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PubMed1978

Ultrastructural localization of alkaline phosphatase in the hypertrophic chondrocyte of the frog.

The ultrastructural localization of alkaline phosphatase was studied in the hypertrophic chondrocyte of the frog (Rana temporaria) by incubating sections of glutaraldehyde fixed tissue in a medium containing sodium beta glycerophosphate and calcium chloride. Control specimens were incubated in substrate free medium. Alkaline phosphatase (orthophosphoric monoester phosphohydrolase) is a high molecular weight glycoprotein that hydrolyses phosphorylated metabolites much as acid phosphatase does except that its action is optimal at an alkaline pH. The results of this investigation showed that alkaline phosphatase activity was present within the cytoplasm and around the plasma membrane of frog hypertrophic chondrocytes. Although only a small proportion of frog hypertrophic chondrocytes demonstrated enzyme activity, there was evidence that this was concentrated within Golgi lamellae and vesicles leaving other organelles unreactive. The finding of alkaline phosphatase activity within Golgi lamellae of hypertrophic chondrocytes is regarded as unusual although postitive reactions within chondrocyte lysosomes have previously been reported (Doty and Schofield, 1976).

Alkaline Phosphatase↗
PubMed1975

Specific effect of 5,6-dihydroxytryptamine on the monoamine fluorophore of the frog's gustatory cells.

A specific formaldehyde-induced yellow fluorescence, suggesting the presence of serotonin-like monoamine has been demonstrated in the gustatory cells of the frog. The fungiform papillae of frogs were examined fluorescence-histochemically after intraperitoneal injection of 5,6-dihydroxytryptamine. The results indicated that the fluorophore of gustatory cells was affected selectively by the drug injection: the yellow fluorescence was transiently enhanced 3 hours after the drug injection, thereafter being reduced rapidly. The effect of 5,6-dihydroxytryptamine was long-lasting with the reduction of the yellow fluorophore persisting at least for the experimental duration of 14 days. A single injection of 6-hydroxydopamine induced a complete depletion of noradrenaline fluorescence from adrenergic nerve terminals, while the fluorescence of gustatory cells was not affected by a high dose of the drug. The present results with pharmacologic treatments further support the view that the gustatory cell of the frog contains a serotonin-like monoamine.

5,6-Dihydroxytryptamine↗
PubMed1988

A cytophotometric analysis of the structure of hypothalamic cell populations in the frog, Rana temporaria (L.), with special reference to seasonal changes in chromatin status.

We used cytophotometry after the Feulgen reaction and UV cytophotometry to measure the DNA content of quiescent cells of the hypothalamic preoptic region (HPR) of adult and juvenile frogs (Rana temporaria) that had been caught in their natural habitat in winter, spring and summer. The histone-to-DNA ratio in cell nuclei was cytophotometrically determined using a combined Feulgen, heparine and alcian-blue staining procedure. The vast majority of HPR cells studied had nuclei with a diploid DNA content. However, we observed great variability in the Feulgen-DNA content of the HPR cell population, which was not detected in the diploid standard (hepatocytes). This heterogeneity in the diploid sample of the HPR cell populations was always greater in prespawning frogs and may have been due to differences in the chromatin arrangement in nuclei. About 1% of cells had a DNA content either ranging between diploid and tetraploid levels (H2C cells) or at the tetraploid level (4C and 2C x 2 cells). The proportion of these cells was not affected by the age of the animals or the annual cycle, thus suggesting that there is no age-related increase in the mean DNA content in the frog HPR. The mean DNA contents of H2C and 4C cells were much higher than those in the standard (hepatocytes). This cannot be simply attributed to the presence of different amounts of nuclear proteins, but rather indicates that at least a certain proportion of the highest DNA contents may be due to a real extra-DNA synthesis.

Animals↗
PubMed1988

Electrophysiological analysis of sodium-transport in the colon of the frog (Rana esculenta). Modulation of apical membrane properties by antidiuretic hormone.

