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Renin-angiotensin system and osmoregulation in the terrestrial chelonian Testudo hermanni gmelin
RENIN-ANGIOTENSIN SYSTEM AND OSMOREGULATION IN THE TERRESTRIAL ~HELONIAN TESTUDO HERMAiVNl GMELIN BIANCAMARIA UVA and MAUROVALLARINO lstituto di Anatomia e Fisiologia Comparate dell’Universita’ di Genova, Italy (Received
27 July 1981)
Abstract-l. Renal renin activity, Na and K plasma urinary levels were studied in Testudu hermunni after salted diet, saline solution administration and furosemide injection. 2. Salt loading depressed renal renin activity whereas injection of the diuretic resulted in sodium loss and enhanced renal renin activity. 3. Results lead one to conclude the presence of a renin-angiotensin system with a direct action upon osmoregulation in these terrestrial chelonians.
INTRODUCTION Evidence for the presence of an angiotensin forming enzyme have been shown in most vertebrates (for a review see Sokabe & Ogawa, 1974). More recently immunoassayable renin activity was demonstrated also in the kidney of the elasmobranch Torpedo marmoraru (Vallarino et al., 1979). Therefore this enzymatic activity is probably lacking only in cyclostomes. The possible physioIogicaI roles of a renin angiotensin system (RAS) have been stated only in mammals (for reviews see Munday & York, 1976; Peach, 1977; Vecsei et al., 1978). including short-term blood pressure regulation and stimulation of aldosterone secretion with effects on glomerular filtration rate and salt water balance. In non-mammalian vertebrates the responses to the production of angjotensin or angiotensin-like substances remain uncertain as far as an effect on osmoregulation might be concerned (Taylor, 1977) and further comparative studies are needed. In reptiles-the class of vertebrates to which the present paper is particularly devoted---the “‘juxtaglomerular apparatus” is only represented by the renin forming juxtaglomerular cells, Iocated in the afferent arterioles close to the glomeruli; a macula densa and an extraglomerular mesangium (the two other components of the juxtaglomerular apparatus in mammals) are absent (Sokabe et al., 1969; Capeili et al., 1970). The presence of a renin-like activity of some reptilian species was demonstrated by Nolly & Fasciolo (1972). A renin-like activity was found in the plasma and in the kidney of a non-poisonous snake, Eluphe quudritkpta, and lower values were determined during the period of hibernation (Seyama et al., 1979). Studies have been carried out on possible chemical differences of angiotensin-like substances in various vertebrates, including only a reptile, the snake E~uph~ c~j~~co~~a (~akajima et af., 1971). However, to our knowledge the evidence for the physiological roles of the RAS in reptiles are limited to a few species. Infusion of homologous kidney extract resulted in a rise of blood pressure and corticosterone secretion in Pwudemys sueannirnsis, but no steroidoge-
nit activity was demonstrated in C&nun sclerops in the same experimentai conditions (Nothstine et al., 1971f. More recently Le Brie & Boeicskevy (1979) demonstrated a significant rise of renal renin content in the water snake Narrix ruxispilora after administration of a diuretic (furosemide) without changes in glomerular filtration rate or plasma electrolytes concentrations. The purpose of the present work was to contribute to this debate with some observations on a terrestrial chelonian Testudo hermanni Gmeiin which appear to depend primarily upon its urinary apparatus for osmotic and ionic regulation, and experiments were undertaken (1) to compare the effects of sodium loading and administration of a diuretic on renal renin activity and (2) to follow in the same conditions sodium and potassium plasmatic and urinary levels as an index of osmoregulatory processes. MATERIALSAND METHODS Tesrudo hrrmanni Gmelin (adult females) specimens {weight ranging from I to 1.5 kg) were acclimatized for several months in the open in a terrarium with a 6Ocm sand layer to allow voluntary burial. Animals were fed with a uniform lettuce diet and water