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Evidence that the frog skin excretes ammonia
03W962Y
Camp. Bmhem. Physd., Vol. 66A. pp. 525 lo 527 0 Pergamon Press Ltd 1980.Printed m Great Britam
EVIDENCE
THAT THE FROG AMMONIA LOU W. FRAZIER
XOiO701.052SSO?.CQO
SKIN EXCRETES
and JOHN C. VANATTA
Department of Physiology, Baylor College of Dentistry, Dallas, Texas 75246: and University of Texas Southwestern Medical School, Dallas, TX 75235. U.S.A. (Receiced 5 October 1979) Abstract-l. The skin of Raw pipiens excretes NH: in an in vitro preparation. 2. The excretion of NH: is increased by loading the animal with NH,Cl (metabolic acidosis). 3. Skins from normal frogs are stimulated to increase excretion of NH,+ by an extract of plasma
obtained from a dog during metabolic acidosis
There are several studies in the literature on the ability of the toad urinary bladder to excrete H+ and NH: and the importance of this organ in the animal’s acid-base balance (Frazier & Vanatta, 1971), (Ludens & Fanestil, 1972). There are no reports in the literature on the ability of the skin of Rana pipiens to excrete NH: or its possible role in the regulation of acid-base balance. Since the frog skin is analogous in some ways to the toad urinary bladder we decided to investigate the possibility of NH: excretion in the frog skin. The purposes of the studies reported are three-fold: (1) to determine if the skin of Rana pipiens excretes NH: ; (2) to determine if the NH: excretion in the skin can be increased by placing the animals in metabolic acidosis; and (3) to see if an extract of plama from another species of animal in metabolic acidosis could stimulate the NH: excretion. We present data giving affirmative answers to all these questions.
MATERIALS AND
METHODS
General The frogs (Rana pipiens) were kept in the laboratory at 23-25°C in deionized water without food from time of receipt until use. After double pithing the frogs, the abdominal or hind-leg skin was removed, cleaned by washing it briefly in the appropriate salt solution and then mounted as a sheet between lucite chambers. Each chamber was 2 ml in volume and the exposed area of the skin on each surface was 3.09 cmz. In seven experiments using skins from normal frogs and six experiments using skins from frogs in metabolic acidosis, ~the serosal and mucosal media were 1.5 mM PO, Ringer solution, pH 6.7-6.8, with 0.6 mM glutamine in Ihe serosal media. In the remainder of the experiments the serosal medid was IOmM PO4 Ringer solution, pH 6.5-6.6. The Ringer solution contained in mM: NaCl. 117.0; KCl, 3.0; CaCI,, 0.9; sodium phosphate was added to the desired concentration with adjustments in the NaCl concentration to maintain the total osmolarity at 237 mOsm/l. After the skins had been mounted in the lucite chambers they were allowed to equilibrate for ls30min with the appropriate solution on each surface. The flux period was then begun and was 222-225 min in duration. Humidified
100% O2 was bubbled into the mucosal bath throughout each experiment. The [NH:] was determined on each mucosal sample at the end of the excretion period. The method of measuring NH: excretion was as previously described (Frazier & Vanatta, 1971). The [NH:] was determined calorimetrically (Chaney & Marbach, 1962). The exposed area of each skin was cut out of the chamber and blotted dry with filter paper and the weight obtained. All excretion rates were normalized and reported as nmol/lOO mg bladder wet weight x minutes. Frogs in normal acid-base balance were fasted and kept in deionized water. Frogs in metabolic acidosis received six doses of 120 mM NH,CI, 0.02 ml/gm body weight, injected into the dorsal lymph sac over a 48 hr period. Esperiments using dog plasma A 10 kg female mongrel dog was anesthetized with chloralose urethane. Acute acidosis was produced by injecting 600 ml of 0.15 N HCI intravenously in 165 min. Heparinized plasma was obtained from blood drawn 4 hr after the onset of the injection of the acid. The plasma was frozen and prepared for use within 4 days before the experiments on the frog skin. Plasma was prepared by adding four volumes of acetone to one volume of plasma, which precipitated the proteins. The mixture was filtered, and the filtrate evaporated under reduced pressure with a maximum temperature of 40°C. When the acetone was removed, 0.05 HCI was added to pH 2.0 and the evaporation continued to dryness, The final volume for reconstituting the solution was calculated based on: (155 mequiv/l)
(V) = (120mequiv/l)
(W)
