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J M Saez

Publications and source records attributed to J M Saez.

At least 73 records · Page 4Linked to original sources
PubMed1993

Identification and characterization of angiotensin-II receptor subtypes in cultured bovine and human adrenal fasciculata cells and PC12W cells.

Angiotensin-II (Ang II) receptor subtypes (AT1 and AT2) were analyzed in bovine adrenal cells (BAC) by binding and cross-linking experiments using [125I]Ang II and [125I]CGP42112, a specific ligand of AT2 receptors. [125I]Ang II binding was reduced by 80% and 20% in the presence of maximal concentrations of the AT1 antagonist losartan (DuP 753) and CGP42112, respectively, whereas [125I]CGP42112 binding was inhibited by Ang II or CGP42112, but not by losartan. In the presence of the reducing agent dithio-1,4-erythritol, the binding of [125I] CGP42112 was increased 2-fold; this was due to an increase in the binding affinity (Kd, 8 +/- 4 x 10(-10) vs. 4.8 +/- 1.2 x 10(-10) M). Cross-linking of [125I]Ang II to BAC in the presence of disuccinimidyl suberate, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed a band of 70,000 +/- 8,000 mol wt (M(r)) under both reducing and nonreducing conditions. This band disappeared when the incubation was performed in the presence of 10(-6) M Ang II or 5 x 10(-8) M CGP42112, but not in the presence of 10(-5) M losartan. Dithio-1,4-erythritol (10 mM) markedly enhanced the band. After cross-linking with 1,5-difluoro-2,4-dinitrobenzene and solubilization of the cells in the presence of protease inhibitors, two radioactive bands were observed with M(r) of 70,000 and 50,000. The first disappeared after the addition of Ang II or CGP42112, whereas the second disappeared in the presence of Ang II or losartan, but not in the presence of CGP42112. Cross-linking of [125I]AngII to either human adrenal fasciculata-reticularis cells, which contain only AT1 sites, or COS-7 cells transfected with human AT1 cDNA revealed a major band of 50,000 M(r) that was blunted by Ang II or losartan, but not by CGP42112. Moreover, cross-linking of [125I]Ang II to PC12W cells, which contain only the AT2 receptor subtype, revealed a single radioactive band of 70,000 Mr that was blunted by CGP42112 but not by losartan. Thus, in both BAC and PC12W cells, the AT2 receptor has a M(r) of 70,000, whereas the AT1 receptor in BAC, human adrenal cells, and cells transfected with human AT1 receptor cDNA has a Mr of 50,000. Therefore, the heterogeneity of the size of the Ang II receptor previously reported after photoaffinity or cross-linking was probably due to only to a variation in the degree of glycosylation between tissues and species, but also to the presence of two different receptor subtypes.

Angiotensin II↗
PubMed1992

Angiotensin-II-induced expression of proto-oncogene (c-fos, jun-B and c-jun) mRNA in bovine adrenocortical fasciculata cells (BAC) is mediated by AT-1 receptors.

We have shown previously that angiotensin-II (A-II) controls proto-oncogene (c-fos, jun-B and c-jun) mRNA accumulation in bovine adrenal fasciculata cells (BAC). Since BAC contain both subtypes (AT-1 and AT-2) of the A-II receptor, we have investigated which subtype was involved in the effect of A-II on proto-oncogene mRNA by using a selective antagonist for AT-1 (DUP 753) and for AT-2 (CGP 42112A). DUP 753, but not CGP 42112A, inhibited the stimulatory effect of A-II on proto-oncogene mRNA, with ID50s of 4 x 10(-7) M, 7 x 10(-7) M and 2 x 10(-6) M for c-fos, jun-B and c-jun, respectively. Neither of the two antagonists by themselves had a direct effect on proto-oncogene mRNA. As the A-II AT-1 receptors are coupled to the phospholipase C system in BAC, we have investigated whether the A-II effects on the proto-oncogenes were mediated by protein kinase C (PKC) or by Ca2+ calmodulin. First, activation of PKC by the phorbol ester, PMA, increased the level of three proto-oncogene mRNAs, whereas calcium ionophore had no effect. Second, staurosporine, a specific inhibitor of PKC, reduced the stimulatory action of A-II on proto-oncogene mRNA by 80-90%, whereas trifluoroperazine, an inhibitor of calmodulin, had no significant effect. These results demonstrate that the effects of A-II on proto-oncogene mRNA are mediated by AT1 receptor subtypes, mainly through activation of the PKC pathway.

