Camp.B&&em.Physiof.Vol. 103A.No. 1, pp.5944, 19%
0300-9629/9255.00+ 0.00 0 1992Pergamon Press Ltd
Printed in Great Britain
IN THE SNAKE
P. F. SILVEIRA,* L. N. SCHIRIPA, E. CARMONAand Z. P. ~CARELLI Serviqo de Fa~acologia, Instituto Butantan, Caixa Postal 65, 05504 S&o Paulo, SP, Brazil. Fax: (55) 011-815-1505 (Received 22 January 1992) Ahstraet-1. There is biochemical and pharmacological evidence to suggest the presence of vasotocin in the blood and plasma of the snake Eothrops jararaca (Bj). 2. XE-64 extracts from Bj blood showed antidiuretic and hypotensive activities in rats and a contractile effect on rat isolated uterus, which was totally dialysable and inhibited by thioglycollate. 3. Extracts from Bj whole plasma presented an antidiuretic activity which was only partially dialysable. 4. The plasma extracts also showed oxytocic properties. 5. When EDTA and SepPak Cl8 extraction were used, a better recovery and characterization of vasotocin by HPLC was obtained. 6. These results indicate the occurrence of free and bound circulating vasotocin in Bj, in an eq~lib~um dependent of its enzymatic hydrolysis.
The neurohypophyseal hormones comprise 11 known structural variants, with arginine vasopressin (AVP) and oxytocin (OT) representing the main nonapeptides in mammals and arginine vasotocin (AVT) and mesotocin (MT) their correspondent hormones in nonmammalian vertebrates (Ervin et al., 1985; Chauvet et al., 1990). However, whereas the occurrence of MT is only partially accepted, AVT has been reported in all investigate species of reptiles. The first description of an ~tidiuretic principle in reptiles was reported in whole hypophysis extracts of Phiiodryas genus snakes (Pot-to and Ferraz, 1942). Later, several authors (Heller and Pickering, 1961; Pickering, 1967; Acher et al., 1968; Bentley, 1968), through chromatographic and pharmacological evidence, had identified this principle as vasotocin; it was synthesized in 1958 by Katsoyannis and Du Vigneaud. More recently, immunological techniques have been used to study AVT in neurohypophyseal, plasma or serum extracts of several nonmammalian vertebrates. After the extraction processes, usually by deproteini~tion involving pr~ipitation associated with gel filtration or ion exchange chromatography, AVT levels have been measured by radioimmunos~y without any evaluation of the biochemical properties and the biological activities of the extract content or of the relationship between AVT and other circulating molecules (Rice, 1982; Nouwen and Kuhn, 1983; Rice et al., 1985; Amason et al., 1986; Shimada et al., 1986). Meanwhile, the use of HPLC has confirmed vasotocin identity in reptile tissues (Michel et al., 1990). In the Brazilian snake Bothrops jararaca, there were no biochemical, pharmacological or immunological data about circulating or tissue vasotocin. *To whom correspondence
Therefore, the present work was undertaken in order to: (1) study some characteristics of Bj AVT using rat diuresis, uterus contraction and blood pressure as bioassays; (2) identify this circulating Bj AVT by analytical reverse phase HPLC. In order to investigate the influences of anticoagulant agents and extraction methods on the AVT recovery, both plasma and blood collected with either heparin or EDTA were used and two extractions methods, with Amberlite IRC-50 XE-64 or Sep-Pak Cl8 columns, were analysed. MATERIALS AND METHODS
Reagents and drugs The reagents and drugs used during this study, and the companies they were obtained from were as follows: ethylenediaminetetraacetic acid-trisodium salt (EDTATitriulex IIII, trichloroacetic acid ffCA). acetic acid. sodium chloride, sodium hydroxide, ’ magnesium sulfate; sodium hydrogen carbonate, calcium chloride dihydrate and ammonium hydroxide (Merck S.A. Industrias Quimicas, Brazil); potassium chloride, anhydrous di-potassium hydrogen phosphate, trifluoroacetic acid (TFA), acetonitrile and urea (E. Merck, Darmstadt, Ge~any); oxytocin, Arg-vasotocin and Lys-vasopressin (Sigma Chemical Co., St Louis, MO, U.S.A.); ethyl ether and methanol (Grupo Qubnica, Brazil); heparin (Liquemine, Roche Laboratories, Brazil); sodium pentobarbital (Nembutal, Abbott Laboratories, Brazil); b-glucose, anhydrous (Mallinckrodt Inc., New York. NY. U.S.A.): XE-64 resin. oreoared from Amberlite IRC-50 (R&hm andHaas Co., U1S.A:) as described by Hits et al. (1953); fresh 4 M sodium thioglycollate (TG) in 0.9% NaCl, prepared from 80% thioglycollic acid (Merck S.A. Industrias Quimicas, Brazil) adjusted to pH 7.0 with 75% sodium hydroxide; Sep-Pak Cl8 cartridge (Waters Associates, Inc., U.S.A.); TSK ODS-12OT (4.6 x 250mm; 5~4m) reverse phase HPLC column (Phannacia, Sweden). Snakes (~~fhrops jararacu, Serpentes, Viperidae, Crotahnae) were collected from the wild and classified by [email protected]
