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Melatonin desensitizing effects on the in vitro responses to MCH, alpha-MSH, isoproterenol and melatonin in pigment cells of a fish (S. marmoratus), a toad (B. ictericus), a frog (R. pipiens), and a lizard (A. carolinensis), exposed to varying photoperiodic regimens.

Melatonin is a weak dose-independent lightening agonist in fish skin, a moderate dose-dependent lightening agonist in toad skin and a potent lightening agent in frog and lizard skins (reversing in a dose-dependent manner the darkening caused by alpha-melanocyte-stimulating hormone). In frog skins, previous exposure to melatonin reduced further lightening actions of the indoleamine, and in toad skins, increasing concentrations of melatonin elicited decreasing lightening responses, suggesting an autodesensitizing action of the hormone. Various concentrations of melatonin diminished the responses to the lightening agonist melanin-concentrating hormone (MCH) in fish skins and to the darkening agonists alpha-MSH in toad, frog and lizard skins and isoproterenol in frog skins. In vitro inhibitory actions of melatonin are mimicked in the absence of the hormone in skin preparations from toads kept in continuous darkness for 48 hr. The lipophylic nature of the indoleamine associated with the results herein described suggests intracellular actions of melatonin on vertebrate pigment cells.

Animals↗

Co-distribution of neuropeptide Y and its C-terminal flanking peptide in the brain and pituitary of the frog Rana ridibunda.

By means of the peroxidase-anti-peroxidase technique, the distribution of neuropeptide tyrosine (NPY) and its C-terminal flanking peptide (C-PON) has been studied on serial sections of the brain and pituitary of the frog Rana ridibunda. Throughout the brain, NPY and C-PON-immunoreactive perikarya exhibited a remarkable co-distribution. These two peptides were found to be co-located within the same cell bodies in various brain regions including the dorsal and ventral pallium, the dorsal and ventral infundibular nuclei and the preoptic nucleus. The distribution of NPY- and C-PON-containing fibers in the brain and pituitary was similar. Sequential double immunohistochemical staining using the indirect immunofluorescence method, showed that NPY and C-PON were actually located within the same nerve processes throughout the frog brain and in the intermediate lobe of the pituitary. These studies indicate that the deduced C-PON sequence is present within the frog precursor to NPY and is formed in vivo in the frog brain. Like NPY, C-PON is transported distally in nerve terminals and is likely released with NPY in various regions of the brain and in the intermediate lobe of the pituitary.

Animals↗

Microsomal monooxygenase system in frog livers.

Liver microsomes prepared from four species of frog, Rana catesbeiana, Rana nigromaculata , Bufo bufo japonicus, and Xenopus laevis, contained cytochrome P-450 and showed NAD(P)H-dependent monooxygenase activities to several foreign chemical compounds tested. The oxidations of the chemical compounds by frog liver microsomes showed significant variations among frog species. The oxidation of 7-ethoxycoumarin was much faster than that by rat liver microsomes. The oxidation of benzo(a)pyrene was significantly induced by 3-methylcholanthrene administration. The monooxygenase activities of frog liver microsomes were more sensitive to cyanide inhibition than those of rat liver microsomes.

Aminopyrine↗

Hepatic ornithine decarboxylase from the frog, Rana negromaculata: dietary induction, purification and some properties.

1. In the liver of the frog, Rana negromaculata, the activity of ornithine decarboxylase (ODC) was induced by dietary stimuli and was rapidly lost upon intraperitoneal injection of cycloheximide or putrescine. 2. Frog liver ODC, purified by DEAE-Cellulofine and immunoaffinity column chromatographies, was used in a comparative study with mouse kidney ODC, also purified by the same method. 3. The purified frog ODC showed three bands on SDS-polyacrylamide gel electrophoretic analysis, as confirmed by [3H]alpha-difluoromethylornithine binding. 4. Frog ODC was found to be similar to mouse enzyme in some properties, for example molecular weight, immunoreactivity and inhibition by rat antizyme, except for a slightly higher Km value for ornithine.

