The potentiating effects of phorbol ester on ACTH-, cholera toxin-, and forskolin-induced cAMP production by cultured bovine adrenal cells is not mediated by the inactivation of alpha subunit of Gi protein

Phorbol ester (PMA) potentiates ACTH-induced cAMP production by both fresh isolated and 7-day-old cultured adrenal cells, but the effect on cultured cells was greater than in fresh cells. In cultured cells the potentiating effects of PMA were dose-dependent and were observed at each effective dose of ACTH without modification of the ED50 for this hormone. These effects of PMA do not seem to be exerted through a modification of the alpha subunit of Gi since pretreatment of the cells with Bordetella pertussis toxin did not modify the action of PMA and since the amount of alpha i in 7-day-old cultured cells was ten times lower than in fresh cells, while the potentiating effect was lower in the latter. Moreover, since PMA still exerted its potentiating action in cells stimulated by maximal concentration of cholera toxin or forskolin either alone or in combination with ACTH, it is likely that its action is not mediated exclusively by the alpha subunit of Gs. Taken together, the present results and those of the literature suggest that this potentiating effect of phorbol ester on effector-induced cAMP production might be mediated by inhibition of the beta-subunit of G proteins.     

Angiotensin II potentiates the effect of ACTH on the production of cyclic AMP by bovine adrenal cells in primary culture

The effect of Angiotensin II (AII) on ACTH-induced cyclic AMP production was studied in bovine adrenocortical cells cultured in a chemically defined serum-free medium. Immediately after collagenase dispersion, AII did not modify either the basal or the ACTH-induced cAMP production by the isolated cells. During cell culture, AII alone did not affect cAMP production. But 2 days after plating, AII increased significantly the ACTH-induced cAMP production by the culture. This potentiating effect increased with the age of culture. Sar1Ala8AII (Saralasin), a potent AII antagonist, inhibited the AII potentiating effect indicating an AII specific action.     

Adrenocorticotropin-induced unresponsiveness in cultured adrenal tumor cells.

Our results demonstrate that adrenocorticotropin (ACTH)-induced refractoriness occurs in cultured adrenal tumor cells. Cells became 85% refractory to ACTH-induced cyclic AMP formation in 20 min and the effect persisted if the hormone remained in the incubation medium. Refractory cells gradually regained hormone-specific responsiveness within 24 h if cultures were incubated in fresh media containing serum. The observed effect is hormone specific since cyclic AMP could not induce unresponsiveness to ACTH. The addition of ACTH plus inhibitors of protein synthesis partially reversed hormone-specific refractoriness. However, preincubation with cycloheximide or diphtheria toxin led to superinduction of ACTH-induced cyclic AMP formation. These experiments suggest that unresponsiveness, following hormonal activation of adrenal cells, may be related to a decrease in hormone-specific binding sites or to synthesis of an adenylate cyclase inhibitor.     

Exogenous steroids alter steroidogenesis in cultured Y-1 adrenal tumor cells by actions preceding cyclic AMP

Using cultured Y-1 mouse adrenal tumor cells which produce 20 alpha-hydroxy-4-pregnen-3-one (20-DHP), it was found that 0.01 mM corticosterone and deoxycorticosterone increased basal and inhibited ACTH-induced 20-DHP production during consecutive 30 and 120 min incubations. Steroid effects were concentration-dependent and reversible. Six other steroids tested did not stimulate 20-DHP production and varied in ability to inhibit ACTH-stimulated steroidogenesis. Experiments demonstrated that 20-DHP production following treatment with cholera toxin, N,0'-dibutyryl cyclic AMP (dbcAMP), or pregnenolone was not inhibited by exogenous steroids. Corticosterone (0.01 mM) increased basal and inhibited ACTH-induced intracellular cyclic AMP (cAMP) production. Cytochalasin D, a microfilament perturbing agent, inhibited steroid-stimulated 20-DHP production, suggesting that ACTH and steroid stimulation mechanisms were similar. These findings taken together suggest that exogenous steroids can alter steroidogenesis by modifying plasma membrane adenylate cyclase activity.     

