In vitro study of frog (Rana ridibunda pallas) interrenal function by use of a simplified perifusion system. V. Influence of adrenocorticotropin upon progesterone production

The dynamics of progesterone production by perifused frog adrenal explants has been investigated under pituitary stimulation. Specific progesterone antibodies have been raised in rabbits and a radioimmunoassay has been developed. The sensitivity of the assay and the low cross-reactivity of the antiserum with the major corticosteroids produced by the interrenal tissue, mainly corticosterone (0.7%) and aldosterone (0.002%), made it possible to measure progesterone concentrations in 150-μl samples of effluent perifusate, without prior extraction of purification. The validity of the method was confirmed on Sephadex LH-20 gel chromatography. A direct effect of temperature variations on progesterone output was demonstrated in the temperature range 5–30°, which strongly suggested that external temperature controls progesterone synthesis by the interrenal gland in vivo. In the presence of distal lobe homogenates a linear log-dose increase in progesterone output was observed. The use of ACTH 1–24 as a reference standard made it possible to compare the biological activity of crude distal lobe homogenates from frog pituitary and synthetic ACTH. The effect of neurointermediate lobe extracts on progesterone production was also studied. At a concentration as low as 0.006 neurointermediate lobe eq/ml, a 2.1-fold increase in progesterone output was observed suggesting a very high concentration of ACTH-like material in frog pars intermedia. As far as is known the present data provide the first unequivocal evidence that, in frog, intact interrenal cells produce significant amounts of progesterone in vitro. Our results demonstrate also that synthetic ACTH and pituitary extracts stimulate progesterone output and that the dynamics of the steroidogenic response to various stimulants was virtually the same with progesterone, corticosterone, or aldosterone.     

In vitro study of frog (Rana radibunda Pallas) interrenal function by use of a simplified perifusion system. I. Influence of adrenocorticotropin upon corticosterone release.

In order to examine the dynamics of the steroidogenic response from amphibian interrenal glands to various stimulae, frog interrenal secretion was studied using a perifusion system technique, Rana ridibunda adrenal fragments were continuously perifused for 10 hr with amphibian culture medium (ACM). Corticosterone release was assayed using a sensitive competitive-binding radioassay. Inhibition curves using either standard corticosterone dissolved in ACM or serial dilutions of effluent perfusate were strictly parallel. When the temperature of the perifusing medium was raised from 5 to 30°, the corticosterone secretion rate increased eightfold, suggesting a possible direct effect of the ambient temperature on corticosterone secretion in the frog. Perifusion of frog interrenal fragments with homologous crude pituitary distal lobe extracts led to a linear log-dose response. When synthetic ^1–24ACTH was used as a reference standard, the perifusion technique proved useful as a semiquantitative assay system for the detection of biologically active pituitary ACTH. Since immunoreactive ACTH is also found in frog intermediate lobes, pars intermedia extracts were assayed for their ability to stimulate corticosterone release in vitro. At a dose as low as 0.006 intermediate lobe equivalent/ml a significant increase in corticosterone release was observed (1.8-fold; P < 0.001). At the lower doses, the effects of intermediate lobe extracts on corticosterone release were dose-related. The maximum effective dose is $\text{1}\text{40}$ intermediate lobe eq/ml. At higher doses (up to $\text{1}\text{5}$ eq/ml) no stronger stimulation could be elicited. Dibutyryl-cyclic-AMP at 10^?2M induced a 3.3-fold increase in corticosterone secretion. From these results, we conclude that the perifusion system technique will prove useful for the study of the mechanisms governing the regulation of frog steroidogenesis in vitro.     