Animals↗

Characterization of oviductal aromatase in the northern leopard frog, Rana pipiens.

Previous work has shown that the oviduct of the northern leopard frog, Rana pipiens, can convert testosterone to estradiol. The present paper examines the characteristics of the aromatase responsible for this reaction and compares it with human placental aromatase. Microsomes were isolated from the oviducts, and aromatase activity was assayed by a tritiated water release method. The Km and the Vmax for androstenedione were 188.1 +/- 30.2 nM and 1.42 +/- 0.11 fmol of estrogen produced/min/mg protein, respectively. Using the same method, human placental aromatase had a Km of 123.0 nM and a Vmax of 113.57 fmol of estrogen produced/min/mg protein. When tested at four temperatures between 15 and 45 degrees C, the frog enzyme showed maximum activity at 37 degrees C. The enzyme had a broad pH optimum between 7.4 and 10.4. The aromatase inhibitor 4-hydroxyandrostenedione inhibited activity by 20% at 0.3 microM and 40% at 0.5 microM. The present study provides additional evidence that an aromatase is present in the frog oviduct. Characterization of this enzyme revealed similarities to human placental aromatase, but the specific activity was much lower in the frog oviduct.

Androstenedione↗

Sprouting and nerve retraction in frog neuromuscular junction during ontogenesis and environmental changes.

Based on recent evidence for a physiological remodeling of neuromuscular contacts (Wernig et al.), a morphometric study was performed on axon- and cholinesterase-stained cutaneous pectoris muscle of frog. The aim of this investigation was to separate changes due to aging, growth, and environmental conditions. Within a single muscle, fiber diameters, synaptic lengths, number of intraterminal branches, and lengths of abandoned gutters differ considerably (with coefficients of variation from 40 to 56%). On the other hand, these parameters are correlated and correlations hold when muscle fibers grow during ontogenesis: large muscle fibers bear larger and more complex junctions than small fibers. Obviously there exist growth regulating interactions between muscle fiber and the presynaptic nerve. To dissociate between age- and growth-related changes muscle fibers of equal diameters in frogs of different age are compared. With increase in age there is an additional increase in abandoned gutters, synaptic length, and complexity independent of muscle fiber growth. Possibly, abandoned gutters accumulate with time and synaptic length increases with age as the net outcome of continual synapse remodeling. When freshly caught frogs (October) were compared with frogs kept under laboratory conditions for a period of 16 weeks (which in addition included a change in season) the number of sprouts in a junction increased by about 2, the average length of presynaptic nerve terminals with small circumscribed contacts increased by 30-150 microns, and abandoned gutters tended to be shorter on fibers with large junctions. The hypothesis is discussed that remodeling is "inherent" to nerve terminals whereby sprouting is counterbalanced and reversed by nerve activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

Neurochemical evidence for afferent GABAergic and efferent cholinergic neurotransmission in the frog vestibule.

Glutamate decarboxylase and choline acetyltransferase activities with magnitudes similar to those of their homologous enzymes in frog nervous tissue were found in homogenates of the frog labyrinth. Transection of the vestibular nerve resulted in a gradual diminution of choline acetyltransferase activity until it reached an 88% decrease 6 weeks after surgery. In contrast, glutamate decarboxylase activity did not suffer any alteration at any time after nerve excision. The presence of their enzymes of synthesis is evidence of the neurotransmitter participation of GABA and acetylcholine in the frog vestibule; the observed decrease of choline acetyltransferase following vestibule nerve excision supports the efferent synaptic bouton localization of choline acetyltransferase. The suggestion that glutamate decarboxylase is located in a cell type (or compartment) that may well be the hair cell is supported by the fact that this enzyme does not suffer any modification after surgery. These results are in accordance with an efferent cholinergic neurotransmission and a putative afferent role of GABA in the frog vestibule.

