- Email: [email protected]
Seasonal kidney and plasma renin concentration in Testudo hermanni gmelin
Camp. Biochem. Phvsiol. Vol. 79A. No. 4. pp. 529-531, 1984
0300-9629184 S3.00 + 0.00 8~ 1984 Pergamon Press Ltd
Printed in Great Bhtain
SEASON.4L KIDNEY AND PLASMA RENIN CONCENTRATION IN TESTUDO HERMANNI GMELIN MAURO
Istituto
(Receirvd Abstract-l. angiotensin conditions
VALLARINO
di Anatomia e Fisiologia Comparate dell’Universit8 di Genova, Via Balbi 5, 16126 &nova. Italy. Telephone: 20-7623
5 March
Seasonal plasma and kidney renin concentration I in the terrestrial chelonian Testudo hrrmanni (different substrates, pH, temperature).
2. Evidence
has been found
phase of the animals: values were obtained
for seasonal
Extensive the
studies
in
mammals
renin-angiotensin
system
have
of the renin levels in relation to the physiological high renal and plasma renin concentration, while lower
demonstrated
its ion water balance (Peach, 1977; Vecsei et al., 1978). In non-mammalian vertebrates the activity of RAS remains a poorly known subject and few experimental studies have been carried out (Nishimura, 1978). Recent findings established in trouts a correlation between RAS and osmotic and urine flow regulation (Arillo et al., 1981), while Nishimura er al. (1981b) demonstrated in fowls that the RAS may not have a substantial role in the control of the blood pressure. The difficulties which arise in a comparative approach are mainly due to the problems concerning the sensitivity as well as the species specificity of the enzymatic assay method. This may also explain the conflicting data offered by various authors, which will be discussed further. In reptiles, previous studies have demonstrated all of the components of the RAS in freshwater turtles, but no correlation was established between hypotension and renin release (Creekmore and Stephens, 1981; Cipolle and Zehr, 1982), while in terrestrial chelonians Uva and Vallarino (1982) have been shown a direct action of the RAS upon osmoregulation. The aim of the present work was to investigate further in some detail a method for renin assay in chelonians, a representative group among reptiles living in various ecological situations and to determine if seasonal changes of renin concentration take place in these animals. action
on blood
pressure
MATERIALS
regulation
(RAS)
was studied by radioimmunoassay of Gmelin under several methodological
variations
active animals demonstrated in hibernating animals
INTRODUCTION
that
1984)
exerts
as well as on
AND METHODS
Females of Tesrudo hrrmanni Gmelin were acclimatized in a terrarium for 2 yr and the experiments were carried out in January (after 2 months of hibernation) and in June-July. when the animals were completely active (temperature ranging between 30 C at daylight and IS ‘C at night). 529
All animals were killed by decapitation. Blood was directly collected in cooled tubes containing NapEDTA (5 mg/ml) and the resulting plasma was dialyzed for 24 hr (4 C) against 5.9 mM Na-EDTA and stored at -20°C until analysis. Kidneys were immediately excised, stored at -2O’C and subsequently thawed at room temperature, minced, weighed and resubmitted twice to freezing and thawing. Each sample was homogenized with distilled water (2 mg/ml) and centrifuged (5 min at 12OOg); the sediment was extracted again with the same amount of water and the combined supernatants were dialyzed overnight (4 C) against 5.9 mM NapEDTA in order to remove catecholamines and to inhibit angiotensinase activity (Nishimura et ul., 1977). Partial purifications were performed on these dialyzed supernatants by acidification, as previously described (Conio rt al., 1980) and these crude extracts (pH 4.3) were stored at -20 C. For renin assay aliquots (50~1) kidney extracts. diluted 1: 12.5 with 0.1 M sodium phosphate buffer, 5.9 mM Na-EDTA (or 50 ~1 of total plasma), were incubated in two different systems, both containing inhibitors of angiotensinase and convertase (4 mM 2,3-dimercaptopropanl-01 and 3.2 mM hydroxyquinoline sulfate in 100 ~1 of the same phosphate buffer). The substrate in the first system was homologous plasma (200 p I), whereas in the second system I nmol of porcine angiotensinogen (Sigma) in 200 jll of the phosphate buffer were