Angiotensin II↗
PubMed1992

Characterization and coupling of angiotensin-II receptor subtypes in cultured bovine adrenal fasciculata cells.

Angiotensin-II (A-II) receptor subtypes and their potential coupling mechanisms were investigated in bovine adrenal fasciculata cells (BAC) in culture, by the use of selective antagonists for AT1 (DUP 753 or Losartan) and AT2 (PD 123177 and CGP 42112A) sites. Competition for [125I]A-II specific binding with AT1 or AT2 selective ligands produced a biphasic displacement curve, suggesting two distinct A-II binding sites. In the presence of PD 123177 (10(-5) M), a concentration at which most of the AT2 sites were saturated, DUP 753 displaced [125I]A-II specific binding in a monophasic manner with an IC50 of 6.2 +/- 1.4 x 10(-7) M. In the presence of DUP 753 (10(-5) M), the displacement produced by CGP 42112A and PD 123177 was also monophasic, with IC50s of 8 +/- 3 x 10(-10) and 4.6 +/- 2.1 x 10(-7) M, respectively. The reducing agent dithio-1,4-erythritol inhibited the binding of [125I]A-II to AT1 (DUP 753 sensitive) sites, but increased its binding to AT2 sites 2-fold. The IC50 values for these two effects were about 0.5 and 3 mM, respectively. The biological effects of A-II in BAC, phosphoinositide hydrolysis and cortisol production, were inhibited in a dose-dependent manner by DUP 753, but not by AT2 antagonists. Similarly, the potentiating action of A-II on corticotropin-induced cAMP production was blocked by DUP 753, but not by AT2 antagonists. These data indicate that BAC contain both receptor subtypes, but that all the known effects of A-II in BAC were induced via the AT1 receptor subtype.

Adrenal Cortex↗
PubMed1992

Regulation of c-fos, c-jun and jun-B messenger ribonucleic acids by angiotensin-II and corticotropin in ovine and bovine adrenocortical cells.

Previous work has shown that corticotropin (ACTH) and angiotensin-II (A-II), in addition to their acute steroidogenic effects, exert long-term influences on adrenal cell differentiated function, stimulatory or inhibitory, respectively. Certain nuclear proto-oncogenes have been implicated in the regulation of gene expression in many cell systems. We have investigated the effects of ACTH and A-II on the levels of c-fos, c-jun, and jun-B messenger RNAs (mRNAs), in bovine and ovine (OAC) adrenal fasciculata cells. In both cell types ACTH produced time- (maximum at 1 h) and dose-dependent (ED50 congruent to 10(-12) M) increase in c-fos (2- to 4-fold) and jun-B (10- to 20-fold) mRNA levels but did not affect c-jun. The concentrations required to induce half-maximal mRNA accumulation and cortisol production were similar. A-II also produced a dose-dependent increase in c-fos and jun-B mRNAs but also in c-jun in both cell types, despite the fact that OAC are resistant to the steroidogenic action of the hormone. The stimulatory effects of A-II on c-fos mRNA were higher than those produced by ACTH, whereas the effects on jun-B were similar but ACTH abolished (OAC) or decreased (bovine adrenal fasciculata cells) the stimulatory effects of A-II on c-jun mRNA. The effects of ACTH and A-II on cortisol production and proto-oncogene mRNAs were in part mimicked by 8 Bromo-cAMP and the phorbol ester phorbol-12-myristate-13 acetate plus calcium ionophore A23187, respectively. In the presence of cycloheximide, which blocks the steroidogenic effects of both hormones, proto-oncogene mRNAs were superinduced by both hormones. This result, together with the fact that dexamethasone failed to affect the mRNA levels suggests that the stimulatory effects of ACTH and A-II on proto-oncogene expression were not related to an autocrine/intracrine action of cortisol. Taken together, these findings show that the proto-oncogene mRNAs in normal adrenal cells are regulated by ACTH and A-II, acting through different intracellular pathways. They also demonstrate differential responsiveness of the Jun family to both hormones. Thus, the opposite long-term action of ACTH and A-II on adrenal cell differentiated function could be mediated by its different initial effects on proto-oncogene expression, in particular in the members of the Jun family.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Opposite vectorial secretion of insulin-like growth factor I and its binding proteins by pig Sertoli cells cultured in the bicameral chamber system.