where, 1/ = initial volume of plasma, and W = volume of water to be added. The purpose of this is to reduce the concentration of total salts from that of the mammalian body fluids to that of toads. A volume of ethanol equal to 0.01 W was added to the residue, and then a volume of water, LY Also, assuming 2.0mM phosphate was present in the initial plasma, the amount of phosphate necessary to make the final phosphate concentration IOmM was added in the form of Na,HP04. The pH of the resulting mixture was adjusted to 6.97 f 0.05 by adding 0.5 NaOH or 0.05 N NaOH. In the experiments on the ammoniuretic preparations from dog plasma, skin of the thighs of the frogs were used exclusively. Paired preparations were used with the skin of one thigh serving as a control, and the skin of the other thigh being used to determine the effect of the plasma factor. The procedures were exactly as given above except that the serosal media of the experimental skin was the prepared plasma, and the serosal media of the control skin
526
Lou W. FRAZIERand JOHY C. VANATTA
Table
I. Excretion
rates of NH;
N
Acid-base state of frog
nmoles
Normal
15
in the frog skin
NH4+ Excretion (100 mg bladder)-'
P valve (min)-1
0.609 + 0.067* < 0.025
Metabolic
Acidosis
17
0.884 + 0.089*
* & SEM.
was 10 mM PO4 Ringer solution with 14, ethanol. tion, the excretion period was 240 min.
In addi-
lower than that of the 15 skins reported above. These frogs were from a different supplier. The reason for these differences was not examined.
Statistics
Statistical analysis was done using the one-tailed Student t-test as reported by Fisher (1936). The one tailed test was appropriate since ammoniuretic activity was present only if the experimental skin excreted more than the control skin. In the first experiments on skins from frogs in normal acid-base balance and metabolic acidosis, the mean f SEM are given. In the experiments on the effects of dog plasma, the mean difference + SEM is reported.
DISCUSSION
RESULTS Effect of metabolic
acidosis on ammonia excretion
All skins excreted ammonia as is shown in Table 1. The average excretion rate for the I5 skins from frogs in normal acid-base balance was 0.609 f 0.067 units, and the average for the 17 skins from frogs in metabolic acidosis was 0.884 _+ 0.089 units (P/2 < 0.025). Thus the normal frog skin excretes ammonia and the rate of excretion is increased in response to an ammonium chloride induced acidosis. Because of the variation of amphibia with various factors, it should be noted that these experiments were done on the same shipment of toads, and we always did controls and experimental observations simultaneously. EfSect of plasmafhctor
on ammorlia e.\-cretiorl
The mean rate of excretion of ammonia of 10 control skins, and the mean of the paired 10 experimental skins are shown in Table 2. The mean difference was 0.230 k 0.04 (P < 0.001). The plasma preparation from the dog clearly stimulated ammonia excretion in the frog skin. The value of the controls of this series is
Table 2. Effect of plasma
Serosal
extract
medium nmoles
10 mM PO4 buffered Rinqer
In 1968 Romeu & Salibian reported ammonia excretion in the skin of the South American frog Leptodactylus ocellatus. Excretion of NH: through the skin has been reported for Rana esculenta (Przylecki, Opienska & Giedroyc, 1922), Xenopus laeuis (Balinsky & Baldwin, 1961). Necturus maculosus (Fanelli & Glodstein, 1964) and the larvae of Amhystoma titrinum (Dietz, Kirschner & Porter, 1967). Thus the finding of ammonia excretion in another species of frog is not too surprising. However, Romeu & Salibian (196X) further note that publications on the subject of ammonia excretion by the skin of amphibia “is scarce and many and very important problems for the understanding of the ionic homeostatic mechanisms in freshwater animals remain almost unexplored”. We were unable to find significant literature on the subject published since that time. The fact that one of the controlling factors in the excretion of ammonia in Rana pipiens is related to the acid-base state of the animal has not been previously reported. The significance of the frog secreting NH,+ through the skin can be considered from different angles. From the standpoint of the physiology of the frog, the skin can assist the kidney in correcting a metabolic acidosis. From the standpoint of the physiologist, this is another model in which ammonia excretion can be studied. In assays for ammoniuretic activity of
from dog on NH:
NH4+ Excretion (100 mg bladder)-1
excretion
in the frog skin
(min)-1
Mean difference + SEM (P value)
0.484*
0.230 10 mM PO4 buffered Ringer + extract of plasma of dog in metabolic acidosis * Average
of IO palred
skms.