used. After the incubation of the mixture (I hr at 37‘C) 0.1 M Tris-HCI buffer, pH 7.4 (150 PI), was added and the reaction was stopped by immersion (20 min) in boiling water. Blanks were prepared by incubation of the mixture under the same conditions except for the temperature (O’C). After centrifugation (IOOOg. IO min) the rate of angiotensin I formation was estimated by a radioimmunoassay method using commercial assay kits (CEA-IRE Sorin). Aliquots of the supernatants (0.1 ml) were added to the following solution: 0.1 M sodium phosphate buffer (pH 7.4) containing 0.3”/, bovine serum albumin; [“‘Ilangiotensin, 0.1 ml (0.025 PCi); antibody against human angiotensin I. 0. I ml. All tubes were equilibrated at 24’C for 20 hr. dextrancharcoal mixture (0.5 ml) was added and after IO min supernatants were separated by centrifugation (I 500 g, IO min at 4 ‘C). The radioactivity was determined on OX-ml aliquots in a y-counter (Packard). A standard curve for angiotensin I was prepared under the same conditions and the ratio between zero standard and total activity was
530
MAURI Table I. Etkts
PH 5 5.5
h.S 7.4
VALLARIN~
of the pH on reni” concentration
Hibernating animals Renal cont. Plasma cont. (ng Agi:ml:hr) (“g A&&) 34 + 2.0 52 & 3.0 76tO5 72 +_0.5
tested wth
heterolopous substrate
Active aniinals Renal cont. Piasma tone. (ng Agl:g;hr) (ng AyI:mi:hr)
0.19 + 2.0 0.10~0.05 i .75 i a.05 1.15+O.OS
lb4 :t 210 144250 210 & 5.0 72 2 4.0
0.84 IO.02
I .02+ 0.02 3.21 * 0.02 I.91 &O.O2
RIA determination of the angiotensin I liberated after incubation with heterolagous substrate. Values are means k SE of 4 determinations for each set of experimental conditions. Statistical significance between active and hibernating animals,
P <
calculated system.
in order to evaluate
RESULTS
0.001:
between pH values.
the binding
capacity
P < 0.01.
of the
AND DISCUSSION
In the first group of experiments carried out on animals killed in the active phase (summer) kidney extracts were incubated with homologous or heterologous substrates and a wide range of pH (5-7.4) was used for the incubation mixture. The results are shown in Table 1. In every case a weak renin activity (or none at ail) was observed after incubation with homologous substrate, whereas a marked liberation of angiotensin I was detectable using porcine angiotensinogen, with a peak of activity at pH 6.5. This different behavior depending on the substrate could be ascribed either to the absence of a renin substrate in the plasma or to plasmatic inhibitors of the renin angiotensinogen reaction. In order to exclude the first hypothesis ahquots of the same supernatants used for radioimmunoassay were injected into anesthetized rats as a qualitative assay and a positive pressor response was obtained. These results demonstrated that a renin substrate is present in the homologous plasma. Further, by adding tortoise plasma (200~1) to the incubation mixture of pig angiotensinogen and kidney extract at different pH values no significant variation in the angiotensin I liberated was detected by RIA assay. In order to ascertain the presence of possible seasonal plasmatic inhibitors, in additional experiments kidney extracts from hibernating animals were incubated with plasma from active animals and kidney extracts from the latter animals were incubated with plasma from animals killed in winter. No change in immunoassayable angiotensin I was observed. It is very likely that angiotensin I liberated from a homologous substrate may possess a different amino acid sequence (Nakajima rr ui., 1971, 1978) without binding capacity to the antibody (anti-human angiotensin) available in commercial kits. Similar observations were made in the eel (Nishimura et al., 1977) and in the fowl (Nishimura et al., 1981a) but not in other cases, in which the radioimmunoassay after incubation with homologous substrate resulted in a better determination of renin activity than that obtained by bioassay (Nishimura et al., 1977: Seyama el crl., 1979; Arillo cf ~1.. 1981). On the other hand, on a large series of non-mammalian vertebrates (Capelii et d., 1970; Taylor, 1977: Le Brie and Boelcskevy, 1979) renin activity on a heterologous substrate was demonstrated, as well as in the present experiments.
