Comp. Biochem. PhysioL Vol. 102B, No. 1, pp. 119-122, 1992

0305-0491/92 $5.00 + 0.00 © 1992 Pergamon Press Ltd

Printed in Great Britain

HYDROLYSIS OF L-CYSTINE-DI-fl-NAPHTHYLAMIDE AND NEUROHYPOPHYSEAL PEPTIDES BY THE PLASMA OF THE SNAKE BOTHROPS JARARACA P. F. SILVEIRA,* L. N. SCHIRIPAand Z. P. PICARELLI Servi¢o de Farmacologia, Instituto Butantan, Caixa Postal 65, 05504 Silo Paulo, SP, Brazil (Fax: 011 815 1505)

(Received 21 August 1991) Abstract--1. Bothrops jararaca plasma or serum hydrolysed L-cystine-di-fl-naphthylamide(CNAase activity) at a degree comparable to that of plasma or serum in pregnant women. 2. In adult snakes, activity was less in males. It was not altered in pregnancy but increased after delivery, being higher at pH 6.4 (unspecificenzymes) than at pH 7.9 (true pregnant woman plasma oxytocinase). 3. Its optimum pH was 5.9, different from that of other known enzymes that hydrolyse the same substrate. 4. Bothrops jararaca plasma also hydrolysed vasopressin, oxytocin and vasotocin. 5. These hydrolysing activities were unexpected for an ovoviviparous reptile.

INTRODUCTION

Oxytocinase, with a restricted distribution in vertebrates (Ryd6n, 1966), occurs in plasma of pregnant primates and of the viviparous snake Natrix rhombi fera (Ehrensing, 1964). In pregnant primates this activity is parallel to an arylamidase activity hydrolysing L-Cystine-di-fl-naphthylamide (CNA) (Hashimoto, 1961; Babuna and Yenen, 1966), a compound that has a chemical bond similar to that of the oxytocin molecule (Ehrensing, 1964)--the S--S bond of N-terminal hemicystine adjacent to tyrosine, also present in vasopressin and in the two neurohypophyseal hormones of reptiles: vasotocin (AVT) and mesotocin (MT). This bond is hydrolysed by the circulating oxytocinase, an aminopeptidase (cystilaminopeptidase, CAP, alpha-aminoacyl-peptide hydrolase; EC 3.4.11.3) of primate placental origin. Since an AVT-like substance has been detected in the blood of the snake Bothrops jararaca (Silveira et al., unpublished data), the presence of enzymes hydrolysing this hormone was investigated. The present work reports the results obtained in this ovoviviparous snake, that belongs to a class with a critical phylogenetic position, as ancestral of birds and mammals. The comparison of CNAase levels in snakes under different physiological conditions is made at pH 6.4 and 7.9, according to Ryd6n (1966), to differentiate between the activity on CNA, similar to that present in pregnant plasma or serum and due to oxytocinase (pH 7.0-7.9), and that due to unspecific enzymes (pH 6.0-6.5). MATERIALS AND M E T H O D S

Snakes Snakes (Bothropsjararaca, Serpentes, Viperidae, Cretaiinae) were collected from the wild, classified by Seq~o de *Author to whom correspondence should be addressed. CBPB 102/I--!

Herpetologia, Instituto Butantan and maintained under controlled environmental conditions (Breno et al., 1990).

