GENERAL

AN0

COMPARATIVE

ENDOCRINOLOGY

28, 413-419 (1976)

ffects of Stress and ACTH on Plasma Corticosterone Cai man Caiman crocodi/~~~ DANIEL

H. GIST AND MARVIN

Levels i

L. KAPLAN

DepartmentofBiologicn1 Sciences, University of Cincinnnti. Citxinnati,

Qllio 45221

Accepted September 9, 1975 Corticosterone levels in blood plasma of the caiman were measured in response to the collection of blood and to the administration of mammalian ACTH. Blood collection resulted in an increase in plasma corticosterone levels; the highest levels of corticosterone were observed 2 hr after the initial sample, and corticosterone levels declined thereafter despite continued blood collection. Prior treatment of caimans with dexamet asone abolished the response to blood collection. Administration of mammalian ACT to dexamethasone-treated caimans resulted in elevated corticosterone levels. The levels of corticosterone measured in plasma did not vary with the amount of ACTM administered. but the duration of the steroidogenic response was dose-dependent.

The requisite hormones for a pituitaryadrenocortical axis (viz pituitary ACTH activity and circulating corticosteroids) are known to occur in representatives of three (Chelonia, Crocodilia, Squamata) out of the four living orders of reptiles, and corticosterone is known to be the major secretory product of reptilian adrenocortical tissue (Sandor, 1972). However, definitive evidence of a steroidogenic action of ACTH upon the reptilian adrenal in Gvo is limited to iguanid t and Bradshaw, 1969; Daugherty allard, 1972; Callard et al.. 1973) amid (Bradshaw, 1975) lizards and the freshwater turtle Chrysemys picta (Callard, 1975). In summary, administration of either mammalian ACTH or reptilian pituitary extracts to intact or dexamethasone-blocked animals elicits a rise in circulating corticosterone levels within 15 to 30 min (Licht and Bradshaw, 1969; Callard et al., 1973). Furthermore, hypophysectomy or pharmacological inhibition of ACTH secretion both result in reduced levels of circulating corticosterone. Evidence for an integral hypothalamic involvement in the control of adrenocortical secretions in lizards is provided by Daugh-

erty and Callard (1972) where lesions in the anterior hypothalamus but not the cere result in a reduction in plasma cortico one concentrations. Significantly, irn~la~t~ of either corticosterone or aldoste the anterior hypothalamus but not tary likewise depress plasma co~coster~~e levels (Callard et al., 1973). Thus in these lizards at least, there is evidence s~~ge~~~~g the existence of a hy~othai~o-~y~o~~yseal-adrenocortical axis which is qu~i~~v~ly similar to that observed in other vertebrates (Jorgensen, 1968; Fortier, 1966). The only attempt to examine the in V~VO steroidogenic response to ACTH in re other than those noted above is that of Nothstine et al. (1971) wko mea costeroid concentrations in mix renal effluent blood of the turtle (Pse~demys sueanniensis) and the caiman (C’c16n?~ti sclevops). Corticosterone was ide~ti~ed as the principal circulating corticosteroid in the dexamethasone-treated caiman, amounts of cortisol and aldoste ever, the levels of these steroids were unaffected by the infusion of either mammalian ACTH or caiman kidney extracts, presumed to contain renin. In the turtle. where only corticosterone was detects 413

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e 1976 by Academic Press. Inc. of reproduction in any form reserved.

414

GIST

AND

mammalian ACTH was without effect whereas turtle kidney extract had both a pressor and a steroidogenic effect. The absence of a steroidogenic action of mammalian ACTH in the caiman is of particular significance, since these reptiles are one of the closest living representatives of the stem reptiles from which both birds and mammals were derived. In view of the implications of these findings in the evolution of adrenocortical controls, this study was undertaken to reexamine in the caiman (Cai~nan crocodilus) the possible role of ACTH in adrenal steroidogenesis. MATERIALS

