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Stimulation of interrenal secretion in amphibia
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
Stimulation
47, 458-466 (1982)
of lnterrenal
I. Direct Effects of Electrolyte C. MASER, Department
of Zoology,
University
Secretion Concentration
P. A. JANSSENS,’ of Karlsruhe,
in Amphibia on Steroid Release
AND W. HANKE*
Kaiserstrasse
12, D-75
Karlsruhe
1, West Germany
Accepted September 29, 1981 Using a perifusion system, the direct regulation of isolated interrenal glands of Rana by electrolyte concentrations was studied. Isolated interrenals reacted for more than 5 hr to mammalian ACTH. Both natural corticosteroids, aldostetone and corticosterone, were released into the perifusion medium in approximately the same amounts. The threshold dose was about 1 mU ACTH given during 5 min. Elevation of Na+ concentration decreased the amounts of both corticosteroids released into the medium while elevation of K+ increased the amounts. The dose-dependent effect was studied in the range 90- 184 mM Na+ and 2.8-27 mM K+. The two corticosteroids responded somewhat differently to changing electrolyte concentrations but there was insufftcient evidence to suggest that the release of the two steroids was regulated differentially. From the time course of steroid release it was clear that production of the hormones was regulated as well as their release. temporaria
The direct regulation of steroid secretion by the mammalian adrenal cortex has often been investigated using in vitro preparations. In mammals, hormone secretion from Zona glomerulosa cells is mainly controlled by the renin-angiotensin system and the extracellular electrolyte concentrations. In contrast, steroid secretion from Zona fasciculata-reticularis cells is influenced mainly by ACTH (Tait and Tait, 1979; Tait et al., 1980; Braley and Williams, 1977, 1980). In Amphibia less is known about the control of adrenal steroidogenesis. The interrenal gland lacks any morphological zonation and secretes mainly corticosterone and aldosterone. Zn viva studies in the bullfrog have shown that mammalian ACTH, frog renin, and hyponatremia stimulate the secretion of both aldosterone and corticosterone while hyperkalemia failed to influence steriodogenesis (Johnston et al.,
1967; Davis et al., 1970; Ulick and Feinholtz, 1968; Braverman et al., 1973). In viva experiments in our laboratory have demonstrated that the corticosterone level in plasma of normal and hypophysectomized juveniles of Xenopus laevis is also influenced by environmental salinity (Maser et al., 1980; Grittmann, 1979). Although evidence exists that extrahypophysial stimuli influence adrenal steroidogenesis in Amphibia as in mammals, only hypophysia; control of the gland has been investigated in vitro (Leboulenger et al., 1978, 1979; Delarue et al., 1979). This paper deals with the direct influence of electrolytes on the interrenal gland of frogs, Rana temporaria. The aim of the study was to investigate the in vitro reaction of the isolated interrenal tissue lacking any endogenous stimulatory influences to changes of electrolyte concentrations. MATERIALS
I On leave of absence from the Department of Zoology, Australian National University, Canberra, ACT 2600, Australia. * To whom requests for reprints should be addressed.
Frogs (i?. temporaria) of about 30 g body weight are purchased from a commercial dealer. The animals were killed by decapitation and the kidney immediately removed. The interrenals on the ventral surface
458 0016~6480/82/080458-09$01.00/O Copyright @ 1982 by Academic Press, Inc. AU rights of reproduction in any form reserved.
AND METHODS
ELECTROLYTES
AND INTERRENAL
of the kidney weredissected under a microscope and
immediately immersedin perifusionmedium.
SECRETION
IN AMPHIBIA
459
The radioactivity of 100~1of this solution plus 8 ml
scintillationfluid wasmeasured to determinethe ex-
Forperifusion thefollowingculturemedium was tractionefficiency. Then200pl of thedissolved exprepared, Minimum Eagle’s medium withHanks’ saltstractor 200pl standard solutionranging from10to modified (MEM, Boehringer Mannheim, West Germany) was diluted in 0.015 M Hepes buffer solution achieving an osmolality of 200 mOsm. pH was adjusted to 7.4. Incubation temperature was fixed at 22 * 1”. The interrenals of one animal were placed within glass wool in a 2.5-ml plastic syringe containing 1 ml of culture medium. The upper end of the syringe was tightly closed by a pierced silicon plug. A silicon tube passing through the plug permitted the medium to flow in. The syringe needle on the lower end allowed the medium to flow off. It was collected by a fraction collector. The flow rate was kept constant by an infusion pump operating at up to 0.16 ml/min. Each experiment started with preperifusion of the interrenals with MEM for 2 hr to overcome the initial steroid output. A constant hormone secretion rate was achieved after this time. Two experimental sets were used: 1. To investigate the time-dependent release of interrenal steroids including maximum reaction and slope of the response, one pair of interrenals were used and fractions of perifusate were taken at 15-min intervals. 2. To establish dose response, 10 pairs of interrenals were tested at the same time. Ten perifusion chambers as described above were supplied simultaneously with culture medium by a IO-way infusion pump. The basal steroid output of each pair of interrenal glands serving as its own control was measured twice prior to stimulation. The steroid response to stimulation was determined during the period of maximal reaction. Fractions were collected and analyzed at 30-min intervals. Different electrolyte concentrations were tested. The normal perifusion medium contains 2.8 a/liter K+ and 85 w/liter Na+ . To investigate the influence of these ions, KC1 or NaCl were added to the medium. Experiments were done with additional 6.3, 15.6,21.1, or 27.3 m&4 KC1 and also with 48, 59, 81, or 94 mM NaCl. The final concentration was always checked by atomic absorption photometry. Determination
of corticosterone
and
aldosterone
was done using high specific RIAs (radioimmunoassays). An RIA chromatogram analysis showed that neither the antibody for corticosterone nor for aldosterone cross-react with any other steroid released by the tissue into the medium. It has been found that the interference is less than 2%. Therefore, chromatography was not necessary before determination. For determination of corticosterone, aliquots of 500 to 1000 ~1 perifusion medium to which had been added 1500 [3H]corticosterone was extracted by cyclohexane dichloromethane 2:l. The extract was dried and redissolved in 500 ~1 5% ethanol.