Adrenocorticotropin-induced unresponsiveness in cultured adrenal tumor cells

Our results demonstrate that adrenocorticotropin (ACTH)-induced refractoriness occurs in cultured adrenal tumor cells. Cells became 85% refractory to ACTH-induced cyclic AMP formation in 20 min and the effect persisted if the hormone remained in the incubation medium. Refractory cells gradually regained hormone-specific responsiveness within 24 h if cultures were incubated in fresh media containing serum. The observed effect is hormone specific since cyclic AMP could not induce unresponsiveness to ACTH. The addition of ACTH plus inhibitors of protein synthesis partially reversed hormone-specific refractoriness. However, preincubation with cycloheximide or diphtheria toxin led to superinduction of ACTH-induced cyclic AMP formation. These experimens suggest that unresponsiveness, following hormonal activation of adrenal cells may be related to a decrease in hormone-specific binding sites or to synthesis of an adenylate cyclase inhibitor.     

Multiple trimeric G-proteins on the trans-Golgi network exert stimulatory and inhibitory effects on secretory vesicle formation.

The role of heterotrimeric G-proteins on the formation of constitutive secretory vesicles (CSVs) and immature secretory granules (ISGs) from the trans-Golgi network (TGN) of PC12 cells was investigated. Using immunofluorescence and subcellular fractionation in conjunction with immunoblotting or ADP-ribosylation by either pertussis toxin or cholera toxin, TGN membranes were found to contain not only several alpha i/alpha o G-protein subunits including apparently alpha i3, but also alpha s. Pertussis toxin treatment of cells, which resulted in the stoichiometric ADP-ribosylation of alpha i/alpha o, a modification known to prevent their coupling to receptors, led to the stimulation of cell-free CSV and ISG formation, suggesting the presence of a guanine nucleotide exchange factor for alpha i/alpha o on the TGN. Mastoparan-7, a peptide known to mimic an activated receptor and to stimulate nucleotide exchange on alpha i/alpha o, inhibited cell-free vesicle formation, an effect abolished by pertussis toxin. In contrast, activation of alpha s by cholera toxin treatment of cells resulted in a stimulation of cell-free CSV and ISG formation. This stimulation could be reversed when the alpha subunits not activated by cholera toxin, i.e. alpha i/alpha o, were activated by GTP gamma S and [AIF4]-. Our results show that both inhibitory and stimulatory trimeric G-proteins on the TGN participate in the regulation of secretory vesicle formation.     

Effects of lectins on cAMP production and steroidogenesis in cultured adrenal tumor cells

The plant lectins, concanavalin A (conA), wheat germ agglutinin (WGA), and phytohemagglutinin (PHA) stimulate steroidogenesis in cultured adrenal tumor cells. ConA maximally stimulated steroidogenesis at 100 μg/ml following an approximate 4 h lag phase. ConA stimulation was completely inhibited by α-methyl-d-mannopyranoside and the WGA effect was prevented by N-acetyl-d-glucosamine. It was also found that conA alone did not cause a measurable increase in either intra- or extracellular cyclic adenosine 3′5′-monophosphate (cAMP) production. In addition, conA when added simultaneously with adrenocorticotropin (ACTH) doubled the intra- and extracellular cAMP production over controls treated with ACTH alone. This enhancement effect was dose dependent. When Y-1 cells were preincubated with conA and then treated with either ACTH or cholera enterotoxin (CT) there was a dose- and time-dependent inhibition of induced cAMP production. In the case of CT, the inhibitory effect occurred even with simultaneous addition of conA and CT. This effect was reversed by addition of both α-methyl-d-mannopyranoside and washing with Eagle's minimal essential medium (MEM) 1 h after CT had bound to its receptor. This reversal was not apparent for the inhibitory effect of conA on ACTH-induced cAMP production which occurred after 2 h of preincubation with conA. These results demonstrate that conA, as well as the other plant lectins, interact with specific membrane receptors to reversibly stimulate steroid production as well as enhancing or inhibiting ligand-induced cAMP production in cultured adrenal tumor cells.     

Thyrotropin effect on the availability of Ni regulatory protein in FRTL-5 rat thyroid cells to ADP-ribosylation by pertussis toxin

Incubation of FRTL-5 rat thyroid cell membranes with [32P]NAD and pertussis toxin results in the specific ADP-ribosylation of a protein of about 40 kDa. This protein has the same molecular mass of the alpha i subunit of the adenylate cyclase regulatory protein Ni and is distinct from proteins ADP-ribosylated by cholera toxin in the same membranes. Prior treatment of FRTL-5 cells with pertussis toxin results in the ADP-ribosylation of Ni, as indicated by the loss of the toxin substrate in the ADP-ribosylation assay performed with membranes prepared from such cells. Preincubation of FRTL-5 cells with thyrotropin causes the same loss; cholera toxin has no such effect. Pertussis toxin, as do thyrotropin and cholera toxin, increases cAMP levels in FRTL-5 cells. Forskolin together with thyrotropin, cholera toxin or pertussis toxin causes a further increase in cAMP levels. Pertussis toxin and thyrotropin are not additive in their ability to increase adenylate cyclase activity, whereas both substances are additive with cholera toxin. A role of Ni in the thyrotropin regulation of the adenylate cyclase activity in thyroid cells is proposed.     