de Herpetologia, Instituto Butantan and maintained under
should be addressed. 59
P. F. SILVEIRAef al.
controlled environmental conditions (Breno ef al., 1990). Wistar rats were maintained in a special room under controlled conditions with a photoperiod of 12:12 L:D. Blood collection
Blood samples were collected by decapitation of nonanaesthetizedsnakes, in polyethylene tub< containing heparin (IO I.U.iml of blood) or 5mM EDTA. Plasma was obtained by ~ntrifugation’at 10,000 rpm, at S”C, for 20 min (Sorvall Superspeed RC 2-E). Blood or plasma pooIs were immediately submitted to chromatographic procedures. Chromatographic procedures
XE-64 column-extraction was performed according to Weinstein et al. (1960) using 45 ml aliquots of Bj whole blood or plasma pools and a column of 2 cm diameter and 1cm length with 7 ml of water on the top. After the sample had passed through the column, 25 ml of water was applied and the active fraction was subsequently eluted with 37 ml of 50% (v/v) acetic acid at room temperature, according to Yoshida et al. (1963). This eluate was dried at 40-50°C on a rotatory evaporator (Rotavapor-R-Buchi) and stored in a vacuum at 5°C. Before using in bioassays, this material was dissolved in deionized water (l/IO of eluate volume) and submitted to the same drying procedure. This operation was repeated once more and then the material was resuspended in an equal volume of 0.6% NaCl (isosmotic with Bj plasma). Sep-Pak C18 column-extraction was performed according to LaRochelle et al. (1980) using 0.1% TFA instead of 4% acetic acid to rinse the column and 75% aqueous acetonitrile containing 0.1% TFA for peptide elution. The effluents were combined, concentrated at 4&5o”C for I5 min on a rotatory evaporator and then lyophilized and stored in a vacuum at 5°C. Characterizution of the extracted material by HPLC
Dried efBuents of XE-64 or Sep-Pak Cl8 columns were dissolved in 0.1% TFA; for samples of 6ml of plasma applied in Sep-Pak CI8, 120~1 of solvent were used; for samnles of 45ml of nlasma or blood submitted to XE-64 column-extraction, f8Opl or 18OOpl of solvent, respectively, were used. Aliquots of 20 ~1 were subjected to reverse _. ODS bonded silica Cl8 .chase HPLC usine_ a fullv_ caDDed column (TSK ODS-120 T). An acetonitrile linear gradient (O-60%) containing 0.1% TFA was applied for 30 min, followed by an isocratic elution with 90% acetonitrile for 5 min. The Beckman System Gold was used with a Model 164 detector and the absorbance was monitored at 214 nm. Synthetic AVT (0.4 pg in 20~1 of 0.1% TFA) was used as standard, The peak amplitude of extracts and standards were used to estimate the AVT content of the samples.
Contractile activity on rat (Holton, 1948) or Bj (Abdalla et al., 1989) isolated uterus was assayed before or 1 min after administration of 40 mM TG. Dipsogenic activity
Dipsogenic activity was evaluated by the water ingestion of male rats (200-300 g), during an 18 hr period. Activity on earotidean blood pressure
Activity on carotidean blood pressure of male rats (280-300 g) was assayed as described by Prezoto ef al. (1991). Statistical analysis
Statistical analysis was performed by unpaired and paired Student’s r-test and variance analysis. RESULTS Figure 1 shows that rat urinary retention increased after administration of heparinized whole plasma as well as after heparinized blood XE-64 cohtmn-extract, in a similar way as it occurs with LVP. However, whereas there is no difference in rat urinary retention of control animals and those injected with
dialysed LVP, a significant difference was found when using dialysed plasma. This rat urinary retention activity was not due to protein concentration since it also occurred in non-protein XE-64 column-extract and not in a control experiment made with a mixture of albumin (40 m&ml) and heparin (10 I.U./ml). In addition, the antidiuretic effect of Bothrops jararaca circulating compound(s) seemed to be unaffected by non-dialysable molecules, since the development of rat antidiuretic response to plasma was similar to that induced by LVP (Fig. 2). As also shown in Table 1,
XE-64 column-extracts from heparinized blood, heparinized plasma or Lys-vasopressin- (LVP) were each dialvsed against 660 volumes of 0.6% NaCl for 18 hr at 5°C in-cello-
(rnlnu~es) Fig. 2. Development of rat antidiuretic responses elicited by i.p. administration of I.Oml of Lys-vasopressin (LVP, 15 mI.U./ml, ( x ), N = 10) or of 1.0 ml Bothropsjmm.xu (Bj) heparinized plasma (O), N = IO). Antidiuresis ratio (means & SEM) is the urinary retention rate (ml of retained water/lOOg of body weight) of experimental over 0.6%
Table 1. Rat antidiuresis induced either by Lysvasopressin (LVP), or heparinized blood, submitted to XE-64 column-extraction, or plasma from Injected material LVP
Plasma Dialysed plasma Blood XE-64
4 8 16 32 64 0.25 0.50
8 7 9 9 8 10 IO
1.08 f 0.07 I .28 If:0.07 1.38&0.11 1.38fO.10 1.36f0.12 1.08 f 0.06 1.17i:o.o5
Values are means f SEM. N = number of rats. *Expressed as antidiuresis ratio (urinary retention rate, in ml of retained water/lOOg of body weight of experimental over 0.6% NaCl i.p. 1.0 ml injected rats). All values were si~iB~ntly different from one (P x0.05, paired Student’s t-test).
