Influence of Melatonin on the Rate of Rana pipiens Tadpole Metamorphosis In Vivo and Regression of Thyroxine-Treated Tail Tips In Vitro

Metamorphosis of Rana pipiens tadpoles may be retarded when the light phase of the light/dark (LD) cycle is shortened or when thyroxine (T₄) is given in the dark because melatonin peaks during the dark. Injection of premetamorphic tadpoles in spontaneous metamorphosis with melatonin (15 μg) retarded tail growth and hindlimb development on 18L:6D but had no significant effect on 6L:18D. During induced metamorphosis (30 μg/liter T₄), melatonin injections retarded tail resorption on 18L:6D and accelerated it on 6L:18D, but did not affect the hindlimb. When melatonin was injected during T₄ immersion at different times in the photophase on 18L:6D (L onset 0800 hr), tail regression was retarded by melatonin at 1430 or 2030 hr. At 0830 hr, shrinkage of tail length was accelerated whereas tail height was not affected. Tail tips in vitro induced to resorb by 0.2 μg/ml T₄ in Niu-Twitty solution regressed more slowly in the presence of melatonin (10 or 15 μg/ml) than with T₄ alone on both 6L:18D and 18L:6D. The findings implicate melatonin in LD cycle effects on tadpole metamorphic rate in vivo , show the importance of the time of melatonin injections, and indicate that melatonin antagonizes the metamorphic action of T₄ at the tissue level.     

Dose-related effects of 17βoestradiol (E2) on liver weight, plasma E2, protein, calcium and thyroid hormone levels, and measurement of the binding of thyroid hormones to vitellogenin in rainbow trout, Salmo gairdneri Richardson

Administration of 17β-oestradiol (E₂) to rainbow trout, in the form of hydrogenated coconut oil implants produced a stable, long-term elevation in plasma E₂ levels. The elevation was doserelated (over the range 1–10mg kg-¹ body weight) both 4 and 8 weeks after implantation. Dose-related increases were also observed with respect to liver weight-body weight ratios and plasma protein levels. Plasma T₃ and total calcium levels were depressed and elevated, respectively, by E₂ treatment but the responses were not linearly related to the dose of E₂ administered; there was no significant effect of E₂ on plasma T₄ levels.
E₂ induced a shift in the binding of T₃ to plasma proteins, with T₃ binding to smaller molecular weight proteins; neither T₄ nor T₃ bound to vitellogenin which was present at high levels in the plasma of E₂-treated fish.     

Correlative Regulation of Nerve Growth Factor Level and Choline Acetyltransferase Activity by Thyroxine in Particular Regions of Infant Rat Brain

Abstract: Effects of thyroxine (T₄) on nerve growth factor (NGF) level and choline acetyltransferase (ChAT) activity of rat brains were investigated. Repetitive intraperitoneal administration of T₄ caused increases in both NGF level and ChAT activity in the frontal cortex, septum, hippocampus, and striatum and decreases in the cerebellum in 2-day-old rats. Only ChAT activity was elevated in the olfactory bulb, and the NGF level remained unchanged there. No changes were observed in the midbrain and pons/medulla. Furthermore, T₄ was effective on the post-natal rats only up to day 11. These results suggest that T₄ plays a role in the developmental regulation of NGF level and ChAT activity in rat brain in a region- and/or stage-specific manner. That (1) changes in NGF level and ChAT activity occurred in regions nearly identical to those that contained NGF-responding neurons, and (2) the change in NGF level in the hippocampus and frontal cortex was followed by the change of ChAT activity after a single injection of T₄ suggest that the effects of T₄ on cholinergic differentiation are, at least in part, mediated via NGF, which itself is quantitatively regulated by T₄.     

