Morpho-functional characterization of cultured pigment cells fromRana esculenta L. Liver

Summary A simple method to isolate and culture liver pigment cells fromRana esculenta L. is described which utilizes a pronase digestion of perfused liver, followed by sedimentation on a Ficoll gradient. A first characterization of isolated and cultured cells is also reported. They show both positivity for nonspecific esterases, and phagocytosis ability, like the cells of phagocytic lineage. Furthermore, after stimulation with a phorbol ester, these cells generate superoxide anions. At phase contrast microscope, liver pigment cells present variability in size, morphology, and in their content of dark-brown granules. Inasmuch as a cell extract obtained from cultured cells exhibits a specific protein band with dopa-oxidase activity, when run on nondenaturing polyacrylamide gel electrophoresis, liver pigment cells fromRana esculenta L. should not be considered as melanophages, but as cells that can actively synthesize melanin. The method presented here seems to be useful to more directly investigate this extra-cutaneous melanin-containing cell system and to clarify its physiologic relevance. This research was partly supported by grant of Ministero della Pubblica Istruzione, Ricerca Scientifica.     

Spleen and Liver Pigmented Macrophages of Rana Esculenta L. A New Melanogenic System?

The present study reports the results of a morpho‐functional analysis of spleen pigmented cells from Rana esculenta L. and comparison with liver melanin‐synthesizing cells, belonging to the macrophage cell lineage. Cytological and cytochemical analyses show that parenchymal pigmented cells of the spleen, like those of the liver, are positive to peroxidase and lipase reactions and have phagocytic properties. The observation of premelanosomes in various stages of differentiation, together with the demonstration of dopa oxidase activity in the melanosome proteins, indicate that spleen pigmented macrophages have endogenous melanogenic ability as do liver pigmented macrophages. Attempts to demonstrate tyrosine‐hydroxylase activity in melanosome protein extracts from frog spleen and liver, using the same protocol as for mammalian tyrosinases, gave negative results. As regards the dopa oxidase activity revealed, some of its properties differ from the typical behaviour observed for tyrosinases from different sources. Peroxidase activity is shown in spleen and liver melanosome proteins with p‐phenylenediamine‐pyrocatechol (PPD‐PC), and not with typical peroxidase substrates. Suitable inhibition tests revealed that dopa oxidase and peroxidase activities might be supported by two different proteins. Liver melanosome extracts display a very strong laccase (dimethoxyphenol‐oxidase) activity but spleen extracts do not. Differences observed in the enzymatic properties of the spleen and liver melanosomes suggest that pigmented macrophages may undergo tissue‐specific differentiation. These preliminary data show that the melanin pathway of pigmented macrophages is different from that of melanocytes and may pave the way to identification of a new melanogenic pathway in vertebrates.     

Melanogenesis in the pigment cells of Rana esculenta L. liver: evidence for tyrosinase-like activity in the melanosome protein fraction

This work demonstrates the presence of a tyrosinase-like activity in the pigment cells of frog Rana esculenta L. liver. The activity was evidenced in the protein melanosome fraction extracted with differential centrifugation methods. The study of this activity, carried out with spectrophotometric methods, indicates 1) the system presents the characteristics of an allosteric enzyme; 2) the grade of cooperativity shows oscillations going from negative cooperativity toward the substrate, evident in the warmest months of the year, to an absence of cooperativity in the coldest months of the year; and 3) the levels of activity of the system also vary according to season, with the highest levels appearing in the coldest months of the year. Given that this extracutaneous system of pigment cells, different from melanocytes, is able to carry out melanogenesis, we suggest its inclusion in the classification of pigment cells.     

