effect of prednisolone on the development of amphibian larvae and the antagonism of litoralon and...

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GENERAL AND COMPARATIVE ENDOCRINOLOGY 38,285-289 (1979) Effect of Prednisolone on the Development of Amphibian Larvae and the Antagonism of Litoralon and Prednisolone LAsz~6 J~ZSEF TGRC~K,~ LAsz~6 FEUER,~ AND GYGRGY CSABA Department of Biology, Semmelweis University of Medicine, 1089 Budapest, Nagyvcirad tdr 4, Hungary, and Chinoin Pharmaceutical and Chemical Works Ltd., 1325 Budapest, Ti, u. I. Hungary Accepted April 4, 1979 Larvae of Rana arvalis Wolterstorffi of developmental stage 26 (Terentiev, 1950; stage X/IV, Taylor and Kollros, 1946) were placed for 2 hr daily in aqueous solution of 10 &ml prednisolone succinate sodium (Di-Adreson-F-aquosum, Organon; prednisolone) or of IO &ml prednisolone + 0.5 pg litoralon (y-L-glutamyl-taurine; glutaurine), a biologically ac- tive substance isolated from the bovine parathyroid and produced synthetically. Body length and development of the larvae were studied. Prednisolone was found to enhance the de- velopment of Rana arvalis Wolterstofi larvae and this effect was antagonized by litoralon. Correlation of this observation with our earlier experimental results implied the possibility that the glucocorticoid and thyroid hormones in some respects antagonize the effects of litoralon and vitamin A. Litoralon (y-L-glutamyl-taurine; glu- taurine) is a lately discovered hormone of the parathyroid (Feuer, 1975). It has a vitamin A-like effect (Feuer et al., 1977), affects the metabolism of vitamin A (Feuer et al., 1977; Feuer, 1977), and modifies the activity of several endocrine glands and target organs (Feuer, 1977). metamorphosis and the possible influence of litoralon on this effect. MATERIALS AND METHODS The appearance of litoralon in phy- logenesis, like that of the parathyroid, probably coincides with the transition from aquatic to terrestrial life (Feuer, 1977). This is why also in our previous experiments, we have chosen the metamorphosis of am- phibia as a model to study the effect of litoralon upon this basic biological process. We have found that litoralon exerts a hor- mone-like balancing effect on the develop- ment and metamorphosis of amphibian larvae and antagonizes the effect of exogene- ous thyroxine (Feuer et al., 1979b). Larvae of Rana arvalis Wolterstorfli (50 larvae per group) were kept in chlorine-free tap water at room temperature of 20-25’. The development of larvae was definied by two methods, namely according to Terentiev (1950) and Taylor and Kollros (1946, hereafter referred to as T-K). The experiments were started with larvae of a late developmental stage (26, T-K-26/N-V) of 27- to 30-mm mean body length. Groups investigated were as follows: (1) control group, (2) prednisolone-treated group (lo&ml prednisolone in the bath, Di-Adreson-F-aquosum, Organon), (3) prednisolone + litoralon-treated group (10 pg pred- nisolone + 0.5 pg litoralon per ml bath). Glucocorticoids are, similarly to thy- roxine, morphogenetic hormones (Csaba, 1977). Our present experiments were aimed at studying the effect of prednisolone, a synthetic water-soluble glucocorticoid, on Larvae were placed into the above solutions for 2 hr daily, otherwise they were kept in chlorine-free tap water. Water was changed daily. The larvae were fed nettle powder, grated apple, and lettuce ad libitum; egg white was given twice a week. Development of the larvae was observed daily and body measurements were made photometrically (Feuer ef al., 1978a). Body length was determined five times. Growth curves of the control and experimental groups were plotted from the mean values of body length. The results were il- lustrated in Terentiev stage diagrams. Statistical significance of the differences was calcu- lated by Student’s f test. RESULTS ’ Deceased on 6 February, 1979. Body length changes, based on determi- * Address all reprint requests to L. Feuer. nations of the mean values, are shown in 285 001~6480/79/070285-05$01.00/O Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any fonp reserved.

