$5.00+ 0.00 0300-9629/92 0 1992PergamonPressplc
Camp.Biochem.Physiot.Vol. 102A,No. 1, pp. 27-31,1992 Printed in Great Britain
ACTIVE AND INACTIVE RENIN OF THE SNAKE BOTHROPS
IN THE PLASMA JARARACA
R. K. GERVITZ,*C. M. W. BRANDIt, S. H. C. MONTEIROand Z. P. PICARELLI Servipo de Farmacologia, Instituto Butantan, C.P. 65, 05504, S&oPaulo, SP, Brazil and TDept. de Bioquimica, Escola Paulista de Medicina, rua 3 de maio, 100. 04044, SBo Paulo, SP, Brazil (Received 2 September 1991)
Abstract-l.
Complementing the work of Gervitz R. K., Hiraichi E., Fichman M. and Lavras A. A. C. Comp. Biochem. Physiol. 86A, 503-507 (1987), conditions have been established for measuring plasma renin activity (PRA) of the venomous snake Bothrops jararacu (Bj). 2. It corresponded to 115.9 + 11.5 ng equivalents of angiotensin II (AII) per ml of plasma (N = 13). 3. PRA did not increase when Bj plasma was submitted to acid-cryetrypsin Bitis arietans venom activation of inactive renin. 4. This may indicate either absence of inactive renin in this plasma or lack of its activation, due to the already demonstrated (Nahas L., Kamiguti A. S., Betti F., Martins I. S. S. and Rodrigues M. I., Comp. Biochem. Physiol. 69A, 739-743, 1981; Prezoto B. C., Hiraichi E., Abdalla F. M. F. and Picarelli Z. P., Comp. Biochem. Physiol. 99C, 135-139, 1991) absence of factor XII.
INTRODUCTION
which is in charge of collecting from nature and classifying the specimens. They were housed under controlled environmental conditions for at least 15 days before use (Breno et al., 1990).
Previous work from this laboratory (Lavras er al., 1980; Gervitz et al., 1987), has demonstrated, in the plasma and kidney of the venomous snake Bothrops jururacu (Bj), components of the renin-angiotensin system similar to those found in mammals, such as a dipeptidylhydrolase (angiotensin I-converting enzyme, ACE), plasma renin substrate, renal renin and angiotensinases. However, the detection of active and/or inactive renin in Bj plasma was still lacking. Whereas active renin was detected in human and in several animal plasmas, inactive renin could only be extensively studied in human plasma. The accurate quantification of inactive renin in animal plasma has proved to be very difficult, partly because of the presence of highly active protease inhibitors. Since plasma kallikrein, one of the proteases supposed to be responsible for the activation of inactive renin (Derkx et al., 1979; Yokosawa et al., 1979), has not been found in Bj plasma (Lavras et al., 1979; Prezoto et al., 1991) it was thought worthwhile to search for renin and inactive renin in this plasma and, if detected, to investigate the possible activation mechanism of prorenin. Therefore, conditions have been established for detecting and measuring the endogenous active renin present in Bj plasma followed by attempts to activate and then measure the inactive renin content. This work reports the results obtained. MATERIALSAND
Plasma Blood samples were obtained by decapitation of the snakes, heparin (10 U/ml blood) being used as anticoagulant. The blood was centrifuged for 30 min at 10°C and 10,000 rpm and the plasma samples obtained were pooled and stored at -20°C. Bj renal renin extract The extract was obtained by the method described by Gervitz et al. (1987). Active renin assay The method for the measurement of plasma renin activity (PRA) was based on that described by Miura (1969). The results were expressed as ng equivalents of angiotensin II (AII) per ml of plasma. For-Bj plasma; the following conditions were established: 1.0 ml of Bi nlasma at uH 5.5 was incubated at 37°C. for 6 hr with 7.5 mM EDTA (Merck), 5 mM PMSF (Sigma)‘and one drop of 5% neomycin sulfate (Sigma). The final volume was adjusted to 1.2 ml with 1.0 M acetate buffer, pH 5.5. The additional steps were similar to those described by Miura (1969), the angiotensins extracted from the incubation medium being assayed on the guinea-pig isolated ileum, on the nephrectomized rat blood pressure and on Bj carotidean blood pressure (Abdalla et al., 1989). In order to confirm that the product assayed was actually a mixture of AI and AH, 3.75 x 10m6M [Sari-Let?] AI1 (synthetized by the Dept. of Biophysics, Escola Paulista de Medicina, Sgo Paulo) was employed as antagonist of AI1 (Boileau et al., 1972) and captopril (0.5 mg/kg) as inhibitor of ACE (Vollmer et al., 1978). Whenever necessary, lOA M pepstatin (Sigma) was used to inhibit renin in the system under study (Guyene et al., 1976).