A two-chamber system has been employed to investigate the vectorial secretion of insulin-like growth factor I (IGF-I) and its binding proteins (IGF-BPs) by pig Sertoli cells. The kinetics of transport of [3H]insulin as well as the transepithelial electrical resistance of filters coated with reconstituted basement membrane, in the absence of presence of Sertoli cells, indicated the formation of a functional barrier by Sertoli cells. The rates of diffusion of [125I]IGF-I or [125I]human CG were slower from the apical (AC) to the basal (BC) compartment than in the opposite direction. However, the IGF-I content and concentration in the BC was twice that seen in the AC. FSH increased the secretion of IGF-I in the BC, and therefore increased the BC/AC ratio. In contrast, most of the IGF-BPs, with apparent molecular sizes of 39-43, 34, 29 and 24 kDa, were secreted in the AC. FSH increased the secretion of IGF-BP 39-43 kDa (BP3). This is the first report of opposite vectorial secretion of IGF-I and its binding protein by any cell type.

Animals↗
PubMed1992

Angiotensin II potentiates agonist-induced 3',5'-cyclic adenosine monophosphate production by cultured bovine adrenal cells through protein kinase C and calmodulin pathways.

Interactions between signal transducing systems may be important in the integrated control of cellular processes in basal and hormonally regulated cells. The cultured bovine adrenal fasciculata cell provides a model to study the interactions between the cAMP and calcium-sensitive phospholipid dependent protein kinase C. In this study, angiotensin II (A-II) and phorbol ester (PMA) potentiated the stimulatory actions of ACTH in a dose-dependent manner on cAMP production. At maximal concentrations, A-II and PMA also potentiated the effects of cholera toxin and forskolin on cAMP production. Both staurosporine, a protein kinase C inhibitor, and desensitization of protein kinase C by a 24-h pretreatment with PMA blunted the effect of PMA, but only partially inhibited (34%) the effect of A-II. Neither nifedipine, a specific calcium channel antagonist, nor pretreatment of cells with pertussis toxin modified the amplifying effects of A-II or PMA. In contrast, trifluoperazine, a calmodulin inhibitor, reduced the potentiating effect of A-II by about 35%, but association with staurosporine blunted its effects. Moreover, the steroidogenic effects of ACTH plus A-II were more than additive, but this synergism was blunted in the presence of both inhibitors. In conclusion, PMA and A-II potentiated agonist-induced cAMP production by bovine adrenal fasciculata cells. The data suggest that the effects of PMA were mediated exclusively by protein kinase C, whereas those of A-II were mediated by both protein kinase C and calmodulin.

Adrenal Glands↗
PubMed1991

Opposite effects of angiotensin-II and corticotropin on bovine adrenocortical cell steroidogenic responsiveness.

