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In tadpoles of the North American bullfrog, Rana catesbeiana, spontaneous and thyroid hormone (T3)-induced metamorphosis is characterized by regression of the tail, which is preceded by a decrease in total protein synthesis in tail tissues. We have demonstrated that thyroid hormone treatment of a tadpole does not affect the synthesis of all proteins equally in the tadpole tail muscle. For example, the synthesis of myosin heavy chains (MHCs) is depressed within 1 day and decreases to 45% of control values after 5 days of T3 treatment, whereas the decreased synthesis of soluble muscle proteins is transient and returns to above control levels by day 5. To determine whether the hormone-induced decrease in MHC synthesis is the result of changes in the transcription of translation of MHC mRNAs, we isolated cDNAs complementary to five different MHC mRNAs from a tail muscle cDNA library and used them to examine the levels of each MHC mRNA in the tail muscle of T3-treated tadpoles. mRNAs that recognize the cDNAs for these five different MHCs are all expressed in the tadpole tail and limb muscles, as well as in the adult leg muscles. MHC mRNAs unique to tadpole tail were not detected. Interestingly, the relative amounts of mRNA for four of the five MHCs increase in tail muscle after T3 treatment of the tadpole, suggesting that repression of MHC gene expression at the protein level does not result from a decrease in the amount of MHC mRNAs. Rather, these results support the contention that the decreased synthesis of MHCs in the tail muscle of T3-treated tadpoles is caused by this hormone, either directly or indirectly, depressing the translation of the MHC mRNAs in this tissue. These results, coupled with the observation that the synthesis of soluble muscle proteins is depressed only in a transient fashion, suggest that T3 may be initiating the expression of a gene(s) that encodes a protein(s) responsible for inhibiting the translation of the MHCs and, perhaps, other structural proteins in the tadpole tail muscle. Whatever the case, the translational regulation of MHC synthesis occurs well before any degradation of the tail tissue is evident and appears to be one of the earliest events in the hormone-induced cell death program of the tadpole tail muscle.

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.

Heat-shock protein 72 (HSP72), the inducible isoform of the HSP70 family, is constitutively expressed in rat hindlimb muscles in proportion to the content of type I muscle fibers. To determine whether this relationship was maintained after fiber transformation, male Sprague-Dawley rats were treated with 3,5,3'-triiodo-DL-thyronine (T3) for 40 days or underwent surgical removal of the left gastrocnemius muscle, after which the left plantaris muscle was allowed to hypertrophy for 30 days. Hypertrophied plantaris muscles exhibited an increased number of type I fibers, type I myosin heavy-chain (MHC) protein, and HSP72 content compared with contralateral muscles. Soleus muscles from rats administered T3 exhibited an increased number of type II fibers, citrate synthase activity, and decreased HSP72 content compared with soleus muscles from controls. These results indicate that the relationship between HSP72 content and type I muscle fiber-MHC composition is maintained when muscles undergo fiber transformation and substantiate that HSP72 content in rat skeletal muscle is not directly linked to a muscle's oxidative capacity.

One of many changes occurring during spontaneous and 3,5,3'-triiodothyronine (T3)-induced metamorphosis of the Rana catesbeiana tadpole is the permanent transition from an ammonotelic, aquatic larva to a ureotelic, terrestrial adult. T3-induced urea production is preceded by T3-induced elevation in the synthesis and level of liver-specific urea cycle enzymes essential for detoxication of ammonia in a terrestrial environment. This report focuses on establishing the effects heat shock (hs) has on the T3-induced expression of genes encoding three essential urea cycle enzymes. We demonstrate that hs stabilizes the intracellular existing levels of carbamyl-phosphate synthetase I (CPS I), the first enzyme in the urea cycle, while concurrently depressing its new synthesis. To establish the effects of hs on CPS I mRNA levels, we characterized cDNAs encoding an amphibian CPS I and demonstrate that it may represent an evolutionary link between microbial CPS and mammalian CPS I. Using this CPS I cDNA and other R. catesbeiana gene-specific probes, we demonstrate that hs depresses the level of T3-induced thyroid hormone receptor beta mRNAs but does not affect the level of T3-induced CPS I, ornithine transcarbamylase, and arginase mRNAs. These results support the contention that the hs response may involve the selective protection of some pre-existing mRNAs and proteins essential for an organism's survival.

Hind limb epidermal cell cultures from stage XII-XV and XVI-XIX tadpoles of the American bullfrog Rana catesbeiana were maintained for 36 and 120 hr in medium containing either fetal calf serum or thyroid hormone (3,3',5-triiodothyronine, T3; 3 x 10(-10) mol/ml). T3 induces the precocious synthesis (within the first 36 hr) of a 59-kDa keratin associated with epidermal stratification in cultures from stages XII-XV. In epidermal cell cultures from stages XVI-XIX, T3 produces an overall pattern of water-insoluble proteins, including keratins, which is strikingly similar to temporally differentiated cultures (120 hr). A 73-kDa protein is among the water-insoluble proteins precociously synthesized by 36-hr-old cultures from stages XVI-XIX treated with T3. This protein, which is not immunoprecipitated by antibodies raised against keratins, corresponds in Mr and pI to a mammalian differentiation-specific desmosomal plaque protein. Immunoprecipitation of keratins from 120-hr-old cultures shows that many of the water-insoluble proteins synthesized are, indeed, keratins. Further, quantitation, by laser densitometry, of immunoprecipitated keratins from 120-hr cultures demonstrates that greater amounts of keratins, particularly the 65 and 59 kDa which are associated with a differentiated epidermis, are present in stages XVI-XIX. This study indicates that epidermal cell cultures from stages XVI-XIX respond more quickly to the differentiating effects of T3.