It is likely the enzyme acts in every case on the Leu-Leu bond and only the liberated angiotensin may be different. The effects of pH on the liberation of angiotensin from the heterologous substrate was also verified in a comparison of renin concentration between active animals and hibernating ones (Table I), both for the kidneys extracts and for the plasma, following the procedure described above. A similar behavior was observed in the two groups of animals; nevertheless, the differences between the activities at pH 6.5 and 7.4 are more marked in active animals. The optimal value of the pH for the enzymatic reaction is coincidental with that found in rainbow trout (Arillo el ul., 1981) and in the toad fish (Nishimura et u/., 1977). whereas different pfl values are reported for the snake ~l~~~le ~~~d~~~jr~u[~(pH 4.5) (Seyama et ~1.. 1979) and for Nutrix tuxispilotu (pH 7.5) (Le Brie and Boelcskevy, 1979). The temperature of the incubation system exerts a clear effect on the liberation of the angiotensin 1, as shown in Fig. 1. The maximum of renin concentration at 37°C is followed by a reduction at 39 C. In other poikilothermic species the optimal temperature of incubation for renin activity is about 20 C (Nishimura et al., 1977; Arillo c’fal.. 1981). However, in the snake Elaphe quudrit~irgutu(Seyama et ui., 1979) the
03
/
-l IOO-
I
i
,”
.,
50
_
$:
.’
_-
r_.
--
----
f
-I,
_*--
i
4/-
’
10
20
30
37 39
ec
Fig. 1. Comparison between renal renin concentrations in active (O--,-.-O) and hibernating (a-----o) animals estimated after incubation of kidney extracts with heteroiogous substrate at different temperatures. Values are means for 5 animals and 4 dete~inations for each animal. The calculations of SE resulted in significant differences by Student’s f-test (P < 0.01) in the range of optimal temperature of incubation, whereas the differences between mean values at 10 and 20 C were slightly significant (P < 0.01).
Renin concentration
in Testudo hermanni Gmelin
renin activity in oitro is higher at 37 than at 20°C. These discrepancies may reflect a species-specificity of the enzyme activity. Also, for other enzymes, e.g. ATPase from skeletal muscles, the optimal temperature of incubation depends on the species of lizard considered and the enzymatic activities are rapidly reduced by a small increase of temperature (Bartholomew, 1968). The temperature is likely to be an important factor for regulation of the enzymatic activity in these poikilothermic species. This may also explain the minor differences observed at 10 and 20°C between active and hibernating animals. Although the data obtained in this work may not reflect true physiological levels, since heterologous substrate has been used, the present study provides evidence for seasonal variations of the renin concentration in Tesfudo. This can be clearly stated and correlated with the physiological phase of the animals when an appropriate procedure is employed. More generally, the method described may be considered a useful tool in studies of renin activity in nonmammalian vertebrates. Among the three experimental conditions that were underlined here the heterologous substrate may be generally accepted: for pH and temperature a preliminary check must be undertaken for individual species. The major advantages of the method are its reliability and sensitivity, since it requires only a few samples and small volumes of extract preparations obtained in a simple way. It allows us to obtain comparable results in different species and to evaluate differences, even if small ones, in experimental programmes for physiological studies in non-mammalian vertebrates. REFERENCES Arillo A., Uva B. and Vallarino M. (1981) Renin activity in rainbow trout (Salmo gairdneri Rich.) and effects of environmental ammonia. Comp. Eiochem. Physiol. 68A, 307-311. Bartholomew G. A. (1968) Body temperature and energy metabolism. In Animal Function: Principles and Adaptations (Edited by Gordon M. S.), pp. 290-354. Macmillan, . _. New
York.
