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PubMed1983

Mitogenic responses of frog lymphocytes to crude and purified preparations of bacterial lipopolysaccharide (LPS).

We have compared in vitro mitogenic responses of frog (Xenopus laevis and Rana pipiens) lymphocytes to various preparations of lipopolysaccharide (LPS). Commercial LPS prepared from E. coli (phenol extraction) and from S. abortus-equi (phenol and TCA extraction procedures) was mitogenic for frog lymphocytes. After reextraction of these LPS preparations with phenol-water, the remaining LPS was either considerably less mitogenic or not mitogenic. Purified E. coli 055:B5 LPS, prepared by phenol water extraction, enzyme treatment and column chromatography, was not mitogenic. Frog cells proliferated poorly or not at all with all concentrations of reextracted or purified LPS tested (0.5-400 micrograms/ml) and at all culture periods examined (days 1-7). All LPS preparations used were mitogenic for CAF1 mouse lymphocytes, whereas reextracted and purified LPS preparations were not mitogenic for lymphocytes from C3H/HeJ cells. Xenopus were also not susceptible to toxicity induced by parenterally administered LPS in concentrations which killed CAF1 mice.

Animals↗
PubMed1987

Inhibition of frog SK effector-target cell binding.

Substances known to inhibit mammalian NK cell activity during the first stage of lysis (i.e., effector cell-target cell binding) will also inhibit frog SK cell activity. We analyzed target cell lysis after conjugate formation between one target cell and at least one effector cell. Using frog SK effector cells and frog allogeneic and mammalian tumor target cells, we demonstrated that EDTA, the Ca+2 and Mg+2 chelating agent, but not EGTA, the Ca+2 chelating agent, inhibited binding. Thus, Mg+2 is required for conjugate formation. The inhibitory effects of EDTA were at least partially reversible following removal of EDTA. Binding also required membrane fluidity since pretreatment of targets with glutaraldehyde prevented effector cell binding. Adding glutaraldehyde after conjugate formation increased lysis. Modification of target cell surface proteins by DMSO and trypsin also inhibited binding of effector and target cells. Substances inhibiting binding decreased lysis as measured by 51Cr-release from target cells. Inhibition of binding between SK effector and target cells adds further support to our view that natural or spontaneous killing of foreign cells may be one of the most primitive immuno-defense mechanisms.

Animals↗
PubMed1987

Non-uniform release at the frog neuromuscular junction: evidence of morphological and physiological plasticity.

The frog neuromuscular junction (NMJ) is a fusiform structure parallel to the muscle fiber with a few secondary and tertiary branches. Both sprouting and regression can occur on the same nerve terminal, suggesting a continuous on-going remodelling of the mature neuromuscular junction. Thus, the frog NMJ is a dynamic structure. Ultrastructural observations of the nerve terminal suggest that the active zones are distributed equally along the mature nerve terminal. Disorganized active zones have however been observed in distal regions. The density of synaptic vesicles is also uniform throughout the whole structure. However, mitochondria appear to be more abundant in the very distal regions of the nerve terminal. The postjunctional folds and the cholinergic receptors are also uniformly distributed along the NMJ. However, during remodelling periods, the distributions of postjunctional folds and of cholinergic receptors are not uniform in the degenerating and regenerating regions. Fig. 1 summarizes these morphological data. The frequency of spontaneous release (MEPPs) at the NMJ is higher in the proximal region than in the distal regions and recent evidence suggests that the mean MEPP amplitude is higher in the proximal than in the distal portions. Evoked transmitter release is also non-uniform along the frog NMJ. As for spontaneous release, it is higher in the proximal regions than in the distal regions. Failures of the active propagation of the PNAP at low safety points, such as the end of the myelinated axon and the branching points, may be one of the mechanisms responsible for unequal evoked release. It is also possible that the PNAP does not actively invade the whole extend of the nerve terminal since Na+ channels are absent from the distal regions. Fig. 2 summarizes these physiological data.

Animals↗
PubMed1982

Visual orienting deficits in frogs with various unilateral lesions.

We have studied the visual prey acquisition behavior of frogs with unilateral optic nerve section, unilateral tectal lobe ablation, and unilateral transverse hemisection at a level between the tectum and the cerebellum. The first two groups of animals oriented normally to stimuli throughout the region of visual field overlap and failed to respond to more peripheral stimuli on one side. Hemisected animals responded to stimuli at all positions in the visual field. For stimuli located contralateral to the lesion, the frogs oriented normally. For ipsilateral stimuli, the frogs oriented forward.

Animals↗
PubMed1993

Interbulbar reciprocal inhibition in frog olfaction.