Sodium transport and apical bioelectrical membrane properties were investigated in frog colonic epithelium in the absence and presence of the antidiuretic hormone arginine-vasotocin (AVT). Apical Na-permeability and intracellular Na-activity were evaluated by analysis of current-voltage relationships in the serosally K-depolarized tissue. Tissue- and apical membrane capacitance were measured by voltages step analysis. The frog colon was found to be a tight epithelium with a transepithelial resistance of 2.63 +/- 0.25 k omega.muF (n = 17). 85-90% of short circuit current (11.2 +/- 1.1 microA.microF.l-1; n = 17) was related to electrogenic Na-transport from mucosa to serosa. Graded doses of amiloride (less than 50 mumol.l-1) induced Michaelis-Menten-type inhibition kinetics. Serosal addition of 10(-6) mol.l-1 AVT induced a significant increase in sodium current (25%), apical sodium permeability (19%) and tissue capacitance (4.3%) whereas intracellular Na-activity remained unchanged. There was a good correlation between increased Na-current and apical Na-permeability. No correlation was found between Na-current and membrane capacitance. Our results demonstrate that in contrast to other species the amphibian colon shows a natriferic reaction to AVT. We suggest that the regulation of Na-transport in frog colon is similar to that in the toad urinary bladder. It is caused by an activation of preexisting apical Na-channels and not by fusion of subapical cytoplasmic vesicles with the apical membrane.

Amiloride↗
PubMed1987

Appropriate conditions to record activation of fast Ca2+ channels in frog skeletal muscle (Rana pipiens).

Frog skeletal muscle fibres possess slow Ca++ channels which are currently studied. Only recently a fast Ca++ current in frog skeletal muscle was reported. This finding is related to the fact that this current is very labile. Here we report the appropriate conditions to record the fast calcium current (ICa-f) in skeletal muscle of the frog Rana pipiens, in isotonic solutions and resting fibre length.

Animals↗
PubMed1988

Noise analysis of cAMP-stimulated Na current in frog colon.

The effects of oxytocin and cAMP on the electrogenic Na+-transport in the short-circuited epithelium of the frog colon (Rana esculenta, Rana temporaria) were investigated. Oxytocin (100 mU.ml-1) elevated the short-circuit current (Isc) transiently by 70% whereas cAMP (1 mmol.l-1) elicited a comparable sustained response. The mechanism of the natriferic action of cAMP was studied by analysing current fluctuations through apical Na+-channels induced by amiloride or CDPC (6-chloro-3,5-diaminopyrazine-2-carboxamid). The noise data were used to calculate Na+-channel density (M) and single apical Na+-current (iNa). iNa-Values obtained with amiloride and CDPC were 1.0 +/- 0.1 pA (n = 5) and 1.1 +/- 0.2 pA (n = 6) respectively and unaffected by cAMP. On the other hand, cAMP caused a significant increase in M from 0.23 +/- 0.08 micron-2 (n = 5) to 0.49 +/- 0.17 micron-2 (n = 5) in the amiloride experiments. In our studies with CDPC we obtained smaller values for M in control (0.12 +/- 0.04 micron-2; n = 6) as well as during cAMP treatment (0.19 +/- 0.06 micron-2; n = 6). However, the cAMP-induced increase in M was also significant. We conclude that cAMP stimulates Na+-transport across the frog colon by activating "silent" apical Na+-channels. Thus, the mechanism of regulation of colonic Na-transport in frogs differs considerably from that in other vertebrates as mammals and birds.

Amiloride↗
PubMed1978

The current voltage relationship of the delayed outward current in the heart of the frog (Rana esculenta) and the tortoise (Testudo germani).

Steady state and non steady state I-V relationships of the resting membrane and delayed rectifying membrane were analysed by applying trapezoid voltage clamp pulses on atrial fibres isolated from the frog and the tortoise. The fibres were disposed in a perfusion chamber for double sucrose gap. In both types of preparation, a component of delayed rectification current was identified which was activated following a comparable time course. The amplitude of the delayed rectification current, when expressed either as normalized to the calculated membrane capacity or to the initial background current, is significantly larger in the frog than in the tortoise. Full activation of the delayed rectifying system can be demonstrated in the tortoise, while in the frog this process is presumably complicated by simultaneous accumulation of K ions in the extracellular region. The difference in magnitude of the delayed outward current has no influence on the duration of action potential which is recorded over the sucrose gap.