Biogenesis of Melanosomes in Kupffer Cells of Proteus anguinus (Urodela,Amphibia)

The ultrastructural characteristics of melanosomes and premelanosomes observed during the biogenesis of melanosomes in liver pigment cells of the neotenic cave salamander Proteus anguinus (Proteidae) are described. It is well known that amphibian liver pigment cells, also known as Kupffer cells (KC), contain melanosomes and are able to synthesize melanin. Liver pigment cells of P. anguinus contain numerous siderosomes and melanosomes. The melanosomes are grouped together within single‐membrane‐bounded bodies, named as ‘clusters of melanosomes’ or ‘melanosomogenesis centers’. Inside such clusters, different structures are present: (1) filament‐like structures, characteristic of the initial stage of melanosome biogenesis, (2) medium electron‐dense melanosomes in different stages of melanization, (3) melanosomes with an electron‐dense cortical area and a less electron‐dense medullar area, and (4) uniformly highly electron‐dense mature melanosomes or melanin granules. Histochemical and cytochemical dihydroxyphenylalanine (DOPA) oxidase reactions in pigment cells were positive. Our results confirm the ability of amphibian KC to synthesize melanin and contribute to this little known subject.     

The Amphibian Kupffer Cells Build and Demolish Melanosomes: An Ultrastructural Point of View

This ultrastructural research was carried out to investigate the nature of the liver pigment cells of anuran and caudate amphibians, the pattern of melanosome ontogenesis, and the demolition processes of old melanosomes. We demonstrate that these liver pigment cells are able to internalize zymosan particles and latex beads; therefore, being professional phagocytes, they, as liver resident macrophages, can be classified as Kupffer cells (KCs). They show “melanosomogenesis centers” in which several maturation stages of premelanosomes are visible; the premelanosomes are formed by two principal components: a filamentous structure that will constitute the “inner” area of the melanosome and a vesicular component, budding from the Trans Golgi Network and that carries enzymes, which will constitute the “cortical area” of the melanosome. Thus the KCs, thanks to the presence of the “melanosomogenesis centers,” are also melanosome producing cells. They are also able to demolish melanosomes by heterophagocytosis and, probably, also by autophagocytosis. In conclusion, we propose a classification of vertebrate pigment cells.     

Different patterns of expression of five neuropeptides in the adrenal gland and kidney of two species of frog

The aim of this study was to demonstrate in the adrenocortical and renal tissues of two species of frog, Rana italica and Rana esculenta, the presence and distribution of five neuropeptides: atrial natriuretic peptide (ANP), Leu-enkephalin (Leu-ENK), neuropeptide Y (NPY), substance P (SP) and vasoactive intestinal peptide (VIP).In anurans, the adrenal medulla is the site for the synthesis, storage and secretion of not only catecholamines but also various peptides. These peptides should not be regarded only as neurotransmitters or modulators for the secretion of catecholamines, but also as hormonal substances that induce systemic effects.All the peptides studied (ANP, Leu-ENK, NPY, SP and VIP) are present in both organs. However, different patterns of expression were observed for some of the peptides in two frogs.Immunopositivity to ANP was found in small clusters of chromaffin cells in both frogs whereas a clear strong positivity was present only in Rana esculenta kidney. Large clusters of chromaffin cells were immunoreactive to Leu-ENK in Rana italica but there were approximately 25% fewer compared to the positive cells present in Rana esculenta. Epithelial cells of renal tubules showed strong immunopositivity to Leu-ENK in Rana esculenta but not in Rana italica. A large number of adrenal cells (70–80%) were immunoreactive to NPY in Rana italica, while in Rana esculenta this peptide was localized in small clusters of chromaffin cells. Both frogs showed many NPY-positive cells in kidney. Many chromaffin cells were found positive to SP and VIP. A strong positivity was also observed in kidney in both frogs. These observations suggest a possible role of these peptides in the control of the physiological functions of adrenal glands and kidney of the two species of frogs studied.     