5000 pg + 100 ~1 [3H]corticosterone (3000 cpm) + 400 ~1 antibody dissolved in borate buffer 1:7000 was incubated on ice for 2 hr. Then 100 ~1 dextrane-coated charcoal was added and the mixture centrifuged for 10 min at 5000 rpm. The radioactivity of the supematant plus 8 ml scintillation fluid was then determined. For determination of aldosterone, aliquots of 300 to 500 ~1 plus 1500 cpm [3H]aldosterone were extracted by dichloromethane, dried, and redissolved in 1000 ~1 0.2% ethylene glycol. Again, the radioactivity of 200 ~1 dissolved extract plus 8 ml scintillation fluid was used to determine the extraction efficiency. Then 300 ~1 dissolved extract or 300 ~1 standard solution ranging from 5 to 5000 pg + 100 ~1 [3H]aldosterone (3000 cpm) + 400 ~1 antibody dissolved in beriglobin borate buffer 1: 10000 was incubated for 5 hr on ice. Then 100 ~1 dextrane-coated charcoal was added, the mixture centrifuged for 10 min at 5000 rpm, and the supematant + 8 ml scintillation fluid used for determination of radioactivity. The amount of corticosterone or aldosterone within the samples was calculated using the standard curves.
RESULTS
Mammalian ACTH stimulated the perifused interrenals to secrete both aldosterone and corticosterone. The time course of the secretion is shown in Fig. 1 from which it is clear that the interrenal can still be stimulated after more than 5 hr in vitro. A study of dose response showed that l10 mU of ACTH is effective (0.8 ml of 1 to 10 mU ACTWml was given). The total amount of hormone secreted depended on the amount of interrenal tissue and other unknown parameters such as season. Therefore in each experiment the basal secretion rate was checked for some time before any variation in incubation conditions was made. The time course of the secretion into the perifusion medium after changing the Na+ concentration is demonstrated in Fig. 2. During the first hours, the amount of each hormone secreted varied between 200 and 400 pg/ml (33.3-66.7 pg/ min). After changing the medium by adding 83 mmol Na+ as NaCl, the secreted amount of both corticosteroids decreased and
460
MASER,JANSSENS, ANDHANKE
r-wm II
ACTH
ACTH
10-3 I u /ml
10-2 I IJ /InI
n
ALDOSTERONE
ACTH IO“ I U /ml
a
CORTICOSTERONE
FIG. 1. Infhtence of different doses of ACTH upon the secretion of aldosterone and corticosterone from the interrenal gland of one animal. Hormones were measured in fractions taken every 15 min. ACTH was given for 5 min. The figure demonstrates the time course of secretion.
reached a very low secretory rate after 1 hr. When the medium was again changed to the initial composition, the secretion rate increased to approximately the initial values. Figure 2 gives an example seiected from several experiments which demonstrates the typical course of events. An increase of the K+ concentration from 2.8 to 24 mM resulted in an increase of aldosterone and corticosterone secretion. Figure 3 shows the time course of the changes. The basal secretion rate in this case was somewhat higher (approx 1 r&ml) than in the experiment demonstrated in Fig. 2. The maximum increase was reached after 0.5 hr for aldosterone and after 1 hr for corticosterone. When the medium was changed back to the initial composition the secretion rates were reduced again to the basal values. Figure 4 gives an example of changes of corticosterone secretion which occurred in a set of experiments (11 incubations in Fig. 4a, 9 incubations in Fig. 4b, 7 incubations in Fig. 4~). In each case 5 determinations were
Lal~l -gz 600
175-92 [mmdlllNQ’
-
I
TIME n
CORTICOSTERONE
m
ALDOSTERONE
2. Influence of two different concentrations of Na+ within the perifusion medium upon the secretion of aldosterone and corticosterone form the interrenal gland of one animal. The Na+ concentration adjusted by using NaCl was given for the whole announced period. Hormones were measured in fractions taken every 1.5min. The figure demonstrates the time course of secretion. FIG.
ELECTROLYTES
AND INTERRENAL
SECRETION
234--2
-2.6
461
IN AMPHIBIA
a-----,
Immol/ll
K’
:‘.: ,::. ii:; :::: r:: :s ;:> :::: ;: 5
6
7
[hrsl
TIME
r-
ALOOSTERONE
a
CORTICOSTERONE
FIG. 3. Influence of two different concentrations of K+ within the perifusion medium upon the secretion of aldosterone and corticosterone from the interrenal gland of one animal. The K+ concentration adjusted by using KC1 was given for the whole announced period. Hormones were measured in fractions taken every 15 min. The figure demonstrates the time course of secretion.
made, 2 before changing the medium and 3 after the change. Each line represents the time course of a single experiment. There was no change in hormone secretion when the composition of the medium was unchanged (control, Fig. 4a). A clear increase occurred in all experiments when the amount of K+ was elevated (Fig. 4b). A reduced secretion rate after increase of the Na+ concentration was found in all experiments shown in Fig. 4c. From such experiments, the changes in secretion rate compared with the basal rate found in two 30-min periods prior to the change of the medium were calculated for different sodium and potassium concentrations. Figure 5 demonstrates the changes of corticosterone and aldosterone secretion depending on the Na+ concentration and Fig. 6 those depending on the K+ concentration. The secretion rate is given for three 30-min periods after the change of the medium. The effect after change of Na+ concentration is strongest in the second and
third periods while in the first period only relatively small changes occur. This clearly demonstrates that the reaction needs about 30 min to be stimulated. After changes of K+ concentration the effect started earlier for the most part. The system looks somewhat exhausted in the third period. All the experiments done with elevated Na+ and lowered K+ concentration in the medium are summarized in Figs. 7 and 8. For Na+ the means of the secretion rates of the second- and third-period after changes of the Na+ concentration are shown. For K+, the mean secretion rates of all three periods after the elevation of the concentration are shown. The elevation of Na+ causes a much more pronounced drop of aldosterone than of corticosterone secretion. Decreased aldosterone secretion is found with Na+ concentrations between 130 and 150 mM while corticosterone secretion is lowered only above 160 m&I.