The long-term effects of angiotensin-II (A-II) and corticotropin (ACTH) on bovine adrenal fasciculata cells (BAC) were studied. Cells were pretreated for 3 days with either A-II or ACTH followed by an examination of the acute steroidogenic response to both hormones as well as the ability to convert several steroid precursors to cortisol and corticosterone. ACTH pretreatment caused a marked increase in cortisol output associated with a decrease in corticosterone secretion in response to both hormones leading to a 50-fold decrease in the corticosterone/cortisol ratio compared to control cells. After incubation with saturating concentrations (5 X 10(-5) M) of 22 R-hydroxycholesterol, pregnenolone or progesterone, ACTH-pretreated cells produced more cortisol than corticosterone whereas the contrary was observed in control cells. However, the conversion of 17 alpha-hydroxyprogesterone and 11-deoxycortisol to cortisol by ACTH-pretreated cells was lower than by control cells. Thus, the main effects of ACTH were a marked increase of 17 alpha-hydroxylase and a small but significant decrease of 21-hydroxylase and 11 beta-hydroxylase activities. A-II pretreatment produced, in a concentration-dependent manner, a down-regulation of its own receptors and homologous and heterologous steroidogenic desensitization. At maximal concentrations (10(-6) M) A-II reduced by 70% its own receptors while the steroidogenic response to A-II and ACTH was reduced by 95% and 75%, respectively. However, the coupling of A-II receptors to phosphoinositide pathway and to Ca2+ influx, as well as its potentiation effect on ACTH-induced cAMP production were similar in control and A-II pretreated cells. Moreover, the conversion of several steroid precursors to corticosterone was similar in control cells and A-II-pretreated cells, whereas the conversion to cortisol was reduced by approximately 30% due mainly to a decrease of 17 alpha-hydroxylase activity. Thus, the marked steroidogenic desensitization induced by A-II is most likely related to some alteration located beyond the activation of the two branches of the phosphoinositide pathway and before the first steps of steroidogenesis.

Adrenal Cortex↗
PubMed1991

Regulation by growth factors of Leydig cell differentiated functions.

In this paper the effects of growth factors on the differentiated function of pig Leydig cells and other steroidogenic cells are reviewed. Two types of action have been observed, i.e. positive or negative acute effects on testosterone secretion, and long-term trophic effects of hCG receptor and responsiveness to hCG. Among the growth factors, insulin-like growth factor I (IGF-I) and transforming growth factor beta (TGF beta-1) are of particular interest. IGF-I is required for the maintenance and probably the expression of differentiated functions of several steroidogenic cells, including the Leydig cells. TGF beta-1 has effects opposite to IGF-I on Leydig cell functions. When considering effects of growth factors on Leydig cells, caution should be taken in extrapolating results obtained in one species to another.

Animals↗
PubMed1991

Characterization and regulation of angiotensin and corticotropin receptors on cultured bovine adrenal cells.

Cultured bovine adrenal fasciculata cells were used to characterize angiotensin II (A-II) and corticotropin (ACTH) receptors and to study their homologous and heterologous regulation. These cells contain one type of high affinity binding sites for A-II (KD congruent to 2.4 +/- 0.3 10(-9) M) and about 100000 sites/cell. Photoaffinity labeling followed by SDS-PAGE under reducing conditions revealed a single macromolecule of apparent MR 65,000. Treatment of cells with increasing concentrations of A-II produced down-regulation of its own receptors and marked homologous and heterologous (ACTH) steroidogenic desensitization. However, the desensitization was not correlated with receptor loss and was mainly due to alterations of the steroidogenic pathway. Pretreatment of cells with ACTH also reduced A-II receptors, but this was not associated with steroidogenic desensitization. Bovine fasciculata cells contain two binding sites for ACTH: one of high affinity (KD congruent to 2.6 +/- 0.4 10(-10) M) and low capacity (2030 +/- 390 sites/cell) and the other of low affinity and high capacity. Affinity cross-linking of ACTH to plasma membranes prepared from adrenal cells revealed a labeled macromolecule of apparent MR 43000. However, cross-linking experiments to intact cells revealed, both under reducing and non-reducing conditions, two labeled macromolecules of apparent MR of 123000 and 43000. Pretreatment of cells with ACTH enhanced its receptor and the cAMP and cortisol responses to further ACTH stimulation. These effects were time- and dose-dependent. The maximal effects were observed at 10(-10) to 10(-9) M. A-II alone had no effect but it blocked partially the stimulatory action of ACTH.

Adrenal Cortex↗
PubMed1991

Cell-cell communication in the testis.