A comparative study of physiological and structural changes at the myoneural junction in two species of frog after transection of the motor nerve

Summary Structural and functional behaviour of motor end-plates after transection of the motor nerve has been studied in two species of frog: Rana esculenta and Rana temporaria. The physiological results show that in both species there is a transient cessation of spontaneous activity followed by a resumption of miniature end-plate potentials (min. e.p.p.s.) after denervation. The characteristics of these potentials (frequency, distribution of amplitudes, time-course) are similar in the two species. However, some differences have been observed: Firstly, the period of silence lasts for 2–4 days in the case of Rana temporaria whereas it is prolonged to about 15 days in Rana esculenta. Secondly, the resumption of min. e.p.p.s. is gradual and after the 10th day of denervation remains constant in Rana temporaria. It is inconstant independent of the period of denervation in Rana esculenta. The morphological results show that the Schwann cell is constantly in contact with the post-synaptic membrane after about 6 days of denervation in both species. It is suggested that either the Schwann cell is capable of functioning for a limited period of time in Rana esculenta or is activated to produce min. e.p.p.s. only in certain cases.     

Über Kanälchenzellen und dunkle Zellen im Nephron von Anuren

Zusammenfassung Die elektronenmikroskopische und enzymcytochemische Untersuchung der Nieren von Rana cancrivora und R. esculenta, Hyla arborea und H. regilla, Bufo carens, B. mauretanicus und B. viridis ergibt, daß im Epithel ihrer Verbindungsstücke verschiedene Zelltypen vorkommen.Die Verbindungsstücke von Rana cancrivora und R. esculenta zeichnen sich — abgesehen von dem Vorhandensein heller Zellen — durch den Besitz sog. Kanälchenzellen aus, die nach Form und Struktur an die Belegzellen der Magendrüsen erinnern. Es handelt sich um sehr mitochondrienreiche, elektronendicht erscheinende Elemente, die durch teilweise verzweigte intrazelluläre Kanälchen mit der Lichtung des Verbindungsstückes kommunizieren. Aus der Oberfläche dieser Canaliculi ragen kurze Cytoplasmafortsätze in das Lumen. An der Basis der Kanälchenzellen bilden zahlreiche Cytoplasmafortsätze ein Labyrinth, das mit den seitlichen Interzellularräumen in Verbindung steht. Die Kanälchenzellen geben eine starke Reaktion auf Bernsteinsäuredehydrogenase und NADH-Diaphorase, die dem Reichtum der Zellen an Mitochondrien entspricht. Unterschiede in der Zahl der Kanälchenzellen bei dem marinen Frosch Rana cancrivora und bei R. esculenta scheinen nicht zu bestehen.Im Verbindungsstück der untersuchten Hyla- und Bufoarten fallen dunkle, d.h. elektronendichte Zellen auf, die zwar viele Mitochondrien enthalten und ein basales Labyrinth bilden, jedoch nicht kanalisiert sind. Auch diese dunklen Zellen geben eine intensive Reaktion auf Bernstein- säuredehydrogenase und NADH-Diaphorase.Die Kanälchenzellen weisen Zeichen einer sekretorischen Aktivität (Bildung von Mukosubstanzen) auf. Die Frage, ob sie außerdem resorptiv tätig sind und ob die dunklen Zellen sowohl sezernieren als auch Ionen aktiv transportieren, kann mit den angewandten Methoden nicht beantwortet werden. Unbekannt ist ferner, ob die Unterschiede im Aufbau der Verbindungsstücke bei den Rana-Arten auf der einen und den Hyla- und sowie Bufo-Arten auf der anderen Seite zu Verschiedenheiten der Harnbildung bei diesen Formen in Beziehung stehen.