2.8 2.6 mom0
m TLt
%
Immol/ll IllvnovlI
K’ No*
‘,
b C
r-l
‘ .!- I/i
. ,i’
FIG. 4. The corticosterone secretion of several interrenal glands. (a) Na+ and K+ concentrations in the perifusion medium were kept constant. (b) The K+ concentration was increased (2.h23.9 mmol/hter K+) indicated by arrow, (c) The Na+ concentration was increased (85-170 mmokliter Na+) indicated by arrow. Fractions were collected every 30 min. The two values before the arrow represent control hormone secretion of each interrenal gland. The three values after the arrow represent (a) continuous rate of hormone release, (b) stimulation, and (c) inhibition of hormone secretion.
05 1
sr-J--l . w. .
a
ELECTROLYTES
>*
J
-
654
AND INTERRENAL
--lob--
-132-
SECRETION
‘l&6-
+170-
463
IN AMPHIBIA
-1a2+
[mmollll
Na*
FIG. 5. Relative change of aldosterone and corticosterone secretion after different Na+ concentrations. For each interrenal gland the mean of the hormone concentrations of two control fractions was defined as 100%. The columns represent the percentage change in hormone release versus control. The reaction was followed for 90 mm. During this period three consecutive fractions were collected, each for 30 min. For each column mean is given f SEM (n = 10).
The elevation of K+ stimulates similar changes in corticosterone and aldosterone secretion. Only at a concentration of about 25 mM corticosterone secretion is much greater than that of aldosterone.
DISCUSSION
These experiments show that the frog interrenal gland does not only react to hypophysial ACTH. It can also be stimu-
--l&0-)
A -I.
--23.9--
--27.3+
hmollll
K*
A
-
FIG. 6. Relative change of aldosterone and corticosterone secretion after different K+ concentrations. For each interrenal gland the mean of the hormone concentrations of two control fractions was defmed as 100%. The columns represent the percentage change in hormone release versus control. The reaction was followed for 90 mm. During this period three consecutive fractions were collected, each for 30 min. For each column mean is given 2 SEM (n = 10).
MASER, JANSSENS,
464
lb0
ii0
I's0
[mmol/ll
Na'
FIG. 7. Conclusions drawn from changes in corticosterone and aldosterone release depending on the Na+ concentration. Each point is calculated using the second and third fractions after the increase of the Na+ concentration. Means are given h SEM (n = 20).
lated directly by the electrolyte concentration of the surrounding medium and other stimulators. The idea of studying the direct regulation of the gland originally came from experiments with hypophysectomized clawed toads, Xenopus laevis, which were acclimated to hyperosmotic salt water (Grittmann, 1979). Such acclimatization studies have shown that both normal and hypophysectomized juvenile clawed toads increased the amount of corticosteroids in blood for a short time, which suggests that extrahypophysial regulation takes place. In animals adapted to salt water in this way the concentration of Na+ in blood LOO 1
CORTI COST ERONE
ALDOSTERONE
lb
2b
3b
Immol/ll
K’
FIG. 8. Conclusions drawn from changes in corticosterone and aldosterone release depending on the K+ concentration. Each point is calculated using the second and third fractions after the increase of the K+ concentration. Means are given k SEM (n = 20).
AND HANKE
plasma was elevated from 110 to 180 m&Y and that of K+ from 4.8 to 5.5 mM. The concentration changes were not exactly mimicked in our in vitro experiments for several reasons. First, the incubation was more effective and the tissue was better conserved in a lower sodium (85 mM) and potassium (2.8 m.M) concentration. Second, a concentration response curve could be followed ranging from 85 to 182 mM Na+ and from 2.8 to 27 mM K+. The hypothesis deduced from the in vivo studies that an elevation of Na+ increases the release of corticosteroids could not be confirmed. On the contrary, Na+ augmentation decreased the amount of corticosteroids released from the tissue. This is in accordance with what is known of mammalian adrenal cortex function. The effect of K+ concentration is also not comparable with the in vivo results in hypophysectomized toads. To increase the amount of corticosteroid release significantly, more than 10 m&f K+ must be added to the incubation medium, which is more than twice that which happened in plasma. Therefore, the results from our in vitro preparations cannot explain the stimulation of the interrenals of hypophysectomized toads in vivo. The difference between the species, in vivo experiments with Xenopus and in vitro with Rana temporaria tissue, also does not give a good explanation. Preliminary studies with Xenopus interrenals gave similar results to those with Rana glands. We shifted from Xenopus to Rana because of the morphological structure of the interrenals. They can more easily be separated and better cleaned from renal tissue when they were taken from Rana than from Xenopus. Other stimulatory factors must be present in vivo beside the electrolytes and ACTH. In further in vitro experiments which are in progress, we have found that angiotensin and arginine vasotocin are also stimulators of adrenal activity (for preliminary details see Hanke, 1981; Hanke and
ELECTROLYTES
AND INTERRENAL
Maser, 1981). Further studies are necessary to determine whether these factors are responsible for the in vivo results. The reactions of the interrenals taken from R. temporaria to mammalian ACTH are in accordance with those found in tissue from Rana ridibunda with a somewhat different perifusion system (Leboulenger et al., 1978, 1979; Delarue et al., 1979). These authors have also shown that the reaction of the inter-renal gland depends on the temperature. Therefore, we kept the temperature constant. Since we also compared the secretion rate before and after stimulation, the dependency on the temperature is not important for our results. The amounts of both hormones increased 15 to 30 min after stimulation. The ratio of both hormones did not change significantly. In our studies on Na+ influence we also added Cl- ions and an additional 19-94 n-&Y Cl-. In those on K+ influence we added between 6.6 and 25 mM Cl+. Since the effects after adding about 20 mM Cl- are not similar in both types of experiments it is improbable that Cl- has definite effects. But this should be proved in another set of experiments. Changes of osmolality are very important. Preliminary experiments have already shown that the osmolality influences the effects of ions. Nevertheless, it is also clear that there is specific action of Na+ and K+ ions, which is independent from the osmolality. Therefore, these investigations must be intensively done to explain the effects. Only a few minutes are needed following the addition of ACTH to increase the corticosteroid release which could last for 2 hr. This suggests that it is not only a fast release of stored material. New synthesis which is also rather fast may occur. There are no clear references in the literature about the distinction between release of stored material and new synthesis, but the time course after stimulation of frog adrenals seems to be similar to that in rat adrenals (Tait et al., 1970).