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Page 1: Effect of prednisolone on the development of amphibian larvae and the antagonism of litoralon and prednisolone

GENERAL AND COMPARATIVE ENDOCRINOLOGY 38,285-289 (1979)

Effect of Prednisolone on the Development of Amphibian Larvae and the Antagonism of Litoralon and Prednisolone

LAsz~6 J~ZSEF TGRC~K,~ LAsz~6 FEUER,~ AND GYGRGY CSABA

Department of Biology, Semmelweis University of Medicine, 1089 Budapest, Nagyvcirad tdr 4, Hungary, and Chinoin Pharmaceutical and Chemical Works Ltd., 1325 Budapest, Ti, u. I. Hungary

Accepted April 4, 1979

Larvae of Rana arvalis Wolterstorffi of developmental stage 26 (Terentiev, 1950; stage X/IV, Taylor and Kollros, 1946) were placed for 2 hr daily in aqueous solution of 10 &ml prednisolone succinate sodium (Di-Adreson-F-aquosum, Organon; prednisolone) or of IO &ml prednisolone + 0.5 pg litoralon (y-L-glutamyl-taurine; glutaurine), a biologically ac- tive substance isolated from the bovine parathyroid and produced synthetically. Body length and development of the larvae were studied. Prednisolone was found to enhance the de- velopment of Rana arvalis Wolterstofi larvae and this effect was antagonized by litoralon. Correlation of this observation with our earlier experimental results implied the possibility that the glucocorticoid and thyroid hormones in some respects antagonize the effects of litoralon and vitamin A.

Litoralon (y-L-glutamyl-taurine; glu- taurine) is a lately discovered hormone of the parathyroid (Feuer, 1975). It has a vitamin A-like effect (Feuer et al., 1977), affects the metabolism of vitamin A (Feuer et al., 1977; Feuer, 1977), and modifies the activity of several endocrine glands and target organs (Feuer, 1977).

metamorphosis and the possible influence of litoralon on this effect.

MATERIALS AND METHODS

The appearance of litoralon in phy- logenesis, like that of the parathyroid, probably coincides with the transition from aquatic to terrestrial life (Feuer, 1977). This is why also in our previous experiments, we have chosen the metamorphosis of am- phibia as a model to study the effect of litoralon upon this basic biological process. We have found that litoralon exerts a hor- mone-like balancing effect on the develop- ment and metamorphosis of amphibian larvae and antagonizes the effect of exogene- ous thyroxine (Feuer et al., 1979b).

Larvae of Rana arvalis Wolterstorfli (50 larvae per group) were kept in chlorine-free tap water at room temperature of 20-25’. The development of larvae was definied by two methods, namely according to Terentiev (1950) and Taylor and Kollros (1946, hereafter referred to as T-K). The experiments were started with larvae of a late developmental stage (26, T-K-26/N-V) of 27- to 30-mm mean body length. Groups investigated were as follows: (1) control group, (2) prednisolone-treated group (lo&ml prednisolone in the bath, Di-Adreson-F-aquosum, Organon), (3) prednisolone + litoralon-treated group (10 pg pred- nisolone + 0.5 pg litoralon per ml bath).

Glucocorticoids are, similarly to thy- roxine, morphogenetic hormones (Csaba, 1977). Our present experiments were aimed at studying the effect of prednisolone, a synthetic water-soluble glucocorticoid, on

Larvae were placed into the above solutions for 2 hr daily, otherwise they were kept in chlorine-free tap water. Water was changed daily. The larvae were fed nettle powder, grated apple, and lettuce ad libitum; egg white was given twice a week. Development of the larvae was observed daily and body measurements were made photometrically (Feuer ef al., 1978a). Body length was determined five times. Growth curves of the control and experimental groups were plotted from the mean values of body length. The results were il- lustrated in Terentiev stage diagrams.

