Interaction of beta-endorphin, naloxone and dopamine: effects on melanocyte-stimulating hormone secretion of amphibian pituitaries in vitro

Neurointermediate lobes from amphibians (Rana pipiens) were incubated in Medium 199 containing dopamine, beta-endorphin or dopamine plus beta-endorphin. Dopamine inhibited melanocyte-stimulating hormone (MSH) secretion as measured by bioassay in hypophysectomized frogs, an effect which was transiently reversed by beta-endorphin. The effects of endorphin were in turn partially suppressed by the opiate antagonist, naloxone hydrochloride. Cells treated with all three agents exhibited expanded rough endoplasmic reticulum and decreased secretory granule content, indicative of peptide release and new synthesis. Beta-Endorphin alone did not stimulate MSH secretion above control levels, and at one time period was seen to reduce MSH secretion. The findings indicate a complex interaction between beta-endorphin and dopamine directly upon MSH secretion at the level of the neurointermediate lobe.     

Effects of neurotransmitters upon the discharge of secretory product from the cutaneous glands of the red-spotted newt.

The effects of sympathetic and parasympathetic agonists and antagonists on discharge of secretory product by the granular and mucous glands were examined in the red-spotted newt, Notopthalmus viridescens viridescens. Observations were made also on the South African clawed toad. Xenopus laevis, the grass frog, Rana pipiens, and the crested newt, Triturus cristatus. In contrast to the granular glands of the South African clawed toad and the grass frog, which were stimulated by alpha-adrenergic agents, those of the red-spotted newt discharge in response to acetylcholine, either in vitro when added to the Holtfreter's solution in which explants were incubated, or in vivo when injected subcutaneously. Granular glands of the crested newt were also dischared in response to subcutaneous injection of acetylcholine. Stimulation of the granular glands by acetylcholine was blocked by atopine but not by tubocurarie, indicating that the cholinergic receptors are muscarinic rather than nicotinic. The mucous glands of the red-spotted newt, on the other hand, did not discharge in response to either acetylcholine or to adrenergic agents.     

Hormone action in newt limb regeneration: insulin and endorphins

Although several hormones have been linked to newt limb regeneration, a cohesive hypothesis as to how these hormones control the process is yet to emerge. A critical review of the traditional approaches and a reevaluation of currently operative assumptions and interpretations of results precede the data on insulin and beta-endorphin. Results from in vivo and in vitro experiments on insulin are summarized, showing that insulin not only promotes various cellular events but also is essential for the expression of the mitogenic effect of nerves on cultured newt limb blastemata. Furthermore, the strong likelihood that insulin may be the common link in promoting limb regeneration in hypophysectomized newts that received pituitary hormone replacement therapy or a nutritional supplement is discussed. The status of beta-endorphin in regeneration is also explored. Data are presented to show that vertebrates with regenerating capacity (newts, tadpoles) have higher levels of plasma beta-endorphin than that found in species where the capacity to regenerate is either restricted (frogs) or totally lost (mammals). beta-Endorphin-like immunoreactivity has been localized in the epidermis of a regenerating newt blastema, as well as in the intermediate lobe of the pituitary gland of axolotl, newt, and Xenopus. A possible opiate connection in vertebrate limb regeneration, in particular, wound healing, is discussed.     

The effects of in vivo and in vitro hyperosmolality on skeletal muscle performance in the amphibians Rana pipiens and Scaphiopus couchii

1. The effect of in vivo and in vitro hyperosmolality on skeletal muscle function was investigated in two species of anuarans Scaphiopus couchii and Rana pipiens. 2. Muscle contractile performance, measured as peak tetanic tension declined to greater degree when tissue dehydration occurred in vitro rather than in vivo, even though tissue water contents were greater in vivo. 3. The muscles from S. couchii, a more dehydration tolerant species than R. pipiens, maintained tension at lower tissue water contents than R. pipiens. 4. Data for the effects of in vivo dehydration on plasma sodium, urea and osmotic concentration, as well as tissue water contents, are also presented for both species.     