METHODS
Animals Snakes (Bothropsjuraracu, Serpentes, Crotalinae) of both sexes (2Ot-600g) were used throughout this study. They came from the Se#o de Herpetologia, Institute Butantan, *To whom correspondence
Measurement of inactive renin Inactive renin is usually measured by in vitro activation followed by assay of the resulting total renin activity by the
should be addressed. 27
28
R. K. GEFWTZ et al.
method described above. The difference in PRA before and after activation is taken as a measure of inactive renin. The in vitro activation of inactive renin was performed by one of the four known methods: (a) acid activation (Atlas et al., 1982FBj plasma was submitted to dialysis for 24 hr. at 4°C. against 0.005 M glycine/HCl buffe;, pH 3.3, containing 0.1M NaCl and 3 mM EDTA. followed bv a second 24 hr dialvsis. at 4°C. against 0.1 M’sodium phosphate, pH 7.4, containing 0.05 G NaCl and 3 mM EDTA. (b) Cryoactivation (Sealey et al., 1976FBj plasma was cryoactivated by storage at -4”C, pH 7.4 for 4 days. (c) Activation by trypsin (Rapelli et al., 1981)--Bj plasma was incubated with tryspin (type-III, from bovine pancreas, Sigma) (8 mg/ml plasma) at 4”C, pH 7.4 for 10 min. SBTI (type-I, Sigma) (16 mg/ml plasma) was then added to interrupt the activation. (d) Activation by Bitis arietans venom (Morris and Taylor, 1987)--l .Oml of Bj plasma plus 2.5 mg of lyophilyzed Bitis arietans venom (kindly supplied by Dr Anne-Marie de Saint-Michel, from Latoxan, France) were incubated at room temperature. At different time intervals, from zero time until 3 hr, aliquots from the incubation mixture were taken and the activation interrupted by addition of 50~1 of 0.2 M EDTA and 50 ~1 of 0.1 M PMSF to inhibit, respectively, the venom metalloprotease and the plasma serinoproteases. Inhibition of trypsin caseinolytic activity by Bj plasma
The evaluation was done by incubating different concentrations of trypsin in Bj plasma, at 4°C and pH 7.4 for 10 min, after which the caseinolytic activity of the enzyme was determined (Kunitz, 1946; Mandelbaum et al., 1982). The results were expressed as percentage of the activity of the enzyme in 0.1 M phosphate buffer, pH 7.4.
RESULTS
PRA
assay
In order to measure PRA in Bj plasma using the method of Miura (1969), initially some experiments for the control of the methodological conditions were undertaken. Firstly, it was shown’that the amount of angiotensin produced and, consequently, the PRA was proportional to the volume of plasma used in the incubation mixture (Table 1). The volume of Bj plasma in the incubates was then fixed as 1.0 ml. In experiments made with two different pools of Bj plasma it was observed that, up to 6 hr of incubation, PRA was proportional to the time of incubation (Fig. l), the yield of angiotensin being already enough to cause effects between 20 and 80% of the maximal response of the guinea-pig ileum to AIL Thus, 6 hr was used as the incubation time. As can be seen in Fig. 2, the product(s) extracted from the incubation mixture was (were) angiotensin(s) since its (their) effect(s) on the guinea-pig ileum was Table I. Relationship between renin activity and volume of Bothrops jararaca plasma present in the incubates (6 hr, 37°C. pH 5.5)
Volumeof plasma (ml) 0.5 1.0 2.0 Values
are means f
Renin activity (ng equiv. AH/ml) 27.8 5 3.5 71.0 * 8.9 158.5 -+ 14.3
SEM (N = 3).
6
3
16
Incubation timr CkwrSl
Fig. 1. Influence of the incubation time on Bothrops jararaca plasma renin activity (PRA>-l.Oml plasma (Pool II O-0, Pool III m---m) incubated for 3,6 or 16 hr at 37”C, pH 5.5 with 7.5 mM EDTA, 5 mM PMSF and one drop of 5% neomycin sulfate: final volume (1.2 ml) adjusted with 1.0 M acetate buffer pH 5.5. The substance(s) released was (were) purified in Dowex 50 W-X4 (I-I+) columns and assayed on the guinea-pig isolated ileum using angiotensin II (AU) as standard. PRA was proportional to the time of incubation, tending to attain a plateau after 6 hr of incubation.
(were) antagonized by Sar. This was confirmed in experiments recording its (their) effect(s) on rat or Bj carotidean blood pressure before and after administration of the antagonist or captopril. Figure 3 shows that the hypertension produced in the rat or Bj by the extracted product(s) decreased after captopril. Plasma renin was the agent responsible for the release of this(these) substance(s), since no contraction was observed on the guinea-pig ileum when the 1ommI
/1
PE
All
Doss
40 20.25 .50
All
All PE PE Sar
50
40
min
15
95
II8
20
25
PE
30 35
H mm #II, loo.
90. 80.
lo. All
All
Dose
50
25 .20 Jo
PE
min
25
30 35
40
Sar
PEAll .2050
45
50 55
Fig. 2. Effect of [Sari-Leu*]Angiotensin II (Sar) upon either the contraction produced on the isolated guinea-pig ileum (above) or the hypertension produced in the anesthetized nephrectomized rat (below) by the substance(s) released in Bothrops jararaca (Bj) plasma, purified under the conditions described in Fig. 1. In the rat, Sar was administered by infusion (1.5 pg/kg/min) and in the guinea-pig ileum, its final concentration was 3.75 x 10m5M. Doses of AI1 in ng and of PE, the product eluted from the Dowex column, in ml eluate (1 ml of eluate corresponds to 0.33 ml Bj plasma). Sar antagonized the effects of both AI1 and PE.
Renin activity in snake plasma
29
Table 2. Plasma renin activity (PRA) of Bothrops jararaco, before and after inactive renin activation by acid, cold trypsin or Bifis orietam venom. Values represent means + SEM
Hg mm
PE
Al
30
4
50
65
70
75 80
All
All
Al
PE
Capt.
