Regulation of IGF-I receptors by corticotropin and angiotensin-II in cultured bovine adrenocortical cells

The effects of angiotensin II (A-II) and corticotropin (ACTH) on insulin-like growth factor-I (IGF-I) receptors of bovine adrenocortical cells were investigated. Pretreatment of cells for 48 h with ACTH or A-II induced in a dose-dependent manner an increase in [125I]IGF-I binding (ED50 congruent to 10(-11)M, Vmax = 10(-10) M with ACTH; ED50 congruent to 3.10(-9) M, Vmax = 10(-7) M with A-II). This resulted from an increase in the number of binding sites without modification of the binding affinity. Pretreatment with 8-Bromo-cAMP (10(-3) M), a phorbol ester (PMA 10(-7) M) + ionophore A23187 (10(-7) M) produced a positive regulation of IGF-I receptors. Glucocorticoids did not mediate the effect of A-II and ACTH on IGF-I receptors. Since previous studies have shown that IGF-I increased ACTH and A-II receptors the present data indicate the existence of a reciprocal positive trophic effect between IGF-I and the two hormones on the regulation of their specific membrane-bound receptors.     

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.     

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)     

Receptors for insulin-like growth factors and insulin on murine fetal cortical brain cells

Fetal murine neuronal cells bear somatomedin receptors which can be classified according to their affinities for IGF-I, IGF-II and insulin. Binding of 125I-IGF-I is half-maximally displaced by 7 ng/ml IGF-I while 15- and 700-fold higher concentrations are required for, respectively, IGF-II and insulin. Linear Scatchard plots of competitive-binding data with IGF-I suggest one single class of type I IGF receptors (Ka = 2.6 X 10(9) M-1; Ro = 4500 sites per cell). The occurrence of IGF-II receptors appears from the specific binding of 125I-IGF-II and competition by unlabeled IGF-II; the IGF-II binding sites display a low affinity for IGF-II and no affinity for insulin. IGF-II also interacts with insulin receptors although 50- to 100-fold less potent than insulin in competing for 125I-insulin binding. The presence of distinct receptors for IGF-I, IGF-II and insulin on fetal neuronal cells is consistent with a role of these peptides in neuronal development, although our data also indicate that IGF-I receptors could mediate the growth promoting effects of insulin.     

Transforming growth factor beta treatment decreases ACTH receptors on ovine adrenocortical cells

Transforming growth factor beta (TGF beta) is a potent inhibitor of adrenocortical cell differentiated functions, whereas corticotropin (ACTH) is the main physiological hormone which acts positively on these functions. We have studied the effects of both TGF beta and ACTH on ovine adrenocortical cell ACTH receptors. Ovine adrenocortical cells contained specific high affinity (Kd = 2.7 +/- 1.6 x 10(-10) M) and low capacity (1190 +/- 120 sites/cell) ACTH receptors. Pretreatment of cells with TGF beta resulted in a time- and dose-dependent (ED50 = 50 pg/ml) decrease of 125I-ACTH1-39 binding. The observed decrease in ACTH binding was due to a 2-3-fold decrease in the number of binding sites without modification of the binding affinity. On the contrary, pretreatment of cells with ACTH caused a 4-4.5-fold increase in the number of ACTH binding sites without an effect on the Kd. When cells were pretreated with both ACTH and TGF beta, TGF beta blocked completely the positive trophic effect of ACTH on its own receptors. The variations in ACTH receptor number were associated with parallel changes on acute ACTH-induced cyclic AMP production. Thus, the effects of TGF beta on ACTH receptor content are likely another important negative action of this peptide on adrenocortical cell differentiation. Moreover, these results suggest that regulation of ACTH receptor number may be one mechanism by which hormones and growth factors control adrenocortical differentiation.     

Effects of IGF-I on proliferation and steroidogenesis of cultured adrenal zona glomerulosa cells

In a primary culture of bovine adrenal zona glomerulosa cells, insulin-like growth factor I (IGF-I)/somatomedin C stimulated DNA synthesis, as measured by [3H] thymidine uptake, at concentrations of 10(-9) and 10(-7) M. IGF-I also prevented ACTH-induced suppression of [3H] thymidine uptake. IGF-I in no way affected aldosterone secretion during short-term exposure to cultured cells, however. Our findings suggest that IGF-I plays an important role in the proliferation of adrenal zona glomerulosa cells.     

Structurally functional organization of corticotropin: lipolytic and steroidogenic activity of some of its fragments]

The influence of ACTH fragments, possessing structural elements, common for certain groups of peptide hormones and kinins--"common" fragments and cluster of basic amino-acids--(Lys 17,18-ACTH 11-18-NH2--I; ACTH 11-13-NH2--II; NH2CO-ACTH18-20-NH2--III) on lipolytic effect of ACTH in rat isolated fat cells and on the steroidogenic effect of ACTH in isolated rat adrenal cells was studied. Fragment I exerts a steroidogenic effect (alpha=0,84) at concentrations of 1--100 microng/ml. At low concentrations (10(-8)--10(-3) microng/ml) fragment I potentiates ACTH-induced steroidogenesis. Fragment I has no effect on the lipolysis;however, it potentiates ACTH-induced lipolysis at concentrations of 10--100 microng/ml. The results obtained support our previous supposition that "common" fragments are essential secondary non-specific active sites of hormones.     