was given ud lihifum. EXperiments were carried out in July (ambient temperature ranging between 3O’C at daylight and I5 C at night) on animals distributed in the following groups: Group I, controls, fed as above described; Group 2-4. animals submitted to a salt diet (lettuce with a 3*/, NaCI) and supplied with salted water (NaCI 14;) for 5, 30 and 50 days respectively; Group 5, saline solutions (1 M NaCI 10ml) were injected intraperitoneally for 3 days. The intervention was performed through a hole drilled ventrally (at the midline between hyo- and hypoplastron plates) and sealed by a cork lubricated with antibiotic ointment; Group 6. a diuretic (Furosemide, Lasix Hoechts) was injected intraperitoneally for 2 days (1Omg~k~day and 2Om~kg/day respectively) following the above described procedure, and in the same way distilled water was supplied after the second injection (10 ml/kg). Urine collections were performed following the procedure described by Bradshaw er u/(1972) for lizards. Animals were housed in cages of appropriate volume. and
449
BIAX( ~MAKIA UVA and MACKO VALLAKIW
450
Table I. Renal renln activity and electrolytes osmotic conditions
Treatments Controls Diet 3”,, NaCl for 5 days 30 days 50 days I M NaCl injections Furosemide injections
Na (mequiv/l) 131 + 3.0 142 146 164 I52 117
& + + * +
1.0* 1.5* 2.0* Is* 2.0*
plasma levels in Jltlkcnt
K (mequiv/l)
Renm activity (ng Agl;g:hr)
4.8 & 0.2 4.4 4.2 4.1 4.4 3.7
_t _t * + _t
0.1 t O.lt o.tt 0. It 0.2t
226 + 2.0 13 37 91 206 512
* 1.0* * 5.0* * 10.0’ +_ 4.0* i 6.0*
Values represent the mean _t SE of 5 determinations on 6 tortoises in each experimental groups. *tStatistically significant differences by Student r-test between treated and control animals (P c O.Ol;.P < 0.05 respectively).
under the wire grid replacing the floor of the upper compartment, a fibre glass layer was fitted as a filter for the urine voided into the lower box. All the animals were killed by decapitation starting from 11 a.m. Groups 5 and 6 were
killed 120 min and 90 min after the last injection respectively. Blood directly collected into lithium-heparinized tubes was centrifuged at 4°C and resulting plasma stored at - 20°C until analysis. Sodium and potassium levels in urine and plasma were determined by Emission Flame Photometry, Reference method (courtesy M. D. S. Zocchi and M. D. G. E. Scafidi, Celesia Hospital, Analyses Laboratories. Genova). Kidney extracts were prepared according to the procedure of Haas er al. (1954) with slight modification previously described (Conio et (II., 1980). Before the partial purification achieved by acidification following the initial step of the above mentioned procedure, the crude extracts were dialyzed overnight against 5.9 mM NaEDTA in order to remove catecholamines and to inhibit angiotensinase activity. For renin assay aliquots (50~1) of kidney extracts were incubated with 0.5 nmol porcine anglotensinogen (Sigma) in 200 111of 0.1 M Na phosphate buffer pH 6.5. Angiotensinase and convertases inhibitors (4 mM 2-3 dimercaptopropan-l-01 and 3.2 mM hydroxyquinoline sulfate in 100 /tl of the same phosphate buffer) were added. At the end of the incubation (1 hr at 37’C) 0.1 M Tris-HCI buffer pH 7.4 was added and the reaction was stopped by immersion in boiling water. For every sample blanks were prepared in the same way except for the incubation temperature (WC). On aliquots of the supernatants obtained by centrifugation the rate of angiotensin I formation was estimated by a radioimmunoassay method (CEA-IRE SORIN commercial
Kits). RESULTS AND DISCUSSION Sodium loading obtained by a salted diet (Groups 2,4 as described in the methods) or by an intraperitoneal injection of NaCl solution (Group 5) resulted in a rise in Na, in a slightly diminished K in plasma and in a depression of renal renin activity (see Table 1). Nevertheless between the two experimental conditions some differences have been observed. In control animals the urinary emission. although varying in a wide range, may be estimated 40ml per day as mean value; the urinary sodium content is very low, under the limits of the method’s sensitivity. However, it must be remembered that in most reptiles the cloaca or the urinary bladder exerts an activity on sodium retention normally under prolactin control