In vitro study of frog (Rana ridibunda Pallas) interrenal function by use of a simplified perifusion system. VIII. Structure-activity relationship of synthetic ACTH fragments and gamma-MSH

The present study was undertaken to determine the structure-activity relationships of ACTH analogs on corticosteroid production by frog adrenal gland. Rana ridibunda interrenal dice were perifused with amphibian culture medium for 10 hr. Corticosterone and aldosterone concentrations were measured in the effluent perifusate using sensitive and specific radioimmunoassay methods. Perifusion of interrenal fragments with increasing concentrations of synthetic human ACTH 1-39 (ranging from 6.25 X 10(-11) to 10(-9) M) led to a linear log-dose increase in both corticosterone and aldosterone secretion. Thus, this model made it possible to compare the steroidogenic potency of several ACTH analogs. Synthetic alpha-MSH and its des-N alpha-acetyl derivative were found to be approximately equipotent, and 5 X 10(3) times less active than authentic ACTH. The short-chain analog ACTH 1-10 was 2 X 10(4) times less potent than ACTH whereas ACTH 4-10 was totally inactive. A fragment of the N-terminal region of the proopiomelanocortin molecule, gamma 3-MSH, caused a dose-related stimulation of steroid secretion. However, in contrast to what has been observed in the rat, gamma 3-MSH did not potentiate the corticotropic action of ACTH on frog interrenal gland. Since processing of proopiomelanocortin in frog intermediate lobe generates high amounts of alpha-MSH and des-N alpha-acetyl alpha-MSH, these results suggest that in amphibians, several peptides other than ACTH may be involved in the control of corticosteroidogenesis.     

In vitro study of frog (Rana ridibunda Pallas) neurointermediate lobe secretion by use of a simplified perifusion system. IV. Interaction between dopamine and thyrotropin-releasing hormone on alpha-melanocyte-stimulating hormone secretion

The interaction between dopamine and TRH on alpha-melanocyte-stimulating hormone (MSH) release from the intermediate lobe of amphibian pituitary has been studied in vitro using the perifusion model. Dopamine (10(-10) to 10(-6) M) was responsible for a dose-related inhibition of alpha-MSH secretion. The inhibitory effect of dopamine (10(-8) and 3.16 X 10(-8) M) was completely abolished in the presence of haloperidol (10(-5) and 10(-6) M, respectively). It has been previously established that, in amphibians, TRH stimulated alpha-MSH release in vitro and that the action of TRH was not mediated via an inhibition of the release of endogenous dopamine (M. C. Tonon, P. Leroux, M. E. Stoeckel, S. Jégou, G. Pelletier, and H. Vaudry, 1986, Endocrinology 112, 133-141). In the present study we demonstrate that TRH (10(-7) M) reverses the inhibitory effect of dopamine (for concentrations ranging from 3.16 X 10(-8) to 10(-6) M) on alpha-MSH secretion and that the effects of TRH and dopamine are additive. Thus, these results indicate that the intracellular events associated with TRH-induced stimulation and dopamine-induced inhibition of alpha-MSH release are not linked together.     

In vitro study of frog (Rana ridibunda pallas) neurointermediate lobe secretion by use of a simplified perifusion system: I. Effect of TRH analogs upon α-MSH release

We have recently shown that the tripeptide l-pyroglutamyl-lhistidyl-l-proline-amide (mammalian TRH) stimulates α-MSH secretion in amphibia. Using a perifusion system technique, we have compared the stereochemical requirements for hormone-receptor interaction of frog melanotrophs with mammalian thyrotrophs and mammatrophs. Of all the analogs tested, only the TRH analog l-N-(2-oxopiperidine-6-yl-carbonyl)-histidyl-thiazolidine-4-carboxamide (MK-771) was equipotent with TRH. All analogs which were known to be TRH agonists in mammals (e.g., [Pic]³-TRH, [Pro-hydrazide]³-TRH) were also relatively active on α-MSH release. Seven analogs were totally inactive on both mammalian pars distalis and frog pars intermedia. The discrepancies concerned only two TRH analogs in which the histidine moiety has been altered [Tyr]²-TRH and [Lys]²-TRH). The biological potencies of these analogs were 17 and 8% on α-MSH release whereas both molecules were devoid of activity in mammals.     