Frogs were submitted in acute experiments to electrical stimulation of both ipsi- and contralateral homologous olfactory nerve bundles subserving the dorsal olfactory mucosae. Mitral layer responses on one side were recorded. When both stimulations were given at different onset times and equal intensity, the ipsilateral mitral layer response fell to a sharp minimum of 42% when the contralateral stimulation was given 25 ms before the ipsilateral one. When both stimuli were adjusted for maximal temporal inhibition and the intensity of the contralateral stimulation was varied, the amplitude of the ipsilateral response decreased as the intensity of the contralateral stimulation increased until intensity equalization was obtained. This interbulbar inhibition was suppressed either by sagittal transection through the interbulbar adhesion or local infusion of bicuculline in the ipsilateral olfactory bulb. It was unaffected by transection of habenular commissure. These findings suggest first, that the neuroreceptors located in the dorsal olfactory mucosae project to both olfactory bulbs, and second, that it exists at mitral layer level of the frog, a reciprocal inhibition which can subserve frog orientation towards proximal odour source.

Animals↗
PubMed1988

Somatostatin modulation of neurally mediated pepsinogen secretion from frog esophageal mucosa.

Frog esophageal mucosa contains peptic glands which are innervated by cholinergic neurons. When incubated in a medium containing 1.5 mM CaCl2, pepsinogen release from esophageal mucosa was increased by a high potassium concentration (55 mM KCl), 1,1-dimethyl-4-phenylpiperazinium (DMPP) or bethanechol. Whereas the response to bethanechol remained little changed, the response to high KCl concentrations or DMPP was abolished in the absence of Ca2+. The stimulatory effects of high KCl concentrations and DMPP were also eliminated by the presence of atropine or somatostatin. Furthermore, pepsinogen release in response to bethanechol was dose-dependently inhibited by somatostatin. Frog esophagus was found to contain somatostatin-like immunoreactivity, with a higher density at the end adjacent to the stomach. Chromatography of mucosa extract on Sephadex G-50 revealed a single peak of somatostatin-like immunoreactivity that coeluted with somatostatin-14. Immunohistochemical staining of the mucosa with peroxidase antiperoxidase technique demonstrated the presence of two varieties of somatostatin-like immunoreactivity-containing cells, one individually dispersed within the intercalated septa and the other in groups within the interlobular septa of the peptic glands. These results seem to indicate that somatostatin or somatostatin-like immunoreactivity may play a modulatory role in neurally mediated pepsinogen secretion in the frog esophagus.

Animals↗
PubMed1991

Benzamide derivatives provide evidence for the involvement of a 5-HT4 receptor type in the mechanism of action of serotonin in frog adrenocortical cells.

We have previously shown that serotonin (5-HT) is a potent stimulator of corticosterone and aldosterone secretion by frog adrenocortical cells and we have demonstrated that the action of 5-HT is not mediated by the classical 5-HT receptor subtypes i.e. 5-HT1, 5-HT2 and 5-HT3. Recently, a non-classical 5-HT receptor (termed 5-HT4) has been characterized using 4-amino-5-chloro-2-methoxy-benzamide derivatives as serotonergic agonists. In the present report, we have investigated the possible involvement of the 5-HT4 receptor subtype in the mechanism of action of 5-HT on steroid secretion. Increasing concentrations of benzamide derivatives (zacopride, cisapride and BRL 24924) gave rise to a dose-related stimulation of corticosteroid production, zacopride being the most potent compound of this series to enhance steroidogenesis. Prolonged administration (230 min) of zacopride induced a rapid increase in corticosterone and aldosterone output followed by a gradual decline of corticosteroid secretion. During prolonged exposure of adrenal tissue to zacopride (10(-5) M), the corticotropic activity of 5-HT (10(-6) M) was totally abolished. The stimulatory effects of 5-HT and zacopride were abolished by the non-selective 5-HT3 antagonist ICS 205 930. In contrast methysergide, a 5-HT1 receptor antagonist, and MDL 72222, a selective 5-HT3 antagonist did not block zacopride-induced corticosteroid secretion. Both 5-HT and zacopride induced a dose-related increase in cAMP production by frog adrenal slices. Taken together, these results indicate that the stimulatory effect of 5-HT on frog adrenocortical tissue is mediated by activation of a 5-HT4 receptor subtype positively coupled to adenylate cyclase.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1981

Cholecystokinin-like immunoreactivity in frog retina: localization, characterization, and biosynthesis.

We sought to localize and characterize cholecystokinin-like immunoreactivity (CCK-LI) in frog retinal tissues, CCK-LI was present in concentrations of 2.20 +/- 0.19 pmol/g wet weight (mean +/- SE, n = 6) in water extracts of frog retina and 0.23 +/- 0.04 pmol/g in acid extracts. The predominant form of CCK-LI co-eluted with the octapeptide of CCK on Sephadex G50 column chromatography. A minor form which eluted as a molecule larger than the 39 amino acid form of CCK was found only in the acid extracts. By immunohistochemistry, CCK-LI was localized to amacrine cells which give rise to processes ramifying in the distal 2/3 of the inner plexiform layer. Time dependent uptake of 35S-methionine into CCK-LI was observed in cultured frog retinas and this uptake was blocked with 0.2 mM cycloheximide. Our findings suggest that the retina may be a useful system for further characterization of the neural function of CCK-LI.