(Brewer & Ensor. 1980). During the salted diet the urinary emission dropped near to zero and no control of sodium content was available. This is in agreement with that described by several authors for saline loaded reptiles (see Bentley, 1971). Urine was collected instead even if in small amount after intraperitoneal injection of saline solution loaded with water and a clear rise in sodium urinary level was observed. This reflects in the first experiments a strong water retention and in the second one the tendency to eliminate the electrolytes excess, in both experiments this corresponds to a lowered plasma renin activity. However, it seems interesting that the reduction in plasma renin activity after administration of a saline solution even if significant did not reach the extent observed after salted diet, in which a more severe salt loading condition was stated. without a contemporary supply of water. The firm correlation between osmotic stress and renin activity is confirmed by the results obtained after administration of a diuretic. While Na and K plasma levels diminished, kidney renin activity showed a clear rise (Table 1). In the same experiment emission of urine was nearly doubled and the total amount of its saline content was very high (Table 2). The rise of sodium in urine has in this case a different explanation in comparison with the effect obtained by saline solution injection. In this last case there is likely to be a close relation with the mechanism of action of the furosemide, involving proximal and distal tubular activity as discussed by Le Brie & Boelcskevy (1979). Electrolytes plasma and urinary levels in the same conditions show a different behaviour in comparison with that obtained by the above menTable
2. Na and K urinary
Na (mequiv:l)
Treatments Controls I M NaCl injections Furosemide injections Values mals).
expressed
levels after osmotic
K (mequlv;‘l)
Trace 181.5 + 2.0* 79.5 +_ 1.4* as in Table
1 (mean
stress
43.5 + I.2 36.5 & 0.9* 18.3 + 2.5* +_ SE of 6 ani-
For volumes of voided urine see in the text (under “Results and Discussion”). * Significant differences
vs control
animals
(P < 0.01).
Renin-angiotensin tioned
authors.
In the herbivorous
system and osmorf :gulation in resruda hermunni Gmelin species
here ob-
served ions’ regulation may be quite different from that present in carnivorous ones, as in the Natrix. At the same time the potassium plasma level decreased and the contemporary decrease of urinary potassium might be explained by a balance between potassium loss and potassium reabsorption in cloaca. it is, however, undoubtable that after Furosemide injection sodium loss resulted in a rise of kidney renin activity. Circulating aldosterone in this species has been
found recently and decrease or rise in aldosterone plasma levels were stated in very simiiar experimenta conditions involving a sodium load or a sodium loss respectively (Uva et al., 1981). All these data led us to conclude the presence in these reptiles of a reninangiotensin-aldosterone system which plays an important physiological role exerting a powerful action in the regulation of salt water balance.
REFERENCES BENTLEY Q. J.
(1971) Endocrines and Osmoregulution, p. 150. Springer-Verlag, Berlin. BREWER K. J. 6% ENSOR D. M. (1980) Hormonal control of osmoregulation in the chelonia. 11The effect of prolactin and corticosterone on Testuda graeca. Gen. camp. Endacr. 42, 3 I O-3 14.
BRADSHAW S. D., SHOEMAKER V. H. & NAGK K. A. (1972) The role of adrenal corticosteroids in the regulation of kidney function in the desert lizard Dipsasuutw dursalis. Camp. Biochrm. Physioi, 43A, 621-635. CAPELLIJ. P.. WESSONL. G. & APONTE G. E. (1970) A phylogenetic study of the renin-angiotensin system. Am. J. Physiol. 218, 1171-I178. CCINIOG., GHIANI P., QATRONE E., TREFJLETTI V., UVA B. & VALLARINOM. (1980) Isolation and characterization of multiple forms of renin from bull kidney. Biochim. hiophys. Acta. 623, 317-328.