Capelli J. P., Wesson
531
L. G. and Aponte G. E. (1970) A phylogenetic study of the renin-angiotensin system. Am. J. Physiol. 218, 1171-l 178. Cipolle M. D. and Zehr J. E. (1982) Characterization of the renin-angiotensin system in the freshwater turtle Pseudemys scripra. Fedn Proc. 41, 1344. Conio G., Ghiani P., Patrone E., Trefiletti V., Uva B. and Vallarino M. (1980) Isolation and characterization of multiple forms of renin from bull kidney. Biochim. hiophys. Arta 632, 317-328. Creekmore J. S. and Stephens G. A. (198 I) Response of the renin-angiotensin system to hypotension in the freshwater turtle. Fedn Proc. 40, 549. Le Brie S. J. and Boelcskevy B. D. (1979) The effect of furosemide on renal function and renin in water snakes. Comp. Biochem. Physiol. 63C, 223-228. Nakajima T., Nakayama T. and Sokabe H. (1971) Examination of angiotensin like substances from renal and extrarenal sources in mammalian and nonmammalian species. Gen. camp. Endocrinol. 11, 458466. Nakajima T., Khosla M. C. and Sakakibara S. (1978) Comparative biochemistry of renins and angiotensins in the vertebrates. Jpn Heart J. 19, 799-805. Nishimura H. (1978) Physiological evolution of the renin-angiotensin system. Jpn Heurf J. 19, 806-822. Nishimura H., Crofton J. T., Norton V. M. and Share L. (1977) Angiotensin generation in teleost fish determined by radioimmunoassay and bioassay. Gen. romp. Endocrinol. 32, 236-247. Nishimura H., Madey M. A.. Mugaas J. N.. Khosla M. C. and Crofton J. T. (198la) Radioimmunoassay of fowl angiotensin I. Gen. camp. Endocrinol. 45, 262-272. Nishimura H., Nakamura Y., Taylor A. A. and Madey M. A. (1981 b) Renin-angiotensin and adrenergic mechanisms in control of blood pressure in fowl. Hypertension 3 (Suppl. l), 141-149. Peach M. J. (1977) Renin-angiotensin system: biochemistry and mechanisms of action. Ph_ysiol. Rec. 57, 313-370. Seyama Y.. lshikawa N. and Yamashita S. (1979) Reninlike activity in the plasma and kidney of a snake. Eluphe quadricirgata. Chem. Pharm. Bull. 27, 2008-20 10. Taylor A. A. (1977) Comparative physiology of the renin-angiotensin system. Fedn Proc. 36, 177fS1780. Uva B. and Vallarino M. (1982) Renin-angiotensin system and osmoregulation in the terrestrial chelonian Testudo hermanni Gmelin. Comp. Biochem. Physiol. 71A, 449-45 I. Vecsei P., Hackenthal E. and Ganten D. (1978) The renin-angiotensin system. Past, present and future. Klin. Wochenschr. 56 (suppl.), 5-2 I.
0300-9629184 S3.00 + 0.00 8~ 1984 Pergamon Press Ltd
Printed in Great Bhtain
SEASON.4L KIDNEY AND PLASMA RENIN CONCENTRATION IN TESTUDO HERMANNI GMELIN MAURO
Istituto
(Receirvd Abstract-l. angiotensin conditions
VALLARINO
di Anatomia e Fisiologia Comparate dell’Universit8 di Genova, Via Balbi 5, 16126 &nova. Italy. Telephone: 20-7623
5 March
Seasonal plasma and kidney renin concentration I in the terrestrial chelonian Testudo hrrmanni (different substrates, pH, temperature).
2. Evidence
has been found
phase of the animals: values were obtained
for seasonal
Extensive the
studies
in
mammals
renin-angiotensin
system
have
of the renin levels in relation to the physiological high renal and plasma renin concentration, while lower
demonstrated
its ion water balance (Peach, 1977; Vecsei et al., 1978). In non-mammalian vertebrates the activity of RAS remains a poorly known subject and few experimental studies have been carried out (Nishimura, 1978). Recent findings established in trouts a correlation between RAS and osmotic and urine flow regulation (Arillo et al., 1981), while Nishimura er al. (1981b) demonstrated in fowls that the RAS may not have a substantial role in the control of the blood pressure. The difficulties which arise in a comparative approach are mainly due to the problems concerning the sensitivity as well as the species specificity of the enzymatic assay method. This may also explain the conflicting data offered by various authors, which will be discussed further. In reptiles, previous studies have demonstrated all of the components of the RAS in freshwater turtles, but no correlation was established between hypotension and renin release (Creekmore and Stephens, 1981; Cipolle and Zehr, 1982), while in terrestrial chelonians Uva and Vallarino (1982) have been shown a direct action of the RAS upon osmoregulation. The aim of the present work was to investigate further in some detail a method for renin assay in chelonians, a representative group among reptiles living in various ecological situations and to determine if seasonal changes of renin concentration take place in these animals. action