Fibroblast growth factor potentiates receptor- and nonreceptor-mediated stimulation of adenylate cyclase in hamster fibroblasts

Basic fibroblast growth factor (FGF) has no effect alone on the basal cAMP synthesis in Chinese hamster fibroblasts (CCL39) but it potentiates (by up to 50%) the stimulation of adenylate cyclase by prostaglandin E1, cholera toxin or forskolin. This potentiating effect is not abolished by pretreatment of the cells with pertussis toxin, which indicates that it is not due to the withdrawal of a tonic inhibition of adenylate cyclase by the pertussis toxin-sensitive inhibitory GTP-binding protein (Gi). Therefore, we conclude that FGF enhances the activation of adenylate cyclase by the stimulatory GTP-binding protein (Gs). Although activation of protein kinase C in CCL39 cells results in a similar potentiation of cAMP production, we provide evidence that the effect of FGF is not mediated by protein kinase C, since (1) the potentiating effects of FGF and phorbol esters are additive and (2) FGF effect persists after down-regulation of protein kinase C. A role of FGF-induced rise in cytoplasmic Ca2+ can also be ruled out because the FGF effect is not mimicked by a Ca2+ ionophore and it persists in Ca2(+)-free medium. Since a similar potentiating effect on cAMP production is elicited by epidermal growth factor, a mitogen known to activate a receptor tyrosine kinase, we suggest that the FGF effect on adenylate cyclase might be mediated by the tyrosine kinase activity that is very likely to be associated with FGF receptors.     

Role of cyclic adenosine 3',5'-monophosphate in mediating the effect of prostaglandin E2 on decidualization in vitro.

When rat endometrial stromal cells from uteri sensitized for decidualization are cultured in vitro, there is an increase in alkaline phosphatase (ALP) activity paralleling that seen in vivo during decidualization. The addition of indomethacin to the culture medium decreases the endogenous production of prostaglandin E2 (PGE2) to below detectable levels and substantially reduces the increase in ALP activity. The addition of either PGE2 or its analog 16,16-dimethyl-PGE2, but not PGF2 alpha or its analog 15(S),15-methyl-PGF2 alpha, overrides this inhibitory effect, suggesting that PGE2 has a specific stimulatory effect upon ALP activity. This in vitro system was used to investigate the role of the cAMP pathway in mediating the stimulatory effect of PGE2 on ALP activity. The data indicate that PGE2 causes an increase in cAMP accumulation by the cells and that the addition of an analog of cAMP or substances which increase the level of cAMP in the cells (1-methyl-3-isobutyl xanthine, cholera toxin, forskolin) causes an increase in ALP activity. Collectively, the results suggest that the stimulatory effect of PGE2 is at least partially mediated by the cAMP pathway.     

The influence of plasma lipoproteins on steroidogenesis of cultured ovine fetal and neonatal adrenal cells

The present study examined the effects of serum and lipoproteins on the function of cultured adrenal cells from 115-127-day-old ovine fetuses and from newborn lambs. On day 1 of culture, corticosteroid output was similar in medium containing 2% horse serum or in serum-free medium, both for fetal and neonatal cells. However, on day 5, cells cultured in the absence of serum produced smaller amounts of these steroids than cells maintained in medium containing serum; the difference was more marked under ACTH1-24 stimulation. Conversely, cAMP production was never lower in the absence than in the presence of serum. When stimulated by ACTH1-24 on day 2 of culture, fetal or neonatal adrenal cells incubated in the presence of a saturating concentration of ovine LDL produced more corticosteroids than cells incubated in serum-free medium; HDL also enhanced ACTH1-24-induced steroidogenesis, but to a lesser extent. VLDL was effective only with neonatal cells. In fetal and neonatal cells cultured for 6 days in ACTH-free medium, VLDL and LDL increased ACTH-induced steroidogenesis, but HDL did not. On the other hand, when cells were cultured in the presence of ACTH1-24, LDL and HDL were equipotent in supporting ACTH1-24-induced steroid output. Three major lipoprotein fractions were observed in serum of fetal and newborn lambs. The concentration of cholesterol was very low in the VLDL fraction of fetuses, but it was similar to that of newborns in LDL. Conversely, 4 times more cholesterol was present in HDL of newborns than in HDL of fetuses. These results suggest that: (i) after several days of cell culture, cholesterol availability is an important limiting factor for the steroidogenesis of cells maintained under serum-free conditions; (ii) both an "LDL pathway" and an "HDL pathway" are operating in adrenal cells from fetal as well as newborn sheep; (iii) LDL and HDL are important physiological sources of cholesterol to support steroidogenesis by fetal and neonatal adrenal cells.     