In addition to the well-established endocrine regulation of testicular functions by gonadotropins, many data accumulated in the last few years indicate that a local control is required for a normal production of androgens and spermatogenesis. In the present paper we review the cell-cell interactions between somatic and germ cells in the testis and their role on the function of each cell type. Also, we will present evidences indicating that some of these interactions are mediated by several growth factors produced and acting within the testis. Moreover, very often the production of these factors are under control of gonadotropins, and in turn the growth factors regulate the sensitivity of testicular cells to these hormones.

Animals↗
PubMed1991

Growth hormone and insulin-like growth factor I treatment increase testicular luteinizing hormone receptors and steroidogenic responsiveness of growth hormone deficient dwarf mice.

To test the hypothesis that insulin-like growth factor (IGF-I) is required for the in vivo development of testicular Leydig cell function, either recombinant human GH [(hGH) (1.5 micrograms/g BW) or recombinant IGF-I (1 microgram/g BW) was injected three times daily into immature Snell dwarf mice (dw/dw) and into phenotypically normal control (Dw/-) for 7 days. In dw/dw mice hGH enhanced significantly body, liver, kidney, and testicular weight. In addition, hGH increased testicular LH receptors and the acute steroidogenic response to human CG, but there was no significant effect on basal plasma testosterone or plasma LH levels. The effects of IGF-I in body and kidney weight were less pronounced than those produced by hGH, but its effects on testicular weight and LH receptors, as well as on the acute steroidogenic response to human CG, were similar to that observed after hGH treatment. In Dw/- mice hGH had no effect on either body or organ weight or on testicular function, despite the fact that it induced a significant increase in plasma IGF-I levels. These results indicated that IGF-I is able to induce the maturation of Leydig cell function and that the effects of hGH on the testis are probably mediated by IGF-I. They also suggest that the delayed puberty associated with GH deficiency or resistance is most likely related to an IGF-I deficiency.

Animals↗
PubMed1991

Regulation of guanine nucleotide binding regulatory proteins in cultured adrenal cells by adrenocorticotropin and angiotensin-II.

In addition to their steroidogenic effect on cultured bovine adrenal fasciculata cells ACTH and angiotensin-II (A-II) have a long term effect on the ability of these cells to respond to subsequent hormonal stimulation. The present work explores the effects of a 72-h pretreatment of adrenal cells with both hormones on the first steps of the mechanism of action of ACTH and A-II and on the amounts of the alpha-subunits of guanine nucleotide binding proteins Gs and Gi. ACTH but not A-II increased acute ACTH or cholera toxin-induced cAMP production. Moreover, ACTH but not A-II enhanced the amount of alpha S protein evaluated by cholera toxin ADP ribosylation, whereas both hormones elevated immunoblotted alpha S. Both hormones increased A-II induced phosphoinositide breakdown and Ca2+ uptake without modification of the A-II potentiating effect on ACTH-induced cAMP production. Treatment of cells with pertussis toxin (PT, 0.5 micrograms/ml) for the last 24 h reduced by 27% the A-II induced phosphoinositide breakdown in A-II pretreated cells but had no significant effect in ACTH-pretreated cells. No effect of PT was observed on A-II induced Ca2+ uptake or on its potentiating action on ACTH-induced cAMP production in ACTH as well as A-II-pretreated cells. Moreover, both hormones increased Gi proteins (40-41 kDa) evaluated by PT ADP ribosylation. Immunoblot analysis revealed that ACTH preferentially enhanced alpha i3, whereas the stimulatory effect of A-II was more marked on alpha i1 and alpha i2. These results indicate that in bovine adrenal fasciculata cells, peptide hormones settle target cell responsiveness not only by regulating the membrane-bound receptors, but also by modulating the level of G proteins coupling these receptors to the intracellular signals.

Adrenal Glands↗
PubMed1991

Regulation of c-fos, c-jun, jun-B, and c-myc messenger ribonucleic acids by gonadotropin and growth factors in cultured pig Leydig cell.