Blood collection Blood was collected in polyethylene tubes containing heparin (10 IU/ml blood) (Liquemine, Roche Laboratories, Brazil) either from the pulmonary artery or by decapitation of B. jararaca snakes anesthetized with sodium pentobarbital (30mg/kg, s.c.) (Nembutal, Abbott Laboratories, Brazil). When blood was collected from snakes in different hormonal conditions, they were only decapitated. Blood pools or individual samples were immediatelycentrifuged at 10,000g, 5°C for 20rain (SorvaU Superspeed RC 2-B) to obtain plasma or left for 24 hr at room temperature to obtain serum. CNAase activity L-Cystine-di-fl-naphthylamide(Sigma, USA) hydrolysis (CNAase activity) was measured according to the method of Babuna and Yenen (1966) and expressed as the absorbance at 565 nm of incubates containing 1 ml of test material and CNA. Incubation proceeded for I hr at 37°C in appropriate buffers, and the values obtained were the mean of duplicate samples. EDTA (Titriplex III, Merck S.A., Ind. Quimicas, Brazil), oxytocin (OT) and Arg-vasotocin (AVT) (Sigma) were added to the incubation mixture to check their influence on CNAase activity ofB. jararaca plasma. The method was checked using plasma from normal women, with pregnancy stage clinically evaluated, provided by the Central Laboratory of S~.o Paulo Hospital, Sio Paulo. Neurohypophyseal hormone hydrolysis Vasopressin, oxytocin and vasotocin hydrolysis were estimated by incubation of B. jararaca plasma with either Lys-vasopressin(LVP) (Sigma), OT or AVT, at pH 7.0-7.5; the reaction was interrupted by heating at 98°C for 15 rain and the pressor activity was assayed on arterial blood pressure of male Wistar rats (280-300g), anesthetized with sodium pentobarbitai (90mg/kg, i,p.), heparinized (1000IU/kg, i.v.) and treated with phenoxybenzamine hydrochloride (3.3 mg/kg, i.v.) (Smith Kline French Labs., USA). Mean blood pressure was recorded through the cannulated carotid artery and a cannulated iliac vein was used for injection of drugs and test materials.

119

P . F . SILVEIRAet al.

120

Table 1. Cystine-di-fl-naphthylamide of plasma (CNAase) hydrolysing activity or serum obtained from blood collected either by puncture of the pulmonary artery (p) or by decapitation (d) of anesthetized Bothrops jararaca snakes (n =6). Values were mean + SEM CNAase activity*

Statistical analysis This was performed with the aid of Student's t-test or variance analysis followed when necessary by the N e w m a n Keuls test. RESULTS S e r u m o r p l a s m a o b t a i n e d f r o m b l o o d collected either by p u n c t u r e o f the p u l m o n a r y a r t e r y o r by d e c a p i t a t i o n o f a n e s t h e t i z e d B. jararaca snakes were equally active o n C N A at p H 6.4 o r 7.9 (Table 1). T h e m e t h o d has s h o w n g o o d a g r e e m e n t by c o m p a r i s o n o f d a t a o b t a i n e d in p r e g n a n t w o m e n p l a s m a to d a t a r e p o r t e d in t h e literature. T h e C N A a s e activity did n o t persist after h e a t i n g B. jararaca p l a s m a in a boiling w a t e r b a t h for 15 min, a n d was also d e p e n d e n t o n the d u r a t i o n o f the i n c u b a t i o n ( d a t a n o t s h o w n ) . T h e o p t i m u m p H for this B. jararaca p l a s m a C N A a s e activity, using T r i s - m a l e a t e buffer, was 5.9; at p H 7.9, the activity was l o w e r w h e n using p h o s p h a t e buffer t h a n w h e n using v e r o n a l o r T r i s - m a l e a t e buffer; at p H 6.4, the activity was similar in T r i s - m a l e a t e a n d p h o s p h a t e buffers. A t p H 5.9, B. jararaca p l a s m a C N A a s e activity was partially i n h i b i t e d at m a x i m u m rate by 5 m M E D T A . This B. jararaca p l a s m a C N A a s e activity was n o t affected by O T at p H 6.4 o r 7.9 o r A V T at p H 5.9, up to 0.9 # M , b u t despite this, B. jararaca p l a s m a s h o w e d A V T - , O T - a n d L V P - d e s t r o y i n g activities (Fig. 1).