HAAS E.. LAMFROMH. & GOLDBLATTH. (1954) A simple
451
method for the extraction and partial purification of renin. Archs Biochem. Bivph,vs. 48, 256-260. LE BRIE S. J. & BO~LCS~~EVY B. D. (1979) The effect of furosemide on renal function and renin in water snakes. Camp. Biochem. Physiol, 63C. 223-228. MUNDAYK. A. & YORKB. G. (1976) The effect of angiotensin on intestinal fluid transport. In f~testjna~ Jon ~ra~sporr (Edited by ROSIN~GNJ. W. L.), pp. 280-285. M.T.P. Press, U.K. NAKAJJMAT., NAKAYAMA T. & SOKABE H. (1971) Examination of angiotensin-like substances from renal and extrarenal sources in m~malian and non mammalian species. Gen. Comp. Endorr. 17, 458-466. NOLLYH. L. & FASCIOLOJ. C. (1972) The renin angiotensin system through the phyjogenet~c scale. Camp. Biochcm. P~~~s~al.41 A, 249-254. NOTHSTINE S. A., DAVIS J. 0. & DE Roes R. M. (1971) Kidney extracts and ACTH on adrenal steroid secretion in a turtle and a crocodilian. Am. J. Physiol. 221, 726-732.
PEACHM. J. (1977) Renin-angiotensin system: biochemistry and mechanisms of action. Physiol. Rer. 57, 331-370. SEYAMAY.. ISHIKAWAM. & YA~~HITA S. (1979) Reninlike activity in the plasma and kidney of a snake, EIaphe yuadricirgata.
Chem. pharm. Bull. 21, 2008-2010.
SOKABEH., OGAWAM.. OCURI M. & NISHIMURA H. (1969) Evolution of the juxtaglomerular apparatus in the vertebrate kidneys. Tex. Rep’I).Biol. Med. 27, 867-885. SOKABE H. & ~CAWA M.-(1974) Comparative studies of the il!xta~lomcrular anoaratus. Int. Re(: CM. 37, 27 l-327. Ti&_&A. A. (1977) comparative physidiogy of the reninangiotensin system. Fedn Proc. Fedn Am. Sacs. exp. Biof. 36;177&178b. UVA B.. VALLARJNOM.. MANDICHA. & ISOLAG. (1981) Plasma aidosterone levels in the female tortoise Tesruda hermanni Gmelin in different experimental conditions. Gm. Camp. Endacr. (in press). VALLARINO M., UVA B., DEPLANOS., ISOLAG. & MANDIW A, (1979) Attivita’renino simile in uteri a diverso rapporto materno-fetale. Boll. Zoo/. Suppl. 46, 272-273. VECSEIP.. HACKENTHAL E. & GANTEN0. (1978) The reninangiotensin-aldosterone system. K lin. Wochenschr. (Sap@ I) 56. 5-2
1.
27 July 1981)
Abstract-l. Renal renin activity, Na and K plasma urinary levels were studied in Testudu hermunni after salted diet, saline solution administration and furosemide injection. 2. Salt loading depressed renal renin activity whereas injection of the diuretic resulted in sodium loss and enhanced renal renin activity. 3. Results lead one to conclude the presence of a renin-angiotensin system with a direct action upon osmoregulation in these terrestrial chelonians.
INTRODUCTION Evidence for the presence of an angiotensin forming enzyme have been shown in most vertebrates (for a review see Sokabe & Ogawa, 1974). More recently immunoassayable renin activity was demonstrated also in the kidney of the elasmobranch Torpedo marmoraru (Vallarino et al., 1979). Therefore this enzymatic activity is probably lacking only in cyclostomes. The possible physioIogicaI roles of a renin angiotensin system (RAS) have been stated only in mammals (for reviews see Munday & York, 1976; Peach, 1977; Vecsei et al., 1978). including short-term blood pressure regulation and stimulation of aldosterone secretion with effects on glomerular filtration rate and salt water balance. In non-mammalian vertebrates the responses to the production of angjotensin or angiotensin-like substances remain uncertain as far as an effect on osmoregulation might be concerned (Taylor, 1977) and further comparative studies are needed. In reptiles-the class of vertebrates to which the present paper is particularly devoted---the “‘juxtaglomerular apparatus” is only represented by the renin forming juxtaglomerular cells, Iocated in the afferent arterioles close to the glomeruli; a macula densa and an extraglomerular mesangium (the two