SECRETION
IN AMPHIBIA
465
It is well known from the in vivo and in vitro studies of several authors (Carstensen et al., 1961; Kraulis and Birmingham, 1964; Johnston et al., 1967; Ulick and Feinholtz,
1968; Chan and Phillips, 1971) that aldosterone and corticosterone are the most important secretory products in Amphibia. No further corticosteroids have been found in this group of animals in appreciable amounts. There is a clear difference compared with mammals. In mammals the amount of aldosterone is much lower than that of other steroids found in the blood. Aldosterone acts in much lower concentration than in Amphibia on Na uptake. One trend in evolution of vertebrates can be seen in an increase of effectiveness (specificity of receptors) of aldosterone. We must conclude that there are no clear differences between control of aldosterone and corticosterone release in our experiments, although the differing responses to sodium concentration may well be worthy of further study. This is in contrast to what is known from mammalian adrenocortical cells. Histological, histochemical, and ultrastructural studies did not provide any evidence that variously differentiated cell types exist in the interrenals of lower vertebrates (for review see Hanke, 1978). Therefore the differences between glomerulosa and fasciculata cells do not exist. Comparing the response it seems that interrenal tissue of Amphibians is more similar to glomerulosa than to fasciculata cells. The biosynthetic pathway from progesterone to corticosterone and on to aldosterone is present in both mammalian glomerulosa and frog inter-renal cells. But the regulation of frog interrenal cells is not as markedly directed to the late biosynthetic pathway including the conversion from corticosterone to aldosterone as has been suggested in mammals. ACKNOWLEDGMENTS The antibodies used in this study were provided by Professor Vecsei, Department of Pharmacology, Uni-
466
MASER, JANSSENS,
versity of Heidelberg. His help and continuous discussions are gratefully acknowledged. The authors thank C. Van Damme for technical help and S. Hassel for typing the manuscript. P.A.J. was supported by a grant from the Alexander von Humboldt-Stiftung.
REFERENCES Braley, L. M., and Williams, G. H. (1977). Rat adrenal cell sensitivity to angiotensin II &24-ACTH, and potassium: a comparative study. Amer. J. Physiol. 233, E402-E406. Braley, L. M., and Williams, G. H. (1980). The effect of unit gravity sedimentation on adrenal steroidogenesis by isolated rat glomerulosa and fasciculata cells. Endocrinology 106, 50-55. Braverman, B., Davis, J. P., and Taylor, A. A. (1973). Sodium depletion and postcaval vein constriction on steroid secretion in the bullfrog. Amer. J. Physiol. 244, 1358- 1362. Carstensen, H., Burgers, A. C. J., and Li, C. H. (l%l). Demonstration of aldosterone and corticosterone as the principal steroids formed in incubates of the American bullfrog (Rana cntesbeiana) and the stimulation of their production by mammalian adrenocorticotropin. Gen. Comp. Endocrinol. 1, 37-50. Ghan, S. W. C., and Phillips, J. G. (1971). Seasonal variations in production in vifro of corticosteroids by the frog (Rana rugulosn) adrenal. J. Endocrinol. 50, 1- 17. Davis, J. O., Urquhart, J., and Higgins, J. T. (1963). The effects of alterations of plasma sodium and potassium concentration on aldosterone secretion. J. Ciin. Invest. 42, 597-609. Delarue, C., Tonon, M. C., Leboulenger, F., Jegou, S., Leroux, P., and Vaudry, H. (1979). In vitro study of frog (Rana ridibunda Pallas) interrenal function by use of a simplified perifusion system. II. Influence of adrenocorticotropin upon aldosterone production. Gen. Comp. Endocrinol. 38, 399-409. Grittmann, M. (1979). Untersuchungen zur Regulation des Interrenalorgans bei Xenopus laevis wahrend hyperosmotischem Stress. Thesis, Univ. of Karlsruhe, Federal Republic of Germany. Hanke, W. (1978). The adrenal cortex of Amphibia. In “General, Comparative and Clinical Endocrinology of the Adrenal Cortex” (I. Chester Jones and
AND HANKE
I. W. Henderson, eds.), Vol. 2, pp. 419-495. Academic Press, London/New York. Hanke, W. (1981). Changes of the biology of the adrenal cortex in the vertebrate evolution. Nova Acta Leopoldina, in press. Hanke, W., and Maser, C. (1981). Regulation of interrenal tissue in Amphibia. “Proceedings of the IXth International Symposium on Comparative Endocrinology,” in press. Johnston, C. I., Davis, J. O., Wright, F. S., and Howard, S. S. (1967). Effects of renin and ACTH on adrenal steroid secretion in the American bullfrog. Amer. J. Physiol. 213, 393-399, Kraulis, I., and Birmingham, M. K. (1964). The conversion of 1l/3-hydroxy-progesterone to corticosterone and other steroid fractions by rat and frog adrenal glands. Acta Endocrinol. 47, 76-84. Leboulenger, F., Delarue, C., Tonon, M. C., Jegou, S., and Vaudry, H. (1978). In vitro study of frog (Rana ridibunda Pallas) interrenal function by use of a simplified perifusion system. I. Influence of adrenocorticotropin upon corticosterone release. Gen. Comp. Endocrinol. 36, 327-338. Leboulenger, F., Delarue, C., Tonon, M. C., Jegou, S., Leroux, P., and Vaudry, H. (1979). Seasonal studies of the interrenal function of the European green frog, in vivo and in vitro. Gen. Comp. Endocrinof. 39, 388-396. Maser, C., Hittler, K., and Hanke, W. (1980). External induction of changes of corticosterone level in Amphibia. Gen. Comp. Endocrinol. 40, 335. Tait, J. F., and Tait, S. A. S. (1979). Recent perspectives on the history of the adrenal cortex. J. Endocrinol. 83, 3P-24P. Tait, S. A. S., Schulster, D., Okamoto, M., Flood, C., and Tait, .I. F. (1970). Production of steroids by in vitro superfusion of endocrine tissue. II. Steroid output from bisected whole, capsular and decapsulated adrenals of normal intact, hypophysectomized and hypophysectomized-nephrectomized rats as a function of tissue on superfusion. Endocrinology 86, 360-382. Tait, J. F., Tait, S. A. S., Bell, J. B. G., Hyatt, P. J., and Williams, B. C. (1980). Further studies on the stimulation of rat adrenal capsular cells: four types of response. J. Endocrinol. 87, 11-27. Ulick, S., and Feinholtz, E. (1968). Metabolism and rate of secretion of aldosterone in the bullfrog. J. Clin. Invest. 47, 2523-2529.