Action Potentials↗
PubMed1983

Effects of guanine nucleotides on vasopressin-induced water flow and sodium transport of the frog bladder.

In the present study, we examined the effects of guanine nucleotides on vasopressin-induced osmotic water flow and sodium transport in the 14-h preincubated frog bladder. We also examined the effects of the adenylate cyclase-cyclic AMP and cyclic AMP-dependent protein kinase system in the bladder's epithelial cells. Gpp(NH)p significantly enhanced vasopressin-induced water flow while it did not affect cyclic AMP-induced water flow. However, Gpp(NH)p did not enhance the vasopressin-induced increment of sodium transport across the frog bladder. The adenylate cyclase activity of the crude homogenate was enhanced by vasopressin, Gpp(NH)p and NaF. The effects of Gpp(NH)p and vasopressin, at their maximum doses, on the enzyme activities were additive, while other combinations were not. Specific Gpp(NH)p binding sites were found in the pellet fraction after 2,400 X g centrifugation. No direct effect on the protein kinase activity was observed in the presence of 10(-6) M nucleotides, such as GTP, GDP, GMP, CTP, UTP, ITP and Gpp(NH)p. Cyclic AMP stimulated the phosphorylation of discrete protein bands, however, Gpp(NH)p did not influence cyclic AMP-dependent protein phosphorylation of crude homogenate of the bladder's epithelial cells. These results suggest the guanine nucleotides stimulate the vasopressin-induced osmotic water flow in frog bladder by enhancing the vasopressin-mediated adenylate cyclase activity, so that accumulated cyclic AMP might activate cyclic AMP-dependent protein kinase.

Adenylyl Cyclases↗
PubMed1988

Trans- and paracellular K+ transport in diluting segment of frog kidney.

In frog diluting segment transepithelial K+ net flux (JKte) occurs via trans- and paracellular transport routes. Inhibition of transcellular K+ transport discloses JKte across the shunt-pathway. By means of K+-sensitive microelectrodes we have measured secretory JKte induced by an acute K+ load, in the diluting segment of the isolated and doubly-perfused frog kidney. Transcellular K+ transport was inhibited by blocking the luminal K+ permeability either directly by barium or indirectly by the diuretic drug amiloride (via intracellular acidification induced by inhibition of Na+/H+ exchange), by the Na+/K+ pump inhibitor ouabain or by inducing an acute acid load. All experimental maneouvers led to a reduction of secretory JKte to about 50% of the control JKte. The apparent permeability coefficient for K+ of this nephron portion after inhibition of transcellular secretory JKte was reduced to a similar extent. We conclude: In frog diluting segment the ratio of trans- over paracellular JKte is close to unity. This ratio represents a minimum estimate because inhibition of the transcellular K+ pathway by barium, amiloride or an acute acid load may have been incomplete. Acidosis and/or amiloride exert large antikaliuretic effects due to the inhibition of the luminal K+ permeability.

Amiloride↗
PubMed1989

The key role of the mitochondria-rich cell in Na+ and H+ transport across the frog skin epithelium.