+ 0.04
(P < OTbOl)
0.714*
527
Frog skin excretes ammonia
plasma, frog skin will be less expensive than the urinary bladder of the toad. The fact that skin from each thigh can be used as paired preparations still allows this paired technique to be used to reduce the effect of variation between animals. In addition, we feel that the fact that there is less percentage variation in the weight of one frog skin
pre~ration compared with its paired preparation, as compared to similar comparisons of two paired toad bladders. We believe that this will reduce the variance of statistics in assays of amminouretic substance, although we have not had sufficient experience with the frog skins to accurately assess this point. We also observed that the skin of Rana p~~iens could acidify the epithelial solution. In these experiments, we were unable to show a difference in the rate of acidification in comparing animals in an ammonium chloride acidosis with those in normal acid-base balance. The fact that the factor present in the plasma of acidotic dogs stimulates ammonia excretion in epithelial structures from two different species lends credence to the theory that such a factor is important in regulating ammonia excretion. A similar factor has also been reported in urine from man in metaboiic acidosis (Melton, Frazier & Vanatta, 1979). Acknowledgements-The expert technical assistance of Mrs Winifred Rhodes is gratefully acknowledged. This investigation was supported in part by U.S. Public Health Service Grant 5SOl-RR05426-14 and by NIH Grant AM18689,
REFERENCES
BAUYSU J. B. & BALDWINE. (1961) The mode of excretion of ammonia and urea in Xetwptrs lueuis. J. ewp. Biol. 38, 695-705. CHANEYA. & MARBACHE. P. (1962) Modified reagents for determination of urea and ammonia. C/in. Chrm. 8, 130-132. DIETZ T. H., KIRSCHNERL, B. & PORTERD, (1967) The roles of sodium transport and anion permeability in generating transepithelial potential differences in larval salamanders. J. exp. Viol. 46, 85-96. FANELLIG. M. & GLODSTEIN, L. (1964) Ammonia excretion in the neotenous newt Nectarus maculasus (Rafinesque). Comp. Biochem. Physioi. 13, 193-204. FISHER R, A. (1936) Statistical methods “for Research Workers, 6th edition pp. 124-125, Oliver & Boyd, London. FRAZIERL. W. & VANATTAJ. C. (1971) Excretion of H’ and NH; by the urinary bladder of the acidotic toad and the effect of short-circuit current on the excretion. Bi~chim. Bioph~fs. Acta 241, 20-29. LUDENSJ. H. & FANESTILD. D. (1972) Acidification of urine by the isolated urinary bladder of the toad. Am. J. Physiol. 223, 1338-1344. MELTONL. B., FRAZIER L. W. & VANA~A J. C. (1979) Stimulation of ammonium ion excretion in the toad urinary bladder by an extract of human urine. Biophps. Acta 585, 53-60. PRZYLECKIS. J., OPIENSKAJ. & GIEDROYCH. (1922) Excretion of nitrogenous compounds by the frog at different temueratures. Arch. internat. Phvsiol. 20, 207-212. ROME; F. G. & SALIB~ANA. (1968) Sodium uptake and ammonia excretion through the ipt t%coskin of the South American frog Leptodactylus ocdfattrs. Lije Sci. 7, 465-470.