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On canaliculi cells and dark cells in the nephron of anurans

Summary The electronmicroscopical and enzymecytochemical investigation of the kidneys of Rana cancrivora, R. esculenta, Hyla arborea, H. regilla, Bufo carens, B. mauretanicus and B. viridis reveals that the epithelium of the connecting tubule (Verbindungsstück) consists of different cell typs.The connecting tubules of Rana cancrivora and R. esculenta are characterized — apart from the occurrence of light cells — by the differentiation of so-called canaliculi cells, the shape and structure of which resemble those of the parietal cells of the gastric glands. These electron dense elements are rich in mitochondria and communicate by partly ramified intracellular canaliculi with the lumen of the connecting tubule. The plasma membrane bordering the canaliculi forms short microvilli-like processes. At the basis of the canaliculated cells numerous cytoplasmic processes form a labyrinth which is in connection with the lateral intercellular space. The canaliculi cells exhibit a strong activity of succinic dehydrogenase and NADH-diaphorase, which corresponds to the abundance of mitochondria in these cells. Differences in the number of canaliculi cells in the marine frog Rana cancrivora and the freshwater species Rana esculenta apparently do not exist.The connecting tubules of the Hyla and Bufo species investigated contain strikingly dark, i.e. electron dense cells, which contain numerous mitochondria, and which possess a basal labyrinth. Canaliculi, however, are lacking. These dark cells, too, give an intensive reaction for succinic dehydrogenase and NADH-diaphorase.The canaliculi cells show features of a secretory activity (production of mucous substances). The question, whether in addition they have a resorptive function and whether the dark cells are secretory and actively ion-transporting elements, cannot be answered by means of the methods applied. Further, it remains unknown whether the differences in the structure of the connecting tubule of the Rana species on the one hand and the Hyla-and Bufo species on the other, are related to differences in the mechanisms of urine production.

Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

A microsatellite‐based method for genotyping diploid and triploid water frogs of the Rana esculenta hybrid complex

Rana esculenta is a hybrid between Rana lessonae (LL) and Rana ridibunda (RR), and hybrids may be diploid (LR) or triploid (LLR or LRR). Genotypes can be roughly determined from erythrocyte size and morphometry in adult frogs, but accurate genotyping requires more labourious methods. Here I demonstrate that both the L and R genomes have specific microsatellite alleles, and that genotype and ploidy can be accurately inferred from the quantitative ratio of PCR‐amplified (polymerase chain reaction‐amplified) genome‐specific alleles. This method greatly facilitates genotyping in DNA studies of the R. esculenta complex and allows analysis of badly preserved samples and embryos.     

Elektronenmikroskopische Untersuchungen am Stirnorgan von Anuren

The ultrastructure of the frontal organ (pineal end-vesicle, Stirnorgan) of Rana temporaria L. and Rana esculenta L. is similar to the submicroscopic organization of the retina and other photosensitive organs. There are five different cell types in the frontal organ: sensory (receptor) cells, ependymal cells, ganglion cells, glial cells and epithelial cells. The ependymal cells may be secretory. There is no evidence for a typical pigment epithelium. The sensory cells have inner and outer segments. The inner segments contain numerous mitochondria, a Golgi complex, filaments, lipid droplets, two centrioles and a fibrillar apparatus (within the connecting piece). The mitochondria are very abundant in the Ellipsoid and Ersatzellipsoid areas (Holmgren) of the inner segment. The outer segment consists of about 60 to 110 discs formed by infoldings of the cell membrane. Most of the sensory cells are cone-like, but there are some elements with rod-like structures. Plexiform areas of the frontal organ contain terminations of the receptor cells, and processes of the nerve cells and glia cells. Synaptic structures have been determined within these areas. Non-medullated and medullated nerve fibers with adjacent glial satellites are observed in the pineal nerve (Nervus pinealis). The anatomical findings are described in detail and discussed in respect to the physiological results of Dodt and Heerd (1962) in Rana temporaria and Rana esculenta.