AND METHODS

Animals. Twenty-six South American caimans (Caiman crocodilus) were obtained from commercial suppliers and housed in a greenhouse under a natural photoperiod for varying periods, in containers previously described (Gist, 1972). The temperature of the greenhouse was regulated at 32 a although the actual air temperature varied according to changing climatic conditions. Caimans used in these experiments were of undetermined sex and ranged in weight from 0.5 to 6 kg. They were fed horsemeat at weekly intervals during the summer months and less frequently in the winter when their appetite was reduced. The concentration of corticosterone in sequential blood samples was used as a measure of adrenocortical response to experimental manipulations. Samples of blood (2-4 ml) were taken via cardiac puncture from unanesthetized caimans, blood was transferred to heparinized centrifuge tubes, and the plasma was separated and stored at -20 ’ until extracted. Initial experiments. Caiman were removed from the greenhouse and placed in an environmental chamber (Sherer Model CEL 37-14) at 28 ‘for 2 hr prior to the withdrawal of the first blood sample. Porcine ACTH (ACTHar, Armour; diluted with reptilian physiological saline to a concentration of 20 units/ml) was administered intraperitoneally at a level of 20 units/kg immediately following the first blood sample. Subsequent samples of blood were taken at 1.5, 30, 60, and 120 min following the injection. Control animals were sampled identically, but no injections were given. Subsequent experiments. A more rigorous maintenance procedure for the caimans was utilized in the remainder of the experiments. Food was withheld from the caimans 10 days prior to their experimental use. Three days prior to the experiment, a caiman

KAPLAN

was removed from the greenhouse and placed in the environmental chamber where it remained, except for sampling, until the end of the experiment. A photoperiod of 12:12 LD was employed and the air temperature of the chamber was maintained at 30 ‘. A tank of water, sufficient in depth so that the caiman could completely submerge, was placed in one half of the chamber. The temperature of the water in the chamber was 269 During the experiments, most animals preferred to remain in the water. Cloaca1 temperatures were recorded during the removal of blood samples, and ranged between 26 and 30”. At the end of the experiment, the cairnan was returned to the greenhouse and following an interval of no less than 3 months was used in subsequent experiments. Preliminary experiments indicated that six intraperitoneal injections of dexamethasone (Decadron; Merck, Sharp, and Dohme, 4 mgiml) spaced 12 hr apart and each consisting of 1 mg dexamethasonekg body wt were sufficient to maintain a “basal” level of circulating corticosterone for the 36-hr period immediately following the last injection. Based on these preliminary studies, this injection protocol was used in all experiments reported herein involving dexamethasone treatment, except in one series (see below) where additional injections were given. When used, ACTH was suspended in reptilian physiological saline and administered as a single intraperitoneal injection given 12 hr following the last dexamethasone injection. Dosages of ACTH employed were 0.3, 3.0, or 30 units ACTH/kg body wt given in a volume of 1 ml. Blood samples were removed from the caiman immediately prior to and at intervals following ACTH administration, each animal serving as its own control. In all experiments samples were taken at the same time of day (initial sample; 9 AM EDT) to minimize the effect of diurnal variations in corticosterone levels. Extraction and quantitation. The extraction procedure of Frankel et a/. (1967) was used with minor modifications; all reagents were of spectroquality grade. Blood plasma (2 ml) was diluted with an equal volume of glass distilled water and 0.01 yCi of [1,2 -3H] corticosterone (New England Nuclear; spec. act. 40-50 PCiimmole diluted to 0.1 &i/ml with ethanol) was added. The mixture was extracted three times with 3 vol of dichloromethane-carbon tetrachloride (l:l), and once with 3 vol of dichloromethane. The combined organic phases were washed once with 5 ml of 0.1 N NaOH, once with 5 ml of 0.2 N acetic acid, once with 5 ml of glass-distilled water and then evaporated to dryness under air at 379 T’he residue was dissolved in 5 ml of 13% EtOH and partitioned against 15 ml of N-hexane. The ethanolic layer was then extracted three times

STRESS AND ACTH IN THE CAIMAN with 5 ml of dichloromethane, the solvent was evaporated to dryness under air, and the residue was concentrated in the bottom of a conical tube. Separation of steroids by paper chromatography and elution of the areas corresponding to authentic corticosterone were essentially as decribed previously (de Roos. 1961). Eluted steroids were dissolved in 10 ml ethanol and a O.5-ml aliquot was removed. The radioactivity was determined in a Packard Tri-carb liquid scintillation counter using a toluene PPO-POPOP cocktail. The counting efficiency was 20-40% and recoveries of added corticosterone averaged 6@-70%. The remainder of the eluted sterioids was evaporated to dryness and dissolved in ethanolic &SO, (H,SO,-50% ethanol. 4X:20). Following incubation for 1 hr ai room temperature, fluorescence was measured with a Turner Model 110 fluorometer using primary filters ?A and 47B. and 2A-12 as a secondary. Eluted blanks were treated identically. The fluorescence was compared to a similarly prepared curve of authentic corticosterone. Authenticity of the corticosterone extracted from caiman plasma was verified by comparing its excitation and emission spectra with that of authentic corticosterone on an Aminco-Bowman spectrofluorometer. RESULTS