AND
COMPARATIVE
ENDOCRINOLOGY
Stimulation
47, 458-466 (1982)
of lnterrenal
I. Direct Effects of Electrolyte C. MASER, Department
of Zoology,
University
Secretion Concentration
P. A. JANSSENS,’ of Karlsruhe,
in Amphibia on Steroid Release
AND W. HANKE*
Kaiserstrasse
12, D-75
Karlsruhe
1, West Germany
Accepted September 29, 1981 Using a perifusion system, the direct regulation of isolated interrenal glands of Rana by electrolyte concentrations was studied. Isolated interrenals reacted for more than 5 hr to mammalian ACTH. Both natural corticosteroids, aldostetone and corticosterone, were released into the perifusion medium in approximately the same amounts. The threshold dose was about 1 mU ACTH given during 5 min. Elevation of Na+ concentration decreased the amounts of both corticosteroids released into the medium while elevation of K+ increased the amounts. The dose-dependent effect was studied in the range 90- 184 mM Na+ and 2.8-27 mM K+. The two corticosteroids responded somewhat differently to changing electrolyte concentrations but there was insufftcient evidence to suggest that the release of the two steroids was regulated differentially. From the time course of steroid release it was clear that production of the hormones was regulated as well as their release. temporaria
The direct regulation of steroid secretion by the mammalian adrenal cortex has often been investigated using in vitro preparations. In mammals, hormone secretion from Zona glomerulosa cells is mainly controlled by the renin-angiotensin system and the extracellular electrolyte concentrations. In contrast, steroid secretion from Zona fasciculata-reticularis cells is influenced mainly by ACTH (Tait and Tait, 1979; Tait et al., 1980; Braley and Williams, 1977, 1980). In Amphibia less is known about the control of adrenal steroidogenesis. The interrenal gland lacks any morphological zonation and secretes mainly corticosterone and aldosterone. Zn viva studies in the bullfrog have shown that mammalian ACTH, frog renin, and hyponatremia stimulate the secretion of both aldosterone and corticosterone while hyperkalemia failed to influence steriodogenesis (Johnston et al.,
1967; Davis et al., 1970; Ulick and Feinholtz, 1968; Braverman et al., 1973). In viva experiments in our laboratory have demonstrated that the corticosterone level in plasma of normal and hypophysectomized juveniles of Xenopus laevis is also influenced by environmental salinity (Maser et al., 1980; Grittmann, 1979). Although evidence exists that extrahypophysial stimuli influence adrenal steroidogenesis in Amphibia as in mammals, only hypophysia; control of the gland has been investigated in vitro (Leboulenger et al., 1978, 1979; Delarue et al., 1979). This paper deals with the direct influence of electrolytes on the interrenal gland of frogs, Rana temporaria. The aim of the study was to investigate the in vitro reaction of the isolated interrenal tissue lacking any endogenous stimulatory influences to changes of electrolyte concentrations. MATERIALS
I On leave of absence from the Department of Zoology, Australian National University, Canberra, ACT 2600, Australia. * To whom requests for reprints should be addressed.
Frogs (i?. temporaria) of about 30 g body weight are purchased from a commercial dealer. The animals were killed by decapitation and the kidney immediately removed. The interrenals on the ventral surface
458 0016~6480/82/080458-09$01.00/O Copyright @ 1982 by Academic Press, Inc. AU rights of reproduction in any form reserved.
AND METHODS
ELECTROLYTES
AND INTERRENAL
of the kidney weredissected under a microscope and
immediately immersedin perifusionmedium.
SECRETION
IN AMPHIBIA
459
The radioactivity of 100~1of this solution plus 8 ml
scintillationfluid wasmeasured to determinethe ex-
Forperifusion thefollowingculturemedium was tractionefficiency. Then200pl of thedissolved exprepared, Minimum Eagle’s medium withHanks’ saltstractor 200pl standard solutionranging from10to modified (MEM, Boehringer Mannheim, West Germany) was diluted in 0.015 M Hepes buffer solution achieving an osmolality of 200 mOsm. pH was adjusted to 7.4. Incubation temperature was fixed at 22 * 1”. The interrenals of one animal were placed within glass wool in a 2.5-ml plastic syringe containing 1 ml of culture medium. The upper end of the syringe was tightly closed by a pierced silicon plug. A silicon tube passing through the plug permitted the medium to flow in. The syringe needle on the lower end allowed the medium to flow off. It was collected by a fraction collector. The flow rate was kept constant by an infusion pump operating at up to 0.16 ml/min. Each experiment started with preperifusion of the interrenals with MEM for 2 hr to overcome the initial steroid output. A constant hormone secretion rate was achieved after this time. Two experimental sets were used: 1. To investigate the time-dependent release of interrenal steroids including maximum reaction and slope of the response, one pair of interrenals were used and fractions of perifusate were taken at 15-min intervals. 2. To establish dose response, 10 pairs of interrenals were tested at the same time. Ten perifusion chambers as described above were supplied simultaneously with culture medium by a IO-way infusion pump. The basal steroid output of each pair of interrenal glands serving as its own control was measured twice prior to stimulation. The steroid response to stimulation was determined during the period of maximal reaction. Fractions were collected and analyzed at 30-min intervals. Different electrolyte concentrations were tested. The normal perifusion medium contains 2.8 a/liter K+ and 85 w/liter Na+ . To investigate the influence of these ions, KC1 or NaCl were added to the medium. Experiments were done with additional 6.3, 15.6,21.1, or 27.3 m&4 KC1 and also with 48, 59, 81, or 94 mM NaCl. The final concentration was always checked by atomic absorption photometry. Determination
of corticosterone
and
aldosterone
was done using high specific RIAs (radioimmunoassays). An RIA chromatogram analysis showed that neither the antibody for corticosterone nor for aldosterone cross-react with any other steroid released by the tissue into the medium. It has been found that the interference is less than 2%. Therefore, chromatography was not necessary before determination. For determination of corticosterone, aliquots of 500 to 1000 ~1 perifusion medium to which had been added 1500 [3H]corticosterone was extracted by cyclohexane dichloromethane 2:l. The extract was dried and redissolved in 500 ~1 5% ethanol.