Material pH 6.4 pH 7.9 Plasma (p) 0.70_+ 0.09 0.35 + 0.05 Plasma (d) 0.65 + 0.07 0.36 + 0.05 Serum (d) 0.65 + 0.05 0.42 + 0.05 *Plasma or serum activity expressed as absorbance/ml/hr; incubation at 37°C in 0.025 M Tris-maleate (pH 6.4) or 0.0225 M veronal (pH 7.9) buffers. At the same pH, there is no significantdifference among the activities of the three types of material obtained from the same snake (variance analysis, P < 0.05). Such activities also d i s a p p e a r e d after h e a t i n g p l a s m a at 98°C for 1 5 m i n a n d were d e p e n d e n t o n the d u r a t i o n o f the i n c u b a t i o n ( d a t a n o t s h o w n ) . C N A a s e activity in p l a s m a o f a d u l t snakes was h i g h e r at p H 6.4 t h a n at p H 7.9 (Table 2). T h e very low activity p r e s e n t in the n e w b o r n s n a k e p l a s m a did n o t p e r m i t verification o f the influence o f p H o n it. T h e activity was h i g h e r in a d u l t females t h a n in a d u l t males a n d d i d n o t c h a n g e d u r i n g p r e g n a n c y b u t it was slightly h i g h e r 14 days after delivery.

110

iSmlnl o= -r-

90

E E

70

50

mU-Kg "~

AVT©

AVT

OT©

OT

LVP©

LVP

16,3

16.3

1666

1666

16.6

16.6

Fig. 1. Decrease of the effects of AVT (Arg-vasotocin; 24.4mlU), OT (oxytocin; 5 IU) or LVP (Lys-vasopressin; 5 IU) incubated with 0.75 ml of Bothrops jararaca plasma (pool of 12 non-pregnant females and one male snake collected by decapitation) and 0.9% NaC1 to a final volume of 1.0 ml, at 37°C for 2 hr, on rat blood pressure, c = Controls at 0 time of incubation. The same dose of B. jararaca plasma had no effect on rat blood pressure. Table 2. Cystine-di-fl-naphthylamide of plasma (CNAase) hydrolysing activity from nonanesthetized and decapitated Bothropsjararacasnakes in different hormonal conditions at pH 6.4 and 7.9. Values were mean + SEM CNAase activityt Material

B. jararaca plasma

B. jararaca

Condition* Non-pregnant females (6) End of pregnancy (4) 14 days after delivery (4) Males (5)

pH 6.4 0.48 _+_0.02§c 0.50 _+0.05§¢ 0.62 _+0.05§a 0.30 + 0.05§b

pH 7.9 0.28 q- 0.01c 0.25___0.02¢ 0.34+ 0.04~ 0.16+ 0.01b

n:~ 6 4 4 5

14-day-old females (10, 2, 6, 7) 0.25 ___0.06b 0.15 + 0.03b 4 pools 14-day-old males (5, 10, 5) 0.19 + 0.05b 0.13 _+0.04b 3 *Number of snakes in parentheses. tPlasma activity expressed as absorbance/ml/hr; incubation at 37°C in 0.025 M Tris-maleate (pH 6.4) or 0.0225 M veronal (pH 7.9) buffers. :~n = number of experiments. §Activity is higher than at pH 7.9 (Student's t-test, P < 0.05). Individual groups marked with different letters were significantly different at the same pH (Newman-Keuls test, P < 0.05).