Camp. Bmhem. Physd., Vol. 66A. pp. 525 lo 527 0 Pergamon Press Ltd 1980.Printed m Great Britam
EVIDENCE
THAT THE FROG AMMONIA LOU W. FRAZIER
XOiO701.052SSO?.CQO
SKIN EXCRETES
and JOHN C. VANATTA
Department of Physiology, Baylor College of Dentistry, Dallas, Texas 75246: and University of Texas Southwestern Medical School, Dallas, TX 75235. U.S.A. (Receiced 5 October 1979) Abstract-l. The skin of Raw pipiens excretes NH: in an in vitro preparation. 2. The excretion of NH: is increased by loading the animal with NH,Cl (metabolic acidosis). 3. Skins from normal frogs are stimulated to increase excretion of NH,+ by an extract of plasma
obtained from a dog during metabolic acidosis
There are several studies in the literature on the ability of the toad urinary bladder to excrete H+ and NH: and the importance of this organ in the animal’s acid-base balance (Frazier & Vanatta, 1971), (Ludens & Fanestil, 1972). There are no reports in the literature on the ability of the skin of Rana pipiens to excrete NH: or its possible role in the regulation of acid-base balance. Since the frog skin is analogous in some ways to the toad urinary bladder we decided to investigate the possibility of NH: excretion in the frog skin. The purposes of the studies reported are three-fold: (1) to determine if the skin of Rana pipiens excretes NH: ; (2) to determine if the NH: excretion in the skin can be increased by placing the animals in metabolic acidosis; and (3) to see if an extract of plama from another species of animal in metabolic acidosis could stimulate the NH: excretion. We present data giving affirmative answers to all these questions.
MATERIALS AND
METHODS
General The frogs (Rana pipiens) were kept in the laboratory at 23-25°C in deionized water without food from time of receipt until use. After double pithing the frogs, the abdominal or hind-leg skin was removed, cleaned by washing it briefly in the appropriate salt solution and then mounted as a sheet between lucite chambers. Each chamber was 2 ml in volume and the exposed area of the skin on each surface was 3.09 cmz. In seven experiments using skins from normal frogs and six experiments using skins from frogs in metabolic acidosis, ~the serosal and mucosal media were 1.5 mM PO, Ringer solution, pH 6.7-6.8, with 0.6 mM glutamine in Ihe serosal media. In the remainder of the experiments the serosal medid was IOmM PO4 Ringer solution, pH 6.5-6.6. The Ringer solution contained in mM: NaCl. 117.0; KCl, 3.0; CaCI,, 0.9; sodium phosphate was added to the desired concentration with adjustments in the NaCl concentration to maintain the total osmolarity at 237 mOsm/l. After the skins had been mounted in the lucite chambers they were allowed to equilibrate for ls30min with the appropriate solution on each surface. The flux period was then begun and was 222-225 min in duration. Humidified
100% O2 was bubbled into the mucosal bath throughout each experiment. The [NH:] was determined on each mucosal sample at the end of the excretion period. The method of measuring NH: excretion was as previously described (Frazier & Vanatta, 1971). The [NH:] was determined calorimetrically (Chaney & Marbach, 1962). The exposed area of each skin was cut out of the chamber and blotted dry with filter paper and the weight obtained. All excretion rates were normalized and reported as nmol/lOO mg bladder wet weight x minutes. Frogs in normal acid-base balance were fasted and kept in deionized water. Frogs in metabolic acidosis received six doses of 120 mM NH,CI, 0.02 ml/gm body weight, injected into the dorsal lymph sac over a 48 hr period. Esperiments using dog plasma A 10 kg female mongrel dog was anesthetized with chloralose urethane. Acute acidosis was produced by injecting 600 ml of 0.15 N HCI intravenously in 165 min. Heparinized plasma was obtained from blood drawn 4 hr after the onset of the injection of the acid. The plasma was frozen and prepared for use within 4 days before the experiments on the frog skin. Plasma was prepared by adding four volumes of acetone to one volume of plasma, which precipitated the proteins. The mixture was filtered, and the filtrate evaporated under reduced pressure with a maximum temperature of 40°C. When the acetone was removed, 0.05 HCI was added to pH 2.0 and the evaporation continued to dryness, The final volume for reconstituting the solution was calculated based on: (155 mequiv/l)
(V) = (120mequiv/l)
(W)