Animals↗
PubMedDate not supplied

Distribution and characterization of cyclo (His-Pro)-like immunoreactivity in anuran (frog) skins.

The distribution of cyclo (His-Pro)-like immunoreactivity in frog skins from seven frog species was examined. The chromatographic elution profile of cyclo (His-Pro)-like immunoreactivity in amphibian skins measured by radioimmunoassay corresponded precisely to that of [3H-Pro]-cyclo (His-Pro) after DEAE-Cellulose, Sephadex G-25 and high-pressure liquid chromatography. The concentrations of cyclo (His-Pro) in frog skins were much higher than the concentrations of TRH previously observed in skin and the concentrations of cyclo (His-Pro) in both brain and gastrointestinal tract.

Animals↗
PubMedDate not supplied

Localization and identification of immunoreactive atrial natriuretic factor (ANF) in the frog ventricle.

The localization of ANF-like immunoreactivity in the ventricle of the frog Rana ridibunda was examined by the indirect immunofluorescence and the immunogold techniques, using an antiserum against synthetic ANF (Arg 101-Tyr 126). At the optic level, an appreciable number of positive cardiocytes was observed in the frog ventricle. Electron microscopic studies showed that all secretory granules present in ventricular cardiocytes contain immunoreactive ANF. The immunoreactive material has been characterized by Sephadex G-50 gel chromatography and reverse phase high performance liquid chromatography (RP-HPLC). After gel filtration, ANF-like immunoreactivity eluted in 3 peaks. The major immunoreactive peak corresponded to high molecular weight material, while one peak co-eluted with synthetic ANF (Arg 101-Tyr 126). Further analysis of frog ventricular extracts by RP-HPLC revealed that the low molecular weight material has the same retention time as synthetic ANF, suggesting a high degree of sequence homology between amphibian and mammalian ANF. These results indicate that in amphibians, ventricular cardiocytes synthesize a peptide immunologically and chemically related to mammalian ANF.

Animals↗
PubMedDate not supplied

Distribution and characterization of endozepine-like immunoreactivity in the central nervous system of the frog Rana ridibunda.

The localization of endozepine-like immunoreactivity in the brain of the frog Rana ridibunda was investigated by indirect immunofluorescence, using an antiserum against synthetic rat octadecaneuropeptide (ODN). A specific immunoreaction was detected in ependymal cells lining the ventricular system of the brain and in circumventricular organs. Numerous immunoreactive cells were found covering the walls of the lateral ventricles in the telencephalon, as well as in the diencephalic and mesencephalic ventricles. In the hypothalamus, both the preoptic nucleus and the infundibular region showed numerous immunopositive cells. Ependymal cells lining the rhomboencephalic fourth ventricle and the central canal of the spinal cord were also immunoreactive. The concentration of endozepine-like immunoreactivity was measured in various regions of the brain using a sensitive and specific radioimmunoassay for rat ODN. The highest levels of ODN-like immunoreactivity were found in the infundibulum, cerebellum and preoptic area. Reverse phase high performance liquid chromatography and radioimmunoassay quantification were used to characterize endozepines in the frog brain. The elution profiles of the different brain regions revealed four major immunoreactive peaks. The present results demonstrate the presence of peptides immunologically related to the endozepine family in the central nervous system of the frog. The localization of immunoreactive endozepines in ependymal cells suggests that these peptides play important neuromodulatory functions in the amphibian brain.

Animals↗
PubMedDate not supplied

Isolation and structural characterization of calcitonin gene-related peptide from the brain and intestine of the frog, Rana ridibunda.

Calcitonin gene-related peptide (CGRP) was isolated in a single molecular form from an extract of the whole brain and from the small intestine of the European green frog, Rana ridibunda. The primary structure of the peptide was established as: Ala-Cys-Asn-Thr-Ala-Thr-Cys-Val-Thr-His10-Arg-Leu-Ala-Asp-Phe-Leu- Ser-Arg-Ser-Gly20-Gly-Met-Ala-Lys-Asn-Asn-Phe-Val-Pro-Thr30- Asn-Val-Gly-Ser-Ala-Phe-NH2. Frog CGRP shows only two amino acid substitutions (Val22-->Met and Gly23-->Ala) compared with chicken CGRP, as deduced from the nucleotide sequence of a cloned cDNA. The Asp14 residue, which leads to enhanced biological potency in chicken CGRP, is also present in the frog peptide. The data show that the structure of CGRP has been strongly conserved during evolution of the vertebrates.