Dose
50
25
50
.4
min
45
50
55
60
All 25
Fig. 3. Effect of captopril (Capt.) upon the hypertension produced in the anesthetized nephrectomized rat by the substance(s) released in Bothrops juraruca plasma incubated and purified under the same conditions as described in Fig. 1. Doses of Al and AI1 in ng, of the product extracted (PE) in ml of eluate (1 ml eluate corresponds to 0.33 ml Bj plasma) and of Capt. in mg/kg. After Capt., the effects of both AI and PE diminished, but that of AI1 remained unaltered.
incubation was carried out in the presence of lo-’ M pepstatin (Fig. 4). Under the experimental conditions used, Bj plasma showed a value of PRA corresponding to 115.9 f 11.5 ng equivalents AI1 per ml of plasma (N = 13).
Activation process
PRA (ng equiv. AH/ml Bj plasma) After Before
Acid (5) Cold (5) Trypsin (4)
108.00 + 140.00 f 101.OO+ 43.25 +
Bitis arktans venom (4)
19.30 27.10 8.40 8.80*
119.90 + 117.70 + 145.00 + 54.12 *
16.90 17.40 27.70 12.90’
Number of plasma pools assayed, in parentheses. Differences between means obtained before and after prorenin activation were not significant (I-test, P < 0.05). ‘Values significantly different from those obtained by the other three activation processes (analysis of variance and Tukey’s test, P < 0.01). Table 3. Trypsin caseinolytic activity inhibition by Bothrops jararaca (Bj) plasma Trypsin concentration (mg/ml Bj plasma)
Caseinolytic activity inhibition %
2 4 8 12
70 56 25 13
Values are given as percentage of inhibition of the activity presented by the same concentration of trypsin in 0.1 M phosphate buffer, pH 7.4.
Plasma inactive renin activation When Bj plasma was submitted to each of the four activating methods, no significant increase in PRA was found (Table 2) (P < 0.05, t-test).
However, the values of PRA obtained before and after treating Bj plasma with Bitis arietans venom were shown to be lower than all the other values determined (P < 0.01, Tukey’s test). When trypsin was used as the activator, the plasma protease inhibitors did not interfere with PRA. The concentration of trypsin used (8 mg/ml plasma) was sufficient to almost surpass the inhibitory effect of Bj plasma, as shown by the results of the determination of the caseinolytic activity at different concentrations of trypsin in Bj plasma (Table 3). In order to find out if these different activation processes could be altering or destroying some
1Omm
components of the renin-angiotensin system such as plasma renin substrate or even plasma renin, Bj plasma was previously submitted to acid dialysis, trypsinization or Bitis arietans venom and further incubated with Bj renal renin extract, since the reaction of renin with renin substrate is species-specific (Nolly and Fasciolo, 1973; Taylor, 1977). Whereas more AI1 was released when this experiment was made with acid-activated Bj plasma (Fig. S), trypsinization and treatment with Bitis arietans venom did not cause any increase
in AIL
I
All
All
T,
All
All
Tz
All
All
Doss
40
40
.25
20
lo
.50
10
20
min
60
65
70
75
90
95
loo
105
Fig. 4. Inhibition of the release of the substance(s) contracting the guinea-pig ileum by 10m5M pepstatin. T, and T2 correspond to incubations made in absence (r, ) or presence (T2) of pepstatin; incubation and purification as in Fig. 1. Doses of AI1 in ng and T, and T2 in ml of eluate (1 ml eluate corresponds to 0.33 ml of Bj plasma).
BjP BjP + R
aBjP aBjP + R
Fig. 5. Release of AI1 by incubation of Bj plasma (BjP) or acid-activated BjP (aBjP) in the absence or presence of renal renin (R). Incubation and purification as in Fig. 1. The increase in the amount of AI1 released in the presence of R indicates that there was excess plasma renin substrate in the incubation medium.