on blood
pressure
MATERIALS
regulation
(RAS)
was studied by radioimmunoassay of Gmelin under several methodological
variations
active animals demonstrated in hibernating animals
INTRODUCTION
that
1984)
exerts
as well as on
AND METHODS
Females of Tesrudo hrrmanni Gmelin were acclimatized in a terrarium for 2 yr and the experiments were carried out in January (after 2 months of hibernation) and in June-July. when the animals were completely active (temperature ranging between 30 C at daylight and IS ‘C at night). 529
All animals were killed by decapitation. Blood was directly collected in cooled tubes containing NapEDTA (5 mg/ml) and the resulting plasma was dialyzed for 24 hr (4 C) against 5.9 mM Na-EDTA and stored at -20°C until analysis. Kidneys were immediately excised, stored at -2O’C and subsequently thawed at room temperature, minced, weighed and resubmitted twice to freezing and thawing. Each sample was homogenized with distilled water (2 mg/ml) and centrifuged (5 min at 12OOg); the sediment was extracted again with the same amount of water and the combined supernatants were dialyzed overnight (4 C) against 5.9 mM NapEDTA in order to remove catecholamines and to inhibit angiotensinase activity (Nishimura et ul., 1977). Partial purifications were performed on these dialyzed supernatants by acidification, as previously described (Conio rt al., 1980) and these crude extracts (pH 4.3) were stored at -20 C. For renin assay aliquots (50~1) kidney extracts. diluted 1: 12.5 with 0.1 M sodium phosphate buffer, 5.9 mM Na-EDTA (or 50 ~1 of total plasma), were incubated in two different systems, both containing inhibitors of angiotensinase and convertase (4 mM 2,3-dimercaptopropanl-01 and 3.2 mM hydroxyquinoline sulfate in 100 ~1 of the same phosphate buffer). The substrate in the first system was homologous plasma (200 p I), whereas in the second system I nmol of porcine angiotensinogen (Sigma) in 200 jll of the phosphate buffer were used. After the incubation of the mixture (I hr at 37‘C) 0.1 M Tris-HCI buffer, pH 7.4 (150 PI), was added and the reaction was stopped by immersion (20 min) in boiling water. Blanks were prepared by incubation of the mixture under the same conditions except for the temperature (O’C). After centrifugation (IOOOg. IO min) the rate of angiotensin I formation was estimated by a radioimmunoassay method using commercial assay kits (CEA-IRE Sorin). Aliquots of the supernatants (0.1 ml) were added to the following solution: 0.1 M sodium phosphate buffer (pH 7.4) containing 0.3”/, bovine serum albumin; [“‘Ilangiotensin, 0.1 ml (0.025 PCi); antibody against human angiotensin I. 0. I ml. All tubes were equilibrated at 24’C for 20 hr. dextrancharcoal mixture (0.5 ml) was added and after IO min supernatants were separated by centrifugation (I 500 g, IO min at 4 ‘C). The radioactivity was determined on OX-ml aliquots in a y-counter (Packard). A standard curve for angiotensin I was prepared under the same conditions and the ratio between zero standard and total activity was
530
MAURI Table I. Etkts
PH 5 5.5
h.S 7.4
VALLARIN~
of the pH on reni” concentration
Hibernating animals Renal cont. Plasma cont. (ng Agi:ml:hr) (“g A&&) 34 + 2.0 52 & 3.0 76tO5 72 +_0.5
tested wth
heterolopous substrate
Active aniinals Renal cont. Piasma tone. (ng Agl:g;hr) (ng AyI:mi:hr)
0.19 + 2.0 0.10~0.05 i .75 i a.05 1.15+O.OS
lb4 :t 210 144250 210 & 5.0 72 2 4.0
0.84 IO.02
I .02+ 0.02 3.21 * 0.02 I.91 &O.O2
RIA determination of the angiotensin I liberated after incubation with heterolagous substrate. Values are means k SE of 4 determinations for each set of experimental conditions. Statistical significance between active and hibernating animals,
P <
calculated system.
in order to evaluate
RESULTS
0.001:
between pH values.
the binding
capacity
P < 0.01.