Amino Acid Sequence↗
PubMed1984

Frog palate mucociliary apparatus: structure, function, and response to formaldehyde gas.

The upper respiratory tract mucociliary apparatus represents one of the first defenses against inhaled noxious materials. The frog palate has been widely used as a model to investigate the mode of action of this apparatus and to study its response to irritant gases. Video analysis was used here for the determination of mucus flow rate and flow patterns, ciliary beat frequency, and the nature of ciliary activity in the in vitro frog palate preparation. The results of studies of time-lapse video recordings were used in conjunction with light microscopic and ultrastructural morphologic investigations to determine functional interactions between cilia, the epiphase, and the periciliary fluid. It was concluded that the cilia enter the epiphase during the effector stroke, that waves may be produced on the under surface of this layer, and that the periciliary fluid is less viscous than, and moves in the same direction as, the epiphase. The response of the frog palate mucociliary apparatus to formaldehyde gas was also studied using an in vitro exposure system. There were distinct concentration-related responses to formaldehyde with initial stimulation, and at higher concentrations, subsequent inhibition of mucociliary function. Stimulation of mucus flow rate was due to increased ciliary activity, while inhibition of flow, which preceded ciliastasis, was attributed to direct effect of formaldehyde on the superficial mucus layer. Ciliastasis on the other hand was considered to provide evidence that the formaldehyde had penetrated the mucus layer and induced direct toxic effects on the underlying epithelial cells.

Animals↗
PubMed1987

Changes in cell membrane fluidity affect the sodium transport across frog skin and its sensitivity to amiloride.

1. 1-5 mM n-hexanol added to the outer (mucosal) medium of isolated skin of the frog Rana temporaria increases the short circuit current (Isc) across it. 2. This effect shows a saturable dependency on the outer sodium concentration, also when NaCl is replaced by Na2SO4. 3. n-Hexanol at a concentration of 1 mM, and cold acclimation of the frogs, which increases the fluidity of epidermal cell membranes, do not affect the sensitivity of Isc to the inhibiting effect of amiloride. 4. n-Hexanol at a concentration (5 mM) which causes a fluidization of cell membrane preparations from isolated frog epidermis also increases the sensitivity of Isc to amiloride. 5. The effects of low concentrations of n-hexanol and of cold acclimation probably depend on an increase of the permeability of apical membranes of epidermal cells to sodium caused by membrane fluidization. At higher concentrations of n-hexanol, a further disordering of the membrane structure occurs with a better access of amiloride to its action sites.

Amiloride↗
PubMed1987

Parathyroid hormone stimulation of renal adenylate cyclase in various vertebrate species: evidence for an effect in the frog.

The effect of [Nle8,18,Tyr34]bPTH-(1-34)amide on renal plasma membrane adenylate cyclase, in the presence of 0.1 mM guanylylimidodiphosphate was measured in rat, chicken, frog and trout. All species showed an enrichment of at least 8-fold (relative to homogenate) in the marker enzyme Na+,K+-ATPase. A significant dose-dependent adenylate cyclase stimulation was found in frog, with affinity values similar to those of rat and chicken (ED50=8, 10 and 3 nM, respectively), but not in trout. The frog response was specific since [Nle8,18,Tyr34]bPTH-(3-34)amide strongly inhibited the agonist-stimulated enzyme. These results suggest the existence of a PTH-like substance in anurans acting via cyclic AMP formation in the kidney.

Adenylyl Cyclases↗
PubMed1988

Characteristics of the water response across the dorsal epithelium of frog tongue.

1. Dorsal epithelium of the frog tongue produced a change in potential difference across the tissue in response to removal of NaCl from the adapting Ringer solution on the mucosa. 2. The response was not caused by an osmotic decrease in the stimulus, and its profile was in many respects similar to that of the receptor potential in frog taste cells. 3. In conclusion, the response may influence or modify water reception in frogs.

Animals↗
PubMed1991

Reabsorption of water and electrolytes in the urinary bladder of intact frogs (genus Rana).

1. Water and electrolyte reabsorption of the urinary bladder epithelia has been studied in intact, fully hydrated frogs (Rana temporaria, R. lessonae, R. ridibunda). 2. The rates of water reabsorption were lower in frogs on wet soil than in those on dry soil and related to the degree of terrestrialism: R. temporaria greater than R. lessonae greater than R. ridibunda. 3. Samples of urine stored up to 24 hr within the urinary bladder were analysed for osmolality and the concentration of urea, ammonia, sodium, potassium, magnesium and calcium. 4. Selective reabsorption of sodium was detected in all species, that of calcium only in R. ridibunda. The efficiency of electrolyte reabsorption was also related to the degree of terrestrialism. 5. In conclusion, in fully hydrated frogs reabsorption by the bladder epithelia contributes significantly to the water and electrolyte conservation.

Absorption↗