamorphosis has been studied by combination of an unlabeled antibody enzyme immunocytochemical technique and an adjacent serial section approach. In prometamorphic tadpoles, immunocytochemical staining for ir-LH-RH was absent from the brain, including a structurally simple median eminence (ME) and perikarya in the anterior preoptic area (aPOA). In metamorphic tadpoles, speckled patches of immunostaining occurred over the outer layer of a modestly developed ME; coincident faint staining of a small number of medial, unpaired cell bodies was localized in the aPOA. In newly metamorphosed juvenile frogs, more diffuse and intense staining of the outer layer of the ME accompanied increased morphological differentiation of this neurohemal area; immunoreactive perikarya again were found in the aPOA, but an increased number of neurons exhibited comparatively greater (moderate) immunostaining. Changes in the quality of immunostaining and in the numbers of cells stained, therefore, were coincident with metamorphic development. Concomitant alterations of ir-LH-RH immunostaining and progressive structural development of the ME suggest a coordinated differentiation of brain neuroendocrine systems during metamorphosis of the bullfrog tadpole.     

Different sensitivity to amiloride of body and tail skins of Rana catesbeiana tadpoles during metamorphosis

Regional differences in potential difference and short-circuit current between the body (dorsal) and the tail skin during metamorphosis of Rana catesbeiana tadpoles were investigated. In body skin, the potential difference and the short-circuit current across the skin develop in two successive steps. At stage XX, the potential difference and the short-circuit current across the body skins were amiloride-insensitive (1st step). At stage XXII, however, amiloride-sensitive potential difference and the short circuit current appeared (2nd step). By contrast, in tail skin the potential difference and the short-circuit current remained amiloride-insensitive (1st step) even at stage XXIII. Since the tail regresses after stage XXIII, the appearance of the second step could not be followed in vivo. To determine whether or not the second step can be induced in the tail, tail skin was cultured under conditions where the skin survives for a much longer period than it does in normally developing tadpoles. Such cultured tail skin generated the amiloride-sensitive potential difference and the short-circuit current and cultured body skin also generated them. Therefore, development of the 2nd step in the tail skin may be delayed in vivo. To characterize the differences between body and tail skin, skins were mutally grafted between body and tail at stage XIII–XV. The body skin grafted on the tail underwent both the 1st and 2nd steps by stage XXII, whereas the tail skin grafted on the body only showed the 1st step by the same stage. These results suggest that the regional specificity of the skin is already established before the prometamorphic stage.Abbreviations CMFS Ca²⁺- and Mg²⁺-free saline - CTS charcoal-treated serum - EDTA ethylene diamine tetra-acetate - I current - PD potential difference - R skin resistance - SCC short-circuit current     

Gene expression profile in the liver of Rana catesbeiana tadpoles exposed to low temperature in the presence of thyroid hormone

Studies of insulin producing β-cells have reported conflicting responses to NF-κB activation, encompassing both pro- and anti-apoptotic effects, possibly reflecting the use of β-cells from different species. Therefore, the aim of this study was to compare the temporal activation of NF-κB in rat and human insulin producing cells and relate this to the dynamics of cell death, STAT-1 activation and the production of nitric oxide (NO). Rat RIN5AH and human islet cells were exposed to the cytokines IL-1β and IFN-γ and the NOS inhibitor aminoguanidine. Cell death, NO production, IκBα phosphorylation, p65 methylation, STAT-1 phosphorylation and cIAP-2 levels were analyzed at different time-points. Cytokine-induced RIN5AH cell death occurred on day 1, and this was paralleled by NF-κB activation, STAT-1 phosphorylation and production of NO. On the other hand, the human islet cells instead died by an NO-independent mechanism on day 3 and 5. This later occurring cell death was associated with a gradual decrease in IκBα phosphorylation and p65 methylation, and a lowered expression of the NF-κB target genes IκBα and cIAP-2. STAT-1 phosphorylation was persistently high during the entire cytokine exposure period in human islet cells. The results favor a pro-survival role of NF-κB and a pro-apoptotic role of STAT-1 in human islet cells. Thus, rodent insulin producing cells may not be suitable as models for human β-cells in the context of cytokine-induced damage.     

Cloning and thyroid hormone regulation of albumin mRNA in Rana catesbeiana tadpole liver.

Thyroid hormones are responsible for the specific biochemical and structural changes that occur during amphibian metamorphosis. In this study we screened a series of cDNAs from a library constructed from T4-treated premetamorphic tadpole liver poly(A)+ RNA in order to identify a clone that could be used to study the influence of T3 on liver-specific gene expression during Rana catesbeiana metamorphosis. The cDNA from one clone exhibited a greater degree of hybridization to liver RNA from thyroid hormone-treated tadpoles than untreated tadpoles and no hybridization to RNA from tail fins of tadpoles of either group. On Northern blots, the mRNA to which the cDNA hybridized was 2.3 kilobases in size. The pattern of hybridization to genomic DNA digested by various restriction enzymes was consistent with the presence of a single gene. Using slot blot analysis we found that the mRNA levels first rose above basal levels only after 5 days of immersion of tadpoles in 12.5 micrograms/liter T3. The mRNA levels increased approximately 10-fold after 7 and 9 days of treatment. Frog livers had mRNA levels that were intermediate between those in untreated tadpoles and tadpoles immersed in T3 for 7 days. Sequence analysis revealed a significant degree of homology to serum albumin and alpha-fetoprotein. While it is known that serum albumin levels rise dramatically during metamorphosis in Rana species, presumably playing a critical role in maintaining water and electrolyte balance during the animals' terrestrial phase, the molecular basis of the induction has not been fully explained.     

Changes in serum lipoproteins during metamorphosis of the bullfrog,Rana catesbeiana

Serum lipoproteins of the bullfrog, Rana catesbeiana, were studied during metamorphosis. Adult bullfrog has essentially one lipoprotein, designated β-lipoprotein. This β-lipoprotein migrates during electrophoresis to β-globulin region and it has a low hydrated density such that it exhibits floatation in a solvent of density 1.063. On the other hand, tadpole serum has one more lipoprotein, designated as α-lipoprotein, in addition to the β-lipoprotein. The α-lipoprotein migrates to the α-globulin region in zone electrophoresis and corresponds to the so called high density lipoprotein judging from ultracentrifugal behavior. Serum α-lipoprotein disappears and β-lipoprotein content decreases during metamorphosis.