Enhancement of Squamous Cell Development in Cultured Skin by Cyclic Adenine Nucleotide and Prostaglandins

The present study tests the hypothesis that agents known to elevate the level of intracellular cyclic adenine nucleotide may direct different epithelial cells onto a pathway of epidermoid (squamous) development and differentiation. We report here that the mixture of dibutyryl cyclic AMP (dbcAMP), prostaglandins E₁, E₂ and B₁, (PG E₁, E₂, B₁), and papaverine (pap) enhances the rate of normal squamous cell development in organ-cultured skin of chick embryos. The three components may act synergistically to elevate the level of intracellular cyclic adenine nucleotide. We recently reported that the same group of agents induces abnormal development (squamous metaplasia) and aberrant differentiation (keratin production) in the normally cuboidal epithelium of cultured whole mammary glands of mice [1]. Thus, dbcAMP, PG E₁, E₂, B₁, and pap are effective in enhancing normal squamous cell development and also in inducing squamous metaplasia de novo in the epithelial components of two different organs of embryonic and adult animals of two classes of vertebrates. The combined findings are suggestive that cyclic adenine nucleotide together with the prostaglandins may act generally on diverse types of epithelia to bring about squamous cell development and a differentiation marked by keratin production.     

CYCLIC NUCLEOTIDE PHOSPHODIESTERASE OF THE BOVINE RETINA: ACTIVITY, SUBCELLULAR DISTRIBUTION AND KINETIC PARAMETERS

Abstract— High phosphodiesterase activity for cyclic AMP and cyclic GMP was found in subcellular fractions of the bovine retina with more rapid hydrolysis of cyclic GMP than cyclic AMP in each fraction. Rod outer segments (ROS) and the supernatant fraction had highest activity. High enzyme activity remained associated with ROS membranes through several steps of purification by gradient centrifugation. A complex kinetic pattern was observed for cyclic AMP hydrolysis by the supernatant fraction yielding two values for K _m; a simple kinetic pattern was observed with cyclic GMP hydrolysis in supernatant and for both cyclic nucleotides in preparations of purified outer segments. Phosphodiesterase activity of outer segments was enhanced by Mg²⁺. Mn²⁺ and inhibited by EDTA. Cyclic AMP had relatively little effect on the hydrolysis of cyclic GMP in supernatant or ROS while cyclic GMP inhibited hydrolysis of cyclic AMP in both fractions.     

Characterization of the Thyroid Hormone Transport System of Cerebrocortical Rat Neurons in Primary Culture

Abstract: The uptake of 3',3,5-triiodo- l -thyronine (T₃) and l -thyroxine (T₄) by primary cultures derived from rat brain hemispheres was studied under initial velocity conditions, at 25°C. Uptake of both hormones was carrier mediated and obeyed simple Michaelis-Menten kinetics. The K _m of T₃ uptake was very similar to that of T₄, and did not vary significantly from day 1 to 4 in culture (310–400 n M ). The maximal velocity ( V _max) of T₃ uptake nearly doubled between day 1 and 4 of culture (41 ± 3 vs. 70 ± 5 pmol/min/mg of DNA, respectively). The V _max of T₄ uptake did not change (28 ± 8 and 31 ± 4 pmol/min/mg of DNA on days 1 and 4, respectively). The rank order of unlabeled thyroid hormone analogues to compete with labeled T₃ or T₄ uptakes were the same (T₃ > T₄ > 3',5',3-triiodo- l -thyronine > 3',3,5-triiodo- d -thyronine > triiodothyroacetic acid), indicating that the transport system is stereospecific. Unlabeled T₄ was a stronger competitor of labeled T₄ uptake than of labeled T₃ uptake, whereas unlabeled T₃ had the same potency for both processes. These results suggest that T₃ and T₄ are transported either by two distinct carriers or by the same carrier bearing separate binding sites for each hormone. They also indicate that the efficiency of T₃ uptake increases during neuronal maturation.     