We have investigated the possibility that the mitochondria-rich (MR) cells participate in sodium and proton transport, when the frog skin epithelium is bathed on its apical side with solutions of low Na+ concentration, by comparing transport rates with morphological observations (MR cell number and MR cell pit surface area). Frogs were adapted to various salinities or the isolated skins were treated with the following hormones, deoxycorticosterone acetate (DOCA), arginine vasotocin (AVT) and oxytocin in order to modify the transport of sodium and hydrogen ions. Adaptation of the frogs (either 3-4 days or 7-10 days) to distilled water, NaCl (50 mmol/l), KCl (50 mmol/l) or Na2SO4 (25 mmol/l) solutions modified the Na+ transport rate and the morphology of the epithelium. The highest Na+ transport rates were found for the animals adapted to the Na+ free solutions and were correlated with an increase in the total MR cell pit surface area (number of MR cells x individual cell pit-surface area). The KCl adaptated group showed the largest increase in sodium and proton transport and also presented a metabolic acidosis as reflected by plasma acidification (pCO2 increase and HCO3- decrease). Proton secretion and sodium absorption were also found to be stimulated by either serosal DOCA addition (10(-6) M) or during acidification of the epithelium by serosally applied CO2. Na+ transport was enhanced by AVT (10(-6) M) or oxytocin (100 mU/ml) when the skin was bathed on its apical side with a high Na+ containing solution (115 mmol/l), whereas these hormones did not exert any effect on Na+ transport when the apical solution was low in Na+ (0.5 mmol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

Tizolemide-induced changes of passive transport components across the basolateral membrane of isolated frog skin.

The effect on transepithelial Na transport of tizolemide was investigated in isolated frog skin (Rana temporaria). It was found that tizolemide (2-5 mM, serosal side) decreased transepithelial Na transport (measured as short circuit current and as net sodium flux) within 60 min to 25-40% of the control level resulting from reduction of the unidirectional sodium influx. Intracellular recording with microelectrodes revealed that these changes were associated with depolarization of the intracellular space to less than 40% of the control values (averaging - 71.7 +/- 5.1 mV) which is a consequence of a decrease in conductance of the basolateral border to about 25% of the control values. The conductance of the apical border was only slightly reduced. It is suggested that tizolemide blocks the partial conductance of potassium at the basolateral border which secondarily diminishes transepithelial Na transport due to a decrease of the driving force for apical border Na entry. A certain degree of inhibition of the Na-K-ATPase by tizolemide cannot be excluded. When vasopressin (ADH) was added to frog skin after treatment with tizolemide, the response was markedly reduced compared to that of untreated control preparations. Under these conditions, the conductance of the basolateral border increased while the apical border remained little influenced by the hormone--opposite to the response of frog skins under control conditions. It is concluded that the mode of action of ADH is more complex than has been recognized hitherto and includes effects at the basolateral border.

Animals↗
PubMed1975

Effect of various lysosomes and endotoxin on vascular permeability in frogs and mice.

Blood-lymph permeability increasing effects of frog liver lysosomes, Escherichia coli 0111 endotoxin, bradykinin and serotonin were demonstrated in frogs with a method developed by the authors. These actions were expressed in a faster dye saturation in the lymph as compared to that of the controls. 2. The method is based on the determinations of concentration of Evans blue transported as protein-bound dye into the lymph. 3. Frog liver and polymorphonuclear leukocyte lysosomes had a capillary permeability increasing action tested by local skin response when injecting Evans blue intravenously in mice. 4. All these phenomena are similar to events described earlier in mammalian systems.

Acid Phosphatase↗
PubMed1986

A comparison of miniature end-plate potentials at normal, denervated, and long-term botulinum toxin type A poisoned frog neuromuscular junctions.

The effects of denervation and long-term botulinum toxin type A (BoTx) poisoning on miniature end-plate potentials (m.e.p.p.s) in the frog were studied with intracellular microelectrode recording. BoTx reduced the frequency of m.e.p.p.s to less than 1% of the level seen in untreated frogs, leaving a large percentage of tiny m.e.p.p.s and slow-rising m.e.p.p.s (slow m.e.p.p.s). Unlike what is observed in the rat, the frequency of slow m.e.p.p.s never increased above the low rate measured in the untreated controls, and in fact slightly but significantly decreased after BoTx. A comparison of the m.e.p.p.s seen after BoTx poisoning (BoTx m.e.p.p.s) and m.e.p.p.s seen after denervation (Schwann m.e.p.p.s) revealed many similarities between the two including amplitude and time-to-peak distributions, temperature Q10 values and responses to several drugs and procedures. However, it was concluded that BoTx m.e.p.p.s do not originate from the Schwann cells because denervation of BoTx-paralysed frogs abolishes all m.e.p.p.s and the drug 4-aminoquinoline affects BoTx m.e.p.p.s and Schwann m.e.p.p.s in opposite ways, increasing the frequency of the former while almost eliminating the latter. BoTx m.e.p.p.s and Schwann m.e.p.p.s probably represent similar processes of secretion which are non-specific in nature, having a lower energy barrier than for normal release and not originating from specialized areas of transmitter release.