Hydroperoxide metabolism of amphibia: tissue catalases of leopard frogs

Catalase activities were measured and compared in liver, kidney, heart, and lung of American Leopard Frogs (Rana pipiens complex). The order of activities was found to be liver greater than kidney greater than heart approximately lung. The liver enzyme was found to be inhibited by aminotriazole, cyanide, and azide and appears to peroxidatively oxidize ethanol.     

Adaptative differential maturation of neurohypophyseal provasotocin in amphibians: presence of hydrin 2(vasotocinyl-glycine) in anura but not in urodela]

Occurrence of hydrin 2 (vasotocinyl-glycine), an intermediate in the provasotocin processing, has been shown in the neurointermediate lobe of the pituitary of semi-aquatic or terrestrial amphibian species belonging to the order Anura, namely Rana esculenta, R. temporaria, R. pipiens (Ranidae), and Bufo bufo, B. marinus, B. ictericus (Bufonidae). In contrast hydrin 2 appears absent in species belonging to the order Urodela, namely the axolotl Ambystoma mexicanum (Ambystomidae) and the pleurodele newt, Pleurodela waltlii (Salamandridae). Whereas water transport through the skin and the urinary bladder is stimulated by vasotocin and hydrin 2 in anuran species, this stimulation is known not to occur in urodele species.     

Amelogenin sequence and enamel biomineralization in Rana pipiens

The amelogenin gene contributes the majority of tooth enamel proteins and plays a significant role in enamel biomineralization. While several mammalian and reptilian amelogenins have been cloned and sequenced, basal vertebrate amelogenin evolution remains to be understood. In order to start elucidating the structure and function of amelogenins in the evolution of enamel, the leopard frog (Rana pipiens) was used as a model. Tissues from Rana pipiens teeth were analyzed for enamel structure and RNA extracts were processed for sequence analysis. Electron microscopy revealed that Rana pipiens enamel contains long and parallel crystals similar to mammalian enamel, while immunoreactions confirmed the site-specific localization of cross-reactive amelogenins in Rana pipiens enamel. Sequencing of amelogenin PCR products revealed a 782bp cDNA with a 546-nucleotide coding sequence encoding 181 amino acids. The homology of the newly discovered Rana pipiens amelogenin nucleotide and amino acid sequence with the published mouse amelogenin was 38.6% and 45%, respectively. These findings report the first complete amelogenin cDNA sequence in amphibians and indicate a close homology between mammalian enamel formation and Rana pipiens enamel biomineralization.     

Species variations in the metabolism of liposoluble organochlorine compounds by hepatic microsomal monooxygenase: comparative kinetics in four vertebrate species

Liposoluble organochlorine compounds were used as substrates in a kinetic study of the hepatic microsomal monooxygenases of the male rat, feral pigeon (Columbia livia), frog (Rana pipiens) and rainbow trout (Salmo gairdnerii). Substrate concentrations were taken down to environmentally realistic levels. Lineweaver-Burk plots gave straight lines for both aldrin (HHDN) and the dieldrin analogue HCE. For both substrates activities followed the order rat greater than feral pigeon greater than trout over the entire concentration range. The metabolism of PCB isomers in microsomes from these uninduced animals was very slow. These results give evidence of cytochrome P-450 forms which can metabolize organochlorine compounds at low substrate concentrations.     

Proinsulin cDNAs from the leopard frog, Rana pipiens: evolution of proinsulin processing

We have isolated a proinsulin cDNA from the Amphibian Rana pipiens. The predicted R. pipiens insulin A- and B-chain amino acid sequences differ from that deduced from the closely related Rana catesbeiana at one residue (Asp for Pro at B2). The R. pipiens and Xenopus laevis proinsulin precursor sequences are of identical length, with the amino acid sequences of the mature A- and B-chains being well conserved. The proinsulin C-peptide amino acid sequence is less well conserved between R. pipiens and X. laevis and also differs in length. The R. pipiens C-peptide is shorter than the homologous X. laevis sequence due to a two amino acid residue truncation. The truncation of the R. pipiens C-peptide compensates for a two amino acid residue extension observed at the N-terminal of the A-chains of insulins from Ranid frogs. A change in the site of proinsulin processing can explain both the C-peptide and A-chain length differences. The evolution of the new proinsulin processing site required two amino acid substitutions.     

