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Plasma arginine vasotocin concentrations in the lizard Varanus gouldii (gray) following water loading, salt loading, and dehydration
GENERAL
AND
Plasma
COMPARATIVE
41, l-6 (1982)
ENDOCRINOLOGY
Arginine Vasotocin Concentrations in the Lizard Varanus gouldii (Gray) following Water Loading, Salt Loading, and Dehydration G. E. RICE’
Zoology
Department,
University
of Western
Australia,
Nedlands,
6009
Western
Australia,
Australia
Accepted July 3, 1981 Plasma arginine vasotocin (AVT) concentrations in salt-loaded, dehydrated, and waterloaded Varanus gouldii were measured by radioimmunoassay and averaged 7.1 2 1.5, 3.9 f 0.3, and 1.6 f 0.4, respectively. Plasma AVT and osmolality were correlated signiticantly (P < 0.001) and this relationship is described by the equation AVT (pglml) = O.OSS(P,, - 276). The plasma osmotic threshold for AVT release was found to be similar to that previously reported for other species.
water loading, salt loading, and dehydration. The renal function of this species under these three treatments has been reported previously (Bradshaw and Rice, 1981; Rice et al., 1982).
The neurohypophyseal hormone arginine vasotocin (AVT) has been isolated from the pituitary glands of all nonmammalian vertebrates examined to date (Sawyer, 1977; Sawyer and Pang, 1975) and in many species it has been implicated in the control of kidney function (Babiker and Rankin, 1979; Bentley, 1974, 1976; Bradshaw, 1975; Bradshaw and Rice, 1981; Dantzler, 1976; Sawyer, 1966, 1977; Shoemaker and Nagy, 1977). Recent studies on reptilian renal physiology have provided evidence that is consistent with AVT acting as the physiological antidiuretic principle (Dantzler et al., 1970; Dantzler, 1976, 1978; Bentley, 1976; Bradshaw, 1978; Bradshaw and Rice, 1981; however, plasma AVT concentrations have yet to be measured in reptiles and it is therefore difficult to distinguish between physiological and pharmacological doses of AVT used to establish its role in the control of renal function. In order to clarify the physiological role of AVT in reptiles and to establish the effects of various osmotic challenges on AVT release, plasma concentrations of AVT were measured in the lizard Vuranus gouldii under conditions of
MATERIALS
AND METHODS
Varanus gouldii (Gray) were collected on Dandaraga Station (27”03’ S, 119”05’ E) 560 km north of Perth, Western Australia. They were maintained in an outside terrarium and fed canned dog food once per week. Water was provided ad libitum. The mean body weight of the experimental animals was 1.85 kg (range 0.90-2.85 kg). During the conditioning and treatment periods, animals were housed in individual cages in a constant cycle room with a 12:12 LD photoperiod and maximum and minimum temperature and relative humidity of 37 and 20” and 60 and 30%, respectively. The three experimental treatments utilised in the present study were: water loading, animals received daily intraperitoneaf injections of distilled water (5 ml000 kg) for a period of 7 days; salt loading, animals received daily intraperitoneal injections of 0.2 ml/l00 g of 2.14 M NaCl for a period of 7 days; and dehydration, animals were denied access to water and food for a period of 10 days which resulted in approx 8-10% reduction in body weight. Plasma sampling procedure. On completion of the conditioning period, 3- to S-ml intracardiac blood samples were collected in chilled 5-ml plastic syringes for electrolyte and hormone determinations. The samples were collected between 0830 and 0930 hr, 24 hr after the final injection of the condition period for salt- and water-loaded animals. All samples analysed were collected within 3 min of handling the animal. They were immediately placed in ice and then centrifuged at Animals
’ Present address: Department of Obstetrics and Gynecology, University of Texas Health Science Center at Dallas, 5523 Harry Hines Blvd., Dallas, TX 75235.
and
treatments.
1 0016~6480/82/050001-06$01.00/0 Copyright 0 1982 by Academic F’ress, Inc. All rights of reproduction in any form reserved.
2
G. E. RICE
2OOOgfor 15 min at 4” within a maximum of IS min of Assay standards. Synthetic AVT (Ferring, 215 collection. The l-ml aliquots of plasma for the meaIU/mg) dissolved in 0.2 M acetic acid and 0.1% BSA surement of AVT concentrations were extracted imwas used for the standards in the assay. It was stored mediately. The remaining plasma was stored at -20 as 600~~1 aliquots in l-ml plastic containers at -20” in until the analysis of electrolyte concentrations and three stock concentrations: 32 &ml, 320 r&ml, and osmolality. 640 pg/ml. This procedure facilitated easy dilution to Field plasma samples were collected during late the eight standards used in the standard curve: 32, 16, spring (November 1979) from V. goal&i on Danaraga 8, 4, 2, 1, 0.5, and 0.25 pg/tube. Fresh standards were Station. Intracardiac blood samples used for the prepared l-2 hr before each assay. analyses were collected within 3 min of sighting the Assay protocol. Plasma AVT concentrations were animal and were hand-centrifuged for S to 6 min in measured using a late-addition, double-antibody assay lithium heparin-coated tubes containing Kwikspin system similar to that described by Koike et al. (1979). granules (Disposable Products, Western Australia). A rabbit antibody raised against 8-arginine vasopresThe plasma was transferred to S-ml plastic containers sin, with a cross-reactivity with oxytocin of ~5% (Fercontaining 100 ~1 of 1% acetic acid (Yagil and Elzion, ring, AB), was used in a 0.5-ml assay system 1979) and then was stored in liquid air until extracted (Weitzman, personal communication). The assay was on returning to Perth, Western Australia. performed at a final antibody dilution of l/250,000. Extraction procedure. The l-ml aliquots of plasma Triplicate SO-w1 aliquots of appropriate standard and were extracted within 1 to 2 hr after collection using a duplicate 200-~1 aliquots of plasma extracts were incumodification of the acetone extraction procedure debated at 4” in 10 x 7S-mm plastic tubes (3DT, Disposscribed by Robertson et al. (1973). Extracts were preable Products) with SO ~1 of AVT antibody and phospared by mixing 1 ml of plasma with 2 ml cold (4”) phate buffer (0.01 M, pH 7.4) to a final volume of 450 acetone (Merck) on a vortex mixer for 30 set to pre- ~1. The SO~1 (looO-2000 cpm) of monoiodinated AVT cipitate the plasma proteins; and after centrifuging at was added 24 hr later, mixed for 15 set on a vortex 2oOOg for 30 min at 4”, the supematant was decanted mixer, and incubated at 4” for a further 72 hr. Free and and mixed with 5 ml of cold (4”) n-hexane (Merck) bound hormone were separated using a second and centrifuged again for 15 min at 2OOOgand 4”. The antibody (donkey antirabbit, Wellcome Laboratories); organic phase was aspirated carefully and discarded. 