Thyrotropin-releasing hormone stimulates the release of melanotropin from frog neurointermediate lobes in vitro

The hypothalamus of Amphibia contains large amounts of tripeptide P-Glu-His-Pro-NH₂ (mammalian thyrotropin-releasing hormone, TRH). However, synthetic TRH is unable to stimulate thyrotropin release from frog pituitary gland. The recent discovery of TRH in the skin of the frog suggests a possible role of this peptide in skin-colour adaptation. Thus we have investigated the role of TRH upon melanotropin (α-MSH) release from perifused frog neurointermediate lobes. A dose related increase in α-MSH release was observed when TRH was added to the perifusion medium. Half-maximum stimulation occurred with the 1 × 10^?8M dose. Theophylline at a dose of 2 × 10^?3M strongly enhanced TRH-induced α-MSH release, indicating that cyclic AMP may be the second messenger. α-MSH releade was not modified by crude homogenates of rat hypothalamus but was significantly reduced when the hypothalamus extracts were preincubated with specific TRH antibodies. As far is known, these results provide the first evidence that P-Glu-His-Pro-NH₂ stimulates the release of α-MSH from frog neurointermediate lobes $\text{in vitro}$. The present findings suggest a possible feedback loop between skin TRH and pituitary MSH in Amphibia.     

Uptake, storage and excretion of chylomicra-bound 3H-alpha-tocopherol by the skin of the rat

Tritium- -tocopherol was incorporated into chylomicra by feeding dl-α-(5 methyl ³H)-tocopherol to rats with thoracic duct cannulae. Tritium α-tocopherol in chylomicra, isolated by flotation through 0.85% NaCl, was injected intravenously into rats and distribution of radioactivity was followed in skin, viscera and carcass. Label measured at $\text{1}\text{12}\text{, 1, 3, 10}\text{and}\text{20}\text{days}$ following injection demonstrated a rapid uptake and prolonged retention in skin compared to viscera and carcass. In fact, relative distribution in skin increased from an average of 10.3% at $\text{1}\text{12}\text{day}$ to 40.1% of the recovered dose at 20 days. Also, the uptake of isotope by skin, expressed as ³H dpm/g lipid, markedly exceeded that of epididymal fat suggesting a preferential distribution to cutaneous tissue. Significant radioactivity was found on the skin surface and hair following injection of ³H-α-tocopherol demonstrating the excretion or secretion of vitamin E compound(s) by the skin.     

Vasculature of the parotoid glands of four species of toads (bufonidae: bufo)

The parotoid glands of toads (Bufonidae) consist of large aggregations of granular glands located between the otic region of the skull and the scapular region. To determine the circulatory pattern of these glands, we perfused the vascular systems of Bufo alvarius, B. marinus, B. terrestris, and B. valliceps with either India ink or Microfil, a fine latex. The perfused glands were studied by gross dissection, microscopic examination, and histology. The vascular patterns of the parotoid glands were compared to the arrangement of vessels in the dorsal skin of Rana sphenocephala (Ranidae), a frog that lacks parotoid glands. The parotoid glands of the four species of toads are supplied with blood by the lateral and dorsal cutaneous arteries and are drained by one or more branches of the internal jugular vein. The dorsal cutaneous artery supplies most of the blood to the parotoid glands in B. terrestris and B. valliceps. In B. alvarius and B. marinus, both the lateral and dorsal cutaneous arteries serve major roles in the blood supply of the glands. These patterns of blood flow have not been described previously for parotoid glands and conflict with earlier accounts for B. alvarius and B. marinus. The arteries and veins associated with the parotoid glands of toads are present in R. sphenocephala, but are arranged differently. In R. sphenocephala, the lateral cutaneous artery supplies the dorsal and lateral skin posterior to the shoulder region, whereas the dorsal cutaneous artery supplies the skin of the shoulder region. In toads, both the lateral and dorsal cutaneous arteries supply the skin of the shoulder region and ramify into subcutaneous capillaries that surround the secretory units of the parotoid glands. Extensive vasculature presumably is important for delivering cholesterol and other precursor molecules to the parotoid glands, where those compounds are converted into toxins.     