Genomic potential of erythroid and leukocytic cells of Rana pipiens analyzed by nuclear transfer into diplotene and maturing oocytes

In order to determine whether differentiated somatic cells maintain genetic totipotency, nuclear transplantations from several differentiated somatic cell types into eggs and oocytes were performed previously in Rana pipiens and Xenopus laevis. The formation of postneurula embryos and tadpoles under the direction of the test nuclei demonstrated their genetic multipotency. In addition, Rana erythrocyte nuclei transplanted to oocytes directed more extensive tadpole development than those injected into eggs. We have extended our studies of the genomic potential of differentiated somatic nuclei from the peripheral blood of Rana pipiens. First, we show that the developmental potential of erythrocyte nuclei injected into oocytes at first meiotic metaphase was greater than those injected into diplotene oocytes. Second, we demonstrate that erythroblast and leukocyte nuclei transplanted to oocytes at first meiotic metaphase promoted more advanced tadpole development than those previously injected into Xenopus eggs. Third, erythrocyte nuclei were more successful in promoting advanced tadpole development compared with erythroblast and leukocyte nuclei. The results show that differentiated somatic nuclei transferred to the cytoplasm of oocytes at first meiotic metaphase display enhanced genomic and developmental potential over those transplanted to diplotene oocytes and eggs, at least for the three nuclear cell types tested from the peripheral blood.     

CNPase activity in the vertebrate retina, retinal pigmented epithelium, and choroid

The activity of the enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase, E.C.3.1.4.37) has been studied in the retina of three vertebrate species. Activity was highest in the goldfish, followed by Xenopus laevis and Rana pipiens. Also, high activity levels were found in goldfish retinal pigment epithelium and choroid, but not in the other two species. When added to in vitro culture systems, 2',3'-cyclic nucleotides were found to have no effect on goldfish cone retinomotor movement, but caused a marked inhibition of Rana pipiens rod outer segment disc membrane shedding. It is suggested that CNPase may play a role in cellular processes requiring membrane structural reorganization.     

Calcium and olfactory transduction

1. Inorganic cations, organic calcium antagonists, and calmodulin antagonists were applied to olfactory epithelia of frogs (Rana pipiens) while recording electroolfactogram (EOG) responses. 2. Inorganic cations inhibited EOGs in a rank order, reflecting their calcium channel blocking potency: La3+ greater than Zn2+ greater than Cd2+ greater than Al3+ greater than Ca2+ greater than Sr2+ greater than Co2+ greater than Ba2+ greater than Mg2+. Barium ion significantly enhanced EOGs immediately following application. 3. Diltiazem and verapamil produced dose-dependent EOG inhibition. 4. Calmodulin antagonists inhibited EOGs without correlation to their anti-calmodulin potency.     