200 ~1 of 1124 dilution of this antibody in phosphate The aqueous phase was evaporated to dryness under buffer was added to each tube and incubated at 4” for a stream of room-temperature air in a 37” water bath. 18 hr; the supernatant was then aspirated, and the The residue was dissolved in 0.5 ml of phsophate buff- precipitate was counted to 1% error with a Prias er, sealed with parafilm (American Can Co.), stored Autogamma counter (Packard). at -20” overnight, and assayed the next day. Analyses. Sodium and potassium concentrations lo&nation. Synthetic 8-arginine vasotocin (Ferring) were determined in I+1 aliquots of plasma with a was iodinated by the chloramine-T oxidation method Varian-Techtron atomic absorption spectropho(Hunter and Greenwood, 1962) adapted for AVT tometer (Type AA-5), using a caesium diluent (1.27 (Weitzman, personal communication). The following g/liter). Chloride concentrations were measured by were added to a reaction vessel: 50 ~1 of 0.5 M phosamperometeric titration with a Buchler Digital chlorphate buffer (pH 7..5), 37 MBq of carrier-free lz51 idometer on lo-p.1 samples. Osmolality was mea(Radiochemical Centre, Amersham), 25 yl of AVT (0.2 sured on 6-~1 samples with a Wescor vapor pressure pg/kl) in 0.2 M acetic acid, and 25 ~1 (50 pg) of osmometer (Model SlOOB). chloramine-T in distilled water. The reaction vial was Statistics. Treatment means were compared using vortexed for 3 set and 15 set after the addition of analysis of variance coupled with a Student-Newchloramine-T, 200 ~1 of 25% bovine serum albumin man-Keuls test. (BSA, Sigma) was added, and the vial was vortexed for 10 sec. The reaction mixture was transferred to a RESULTS 0.9 x 2S-cm G2S Sephadex column and eluted with 0.2 M acetic acid containing 0.1% BSA. The column Assay Characteristics was prewashed with 2% BSA in 0.2 M acetic acid. Using the late-addition double-antibody Fractions of 1 ml were collected by an LKB fraction assay technique, the minimal limit of decollector and 2S-~1 aliquots were counted with a Prias tection of the assay, defined as the least Autogamma counter. Undamaged monoiodinated of unlabelled hormone that AVT was identified by its ability to bind to antibody (in concentration excess). The three postpeak fractions from the profile can be distinguished by an error estimate of of iodinated AVT were pooled and rechromatographed two standard deviations from a sample on a 0.9 x 2S-cm Sephadex G2S column and eluted containing no hormone (Chard, 1978), avwith 0.2 M acetic acid and 0.1% BSA. The l-ml fractions were collected and the second and third postpeak eraged 0.92 pg/ml for extracted plasma esfractions of monoiodinated AVT were pooIed and timated from three assays, and 0.21 k 0.14 stored at -20” as 50-~1 aliquots. pg/tube for the normal standard curve esti-
PLASMA
AVT
CONCENTRATIONS
Logit b 0
-
-1
-
-2
-
-3 025
0.5
1 NT
2
4
8
16
(W/tube)
FIG. 1. Logit-log transformation of the standard curves from 10 assays. The mean ? standard error is reported; r = 0.998, y =- -1.258x + 0.70.
mated from 10 assays. Figure 1 presents a logit-log transformation (Rodbard and Lewald, 1970) of the standard curves from 10 assays. The regression coefficient associated with this linear transformation was 0.998 (P -=c0.05).
In two assays, six plasma replicates were assayed to assess the intraassay coefficient of variation, and in three assays duplicate extractions of a standard plasma were assayed to assess the interassay coefficient of variation. These coefficients average 9.4 and 16.4%, respectively. In two assays, duplicates of known amounts of standard AVT (64, 32, 16, 8,
PLASMA SODIUM, CONCENTRATIONS
Treatment Water loading (II = 6) Salt loading (n = 6) Dehydration (n = 6) Field samples (n = 15)
IN
and 4 pg) were added to bentonite-extracted plasma (Skowsky et al., 1974) and assayed. The recoveries of standard AVT average 105.1 + 0.16% when corrected for extraction losses (n = 20). The recovery of AVT, estimated for each assay from the extraction of six replicates of a standard plasma to which a known amount of radiolabelled hormone had been added, averaged 72.5 2 3.1% (from 10 assays). The plasma AVT concentrations reported are uncorrected from recovery. The cross-reactivity of the assay system with oxytocin was assessed (Chard, 1973) and was CO. 1%. The cross-reactivity of the assay system with mesotocin was not assessed. Plasma Electrolyte Concentrations and Osmolality
Plasma sodium, potassium, and chloride ion concentrations and osmolality for water-loaded, salt-loaded, dehydrated, and field-sampled V. gouldii are presented in Table 1. Osmolality significantly increased in both dehydrated and salt-loaded animals when compared with water-loaded animals (P < O.OOl), and the values are similar to those reported by Bradshaw and Rice (1981) and Rice et al. (1982) for this species. The plasma osmolality of field-sampled animals were not significantly different from the
TABLE 1 POTASSIUM, AND CHLORIDE ION CONCENTRATIONS FOR WATER-LOADED, SALT-LOADED, DEHYDRATED,
Na+ (meq/liter)
K+ (meq/liter)
Cl(meq/liter)
*135.9 + 2.48 P < 0.001 176.1 2 6.50
n 4.34 2 0.19 NS 5.42 k 0.34
*135.3 f 3.9 P < 0.01 165.4 k 5.8 P < 0.01 *146.4 2 6.9 NS 138.4 k 5.2
P < 0.025
NS
*156.3 ? 3.41 NS 146.6 f 3.82
n 4.84 k 0.55 NS 4.76 k 0.23
3
Varanus gouldii
AND OSMOLALITY AND AVT AND FIELD Vat-anus gouldii
Osmolality (mOsm/kg) *274 z!z10.5 P < 0.001 375 + 7.4 P < 0.001 *325 5 8.5 NS 321 2 5.2
AVT (pdml) *1.6 f 0.4 0.001 7.1 t 1.5 P < 0.001 *3.9 Ii 0.3 NS 3.5 t 0.4
P <
Note. The mean 2 SE is reported. Statistical comparisons between the means for water-loaded, salt-loaded, and dehydrated animals were made by the Student-Newman-Keuls test. Student’s t tests were used to compare the field sample means with the means for dehydrated animals; n is the number of animals. (1NS, not significant. *P < 0.01.