Interactions of thyrotropin releasing hormone and morphine sulfate in rodents.

Thyrotopin releasing hormone (TRH) produces “wet dog shakes” in rats similar to those observed during morphine withdrawal. The shaking behavior precipitated by morphine abstinence can be exacerbated by TRH administration while the other components of the morphine withdrawal syndrome remain unchanged. Morphine, chlorpromazine, apomorphine, and Δ⁹-tetrahydrocannabinol effectively block shakes induced by either TRH administration or morphine withdrawal. These results suggest the possibility that endogenous TRH may be associated with the “wet dog shakes” observed as a portion of morphine's abstinence syndrome in rats. However, TRH is unable to alter the stereospecific binding of morphine $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$, and naloxone fails to potentiate the number of TRH-induced shakes. TRH has no antinociceptive properties, and it cannot alter those of morphine. These data suggest that more than one neuromechanism may be responsible for shaking behavior in rats.     

The thermodynamic degree of coupling between metabolism and sodium transport in frog skin

The tightness of coupling between two processes is advantageously evaluated by the thermodynamic degree of coupling $\text{q}$, varying in absolute value from zero for uncoupled processes to unity for processes which are related stoichiometrically. Two methods for the determination of $\text{q}$ in the active pathway in frog skin have been developed, employing amiloride to abolish active sodium transport. The values of $\text{q}$ in 6 frog skins varied, but were always less than unity (mean $\text{0.79 ± 0.06}$ S.E. according to one method, $\text{0.78 ± 0.06}$ S.E. according to the other). This indicates that metabolism and sodium transport are incompletely coupled in this tissue even when passive transepithelial leakage pathways are taken into account.     

Early appearance in neural crest and crest-derived cells of an antigenic determinant present in avian neurons

A monoclonal antibody (“$\text{E}\text{C8}$”) against chicken dorsal root ganglion cells has been produced. The epitope (antigenic determinant) to which this antibody binds appears in neuronal cells—of both the peripheral and central nervous systems—and in a limited number of nonneuronal cell types in avian embryos. The epitope is intracellular and is probably part of a protein as judged by its susceptibility to proteases. This epitope appears very early in neuronal development. It may be detected in brain, spinal cord, and ventral root nerve fibers of Hamburger-Hamilton stage 16 chicken embryos (51–56 hr of incubation). At this same age, $\text{E}\text{C8}\text{-}\text{immunoreactive}$ cells can be found in the neural crest migratory space between the neural tube and the somite about a day before dorsal root ganglia begin to coalesce. Since some cultured neural crest cells (but not somitic mesenchymal cells) also express this epitope, we propose that the $\text{E}\text{C8}$ monoclonal antibody identifies an early differentiating subpopulation of neural crest cells which express this putative neuronal trait soon after the time of cessation of migration in vivo.     

Hormone-treated CHO cells exit the cell cycle in the G2 phase

In order to localize and identify receptor structures, the binding of radiolabeled thyrotropin releasing hormone (TRH) to thyrotropin (TSH)-secreting cells from rat and bovine anterior pituitaries and from a mouse TSH-secreting tumor was studied $\text{in vitro}$. The binding of TRH to rat anterior pituitaries increased linearly with the log of TRH concentration in the incubation medium. Plasma membranes were the only subcellular fractions isolated after incubation from bovine anterior pituitary and the TSH tumor which bound detectable quantities of TRH.     

Effect of sanguinarine,a benzophenanthridine alkaloid,on frog skin potential difference and short circuit current

Sanguinarine, a benzophenanthridine alkaloid, causes an initial stimulation of frog skin short circuit current $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ when present in the mucosal bathing medium at 10^?4 M. The stimulation is accompanied by an increase in spontaneous potential difference (PD) and increase in D.C. resistance. No effects are seen with sanguinarine in the serosal bathing medium. The initial stimulation is followed by a decrease in $\text{I}{}_{\mathrm{<mtext>se</mtext>}}$ and PD, but a continued increase in resistance. In skins whose initial spontaneous PD is high, no initial stimulation in ${}_{\mathrm{<mtext>se</mtext>}}$ and PD is seen; however, clamping these skins to a lower potential does not alter their initial inhibitory response to sanguinarine. Likewise, clamping the lower potential skins to higher potential does not alter their initial stimulatory response. Sanguinarine seems to be acting on the permeability barriers at the outer surface of the frog skin.     