R. K. GERVITZet al.
30 DISCUSSION
Complementing the work of Gervitz et al. (1987) which demonstratred the presence of some components of the renin-angiotensin system in plasma and kidney of the venomous snake Bothrops jararaca, the present data have proved the existence of renin activity in the snake plasma. However, it was not possible to demonstrate the presence of inactive renin in this plasma, when using the in vitro activation processes of acid dialysis, cryoactivation and limited proteolysis with trypsin or Bitis arietans venom. The lack of activation of Bj plasma by acid treatment does not seem to be due to a plasma renin substrate deficiency. Contrary to the described susceptibility of human angiotensinogen to acidification (Printz et al., 1977), Bj plasma renin substrate seems to resist this process, since acid-treated Bj plasma further released angiotensin when incubated with Bj renal renin extract. Regarding c~oactivation, it is already known not to occur in plasmas lacking Hageman factor and/or kallikrein (Sealey and Atlas, 1984) as is the case with Bothrops jararaca plasma (Prezoto et al., 1991). When trypsin was used as activator, Sealey and Atlas (1984) observed that its proteolytic action must be strictly controlled since concurrent destruction of active renin may occur. On the other hand, Johannessen et al. (1990) reported the destruction of rat angiotensinogen by trypsin and Dzau et af. (1986) as well as Gallagher ef al. (1980) observed that when dog plasma was treated with trypsin, digestion of the endogenous renin substrate was sufficient to cause a decrease in dog PRA. In turtles, Cipolle et al. (1984) also did not discard the possibility of destruction of a significant portion of plasma renin substrate by trypsin. In the present experiments if trypsin did cause alterations in plasma renin or its substrate, these must be discrete, since addition of Bj renal extract to trypsin-treated Bj plasma did not increase the values of previous results, these results being also similar to those obtained with only acid- or cold-treated Bj plasma (Table 2). When Bj plasma was treated with Bitis arietuns venom, PRA values, before and after treatment, were lower than all other values obtained (P < 0.05, Tukey’s test). Actually, since the venom is a complex mixture of enzymes, this could be caused by the digestion not only of the plasma renin substrate but also of the endogenous renin. Therefore, the lack of activation of inactive renin in Bj plasma actually seems to be due to the already demonstrated absence of factor XII in this plasma (Nahas et al., 1981; Prezoto et al., 1991) and reinforces the hypothesis that kallikrein, a factor XII-dependent enzyme, is responsible for the activation of inactive renin (Derkx et al., 1979; Yokosawa et al., 1979). As opposed to the reports stating that in humans 90% of renin circulates as inactive renin or prorenin (Cooper et al., 1977; Sealey et al., 1980), the present data could also indicate that there is no circulating inactive renin in Bj. It is known that the kidney is the major source of circulating renin, stored in juxtaglomerular granules. Recently, Ikeda et al.
(1991) using subcellular fractions of dog kidney cortex, located a renin processing enzyme in these granules. Therefore, it is possible that inactive renin synthetized in the kidney is processed to the active form in the juxtaglomerular cells before reaching the circulation. To confirm such a hypothesis in Bothrops jararaca, it will be necessary to look for inactive renin and its processing enzyme in the kidney of this snake. Ae&n#wiedge~~rs-The authors thank Mr Wilson de Barros D’Avila for technical assistance, Miss Wanda Regina Carrella da Silva for the typing and Dr Hanna A. Rothschild for revising the English version. Part of the guinea-pig ileum assays were made by F. M. F. Abdalla, recipient of a CNPq Scholarship (Proc. 824102/88-Q
REFERENCES
Abdalla F. M. F., Hiraichi E., Picarelli 2. P. and Prezoto 3. C. (1989) Kallikrein-kinin system in the plasma of the snake Bothrops jararaca. Br. J. Pharmac. 98, 252-258. Atlas S. A., Hesson T. E., Sealey J. E. and Laragh J. H. (1982) Reversible acid-activation of inactive renin: evidence favouring a unimolecular reaction. C&z. Sci. 63, 167s-1’70s. Boileau J. C., Crexells C. and Biron P. (1972) Dissociation constant of angiotensin II as determined by the use of an irreversible specific inhibitor (I-Sar-8-Leu-ATII). Rev. Can. Biol. 31, 73-77.
Breno M. C., Yamanouye N., Prezoto B. C., Lazari M. F. M., Toffoletto 0. and Picarelli Z. P. (1990) Maintenance of the snake Bothropsjararaca @Vied; 182h) in captivity. The Snake 22, 126-130.
Cipolle M. D. and Zehr J. E. (1984) Characterization of the renin-an~otensin system in the turtle Pseudemys scripta. Am. J. Physiol. 247, RlS-R23. Cooper R. M., Murray Y. G. E. and Osmond D. H. (1977) Trypsin induced activation of renin precursor in plasma of normal and anephric man. Circ. Rex 40 (Suppl. I), 1171-1179. Derkx F. H. M., Bouma B. N., Tan-Tjiong H. L. and Schalekamp M. A. D. H. (1979) Activators of inactive renin fprorenin) in human plasma: their connection with kinin formation, coagulation and fibrinolysis. Clin. Sci. 57, 89s-92s.
Dzau V. J., Wilcox C. S., Sands K. and Dunckel P. (1986) Dog inactive renin: biochemical characte~~tion and secretion into renal plasma and lymph. Am. J. Physiol. 250, E5.5-E61. Gallagher J. F., Laragh J. H., Atlas S. A. and Sealey J. E. (1980) Effect of trypsin or acid treatment on dog plasma renin activity measurement. Endocrinology 107, 147-154. Gervitz R. K., Hiraichi E., Fichman M. and Lavras A. A. C. (1987) The Knin-an~otensin system in the snake Bothropsjararaca (Serpentes, Crotalinae). Camp. Biochem. Physiol. 86A, 503m--507.
Guyene T. T., Devaux C., Menard J. and Corvol P. (1976) I~ibition of human plasma renin activity by pepitatin. J. Gin. Endow. Metab. 43. 1301-1306.
Ikeda M., Oda K., Tsuji E., Noda K., Sasaguri M., Ideishi M. and Arakawa K. (1991) Human renin activation by protease from the renin granule fraction of the dog kidney cortex. Life Science 48, 9-17. Johannessen A., Nielsen A. H. and Poulsen K. (1990). Measurement of inactive renin in rat plasma: effect of n~hr~omy and sialoaden~tomy bn the plasma concentration. J. Hypertension 8, 345-349. Kunitz M. (1946) Cryiialline soybean trypsin inhibitor. II.
tieneral propertles J. gen. Physiol. 30, 306-310.