other components of the juxtaglomerular apparatus in mammals) are absent (Sokabe et al., 1969; Capeili et al., 1970). The presence of a renin-like activity of some reptilian species was demonstrated by Nolly & Fasciolo (1972). A renin-like activity was found in the plasma and in the kidney of a non-poisonous snake, Eluphe quudritkpta, and lower values were determined during the period of hibernation (Seyama et al., 1979). Studies have been carried out on possible chemical differences of angiotensin-like substances in various vertebrates, including only a reptile, the snake E~uph~ c~j~~co~~a (~akajima et af., 1971). However, to our knowledge the evidence for the physiological roles of the RAS in reptiles are limited to a few species. Infusion of homologous kidney extract resulted in a rise of blood pressure and corticosterone secretion in Pwudemys sueannirnsis, but no steroidoge-
nit activity was demonstrated in C&nun sclerops in the same experimentai conditions (Nothstine et al., 1971f. More recently Le Brie & Boeicskevy (1979) demonstrated a significant rise of renal renin content in the water snake Narrix ruxispilora after administration of a diuretic (furosemide) without changes in glomerular filtration rate or plasma electrolytes concentrations. The purpose of the present work was to contribute to this debate with some observations on a terrestrial chelonian Testudo hermanni Gmeiin which appear to depend primarily upon its urinary apparatus for osmotic and ionic regulation, and experiments were undertaken (1) to compare the effects of sodium loading and administration of a diuretic on renal renin activity and (2) to follow in the same conditions sodium and potassium plasmatic and urinary levels as an index of osmoregulatory processes. MATERIALSAND METHODS Tesrudo hrrmanni Gmelin (adult females) specimens {weight ranging from I to 1.5 kg) were acclimatized for several months in the open in a terrarium with a 6Ocm sand layer to allow voluntary burial. Animals were fed with a uniform lettuce diet and water was given ud lihifum. EXperiments were carried out in July (ambient temperature ranging between 3O’C at daylight and I5 C at night) on animals distributed in the following groups: Group I, controls, fed as above described; Group 2-4. animals submitted to a salt diet (lettuce with a 3*/, NaCI) and supplied with salted water (NaCI 14;) for 5, 30 and 50 days respectively; Group 5, saline solutions (1 M NaCI 10ml) were injected intraperitoneally for 3 days. The intervention was performed through a hole drilled ventrally (at the midline between hyo- and hypoplastron plates) and sealed by a cork lubricated with antibiotic ointment; Group 6. a diuretic (Furosemide, Lasix Hoechts) was injected intraperitoneally for 2 days (1Omg~k~day and 2Om~kg/day respectively) following the above described procedure, and in the same way distilled water was supplied after the second injection (10 ml/kg). Urine collections were performed following the procedure described by Bradshaw er u/(1972) for lizards. Animals were housed in cages of appropriate volume. and
449
BIAX( ~MAKIA UVA and MACKO VALLAKIW
450
Table I. Renal renln activity and electrolytes osmotic conditions
Treatments Controls Diet 3”,, NaCl for 5 days 30 days 50 days I M NaCl injections Furosemide injections
Na (mequiv/l) 131 + 3.0 142 146 164 I52 117
& + + * +
1.0* 1.5* 2.0* Is* 2.0*
plasma levels in Jltlkcnt
K (mequiv/l)
Renm activity (ng Agl;g:hr)
4.8 & 0.2 4.4 4.2 4.1 4.4 3.7
_t _t * + _t
0.1 t O.lt o.tt 0. It 0.2t
226 + 2.0 13 37 91 206 512
* 1.0* * 5.0* * 10.0’ +_ 4.0* i 6.0*
Values represent the mean _t SE of 5 determinations on 6 tortoises in each experimental groups. *tStatistically significant differences by Student r-test between treated and control animals (P c O.Ol;.P < 0.05 respectively).