In vitro study of frog (Rana ridibunda Pallas) interrenal function by use of a simplified perifusion system III. Influence of previous hypophysectomy or dexamethasone treatment of donors upon corticosterone and aldosterone production

Effects of long-term hypophysectomy and dexamethasone treatment on interrenal function of Rana ridibunda have been studied in vitro by use of a perifusion system technique. Hypophysectomy significantly reduced secretory activity of interrenal tissue (nearly 60% reduction for both corticosterone and aldosterone biosynthesis) but did not affect interrenal responsiveness to crude homologous pituitary extract and did not modify the ratio aldosterone/corticosterone as well. Daily injections of 1 mg dexamethasone during 20 days diminished both corticosterone and aldosterone productions by interrenal tissue to about 40% of those of glands from normal animals. Dexamethasone treatment did not alter interrenal responsiveness to crude homologous pituitary extract. In addition, daily injections of 0.6% NaCl solution for 18 days (0.25 ml/animal) resulted in a specific decrease in aldosterone secretion. Aldosterone/corticosterone secretion ratios were 2.1 in normal frogs, 2.3 in hypophysectomized frogs, 2.2 in sham-hypophysectomized frogs, and 1.9 in dexamethasone-treated frogs. This ratio fell to 1.3 in saline-treated frogs. These in vitro results are consistent with previous in vivo data on anuran pituitary-interrenal relationships which had demonstrated partial independence of interrenal function from pituitary control. They also provide evidence that alteration of electrolyte balance specifically modifies aldosterone production without significant change in corticosterone biosynthesis.     

In vitro study of frog (Rana ridibunda pallas) neurointermediate lobe secretion by use of a simplified perifusion system: III. Effect of neuropeptides on α-MSH secretion

It has been previously demonstrated that thyrotropin-releasing hormone (TRH) stimulates in vitro the release of α-melanocyte-stimulating hormone (α-MSH) in frog. In the present study, the effects of various neuropeptides on spontaneous and/or TRH-induced α-MSH secretion were investigated, using a well-defined perifusion system technique. Vasoactive intestinal peptide, (VIP) a neurohormone which stimulates TRH target cells in mammals, was totally devoid of effect on frog melanotrophs although VIP-like material could be detected in neurointermediate lobe extracts. Somatostatin-like immunoreactive material was found in high concentrations in the frog neurointermediate lobe complex, but synthetic somatostatin (from 10^?10 to 10^?6M) did not modify the spontaneous release of α-MSH. At doses of 10^?8 and 10^?6M, synthetic somatostatin did not modify TRH-induced α-MSH secretion. Morphine (10^?5M) and opioid peptides (10^?10 to 10^?6M) had no effect on spontaneous α-MSH secretion. In addition, methionine enkephalin (10^?5M) did not modify the stimulatory effect of TRH on α-MSH secretion. From these results we conclude that, among the neuropeptides which modulate prolactin secretion in mammals, only TRH is involved in α-MSH secretion in the frog.     

In vitro study of frog (Rana ridibunda pallas) neurointermediate lobe secretion by use of a simplified perifusion system: II. Lack of action of thyroxine on TRH-induced α-MSH secretion

Thyrotropin-releasing hormone (TRH) stimulates α-melanocyte-stimulating hormone (α-MSH) secretion in amphibia as well as thyrotropin-stimulating hormone (TSH) and prolactin secretions in mammals. Since thyroid hormones regulate the stimulatory effect of TRH on TSH and prolactin, the possible role of thyroxine (T₄) in the control of α-MSH secretion in amphibia, has been investigated. Neurointermediate lobes of Rana ridibunda were perifused in amphibian culture medium for 7 hr and the amounts of α-MSH released into the effluent perfusate were measured by radioimmunoassay. In vivo treatment with T₄ (0.5 mg/kg twice a day for 9 days) did not modify the in vitro response of the neurointermediate lobes to TRH (10^?9 to 10^?7M). In addition, prolonged infusion of T₄in vitro did not alter spontaneous and TRH-induced α-MSH release. In spite of the inhibitory effect of T₄ on TRH-induced TSH and prolactin secretions in mammals, the present data show that, in frogs, thyroid hormone does not modulate the stimulation of α-MSH secretion induced by TRH.     