4
G. E. RICE
values observed for dehydrated animals. The increases in plasma osmolality are due primarily to increased NaCl concentrations and these changes are of more significant proportions in salt-loaded animals (P < 0.001). Plasma potassium ion concentrations were unaltered by the treatments and were not significantly different from the values reported for the field animals. Plasma AVT Concentrations
Plasma AVT concentrations with the three treatments are also shown in Table 1 and range from below the limit of detection of the assay (0.92 pg/ml) to 13.5 pg/ml (uncorrected for recovery). AVT concentrations significantly increased with both salt loading and dehydration, when compared with the concentration observed for water-loaded animals (P < 0.001). This increase was significantly greater in the case of salt loading (P < 0.001). The increase in plasma AVT concentrations are consistent with the rise in plasma osmolality and the relationship between these two parameters is presented in Fig. 2. The data presented are for 6 water-loaded, 6 salt-loaded, 6 dehydrated, and 15 field V. goufdii (n = 33). A significant positive regression between plasma osmolality and AVT concentrations was obtained (r = 0.776, P < 0.001) and is described by the equation AVT (pgml) = O.OSS(P,,, - 276). DISCUSSION
The present study reports the first radioimmunoassay measurements of plasma arginine vasotocin concentrations for a reptile and documents the effects of dehydration, chronic sodium chloride loading, and water loading on the circulating levels of this hormone. In the lizard V. gouldii, circulating levels ranged from 1 to 18 pg/ml (when corrected for recovery, see Materials and Methods) and were related significantly to plasma osmolality . Bradshaw and Rice (1981) reported renal changes in V. gouldii in response to
-11351
[ AVT (pglml)
9 y
6 5 4 3 2 1
0
.
.*
. . *. *. .. .*. 260
. 280
.
.
..
:
.
* :
. .
. . xx)
320
340
Kkmlhg
3M)
380
403
HP0
FIG. 2. The relationship between plasma AVT concentrations (pg/ml) and plasma osmolality (mOsm/kg H,O). The regression line fitted to the data is described by the equation AVT (pg/ml) = 0.085 (P,,, - 276), r = 0.776, P < 0.001.
salt loading and dehydration that were consistent with the release of AVT into the circulation and its antidiuretic action. Glomerular filtration rate, relative free water clearance (CH2JCin), and urine volume significantly decreased, and fractional reabsorption of water significantly increased. Similarly, acute exogenously administered AVT evoked an antidiuresis due to a fall in glomerular filtration rate and increased tubular water permeability; both of these effects were abolished completely by the subsequent injection of probenecid, a competitive inhibitor of AVT action in the lower vertebrates (Dantzler et al., 1970). The antidiuresis associated with dehydration was of a more tubular response when compared with salt-loaded animals, and these data were interpreted as indicating higher circulating levels of AVT with salt loading than those which occur with dehydration. The data from the present study confirm this interpretation; salt loading resulted in an 80% increase above the values observed with dehydration. In the few species where plasma AVT concentrations have been measured, similar increases in concentration have been observed. In dehydrated Rana catesbeiana, plasma AVT concentrations increased from about 5 pg/ml in normally hydrated individuals to 60 pg/ml (Rosenbloom and Fisher, 1974; Pang et al. 1974; Sawyer
PLASMA
AVT
CONCENTRATIONS
and Pang, 1975; Pang, 1977). In the domestic fowl, Koike et al. (1977) reported a mean plasma concentration of 4.4 $/ml for hydrated hens and this value increased to 18 pglml with 96-hr water deprivation. Similar values have been observed in the Pekin duck; in normally hydrated birds, plasma AVT concentration was 5.1 fmol/ml and with salt water adaptation the mean concentration increased to 22.7 fmol/ml (Mohring et al., 1980). Plasma AVT concentrations in V. god&i were correlated significantly with plasma osmolality and this relationship has previously been demonstrated in the chicken (Koike et al., 1977, 1979) and the Pekin duck (M&ring et al., 1980). Acute 5% saline loading of R. catesbeiana, although producing an antidiuresis and elevated plasma osmolality, did not increase circulating AVT centrations above control values, and it was suggested that in this species plasma AVT concentration and osmolality are only “loosely” related. The linear regression equation fitted to the plot of plasma AVT concentrations and osmolality provides estimates of the sensitivity and the threshold of the osmotically mediated AVT release mechanism. In V. gouldii, the slope of the regression line is less than that observed for birds or mammals, indicating a lower sensitivity of the AVT release mechanism to changes in the plasma osmotic pressure. In this species, plasma osmolality. In this species, plasma AVT increased by 1 pg/ml for an increase in plasma osmolality of 11.9 m&m/kg. The threshold of plasma osmolality, determined by the intercept of the regression line with the abscissa, was 270 mOsm/kg and is similar to the values reported for both birds (Koike et al., 1977, 1979, Mohring et al., 1980) and mammals (Robertson et al., 1973; Dunn et al., 1973; Moses and Miller, 1974). The AVT levels measured in plasma samples collected from animals in the field are in good agreement with the laboratorv
IN
Varanus
5
gouldii
data. The plasma AVT concentrations in field animals during late spring are not significantly different from the values observed in laboratory-dehydrated animals. As the daily maximum temperatures on Dandaraga Station during November 1979 averaged 38” and as surface water was not available during the study period, it is reasonable to suggest that the animals collected were mildly dehydrated. Plasma electrolyte concentration and osmolality of the field animals were also not significantly different from the values reported for the dehydrated animals. ACKNOWLEDGMENTS This work was supported by a research grant from the University of Western Australia. Especial thanks are due to Dr. R. E. Weitzman for providing AVP antibody and helpful suggestions on the assay methodology, and to Dr. E. Horgan and Dr. G. Martin for their assistance during the development of the assay. I am most grateful to Professor S. D. Bradshaw for his valuable comments and discussion of this work. Expert technical assistance was provided by Mr. .I. R. Beesley. The author was in receipt of a Commonwealth Postgraduate Award.