Aminoquinolines↗
PubMed1985

Eye-specific segregation of optic afferents in mammals, fish, and frogs: the role of activity.

Eye-specific patches or stripes normally develop in the visual cortex and superior colliculus of many (but not all) mammals and are also formed, after surgically produced binocular innervation, in the optic tectum of fish and frogs. The segregation of ocular dominance patches or columns has been studied using a variety of anatomical pathway-tracing techniques, by electrophysiological recording of postsynaptic units or field potentials, and by the 2-deoxyglucose method following visual stimulation of only one eye. In the tectum of both fish and frogs and in the cortex and colliculus of mammals, eye-specific patches develop from initially diffuse, overlapping projections. Of the various mechanisms that might cause such segregation, the evidence favors an activity-dependent process that stabilizes synapses from the same eye because of their correlated activity. First, several environmental manipulations affect the segregation of afferents in visual cortex: strabismus and alternate monocular exposure apparently enhance segregation, whereas dark rearing slows the segregation process, and monocular deprivation causes the experienced eye to form larger patches at the expense of those of the deprived eye. Second, blocking activity in both eyes is effective in preventing the segregation both in the tectum of fish and frog and in the visual cortex of cat. With the eyes blocked, alternate stimulation of the optic nerves permits the segregation of ocular dominance, at least onto single cells in the cat visual cortex. These findings are discussed in terms of an activity-dependent stabilization of those synapses having correlated activity (those from neighboring ganglion cells within one eye) but not of those lacking correlated activity (those from left and right eyes). We suggest that the eye-specific patches represent a compromise between total segregation of the projections from the two eyes and the formation of a single continuous retinotopic map across the surface of the cortex or tectum.

Animals↗
PubMed1995

Molecular characterization of a centromeric satellite DNA in the hemiclonal hybrid frog Rana esculenta and its parental species.

Hybrid water frogs Rana esculenta reproduce by hybridogenesis: one parental genome (of Rana lessonae) is excluded in the germ line, the other (of Rana ridibunda) is clonally transmitted to haploid gametes. The two parental species differ in that the amount of centromeric heterochromatin revealed by differential staining is much higher in Rana ridibunda. An abundant, tandemly arrayed, centromeric satellite DNA, designated RrS1, is revealed in Rana ridibunda genomes by the restriction endonuclease Stul, which generates a major repetitive sequence fragment of 300 and a minor one of 200 bp. This AT-rich (68%) satellite family is located at the centromeres of the five largest chromosomes (1-5) and of a medium to small heterobrachial one (8 or 9); it thus constitutes only part of the centromeric heterochromatin that characterizes all Rana ridibunda chromosomes. RrS1 represents about 2.5% of the genome of Rana ridibunda; it may represent as little as 0.2% of the genome of Rana lessonae, and cannot be detected in Xenopus laevis frogs or Salamandra salamandra and Triturus carnifex salamanders. Segments of the satellite sequence are similar to sequences of yeast centromeric DNA element CDEIII and of the mammalian CENP-B box. A role for RrS1 and other centromeric satellite DNAs in the germ line genome exclusion of the hybridogenetic frog hybrids, although suggested, has not yet been demonstrated.

Animals↗
PubMed1994

The development of Hepatozoon sipedon sp. nov. (Apicomplexa: Adeleina: Hepatozoidae) in its natural host, the Northern water snake (Nerodia sipedon sipedon), in the culicine vectors Culex pipiens and C. territans, and in an intermediate host, the Northern leopard frog (Rana pipiens).