Seasonal study of the interrenal function of the European green frog, in vivo and in vitro.

The existence of seasonal changes in the secretory activity of the interrenal glands in European Green frogs has been investigated in vivo, in a natural environment, and in vitro, using a perifusion system technique. In vivo, plasma corticosterone levels have been systematically measured at six different moments during a 24-hr period (03, 07, 11, 15, 19, and 23 hr), for almost all the months, during 2 consecutive years. In this part of the study, corticosterone concentrations were assayed in duplicate, in an average of seven animals at each of the six definite moments of the 24-hr cycle. Significant fluctuations in circulating corticosterone have been recorded: a single peak in late spring (May and June) coinciding with the spawning period; a decrease during summer and autumn; the nadir in the depth of winter (February); and a sharp increase at the emergence from hibernation (end of March). The amplitude of the fluctuations ranged from 0.28 ± 0.07 μg/100 ml in late February to 2.68 ± 0.36 μg/100 ml in late May. No significant difference in plasma corticosterone concentrations was observed between males and females. The in vitro studies consisted of comparing the responses of perifused frog interrenal tissue to increasing doses of homologous pituitary extracts, at various times of the year. According to the date of the experiment, important differences in maximum corticosterone snd aldosterone secretions were recorded. As an example, under submaximum ACTH stimulations, 188 and 193% increases in corticosterone and aldosterone outputs, respectively, were recorded on June 4th, whereas the corresponding figures on October 17th were 38 and 54%. The maximum capacity of interrenal tissue to secrete corticosterone and aldosterone after stimulation by homologous pituitary extract was observed in May and June, whereas the minimum capacity was recorded in winter. These results are in complete agreement with our in vivo data. From the present results, we conclude that corticosterone production undergoes seasonal fluctuations in European Green frog. Since the fluctuations cannot simply originate from variations of ambient temperature (F. Leboulenger, C. Delarue, M. C. Tonon, S. Jegou, and H. Vaudry, 1978, Gen. Comp. Endocrinol.36, 327–338), our in vitro results support the view that plasma corticosterone rhythms are due, at least in part, to seasonal variations of interrenal sensitivity to ACTH.     

Actions of angiotensin II antagonists upon aldosterone production by isolated adrenal glomerulosa cells.

The biological activities of angiotensin II antagonists upon basal and angiotensin II-stimulated aldosterone production were evaluated in an isolated canine glomerulosa cell preparation. The most potent competitive antagonist of angiotensin II-stimulated aldosterone production was the [Sar1, Ile8]derivative of angiotensin II. However, this peptide was also a partial agonist at concentrations required to inhibit the steroidogenic effect of angiotensin II on dog adrenal cells, and never reduced aldosterone production to basal levels. On a molar basis, the [Sar1, Ala8] and [Sar1, Gly8]derivatives of angiotensin II were relatively less potent as competitive inhibitors of angiotensin II-stimulated aldosterone production. However, the [Ala8] and [Gly8]-analogues did not exhibit significant agonist activity and were therefore more effective antagonists of angiontensin II-stimulated aldosterone production. These results suggest that increased length of the aliphatic side chain at the C-terminus of angiotensin II antagonists is accompanied by enhanced affinity for the receptor site, but also by increased agonist activity upon aldosterone synthesis. The actions of angiotensin II and [Des-Asp1]angiotensin II upon aldosterone production were inhibited identically and completely by [Sar1, Ala8]angiotensin II, and identically, though incompletely, by lower concentrations of [Sar1, Ile8]angiotensin II. The heptapeptide antagonist [Des-Asp1, Ile8]angiotensin II was much less potent than [Sar1, Ile8]angiotensin II as an inhibitor of the actions of both the heptapeptide and octapeptide agonists. The antagonist activity of six angiotensin II analogues at the adrenal level, determined by the concentration required for 50% inhibition of maximum aldosterone secretion, correlated well with their antagonist activity measured upon isolated smooth muscle. These observations demonstrate that the octapeptide antagonists are more effective than the heptapeptide antagonists upon angiotensin II-stimulated aldosterone production, and that angiotensin II receptors in smooth muscle and adrenal cortex exhibit generally similar responses to angiotensin II antagonists. Also, these results do not support the proposal that the [Des-Asp1]heptapeptide is an important intermediate in the action of angiotensin II upon adolesterone production in the adrenal glomerulosa cells. The production of aldosterone by dispersed zona glomerulosa cells in vitro provides a highly sensitive and biologically appropriate response for evaluation of the agonist and antagonist properties of angiotensin II analogues upon the adrenal gland.     