Renin activity in snake plasma Lavras A. A. C., Fichman M., Hiraichi E., Boucault M. A., Tobo T., Schmuziger P., Nahas L. and Picarelli Z. P. (1979) Deficiency of kallikrein-kinin system and presence of potent kininase activity in the plasma of Bothrops jararuca (Serpentes, Crotalinae). C@ncia e Cultura 31, 168-174. Lavras A. A. C., Fichman M., Hiraichi E., Tobo T. and Boucault M. A. (1980) The kininases of Borhrops jararaca. Acla Physiol. Lutinoam. 30, 267-274.
Mandelbaum F. R., Reich1 A. P. and Assakura M. T. (1982) Isolation and characterization of a proteolytic enzyme from the venom of the snake Bothrops jararaca (jararaca). Toxicon 20, 955-972.
Morris B. J. and Taylor J. E. (1987) Activation by puff adder venom of inactive renin in normal and hypertensive rat plasma. C/in. exp. Pharmac. Physiol. 14, 23-41.
Miura Y. (1969) Improved method for measurement of renin activity in human plasma. Tohoku J. exp. Med. 98, 125-134.
Nahas L., Kamiguti A. S., Betti F., Martins I. S. S. and Rodrigues M. I. (1981) Blood coagulation mechanism in the snakes Waglerophis merremii and Bothrops jararaca. Comp. Biochem. Physiol. 69A, 739-743.
Nolly H. L. and Fasciolo J. C. (1973) The specificity of the renin-angiotensinogen reaction through the phylogenetic scale. Comp. Biochem. Physiol. 44A, 6399645. Prezoto B. C., Hiraichi E., Abdalla F. M. F. and Picarelli Z. P. (1991) Activation of the kallikrein-kinin system
31
in plasma of some Brazilian snakes. Comp. Biochem. Physiol. 99C, 135-139.
Printz M. P., Printz J. M. and Dworschack R. T. (1977) Human angiotensinogen. J. biol. Chem. 252, 1654-1662. Rapelli A., Glorioso N., Madeddu P., Dessi-Fulgheri P., Fois G., Lenguini L., Meloni F. and Palenno M. (1981) Trypsin activation of inactive renin in human plasma. An assessment of some methodological aspects. C/in. exp. Hypertension 3, 299-3 18.
Sealey J. E. and Atlas S. A. (1984) Inactive renin: speculations concerning its secretion and activation. J. Hypertension 2 (suppl. 1), 115-123. Sealey J. E., Atlas S. A. and Laragh J. H. (1980) Prorenin and other large molecular weight forms of renin. Endocr. Rev. 1, 365-391. Sealey J. E., Moon C., Laragh J. H. and Alderman M. (1976) Plasma prorenin: cryoactivation and relationship to renin substrate in normal subjects. Am. J. Med. 61, 73 l-738. Taylor A. A. (1977) Comparative physiology of the renin-angiotensin system. Fed. Proc. 35, 1776-1780. Vollmer R. R., Boccagno J. H., Harris D. N. and Mm-thy V. S. (1978) Hypertension induced by inhibition of angiotensin converting enzyme in pentobarbital anesthetized dogs. Eur. J. Pharmac. 51, 39-45. Yokosawa N., Takahashi N., Inagami T. and Page L. D. (1979) Isolation of completely inactive plasma prorenin and its activation by kallikreins. Biochim. bioohvs. a . Acta 569, 211-219. _
Camp.Biochem.Physiot.Vol. 102A,No. 1, pp. 27-31,1992 Printed in Great Britain
ACTIVE AND INACTIVE RENIN OF THE SNAKE BOTHROPS
IN THE PLASMA JARARACA
R. K. GERVITZ,*C. M. W. BRANDIt, S. H. C. MONTEIROand Z. P. PICARELLI Servipo de Farmacologia, Instituto Butantan, C.P. 65, 05504, S&oPaulo, SP, Brazil and TDept. de Bioquimica, Escola Paulista de Medicina, rua 3 de maio, 100. 04044, SBo Paulo, SP, Brazil (Received 2 September 1991)
Abstract-l.
Complementing the work of Gervitz R. K., Hiraichi E., Fichman M. and Lavras A. A. C. Comp. Biochem. Physiol. 86A, 503-507 (1987), conditions have been established for measuring plasma renin activity (PRA) of the venomous snake Bothrops jararacu (Bj). 2. It corresponded to 115.9 + 11.5 ng equivalents of angiotensin II (AII) per ml of plasma (N = 13). 3. PRA did not increase when Bj plasma was submitted to acid-cryetrypsin Bitis arietans venom activation of inactive renin. 4. This may indicate either absence of inactive renin in this plasma or lack of its activation, due to the already demonstrated (Nahas L., Kamiguti A. S., Betti F., Martins I. S. S. and Rodrigues M. I., Comp. Biochem. Physiol. 69A, 739-743, 1981; Prezoto B. C., Hiraichi E., Abdalla F. M. F. and Picarelli Z. P., Comp. Biochem. Physiol. 99C, 135-139, 1991) absence of factor XII.
INTRODUCTION
which is in charge of collecting from nature and classifying the specimens. They were housed under controlled environmental conditions for at least 15 days before use (Breno et al., 1990).