REFERENCES Babiker, M. M., and Rankin, J. C. (1979). Renal and vascular effects of neurohypophysial hormones in the African lungfish Protopterus annectens (Owen). Gen. Comp. Endocrinol. 37, 26-34. Bentley, P. J. (1974). Actions of neurohypophysial peptides in amphibians, reptiles and birds. In “Handbook of Physiology” (S. R. Geiger, ed.), pp. 19-28. Amer. Physiol. Sot. Washington, D.C. Bentley, P. J. (1976). Osmoregulation. In “Biology of Reptilia” (C. Gans and W. R. Dawson, eds.), Vol. V, pp. 365-412. Academic Press, New York/ London. Bradshaw, S. D. (1975). Osmoregulation and pituitary-adrenal function in desert reptiles. Gen. Comp.
Endocrinol.
25,
230-248.
Bradshaw, S. D. (1978). Volume regulation in desert reptiles and its control by pituitary and adrenal hormones. In “Osmotic and Volume Regulation” (C. B. Jorgensen and E. Skadhauge, eds.), pp. 38-59. Munksgaard, Copenhagen. Bradshaw, S. D., and Rice, G. E. (1981). The effects of pituitary and adrenal hormones on renal and post-renal reabsorption of water and electrolytes in the lizard Varanus gouldii (Gray). Gen. Comp. Endocrinol.
44,
89-94.
6
G. E. RICE
Chard, T. (1973). The radioimmunoassay of oxytocin and vasopressin. J. Endocrinol. 58, 143- 160. Chard, T. (1978). An introduction to radioimmunoassay and related techniques. In “Laboratory Techniques in Biochemistry and Molecular Biology” (T. S. Work and E. Work, eds.), New-Holland Publ., Amsterdam/NewYork/Oxford. Dantzler, W. H. (1976). Renal function (with special emphasis on nitrogen excretion). In “Biology of the Reptilia” (C. Cans and W. R. Dawson, eds.), Vol. V, pp. 447-503. Academic Press, New York/London. Dantzler, W. H. (1978). Some renal and glomerular and tubular mechanisms involved in osmotic and volume regulation in reptiles and birds. In “Osmotic and Volume Regulation,” (C. B. Jorgensen and E. Skadhauge, eds.) pp. 187-201. Alfred Benzon Symp. XI, Munksgaard. Dantzler, W. H., Schaffner, D. P., Chiu, P. J. S., and Sawyer, W. H. (1970). Probenecid inhibition of arginine vasotocin in snakes, frogs, and on frog bladdersin virro. Amer. J. Physiol. 218,929-936. Dunn, F. L., Brenna, T. J., Nelson, A. E., and Robertson, G. L. (1973). The role of blood osmolality and volume in regulating vasopression secretion in the rat. J. C/in. Invesf. 52, 3213-3219. Hunter, W. M., and Greenwood, F. C. (1962). Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature (London) 194, 495-496. Koike, T. I., Pryor, L. R., Neldon, H. L., and Venable, R. S. (1977). Effect of water deprivation of plasma radioimmunoassayable arginine vasotocin in conscious chickens (Callus domesticus). Gen. Corn. Endocrinol. 33, 359-364. Koike, T. I., Pryor, L. R., and Neldon, H. L. (1979). Effect of saline infusion on plasma immunoreactive vasotocin in conscious chickens (Gallus domesticus). Gen. Comp. Endocrinol. 37, 451-458. Mohring, J., Schoun, J., Simon-Oppermann, C., and Simon, E. (1980). Radioimmunoassay for arginine vasotocin (AVT) in serum of Pekin ducks: AVT concentrations after adaptation to fresh water and salt water. Pfluegers Arch. 387, 91-97. Moses, A. M., and Miller, M. (1970). Osmotic influences on the release of vasopressin. In “Handbook of Physiology” (E. Knobil and W. H.
Sawyer, eds.), Section 7, pp. 225-255. Amer. Physiol. Sot., Washington, D.C. Pang, P. K. T. (1977). Osmoregulatory functions of neurohypophysial hormones in fishes and amphibians. Amer. Zool. 17, 739-749. Pang, P., Sawyer, W. H., and Casals, C. (1974). Circulating levels of arginine vasotocin (AVT) in lower vertebrates. Amer. Zool. 14, 1244. Rice, G. E., Bradshaw, S. D., and Prendergast, F. J. (1982). The effects of bilateral adrenalectomy on renal function in the lizard Varanus gouldii (Gray). Gen. Comp. Endocrinol., in press. Robertson, G. L., Mahr, E. A., Athar, S., and Sinha, T. (1973) Development in clinical application of a new method for the radioimmunoassay of arginine vasotocin in human plasma. J. Clin. Invest. 52, 2340-2352. Rodbard, D., and Lewald, J. E. (1970). Computer analysis of radioligand assay and radioimmunoassay data. Acta Endocrinol. Suppl. 147 64, 79. Rosenbloom, A. A., and Fisher, D. A. (1974). Radioimmunoassay of arginine vasotocin. Endocrinology 95, 1726-1732. Sawyer, W. H. (1966). Diuretic and natriuretic responses of the lunglish (Protopterus aethiopicus) to arginine vasotocin. Amer. J. Physiol. 210, 191-197. Sawyer, W. H. (1977). Evolution of neurohypophysial hormones and their receptors. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 36, 1842-1847. Sawyer, W. H., and Pang, P. K. T. (1975) Endocrine adaptation to osmotic requirements of the environment. Endocrine factors in osmoregulation by lungtishes and amphibians. Gen. Comp. Endocrinol. 25, 224-229. Shoemaker, V. H., and Nagy, K. A. (1977) Osmoregulation in amphibians and reptiles. Annu. Rev. Physiol. 39, 449-471. Skowsky, W. R., Rosenbloom, A. A., and Fisher, D. A. (1974). Radioimmunoassay measurement of arginine vasopressin in serum: Development and application. J. Clin. Endocrinol. Metah. 38, 278-287. Yagil, R., and Elzion, Z. (1979). The role of antidiuretic hormone and aldosterone in the dehydrated and rehydrated camel. Comp. Biochem. Physiol. 63A, 275-273.