Stimulatory effect of a GnRH agonist (buserelin) in in vitro and in vivo testosterone production by the frog (Rana esculenta) testis

The summary testicular effects of an agonistic analogue of gonadotropin-releasing hormone (buserelin, GnRHa) have been studied in vitro and in vivo in the frog, Rana esculenta. During 3 h incubation GnRHa (8 X 10(-7) M) potentiated pituitary factors in stimulating testosterone production by minced testes in vitro. After 6 h of incubation 8 X 10(-7) M GnRHa stimulated maximal testosterone output. Testes of 10-day hypophysectomized animals did not show any GnRHa effect in vitro. In vivo, a direct effect of GnRHa on testicular testosterone production was demonstrated in hypophysectomized animals, although this effect was temperature-dependent, requiring the frog to be maintained at a high temperature (24 degrees C). No effect of GnRHa was detectable in frogs kept at a low temperature (4 degrees C).     

In vitro stimulation of cyclic-AMP production in Rana catesbeiana ovaries by homologous gonadotropins.

In vitro effects of purified Rana LH and FSH on cyclic AMP accumulation were examined in ovaries of immature and mature bullfrog, Rana catesbeiana, at different states of the sexual cycle. Rana FSH was active with clear dose dependency only in immature ovaries but was less potent than Rana LH. Mature females did not respond to Rana FSH. Rana LH always increased the cAMP accumulation in mature females but the response was lowest with prespawning and postspawning females.     

Gonadotropin-releasing hormone stimulates biosynthesis of prostaglandin F2 alpha by the interrenal gland of the water frog, Rana esculenta, in vitro.

The present study was carried out to evaluate the in vitro effects of mammalian gonadotropin-releasing hormone (mGnRH) on the production of prostaglandin F2 alpha (PGF2 alpha) and sex steroids (progesterone, androgens, and 17 beta-estradiol) by the interrenal gland of male and female Rana esculenta during three different periods of the sexual annual cycle. In both sexes, mGnRH induced a significant increase in PGF2 alpha in the incubation medium in all examined periods. Progesterone and androgens were undetectable, while 17 beta-estradiol was significantly increased by mGnRH in interrenals incubated during the postreproductive period in both sexes. These results suggest that R. esculenta interrenals could be a GnRH-dependent PGF2 alpha-secreting tissue. In addition, the simultaneous increase in PGF2 alpha and estradiol from postreproductive cultured interrenals support the notion that mGnRH-induced estradiol synthesis is mediated through PGF2 alpha formation. This finding, taken together with other previous studies, strongly suggests that the end of the breeding period in R. esculenta depends on GnRH-induced PGF2 alpha-mediated enhancement of estradiol synthesis in a steroidogenetic organ (probably interrenals).     

Demonstration of aldosterone and corticosterone as the principal steroids formed in incubates of adrenals of the American bullfrog (Rana catesbeiana) and stimulation of their production by mammalian adrenocorticotropin

Aldosterone and corticosterone were found to be the only Δ⁴-3-ketosteroids produced to any significant extent in vitro by adrenal tissue from the American bullfrog. The production of aldosterone was stimulated highly by bovine adrenocorticotropin as well as by the frog's own anterior pituitaries. The amount of aldosterone produced by the bullfrog adrenal tissue under the stimulation of highly purified bovine ACTH was about four times the amount of corticosterone.The material from which the steroids were isolated was purified by solvent partitioning and reversed phase chromatography on purified acetylated filter paper according to a newly developed method. The identity of the isolated material with aldosterone was confirmed by the location of the ultraviolet absorption maximum at 239 mμ, by the fact that a characteristic spectrum was obtained before and after heating in sulfuric acid, on the basis of a positive reaction with blue tetrazolium and of the absence of any reaction with the Porter-Silber reaction, and by the results of bioassay in the Na²⁴-K⁴² test.     