Investigations on Myelination In Vitro: Regulation of 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase by Thyroid Hormone in Cultures of Dissociated Brain Cells from Embryonic Mice

Abstract: The direct influence of l -3,3',5-triiodothyronine (T₃) on the development of 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37, CNPase) is demonstrated by using an in vitro culture system of dissociated embryonic mouse brain cells. Serum from a thyroidectomized calf, which contained low levels of T₃ (31 ng/100 ml), and thyroxine, T₄ (<1 μg/ml), was used in the culture medium in place of normal calf serum (T₃, 103 ng/100 ml; T₄, 5.7 μg/ml) to render the culture responsive to exogenously added T₃. The lower levels of enzyme activity observed in the presence of such a deficient medium could be restored to normal values by T₃ supplementation. Half-maximal effect was obtained with 2.5 ± 10⁻⁹ m -T₃. Three days of hormone treatment resulted in the maximal stimulation of CNPase. T₄ was less effective in inducing CNPase activity and the inactive analog of the hormone, reverse T₃ (3,3',5'-T₃) was ineffective. The morphological appearance of the cells was characterized by deformed (smaller size and less in number) reaggregates in the cultures, lacking hormone.     

Changes in thyroid function and gonadotrophin activity during sexual development in ducks Anas sp. (Aves: Anatidae)

Age-related changes in plasma iodohormone concentrations in male and female Aylesbury and Khaki Campbell ducklings were determined between the second week after hatching and sexual maturity.
In both sexes and strains the concentration of total thyroxine (T₄) and the free thyroxine index (FT₄1) were low during the first 6–8 weeks after hatching. A marked increase in the T₄ and FT₄1 occurred when the ducklings were nine weeks old; T₄ progressively increased over the following four weeks and remained high thereafter. The T₄ concentration in both sexes was directly related to age.
Concentrations of total tri-iodothyronine (T₃) were more variable than T₄ but were highest before sexual maturation and inversely age-related. An age-related decline in free tri-iodothyronine index (FT₃1) was observed during the first nine weeks.
The possibility that these maturational changes in thyroid function may reflect changes in gonadal activity was assessed by measuring the concentration of luteinizing hormone (LH) in the same plasma samples. Although plasma LH increased with age, in all cases the increase occurred after changes in the thyroid hormones were observed. Moreover, the maturational pattern of LH was related to sex and strain whereas thyroid function was unaffected by sex or strain.
These results demonstrate reciprocal changes in plasma T₃ and T₄ levels in ducks during sexual development, although the stimuli responsible and physiological significance are uncertain.     

Two Intracellular Signaling Pathways for the Dopamine D3 Receptor: Opposite and Synergistic Interactions with Cyclic AMP

Abstract: As cerebral neurons express the dopamine D₁ receptor positively coupled with adenylyl cyclase, together with the D₃ receptor, we have investigated in a heterologous cell expression system the relationships of cyclic AMP with D₃ receptor signaling pathways. In NG108-15 cells transfected with the human D₃ receptor cDNA, dopamine, quinpirole, and other dopamine receptor agonists inhibited cyclic AMP accumulation induced by forskolin. Quinpirole also increased mitogenesis, assessed by measuring [³H]thymidine incorporation. This effect was blocked partially by genistein, a tyrosine kinase inhibitor. Forskolin enhanced by 50–75% the quinpirole-induced [³H]thymidine incorporation. This effect was maximal with 100 n M forskolin, occurred after 6–16 h, was reproduced by cyclic AMP-permeable analogues, and was blocked by a protein kinase A inhibitor. Forskolin increased D₃ receptor expression up to 135%, but only after 16 h and at concentrations of >1 µ M . Thus, in this cell line, the D₃ receptor uses two distinct signaling pathways: it efficiently inhibits adenylyl cyclase and induces mitogenesis, an effect possibly involving tyrosine phosphorylation. Activation of the cyclic AMP cascade potentiates the D₃ receptor-mediated mitogenic response, through phosphorylation by a cyclic AMP-dependent kinase of a yet unidentified component. Hence, transduction of the D₃ receptor can involve both opposite and synergistic interactions with cyclic AMP.     