Recent in vivo and in vitro studies with polyribosomes from the tail muscle of T3-treated tadpoles establish that this hormone initiates a regulating effect on tadpole tail muscle which operates at the translational level and results in an overall decreased rate of protein synthesis (Saleem, M. & Atkinson, B. G. (1978) J. Biol. Chem. 253, 1378-1384). This hormone-induced decrease in the rate of protein synthesis is partially, if not wholly, due to the presence of a sarcoplasmic factor(s) inhibiting ribosomal translational efficiency. This research employs the use of a reconstituted, cell-free polypeptide synthesizing system as a means to substantiate the presence of an inhibitor and further elucidate the mechanism by which this inhibitory factor(s) depresses protein synthesis. The results of this study further demonstrate the presence of an inhibitor of protein synthesis in the tail muscle sarcoplasm of T3-treated tadpoles and suggest that this depressed synthetic activity results from an interaction of the inhibitor with ribosomal or polyribosomal constituents.

Nuclei and chromatin isolated in the presence of calcium or magnesium from Rana catesbeiana liver tissue exhibit considerable endogenous deoxyribonuclease activity. This activity is present in liver nuclei isolated from froglets as well as in liver nuclei isolated from untreated and thyroid hormone treated premetamorphic tadpoles. Nuclei and chromatin isolated in the absence of divalent cations and in the presence of spermine exhibit no detectable expression of the endogenous deoxyribonuclease activity. The endogenous deoxyribonuclease present, but not expressed, in spermine-isolated tadpole liver nuclei or chromatin is salt extractable. Once dialyzed, the salt-extracted deoxyribonuclease is activated by calcium or magnesium. This deoxyribonuclease shows maximal enzyme activity in 15 mM calcium at pH 8.0 or in 15 mM magnesium at pH 7.4. After Ca2+ activation, deoxyribonuclease activity is maximally inhibited by amounts of spermine similar to that required to completely inhibit DNase I. Destruction of the salt-extracted deoxyribonuclease activity by treatment with proteinase K or heat suggests that it is of a proteinaceous nature. The localization and nature of this enzyme activity established that it is associated with the salt-soluble proteins affiliated with tadpole and froglet liver chromatin.

Epidermal cells from the hind limb of premetamorphic tadpoles of the American bullfrog Rana catesbeiana (stages IX-XI) were maintained in primary culture for 120 hr. Cultures, maintained for 36 hr in medium supplemented with L-triiodothyronine (T3; 3 x 10(-10) mol T3/ml medium), synthesize water-insoluble proteins with Mrs of 59, 50, 48, and 43. The 59 and 48 kDa proteins synthesized by T3-supplemented cultures are not detectably synthesized by 36-hr-old cell cultures without added T3, but they are synthesized by both hormone-supplemented and unsupplemented cultures after 120 hr. These two proteins have Mrs and pIs which correspond with keratins detected in stratifying mammalian epidermis and are immunoprecipitated by antibodies prepared against human keratins. These observations indicate that T3 induces epidermal cell cultures from the hind limb of premetamorphic tadpoles to precociously synthesize water-insoluble proteins which (1) have Mrs and pIs similar to keratins from differentiating amphibian epidermal tissue and (2) are biochemically and immunologically similar to keratins associated with the differentiation of mammalian epidermal tissue.

Myosin heavy chain (MHC) and tropomyosin (Tm) have been isolated from limb muscles of the North American bullfrog, Rana catesbeiana, and injected into rabbits to raise monospecific antibodies. These antibodies were used to study the localization and synthesis of myosin heavy chain and tropomyosin in the limb buds of premetamorphic (stage VI-VII) tadpoles treated with triiodothyronine (T3) to induce metamorphosis. Indirect immunofluorescence localization detects the accumulation of both MHC and Tm in the developing thigh region within 24 h of T3 treatment. During the subsequent 48 h, the accumulation of these proteins is enhanced in the thigh and progresses from thigh to the distal regions of the limb. Quantitative immunochemical determinations indicate that within 24 h of T3 treatment, synthesis of Tm and MHC are increased 23-fold and 6-fold, respectively. Following 5 days of T3 treatment, the synthetic rates of Tm and MHC are 266 and 70 times the control values, respectively. Both methods suggest that Tm is synthesized and accumulated at a greater rate than myosin heavy chain. These observations suggest that T3 promotes the differentiation of muscle in the limb buds of premetamorphic tadpoles and that limb development promoted by T3 in tadpoles is similar to that described during the embryonic development of higher vertebrates.