pH dependence of the acetylcholine receptor channel: a species variation

The effects of pH changes on the miniature endplate current (mepc) and on endplate current fluctuations (acetylcholine [ACh] noise) were examined at the neuromuscular junction in vitro in two species of frogs. In Rana pipiens the relationship between the decay time constant of the mepc (tau') and pH had a symmetrical bell shape; the value of tau' being largest at pH 7 and decreasing at more acid or more alkaline pH. In acid pH the mepc amplitude (A) decreased relative to its value at pH 7, and in alkaline pH A increased. In Rana ridibunda a narrower and asymmetric bell-shaped dependence of tau' on pH, having a maximum of pH 5.5, was found. The mepc amplitude was again reduced in acid pH but had a peak at pH 5.5. Also, its value at pH 9 was larger than at pH 7. These results were obtained with a number of different buffers and were not found to be sensitive to the nature of the buffer chosen. By performing ACh-noise analysis we found that in Rana pipiens at acid pH (5.5-5.0), the single channel conductance (gamma) and the single channel open time (tau) were significantly reduced relative to their value at pH 7. However, in Rana ridibunda at acid pH (5.4) gamma was unchanged and tau was markedly increased relative to their values at pH 7. The results can be explained quantitatively by electrostatic interaction between two fixed and titratable ionic groups and a mobile charge in the receptor molecule. The model fits the data for groups having pKs approximately 4.8 and approximately 9.8 for Rana pipiens and approximately 4.6 and approximately 6.3 for Rana ridibunda. The groups can be tentatively identified as amino acid residues; glutamic or aspartic and lysine or tyrosine for Rana pipiens; glutamic or aspartic and histidine for Rana ridibunda. The difference in the fitted values of the other model parameters for these two species can be attributed to differences in the spatial configuration of the charged groups.     

Cytonuclear discordance across a leopard frog contact zone

We conducted a genetic analysis of the extent of hybridization within and outside a contact zone between two North American leopard frogs, Rana blairi and Rana pipiens by comparing distribution patterns of mitochondrial and nuclear haplotypes. The contact zone, located between South Dakota, Nebraska, and Iowa, USA, was previously examined using morphological data in the 1970s, leading to the conclusion that hybridization was rare between R. pipiens and R. blairi. Our genetic analysis of 51 populations (611 samples) shows strong cytonuclear discordance. Mitochondrial-haplotype distribution matches the same pattern as the documented species spatial distributions based on morphology. However, the geographic distribution of the nuclear haplotypes reveals asymmetrical swamping of the R. pipiens nuclear haplotypes by R. blairi haplotypes. Phylogenetic analyses of both mitochondrial and nuclear markers provide strong evidence for the presence of R. blairi-R. pipiens introgression for the nuclear marker. A pattern of mitochondrial isolation with nuclear introgression is extremely unusual, and predicted to occur much less often than the reverse.     

Phylogenetic relationships of leopard frogs (Rana pipiens complex) from an isolated coastal mountain range in southern Sonora, Mexico

Mitochondrial DNA sequence data from the control region and 12S rRNA in leopard frogs from the Sierra El Aguaje of southern Sonora, Mexico, together with GenBank sequences, were used to infer taxonomic identity and provide phylogenetic hypotheses for relationships with other members of the Rana pipiens complex. We show that frogs from the Sierra El Aguaje belong to the Rana berlandieri subgroup, or Scurrilirana clade, of the R. pipiens group, and are most closely related to Rana magnaocularis from Nayarit, Mexico. We also provide further evidence that Rana magnaocularis and R. yavapaiensis are close relatives.     

Effects of acid stress in adult Rana pipiens

The decline in frog populations is a well-recognized worldwide phenomenon and infectious disease has been implicated as a major cause in the global decline of amphibian populations. Rana pipiens are disappearing from many habitats where they used to flourish, and environmental acidification has been considered as a possible contributor to this disappearance. We present a model that integrates the results of several experiments on the effects of acid exposure on natural resistance and mortality of adult Rana pipiens. These studies suggest that different components of the natural defense mechanisms of these frogs have different acid sensitivities. We have shown previously that exposure to pH 5.5 leads to a reduction in splenic white blood cell number, viability, and to colonization of the spleen with both Gram positive and Gram negative bacteria. In this paper we show that exposure to pH 6.0 did not affect the number or viability of splenic white blood cells but did result in colonization of the spleen by bacteria. We also show that cold exposure by itself does not cause a systemic bacterial infection in adult Rana pipiens, but acid stress following cold exposure does. The data presented in this paper provide empirical evidence to support the hypothesis that acid stress may be a contributor to the decline of Rana pipiens in the northeastern region of the United States.     