Effects of prostaglandin F2 alpha on agonist-induced progesterone production in cultured bovine luteal cells

The present study examines the effects of prostaglandin F2 alpha (PGF2 alpha) on basal and agonist-stimulated progesterone (P4) production utilizing long-term, serum-free cultures of bovine luteal cells. During the first 24 h of culture, PGF2 alpha had no significant effect on P4 production, and was unable to inhibit either luteinizing hormone (LH)- or dibutyryl cAMP (dbcAMP)-stimulated increases in P4. Treatment with PGF2 alpha on Day 1 produced a moderate, nonsignificant (P greater than 0.05) inhibition of cholera toxin (CT)- and forskolin (FKN)-stimulated P4 synthesis. Beyond Day 1 of culture (Days 3-11), PGF2 alpha continued to have no significant effect on basal P4 production, but suppressed all stimulatory effects of LH, dbcAMP, CT and FKN. Treatment with indomethacin inhibited prostaglandin synthesis by the cultured cells and also elevated levels of P4 from Days 3 to 11 of culture. Concurrent treatment with PGF2 alpha suppressed the steroidogenic effect of indomethacin. From these studies it was concluded that in cultured bovine luteal cells, PGF2 alpha does not affect basal P4 production, but is able to inhibit agonist-stimulated P4 production at a site beyond the accumulation of cAMP. This inhibitory effect is not apparent during the first 24 h of culture, but appears after Day 1 and persists throughout the remaining 10 days of the culture period.     

Adrenal adenylate cyclase and steroidogenic activities of 63 day old ovine fetuses: in vitro effects of ACTH1-24 and forskolin

This study examines the activity of the adenylate cyclase system and that of some enzymes of the steroidogenic pathway of adrenal cells from 62-63 day old ovine fetuses. Synthetic corticotropin (ACTH1-24), cholera toxin and forskolin stimulated both cAMP and corticoid productions by freshly isolated adrenal cells. The cAMP response to ACTH1-24 was lower than that to forskolin. However, forskolin-induced steroidogenesis was significantly lower than the ACTH1-24-induced steroid output. Freshly isolated cells metabolized quickly [14C]-labeled pregnenolone mainly through the 17-deoxy pathway. The amounts of cortisol and of corticosterone formed, in the presence of exogenous pregnenolone, were roughly 15-fold higher than under maximal stimulation by ACTH1-24. When the cells were cultured for 6 days in the absence or presence of ACTH1-24 (10(-8) M) or forskolin (10(-5) M), a small development of the cAMP response to these factors was observed in the course of the experiment. However, the mechanism of this development appeared different, according to the conditions of culture. The amounts of corticosterone secreted on day 6 by ACTH1-24- or forskolin-treated cells were 2- to 4-fold higher than on day 1, whereas cortisol outputs were much lower on day 6 than on day 1. The response to ACTH1-24 of cells maintained in ACTH-free media decreased dramatically during the culture in terms of both cortisol and of corticosterone. On day 6 of the experiment, the metabolism of [14C]pregnenolone was lower than on day 1 under all 3 conditions of culture. Only the 3 beta-hydroxysteroid dehydrogenase/isomerase activity could be maintained by continuous treatment with forskolin. However, both ACTH1-24 and forskolin enhanced the production of pregnenolone from an endogenous substrate. In conclusion, these results present evidence that: 1) the adenylate cyclase system is not a bottleneck in the steroidogenic response to ACTH1-24 of freshly isolated adrenal cells from 62-63 day old ovine fetuses; 2) the main rate-limiting step for steroidogenesis by these cells is the availability of pregnenolone; 3) neither ACTH1-24 nor forskolin is able to maintain the activity of most enzymes involved in the metabolization of pregnenolone by cultured cells while increasing pregnenolone availability; 4) some inhibiting factors are involved in the loss of adrenal cells responsiveness to ACTH between days 50 and 100 of gestation, and they probably act mainly on the adenylate cyclase system.     