Snake CNAase activity DISCUSSION

Most of the studies conducted in men and nonpregnant normal women have not detected oxytocinase activity in their plasma or serum (Tuppy, 1968). However, despite the considerable amount of vasopressin-destroying activity reported in their serum, plasma vasopressin was shown to be inactivated by binding to proteins and not by such activity (Werle, 1960; Arimura and Yamaguchi, 1964). Furthermore, in other non-primate mammals, vasopressinase activity seems to be restricted to serum, with evidence that its activation would occur in the course of blood coagulation (Heller, 1960). In B.jararaca, the present data show plasma LVP-, OT- and AVT-hydrolysing activities that could not be attributed to the same serum-restricted vasopressinase. In addition to these activities, B. jararaca showed a concomitant CNAase activity in plasma and serum. Concerning this CNAase activity in B. jararaca serum, it is necessary to consider the absence of Factor XII and some other differences in the blood coagulation process of this snake (Nahas et al., 1981; Prezoto et al., 1991) resulting in a delayed clot formation which renders the process used to obtain serum favourable for proteolysis. Therefore, similar levels of CNAase in B. jararaca serum and plasma could then be due to its high stability. However, its gradual activation during serum formation should also be considered. In human plasma or serum, oxytocinase also showed a remarkable stability; serum from pregnant women does not loose its oxytocinase activity to any significant extent when heated at 37°C for 24 hr (Tuppy, 1968). In human pregnant plasma but not in tissues other than the placenta, a correlation has been established between CNAase and oxytocinase activities (Ryd6n, 1966). The use of CNA for the estimation of plasma oxytocinase is much more accurate than the biological methods (Tuppy, 1968). Despite B. jararaca plasma activity on the reported neurohyphyseal peptides, there was no detectable inhibition of CNAase in the presence of oxytocin or vasotocin, at the employed concentrations. In addition, B. jararaca plasma level of CNAase activity was always higher at pH 6.4, except in newborn snakes. Therefore, the interference of enzyme(s) other than primate oxytocinase seemed to be responsible for these activities. However, B. jararaca plasma CNAase at pH 7.9 is still at a level comparable to that of serum from pregnant women. Our data show that in male snakes it corresponded to that of human female serum at 8-12 weeks of pregnancy and in female snakes it is as high as that in human female serum at 12-20 weeks of pregnancy (Babuna and Yenen, 1966). Nonpregnant women have only traces of plasma oxytocinase activity; the values change as a function of the menstrual cycle, being very low during menstruation and relatively higher near ovulation; at this phase, the level is similar to that found in normal men after a period of daily injections of 5 mg of stilbestrol (Page, 1946, 1947). During pregnancy, woman serum oxytocinase activity measured by hydrolysis of CNA (Babuna and Yenen, 1966) or L-cystine-di-pnitroanilide (Majkic-Singh et al., 1982) increases. Hormonal control, similar to that described in the above-mentioned human situations or as occurs with

121

mammalian brain arylamidases (Gandarias et al., 1989) and with peptide hormones of fetal and/or maternal origin (Kurauchi et al., 1989), also seemed to influence B. jararaca plasma CNAase. The low CNAase level in newborn snakes and the higher CNAase activity found in adult snakes at acid pH, suggest that this activity is more affected by hormones at this pH. In addition, the optimum pH of B. jararaca plasma CNAase is in the acid zone, different from that found in the plasma of pregnant or non-pregnant women and ofNatrix rhombifera. As in women and N. rhombifera, in B. jararaca the level of CNAase activity did not seem to be directly related to neurohypophyseal hormone metabolism during pregnancy. The viviparity also did not seem to be a necessary condition for this activity. In normal men and pregnant women the oxytocin half-life did not differ (Amico et al., 1984). In N. rhombifera, a viviparous snake (Ehrensing, 1964) and in B. jararaca, an ovoviviparous snake, CNAase activity occurred in males and females and did not change during pregnancy. Furthermore, whereas in B. jararaca the level of plasma CNAase slightly increased 14 days after delivery, in women the plasma CNAase decreased progressively until insignificant levels were attained 28 days after delivery (Page, 1946). Therefore, despite the lack of information about enzyme specificity and origin, our results showed clearly, in B. jararaca plasma, a CNAase activity and an ability to hydrolyse neurohypophyseal hormones, which seemed to be different from that of pregnant primate oxytocinase and that of serum-restricted vasopressinase, two uncommon facts in the phylogenetic scale, with unknown physiological significance.