lrzitial experiments. Sequential sampling of blood from the same caiman allowed the monitoring of plasma corticosterone levels over varying time intervals. Figure 1 compares the plasma corticosterone levels in untreated caimans with those receiving a single injection of 20 units/kg ACTH over a 2-h period. As evidenced by the large standard errors. there was considerable variation both between animals as well as within the same animal during the sampling

ma corticosterone levels in both groups rose during the sampling period and at the end of 2 r were significantly higher (1” < .05) than at the beginning of the experiment. However, no difference was observed between those caimans receiving ACTH and those receiving no injection. These initial experiments suggested that handling and/or sequential blood removal itself could be responsible for the observed rise in corticosterone levels. ~l~~s@~~ent experiments. Untreated cai-

16

415

r

FIG. I. Plasma corticosterone levels in sequential blood samples from untreated caimans and caimans receiving a single intraperitoneal injection of ACTH immediately following the initiai blood sample. Data are presented as the mean concentration = the standard error.

mans sampled over a 4-hr period e~~~b~ted elevations in plasma corticosterone levels at 2 and 4 hr following the initial sample (Table 1). maximum ConcentraFions of plasma corticosterone were observed at 2 hr, and these declined slightly at 4 hr. C&mans sampled following dexamethaso~e administration did not exhibit an ~~~vati~~ in corticosterone levels over a 6-hr sampling period. Corticosterone levels were not. however, depressed following dexarn~t~~sone administration e In order to determine whether dexamet asone-treated caimans were capable of responding to exogenous ACTH, untreated caimans were sampled over a 6-m period; l& hr later dexamethasone t initiated and continued for 7 fourth day of treatment, the &mans were sampled during a 6-hr period and on 1 seventh day they were sampled a 24-hr period fo~owi~g a single injection of 3 units ACTWkg. The results of these experiments are presented in Fig. 2. Following the period of dexamethasone treatment, during which plasma levels were unaltered by bl caimans responded to exogenous A

416

GIST AND KAPLAN

TABLE 1 EFFECTSOF DEXAMETHASONEON PLASMA CORTICOSTERONE LEVELS INTHE CAIMAN Cairnan Hours following initial sample

n No treatment Dexamethasone*

7 4

crocodilus

0

2

4

6

2.20+- .47” 2.43 2 .25

6.745 1.75 2.32 + .44

5.74c .15 2.392 .41

2.332 .31

” Data are presented as the mean corticosterone concentration -r- the standard error. * A total of 6 mg/Kg dexamethasone was given in six equal injections spaced 12 hr apart. The second collection period was initiated 12 hr following the last injection.

increasing plasma corticosterone levels. The response was evident at 2 hr, reached a maximum at 6 hr, and only after 24 hr did corticosterone levels return to near preinjection levels. Although the maximum corticosterone levels observed in response to ACTH injections were similar to those attained following blood collection from the same caimans prior to dexamethasone treatment, the time required to attain maximum concentrations was shorter (2 hr) in untreated caimans . The responses of dexamethasone-blocked &mans to varying dosages of ACTH are presented in Fig. 3. Caimans receiving reptilian physiological saline exhibited little change in plasma corticosterone levels, but those receiving 0.3, 3.0, or 30 units ACTH/

kg responded with increasing plasma corticosterone concentrations. During the first 6 hr of the response, no difference could be detected among the various dose levels, but after 6 hr, plasma corticosterone levels in animals receiving 0.3 or 3.0 units ACTHIkg began to decline and by the end of the 24hr sampling period had returned to near preinjection levels. Plasma corticosterone levels in caimans receiving 30 units ACTH/kg, however, continued to rise, peaked at 12 hr, and at 24 hr were still sixfold higher than prior to ACTH administration. DISCUSSION

Concentrations of corticosterone recorded in undisturbed (i.e., no prior injections or blood collections) caimans were in

1Ll FIG. 2. Plasma were first sampled following a single Data are presented

corticosterone levels in sequential blood samples taken from the same caiman. Animals without prior treatment, then again following dexamethasone administration, and finally intraperitoneal injection of ACTH given immediately following the initial blood sample. as the mean concentration from three animals 2. the standard error.