Modulation of meiotic arrest in mouse oocytes by guanyl nucleotides and modifiers of G-proteins.

Guanyl nucleotide binding-proteins, or G-proteins, are ubiquitous molecules that are involved in cellular signal transduction mechanisms. Because a role has been established for cAMP in meiosis and G-proteins participate in cAMP-generating systems by stimulating or inhibiting adenylate cyclase, the present study was conducted to examine the possible involvement of G-proteins in the resumption of meiotic maturation. Cumulus cell-free mouse oocytes (denuded oocytes) were maintained in meiotic arrest in a transient and dose-dependent manner when microinjected with the nonhydrolyzable GTP analog, GTP gamma S. This effect was specific for GTP gamma S, because GppNHp, GTP, and ATP gamma S were without effect. Three compounds, known to interact with G-proteins, were tested for their ability to modulate meiotic maturation: pertussis toxin, cholera toxin, and aluminum fluoride (AlF4-). Pertussis toxin had little effect on maturation in either cumulus cell-enclosed oocytes or denuded oocytes when meiotic arrest was maintained with dibutyryl cAMP (dbcAMP) or hypoxanthine. Cholera toxin stimulated germinal vesicle breakdown (GVB) in cumulus cell-enclosed oocytes during long-term culture, but its action was inhibitory in denuded oocytes. AlF4- stimulated GVB in both cumulus cell-enclosed oocytes and denuded oocytes when meiotic arrest was maintained with hypoxanthine but was much less effective in dbcAMP-arrested oocytes. In addition, AlF4- abrogated the inhibitory action of cholera toxin in denuded oocytes and also that of follicle-stimulating hormone (FSH) in cumulus cell-enclosed oocytes. Cholera toxin or FSH alone each stimulated the synthesis of cAMP in oocyte-cumulus cell complexes, whereas pertussis toxin or AlF4- alone were without effect. Both cholera toxin and AlF4- augmented the stimulatory action of FSH on cAMP. These data suggest the involvement of guanyl nucleotides and G-proteins in the regulation of GVB, although different G-proteins and mediators may be involved at the oocyte and cumulus cell levels. Cholera toxin most likely acts by ADP ribosylation of the alpha subunit of Gs and increased generation of cAMP, whereas AlF4- appears to act by antagonizing a cAMP-dependent step.     

Modulation of responsiveness to cAMP stimulating agonists by phorbol ester in fetal rat osteoblasts.

We studied the effect of activation of protein kinase C (PKC) by a phorbol ester on cAMP accumulation in fetal rat osteoblasts. Activation of PKC by phorbol 12-myristate 13-acetate (PMA) caused a potentiation of cAMP accumulation induced by parathyroid hormone (PTH), forskolin, and cholera toxin. The results suggest that the potentiating effect of PMA on PTH-induced cAMP accumulation was not due to an effect on the PTH-receptor nor to an effect on cAMP degradation, as the effect of PMA persisted in the presence of a phosphodiesterase inhibitor. Pretreatment of the cells with pertussis toxin did not prevent the action of PMA, indicating that PMA does not act via the inhibitory G-protein. PMA had a biphasic effect on prostaglandin E2 (PGE2)-induced cAMP accumulation; i.e., at concentrations greater than or equal to 10(-6) M, PMA potentiated the PGE2-induced cAMP response but PMA attenuated cAMP accumulation induced by concentrations of PGE2 less than or equal to 5.10(77) M. From our data we conclude that PKC can interact with a stimulated cAMP pathway in a stimulatory and inhibitory manner. Potentiation of cAMP accumulation is probably due to modification of the adenylate cyclase complex, whereas attenuation of stimulated cAMP accumulation appears to be due to an effect on a different site of the cAMP generating pathway, which may be specific to PGE2-induced cAMP accumulation.     