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Durchgeführt mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Annual cycle of activity of the neurocytes of the ventral lateral septum in the brainof Rana esculenta L.∗

Abstract

Sexually mature male and femaleRana esculenta L. were captured in their natural habitat in six phases of the annual cycle. Nuclear volumes of neurocytes in the ventral lateral septum (VLS) were found to fluctuate distinctly in the course of the year. In both sexes nuclear volumes of neurocytes were maximal in the phases preceding the breeding season (3rd decade of January, and 1st decadeof April), and minimal in the phaseat the beginning of hibernation (3rd decade of October). The results lead to the assumption that the VLS ofRana esculenta may have an active role in controlling gonadotropin release.     

Influence of Hyperthyroidism on the Activity of Liver Nitric Oxide Synthase in the Rat

Hyperthyroidism enhances the prooxidant activity of the liver by elevating superoxide radical and/or hydrogen peroxide generation in microsomal, mitochondrial, and peroxisomal fractions, with an increased respiratory burst of Kupffer cells. In this study, the influence of daily doses of 0.1 mg 3,3′,5-triiodothyronine (T₃)/kg for three consecutive days on liver nitric oxide (NO) synthase (NOS) was assessed, as a possible contributory mechanism to T₃-induced liver prooxidant activity. Thyroid calorigenesis was paralleled by a progressive increment in the rate of NO generation, with significant increases after 2 (47%) and 3 days (70%) of T₃treatment, and a net 45% (P< 0.05) enhancement in theN^G-methyl-l-arginine-sensitive NO production, compared to control values. These enhancement effects were reversed to control levels after 3 days of hormone withdrawal, concomitantly with the normalization of hepatic respiration. Enhancement of liver NOS activity in hyperthyroid animals was diminished by 27% (P< 0.05) by the selectivein vivoinactivation of Kupffer cells by gadolinium chloride (GdCl₃), without direct actions of GdCl₃on the enzyme. These data demonstrate that hyperthyroidism leads to a significant and reversible enhancement in rat liver NOS activity, an effect that is exerted at hepatocyte and Kupffer cell levels, thus representing an additional source of prooxidants to those of reactive oxygen species.     

Ultrastructural changes in dermal melanophores in the process of pigment granule migration

The results of electron microscope investigations on dermal melanophores of Rana temporaria L. during migration of pigment granules are presented. It was shown that in comparison to the previous observations dermal melanophores are flat cells without branches. Ultrastructural differences have been demonstrated in dermal melanophores during migration of pigment granules. During melanosome dispersion membrane vesicle bodies are seen in the cytoplasm to be inserted in the melanophore membrane.     

Spleen and liver pigmented macrophages of Rana esculenta L. A new melanogenic system?

The present study reports the results of a morpho-functional analysis of spleen pigmented cells from Rana esculenta L. and comparison with liver melanin-synthesizing cells, belonging to the macrophage cell lineage. Cytological and cytochemical analyses show that parenchymal pigmented cells of the spleen, like those of the liver, are positive to peroxidase and lipase reactions and have phagocytic properties. The observation of premelanosomes in various stages of differentiation, together with the demonstration of dopa oxidase activity in the melanosome proteins, indicate that spleen pigmented macrophages have endogenous melanogenic ability as do liver pigmented macrophages. Attempts to demonstrate tyrosinehydroxylase activity in melanosome protein extracts from frog spleen and liver, using the same protocol as for mammalian tyrosinases, gave negative results. As regards the dopa oxidase activity revealed, some of its properties differ from the typical behaviour observed for tyrosinases from different sources. Peroxidase activity is shown in spleen and liver melanosome proteins with p-phenylenediamine-pyrocatechol (PPD-PC), and not with typical peroxidase substrates. Suitable inhibition tests revealed that dopa oxidase and peroxidase activities might be supported by two different proteins. Liver melanosome extracts display a very strong laccase (dimethoxyphenoloxidase) activity but spleen extracts do not. Differences observed in the enzymatic properties of the spleen and liver melanosomes suggest that pigmented macrophages may undergo tissue-specific differentiation. These preliminary data show that the melanin pathway of pigmented macrophages is different from that of melanocytes and may pave the way to identification of a new melanogenic pathway in vertebrates.     