5000 pg + 100 ~1 [3H]corticosterone (3000 cpm) + 400 ~1 antibody dissolved in borate buffer 1:7000 was incubated on ice for 2 hr. Then 100 ~1 dextrane-coated charcoal was added and the mixture centrifuged for 10 min at 5000 rpm. The radioactivity of the supematant plus 8 ml scintillation fluid was then determined. For determination of aldosterone, aliquots of 300 to 500 ~1 plus 1500 cpm [3H]aldosterone were extracted by dichloromethane, dried, and redissolved in 1000 ~1 0.2% ethylene glycol. Again, the radioactivity of 200 ~1 dissolved extract plus 8 ml scintillation fluid was used to determine the extraction efficiency. Then 300 ~1 dissolved extract or 300 ~1 standard solution ranging from 5 to 5000 pg + 100 ~1 [3H]aldosterone (3000 cpm) + 400 ~1 antibody dissolved in beriglobin borate buffer 1: 10000 was incubated for 5 hr on ice. Then 100 ~1 dextrane-coated charcoal was added, the mixture centrifuged for 10 min at 5000 rpm, and the supematant + 8 ml scintillation fluid used for determination of radioactivity. The amount of corticosterone or aldosterone within the samples was calculated using the standard curves.
RESULTS
Mammalian ACTH stimulated the perifused interrenals to secrete both aldosterone and corticosterone. The time course of the secretion is shown in Fig. 1 from which it is clear that the interrenal can still be stimulated after more than 5 hr in vitro. A study of dose response showed that l10 mU of ACTH is effective (0.8 ml of 1 to 10 mU ACTWml was given). The total amount of hormone secreted depended on the amount of interrenal tissue and other unknown parameters such as season. Therefore in each experiment the basal secretion rate was checked for some time before any variation in incubation conditions was made. The time course of the secretion into the perifusion medium after changing the Na+ concentration is demonstrated in Fig. 2. During the first hours, the amount of each hormone secreted varied between 200 and 400 pg/ml (33.3-66.7 pg/ min). After changing the medium by adding 83 mmol Na+ as NaCl, the secreted amount of both corticosteroids decreased and
460
MASER,JANSSENS, ANDHANKE
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FIG. 1. Infhtence of different doses of ACTH upon the secretion of aldosterone and corticosterone from the interrenal gland of one animal. Hormones were measured in fractions taken every 15 min. ACTH was given for 5 min. The figure demonstrates the time course of secretion.
reached a very low secretory rate after 1 hr. When the medium was again changed to the initial composition, the secretion rate increased to approximately the initial values. Figure 2 gives an example seiected from several experiments which demonstrates the typical course of events. An increase of the K+ concentration from 2.8 to 24 mM resulted in an increase of aldosterone and corticosterone secretion. Figure 3 shows the time course of the changes. The basal secretion rate in this case was somewhat higher (approx 1 r&ml) than in the experiment demonstrated in Fig. 2. The maximum increase was reached after 0.5 hr for aldosterone and after 1 hr for corticosterone. When the medium was changed back to the initial composition the secretion rates were reduced again to the basal values. Figure 4 gives an example of changes of corticosterone secretion which occurred in a set of experiments (11 incubations in Fig. 4a, 9 incubations in Fig. 4b, 7 incubations in Fig. 4~). In each case 5 determinations were
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2. Influence of two different concentrations of Na+ within the perifusion medium upon the secretion of aldosterone and corticosterone form the interrenal gland of one animal. The Na+ concentration adjusted by using NaCl was given for the whole announced period. Hormones were measured in fractions taken every 1.5min. The figure demonstrates the time course of secretion. FIG.
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FIG. 3. Influence of two different concentrations of K+ within the perifusion medium upon the secretion of aldosterone and corticosterone from the interrenal gland of one animal. The K+ concentration adjusted by using KC1 was given for the whole announced period. Hormones were measured in fractions taken every 15 min. The figure demonstrates the time course of secretion.
made, 2 before changing the medium and 3 after the change. Each line represents the time course of a single experiment. There was no change in hormone secretion when the composition of the medium was unchanged (control, Fig. 4a). A clear increase occurred in all experiments when the amount of K+ was elevated (Fig. 4b). A reduced secretion rate after increase of the Na+ concentration was found in all experiments shown in Fig. 4c. From such experiments, the changes in secretion rate compared with the basal rate found in two 30-min periods prior to the change of the medium were calculated for different sodium and potassium concentrations. Figure 5 demonstrates the changes of corticosterone and aldosterone secretion depending on the Na+ concentration and Fig. 6 those depending on the K+ concentration. The secretion rate is given for three 30-min periods after the change of the medium. The effect after change of Na+ concentration is strongest in the second and
third periods while in the first period only relatively small changes occur. This clearly demonstrates that the reaction needs about 30 min to be stimulated. After changes of K+ concentration the effect started earlier for the most part. The system looks somewhat exhausted in the third period. All the experiments done with elevated Na+ and lowered K+ concentration in the medium are summarized in Figs. 7 and 8. For Na+ the means of the secretion rates of the second- and third-period after changes of the Na+ concentration are shown. For K+, the mean secretion rates of all three periods after the elevation of the concentration are shown. The elevation of Na+ causes a much more pronounced drop of aldosterone than of corticosterone secretion. Decreased aldosterone secretion is found with Na+ concentrations between 130 and 150 mM while corticosterone secretion is lowered only above 160 m&I.