Seasonal changes of thyroid hormones in field-collected Atlantic cod in relation to condition indices, water temperature and photoperiod

Serum T₄ and T₃ in wild Atlantic cod Gadus morhua ranged from 1 to 12 ng ml⁻¹ and from 2 to 27 ng ml⁻¹ respectively over a 3-year period. In general, the concentrations increased from summer (T₃) or early autumn (T₄) to maxima in mid-winter and declined abruptly during spring. The T₄/T₃ monthly means were lowest in summer and highest in winter. The seasonal patterns of thyroid hormones were weakly correlated with changes in water temperature. However, both T₄ and T₃ co-varied simultaneously with photoperiod. In addition, T₃ was correlated with the hepatosomatic index and condition factor during summer and autumn. It is suggested that the seasonal changes in the release of T₄ from the thyroid were photoperioddriven, and that the course of T3 was regulated by the metabolic state of the fish during the somatic growth period.     

[3H]GLYCOGEN HYDROLYSIS IN BRAIN SLICES: RESPONSES TO NEUROTRANSMITTERS AND MODULATION OF NORADRENALINE RECEPTORS

Abstract— Different agents have been investigated for their effects on [C³H]glycogen synthesized in mouse cortical slices. Of these noradrenaline, serotonin and histamine induced clear concentration-dependent glycogenolysis.
[C³H]Glycogen hydrolysis induced by noradrenaline appears to be mediated by beta-adrenergic receptors because it is completely prevented by timolol, while phentolamine is ineffective. It seems to involve cyclic AMP because it is potentiated in the presence of isobutylmethylxanthine; in addition dibutyryl cyclic AMP (but not dibutyryl cyclic GMP) promotes glycogenolysis.
Lower concentrations of noradrenaline were necessary for [C³H]glycogen hydrolysis (EC₅₀= 0.5μM) than for stimulation of cyclic AMP accumulation (EC₅₀= 8μM).
After subchronic reserpine treatment the concentration-response curve to noradrenaline was significantly shifted to the left (EC₅₀= 0.09 ± 0.02 μM as compared with 0.49 ± 0.08 μM in saline-pretreated mice) without modifications of either the basal [C³H]glycogen level, maximal glycogenolytic effect, or the dibutyryl cAMP-induced glycogenolytic response.
In addition to noradrenaline, clear concentration-dependent [³H]glycogen hydrolysis was observed in the presence of histamine or serotonin. In contrast to the partial [³H]glycogen hydrolysis elicited by these biogenic amines, depolarization of the slices by 50 mM K⁺ provoked a nearly total [C³H)glycogen hydrolysis.     

Role of Ca2+ in Differentiation Mediated by Nerve Growth Factor and Dibutyryl Cyclic AMP in PC12 Cells

Abstract: Nerve growth factor (NGF) and dibutyryl cyclic AMP (dbcAMP) have synergistic effects on the neurite outgrowth of rat pheochromocytoma PC12 cells. The sites of interaction between NGF and dbcAMP have been studied extensively; however, the role of Ca²⁺ in differentiation induced by the two agents remains unclear. To understand whether intracellular Ca²⁺ is involved in the differentiation induced by the two agents, PC12 cells were treated with NGF, dbcAMP, or NGF plus dbcAMP for 2 days, and then effects on neurite outgrowth, ATP-induced Ca²⁺ influx, and Ca²⁺ mobilization from intracellular Ca²⁺ pools were examined. NGF or dbcAMP alone enhanced neurite outgrowth and Ca²⁺ accumulation by nonmitochondrial Ca²⁺ pools or the thapsigargin (TG)-sensitive Ca²⁺ pool. The dbcAMP acted synergistically with NGF to increase neurite outgrowth and to enlarge the TG-sensitive Ca²⁺ pool. The synergistic effect occurred within the first hour of treatment with dbcAMP plus NGF. On the other hand, dbcAMP abolished NGF's ability to enhance ATP-induced influx of extracellular Ca²⁺. Therefore, NGF and dbcAMP induced different effects on Ca²⁺ signaling pathways through two different but interacting pathways. In PC12 cells pretreated with TG to deplete the TG-sensitive Ca²⁺ pool, the dbcAMP- or dbcAMP plus NGF-mediated neurite outgrowth was significantly inhibited, whereas NGF-mediated neurite outgrowth was not affected by TG pretreatment. Our results suggest that the intracellular nonmitochondrial Ca²⁺ pools were changed in the differentiation process and were necessary for the synergistic effect of NGF and dbcAMP.     