Statistical significance of the differences was calcu- lated by Student’s f test.

RESULTS

’ Deceased on 6 February, 1979. Body length changes, based on determi- * Address all reprint requests to L. Feuer. nations of the mean values, are shown in

285 001~6480/79/070285-05$01.00/O Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any fonp reserved.

Page 2: Effect of prednisolone on the development of amphibian larvae and the antagonism of litoralon and prednisolone

286 TOROK, FEUER, AND CSABA

I I I

b :“3 5”, zo 25 30 63 68 73

days

FIG. 1. Development of amphibian larvae on the effects of prednisolone (10 &ml) and prednisolone (10 j&ml) + litoralon (0.5 &ml).

Fig. 1. Body length of the control animals increased until the 15th experimental day (58 days of larval age) and then it began to decrease. Under the effect of prednisolone growth proceeded only until the fifth ex- perimental day (48 days of larval age) and even then to a significantly (P < 0.05) lesser degree; after this body length abruptly de- creased. Significantly (P < 0.01) lower val- ues than the control were obtained at each further occasion. The slopes of the curves are very similar to those of the groups treated with 0.01 pg/ml T3 (triio-

dothyronine, Merck) (Feuer er al., 1979b). The stimulatory effect of prednisolone on

morphogenesis is readily seen from com- parison of the stage diagrams (Figs. 2 and 3). Most conspicuous are the data of the period between Days 5 and 23 of the ex- periment. For instance, while in the control group nearly half of the population be- longed to stage 26 (T-K-26/IV-V) by the fifth experimental day, in the treated group the majority of the tadpoles stage 27 (T-K-27/X) and some of them even 28 (T-K-2WXIV) in the same period. On the 13th day the

Control group

27 Terentrev stage. [I26

n 027 El 28 I 29 I 30

27

48 56 64

FIG. 2. Stage (Terentiev) diagram of control group.

Page 3: Effect of prednisolone on the development of amphibian larvae and the antagonism of litoralon and prednisolone

PREDNISOLONE, LITORALON, AND FROG DEVELOPMENT 287

Prednisolone treated group

4a 56 64 Experfmentol 66 72 da&s

Age of hrvoe rn days

FIG. 3. Stage (Terentiev) diagram of prednisolone (10 &ml)-treated group.

majority of the larvae of the control group was in stage 27 (T-K-27/X), while in the prednisolone treated groups stage 28 (T-K- 28/XIV) dominated.

Antagonism between prednisolone and litoralon was noticeable as early as on the fifth experimental day and was evident dur- ing the entire period of observation (Fig.

tion of the animals was in complete agree- ment with the above findings (Fig. 4.).

The developmental stage of the larvae treated with prednisolone + litoralon was constantly between those of the control and the prednisolone treated groups, being nearer to the control values until the 30th day of treatment.

1.). Body length of the larvae treated with the DISCUSSION

combination of prednisolone + litoralon Glucocorticoids can enhance (Gorbman, differed significantly from both the controls 1964; Kobayashi and Okubo, 1954) or in- (P < 0.02) and those treated with pred- hibit (Gorbman, 1964; Dastoli and Tector, nisolone only (P < 0.01). Stage distribu- 1959; Kobayashi, 1958) metamorphosis.

Predtvsoion + I,toralon treated group

Tcrenttev s toge :: 26

0 27 Rza B 29 I 30

4.3 56 64 66 Expe#me”to, days76

Age of lorvoe rn days

FIG. 4. Stage (Terentiev) diagram of prednisolone (10 &ml)- + litoralon (0.5 kg/ml&treated group.

Page 4: Effect of prednisolone on the development of amphibian larvae and the antagonism of litoralon and prednisolone

288 I%RiijK, FEUER, AND CSABA

According to Kaltenbach (1968) “incon- sistencies among reports may be due to differences in the concentrations of thyroxine and steroids administered, the order and manner of their administration or the species and stage of amphibian used for testing.”