where, 1/ = initial volume of plasma, and W = volume of water to be added. The purpose of this is to reduce the concentration of total salts from that of the mammalian body fluids to that of toads. A volume of ethanol equal to 0.01 W was added to the residue, and then a volume of water, LY Also, assuming 2.0mM phosphate was present in the initial plasma, the amount of phosphate necessary to make the final phosphate concentration IOmM was added in the form of Na,HP04. The pH of the resulting mixture was adjusted to 6.97 f 0.05 by adding 0.5 NaOH or 0.05 N NaOH. In the experiments on the ammoniuretic preparations from dog plasma, skin of the thighs of the frogs were used exclusively. Paired preparations were used with the skin of one thigh serving as a control, and the skin of the other thigh being used to determine the effect of the plasma factor. The procedures were exactly as given above except that the serosal media of the experimental skin was the prepared plasma, and the serosal media of the control skin
526
Lou W. FRAZIERand JOHY C. VANATTA
Table
I. Excretion
rates of NH;
N
Acid-base state of frog
nmoles
Normal
15
in the frog skin
NH4+ Excretion (100 mg bladder)-'
P valve (min)-1
0.609 + 0.067* < 0.025
Metabolic
Acidosis
17
0.884 + 0.089*
* & SEM.
was 10 mM PO4 Ringer solution with 14, ethanol. tion, the excretion period was 240 min.
In addi-
lower than that of the 15 skins reported above. These frogs were from a different supplier. The reason for these differences was not examined.
Statistics
Statistical analysis was done using the one-tailed Student t-test as reported by Fisher (1936). The one tailed test was appropriate since ammoniuretic activity was present only if the experimental skin excreted more than the control skin. In the first experiments on skins from frogs in normal acid-base balance and metabolic acidosis, the mean f SEM are given. In the experiments on the effects of dog plasma, the mean difference + SEM is reported.
DISCUSSION
RESULTS Effect of metabolic
acidosis on ammonia excretion
All skins excreted ammonia as is shown in Table 1. The average excretion rate for the I5 skins from frogs in normal acid-base balance was 0.609 f 0.067 units, and the average for the 17 skins from frogs in metabolic acidosis was 0.884 _+ 0.089 units (P/2 < 0.025). Thus the normal frog skin excretes ammonia and the rate of excretion is increased in response to an ammonium chloride induced acidosis. Because of the variation of amphibia with various factors, it should be noted that these experiments were done on the same shipment of toads, and we always did controls and experimental observations simultaneously. EfSect of plasmafhctor
on ammorlia e.\-cretiorl
The mean rate of excretion of ammonia of 10 control skins, and the mean of the paired 10 experimental skins are shown in Table 2. The mean difference was 0.230 k 0.04 (P < 0.001). The plasma preparation from the dog clearly stimulated ammonia excretion in the frog skin. The value of the controls of this series is
Table 2. Effect of plasma
Serosal
extract
medium nmoles
10 mM PO4 buffered Rinqer
In 1968 Romeu & Salibian reported ammonia excretion in the skin of the South American frog Leptodactylus ocellatus. Excretion of NH: through the skin has been reported for Rana esculenta (Przylecki, Opienska & Giedroyc, 1922), Xenopus laeuis (Balinsky & Baldwin, 1961). Necturus maculosus (Fanelli & Glodstein, 1964) and the larvae of Amhystoma titrinum (Dietz, Kirschner & Porter, 1967). Thus the finding of ammonia excretion in another species of frog is not too surprising. However, Romeu & Salibian (196X) further note that publications on the subject of ammonia excretion by the skin of amphibia “is scarce and many and very important problems for the understanding of the ionic homeostatic mechanisms in freshwater animals remain almost unexplored”. We were unable to find significant literature on the subject published since that time. The fact that one of the controlling factors in the excretion of ammonia in Rana pipiens is related to the acid-base state of the animal has not been previously reported. The significance of the frog secreting NH,+ through the skin can be considered from different angles. From the standpoint of the physiology of the frog, the skin can assist the kidney in correcting a metabolic acidosis. From the standpoint of the physiologist, this is another model in which ammonia excretion can be studied. In assays for ammoniuretic activity of
from dog on NH:
NH4+ Excretion (100 mg bladder)-1
excretion
in the frog skin
(min)-1
Mean difference + SEM (P value)
0.484*
0.230 10 mM PO4 buffered Ringer + extract of plasma of dog in metabolic acidosis * Average
of IO palred
skms.