MODULATION OF CYCLIC AMP LEVELS IN THE BOVINE SUPERIOR CERVICAL GANGLION BY PROSTAGLANDIN E, AND DOPAMINE

Abstract— Dopamine, norepinephrine, carbamylcholine and PGE₁ (prostaglandin E₁). increased cyclic AMP concentrations in slices of bovine superior cervical ganglia. PGF_1α was less effective and neither PGE₂ nor PGF_2α had any effect. Dopamine and PGE, alone or in combination, did not modify low K _m cyclic AMP phosphodiesterase activity. Combinations of dopamine and PGE, showed a marked synergistic effect, increasing ganglionic cyclic AMP to a much greater extent than that observed when the two compounds were tested alone. Norepinephrine (10 μ M) , which increased cyclic AMP as much as 10 μ m -dopamine, showed no synergistic effect when tested in the presence of PGE₁ or other PGs. Phentolamine, fluphenazine and triflupromazine blocked the dopamine effect without suppressing its synergism with PGE₁ Adenylate cyclase of synaptosomes isolated from the ganglia under a variety of experimental conditions appeared to be as responsive to PGE₁ as the slices, but it was poorly stimulated by dopamine and was not synergistically modulated by dopamine in the presence of PGE₁
These and other data are interpreted as indicating the presence of both a PGE₁-sensitive and a PGE₁-modulated dopamine-sensitive adenylate cyclase in the cervical ganglion. These adenylate cyclases are tentatively assigned to pre- and post-synaptic structures respectively.     

Plasma and ovarian thyroxine levels in relation to sexual maturation and gestation in female Sebastes inermis

Plasma T₄ (L-thyroxine) concentrations in the viviparous rockfish Sebastes inermis showed a peak (119·3±27·8 ng ml⁻¹) during the development of embryos. Ovarian T₄ concentrations increased during vitellogenesis and final maturation and decreased during embryogenesis.However, total T₄ content within the ovary increased continuously up to the larval stages. Plasma oestradiol-17β, (E₂) concentrations peaked at the final maturation stage and then recovered to the level seen during the non-reproductive periods. Plasma testosterone (T) concentrations showed a peak (5·33±1·08 ng ml⁻¹) at the embryo stage and high levels were maintained throughout the gestation period. Plasma T₄ concentrations during the embryo stage and ovarian T₄ content during vitellogenesis were much higher than the levels reported in oviparous fishes. These data suggest that in viviparous rockfish, T₄ may be required for sustaining gestation or embryo development within the maternal body. In addition, the high content of total T₄ in the ovary implies that there probably does exist a maternal-embryo relationship during gestation as well as during vitellogenesis.     

Seasonal and diurnal rhythms of thyroidal status in the rainbow trout, Salmo gairdneri Richardson

The concentrations of thyroxine (T₄) and triiodothyronine (T₃) in the blood plasma of rainbow trout ( Salmo gairdneri Richardson) at intervals throughout the year have been measured by a chemically specific gas-liquid chromatographic (GLC) method. Mean hormone levels showed a seasonal variation, maximal levels of both hormones occurring in winter and minimal concentrations in mid summer. An apparent secondary maximum in mean T₄ and T₃ concentrations was observed in spring. (T₄)/(T₃) ratios have been found to be highest in winter and lowest in summer. A radioimmunoassay procedure, validated by GLC analyses, revealed the presence of a diurnal rhythm in serum T₄ levels of trout sampled in September and in April.     