12.5 +_0.9 12.1 t 1.I 12.2 * 1.1 14.3 + 1.7
2nd period 13.5 f 0.6 14.0+ I.1 12.7 + I .8 14.9 _c 1.6
lower activity than whole plasma, blood XE-64 column-extracts have half the activity of whole plasma. In addition, XE-64 column-extracts from blood or plasma induced rat uterus contraction which was inhibited by TG and was dialysable (Fig. 3). Furthermore, blood XE-64 column-extract also contracted Buthrops jararaca uterus (data not shown). The hypotension produced in rats by this blood XE-64 column-extract was an unexpected result which was not observed neither with whole plasma,
NaCl injected rats. No significant different responses were obtained, at each time, between LVP and Bj plasma (P < 0.05, Student’s t-test).
part of this plasma antidiuretic activity was dialysable. Table 2 shows that there was no difference among the effects induced by LVP, plasma and 0.6% NaCl on rat dipsogenic activity. Dose-dependent antidiuretic responses to LVP and plasma were shown in Table 1. The comparison of the antidiuresis ratios induced by plasma (dialysed and non-dialysed) or by blood XE-64 column-extract is indicative of the amount of antidiuretic compound(s) existing in these materials: whereas dialysed plasma has four times
NaCl-LVP LVP-NaCI NaCl-plasma Plasma-NaCI
Values are means i SEM. *Expressed as water ingestion (ml/100 g of body weight). Number of animals per group: 6. Independent of the material and of the order of injection there was no significant difference among the groups of rats (P < 0.05, analysis of variance).
Order of injection
. .. . * .., l
iOT Of TG 6 -IO
. * 1 OT
Fig. 3. Inhibition, by 40 mM sodium thioglycollate (TG) or dialysis, of the contractile activity upon rat uterus, presented by XE-64 column-extracts from heparinized blood and plasma collected from decapitated ~~rh~op~~~~u?~cu snakes. OT, 0.6 m1.U. oxytocin; a and a’, 0.2 and 0.05 ml of XE-64 extracts from blood; b, 0.1 ml of dialysed blood extract; c, 0.1 ml of plasma extract.
SILVEIRA et al.
Bothrops jararaca snake is unable to promote an endogenous release of antidiuretic hormone in rats. Therefore, it was supposed that compounds related to vasotocin could be responsible for the antidiuretic effect of the snake plasma. In order to further investigate the vasotocinlike substance in Bothrops jararaca, blood and plasma extracts were prepared in XE-64 columns. As expected, these co~umn~xtracts of snake blood presented antidiuretic activity in rats but, in contrast to extracts of dog blood (Weinstein er al., 1960), they caused hypotension in rats. However, this effect could not be considered the origin of the rat antidiuresis since it was a rapid response and it has not been
Fig. 4. Effect of XE-64 column-extracts of Bothrops juraraca heparinized blood or plasma on the mean carotidean rat blood pressure. LVP (Lys-vasopressin), 16.7 fig/kg; H (histamine), 16.7 peg/kg; a and a’ (blood extract), 0.66 and 0.33 ml/kg; b (whole plasma), 0.33 ml/kg; c (control -0.6% NaCl submitted to XE-64 column-extraction), 0.33 ml/kg; v (0.6% NaCI), 0.33 ml/kg; d and d’ (plasma extract), 0.66 and 0.33 ml/kg.