Previous work from this laboratory (Lavras er al., 1980; Gervitz et al., 1987), has demonstrated, in the plasma and kidney of the venomous snake Bothrops jururacu (Bj), components of the renin-angiotensin system similar to those found in mammals, such as a dipeptidylhydrolase (angiotensin I-converting enzyme, ACE), plasma renin substrate, renal renin and angiotensinases. However, the detection of active and/or inactive renin in Bj plasma was still lacking. Whereas active renin was detected in human and in several animal plasmas, inactive renin could only be extensively studied in human plasma. The accurate quantification of inactive renin in animal plasma has proved to be very difficult, partly because of the presence of highly active protease inhibitors. Since plasma kallikrein, one of the proteases supposed to be responsible for the activation of inactive renin (Derkx et al., 1979; Yokosawa et al., 1979), has not been found in Bj plasma (Lavras et al., 1979; Prezoto et al., 1991) it was thought worthwhile to search for renin and inactive renin in this plasma and, if detected, to investigate the possible activation mechanism of prorenin. Therefore, conditions have been established for detecting and measuring the endogenous active renin present in Bj plasma followed by attempts to activate and then measure the inactive renin content. This work reports the results obtained. MATERIALSAND
Plasma Blood samples were obtained by decapitation of the snakes, heparin (10 U/ml blood) being used as anticoagulant. The blood was centrifuged for 30 min at 10°C and 10,000 rpm and the plasma samples obtained were pooled and stored at -20°C. Bj renal renin extract The extract was obtained by the method described by Gervitz et al. (1987). Active renin assay The method for the measurement of plasma renin activity (PRA) was based on that described by Miura (1969). The results were expressed as ng equivalents of angiotensin II (AII) per ml of plasma. For-Bj plasma; the following conditions were established: 1.0 ml of Bi nlasma at uH 5.5 was incubated at 37°C. for 6 hr with 7.5 mM EDTA (Merck), 5 mM PMSF (Sigma)‘and one drop of 5% neomycin sulfate (Sigma). The final volume was adjusted to 1.2 ml with 1.0 M acetate buffer, pH 5.5. The additional steps were similar to those described by Miura (1969), the angiotensins extracted from the incubation medium being assayed on the guinea-pig isolated ileum, on the nephrectomized rat blood pressure and on Bj carotidean blood pressure (Abdalla et al., 1989). In order to confirm that the product assayed was actually a mixture of AI and AH, 3.75 x 10m6M [Sari-Let?] AI1 (synthetized by the Dept. of Biophysics, Escola Paulista de Medicina, Sgo Paulo) was employed as antagonist of AI1 (Boileau et al., 1972) and captopril (0.5 mg/kg) as inhibitor of ACE (Vollmer et al., 1978). Whenever necessary, lOA M pepstatin (Sigma) was used to inhibit renin in the system under study (Guyene et al., 1976).
METHODS
Animals Snakes (Bothropsjuraracu, Serpentes, Crotalinae) of both sexes (2Ot-600g) were used throughout this study. They came from the Se#o de Herpetologia, Institute Butantan, *To whom correspondence
Measurement of inactive renin Inactive renin is usually measured by in vitro activation followed by assay of the resulting total renin activity by the
should be addressed. 27
28
R. K. GEFWTZ et al.
method described above. The difference in PRA before and after activation is taken as a measure of inactive renin. The in vitro activation of inactive renin was performed by one of the four known methods: (a) acid activation (Atlas et al., 1982FBj plasma was submitted to dialysis for 24 hr. at 4°C. against 0.005 M glycine/HCl buffe;, pH 3.3, containing 0.1M NaCl and 3 mM EDTA. followed bv a second 24 hr dialvsis. at 4°C. against 0.1 M’sodium phosphate, pH 7.4, containing 0.05 G NaCl and 3 mM EDTA. (b) Cryoactivation (Sealey et al., 1976FBj plasma was cryoactivated by storage at -4”C, pH 7.4 for 4 days. (c) Activation by trypsin (Rapelli et al., 1981)--Bj plasma was incubated with tryspin (type-III, from bovine pancreas, Sigma) (8 mg/ml plasma) at 4”C, pH 7.4 for 10 min. SBTI (type-I, Sigma) (16 mg/ml plasma) was then added to interrupt the activation. (d) Activation by Bitis arietans venom (Morris and Taylor, 1987)--l .Oml of Bj plasma plus 2.5 mg of lyophilyzed Bitis arietans venom (kindly supplied by Dr Anne-Marie de Saint-Michel, from Latoxan, France) were incubated at room temperature. At different time intervals, from zero time until 3 hr, aliquots from the incubation mixture were taken and the activation interrupted by addition of 50~1 of 0.2 M EDTA and 50 ~1 of 0.1 M PMSF to inhibit, respectively, the venom metalloprotease and the plasma serinoproteases. Inhibition of trypsin caseinolytic activity by Bj plasma
The evaluation was done by incubating different concentrations of trypsin in Bj plasma, at 4°C and pH 7.4 for 10 min, after which the caseinolytic activity of the enzyme was determined (Kunitz, 1946; Mandelbaum et al., 1982). The results were expressed as percentage of the activity of the enzyme in 0.1 M phosphate buffer, pH 7.4.