of the
AND DISCUSSION
In the first group of experiments carried out on animals killed in the active phase (summer) kidney extracts were incubated with homologous or heterologous substrates and a wide range of pH (5-7.4) was used for the incubation mixture. The results are shown in Table 1. In every case a weak renin activity (or none at ail) was observed after incubation with homologous substrate, whereas a marked liberation of angiotensin I was detectable using porcine angiotensinogen, with a peak of activity at pH 6.5. This different behavior depending on the substrate could be ascribed either to the absence of a renin substrate in the plasma or to plasmatic inhibitors of the renin angiotensinogen reaction. In order to exclude the first hypothesis ahquots of the same supernatants used for radioimmunoassay were injected into anesthetized rats as a qualitative assay and a positive pressor response was obtained. These results demonstrated that a renin substrate is present in the homologous plasma. Further, by adding tortoise plasma (200~1) to the incubation mixture of pig angiotensinogen and kidney extract at different pH values no significant variation in the angiotensin I liberated was detected by RIA assay. In order to ascertain the presence of possible seasonal plasmatic inhibitors, in additional experiments kidney extracts from hibernating animals were incubated with plasma from active animals and kidney extracts from the latter animals were incubated with plasma from animals killed in winter. No change in immunoassayable angiotensin I was observed. It is very likely that angiotensin I liberated from a homologous substrate may possess a different amino acid sequence (Nakajima rr ui., 1971, 1978) without binding capacity to the antibody (anti-human angiotensin) available in commercial kits. Similar observations were made in the eel (Nishimura et al., 1977) and in the fowl (Nishimura et al., 1981a) but not in other cases, in which the radioimmunoassay after incubation with homologous substrate resulted in a better determination of renin activity than that obtained by bioassay (Nishimura et al., 1977: Seyama el crl., 1979; Arillo cf ~1.. 1981). On the other hand, on a large series of non-mammalian vertebrates (Capelii et d., 1970; Taylor, 1977: Le Brie and Boelcskevy, 1979) renin activity on a heterologous substrate was demonstrated, as well as in the present experiments.
It is likely the enzyme acts in every case on the Leu-Leu bond and only the liberated angiotensin may be different. The effects of pH on the liberation of angiotensin from the heterologous substrate was also verified in a comparison of renin concentration between active animals and hibernating ones (Table I), both for the kidneys extracts and for the plasma, following the procedure described above. A similar behavior was observed in the two groups of animals; nevertheless, the differences between the activities at pH 6.5 and 7.4 are more marked in active animals. The optimal value of the pH for the enzymatic reaction is coincidental with that found in rainbow trout (Arillo el ul., 1981) and in the toad fish (Nishimura et u/., 1977). whereas different pfl values are reported for the snake ~l~~~le ~~~d~~~jr~u[~(pH 4.5) (Seyama et ~1.. 1979) and for Nutrix tuxispilotu (pH 7.5) (Le Brie and Boelcskevy, 1979). The temperature of the incubation system exerts a clear effect on the liberation of the angiotensin 1, as shown in Fig. 1. The maximum of renin concentration at 37°C is followed by a reduction at 39 C. In other poikilothermic species the optimal temperature of incubation for renin activity is about 20 C (Nishimura et al., 1977; Arillo c’fal.. 1981). However, in the snake Elaphe quudrit~irgutu(Seyama et ui., 1979) the
03
/
-l IOO-
I
i
,”
.,
50
_
$:
.’
_-
r_.
--
----
f
-I,
_*--
i
4/-
’
10
20
30
37 39
ec
Fig. 1. Comparison between renal renin concentrations in active (O--,-.-O) and hibernating (a-----o) animals estimated after incubation of kidney extracts with heteroiogous substrate at different temperatures. Values are means for 5 animals and 4 dete~inations for each animal. The calculations of SE resulted in significant differences by Student’s f-test (P < 0.01) in the range of optimal temperature of incubation, whereas the differences between mean values at 10 and 20 C were slightly significant (P < 0.01).