Acetylcholinesterase↗

Immunohistochemical localization of delta sleep-inducing peptide-like immunoreactivity in the central nervous system and pituitary of the frog Rana ridibunda.

The purpose of the present study was to investigate the distribution of delta sleep-inducing peptide in the brain and pituitary of the frog Rana ridibunda and to determine the possible effect of this nonapeptide on adrenocorticotropic hormone and corticosteroid secretion. Delta sleep-inducing peptide-like immunoreactive fibres were observed throughout the brain of the frog. These fibres generally exhibited the characteristics of glial cell processes. Scarce delta sleep-inducing peptide-positive fibres were seen in the olfactory bulb and in the periventricular areas of the telencephalon. In the diencephalon, numerous delta sleep-inducing peptide-containing processes were noted in the preoptic nucleus, the infundibular nuclei and the median eminence. A few cerebrospinal fluid-contacting cells were visualized in the ventral nucleus of the infundibulum. Delta sleep-inducing peptide-positive fibres were also observed in the mesencephalon, radiating through the different layers of the tectum. In the cerebellum, all Purkinje cells exhibited delta sleep-inducing peptide-like immunoreactivity. More caudally, numerous delta sleep-inducing peptide-positive fibres were noted in the vestibular nucleus of the rhombencephalon. A dense network of delta sleep-inducing peptide-containing fibres was seen in the pars nervosa of the pituitary. In the distal lobe, a population of endocrine cells located in the anteroventral region contained delta sleep-inducing peptide-immunoreactive material. Labelling of consecutive sections of the pituitary by delta sleep-inducing peptide and adrenocorticotropic hormone antiserum revealed that a delta sleep-inducing peptide-related peptide is expressed in corticotroph cells. The possible role of delta sleep-inducing peptide in the control of adrenocorticotropic hormone and corticosteroid release was studied in vitro, using the perifusion system technique. Administration of graded doses of delta sleep-inducing peptide (from 10(-8) to 10(-6) M) to perifused frog anterior pituitary cells did not affect the spontaneous release of adrenocorticotropic hormone. In addition, prolonged infusion of delta sleep-inducing peptide (10(-6) M) did not alter the stimulatory effect of corticotropin-releasing factor (10(-7) M) on adrenocorticotropic hormone secretion. Similarly, exposure of frog interrenal slices to delta sleep-inducing peptide did not induce any modification of spontaneous or adrenocorticotropic hormone-evoked secretion of corticosterone and aldosterone. Our results provide the first evidence for the presence of a delta sleep-inducing peptide-related peptide in lower vertebrates. The occurrence of delta sleep-inducing peptide-like immunoreactivity in specific areas of the brain suggests that the peptide may act as a neuromodulator.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenocorticotropic Hormone↗

Effects of spike discharge history on discharge probability and latency in frog basilar papilla units.

Gaumond et al. [(1982) J. Neurophysiol. 48, 856-873] showed in the cat that a multiplicative-intensity model can generally account quite well for reduction of the probability of an auditory-nerve spike by another spike preceding it by 4 to 25 ms, and that for smaller separations there is also an increased latency of the following spike. Bosch [(1990) D. Sc. Dissertation, Washington University, St. Louis, MO] made important improvements in experimental design and estimation techniques for studying these effects, and confirmed their presence in the gerbil. However, direct application of these methods to the frog does not yield reliable estimates. A clearer separation of discharge probability and latency effects in frog basilar papilla units is provided by the paired-click paradigm used in this study, which is applicable to low-spontaneous-rate units that generally respond to click stimuli with zero or one spike within a short interval following the click. The results confirm the existence in the frog of both spike-probability and spike-latency effects that are qualitatively similar to those found in mammals, although the absolute refractory time is much longer in frog, and the relative refractory time usually shorter. The paired-click paradigm also reveals a stimulus-history effect at stimulus levels which are near threshold: when there is no response to the first click, responses to the second click occur with increased probability and reduced latency.