AND
Plasma
COMPARATIVE
41, l-6 (1982)
ENDOCRINOLOGY
Arginine Vasotocin Concentrations in the Lizard Varanus gouldii (Gray) following Water Loading, Salt Loading, and Dehydration G. E. RICE’
Zoology
Department,
University
of Western
Australia,
Nedlands,
6009
Western
Australia,
Australia
Accepted July 3, 1981 Plasma arginine vasotocin (AVT) concentrations in salt-loaded, dehydrated, and waterloaded Varanus gouldii were measured by radioimmunoassay and averaged 7.1 2 1.5, 3.9 f 0.3, and 1.6 f 0.4, respectively. Plasma AVT and osmolality were correlated signiticantly (P < 0.001) and this relationship is described by the equation AVT (pglml) = O.OSS(P,, - 276). The plasma osmotic threshold for AVT release was found to be similar to that previously reported for other species.
water loading, salt loading, and dehydration. The renal function of this species under these three treatments has been reported previously (Bradshaw and Rice, 1981; Rice et al., 1982).
The neurohypophyseal hormone arginine vasotocin (AVT) has been isolated from the pituitary glands of all nonmammalian vertebrates examined to date (Sawyer, 1977; Sawyer and Pang, 1975) and in many species it has been implicated in the control of kidney function (Babiker and Rankin, 1979; Bentley, 1974, 1976; Bradshaw, 1975; Bradshaw and Rice, 1981; Dantzler, 1976; Sawyer, 1966, 1977; Shoemaker and Nagy, 1977). Recent studies on reptilian renal physiology have provided evidence that is consistent with AVT acting as the physiological antidiuretic principle (Dantzler et al., 1970; Dantzler, 1976, 1978; Bentley, 1976; Bradshaw, 1978; Bradshaw and Rice, 1981; however, plasma AVT concentrations have yet to be measured in reptiles and it is therefore difficult to distinguish between physiological and pharmacological doses of AVT used to establish its role in the control of renal function. In order to clarify the physiological role of AVT in reptiles and to establish the effects of various osmotic challenges on AVT release, plasma concentrations of AVT were measured in the lizard Vuranus gouldii under conditions of
MATERIALS
AND METHODS
Varanus gouldii (Gray) were collected on Dandaraga Station (27”03’ S, 119”05’ E) 560 km north of Perth, Western Australia. They were maintained in an outside terrarium and fed canned dog food once per week. Water was provided ad libitum. The mean body weight of the experimental animals was 1.85 kg (range 0.90-2.85 kg). During the conditioning and treatment periods, animals were housed in individual cages in a constant cycle room with a 12:12 LD photoperiod and maximum and minimum temperature and relative humidity of 37 and 20” and 60 and 30%, respectively. The three experimental treatments utilised in the present study were: water loading, animals received daily intraperitoneaf injections of distilled water (5 ml000 kg) for a period of 7 days; salt loading, animals received daily intraperitoneal injections of 0.2 ml/l00 g of 2.14 M NaCl for a period of 7 days; and dehydration, animals were denied access to water and food for a period of 10 days which resulted in approx 8-10% reduction in body weight. Plasma sampling procedure. On completion of the conditioning period, 3- to S-ml intracardiac blood samples were collected in chilled 5-ml plastic syringes for electrolyte and hormone determinations. The samples were collected between 0830 and 0930 hr, 24 hr after the final injection of the condition period for salt- and water-loaded animals. All samples analysed were collected within 3 min of handling the animal. They were immediately placed in ice and then centrifuged at Animals
’ Present address: Department of Obstetrics and Gynecology, University of Texas Health Science Center at Dallas, 5523 Harry Hines Blvd., Dallas, TX 75235.
and
treatments.
1 0016~6480/82/050001-06$01.00/0 Copyright 0 1982 by Academic F’ress, Inc. All rights of reproduction in any form reserved.
2
G. E. RICE
2OOOgfor 15 min at 4” within a maximum of IS min of Assay standards. Synthetic AVT (Ferring, 215 collection. The l-ml aliquots of plasma for the meaIU/mg) dissolved in 0.2 M acetic acid and 0.1% BSA surement of AVT concentrations were extracted imwas used for the standards in the assay. It was stored mediately. The remaining plasma was stored at -20 as 600~~1 aliquots in l-ml plastic containers at -20” in until the analysis of electrolyte concentrations and three stock concentrations: 32 &ml, 320 r&ml, and osmolality. 640 pg/ml. This procedure facilitated easy dilution to Field plasma samples were collected during late the eight standards used in the standard curve: 32, 16, spring (November 1979) from V. goal&i on Danaraga 8, 4, 2, 1, 0.5, and 0.25 pg/tube. Fresh standards were Station. Intracardiac blood samples used for the prepared l-2 hr before each assay. analyses were collected within 3 min of sighting the Assay protocol. Plasma AVT concentrations were animal and were hand-centrifuged for S to 6 min in measured using a late-addition, double-antibody assay lithium heparin-coated tubes containing Kwikspin system similar to that described by Koike et al. (1979). granules (Disposable Products, Western Australia). A rabbit antibody raised against 8-arginine vasopresThe plasma was transferred to S-ml plastic containers sin, with a cross-reactivity with oxytocin of ~5% (Fercontaining 100 ~1 of 1% acetic acid (Yagil and Elzion, ring, AB), was used in a 0.5-ml assay system 1979) and then was stored in liquid air until extracted (Weitzman, personal communication). The assay was on returning to Perth, Western Australia. performed at a final antibody dilution of l/250,000. Extraction procedure. The l-ml aliquots of plasma Triplicate SO-w1 aliquots of appropriate standard and were extracted within 1 to 2 hr after collection using a duplicate 200-~1 aliquots of plasma extracts were incumodification of the acetone extraction procedure debated at 4” in 10 x 7S-mm plastic tubes (3DT, Disposscribed by Robertson et al. (1973). Extracts were preable Products) with SO ~1 of AVT antibody and phospared by mixing 1 ml of plasma with 2 ml cold (4”) phate buffer (0.01 M, pH 7.4) to a final volume of 450 acetone (Merck) on a vortex mixer for 30 set to pre- ~1. The SO~1 (looO-2000 cpm) of monoiodinated AVT cipitate the plasma proteins; and after centrifuging at was added 24 hr later, mixed for 15 set on a vortex 2oOOg for 30 min at 4”, the supematant was decanted mixer, and incubated at 4” for a further 72 hr. Free and and mixed with 5 ml of cold (4”) n-hexane (Merck) bound hormone were separated using a second and centrifuged again for 15 min at 2OOOgand 4”. The antibody (donkey antirabbit, Wellcome Laboratories); organic phase was aspirated carefully and discarded. 