Treatment of primary cultures of calf adrenal glomerulosa cells with adrenocorticotropin (ACTH) and phorbol esters: a comparative study of the effects on aldosterone production and ACTH signaling system

We studied the mechanism that underlies the desensitization of calf adrenal glomerulosa cells induced by 4 h of ACTH treatment. In control cells, acute ACTH treatment provoked sizeable increases in aldosterone, cAMP, and diacylglycerol, and translocated protein kinase-C from cytosol to membrane. In desensitized cells, acute ACTH effects on aldosterone and cAMP decreased by 25-60%, and diacylglycerol levels and protein kinase-C translocation were persistently stimulated and not substantially affected by further acute ACTH treatment. After 4 h of treatment with 1 microM phorbol 12-myristate 13-acetate (PMA) there were no acute effects of ACTH on the production of aldosterone, cAMP, or diacylglycerol or on protein kinase-C, which was already strongly translocated. These results suggest that ACTH-mediated desensitization of calf adrenal glomerulosa cells may be at least partially mimicked by long term treatment with phorbol esters and could be due to ACTH-induced increases in diacylglycerol-protein kinase-C signaling.     

In vivo and in vitro stimulatory effect of a gonadotropin-releasing hormone analog (HOE 766) on spermatogonial multiplication in the frog, Rana esculenta

The effects of a GnRH analog (GnRHA), D-Ser-t-Bu6,desGly-NH2(10) (HOE 766) on spermatogenesis were analyzed in the frog, Rana esculenta. Intact animals caught at two different periods of the year (January and March) were treated with HOE 766 (GnRHA, 45 ng/g BW) at low (4 +/- 1 C) and high (22 +/- 2 C) temperatures. Hypophysectomized frogs were used also and, in addition to GnRHA, these animals were treated with crude pars distalis homogenate. In vitro incubations were carried out at 15 C, for 0, 6, and 24 h with the addition of 1 microgram GnRHA. Half of each testis was used as the untreated control. Histological sections of the testes were analyzed for the evaluation of the mitotic index of the primary spermatogonia. Intact March animals had mitotic indices higher than January animals. GnRHA treatment elicited an increase of the mitotic index in both intact and hypophysectomized animals. High temperature potentiated the GnRHA effect while low temperature favored pars distalis treatment. In conclusion, the present results are consistent with the fact that in the frog, R. esculenta, the magnitude of spermatogonial proliferation is temperature dependent, and for the first time it is shown that GnRH-like substances have direct stimulatory effect on the mitotic activity of the primary spermatogonia in a vertebrate.     

Regulation of androgen production by frog (Rana esculenta) testis: an in vitro study on the effects exerted by estradiol, 5 alpha-dihydrotestosterone, testosterone, melatonin, and serotonin

The possible role of estradiol-17 beta (E2), testosterone (T), 5 alpha-dihydrotestosterone (DHT), melatonin, and serotonin on the regulation of androgen (A) production by the frog, Rana esculenta, testes was studied in vitro. E2 (10(-6) M) inhibited A production whether alone or in combination with oLH (20 micrograms) after 6 hr incubation. After 24 hr incubation. A production was reduced by E2 concentration of around 10(-6) and 10(-9) M. Melatonin and serotonin did not induce any change whichever experimental condition was used. Preincubation for 6 hr with 10(-6) M T or DHT enhanced the oLH-stimulated A production after 18 hr incubation. These data suggest that steroids may regulate their intratesticular levels without passing into the blood stream.