Acoustic Stimulation↗

Effect of vanadate and of removal of extracellular Ca2+ and Na+ on tension development and 45Ca efflux in rat and frog myocardium.

Vanadate in the range 0-5 mM has positive inotropic effects on myocardial strips of frog and to a lesser extent on those of rat. Inhibiting the sarcolemmal Na+, Ca2+ exchange by a solution free of Ca2+ and Na+ caused a drop in 45Ca efflux and a transient increase in resting tension. These effects were more expressed for the frog than for the rat myocardium, which suggests that the Na+ for Ca2+ exchange across the cell membrane is more important in the frog than in the rat myocardium. A subsequent addition of vanadate at 2 or 5 mM had no effect on 45Ca efflux, while it increased the resting tension. This increase was higher for the frog than for the rat myocardium. These results suggest that the inotropic effects of vanadate may be due to an effect on membrane-bound Ca2+-ATPase.

Animals↗

Temperature and adrenoceptors in the frog heart.

1. Cardiac adrenergic receptors in a frog, Rana tigrina, were examined in winter and summer months using isolated atria preparation maintained at 24 degrees, 14 degrees and 6 degrees C. Treatments included an examination of the atrial responses to selective alpha and beta adrenergic agonists (phenylephrine and isoproterenol respectively) and antagonists (phentolamine and propranolol). 2. Basal atrial beating rates differed between summer and winter months and increased with temperature. 3. Phenylephrine produced dose-dependent increases in the atrial beating rate and tension in the winter frogs only at 6 degrees C. These increases were blunted by phentolamine. 4. Isoproterenol produced positive chronotropic effects of 14 degrees and 24 degrees C but not at 6 degrees C in both summer and winter frogs; these effects were abolished by propranolol. Further, at 6 degrees C, the contractile response of the atrial tissue to isoproterenol was very sensitive. 5. Data suggests that the alpha adrenoceptor might be physiologically important to the frog in the low temperature environment of the cold season, during which period the cardiac beta adrenergic activity would be minimal or even absent.

Animals↗

Stizolobic acid on frog spinal cord; possible species dependent activation of excitatory amino acid receptors.

1. We examined the effects of stizolobic acid, an amino acid isolated from a plant, Stizolobium hassjoo, on the binding of [3H]glutamic acid and [3H]kainic acid to synaptosomes from frog spinal cords and on the depolarization at the ventral roots of frog spinal cords. 2. Stizolobic acid inhibited the binding of [3H]kainic acid more potently than that of [3H]glutamic acid. 3. Among stizolobic acid derivatives, 3-Br-stizolobic acid was the most potent inhibitor of the binding of [3H]kainic acid, but the inhibitory potency was 100 times weaker than that of kainic acid. 4. Stizolobic acid and its derivatives could cause depolarization of the ventral root of frog spinal cord in a dose dependent manner, and 3-Br-stizolobic acid was a more potent inducer of depolarization than kainic acid, but the dose dependency of 3-Br-stizolobic acid was a little different from that of kainic acid. 5. The results suggest that stizolobic acid and its analogues act as a kainic acid agonist in frog spinal cord. 6. The present results and others indicate that stizolobic acid may interact with the different types of excitatory amino acid receptors dependent on the species of animals.

Amino Acids, Dicarboxylic↗

Experimental seizures in the frog (Rana pipiens).