Other authors (Weissmann and Thomas, 1963; Wenzel and Acosta, 1973) have shown that cortisone, as a lysosomal stabi- lizer, antagonizes the lysosomal activating effect of vitamin A. This antagonism, which takes place at a subcellular level, is also responsible for several other glucocorticoid effects antagonistic to vitamin A. Thus, hydrocortisone inhibits the increased sul- fate metabolism of the skin produced by vitamin A (Barker et al., 1964) and sus- pends the anticarcinogenic effect of vitamin A (Shamberger, 1971). At the same time vitamin A prevents (Cohen and Cohen, 1973) or antagonizes (Brazenor and Stephens, 1973) the immunosuppressive effect of glucocorticoids.

Our own investigations demonstrated that litoralon potentiated the effect of vita- min A and antagonized that of prednisolone in tissue and cell cultures of the rat thymus (Feuer et al., 1978~). Litoralon, adminis- tered by itself, considerably increased thymocyte proliferation and macrophage reactions in thymus tissue and cell cultures (Feuer et al., 1978b) and enhances the lysosomal enzyme activity during amphi- bian metamorphosis (Feuer et al., 1979a).

This seems to indicate that litoralon and glucocorticoids both affect the vitamin A-thyroid hormone axis but in opposite di- rection. While litoralon potentiates the ef- fects of vitamin A and antagonizes those of the hormones T3 and Tq, glucocorticoids on the contrary antagonize the effect of vita- min A and facilitate the hormonal actions of

and T, effects and this is how the action of thyroid hormones is increased. It also must be taken into consideration that the effect of thyroid hormones has been considerably extended during evolution. Their effect on metabolic rate, heat production, and oxy- gen consumption is not present in the poi- kilotherm animals and appears only in birds and mammals.

Propionic acid analogs of T3 and T, have a still much stronger effect on the metamorphosis of amphibia while their ef- fect on metabolic rate lags far behind that of thyroid hormones (Foldes, 1976).

The present experiments, in which body length curves of the prednisolone-treated larvae correspond to those of animals treated with T3 in previous experiments (Feuer et al., 1979b) and the growth curves of the animals treated with the combination of prednisolone + litoralon are similar to those subjected to triiodothyronine + litoralon (Feuer et al., 1979b), seem to con- firm the above suggested correlation of these hormones. Further investigations are however required in support of this conclu- sion.

REFERENCES Barker, S. A., Cruickshank. C. N. D., and Webb, T.

(1964). The effect of vitamin A, hydrocortisone and citral upon sulphate metabolism in skin. Exp. Cell. Res. 35, 255-261.

Brazenor, G., and Stephens, F. 0. (1973). The effect of vitamin A on the immune suppressive action of corticone on skin homografts in mice. Ausfr. N. Z. J. Surg. 42, 314-317.

Cohen. B. E., and Cohen, 1. K. (1973). Vitamin A adjuvant and steroid antagonist immune response. J. Immunol. 111, 1376- 1380.

Csaba, G. (1977). Hormonal regulation: morphogenet- ic and adaptive systems. Bid. Rev. 52, 295-303.

Dastoli, F. R., and Tector A. J. (1959). The effect of cortisone on amphibian metamorphosis. J. Exp. Zool. 87, 259-277.

Feuer L. (1975). B. P. 1,404.225; (1977). U.S.P.

T3 and T,. It cannot be excluded however, 4,001,396.

that the permissive effect of glucocorticoids Feuer, L. (1977). Theoretical background of the rec-

(Ingle, 1954; Sayers and Travis, 1971) has ognition of a new bioactive substance, litoralon, isolated from the parathyroid. Further theoretical

also some role in the manifestation of T3 considerations. Biologic 25, (I), 3-33.