Energy liberation and chemical change in frog skeletal muscle during single isometric tetanic contractions

Recent data obtained from Rana temporaria sartorius muscles during an isometric tetanus indicate that the time-course of phosphocreatine (PC) splitting cannot account for the total energy (heat + work) liberation (Gilbert et al. 1971. J. Physiol. (Lond.) 218:)63). As this conclusion is important to an understanding of the chemical energetics of contraction, similar experments were performed on unpoisoned, oxygenated Rana pipiens sartorius muscles. The muscles were tetanized (isometrically) at 0 degrees C for 0.6, 1, or 5 s; metabolism was rapidly arrested by freezing the muscles with a specially designed hammer apparatus, and the frozen muscles were chemically analyzed. Comparable myothermal measurments were made on frogs from the same batch. Results of these experiments indicate: (a) The energy liberation parallels the PC and ATP breakdown with a proportionality constant of 10.7 kcal/mol; (b) comparably designed experiments with sartorius muscles of R. temporaria revealed that the ratio of energy liberation to PC splitting was significantly greater than that observed in R. pipiens sartorius muscles; (c) there is no systematic difference between experiments in which metabolism was arrested by the hammer apparatus and others using a conventional immersion technique.     

Reduction of calcium inactivation of sarcoplasmic reticulum calcium release by fura-2 in voltage-clamped cut twitch fibers from frog muscle

Cut fibers from Rana temporaria and Rana pipiens (striation spacing, 3.9-4.2 microns) were mounted in a double Vaseline-gap chamber and studied at 14 degrees C. The Ca indicator purpurate-3,3' diacetic acid (PDAA) was introduced into the end pools and allowed to diffuse into the optical recording site. When the concentration at the site exceeded 2 mM, step depolarizations to 10 mV were applied and the [Ca] transient measured with PDAA was used to estimate Ca release from the sarcoplasmic reticulum (SR) (Baylor, S. M., W. K. Chandler, and M. W. Marshall. 1983. Journal of Physiology. 344:625-666). With depolarization, the rate of SR Ca release increased to an early peak and then rapidly decreased several-fold to a quasi-steady level. The total amount of Ca released from the SR at the time of peak rate of release appeared to be independent of SR Ca content, consistent with the idea that a single activated channel might pass, on average, a fixed number of ions, independent of the magnitude of the single channel flux. A possible explanation of this property is given in terms of locally induced Ca inactivation of Ca release. The solution in the end pools was then changed to one with PDAA plus fura-2. SR Ca release was estimated from the [Ca] transient, as before, and from the delta [Cafura-2] signal. On average, 2-3 mM fura-2 increased the quasi-steady level of the rate of SR Ca release by factors of 6.6 and 3.8, respectively, in three fibers from Rana temporaria and three fibers from Rana pipiens. The peak rate of release was increased in five of the six fibers but to a lesser extent than the quasi-steady level. In all fibers, the amplitude of the free [Ca] transient was markedly reduced. These increases in the rate of SR Ca release are consistent with the idea that Ca inactivation of Ca release develops during a step depolarization to 10 mV and that 2-3 mM fura-2 is able to reduce this inactivation by complexing Ca and thereby reducing free [Ca]. Once the concentration of fura-2 becomes sufficiently large, a further increase reduces the rate of SR Ca release. On average, 5-6 mM fura-2 increased the quasi-steady rate of release, compared with 0 mM fura-2, by 6.5 and 2.9, respectively, in four fibers from Rana temporaria and three from Rana pipiens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vitamin A enhances forelimb regeneration in juvenile leopard frogs Rana pipiens

The effect of retinoids on forelimb regeneration in the leopard frog, Rana pipiens, was examined by the topical application of a concentrated solution of vitamin A palmitate to the amputated left limb stumps of 30 froglets. This treatment resulted in the enhancement of regeneration; regenerative outgrowths of experimental frogs were more frequent, larger in size, and had improved morphology compared to their untreated control counterparts. We conclude that vitamin A palmitate, topically administered in concentrated dosages, exerts a positive effect on the regenerative capacity of Rana pipiens.