control column extracts (0.6% NaCl), nor with plasma XE-64 column-extracts (Fig. 4). AVT peptide was identified by reverse phase HPLC, in heparinized blood and plasma-EDTA XE-64 column-extracts (Fig. 5B), as well as in heparinized plasma and plasma-EDTA Sep-Pak Cl 8 column-extracts (Fig. 5C), since a peak at the same retention time (20.8 mitt) of synthetic AVT was obtained. AVT content of plasma-EDTA XE-64 column-extracts was estimated as 10 times lower than that of heparinized blood XE-64 column-extract or heparinized plasma as well as plasma-EDTA Sep-Pak Cl8 column-extracts. However, AVT was undetectable, by the same HPLC analysis, in the heparinized plasma XE-64 column-extract (Fig. 5A).
The present work demonstrated Bothrops jararaca plasma antidiuretic activity in rats with a time-course similar to that shown by the synthetic peptide Lysvasopressin. However, whereas Lys-vasopressin antidiuretic activity was completely dialysable, this plasma antidiuretic activity was only partially dialysable, suggesting an interaction between the snake plasma antidiuretic compound(s) and larger molecules. In addition, Lys-vasopressin or plasma did not modify water ingestion in normally hydrated rats. Since overhydrated rats were used in the antidiuresis assays, this last result suggested that the plasma of
Fig. 5. HPLC analysis of Bothrops jararuca (Bj) plasma, collected with heoarin (hen) and submitted to XE-64 extraction (profile A)*or c&&ted with EDTA and submitted either to the same extraction (profile B) or to Sep-Pak Cl8 extraction (profile C). Bj hep blood XE-64 column-extracts showed similar pattern to profile B and Bj hep plasma
Sep-Pak C18 similar to profile C. See text for details. Vasotocin retention time (20.8 min) is indicated by arrows.
demonstrated in plasma XE-64 column-extracts. Both blood or plasma XE-64 column-extracts produced rat uterus contractions which were inhibited by thioglycollate. As this agent is able to cleave the disulphide bond of neurohypophyseal peptides and to interact with their receptors (Fitzpatrick and Bentley, 1968), this result confirmed the existence of a vasotocin-like compound in these extracts. In addition, these blood column-extracts lost their oxytocic activity after dialysis, suggesting that it is due to small molecules. Reverse phase HPLC has been successfully used to separate vasopressin and vasotocin (Blevins et al., 1980) as well as to identify either vasopressin, in marsupials (Bathgate et al., 1990) in rat and in human plasma (LaRochelle et al., 1980) or vasotocin in ovine fetal blood, urine and amniotic fluid (Ervin et al., 198.5) in amphibians (Rouille et al., 1989) and in snake pituitary (Niche1 et al., 1990). In the present work, Arg-vasotocin was detected by HPLC analysis, in XE-64 column-extracts of heparinized blood but not of heparinized plasma. This finding agrees with the low oxytocic activity of these heparinized plasma extracts when compared to that of heparinized blood extracts. As recently described by Silveira et al. (1991), plasma of Bothrops jururac~ has a strong hydrolytic activity on vasotocin, on related peptides and on L-cystine-di-/3-naphthylamide, this last inhibited by EDTA. Actually, when EDTA was used for collecting blood, Arg-vasotocin was identified in the corresponding plasma XE-64 columnextracts. Furthermore, HPLC analysis revealed that Sep-Pak C 18 column-extraction of plasma provided a higher recovery of this peptide than the XE-64 column-extraction. On the other hand, quantitative data for circulating vasotocin in the snake ~ufhrops juraraca could not be obtained since hydration conditions, stage of reproductive cycle and other known factors that influence vasotocin levels, could not be controlled in the present study. Also, the use of pharmacological or immunological tests for measuring vasotocin showed some restrictions when the partially purified columnextracts where analysed. Biologically and immunologically active products resulting from vasotocin degradation and undamaged or hydrolysed mesotocin could exist in these extracts. In addition, as suggested for oxytocin (Kumaresan et al., 1969), vasotocin could have different sites of biological and immunological activities which could be differently affected by inactivating agents. Therefore, further studies with more accurate evaluation of vasotocin circulating in this snake, through its pharmacological effects, could be made after its purification using, as an appropriate step, the Sep-Pak Cl8 column-extraction and the inhibition of vasotocin damage by EDTA or other agents. Another possibility is the radioimmunoassay measurement of Bathrops juraraca vasotocin directly in these Sep-Pak Cl8 column-extracts since the recovery of this peptide in this procedure and the crossreaction between related compounds with vasotocin antibodies have been evaluated. Ackno~Zedgemenfs-The authors are grateful to Mr Wilson de Barros D’Avila for his skilled technical assistance and Mrs Wanda R. Carrella da Silva for typing the manuscript,
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