Effect of thyrotropin releasing hormone on the camp content in circumesophageal ganglia of lymnaea emarginata

The effect and metabolic fate of thyrotropin releasing hormone on the cAMP content of $\text{in}$$\text{vitro}$ incubated ganglia from the pond snail $\text{Lymnaea}$$\text{emarginata}$ was studied. It was found that TRH caused an increase in the cAMP content of parietal ganglia, a decrease in the cAMP content of cerebral ganglia and no change in the other circumesophageal ganglia. $\text{In}$$\text{vitro}$ incubated ganglia did not accumulate or degrade significant amounts of ³H-TRH.     

Hydrocortisone increases the number of receptors for thyrotropin-releasing hormone on pituitary cells in culture.

Hydrocortisone (cortisol) increased the binding of thyrotropin-releasing hormone (TRH) to specific membrane receptors in 4 clonal strains of rat pituitary cells. At the highest effective cortisol concentration (3–5 × 10^?6 M), the increase was observed within 6–8 hr and became maximal (140 to 160% of control binding) by 18–24 hr. Half-maximum stimulation occurred in serum-containing medium at 9 × 10^?8 M cortisol, and a significant increase in TRH binding was seen at 3 × 10^?8 M. Equilibrium binding studies showed that enhanced TRH binding was explained by an increase in receptor number with no change in affinity. Similar effects were seen with Dexamethasone, but no increase in TRH binding was noted when testosterone, methyltestosterone, progesterone, estradiol or the antiestrogen Lilly 88571 were added to the culture medium. Cortisol treatment did not cause the appearance of specific TRH binding sites in cell strains previously shown to lack receptors for the tripeptide (F₄C₁, GH₁2C₁ and R₅ cells). When added cortisol was removed from medium, receptor number decayed to control values with a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of about 30 hr. Previous studies have shown that TRH receptors in GH-cells can be down-modulated by TRH and thyroid hormones; the present findings demonstrate that glucocorticoid hormones can increase the number of TRH receptors in GH-cells.     

Circadian rhythm of histamine in the pineal gland

The stimulating effects of 2-guanidinbenzimidazole and phentolamine on sodium transport through the isolated skin of the frog $\text{Rana esculenta}$ are described. These substances only act when added to the epithelial side, suggesting that they affect the permeability of the external barrier of sodium compartment. The role of the imidazole group in the activation of sodium transport is discussed.     

An improved synthesis of thyrotropin releasing hormone (TRH) and crystallization of the tartrate

An improved synthesis of thyrotropin releasing hormone (TRH), pGlu-His-Pro-NH₂, is reported. Z-pGlu-ONB (N-hydroxy-5-norbornene-2,3-dicarboximide ester) was reacted with H-His-OH to yield a crystalline Z-pGlu-His-OH which was coupled with H-Pro-NH₂ by the $\text{HONB}\text{DCC}$ method to give Z-pGlu-His-Pro-NH₂ as a fine crystal. Hydrogenation of this protected tripeptide yielded pure TRH nearly quantitatively. The optical purity of TRH thus obtained was confirmed by the method L- and D- amino acid oxidase digestion. The crystallization of TRH was achieved as a tartrate, and the properties of the crystalline TRH-tartrate are described.     

Localization of prolactin receptor in the dorsal and ventral skin of the frog (Rana ridibunda)

The dorsal and ventral skin in amphibians plays an important role in osmoregulation. Prolactin hormone is involved in regulation of amphibian skin functions, such as water and electrolyte balance. Therefore, amphibians may be useful as a model for determining the sites of the prolactin receptor. In this study, prolactin receptor was detected in frog dorsal and ventral skin using immunohistochemical staining method. Prolactin receptor immunoreactivity was localized in all epidermal layers except stratum corneum of dorsal skin epidermis, stratum germinativum layer of ventral skin epidermis, myoepithelial cells, secretory epithelium and secretory channel cells of granular glands in both skin regions. The mucous glands and secretory granules of granular glands did not show immunoreactivity for the prolactin receptor. According to our immunohistochemical results, the more widespread detection of prolactin receptor in dorsal skin epidermis indicates that prolactin is more effective in dorsal skin. Presence of prolactin receptors in epidermis points out its possible osmoregulatory effect. Moreover, detection of receptor immunoreactivity in various elements of poison glands in the dermis of both dorsal and ventral skin regions suggests that prolactin has a regulatory effect in gland functions.     