200 ~1 of 1124 dilution of this antibody in phosphate The aqueous phase was evaporated to dryness under buffer was added to each tube and incubated at 4” for a stream of room-temperature air in a 37” water bath. 18 hr; the supernatant was then aspirated, and the The residue was dissolved in 0.5 ml of phsophate buff- precipitate was counted to 1% error with a Prias er, sealed with parafilm (American Can Co.), stored Autogamma counter (Packard). at -20” overnight, and assayed the next day. Analyses. Sodium and potassium concentrations lo&nation. Synthetic 8-arginine vasotocin (Ferring) were determined in I+1 aliquots of plasma with a was iodinated by the chloramine-T oxidation method Varian-Techtron atomic absorption spectropho(Hunter and Greenwood, 1962) adapted for AVT tometer (Type AA-5), using a caesium diluent (1.27 (Weitzman, personal communication). The following g/liter). Chloride concentrations were measured by were added to a reaction vessel: 50 ~1 of 0.5 M phosamperometeric titration with a Buchler Digital chlorphate buffer (pH 7..5), 37 MBq of carrier-free lz51 idometer on lo-p.1 samples. Osmolality was mea(Radiochemical Centre, Amersham), 25 yl of AVT (0.2 sured on 6-~1 samples with a Wescor vapor pressure pg/kl) in 0.2 M acetic acid, and 25 ~1 (50 pg) of osmometer (Model SlOOB). chloramine-T in distilled water. The reaction vial was Statistics. Treatment means were compared using vortexed for 3 set and 15 set after the addition of analysis of variance coupled with a Student-Newchloramine-T, 200 ~1 of 25% bovine serum albumin man-Keuls test. (BSA, Sigma) was added, and the vial was vortexed for 10 sec. The reaction mixture was transferred to a RESULTS 0.9 x 2S-cm G2S Sephadex column and eluted with 0.2 M acetic acid containing 0.1% BSA. The column Assay Characteristics was prewashed with 2% BSA in 0.2 M acetic acid. Using the late-addition double-antibody Fractions of 1 ml were collected by an LKB fraction assay technique, the minimal limit of decollector and 2S-~1 aliquots were counted with a Prias tection of the assay, defined as the least Autogamma counter. Undamaged monoiodinated of unlabelled hormone that AVT was identified by its ability to bind to antibody (in concentration excess). The three postpeak fractions from the profile can be distinguished by an error estimate of of iodinated AVT were pooled and rechromatographed two standard deviations from a sample on a 0.9 x 2S-cm Sephadex G2S column and eluted containing no hormone (Chard, 1978), avwith 0.2 M acetic acid and 0.1% BSA. The l-ml fractions were collected and the second and third postpeak eraged 0.92 pg/ml for extracted plasma esfractions of monoiodinated AVT were pooIed and timated from three assays, and 0.21 k 0.14 stored at -20” as 50-~1 aliquots. pg/tube for the normal standard curve esti-
PLASMA
AVT
CONCENTRATIONS
Logit b 0
-
-1
-
-2
-
-3 025
0.5
1 NT
2
4
8
16
(W/tube)
FIG. 1. Logit-log transformation of the standard curves from 10 assays. The mean ? standard error is reported; r = 0.998, y =- -1.258x + 0.70.
mated from 10 assays. Figure 1 presents a logit-log transformation (Rodbard and Lewald, 1970) of the standard curves from 10 assays. The regression coefficient associated with this linear transformation was 0.998 (P -=c0.05).
In two assays, six plasma replicates were assayed to assess the intraassay coefficient of variation, and in three assays duplicate extractions of a standard plasma were assayed to assess the interassay coefficient of variation. These coefficients average 9.4 and 16.4%, respectively. In two assays, duplicates of known amounts of standard AVT (64, 32, 16, 8,
PLASMA SODIUM, CONCENTRATIONS
Treatment Water loading (II = 6) Salt loading (n = 6) Dehydration (n = 6) Field samples (n = 15)
IN
and 4 pg) were added to bentonite-extracted plasma (Skowsky et al., 1974) and assayed. The recoveries of standard AVT average 105.1 + 0.16% when corrected for extraction losses (n = 20). The recovery of AVT, estimated for each assay from the extraction of six replicates of a standard plasma to which a known amount of radiolabelled hormone had been added, averaged 72.5 2 3.1% (from 10 assays). The plasma AVT concentrations reported are uncorrected from recovery. The cross-reactivity of the assay system with oxytocin was assessed (Chard, 1973) and was CO. 1%. The cross-reactivity of the assay system with mesotocin was not assessed. Plasma Electrolyte Concentrations and Osmolality
Plasma sodium, potassium, and chloride ion concentrations and osmolality for water-loaded, salt-loaded, dehydrated, and field-sampled V. gouldii are presented in Table 1. Osmolality significantly increased in both dehydrated and salt-loaded animals when compared with water-loaded animals (P < O.OOl), and the values are similar to those reported by Bradshaw and Rice (1981) and Rice et al. (1982) for this species. The plasma osmolality of field-sampled animals were not significantly different from the
TABLE 1 POTASSIUM, AND CHLORIDE ION CONCENTRATIONS FOR WATER-LOADED, SALT-LOADED, DEHYDRATED,
Na+ (meq/liter)
K+ (meq/liter)
Cl(meq/liter)
*135.9 + 2.48 P < 0.001 176.1 2 6.50
n 4.34 2 0.19 NS 5.42 k 0.34
*135.3 f 3.9 P < 0.01 165.4 k 5.8 P < 0.01 *146.4 2 6.9 NS 138.4 k 5.2
P < 0.025
NS
*156.3 ? 3.41 NS 146.6 f 3.82
n 4.84 k 0.55 NS 4.76 k 0.23
3
Varanus gouldii
AND OSMOLALITY AND AVT AND FIELD Vat-anus gouldii
Osmolality (mOsm/kg) *274 z!z10.5 P < 0.001 375 + 7.4 P < 0.001 *325 5 8.5 NS 321 2 5.2
AVT (pdml) *1.6 f 0.4 0.001 7.1 t 1.5 P < 0.001 *3.9 Ii 0.3 NS 3.5 t 0.4
P <
Note. The mean 2 SE is reported. Statistical comparisons between the means for water-loaded, salt-loaded, and dehydrated animals were made by the Student-Newman-Keuls test. Student’s t tests were used to compare the field sample means with the means for dehydrated animals; n is the number of animals. (1NS, not significant. *P < 0.01.