The nuclear protooncogenes have been implicated in the coordinate regulation of gene expression during cell proliferation and differentiation. Previous work has shown that LH and human h CG as well as several growth factors including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), transforming growth factor-beta, and insulin-like growth factor-I play a role in Leydig cell differentiated functions. To evaluate the possibility that protooncogenes mediate long term effects of these factors, their action on the levels of c-fos, c-jun, jun-B, and c-myc messenger (m) RNAs was studied. hCG (10(-9) M) produced a time-dependent increase in c-fos (9-fold), jun-B (18-fold) and c-myc (5-fold) mRNA levels but did not affect c-jun. The concentration of hCG required for half-maximal stimulation (ED50 = 7 +/- 4 x 10(-12) M) was similar to that required to induce half-maximal testosterone production. At optimal concentrations, the effects of EGF and bFGF on c-fos and jun-B mRNAs were lower than those induced by hCG, but their effects on c-myc mRNA were higher. In addition, they stimulated c-jun. Moreover, EGF and bFGF potentiated the effects of hCG on c-fos and jun-B, whereas hCG potentiated the action of growth factors on c-jun. Transforming growth factor-beta increased only jun-B mRNA, whereas insulin-like growth factor I increased c-fos, jun-B, and c-myc but less effectively than hCG. Lastly, the phorbol ester phorbol 12-myristate 13-acetate increased the level of the four protooncogene mRNAs, and its effects on c-fos and c-myc were significantly higher than those produced by hCG. These data indicate that the regulation of protooncogene mRNAs in normal Leydig cells is multifactorial. They also show differential responsiveness of the members of the Jun family to several factors. Our results are consistent with the hypothesis that the Fos and Jun families of regulatory proteins could play a role in mediating long term responses to the complex array of hormones and growth factors to which Leydig cells are exposed in vivo.

Animals↗
PubMed1991

Human proopiomelanocortin-(79-96), a proposed cortical androgen-stimulating hormone, does not affect steroidogenesis in cultured human adult adrenal cells.

The existence of a cortical androgen-stimulating hormone (CASH), distinct from ACTH, regulating the secretion of human adrenal androgens has long been postulated. Recently, it has been reported that an 18-amino acid peptide, corresponding to the first part of the joining peptide of proopiomelanocortin [POMC-(79-96)], was able to stimulate the secretion of dehydroepiandrosterone from cultured human adult adrenocortical cells, but had no effect on cortisol production. We have studied the acute and long term effects of ACTH (10(-11) and 10(-9) M), CASH-18 (10(-8) M), or both on cortisol and dehydroepiandrosterone sulfate by human adult adrenocortical cells. Although ACTH increased steroid secretion and enhanced the steroidogenic responsiveness to further ACTH stimulation, CASH-18 alone or together with ACTH (10(-11) or 10(-9) M) had no effect. In addition, we were unable to demonstrate any specific binding of [125I]CASH-18 to human adrenocortical cells, although [125I] ACTH-(1-39) binds specifically to the same cell preparation.

Adrenal Glands↗
PubMed1991

ACTH and angiotensin II regulation of insulin-like growth factor-I and its binding proteins in cultured bovine adrenal cells.