under the wire grid replacing the floor of the upper compartment, a fibre glass layer was fitted as a filter for the urine voided into the lower box. All the animals were killed by decapitation starting from 11 a.m. Groups 5 and 6 were
killed 120 min and 90 min after the last injection respectively. Blood directly collected into lithium-heparinized tubes was centrifuged at 4°C and resulting plasma stored at - 20°C until analysis. Sodium and potassium levels in urine and plasma were determined by Emission Flame Photometry, Reference method (courtesy M. D. S. Zocchi and M. D. G. E. Scafidi, Celesia Hospital, Analyses Laboratories. Genova). Kidney extracts were prepared according to the procedure of Haas er al. (1954) with slight modification previously described (Conio et (II., 1980). Before the partial purification achieved by acidification following the initial step of the above mentioned procedure, the crude extracts were dialyzed overnight against 5.9 mM NaEDTA in order to remove catecholamines and to inhibit angiotensinase activity. For renin assay aliquots (50~1) of kidney extracts were incubated with 0.5 nmol porcine anglotensinogen (Sigma) in 200 111of 0.1 M Na phosphate buffer pH 6.5. Angiotensinase and convertases inhibitors (4 mM 2-3 dimercaptopropan-l-01 and 3.2 mM hydroxyquinoline sulfate in 100 /tl of the same phosphate buffer) were added. At the end of the incubation (1 hr at 37’C) 0.1 M Tris-HCI buffer pH 7.4 was added and the reaction was stopped by immersion in boiling water. For every sample blanks were prepared in the same way except for the incubation temperature (WC). On aliquots of the supernatants obtained by centrifugation the rate of angiotensin I formation was estimated by a radioimmunoassay method (CEA-IRE SORIN commercial
Kits). RESULTS AND DISCUSSION Sodium loading obtained by a salted diet (Groups 2,4 as described in the methods) or by an intraperitoneal injection of NaCl solution (Group 5) resulted in a rise in Na, in a slightly diminished K in plasma and in a depression of renal renin activity (see Table 1). Nevertheless between the two experimental conditions some differences have been observed. In control animals the urinary emission. although varying in a wide range, may be estimated 40ml per day as mean value; the urinary sodium content is very low, under the limits of the method’s sensitivity. However, it must be remembered that in most reptiles the cloaca or the urinary bladder exerts an activity on sodium retention normally under prolactin control
(Brewer & Ensor. 1980). During the salted diet the urinary emission dropped near to zero and no control of sodium content was available. This is in agreement with that described by several authors for saline loaded reptiles (see Bentley, 1971). Urine was collected instead even if in small amount after intraperitoneal injection of saline solution loaded with water and a clear rise in sodium urinary level was observed. This reflects in the first experiments a strong water retention and in the second one the tendency to eliminate the electrolytes excess, in both experiments this corresponds to a lowered plasma renin activity. However, it seems interesting that the reduction in plasma renin activity after administration of a saline solution even if significant did not reach the extent observed after salted diet, in which a more severe salt loading condition was stated. without a contemporary supply of water. The firm correlation between osmotic stress and renin activity is confirmed by the results obtained after administration of a diuretic. While Na and K plasma levels diminished, kidney renin activity showed a clear rise (Table 1). In the same experiment emission of urine was nearly doubled and the total amount of its saline content was very high (Table 2). The rise of sodium in urine has in this case a different explanation in comparison with the effect obtained by saline solution injection. In this last case there is likely to be a close relation with the mechanism of action of the furosemide, involving proximal and distal tubular activity as discussed by Le Brie & Boelcskevy (1979). Electrolytes plasma and urinary levels in the same conditions show a different behaviour in comparison with that obtained by the above menTable
2. Na and K urinary
Na (mequiv:l)
Treatments Controls I M NaCl injections Furosemide injections Values mals).
expressed
levels after osmotic
K (mequlv;‘l)
Trace 181.5 + 2.0* 79.5 +_ 1.4* as in Table
1 (mean
stress
43.5 + I.2 36.5 & 0.9* 18.3 + 2.5* +_ SE of 6 ani-
For volumes of voided urine see in the text (under “Results and Discussion”). * Significant differences
vs control
animals
(P < 0.01).
Renin-angiotensin tioned
authors.
In the herbivorous
system and osmorf :gulation in resruda hermunni Gmelin species
here ob-
served ions’ regulation may be quite different from that present in carnivorous ones, as in the Natrix. At the same time the potassium plasma level decreased and the contemporary decrease of urinary potassium might be explained by a balance between potassium loss and potassium reabsorption in cloaca. it is, however, undoubtable that after Furosemide injection sodium loss resulted in a rise of kidney renin activity. Circulating aldosterone in this species has been
found recently and decrease or rise in aldosterone plasma levels were stated in very simiiar experimenta conditions involving a sodium load or a sodium loss respectively (Uva et al., 1981). All these data led us to conclude the presence in these reptiles of a reninangiotensin-aldosterone system which plays an important physiological role exerting a powerful action in the regulation of salt water balance.