Acknowledgements--The authors are grateful to Mr Wilson de Barros D'Avila for his skilled technical assistance and Miss Wanda R. Carrella da Silva for typing the manuscript. Thanks also to Misses Maria C. Breno and Norma Yamanouye for information about the snakes and for maintaining them under controlled conditions and to Mr Benedito C. Prezoto for helping in blood collection from anesthetized snakes. REFERENCES

Amico J. A., Seitchik J. and Robinson A. G. (1984) Studies of oxytocin in plasma of women during hypocontractile labor. J. clin. Endocr. MetaboL 58, 274-279. Arimura A. and Yamaguchi T. (1964) Serum proteinvasopressin binding and its influence on the pressor activity of the hormone. Jap. J. Physiol. t4, 90-101. Babuna C. and Yenen E. (1966) Enzymatic determination of placental function: a rapid method. Am. Z Obstet. GynecoL 95, 925-934. Breno M. C., Yamanouye N., Prezoto B. C., L~izari M. F. M., Toffoletto O. and Picarelli Z. P. (1990) Maintenance of the snake Bothropsjararaca (Weid, 1824) in captivity. Snake 22, 126-130. Ehrensing R. (1964) Plasma oxytocinase,cystine and leucine naphthylamidases of snake, turtle and chicken. Proc. Soc. exp. Biol. Med. 117, 370-373. Gandarias J. M. de, Ramirez M., Zulaica J. and Casis L. (1989) Aminopeptidase (Arylamidase) activity in discrete areas of the rat brain: sex differences. Horm. Metabol. Res. 21, 285-286. Hashimoto T. (1961) Studies on 1-cystine-aminopeptidase. Part I. Biochemicalaspects. J. Jap. Obstet. GynecoL Soc. 8, 87-95.

122

P.F. SILVEIRAetal.

Heller H. (1960) Inactivation of vasopressin and oxytocin in vivo. In Polypeptides which Affect Smooth Muscles and Blood Vessels (Edited by M. Schachter), pp. 89-90. Pergamon Press, Oxford. Kurauchi O., Mizutani S., Nomura S., Hotta R., Ito Y., Asada Y., Furuhashi M., Kasugai M. and Tomoda Y. (1989) Effects of progesterone and estradiol on aminopeptidase A (APP) and leucine aminopeptidase (LAP) activities in the pregnancy serum and placenta of the rat. Expl clin. Endocr. 93, 90-94. Majkic-Singh N., Vukovic A., Spasic S., Ruzic A., Stojanov M. and Berk6s I. (1982) Oxytocinase (CAP) activity in serum during normal pregnancy. Clin. Biochem. 15, 152-153. 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.

Page E. W. (1946) The value of plasma pitocinase determinations in obstetrics. Am. J. Obstet. Gynecol. 52, 1014-1022. Page E. W. (1947) A blood test for estimating the week of pregnancy. Science 105, 292-293. Prezoto B. C., Hiraichi E., Abdalla F. M. F. and Picarelli Z. P. (1991) Activation of the kallikrein-kinin system in plasma of some brazilian snakes. Comp. Biochem. Physiol. 99C, 135-139. Ryd6n G. (1966) Cystine aminopeptidase and oxytocinase activity in pregnancy. Acta Obstet. Gynecol. Scand. XLV (Suppl. 3), 4-105. Tuppy H. (1968) The influence of enzymes on neurohypophysial hormones and similar peptides. Hand. exp. Pharmac. XXIII, 67-129. Werle E. (1960) Discussion remark. In Polypeptides which Affect Smooth Muscles and Blood Vessels (Edited by M. Schachter), pp. 89-90. Pergamon Press, Oxford.

Hydrolysis of L-cystine-di-beta-naphthylamide and neurohypophyseal peptides by the plasma of the snake Bothrops jararaca.

1. Bothrops jararaca plasma or serum hydrolysed L-cystine-di-beta-naphthylamide (CNAse activity) at a degree comparable to that of plasma or serum in ...
324KB Sizes 0 Downloads 0 Views