In vitro effect of insulin and insulin-like growth factor-I on cell multiplication and adrenocorticotropin responsiveness of fetal adrenal cells

The present study examined the effects of both insulin and insulin-like growth factor-I (IGF-I) on cell division and specific functions of cultured adrenocortical cells from 100- to 122-day-old ovine fetuses. When culture was performed in a serum-free medium containing transferrin and ascorbic acid, the number of cells increased only slightly (1.2-fold) over a 4-day period. Addition of insulin or IGF-I in the culture medium enhanced the number of cells counted on Day 5. The effect of both peptides was dose-dependent, but 10 ng/ml IGF-I was as potent as 10 micrograms/ml insulin. The acute cyclic adenosine 3',5'-monophosphate (cAMP) and steroidogenic responses to adrenocorticotropin (ACTH1-24) decreased in fetal cells cultured in the absence of insulin or ACTH. Insulin at micromolar concentrations not only prevented this decrease but enhanced the acute ACTH1-24-induced cAMP output on Day 5 over that observed on Day 2. Treatment of fetal cells for 4 days with increasing concentrations of insulin or IGF-I enhanced the acute cAMP and steroidogenic responses (3- to 4-fold) to ACTH1-24 over that of control cells. The ED50 of IGF-I was about 3 ng/ml (congruent to 0.4 nM) whereas that of insulin was about 10 ng/ml (1.7 nM). However, a second plateau was apparent at concentrations of insulin above 1 microgram/ml. The acute cholera toxin stimulation of cAMP production of cells cultured in the absence of insulin or ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of several pro-opiomelanocortin derived peptides on steroidogenesis in ovine and bovine adrenal cells

The effects of several peptides derived from the amino-terminal end of proopiomelanocortin (N-POMC) alone or in combination with ACTH on ovine and bovine adrenal cell steroidogenesis have been studied. Neither porcine N-POMC1-61 amide, nor gamma 3-MSH, nor been studied. Neither porcine N-POMC1-61 amide, nor gamma 3-MSH, nor Lys-gamma 3-MSH alone had any steroidogenic effect while porcine N-POMC1-80 alone had a week but significant steroidogenic effect on isolated adrenal, the effect being more pronounced on cultural adrenal cells. The potentiation by N-POMC peptides of the steroidogenic action of ACTH was investigated using both freshly isolated and cultured adrenal cells. At 10(-8) M N-POMC1-61 amide, gamma 3-MSH and Lys-gamma 3-MSH did not modify the ACTH dose-response for steroids (gluco- and mineralocorticoids) and cAMP production. Likewise, the stimulatory effect of 10(-12) M ACTH was not modified by increasing concentrations (10(-11) to 10(-8) M) of N-POMC1-61 amide or gamma 3-MSH. On the other hand, an additive effect of 10(-8) M N-POMC1-80 on the steroidogenic action of low concentration of ACTH was observed. This again was more pronounced in cultured adrenal cells. The same effects were observed when increasing concentrations of this peptide (10(-9) and 10(-8) M) were added together with 10(-12) M ACTH. This result indicates that the potentiating effects of N-POMC derived peptides on the steroidogenic effect of ACTH which has been described on rat and human adrenal, is not a general phenomenon in mammals.     

Hormonal regulation of insulin-like growth factor I secretion by bovine adrenal cells

The role of insulin-like growth factor I (IGF-I) on the specific function of several steroidogenic cells has been recently reported. Since IGF-I is produced by several tissues, we have investigated whether bovine adrenal cells secrete this peptide. Purification of conditioned medium from adrenal cells incubated with [35S]methionine through affinity chromatography (monoclonal anti-IGF-I antibody), high pressure liquid chromatography, and polyacrylamide gel electrophoresis revealed a single band of similar Mr as pure recombinant IGF-I. Moreover, the purified adrenal-secreted IGF-I displaced bound 125I-IGF-I to its adrenal receptors, and pretreatment of adrenal cells with the purified peptide enhanced the acute corticotropin (ACTH)-induced cAMP production as recombinant IGF-I. The basal secretion of IGF-I (6 +/- 1 ng/48 h/10(6) cells) was stimulated 3-, 4.5-, and 9.5-fold by fibroblast growth factor, angiotensin II (A-II), and ACTH, respectively, but not by growth hormone. The stimulatory effects of A-II and ACTH were dose-dependent (ED50 congruent to 2.5 x 10(-8) and 1.5 x 10(-10) M, respectively), and the effects of both hormones were additive. Glucocorticoids were not the mediators of the effect of the two hormones on IGF-I secretion, since inhibition of their steroidogenic action by aminoglutethimide did not significantly modify IGF-I secretion. An immunoreactive IGF-I material was also secreted by mouse adrenal tumor cell line Y-1, but the stimulatory effect of ACTH was only 2-fold, and there was no effect of A-II. Since bovine adrenal cells contain specific IGF-I receptors and this peptide is required for the maintenance of some adrenal cell-specific function, the present data suggest that IGF-I may act in an autocrine fashion to stimulate adrenal cell differentiation stimulated by ACTH and A-II.     