Über das Reaktionsspektrum von Rezeptoren aus der Riechschleimhaut von Wasserfröschen (Rana esculenta)

Zusammenfassung Aus dem Riechepithel von Rana esculenta wurden mit metallgefüllten Mikropipetten Aktionspotentiale einzelner Rezeptoren abgeleitet (Dauer 4–7 msec, maximale Frequenz 10 Imp./sec). Die Reaktionen von 25 Zellen auf 28 Duftstoffe werden beschrieben.Fast alle daraufhin geprüften Zellen reagieren auf Oenanthaldehyd, Amylacetat, Capronsäureäthylester und Caprylsäureäthylester mit einer Erhöhung der Impulsfrequenz (Grundspektrum). Außerdem haben die Rezeptoren ein variables Beispektrum geringen Umfanges.

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Odor specificities of receptors in the olfactory epithelium of the frog Rana esculenta

Summary Action potentials from single receptors in the olfactory epithelium of water frogs (Rana esculenta) have been recorded by means of metal filled glass pipettes. The reactions of 25 cells to stimulation with 28 odorous substances are described. All cells so far tested showed an increase in spike frequency when stimulated with heptanal, amyl acetate, the ethyl ester of hexanoic acid, and the ethyl ester of octanoic acid, (basic spectrum). An additional small but variable reaction spectrum (side spectrum) was recorded.

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Herrn Prof. Dr. H. Autrum zum 60. Geburtstag gewidmet.     

On the biology of Amphibia in the ecosystem of Lake Glubokoe

In Lake Glubokoe, the Amphibia are represented by five species. Two numerous species, Rana temporaria L. and Bufo bufo L., spawn in the lake and appear to be a significant component of its ecosystem. Thus, in autumn R. temporaria is a common food for predatory fish (pike, perch). During their wintering R. temporaria participate in the consumption of oxygen from the water. They may be a source of dangerous infections. Their tadpoles and those of B. bufo feed in the lake and annually remove substances from it. Adult R. temporaria in autumn, and the young of the year of R. temporaria and of B. bufo influence the population numbers of small littoral invertebrates. Several dozen pairs of Rana terrestris Andrzejw are observed to spawn regularly. Rana esculenta L. and Rana ridibunda Pallas are scarce. The influence of introducing a nature preserve on the numbers of Amphibia in Lake Glubokoe is discussed.     

ASYMMETRIC COMPETITION IN MIXED POPULATIONS OF TADPOLES OF THE HYBRIDOGENETIC: RANA ESCULENTA COMPLEX

Hybridogenetic Rana esculenta tadpoles display tolerance to extreme environmental conditions and fit criteria of the “general-purpose” genotype. A trade-off between generality and competitive ability is assumed to occur in asexual species, but the evidence remains unclear. The purpose of my experiment was to test the competitive ability of hemiclonal hybrid Rana esculenta tadpoles relative to the parental species Rana lessonae. Mixed and single genotype populations of R. esculenta and R. lessonae tadpoles were reared at three densities in artificial ponds. Survival of R. esculenta was higher than for R. lessonae tadpoles, but did not differ among densities. Body size at metamorphosis was the same between genotypes, but decreased with increasing density. Larval period was not affected by density, but R. esculenta tended to metamorphose earlier than R. lessonae. Percentage of individuals metamorphosing was higher for R. esculenta at both medium and high densities, but the same as R. lessonae at the low density. The difference in survival, body size, and larval period between tadpoles reared in single and mixed genotype populations was unaffected by genotype or density. The difference in the percentage of metamorphs, however, was strongly affected. The percentage of hybrids metamorphosing was 9% above the responses of single genotype populations at the highest density. Conversely, the percentage of R. lessonae metamorphosing was 12% below the responses of single genotype populations at the same density. Hybrid success in this experiment further supports the criterion of a “general-purpose” genotype without assumptions of reduced competitive ability.