RESULTS
PRA
assay
In order to measure PRA in Bj plasma using the method of Miura (1969), initially some experiments for the control of the methodological conditions were undertaken. Firstly, it was shown’that the amount of angiotensin produced and, consequently, the PRA was proportional to the volume of plasma used in the incubation mixture (Table 1). The volume of Bj plasma in the incubates was then fixed as 1.0 ml. In experiments made with two different pools of Bj plasma it was observed that, up to 6 hr of incubation, PRA was proportional to the time of incubation (Fig. l), the yield of angiotensin being already enough to cause effects between 20 and 80% of the maximal response of the guinea-pig ileum to AIL Thus, 6 hr was used as the incubation time. As can be seen in Fig. 2, the product(s) extracted from the incubation mixture was (were) angiotensin(s) since its (their) effect(s) on the guinea-pig ileum was Table I. Relationship between renin activity and volume of Bothrops jararaca plasma present in the incubates (6 hr, 37°C. pH 5.5)
Volumeof plasma (ml) 0.5 1.0 2.0 Values
are means f
Renin activity (ng equiv. AH/ml) 27.8 5 3.5 71.0 * 8.9 158.5 -+ 14.3
SEM (N = 3).
6
3
16
Incubation timr CkwrSl
Fig. 1. Influence of the incubation time on Bothrops jararaca plasma renin activity (PRA>-l.Oml plasma (Pool II O-0, Pool III m---m) incubated for 3,6 or 16 hr at 37”C, pH 5.5 with 7.5 mM EDTA, 5 mM PMSF and one drop of 5% neomycin sulfate: final volume (1.2 ml) adjusted with 1.0 M acetate buffer pH 5.5. The substance(s) released was (were) purified in Dowex 50 W-X4 (I-I+) columns and assayed on the guinea-pig isolated ileum using angiotensin II (AU) as standard. PRA was proportional to the time of incubation, tending to attain a plateau after 6 hr of incubation.
(were) antagonized by Sar. This was confirmed in experiments recording its (their) effect(s) on rat or Bj carotidean blood pressure before and after administration of the antagonist or captopril. Figure 3 shows that the hypertension produced in the rat or Bj by the extracted product(s) decreased after captopril. Plasma renin was the agent responsible for the release of this(these) substance(s), since no contraction was observed on the guinea-pig ileum when the 1ommI
/1
PE
All
Doss
40 20.25 .50
All
All PE PE Sar
50
40
min
15
95
II8
20
25
PE
30 35
H mm #II, loo.
90. 80.
lo. All
All
Dose
50
25 .20 Jo
PE
min
25
30 35
40
Sar
PEAll .2050
45
50 55
Fig. 2. Effect of [Sari-Leu*]Angiotensin II (Sar) upon either the contraction produced on the isolated guinea-pig ileum (above) or the hypertension produced in the anesthetized nephrectomized rat (below) by the substance(s) released in Bothrops jararaca (Bj) plasma, purified under the conditions described in Fig. 1. In the rat, Sar was administered by infusion (1.5 pg/kg/min) and in the guinea-pig ileum, its final concentration was 3.75 x 10m5M. Doses of AI1 in ng and of PE, the product eluted from the Dowex column, in ml eluate (1 ml of eluate corresponds to 0.33 ml Bj plasma). Sar antagonized the effects of both AI1 and PE.
Renin activity in snake plasma
29
Table 2. Plasma renin activity (PRA) of Bothrops jararaco, before and after inactive renin activation by acid, cold trypsin or Bifis orietam venom. Values represent means + SEM
Hg mm
PE
Al
30
4
50
65
70
75 80
All
All
Al
PE
Capt.
Dose
50
25
50
.4
min
45
50
55
60
All 25
Fig. 3. Effect of captopril (Capt.) upon the hypertension produced in the anesthetized nephrectomized rat by the substance(s) released in Bothrops juraruca plasma incubated and purified under the same conditions as described in Fig. 1. Doses of Al and AI1 in ng, of the product extracted (PE) in ml of eluate (1 ml eluate corresponds to 0.33 ml Bj plasma) and of Capt. in mg/kg. After Capt., the effects of both AI and PE diminished, but that of AI1 remained unaltered.
incubation was carried out in the presence of lo-’ M pepstatin (Fig. 4). Under the experimental conditions used, Bj plasma showed a value of PRA corresponding to 115.9 f 11.5 ng equivalents AI1 per ml of plasma (N = 13).
Activation process
PRA (ng equiv. AH/ml Bj plasma) After Before
Acid (5) Cold (5) Trypsin (4)
108.00 + 140.00 f 101.OO+ 43.25 +
Bitis arktans venom (4)
19.30 27.10 8.40 8.80*
119.90 + 117.70 + 145.00 + 54.12 *
16.90 17.40 27.70 12.90’
Number of plasma pools assayed, in parentheses. Differences between means obtained before and after prorenin activation were not significant (I-test, P < 0.05). ‘Values significantly different from those obtained by the other three activation processes (analysis of variance and Tukey’s test, P < 0.01). Table 3. Trypsin caseinolytic activity inhibition by Bothrops jararaca (Bj) plasma Trypsin concentration (mg/ml Bj plasma)
Caseinolytic activity inhibition %
2 4 8 12
70 56 25 13
Values are given as percentage of inhibition of the activity presented by the same concentration of trypsin in 0.1 M phosphate buffer, pH 7.4.
Plasma inactive renin activation When Bj plasma was submitted to each of the four activating methods, no significant increase in PRA was found (Table 2) (P < 0.05, t-test).