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FIG. 4. The corticosterone secretion of several interrenal glands. (a) Na+ and K+ concentrations in the perifusion medium were kept constant. (b) The K+ concentration was increased (2.h23.9 mmol/hter K+) indicated by arrow, (c) The Na+ concentration was increased (85-170 mmokliter Na+) indicated by arrow. Fractions were collected every 30 min. The two values before the arrow represent control hormone secretion of each interrenal gland. The three values after the arrow represent (a) continuous rate of hormone release, (b) stimulation, and (c) inhibition of hormone secretion.
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FIG. 5. Relative change of aldosterone and corticosterone secretion after different Na+ concentrations. For each interrenal gland the mean of the hormone concentrations of two control fractions was defined as 100%. The columns represent the percentage change in hormone release versus control. The reaction was followed for 90 mm. During this period three consecutive fractions were collected, each for 30 min. For each column mean is given f SEM (n = 10).
The elevation of K+ stimulates similar changes in corticosterone and aldosterone secretion. Only at a concentration of about 25 mM corticosterone secretion is much greater than that of aldosterone.
DISCUSSION
These experiments show that the frog interrenal gland does not only react to hypophysial ACTH. It can also be stimu-
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FIG. 6. Relative change of aldosterone and corticosterone secretion after different K+ concentrations. For each interrenal gland the mean of the hormone concentrations of two control fractions was defmed as 100%. The columns represent the percentage change in hormone release versus control. The reaction was followed for 90 mm. During this period three consecutive fractions were collected, each for 30 min. For each column mean is given 2 SEM (n = 10).
MASER, JANSSENS,
464
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FIG. 7. Conclusions drawn from changes in corticosterone and aldosterone release depending on the Na+ concentration. Each point is calculated using the second and third fractions after the increase of the Na+ concentration. Means are given h SEM (n = 20).
lated directly by the electrolyte concentration of the surrounding medium and other stimulators. The idea of studying the direct regulation of the gland originally came from experiments with hypophysectomized clawed toads, Xenopus laevis, which were acclimated to hyperosmotic salt water (Grittmann, 1979). Such acclimatization studies have shown that both normal and hypophysectomized juvenile clawed toads increased the amount of corticosteroids in blood for a short time, which suggests that extrahypophysial regulation takes place. In animals adapted to salt water in this way the concentration of Na+ in blood LOO 1
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FIG. 8. Conclusions drawn from changes in corticosterone and aldosterone release depending on the K+ concentration. Each point is calculated using the second and third fractions after the increase of the K+ concentration. Means are given k SEM (n = 20).
AND HANKE
plasma was elevated from 110 to 180 m&Y and that of K+ from 4.8 to 5.5 mM. The concentration changes were not exactly mimicked in our in vitro experiments for several reasons. First, the incubation was more effective and the tissue was better conserved in a lower sodium (85 mM) and potassium (2.8 m.M) concentration. Second, a concentration response curve could be followed ranging from 85 to 182 mM Na+ and from 2.8 to 27 mM K+. The hypothesis deduced from the in vivo studies that an elevation of Na+ increases the release of corticosteroids could not be confirmed. On the contrary, Na+ augmentation decreased the amount of corticosteroids released from the tissue. This is in accordance with what is known of mammalian adrenal cortex function. The effect of K+ concentration is also not comparable with the in vivo results in hypophysectomized toads. To increase the amount of corticosteroid release significantly, more than 10 m&f K+ must be added to the incubation medium, which is more than twice that which happened in plasma. Therefore, the results from our in vitro preparations cannot explain the stimulation of the interrenals of hypophysectomized toads in vivo. The difference between the species, in vivo experiments with Xenopus and in vitro with Rana temporaria tissue, also does not give a good explanation. Preliminary studies with Xenopus interrenals gave similar results to those with Rana glands. We shifted from Xenopus to Rana because of the morphological structure of the interrenals. They can more easily be separated and better cleaned from renal tissue when they were taken from Rana than from Xenopus. Other stimulatory factors must be present in vivo beside the electrolytes and ACTH. In further in vitro experiments which are in progress, we have found that angiotensin and arginine vasotocin are also stimulators of adrenal activity (for preliminary details see Hanke, 1981; Hanke and
ELECTROLYTES
AND INTERRENAL
Maser, 1981). Further studies are necessary to determine whether these factors are responsible for the in vivo results. The reactions of the interrenals taken from R. temporaria to mammalian ACTH are in accordance with those found in tissue from Rana ridibunda with a somewhat different perifusion system (Leboulenger et al., 1978, 1979; Delarue et al., 1979). These authors have also shown that the reaction of the inter-renal gland depends on the temperature. Therefore, we kept the temperature constant. Since we also compared the secretion rate before and after stimulation, the dependency on the temperature is not important for our results. The amounts of both hormones increased 15 to 30 min after stimulation. The ratio of both hormones did not change significantly. In our studies on Na+ influence we also added Cl- ions and an additional 19-94 n-&Y Cl-. In those on K+ influence we added between 6.6 and 25 mM Cl+. Since the effects after adding about 20 mM Cl- are not similar in both types of experiments it is improbable that Cl- has definite effects. But this should be proved in another set of experiments. Changes of osmolality are very important. Preliminary experiments have already shown that the osmolality influences the effects of ions. Nevertheless, it is also clear that there is specific action of Na+ and K+ ions, which is independent from the osmolality. Therefore, these investigations must be intensively done to explain the effects. Only a few minutes are needed following the addition of ACTH to increase the corticosteroid release which could last for 2 hr. This suggests that it is not only a fast release of stored material. New synthesis which is also rather fast may occur. There are no clear references in the literature about the distinction between release of stored material and new synthesis, but the time course after stimulation of frog adrenals seems to be similar to that in rat adrenals (Tait et al., 1970).