Renin concentration
in Testudo hermanni Gmelin
renin activity in oitro is higher at 37 than at 20°C. These discrepancies may reflect a species-specificity of the enzyme activity. Also, for other enzymes, e.g. ATPase from skeletal muscles, the optimal temperature of incubation depends on the species of lizard considered and the enzymatic activities are rapidly reduced by a small increase of temperature (Bartholomew, 1968). The temperature is likely to be an important factor for regulation of the enzymatic activity in these poikilothermic species. This may also explain the minor differences observed at 10 and 20°C between active and hibernating animals. Although the data obtained in this work may not reflect true physiological levels, since heterologous substrate has been used, the present study provides evidence for seasonal variations of the renin concentration in Tesfudo. This can be clearly stated and correlated with the physiological phase of the animals when an appropriate procedure is employed. More generally, the method described may be considered a useful tool in studies of renin activity in nonmammalian vertebrates. Among the three experimental conditions that were underlined here the heterologous substrate may be generally accepted: for pH and temperature a preliminary check must be undertaken for individual species. The major advantages of the method are its reliability and sensitivity, since it requires only a few samples and small volumes of extract preparations obtained in a simple way. It allows us to obtain comparable results in different species and to evaluate differences, even if small ones, in experimental programmes for physiological studies in non-mammalian vertebrates. REFERENCES Arillo A., Uva B. and Vallarino M. (1981) Renin activity in rainbow trout (Salmo gairdneri Rich.) and effects of environmental ammonia. Comp. Eiochem. Physiol. 68A, 307-311. Bartholomew G. A. (1968) Body temperature and energy metabolism. In Animal Function: Principles and Adaptations (Edited by Gordon M. S.), pp. 290-354. Macmillan, . _. New
York.
Capelli J. P., Wesson
531
L. G. and Aponte G. E. (1970) A phylogenetic study of the renin-angiotensin system. Am. J. Physiol. 218, 1171-l 178. Cipolle M. D. and Zehr J. E. (1982) Characterization of the renin-angiotensin system in the freshwater turtle Pseudemys scripra. Fedn Proc. 41, 1344. Conio G., Ghiani P., Patrone E., Trefiletti V., Uva B. and Vallarino M. (1980) Isolation and characterization of multiple forms of renin from bull kidney. Biochim. hiophys. Arta 632, 317-328. Creekmore J. S. and Stephens G. A. (198 I) Response of the renin-angiotensin system to hypotension in the freshwater turtle. Fedn Proc. 40, 549. Le Brie S. J. and Boelcskevy B. D. (1979) The effect of furosemide on renal function and renin in water snakes. Comp. Biochem. Physiol. 63C, 223-228. Nakajima T., Nakayama T. and Sokabe H. (1971) Examination of angiotensin like substances from renal and extrarenal sources in mammalian and nonmammalian species. Gen. camp. Endocrinol. 11, 458466. Nakajima T., Khosla M. C. and Sakakibara S. (1978) Comparative biochemistry of renins and angiotensins in the vertebrates. Jpn Heart J. 19, 799-805. Nishimura H. (1978) Physiological evolution of the renin-angiotensin system. Jpn Heurf J. 19, 806-822. Nishimura H., Crofton J. T., Norton V. M. and Share L. (1977) Angiotensin generation in teleost fish determined by radioimmunoassay and bioassay. Gen. romp. Endocrinol. 32, 236-247. Nishimura H., Madey M. A.. Mugaas J. N.. Khosla M. C. and Crofton J. T. (198la) Radioimmunoassay of fowl angiotensin I. Gen. camp. Endocrinol. 45, 262-272. Nishimura H., Nakamura Y., Taylor A. A. and Madey M. A. (1981 b) Renin-angiotensin and adrenergic mechanisms in control of blood pressure in fowl. Hypertension 3 (Suppl. l), 141-149. Peach M. J. (1977) Renin-angiotensin system: biochemistry and mechanisms of action. Ph_ysiol. Rec. 57, 313-370. Seyama Y.. lshikawa N. and Yamashita S. (1979) Reninlike activity in the plasma and kidney of a snake. Eluphe quadricirgata. Chem. Pharm. Bull. 27, 2008-20 10. Taylor A. A. (1977) Comparative physiology of the renin-angiotensin system. Fedn Proc. 36, 177fS1780. Uva B. and Vallarino M. (1982) Renin-angiotensin system and osmoregulation in the terrestrial chelonian Testudo hermanni Gmelin. Comp. Biochem. Physiol. 71A, 449-45 I. Vecsei P., Hackenthal E. and Ganten D. (1978) The renin-angiotensin system. Past, present and future. Klin. Wochenschr. 56 (suppl.), 5-2 I.