Modulation of cyclic AMP formation and progesterone secretion by human chorionic gonadotropin, epinephrine, buserelin and prostaglandins in normal or human chorionic gonadotropin desensitized rat immature luteal cells in monolayer culture

It is now well recognized that hCG-induced luteolysis is associated with hCG-induced desensitization, but the physiological significance of luteal cell GnRH, PGs and beta-receptors is still undefined. Therefore, we intend in this study to observe the effects of prostaglandin F2 alpha and prostaglandin E2 and the interactions between epinephrine, a potent LHRH agonist [(D-Ser-(TBu)6, des-Gly-NH10(2) LHRH ethylamide: Buserelin] and hCG in normal and in vitro hCG-desensitized rat immature luteal cells in monolayer culture, on basal, hCG or cholera toxin stimulated intracellular and extracellular cAMP and progesterone secretion. The present report shows that incubation of immature rat luteal cells in monolayer culture with Buserelin, led to 25-50% inhibition of the epinephrine-as well as PGE2-induced cAMP and progesterone responses. The LHRH agonist can also reverse the stimulatory effects of cholera toxin in the presence of hCG and led with PGF2 alpha, to additive inhibitory effects on extracellular cAMP accumulation induced by cholera toxin. Both Buserelin and PGF2 alpha can reverse the hCG-induced cAMP and progesterone release but no effect could be observed when the incubation was carried out with either substance in the absence of hCG. Prostaglandin E2, in acute conditions of incubation, seems to share agonist properties with hCG when both were incubated with luteal cells. Buserelin reversed the stimulatory effects of PGE2, hCG, epinephrine and cholera toxin on cAMP and progesterone responses to these substances. These results suggest that Buserelin and PGF2 alpha have luteolytic-like effects and that there may be a complementary action for the two substances. Preincubation of rat luteal cells in monolayer culture with 1 nM hCG for a 24 h period led to the inhibition of cAMP and progesterone responses after a subsequent exposure to hCG and epinephrine. Luteal cells were no longer responsive to hCG while the presence of epinephrine in hCG-desensitized cells led to a 40% stimulation of cAMP and progesterone production. These observations suggest that occurred a partial alteration of the N component activity of the adenylyl cyclase system.     

Treatment of intact striatal neurones with cholera toxin or 8-bromoadenosine 3',5'-(cyclic)phosphate decreases the ability of pertussis toxin to ADP-ribosylate the alpha-subunits of inhibitory and other guanine-nucleotide-binding regulatory proteins, Gi and Go. Evidence for two distinct mechanisms.

Using primary cultures of striatal neurones from the mouse embryo, we showed that treatment of intact cells with cholera toxin (5 micrograms/ml, 22 h) decreases the subsequent ADP-ribosylation of the alpha subunit of the guanine-nucleotide-binding regulatory protein Go (Go alpha) and the alpha subunit of the inhibitory guanine-nucleotide-binding regulatory protein (Gi alpha) of adenylate cyclase, which is catalyzed in vitro on neuronal membranes by pertussis toxin. The inhibitory effect of cholera toxin could not only be attributed to an increased production of cAMP in neurones. Treatment of cells with 0.1 microM 8-bromoadenosine 3',5'-(cyclic)phosphate (BrcAMP) for 16 h, or with 0.1 mM BrcAMP for 5 min, mimicked the effect of cholera toxin on the ADP-ribosylation of Go alpha and Gi alpha in vitro. However, the two agents seem to act through distinct mechanisms. The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine prevented the action of Br8cAMP but not that of cholera toxin. In addition, measurements of the pI of the Go alpha deduced from immunoblots of two-dimensional gels performed using a specific antibody directed against Go alpha suggest that treatment of neurones with cholera toxin induces ADP-ribosylation of Go alpha in intact cells, while BrcAMP does not.