We investigated the effects of chemical convulsants in the leopard frog. Systemic kainic acid (5-20 mg/kg) caused limbic-like seizures, with staring, catatonia, fasciculations, and severe motor seizures, which were almost always lethal. Intracerebral electroencephalographic (EEG) recordings showed spike or spike-and-wave patterns at 6-8 Hz that decreased in frequency and increased in amplitude, maximal at an electrode in the midline olfactory/telencephalic (OLF-M) region. With time, an interictal pattern of 100-200 microV periodic spikes developed, followed by diffuse suppression of all brain activity. Seizures induced by pentylenetetrazole (150-450 mg/kg) and bicuculline (5-10 mg/kg) were characterized by the abrupt onset of motor activity, which continued intermittently for several hours, followed by recovery. EEG recordings in animals treated with pentylenetetrazole showed rhythmic spike-and-wave bursts at 1.5-3 Hz that were maximal at OLF-M. Recordings from frogs treated with bicuculline showed repetitive 3-6 Hz spike-and-wave discharges maximal at OLF-M that were nearly constant in amplitude and at times became continuous. Strychnine (1-5 mg/kg) caused reversible seizures characterized by tonic extensions of the extremities, that seemed to originate in the spinal cord. Frogs with recurrent seizures from systemic cis-diamminedichloroplatinum II showed 4-8 Hz rhythmic spike-and-wave activity that gradually slowed in frequency and increased in amplitude. Thus, the frog's reactivity to convulsive agents is similar to that of mammals.

Animals↗

Pharmacological profile of the tachykinin receptor involved in the stimulation of corticosteroid secretion in the frog Rana ridibunda.

It has recently been shown that the adrenal gland of the frog Rana ridibunda is densely innervated by a network of fibers containing two novel tachykinins, i.e. ranakinin (the counterpart of substance P) and [Leu3, Ile7]neurokinin A. Both ranakinin and [Leu3, Ile7]neurokinin A stimulate corticosteroid secretion from frog adrenal glands in vitro. In the present study, we have investigated the pharmacological profile of the receptors involved in the stimulatory action of ranakinin on perifused frog adrenal slices. The selective NK-1 receptor antagonists [D-Pro4, D-Trp7,9]substance P 4-11 and CP-96,345, did not affect the stimulatory action of ranakinin. The selective NK-1 agonist substance P 6-11 had no effect on corticosteroid secretion. The non-peptidic NK-1 receptor antagonist RP 67580 significantly reduced the stimulatory effect of ranakinin on corticosterone and aldosterone secretion by 57 and 55%, respectively. In addition, the dual NK-1/NK-2 receptor antagonist FK-224 significantly inhibited the effect of ranakinin on corticosterone (- 80%) and aldosterone secretion (- 95%). Finally, the amphiphilic analogue of substance P, [D-Pro2, D-Phe7, D-Trp9]substance P, had no effect on corticosteroid secretion. These data suggest that in the frog adrenal gland the stimulatory action of ranakinin on steroid secretion is mediated by a novel type of receptor which differs substantially from the mammalian NK-1 receptor subtype.

Adrenal Cortex Hormones↗

Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of new therapeutic agents.

Granular glands in the skins of frogs of the genus Rana, a widely distributed group with over 250 species, synthesize and secrete a remarkably diverse array of peptides with antimicrobial activity that are believed to have arisen as a result of multiple gene duplication events. Almost without exception, these components are hydrophobic, cationic and form an amphipathic alpha-helix in a membrane-mimetic solvent. The peptides can be grouped into families on the basis of structural similarity. To date, brevinin-1, esculentin-1, esculentin-2, and temporin peptides have been found in ranid frogs of both Eurasian and North American origin; ranalexin, ranatuerin-1, ranatuerin-2 and palustrin peptides only in N. American frogs; and brevinin-2, tigerinin, japonicin, nigrocin and melittin-related peptides only in Eurasian frogs. It is generally assumed that this structurally diversity serves to protect the organism against a wide range of pathogens but convincing evidence in support of this hypothesis is still required. The possibility that "antimicrobial peptides" fulfill additional or alternative biological functions should not be rejected. The molecular heterogeneity of the peptide families, particularly brevinin-1, brevinin-2 and ranatuerin-2, may be exploited for the purposes of unequivocal identification of specimens and for an understanding of phylogenetic interrelationships between species. The broad-spectrum antibacterial and antifungal activities of certain peptides, for example esculentin-1, ranalexin-1 and ranatuerin, together with their relatively low hemolytic activity, make them candidates for development into therapeutically useful anti-infective agents.