Distribution and characterization of cyclo(His-Pro)-like immunoreactivity in the rat gastrointestinal tract

The distribution of cyclo(His-Pro), thyrotropin-releasing hormone (TRH) and $\text{pyroglutamate aminopeptidase}$ activity was examined in the rat gastrointestinal (GI) tract. Cyclo(His-Pro)-like immunoreactivity was present in the following order of distribution (fmoles/mg protein): caecum > colon = jejunum = ileum > stomach = duodenum = rectum, and was immunologically and chromatographically identical with the authentic cyclo(His-Pro). Cyclo(His-Pro) concentrations showed significantly positive correlations with TRH concentrations, but not with $\text{pyroglutamate aminopeptidase}$ activities, in most tissues of the GI tract, suggesting a precursor role of TRH for gut cyclo(His-Pro). These data suggest that cyclo(His-Pro) may be involved in regulating rat GI functions.     

Peridermal cell patterning in the feather-forming skin of the chick embryo

The shape, distribution, and orientation of peridermal cells were examined in the dorsolumbar skin of $\text{7}\text{1}\text{2}\text{-}\text{day}$ chick embryos. Most feather rudiments of middorsal and lateral rows showed a marked cephalocaudal polarity. A similar polarity was found in the prospective rudiments of skin areas lateral to the last-formed row. On the cranial slope and apex of rudiments, cells are convex and predominantly elongated at right angles with respect to the cephalocaudal axis, whereas on the caudal slope, most cells are flat, polygonal, surrounded by a border-line ridge, and oriented predominantly with their long axis parallel to the cephalocaudal axis. The significance of this pattern is discussed in view of the fact that the epidermis is the determinant tissue in feather orientation.     

Stimulatory effect of prostaglandin E1 on thyroliberin-induced α-melanotropin release from perifused neuro-intermediate lobes of frog pituitary gland

A possible direct effect of prostaglandins on α-melanotropin (α-MSH) release at the level of the intermediate lobe of the frog pituitary was investigated $\text{in vitro}$ using a perifusion system technique. The effect of prostaglandins was studied on both spontaneous and TRH-stimulated α-MSH secretion. No significant effect of PGE₁, PGE₂, PGF_1α or PGF_2α on basal release of α-MSH could be detected. Indomethacin did not alter the α-MSH release induced by TRH. Conversely a significant increase in TRH-induced α-MSH secretion was observed in the presence of 1 x 10^?6M PGE₁. This magnifying effect was directly related to the concentration of TRH for doses ranging from 1 x 10^?8M to 1 x 10^?6M.     

Histology and lectin-binding patterns in the skin of the terrestrial horned frog Ceratophrys ornata

The terrestrial horned frog, Ceratophrys ornata, lives on a wet substratum and absorbs water through the ventral epidermis; water is lost by evaporation from the dorsal skin. Thus, this species may be useful as a model for determining whether or not skin histology and lectin-binding patterns, indicative of glycoconjugates, are related to skin functions such as osmoregulation and water balance. With this in mind, a histological and lectin-histochemical study was carried out on dorsal and ventral skin of aquatic tadpoles and of a young terrestrial frog of C. ornata. Sections of skin were stained with various dyes to demonstrate general histological features and with two horseradish peroxidase (HRP)-conjugated lectins, Ulex europaeus agglutinin (UEA 1) and soybean agglutinin (SBA) which bind to specific terminal sugar residues of glycoconjugates, namely L-fucose and N-acetyl-D-galactosamine or D-galactose, respectively. In early stage tadpoles both lectin-binding patterns were similar in the bilaminar epidermis of dorsal and ventral skin (i.e., each lectin stained the apical cell layer). However, metamorphic changes resulted in a young frog with typical adult-type skin composed of a stratified squamous epidermis and three distinct types of glands containing glycoconjugates in their secretions. Strikingly different lectin-binding patterns were evident in the epidermis from dorsal and ventral regions of the body. The epidermis from the dorsal region was stained by both lectins; in contrast, that from the ventral region although stained strongly by HRP-SBA, did not react with HRP-UEA 1 indicating that few, if any, fucose residues were present in the ventral epidermis. These findings, as suggested in the discussion, indicate that different glycoconjugate patterns in dorsal and ventral skin may be associated with the regulation of water balance in the frog.     