Page 5: Effect of prednisolone on the development of amphibian larvae and the antagonism of litoralon and prednisolone

PREDNISOLONE, LITORALON, AND FROG DEVELOPMENT 289

Feuer, L., Banyai, B., and Hercsel, J. (1977). Influ- ence of protein free aqueous extract of parathyroid powder on serum vitamin A level in rats. Experientia 33, 1005-- 1006.

Feuer, L., Kovacs, P., and Csaba, C. (1979a). The effect of litoralon (y-r-glutamyl-taurine) on the lysosomal activity of mesenchymal cells and mac- rophages. Cotnp. Biochem. Physioi., in press.

Feuer, L., Torok, L. J., and Csaba, G. (1979b). The triiodothyronine antagonistic effect of gamma-r- glutamyltaurine (litoralon). Endokrinologie, in press.

Feuer, L., Torok, L. J., Kapa, E., and Csaba, G. (1978a). The effect of gamma-t-glutamyl-taurine (litoralon) on the amphibian metamorphosis. Comp. B&hem. Physial. 61, 67-71.

Feuer, L., Torok, O., and Csaba, G. (1978b). Effect of glutaurine, a newly discovered parathyroid hor- mone on rat thymus cultures. Acto Morphol. Acad. Sci. Hung. 26(2), 87-94.

Feuer, L., Torok. O., and Csaba, G. (1978c). Effect of glutaurine on vitamin A and prednisolone treated thymus cultures. Acta Morphol. Acad. Sci. Hung. 26(2), 75-85.

Foldes, I. (1976). “Exophthalmic goiter,” p. 19. Hung. Acad. Sci., Budapest.

Gorbman, A. (1964). Endocrinology of the amphibian. In: “Physiology of the Amphibia” (J. A. Moore, ed.), p. 410. Academic Press, New York London.

Ingle, D. J. (1954). Permissive action of hormones. J. C/in. Endocrinol. Metab. 14, 1272- 1274.

Kaltenbach, J. C. (1968). Nature of hormone action in amphibian metamorphosis. In “Metamorphosis, A Problem in Developmental Biology” (W. Etkin

and L. I. Gilbert, eds.), pp. 423-424 North- Holland, Amsterdam Appleton-Century, New York.

Kobayashi, H. (1958). Effect of desoxycorticosterone acetate on metamorphosis induced by thyroxine in anuran tadpoles. Endocrinology 62, 371-377.

Kobayashi, H., and Okubo, K. (1954). Effects of de- soxycorticosterone acetate on broodines, molting and pituitary lactogen content in the canary and on metamorphosis of the toad tadpole. Annot. 2001. Japan. 27, 173- 179.

Sayers, Cl., and Travis, R. H. (1971). Adrenocortico- tropic hormone: Adrenocortical steroids and their synthetic analogs. In “The Pharmacological Basis of Therapeutics (L. S. Goodman and A. Gilman, eds.), pp. 1604 1642 Macmillan, London.

Shamberger, R. J. (1971). Inhibitory effect of vitamin A on carcinogenesis. J. Nat. Cancer Invest. 47, 667-673.

Taylor, A. C., and Kollros, J. J. (1946). Stages in the normal development of Rana pipiens larvae. Anat. Rec. 94, 7-23.

Terentiev, P. B. (1950). “Liagushka [“The frog”], p. 59-67 Sovietskaia Nauka., Moscow.

Weissmann, G., and Thomas, L. J. (1963). Studies on lysosomes. II. The effect of cortisone on the re- lease of acid hydrolases from large granule frac- tion of rabbit liver induced by an excess of vitamin A. J. C/in. Invest. 42, 661-669.

Wenzel, D. G., and Acosta, D. (1973). Permeability of lysosoma and mitochondria in cultured rat heart muscle and endotheloid cells as affected by vita- min A, chlorpromazine, amphotericin B and clotibrate. Res. Commun. Chum. Path&. Phar- macol. 6, 689-700.