Amino Acid Sequence↗

Activation and repolarization patterns in the ventricular epicardium under sinus rhythm in frog and rabbit hearts.

Our study compared the contributions of activation sequence and local repolarization durations distribution in the organization of epicardial repolarization in animals with fast (rabbit) and slow (frog) myocardial activation under sinus rhythm. Activation times, repolarization times and activation-recovery intervals (ARI) were obtained from ventricular epicardial unipolar electrograms recorded in 13 Chinchilla rabbits (Oryctolagus cuniculus) and 10 frogs (Rana temporaria). In frogs, depolarization travels from the atrioventricular ring radially. ARIs increased progressively from the apex to the middle portion and finally to the base (502+/-75, 557+/-73, 606+/-79 ms, respectively; P<0.01). In rabbits, depolarization spread from two epicardial breakthroughs with the duration of epicardial activation being lower than that in frogs (17+/-3 vs. 44+/-18 ms; P<0.001). ARI durations were 120+/-37, 143+/-45, and 163+/-40 ms in the left ventricular apex, left, and right ventricular bases, respectively (P<0.05). In both species, repolarization sequence was directed from apex to base according to the ARI distribution with dispersion of repolarization being higher than that of activation (P<0.001). Thus, excitation spread sequence and velocity per se do not play a crucial role in the formation of ventricular epicardial repolarization pattern, but the chief factor governing repolarization sequences is the distribution of local repolarization durations.

Animals↗

Glucose and free amino acids in the blood of lampreys (Lampetra fluviatilis L.) and frogs (Rana temporaria L.) under prolonged starvation.

The content variation dynamics of glucose and free amino acids in blood plasma was followed for lampreys and frogs from autumn till spring, when the exogenous feeding is switched off. In October, the glucose level is rather high (8-10 mM) in blood plasma of both lampreys and frogs. It falls by 50% during winter and falls to a critically low level (1-2 mM) during spring. The lamprey plasma amino acid levels increase by 74% from November to April and thus reach the lower limit known for mammals. The amount of free amino acids in frog plasma decreases by 40% by spring in comparison with the values in autumn. More intensive proteolysis in lamprey tissues compared with that in frog tissues has been confirmed by quantitatively determining leucine, isoleucine, and valine in the blood of these animals. Besides these three amino acids, alanine, glycine, lysine, threonine and, in certain periods, tyrosine have been found to be quantitatively significant in the plasma of both animals.

Amino Acids↗

Maternal transfer of organochlorine pesticides, polychlorinated dibenzo-p-dioxins, dibenzofurans, and coplanar polychlorinated biphenyls in frogs to their eggs.

Previous study confirmed that there were sexual differences in concentrations of organochlorine pesticides and dioxins in frogs during the breeding season. The present study utilized the egg masses from the previous study to determine the cause(s) of the sexual differences. When concentrations of the detected chemicals were compared between the female frogs and their eggs, it was found that the concentrations in the eggs were several times those in their mothers. The results indicated that maternal transfer reduced tissue concentrations in females. Since the weight of each egg mass was half that of the mother, two-thirds of the chemicals in a female frog were transferred to its eggs. In addition, the degree of maternal transfer differed among compounds. Maternal transfer of PCDDs and PCDFs with four or five chlorine atoms and coplanar PCBs followed the fugacity model. However, maternal transfer decreased for PCDDs/DFs with six or more chlorines as the octanol-water partition coefficient increased. From these results, maternal transfer was confirmed as the cause of sexual differences in concentrations of organochlorine pesticides and dioxins in adult frogs during the breeding season.

Animals↗