However, the values of PRA obtained before and after treating Bj plasma with Bitis arietans venom were shown to be lower than all the other values determined (P < 0.01, Tukey’s test). When trypsin was used as the activator, the plasma protease inhibitors did not interfere with PRA. The concentration of trypsin used (8 mg/ml plasma) was sufficient to almost surpass the inhibitory effect of Bj plasma, as shown by the results of the determination of the caseinolytic activity at different concentrations of trypsin in Bj plasma (Table 3). In order to find out if these different activation processes could be altering or destroying some
1Omm
components of the renin-angiotensin system such as plasma renin substrate or even plasma renin, Bj plasma was previously submitted to acid dialysis, trypsinization or Bitis arietans venom and further incubated with Bj renal renin extract, since the reaction of renin with renin substrate is species-specific (Nolly and Fasciolo, 1973; Taylor, 1977). Whereas more AI1 was released when this experiment was made with acid-activated Bj plasma (Fig. S), trypsinization and treatment with Bitis arietans venom did not cause any increase
in AIL
I
All
All
T,
All
All
Tz
All
All
Doss
40
40
.25
20
lo
.50
10
20
min
60
65
70
75
90
95
loo
105
Fig. 4. Inhibition of the release of the substance(s) contracting the guinea-pig ileum by 10m5M pepstatin. T, and T2 correspond to incubations made in absence (r, ) or presence (T2) of pepstatin; incubation and purification as in Fig. 1. Doses of AI1 in ng and T, and T2 in ml of eluate (1 ml eluate corresponds to 0.33 ml of Bj plasma).
BjP BjP + R
aBjP aBjP + R
Fig. 5. Release of AI1 by incubation of Bj plasma (BjP) or acid-activated BjP (aBjP) in the absence or presence of renal renin (R). Incubation and purification as in Fig. 1. The increase in the amount of AI1 released in the presence of R indicates that there was excess plasma renin substrate in the incubation medium.
R. K. GERVITZet al.
30 DISCUSSION
Complementing the work of Gervitz et al. (1987) which demonstratred the presence of some components of the renin-angiotensin system in plasma and kidney of the venomous snake Bothrops jararaca, the present data have proved the existence of renin activity in the snake plasma. However, it was not possible to demonstrate the presence of inactive renin in this plasma, when using the in vitro activation processes of acid dialysis, cryoactivation and limited proteolysis with trypsin or Bitis arietans venom. The lack of activation of Bj plasma by acid treatment does not seem to be due to a plasma renin substrate deficiency. Contrary to the described susceptibility of human angiotensinogen to acidification (Printz et al., 1977), Bj plasma renin substrate seems to resist this process, since acid-treated Bj plasma further released angiotensin when incubated with Bj renal renin extract. Regarding c~oactivation, it is already known not to occur in plasmas lacking Hageman factor and/or kallikrein (Sealey and Atlas, 1984) as is the case with Bothrops jararaca plasma (Prezoto et al., 1991). When trypsin was used as activator, Sealey and Atlas (1984) observed that its proteolytic action must be strictly controlled since concurrent destruction of active renin may occur. On the other hand, Johannessen et al. (1990) reported the destruction of rat angiotensinogen by trypsin and Dzau et af. (1986) as well as Gallagher ef al. (1980) observed that when dog plasma was treated with trypsin, digestion of the endogenous renin substrate was sufficient to cause a decrease in dog PRA. In turtles, Cipolle et al. (1984) also did not discard the possibility of destruction of a significant portion of plasma renin substrate by trypsin. In the present experiments if trypsin did cause alterations in plasma renin or its substrate, these must be discrete, since addition of Bj renal extract to trypsin-treated Bj plasma did not increase the values of previous results, these results being also similar to those obtained with only acid- or cold-treated Bj plasma (Table 2). When Bj plasma was treated with Bitis arietuns venom, PRA values, before and after treatment, were lower than all other values obtained (P < 0.05, Tukey’s test). Actually, since the venom is a complex mixture of enzymes, this could be caused by the digestion not only of the plasma renin substrate but also of the endogenous renin. Therefore, the lack of activation of inactive renin in Bj plasma actually seems to be due to the already demonstrated absence of factor XII in this plasma (Nahas et al., 1981; Prezoto et al., 1991) and reinforces the hypothesis that kallikrein, a factor XII-dependent enzyme, is responsible for the activation of inactive renin (Derkx et al., 1979; Yokosawa et al., 1979). As opposed to the reports stating that in humans 90% of renin circulates as inactive renin or prorenin (Cooper et al., 1977; Sealey et al., 1980), the present data could also indicate that there is no circulating inactive renin in Bj. It is known that the kidney is the major source of circulating renin, stored in juxtaglomerular granules. Recently, Ikeda et al.
(1991) using subcellular fractions of dog kidney cortex, located a renin processing enzyme in these granules. Therefore, it is possible that inactive renin synthetized in the kidney is processed to the active form in the juxtaglomerular cells before reaching the circulation. To confirm such a hypothesis in Bothrops jararaca, it will be necessary to look for inactive renin and its processing enzyme in the kidney of this snake. Ae&n#wiedge~~rs-The authors thank Mr Wilson de Barros D’Avila for technical assistance, Miss Wanda Regina Carrella da Silva for the typing and Dr Hanna A. Rothschild for revising the English version. Part of the guinea-pig ileum assays were made by F. M. F. Abdalla, recipient of a CNPq Scholarship (Proc. 824102/88-Q
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