Weak acid accumulation in the serosal extracellular compartment of the frog gastric mucosa

The dimethyloxazolidine dione distribution in the extracellular compartments of the frog gastric mucosa was analyzed by washout kinetics. The volumes of the two extracellular compartments, serosal and mucosal, were estimated by inulin washout as $\text{0.435 ± 0.019}$ and $\text{0.176 ± 0.018 μl/μl}$ tissue water, respectively. In the serosal extracellular space, significant dimethyloxazolidine dione accumulations of $\text{2.63 ± 0.25}$, $\text{2.28 ± 0.16}$, and $\text{1.86 ± 0.08}$ times that of the bathing media were found for bathing solutions with pH values of 6.9, 7.4, and 7.9 respectively. A high pH of the serosal extracellular fluid by itself could not account for the high values of dimethyloxazolidine dione accumulation. A difference in the total dimethyloxazolidine dione accumulation requires: (a) the existence of differences in the pH values and also the existence of a difference in the diffusion coefficient of the two forms of dimethyloxazolidine dione; or (b), a binding of one of the two forms, i.e., binding of dimethyloxazolidine dione form by fixed charges.     

Ontogeny of the retina and optic nerve of Xenopus laevis: IV. Ultrastructural evidence of early ganglion cell differentiation

Ultrastructural evidence indicates that Xenopus retinal ganglion cell axons differentiate early, between stages 28 and 32. Light microscope studies indicated the presence of argryophilic material in the ventral retina and optic stalk of early embryos. Ultrastructural analysis of this region confirmed the presence of axons in the stalk and interstices of ventral retinal cells. Axons containing aligned microtubules and neurofilaments and elongated mitochondria with a paucity of other cell inclusions are found with increasing frequency in the ventral retina from stages 28 through $\text{33}\text{34}$. Central and dorsal regions of the retinas examined show little or no evidence of axons. A discrete, small bundle of axons is found in the optic stalk of stage 28 embryos and by stage $\text{30}\text{31}$ the number of axons in bundles has increased, suggesting early fasciculation. Between stages 28 and $\text{33}\text{34}$ (± 12 hr) extracellular space surrounding early axons diminishes and processes from neuroretinal cells in contact with axons surround developing axon bundles. The evidence presented suggests that axon initiation occurs in stages much earlier than previously reported. Other investigators have failed to detect ganglion cell differentiation prior to stage 32 possibly because they examined regions of the retina with few axons. Thus, experiments which rotate the retina in the orbit may have to be reevaluated since regenerating axons may use previously established pathways to organize and “home in” on tectal target cells.     

Plasmodium berghei: Thyroid hyperplasia and thyroid hyperactivity in mice

Statistically significant (P < 0.05) thyroid hyperactivity (¹³¹I% uptakes) occurs on certain days in Plasmodium berghei infected C3H mice. Male mice thyroid glands are made more hyperactive by the malaria than the female glands. Hyperactive thyroid glands may be at least one cause of hypocholesterolemia in plasmodium-infected rodents. Thyroid hyperplasia was found only in experimental mice ($\text{6}\text{20}$ males; $\text{8}\text{20}$ females). A hyperactive thyroid gland appears to be an unreported aspect of experimental acute P. berghei infections in mice. The hyperthyroidism may be due to possible toxic substances acting either directly on the thyroid gland, or indirectly on the hypothalamus affecting TSH production.