+ 0.04
(P < OTbOl)
0.714*
527
Frog skin excretes ammonia
plasma, frog skin will be less expensive than the urinary bladder of the toad. The fact that skin from each thigh can be used as paired preparations still allows this paired technique to be used to reduce the effect of variation between animals. In addition, we feel that the fact that there is less percentage variation in the weight of one frog skin
pre~ration compared with its paired preparation, as compared to similar comparisons of two paired toad bladders. We believe that this will reduce the variance of statistics in assays of amminouretic substance, although we have not had sufficient experience with the frog skins to accurately assess this point. We also observed that the skin of Rana p~~iens could acidify the epithelial solution. In these experiments, we were unable to show a difference in the rate of acidification in comparing animals in an ammonium chloride acidosis with those in normal acid-base balance. The fact that the factor present in the plasma of acidotic dogs stimulates ammonia excretion in epithelial structures from two different species lends credence to the theory that such a factor is important in regulating ammonia excretion. A similar factor has also been reported in urine from man in metaboiic acidosis (Melton, Frazier & Vanatta, 1979). Acknowledgements-The expert technical assistance of Mrs Winifred Rhodes is gratefully acknowledged. This investigation was supported in part by U.S. Public Health Service Grant 5SOl-RR05426-14 and by NIH Grant AM18689,
REFERENCES
BAUYSU J. B. & BALDWINE. (1961) The mode of excretion of ammonia and urea in Xetwptrs lueuis. J. ewp. Biol. 38, 695-705. CHANEYA. & MARBACHE. P. (1962) Modified reagents for determination of urea and ammonia. C/in. Chrm. 8, 130-132. DIETZ T. H., KIRSCHNERL, B. & PORTERD, (1967) The roles of sodium transport and anion permeability in generating transepithelial potential differences in larval salamanders. J. exp. Viol. 46, 85-96. FANELLIG. M. & GLODSTEIN, L. (1964) Ammonia excretion in the neotenous newt Nectarus maculasus (Rafinesque). Comp. Biochem. Physioi. 13, 193-204. FISHER R, A. (1936) Statistical methods “for Research Workers, 6th edition pp. 124-125, Oliver & Boyd, London. FRAZIERL. W. & VANATTAJ. C. (1971) Excretion of H’ and NH; by the urinary bladder of the acidotic toad and the effect of short-circuit current on the excretion. Bi~chim. Bioph~fs. Acta 241, 20-29. LUDENSJ. H. & FANESTILD. D. (1972) Acidification of urine by the isolated urinary bladder of the toad. Am. J. Physiol. 223, 1338-1344. MELTONL. B., FRAZIER L. W. & VANA~A J. C. (1979) Stimulation of ammonium ion excretion in the toad urinary bladder by an extract of human urine. Biophps. Acta 585, 53-60. PRZYLECKIS. J., OPIENSKAJ. & GIEDROYCH. (1922) Excretion of nitrogenous compounds by the frog at different temueratures. Arch. internat. Phvsiol. 20, 207-212. ROME; F. G. & SALIB~ANA. (1968) Sodium uptake and ammonia excretion through the ipt t%coskin of the South American frog Leptodactylus ocdfattrs. Lije Sci. 7, 465-470.