4
G. E. RICE
values observed for dehydrated animals. The increases in plasma osmolality are due primarily to increased NaCl concentrations and these changes are of more significant proportions in salt-loaded animals (P < 0.001). Plasma potassium ion concentrations were unaltered by the treatments and were not significantly different from the values reported for the field animals. Plasma AVT Concentrations
Plasma AVT concentrations with the three treatments are also shown in Table 1 and range from below the limit of detection of the assay (0.92 pg/ml) to 13.5 pg/ml (uncorrected for recovery). AVT concentrations significantly increased with both salt loading and dehydration, when compared with the concentration observed for water-loaded animals (P < 0.001). This increase was significantly greater in the case of salt loading (P < 0.001). The increase in plasma AVT concentrations are consistent with the rise in plasma osmolality and the relationship between these two parameters is presented in Fig. 2. The data presented are for 6 water-loaded, 6 salt-loaded, 6 dehydrated, and 15 field V. goufdii (n = 33). A significant positive regression between plasma osmolality and AVT concentrations was obtained (r = 0.776, P < 0.001) and is described by the equation AVT (pgml) = O.OSS(P,,, - 276). DISCUSSION
The present study reports the first radioimmunoassay measurements of plasma arginine vasotocin concentrations for a reptile and documents the effects of dehydration, chronic sodium chloride loading, and water loading on the circulating levels of this hormone. In the lizard V. gouldii, circulating levels ranged from 1 to 18 pg/ml (when corrected for recovery, see Materials and Methods) and were related significantly to plasma osmolality . Bradshaw and Rice (1981) reported renal changes in V. gouldii in response to
-11351
[ AVT (pglml)
9 y
6 5 4 3 2 1
0
.
.*
. . *. *. .. .*. 260
. 280
.
.
..
:
.
* :
. .
. . xx)
320
340
Kkmlhg
3M)
380
403
HP0
FIG. 2. The relationship between plasma AVT concentrations (pg/ml) and plasma osmolality (mOsm/kg H,O). The regression line fitted to the data is described by the equation AVT (pg/ml) = 0.085 (P,,, - 276), r = 0.776, P < 0.001.
salt loading and dehydration that were consistent with the release of AVT into the circulation and its antidiuretic action. Glomerular filtration rate, relative free water clearance (CH2JCin), and urine volume significantly decreased, and fractional reabsorption of water significantly increased. Similarly, acute exogenously administered AVT evoked an antidiuresis due to a fall in glomerular filtration rate and increased tubular water permeability; both of these effects were abolished completely by the subsequent injection of probenecid, a competitive inhibitor of AVT action in the lower vertebrates (Dantzler et al., 1970). The antidiuresis associated with dehydration was of a more tubular response when compared with salt-loaded animals, and these data were interpreted as indicating higher circulating levels of AVT with salt loading than those which occur with dehydration. The data from the present study confirm this interpretation; salt loading resulted in an 80% increase above the values observed with dehydration. In the few species where plasma AVT concentrations have been measured, similar increases in concentration have been observed. In dehydrated Rana catesbeiana, plasma AVT concentrations increased from about 5 pg/ml in normally hydrated individuals to 60 pg/ml (Rosenbloom and Fisher, 1974; Pang et al. 1974; Sawyer
PLASMA
AVT
CONCENTRATIONS
and Pang, 1975; Pang, 1977). In the domestic fowl, Koike et al. (1977) reported a mean plasma concentration of 4.4 $/ml for hydrated hens and this value increased to 18 pglml with 96-hr water deprivation. Similar values have been observed in the Pekin duck; in normally hydrated birds, plasma AVT concentration was 5.1 fmol/ml and with salt water adaptation the mean concentration increased to 22.7 fmol/ml (Mohring et al., 1980). Plasma AVT concentrations in V. god&i were correlated significantly with plasma osmolality and this relationship has previously been demonstrated in the chicken (Koike et al., 1977, 1979) and the Pekin duck (M&ring et al., 1980). Acute 5% saline loading of R. catesbeiana, although producing an antidiuresis and elevated plasma osmolality, did not increase circulating AVT centrations above control values, and it was suggested that in this species plasma AVT concentration and osmolality are only “loosely” related. The linear regression equation fitted to the plot of plasma AVT concentrations and osmolality provides estimates of the sensitivity and the threshold of the osmotically mediated AVT release mechanism. In V. gouldii, the slope of the regression line is less than that observed for birds or mammals, indicating a lower sensitivity of the AVT release mechanism to changes in the plasma osmotic pressure. In this species, plasma osmolality. In this species, plasma AVT increased by 1 pg/ml for an increase in plasma osmolality of 11.9 m&m/kg. The threshold of plasma osmolality, determined by the intercept of the regression line with the abscissa, was 270 mOsm/kg and is similar to the values reported for both birds (Koike et al., 1977, 1979, Mohring et al., 1980) and mammals (Robertson et al., 1973; Dunn et al., 1973; Moses and Miller, 1974). The AVT levels measured in plasma samples collected from animals in the field are in good agreement with the laboratorv
IN
Varanus
5
gouldii
data. The plasma AVT concentrations in field animals during late spring are not significantly different from the values observed in laboratory-dehydrated animals. As the daily maximum temperatures on Dandaraga Station during November 1979 averaged 38” and as surface water was not available during the study period, it is reasonable to suggest that the animals collected were mildly dehydrated. Plasma electrolyte concentration and osmolality of the field animals were also not significantly different from the values reported for the dehydrated animals. ACKNOWLEDGMENTS This work was supported by a research grant from the University of Western Australia. Especial thanks are due to Dr. R. E. Weitzman for providing AVP antibody and helpful suggestions on the assay methodology, and to Dr. E. Horgan and Dr. G. Martin for their assistance during the development of the assay. I am most grateful to Professor S. D. Bradshaw for his valuable comments and discussion of this work. Expert technical assistance was provided by Mr. .I. R. Beesley. The author was in receipt of a Commonwealth Postgraduate Award.