The life cycle of Hepatozoon sipedon sp. nov. was studied in two snake species, the Northern water snake and the Eastern garter snake, in its mosquito hosts Culex pipiens and C. territans, and in the Northern leopard frog. Gametogenesis, fertilization and sporogony occurred within fat body cells in the haemocoel of mosquitoes that had fed on infected water snakes. Mature oocysts averaging 263 microns in diameter and containing more than 500 sporocysts were observed in mosquitoes 28 days post-feeding. Each sporocyst enclosed eight sporozoites. Dizoic cysts were found in the liver of frogs that had been fed infected mosquitoes seven days previously. Two rounds of merogony in various internal organs and intraerythrocytic gamonts were observed in snakes that had been fed frogs which had been orally inoculated with infected mosquitoes. Developmental stages were not seen in snakes that were fed infected mosquitoes directly. A comparison of this life cycle with those described for other Hepatozoon species infecting snakes is presented with reference to the different modes of transmission featured by these parasites.

Animals↗
PubMed1991

Teratogenesis, toxicity, and bioconcentration in frogs exposed to dieldrin.

Teratogenesis, acute and chronic toxicity, growth and bioconcentration were investigated in various life stages (embryos, tadpoles, juveniles, adults) of the frogs Xenopus laevis (African clawed frog), Rana catesbeiana (bullfrog) and Rana pipiens (leopard frog) exposed to aqueous dieldrin in static-renewal and flow-through systems in a study on the development of wildlife-based water quality criteria. R. catesbeiana was the most sensitive tadpole in acute tests; X. laevis was the most sensitive in embryo-larval and chronic tadpole tests. Tadpole 96-h LC50s ranged from 40.4 to 49.5 micrograms/L dieldrin for X. laevis, from 8.7 to 30.3 micrograms/L for R. catesbeiana and was 71.3 micrograms/L for R. pipiens. The 24-day LC50 for X. laevis tadpoles was 5.5 micrograms/L dieldrin; the 28-day LC50 for R. pipiens tadpoles was 8.3 micrograms/L. Adult R. pipiens had a 28-day LC50 of 53.4 micrograms/L dieldrin. Gross spinal deformities in embryo-larval tests were observed at dieldrin concentrations as low as 1.3 micrograms/L after 10-days exposure to X. laevis and at 25.4 micrograms/L for a 21-day exposure to R. catesbeiana. Mean X. laevis 14 to 21-day LOAEL (Lowest Observed Adverse Effect Level) and NOAEL (No Observed Adverse Effect Level) values for embryo-larval tests (25.5 and 11.0 micrograms/L dieldrin, respectively) were virtually the same as the 21-day single test values obtained for R. catesbeiana (25.1 and 11.0 micrograms/L dieldrin). Mean 14 to 24-day X. laevis LOAEL and NOAEL values for tadpole chronic tests (1.6 and less than 1.4 micrograms/L dieldrin, respectively) were lower than the 28-day single test values obtained for R. pipiens (4.1 and 1.9 micrograms/L, respectively). Tissue dieldrin levels at the LC50, LOAEL and NOAEL in the tadpole acute tests were fairly similar between X. laevis (11, 24 and 7 micrograms/g, respectively) and R. catesbeiana (means of 8.6, 12.0 and 1.2 micrograms/g, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Effects of pentachlorophenol-contaminated food organisms on toxicity and bioaccumulation in the frog Xenopus laevis.

Sub-adult African clawed frogs (Xenopus laevis) were fed pentachlorophenol (PCP)-injected mealworms containing 64.8 to 2604 ug of PCP per gram of worm for 27 days. There was no mortality and no significant bioaccumulation of PCP in the frogs. After three weeks, frogs fed 2,604 micrograms/g of PCP ceased eating. The no observed adverse effects level (NOAEL) based on significantly reduced food consumption (PCP-injected mealworms) was 638 micrograms/g. This corresponded to a NOAEL based on PCP intake of about 8 micrograms PCP/g frog/day. A toxicity threshold model estimated that about 800 micrograms/L of waterborne PCP may be a threshold for adverse effects in Xenopus or similar amphibians. Further study is needed to verify threshold estimates.

Animals↗