Insulin-like growth factor-I (IGF-I) is required for the maintenance of differentiated functions of bovine adrenal fasciculata cells in culture. We have investigated, by immunocytochemistry, the presence of IGF-I in cells cultured in the absence or presence of ACTH and angiotensin II (AII), as well as the secretion of IGF-I and its binding proteins (IGFBPs). In control cultures, very few cells were specifically stained with the anti-IGF-I serum. Following 2 days of treatment with AII (1 microM) or ACTH (10 nM) the number of stained cells increased by 5- and 14-fold respectively. In all cases the staining was specific, since it was abolished when non-immune rabbit serum replaced the anti-IGF serum or when the anti-IGF-I serum was preincubated with saturating concentrations of the peptide. Under the same experimental conditions the secretion of IGF-I into the medium, evaluated by a specific radioimmunoassay, was increased two- and sevenfold by AII and ACTH respectively. Using the method of Western ligand blotting, the major form of IGFBP secreted by control adrenal cells was found to be a 38-42 kDa doublet protein. Two minor forms with apparent molecular weights of 28-31 kDa and 24 kDa have also been identified. Following acid-ethanol extraction of the conditioned medium, all the IGFBPs were recovered in the pellet, whereas most of the IGF-I was in the supernatant. ACTH and, to a lesser extent, AII pretreatment increased the 38-42 kDa IGFBP by several fold, decreased the 28-31 kDa IGFBP and had no effect on the 24 kDa IGFBP. In conclusion, these results demonstrate (i) that bovine adrenal cells contain IGF-I-like immunoreactive material, (ii) that the stimulatory effects of ACTH and AII on IGF-I secretion by bovine adrenal cells are due mainly to an increase in the number of IGF-I-producing cells and (iii) that ACTH and AII modulate the secretion of IGFBP by adrenal cells. Although the roles of IGFBPs have not been defined in adrenal cells, they are capable of modulating the biological action of IGFs in other cell cultures. Regulation of both IGF-I and its binding proteins by the two specific hormones ACTH and AII suggests important roles for these binding proteins in modulating the action of IGF-I in bovine adrenal cell function.

Adrenal Cortex↗
PubMed1991

[Demonstration of the secretion of IGF-I and of its binding proteins by bovine adrenal fasciculata cells in culture. Immunocytochemistry, radioimmunoassay and ligand blotting].

Insulin-like growth factor I (IGF-I) is required for the maintenance of differentiated functions of bovine adrenal fasciculata cells in culture. We have investigated, by immunocytochemistry, the presence of IGF-I in cells cultured in the absence or presence of corticotropin (ACTH) and angiotensin II (A-II), as well as the secretion of IFG-I and its binding proteins (IGF-BP). In control cultures, very few cells were specifically stained with the anti-IGF-I serum. Following 2 days treatment with A-II (10(-6)M) or ACTH (10(-8)M) the number of stained cells increased 5 and 14 fold, respectively. In all cases the staining was specific since it was abolished when non-immune rabbit serum replaced the anti-IGF serum or when the anti-IGF-I serum was preincubated with saturating concentrations of the peptide. Under the same experimental conditions the secretion of IGF-I in the medium, evaluated by a specific radioimmunoassay, was increased 2- and 7-fold by A-II and ACTH, respectively. Using the method of western ligand blot, we found that the major form of IGF-BP secreted by control adrenal cells is a 38-42 kDa doublet protein. Two minor forms with apparent mol wt of 28-31 kDa and 24 kDa have also been identified. Following acid-ethanol extraction of the conditioned medium all the IGF-BP were recovered in the pellet, whereas most of the IGF-I was in the supernatant. ACTH and, to a lesser extent.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Control of production of insulin-like growth factor I by pig Leydig and Sertoli cells cultured alone or together. Cell-cell interactions.

The production of insulin-like growth factor I (IGF-I) by pig Leydig cells and pig Sertoli cells cultured alone or together was investigated. Human chorionic gonadotropin (hCG) and basic fibroblast growth factor (FGF) stimulate in a dose-dependent manner IGF-I production by Leydig cells. At maximal concentrations the effects of both factors were almost additive. Insulin at micromolar concentrations enhanced IGF-I production and potentiated the effects of hCG and FGF. The secretion of IGF-I by Sertoli cells was stimulated by FSH and FGF. Under basal conditions, the production of IGF-I by the coculture was similar to the addition of the production by each cell cultured alone. In contrast, in the presence of hCG, FSH or FGF, the production of IGF-I by the coculture largely exceeded that expected from the monocultures. Moreover, stimulation of the coculture with both hCG and FSH resulted in a further increase in IGF-I production. These results indicate that Leydig as well as Sertoli cells secrete IGF-I and that the secretion of both cell types is stimulated by the corresponding gonadotropin. In addition, they indicate that Leydig-Sertoli interactions play a role in the control of IGF-I production, supporting the contention that this growth factor plays an important role in the paracrine and autocrine control of testicular functions.

Animals↗