SECRETION
IN AMPHIBIA
465
It is well known from the in vivo and in vitro studies of several authors (Carstensen et al., 1961; Kraulis and Birmingham, 1964; Johnston et al., 1967; Ulick and Feinholtz,
1968; Chan and Phillips, 1971) that aldosterone and corticosterone are the most important secretory products in Amphibia. No further corticosteroids have been found in this group of animals in appreciable amounts. There is a clear difference compared with mammals. In mammals the amount of aldosterone is much lower than that of other steroids found in the blood. Aldosterone acts in much lower concentration than in Amphibia on Na uptake. One trend in evolution of vertebrates can be seen in an increase of effectiveness (specificity of receptors) of aldosterone. We must conclude that there are no clear differences between control of aldosterone and corticosterone release in our experiments, although the differing responses to sodium concentration may well be worthy of further study. This is in contrast to what is known from mammalian adrenocortical cells. Histological, histochemical, and ultrastructural studies did not provide any evidence that variously differentiated cell types exist in the interrenals of lower vertebrates (for review see Hanke, 1978). Therefore the differences between glomerulosa and fasciculata cells do not exist. Comparing the response it seems that interrenal tissue of Amphibians is more similar to glomerulosa than to fasciculata cells. The biosynthetic pathway from progesterone to corticosterone and on to aldosterone is present in both mammalian glomerulosa and frog inter-renal cells. But the regulation of frog interrenal cells is not as markedly directed to the late biosynthetic pathway including the conversion from corticosterone to aldosterone as has been suggested in mammals. ACKNOWLEDGMENTS The antibodies used in this study were provided by Professor Vecsei, Department of Pharmacology, Uni-
466
MASER, JANSSENS,
versity of Heidelberg. His help and continuous discussions are gratefully acknowledged. The authors thank C. Van Damme for technical help and S. Hassel for typing the manuscript. P.A.J. was supported by a grant from the Alexander von Humboldt-Stiftung.
REFERENCES Braley, L. M., and Williams, G. H. (1977). Rat adrenal cell sensitivity to angiotensin II &24-ACTH, and potassium: a comparative study. Amer. J. Physiol. 233, E402-E406. Braley, L. M., and Williams, G. H. (1980). The effect of unit gravity sedimentation on adrenal steroidogenesis by isolated rat glomerulosa and fasciculata cells. Endocrinology 106, 50-55. Braverman, B., Davis, J. P., and Taylor, A. A. (1973). Sodium depletion and postcaval vein constriction on steroid secretion in the bullfrog. Amer. J. Physiol. 244, 1358- 1362. Carstensen, H., Burgers, A. C. J., and Li, C. H. (l%l). Demonstration of aldosterone and corticosterone as the principal steroids formed in incubates of the American bullfrog (Rana cntesbeiana) and the stimulation of their production by mammalian adrenocorticotropin. Gen. Comp. Endocrinol. 1, 37-50. Ghan, S. W. C., and Phillips, J. G. (1971). Seasonal variations in production in vifro of corticosteroids by the frog (Rana rugulosn) adrenal. J. Endocrinol. 50, 1- 17. Davis, J. O., Urquhart, J., and Higgins, J. T. (1963). The effects of alterations of plasma sodium and potassium concentration on aldosterone secretion. J. Ciin. Invest. 42, 597-609. Delarue, C., Tonon, M. C., Leboulenger, F., Jegou, S., Leroux, P., and Vaudry, H. (1979). In vitro study of frog (Rana ridibunda Pallas) interrenal function by use of a simplified perifusion system. II. Influence of adrenocorticotropin upon aldosterone production. Gen. Comp. Endocrinol. 38, 399-409. Grittmann, M. (1979). Untersuchungen zur Regulation des Interrenalorgans bei Xenopus laevis wahrend hyperosmotischem Stress. Thesis, Univ. of Karlsruhe, Federal Republic of Germany. Hanke, W. (1978). The adrenal cortex of Amphibia. In “General, Comparative and Clinical Endocrinology of the Adrenal Cortex” (I. Chester Jones and
AND HANKE
I. W. Henderson, eds.), Vol. 2, pp. 419-495. Academic Press, London/New York. Hanke, W. (1981). Changes of the biology of the adrenal cortex in the vertebrate evolution. Nova Acta Leopoldina, in press. Hanke, W., and Maser, C. (1981). Regulation of interrenal tissue in Amphibia. “Proceedings of the IXth International Symposium on Comparative Endocrinology,” in press. Johnston, C. I., Davis, J. O., Wright, F. S., and Howard, S. S. (1967). Effects of renin and ACTH on adrenal steroid secretion in the American bullfrog. Amer. J. Physiol. 213, 393-399, Kraulis, I., and Birmingham, M. K. (1964). The conversion of 1l/3-hydroxy-progesterone to corticosterone and other steroid fractions by rat and frog adrenal glands. Acta Endocrinol. 47, 76-84. Leboulenger, F., Delarue, C., Tonon, M. C., Jegou, S., and Vaudry, H. (1978). In vitro study of frog (Rana ridibunda Pallas) interrenal function by use of a simplified perifusion system. I. Influence of adrenocorticotropin upon corticosterone release. Gen. Comp. Endocrinol. 36, 327-338. Leboulenger, F., Delarue, C., Tonon, M. C., Jegou, S., Leroux, P., and Vaudry, H. (1979). Seasonal studies of the interrenal function of the European green frog, in vivo and in vitro. Gen. Comp. Endocrinof. 39, 388-396. Maser, C., Hittler, K., and Hanke, W. (1980). External induction of changes of corticosterone level in Amphibia. Gen. Comp. Endocrinol. 40, 335. Tait, J. F., and Tait, S. A. S. (1979). Recent perspectives on the history of the adrenal cortex. J. Endocrinol. 83, 3P-24P. Tait, S. A. S., Schulster, D., Okamoto, M., Flood, C., and Tait, .I. F. (1970). Production of steroids by in vitro superfusion of endocrine tissue. II. Steroid output from bisected whole, capsular and decapsulated adrenals of normal intact, hypophysectomized and hypophysectomized-nephrectomized rats as a function of tissue on superfusion. Endocrinology 86, 360-382. Tait, J. F., Tait, S. A. S., Bell, J. B. G., Hyatt, P. J., and Williams, B. C. (1980). Further studies on the stimulation of rat adrenal capsular cells: four types of response. J. Endocrinol. 87, 11-27. Ulick, S., and Feinholtz, E. (1968). Metabolism and rate of secretion of aldosterone in the bullfrog. J. Clin. Invest. 47, 2523-2529.