STRESS

AND

ACTH

IN THE

CAIMAN

414

FIG. 3. Effects of ACTH on plasma corticosterone levels in sequential blood samples from the same dexamethasone-treated caiman. ACTH or saline was administered as a single intraperitoneai injection immediately following the initial blood sample. Each bar represents the mean concentration from three caimans -c the standard error except the saline group where n = 4.

the range of 2-5 &lo0 ml plasma. These values agree well with those reported for other reptiles (Licht and Bradshaw, 1969; Daugherty and Callard, 1972; Callard et al., 1973; Callard, 1975; Bradshaw, 1975), but are lower than those reported in postcaval blood of the same caiman (Nothstine et al., 1971). Although concentration differences are to be expected between postcaval and peripheral blood, it is difficult to predict their magnitude in the absence of estimates of postcaval and total blood flow, pammeters not measured in this study. Blood collection from the caiman resulted in an increase in plasma corticosterone concentlrations. Since prior treatment with dexamethasone abolished this response, it may be concluded that blood collection in the caiman is a stress which is capable of eliciting the release of endogenous corticotropin. The ability of less traumatic stimuli such as handling to provoke elevations of circulating corticosteroids is well-documented in teleosts (Leloup-Hatey, 1958; Fagerlund , 1967)) and hypophysectomy or pnior treatment with dexamethasone eliminates this response (Donaldson and Mcride, 1967; Fagerlund and McBride, 1969). There is only limited information regardthe effects of stress upon reptilian enocortical tissue. Confinement is reported to result in elevated adrenal weights

in Scelopovus cyanogenys (Callard and Chester Jones, 1971) and elevated plasma corticosterone levels in Arnp~i~ol~~~~~ ineTmis (Bradshaw, 1975). In the present stu the steroidogenic response to blood collection in the caiman was a transitory one; maximum concentrations of co~icostero~e were observed at 2 hr and declined thereafter despite continued sampling u In the mammal, the steroidogenic to stress is also transient, the paragon o the elevation of corticosteroids being dependent upon the type of stress administered (Dallman and Jones, 1973a.; Alle al., 1973). Furthermore, additional stresses applied at intervals following an initial stress further augment corticosteroid le man and Jones, 1973b). Such appear to be the case in the ca.i even with continued blood collections over a 6-hr period, plasma c~~co~tero~~ levels still declined after 2 hr. These obse~ati~~~ could be accounted for by a negative feedback by corticosterone and/or al to suppress endo~e~ous ACTH Such a feedback has been suggested ‘by Callard et al. (1973), based on re~~cti~~.s in plasma corticos.&erone levels ~oIlQ~i~g implantation of either of these ~o~o~~~ into the anterior hypothalamic region of

418

GIST AND KAPLAN

blood volume is reported to augment plasma corticosterone levels (Callard, 1975). This response, known to operate in mammals via the release of pituitary ACTH (Redgate, 1966), occurs in the turtle in the absence of the pituitary and even in hypophysectomized animals treated with dexamethasone. In the present study, no significant change in corticosterone levels was observed during blood collection from dexamethasone-treated animals. It should be noted, however, that the caiman is capable of a rapid water uptake through the skin (Bentley and Schmidt-Nielsen, 1965) and that in the present study most of the caimans preferred to remain in water during the sampling periods. Such behavior might compensate for a reduced blood volume. Administration of ACTH to dexamethasone-treated caimans was followed by an elevation of plasma corticosterone levels. Unlike the results of Nothstine et al. (1971) caimans in the present study responded to as little as 0.3 units ACTH/kg. A similar sensitivity to porcine ACTH is reported for the turtle C. picta (Callard, 1975). The differing results possibly stem from the short period of dexamethasone infusion used by Nothstine et al. (1971) and/or the surgery prior to blood collections. Our results suggest that such manipulations may be stressful in themselves and might account for both the high levels of corticosterone reported and the absence of an additional response to ACTH infusion. The magnitude of the increase in plasma corticosterone levels was not correlated with the amount of ACTH given during the initial 6 hr of the response. The duration of the steroidogenic response, however, was dependent on ACTH dosage; corticosterone levels returned to preinjection levels within 12 hr in caimans receiving 0.3 units ACTH/kg, whereas those receiving 30 units ACTH/kg retained elevated plasma corticosterone levels 24 hr after hormone administration. These results are difficult to reconcile in view of the dose-dependent steroi-