REFERENCES Babiker, M. M., and Rankin, J. C. (1979). Renal and vascular effects of neurohypophysial hormones in the African lungfish Protopterus annectens (Owen). Gen. Comp. Endocrinol. 37, 26-34. Bentley, P. J. (1974). Actions of neurohypophysial peptides in amphibians, reptiles and birds. In “Handbook of Physiology” (S. R. Geiger, ed.), pp. 19-28. Amer. Physiol. Sot. Washington, D.C. Bentley, P. J. (1976). Osmoregulation. In “Biology of Reptilia” (C. Gans and W. R. Dawson, eds.), Vol. V, pp. 365-412. Academic Press, New York/ London. Bradshaw, S. D. (1975). Osmoregulation and pituitary-adrenal function in desert reptiles. Gen. Comp.
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25,
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Bradshaw, S. D. (1978). Volume regulation in desert reptiles and its control by pituitary and adrenal hormones. In “Osmotic and Volume Regulation” (C. B. Jorgensen and E. Skadhauge, eds.), pp. 38-59. Munksgaard, Copenhagen. Bradshaw, S. D., and Rice, G. E. (1981). The effects of pituitary and adrenal hormones on renal and post-renal reabsorption of water and electrolytes in the lizard Varanus gouldii (Gray). Gen. Comp. Endocrinol.
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G. E. RICE
Chard, T. (1973). The radioimmunoassay of oxytocin and vasopressin. J. Endocrinol. 58, 143- 160. Chard, T. (1978). An introduction to radioimmunoassay and related techniques. In “Laboratory Techniques in Biochemistry and Molecular Biology” (T. S. Work and E. Work, eds.), New-Holland Publ., Amsterdam/NewYork/Oxford. Dantzler, W. H. (1976). Renal function (with special emphasis on nitrogen excretion). In “Biology of the Reptilia” (C. Cans and W. R. Dawson, eds.), Vol. V, pp. 447-503. Academic Press, New York/London. Dantzler, W. H. (1978). Some renal and glomerular and tubular mechanisms involved in osmotic and volume regulation in reptiles and birds. In “Osmotic and Volume Regulation,” (C. B. Jorgensen and E. Skadhauge, eds.) pp. 187-201. Alfred Benzon Symp. XI, Munksgaard. Dantzler, W. H., Schaffner, D. P., Chiu, P. J. S., and Sawyer, W. H. (1970). Probenecid inhibition of arginine vasotocin in snakes, frogs, and on frog bladdersin virro. Amer. J. Physiol. 218,929-936. Dunn, F. L., Brenna, T. J., Nelson, A. E., and Robertson, G. L. (1973). The role of blood osmolality and volume in regulating vasopression secretion in the rat. J. C/in. Invesf. 52, 3213-3219. Hunter, W. M., and Greenwood, F. C. (1962). Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature (London) 194, 495-496. Koike, T. I., Pryor, L. R., Neldon, H. L., and Venable, R. S. (1977). Effect of water deprivation of plasma radioimmunoassayable arginine vasotocin in conscious chickens (Callus domesticus). Gen. Corn. Endocrinol. 33, 359-364. Koike, T. I., Pryor, L. R., and Neldon, H. L. (1979). Effect of saline infusion on plasma immunoreactive vasotocin in conscious chickens (Gallus domesticus). Gen. Comp. Endocrinol. 37, 451-458. Mohring, J., Schoun, J., Simon-Oppermann, C., and Simon, E. (1980). Radioimmunoassay for arginine vasotocin (AVT) in serum of Pekin ducks: AVT concentrations after adaptation to fresh water and salt water. Pfluegers Arch. 387, 91-97. Moses, A. M., and Miller, M. (1970). Osmotic influences on the release of vasopressin. In “Handbook of Physiology” (E. Knobil and W. H.
Sawyer, eds.), Section 7, pp. 225-255. Amer. Physiol. Sot., Washington, D.C. Pang, P. K. T. (1977). Osmoregulatory functions of neurohypophysial hormones in fishes and amphibians. Amer. Zool. 17, 739-749. Pang, P., Sawyer, W. H., and Casals, C. (1974). Circulating levels of arginine vasotocin (AVT) in lower vertebrates. Amer. Zool. 14, 1244. Rice, G. E., Bradshaw, S. D., and Prendergast, F. J. (1982). The effects of bilateral adrenalectomy on renal function in the lizard Varanus gouldii (Gray). Gen. Comp. Endocrinol., in press. Robertson, G. L., Mahr, E. A., Athar, S., and Sinha, T. (1973) Development in clinical application of a new method for the radioimmunoassay of arginine vasotocin in human plasma. J. Clin. Invest. 52, 2340-2352. Rodbard, D., and Lewald, J. E. (1970). Computer analysis of radioligand assay and radioimmunoassay data. Acta Endocrinol. Suppl. 147 64, 79. Rosenbloom, A. A., and Fisher, D. A. (1974). Radioimmunoassay of arginine vasotocin. Endocrinology 95, 1726-1732. Sawyer, W. H. (1966). Diuretic and natriuretic responses of the lunglish (Protopterus aethiopicus) to arginine vasotocin. Amer. J. Physiol. 210, 191-197. Sawyer, W. H. (1977). Evolution of neurohypophysial hormones and their receptors. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 36, 1842-1847. Sawyer, W. H., and Pang, P. K. T. (1975) Endocrine adaptation to osmotic requirements of the environment. Endocrine factors in osmoregulation by lungtishes and amphibians. Gen. Comp. Endocrinol. 25, 224-229. Shoemaker, V. H., and Nagy, K. A. (1977) Osmoregulation in amphibians and reptiles. Annu. Rev. Physiol. 39, 449-471. Skowsky, W. R., Rosenbloom, A. A., and Fisher, D. A. (1974). Radioimmunoassay measurement of arginine vasopressin in serum: Development and application. J. Clin. Endocrinol. Metah. 38, 278-287. Yagil, R., and Elzion, Z. (1979). The role of antidiuretic hormone and aldosterone in the dehydrated and rehydrated camel. Comp. Biochem. Physiol. 63A, 275-273.