REFERENCES BENTLEY Q. J.
(1971) Endocrines and Osmoregulution, p. 150. Springer-Verlag, Berlin. BREWER K. J. 6% ENSOR D. M. (1980) Hormonal control of osmoregulation in the chelonia. 11The effect of prolactin and corticosterone on Testuda graeca. Gen. camp. Endacr. 42, 3 I O-3 14.
BRADSHAW S. D., SHOEMAKER V. H. & NAGK K. A. (1972) The role of adrenal corticosteroids in the regulation of kidney function in the desert lizard Dipsasuutw dursalis. Camp. Biochrm. Physioi, 43A, 621-635. CAPELLIJ. P.. WESSONL. G. & APONTE G. E. (1970) A phylogenetic study of the renin-angiotensin system. Am. J. Physiol. 218, 1171-I178. CCINIOG., GHIANI P., QATRONE E., TREFJLETTI V., UVA B. & VALLARINOM. (1980) Isolation and characterization of multiple forms of renin from bull kidney. Biochim. hiophys. Acta. 623, 317-328.
HAAS E.. LAMFROMH. & GOLDBLATTH. (1954) A simple
451
method for the extraction and partial purification of renin. Archs Biochem. Bivph,vs. 48, 256-260. LE BRIE S. J. & BO~LCS~~EVY B. D. (1979) The effect of furosemide on renal function and renin in water snakes. Camp. Biochem. Physiol, 63C. 223-228. MUNDAYK. A. & YORKB. G. (1976) The effect of angiotensin on intestinal fluid transport. In f~testjna~ Jon ~ra~sporr (Edited by ROSIN~GNJ. W. L.), pp. 280-285. M.T.P. Press, U.K. NAKAJJMAT., NAKAYAMA T. & SOKABE H. (1971) Examination of angiotensin-like substances from renal and extrarenal sources in m~malian and non mammalian species. Gen. Comp. Endorr. 17, 458-466. NOLLYH. L. & FASCIOLOJ. C. (1972) The renin angiotensin system through the phyjogenet~c scale. Camp. Biochcm. P~~~s~al.41 A, 249-254. NOTHSTINE S. A., DAVIS J. 0. & DE Roes R. M. (1971) Kidney extracts and ACTH on adrenal steroid secretion in a turtle and a crocodilian. Am. J. Physiol. 221, 726-732.
PEACHM. J. (1977) Renin-angiotensin system: biochemistry and mechanisms of action. Physiol. Rer. 57, 331-370. SEYAMAY.. ISHIKAWAM. & YA~~HITA S. (1979) Reninlike activity in the plasma and kidney of a snake, EIaphe yuadricirgata.
Chem. pharm. Bull. 21, 2008-2010.
SOKABEH., OGAWAM.. OCURI M. & NISHIMURA H. (1969) Evolution of the juxtaglomerular apparatus in the vertebrate kidneys. Tex. Rep’I).Biol. Med. 27, 867-885. SOKABE H. & ~CAWA M.-(1974) Comparative studies of the il!xta~lomcrular anoaratus. Int. Re(: CM. 37, 27 l-327. Ti&_&A. A. (1977) comparative physidiogy of the reninangiotensin system. Fedn Proc. Fedn Am. Sacs. exp. Biof. 36;177&178b. UVA B.. VALLARJNOM.. MANDICHA. & ISOLAG. (1981) Plasma aidosterone levels in the female tortoise Tesruda hermanni Gmelin in different experimental conditions. Gm. Camp. Endacr. (in press). VALLARINO M., UVA B., DEPLANOS., ISOLAG. & MANDIW A, (1979) Attivita’renino simile in uteri a diverso rapporto materno-fetale. Boll. Zoo/. Suppl. 46, 272-273. VECSEIP.. HACKENTHAL E. & GANTEN0. (1978) The reninangiotensin-aldosterone system. K lin. Wochenschr. (Sap@ I) 56. 5-2
1.