dogenic action of ACTH in Anolis (Licht and Bradshaw, 1%9) and in C. picta (Callard, 1975) under similar conditions; however, it should be noted that these investigators examined the adrenal response to ACTH over a limited period of time not exceeding 2 hr. The concentrations of corticosterone observed in the caiman following the administration of either 0.3 or 3.0 units of ACTH were both similar in magnitude to those observed following blood collection alone in untreated animals. Thus it is unlikely that the difference in length of the response between these two dosages is a pharmacological one. The temperature at which these experiments were conducted, 30”, is lower than the preferred temperature of C. crocodilus (349, recently reported by Diefenbach (1975). Sub-optimal temperatures are known to prolong and diminish the adrenocortical response to exogenous ACTH in A. carolinensis (Licht and Bradshaw, 1969) and D. dorsalis (Callard et al., 1973), and might account in part for the extended responses observed in the caiman. The dose-dependent longevity of the corticosterone response to ACTH might also be explained on the basis of a slow degradation of circulating corticosterone. The hepatic enzymes associated with steroid catabolism in mammals are not readily induced by high corticosteroid levels (Yates et al., 1959), and a low activity of these enzymes in the caiman could account for the prolonged response seen in this study. ACKNOWLEDGMENTS We are grateful to Dr. Wendel W. Leavitt in whose laboratory some of the initial experiments were conducted. This study was supported in part by National Science Foundation Grant GB-14987.

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AND

ACTH

Bentley, P. J., and Schmidt-Nielsen, K. (1965). Permeability to water and sodium of the crocodilian, Caimnn sclerops. J. Cell. Camp. Physiol. 66.303-310. Bradshaw, S. D. (1975). Osmoregulation and pituitary-adrenal function in desert reptiles. Gen. Camp. End0u+ifrol. 25, 230-248. Callard. G.V. (1975). Control of the interrenal gland of the freshwater turtle Chrysemys picta in viva and in vitro. Gen. Comp. Endocrinol. 25, 323331. Callard. I. P., Ghan, S. W. C., and Callard, G. V. (1973). Hypothalamic-pituitary-adrenal relationships in reptiles. In “Brain-Pituitary-Adrenal Interrelationships” (A. Brodish and E. S. Redgate. eds.), pp. 270-292. S. Karger, Basel. Dallman. M. F.y and Jones, M. T. (1973a). Corticosteroid feedback control of stress-induced ACTH secretion. In “Brain-Pituitary-Adrenal Interrelationships” (A. Brodish and E. S. Redgate, eds.). pp. 176196. S. Karger, Basel. Dallman. M. F., and Jones. M. T. (1973b). Corticosteroid feedback control of ACTH secretion: effect of stress-induced corticosterone secretion on subsequent stress responses in the rat. Endocrinology

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Fortier. C. (1966). Nervous control of ACTH secretion. in “The Pituitary Gland” (6. W. Harris and B. T. Donovan. eds.), Vo!. 2, pp. 195-234. University of California Press. Berkeley. Frankel. A. I.. Cook. B.. Graber. J. W.. and Nalbandov. A. V. (1967). Determination of corticosterone in plasma by fluoromettic techniques. Ei~docrinolog); 80, 181-194. Gist. D. H. (1972). Effects of mammalian ACTH on liver and muscle glycogen levels in the South American caiman (Cairnan scieropsf. Gen. Camp. Endocrinol. 19, I-6. Jorgenson, Cc. B. (1968). Central nervous contra! of adenohypophyseai function. In ‘. Perspectives in Endocrinology: Hormones in the Lives of Lower Vertebrates” (F. J.W. Barrington and C. B. Jorgensen. eds.), pp. 469-S41. Academic Press, New York. Leloup-Hatey. J. (19%). Influence de I’agitation motrice sur la teneur du plasma I7-hydroxycorticosteroides d’un teleosteen: la carpe (Cyprinus curpi

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Effects of stress and ACTH on plasma corticosterone levels in the caiman Caiman crocodilus.

GENERAL AN0 COMPARATIVE ENDOCRINOLOGY 28, 413-419 (1976) ffects of Stress and ACTH on Plasma Corticosterone Cai man Caiman crocodi/~~~ DANIEL H...
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