Migration tendency in juvenile steelhead trout, Salmo gairdneri Richardson, injected with thyroxine and thiourea

Juvenile steelhead trout, Salmo gairdneri injected with thyroxine (T₄) near the time of seaward migration exhibited a significantly lower migration tendency than untreated controls of fish injected with saline, thiourea, or a combination of T₄ and thiourea. Fish injected with thiourea alone or in combination with T₄ prior to the time of maximum migration tendency showed enhanced migration over untreated and saline injected controls and those injected with T₄ alone. Injections of T₄ or a combination of T₄ and thiourea elevated plasma T₄ and tri-iodothyronine (T₃) concentrations, while injection of thiourea alone depressed thyroid hormone levels relative to saline-injected controls. Plasma concentrations of T₃ and T₄ returned to control levels within 10 days in all groups. We suggest that thyroid hormones are antagonistic to mechanisms underlying seaward migration of steelhead trout and that these antagonistic effects must be overcome before migration tendency is expressed.     

Triiodothyronine Is a High-Affinity Inhibitor of Amino Acid Transport System L1 in Cultured Astrocytes

Abstract: The relationship between the transport of thyroid hormones and that of amino acids was examined by measuring the uptake of amino acids that are characteristic substrates of systems L, A, and N, and the effect of 3,3',5-triiodo-L-thyronine (T₃) on this uptake, in cultured astrocytes. Tryptophan and leucine uptakes were rapid, Na⁺-independent, and efficiently inhibited by T₃ (half-inhibition at ∼ 2 μ M ). Two Na⁺-independent L-like systems (L₁ and L₂), common to leucine and aromatic amino acids, were characterized kinetically. System L₂ had a low affinity for leucine and tryptophan ( K _m= 0.3–0.9 m M ). The high-affinity system L₁ ( K _m∼ 10 μ M for both amino acids) was competitively inhibited by T₃ with a K _i of 2–3 μ M (close to the T₃ transport K _m). Several T₃ analogues inhibited system L₁ and the T₃ transport system similarly. Glutamine uptake and α-(methylamino)isobutyric acid uptake were, respectively, two and 200 times lower than tryptophan and leucine uptakes. T₃ had little effect on the uptakes of glutamine and α-(methylamino)isobutyric acid. The results indicate that the T₃ transport system and system L₁ are related.     

Seasonal changes in thyroidal status in the plaice, Pleuronectes platessa L.

A chemically specific analysis for plasma thyroid hormones has been used to disclose a seasonal bimodality in the concentrations of thyroxine (T₄) and triiodothyronine (T₃) in the plaice; maxima in both hormone levels occur in winter and in summer. The ratio of T₄ concentrations to T₃ varies seasonally being at a minimum in summer. A highly significant correlation of T₄ plasma levels with the landings per unit fishing effort for the same area has been observed.     

Physiological response of Pinus halepensis needles under ozone and water stress conditions

The aim of this study was to evaluate how physiological processes of potted Pinus halepensis plants, grown under controlled conditions, were affected by ozone (O₃) and/or water stress, integrating the gas exchange and biochemical data with fluorescence OJIP polyphasic transient data. Plants submitted to only water stress (T₁) and with ozone (T₃) showed a strong decrease in stomatal conductance and gas exchange, coinciding with a reduction of maximum yield of photochemistry ( φ _po) and very negative values of leaf water potential. Simultaneously, a great increase of both PSII antenna size, indicated by absorption per reaction centre, and electron transport per reaction centre were found. The reduction of photosynthesis in the O₃-treated plants (T₂) by a slowing down of the Calvin cycle was supported by the increase of related fluorescence parameters such as relative variable fluorescence, heat de-excitation constant, energy de-excitation by spillover, and the decrease of φ _po. We suggest an antagonistic effect between the two stresses to explain the delayed ozone-induced decrease of stomatal conductance values for T₃ with respect to T₁ plants, by an alteration of the physiological mechanisms of stomatal opening, which involve the increase of intra-cellular free-calcium induced by ABA under co-occurring water shortage. We emphasise the importance of considering the intensity of the individual stress factor in studies concerning the interaction of stresses.