Insulin-like growth factor I (IGF I) induces cortisol production in bovine adrenocortical cells in primary culture

The effects of a physiological dose of IGF I (40 ng/ml approximately 5 x 10(-9) M) on steroidogenesis were studied in bovine adrenal fasciculata cells cultured in serum-free McCoy's medium. They were compared with those of a single dose of ACTH (0.25 ng/ml approximately 10(-10) M) at approximately the concentration inducing half-maximal stimulation. With IGF I, steroidogenesis commenced after 48 h culture and progressively increased throughout the 96-h test period. Expressed as stimulated level/control level ratios, glucocorticoid (cortisol + corticosterone) responses to IGF I after 4 days' culture (2.41 +/- 0.20 (SEM) n = 9) were similar to those obtained with ACTH (2.59 +/- 0.18, n = 9). A combination of the two peptides had a synergistic effect (5.95 +/- 0.79, n = 5). The cortisol/corticosterone ratio increased in the presence of IGF I from 1 +/- 0.19 to 1.76 +/- 0.45 (n = 7, P less than 0.02), although less so than in the presence of ACTH (5.50 +/- 0.98). Moreover, cortisol production was accompanied by androstenedione production (2.36 ng/10(6) cells, n = 3) similar to that induced by ACTH (2.10 ng/10(6) cells, n = 3). These findings together suggest stimulation of 17 alpha-hydroxylase activity. Cell multiplication was unaffected by IGF I. [3H]Thymidine incorporation into DNA reached only 193% +/- 17 (SEM) (n = 4) of control levels, whereas with ACTH it dropped to 60% +/- 5. Our findings show that IGF I alone has no mitogenic effect on adrenocortical cells in vitro, but that it is capable of inducing differentiated steroidogenesis.     

Interactions between follicle-stimulating hormone and growth factors in modulating secretion of steroids and inhibin-related peptides by nonluteinized bovine granulosa cells

The aim was to investigate potential interactions between FSH and intraovarian growth factors in modulating secretion of inhibin A (inh A), activin A (act A), follistatin (FS), estradiol (E2), and progesterone (P4) by bovine granulosa cells cultured under conditions in which a nonluteinized FSH-responsive phenotype is maintained. Cells from 4- to 6-mm follicles were cultured in serum-free medium containing insulin (10 ng/ml) and androstenedione (10(-7) M), and effects of ovine FSH (0.037-3 ng/ml) were tested alone and in combination with insulin-like growth factors (IGF) (LR3 IGF-I analogue; 2-50 ng/ml) and epidermal growth factor (EGF; 0.1-10 ng/ml). Medium was changed every 48 h and cultures ended after 144 h, when cell number was determined. Between 48-96 h and 96-144 h, FSH promoted (P < 0.0001) increases in output of inh A (6-fold), act A (15-fold), FS (6-fold), and E2 (18-fold), with maximal responses (in parentheses) elicited by 0.33 ng/ml FSH during the final period. Higher FSH doses (1 and 3 ng/ml) gave reduced responses for each of the above hormones, whereas P(4) output was maximal (3-fold) at these doses. FSH promoted a slight increase in cell number ( approximately 1.7-fold; P < 0.001). LR3 IGF-I alone markedly increased (P < 0.0001) output of inh A (8-fold), act A (41-fold), FS (12-fold), and E2 (18-fold); this was accompanied by modest increases (P < 0.01) in P4 output ( approximately 2.5-fold) and cell number ( approximately 2-fold). Whereas FSH enhanced inh A, act A, FS, and E2 secretion evoked by lower doses of LR3 IGF-I, it suppressed (P < 0.001) the response to the highest dose. EGF alone promoted a 1.7-fold increase in cell number (P < 0.001) without affecting hormone release; however, it abolished (P < 0.001) FSH-induced secretion of inh A, act A, FS, and E2. Both FSH alone and LR3 IGF-I alone dose-dependently increased the act A:FS ratio ( approximately 3-fold; P < 0.005) and act A:inh A ratio (3-fold to 6-fold; P < 0.001), suggesting that both factors selectively raise activin "tone" and that this could be a key requirement for FSH and IGF-induction of follicular E2 production. This hypothesis was reinforced by the finding that addition of FS, to reduce the act A:FS ratio and sequester secreted activin, markedly suppressed (P < 0.001) FSH (3-fold)-, and LR3 IGF-I (2-fold)-induced E2 output.     

On the role of intra-adrenal unesterified cholesterol in the steroidogenic effect of corticotropin.

Since ACTH (corticotropin) increases intra-adrenal and intramitochondrial free cholesterol levels, the relative importance of these effects during ACTH-induced steroidogenesis was examined. Rats were treated in vivo with ACTH plus aminoglutethimide to increase free cholesterol (2--3-fold), and the latter was tested as a steroidogenic factor after removal of aminoglutethimide blockade and subsequent prolonged incubation of the cholesterol-rich adrenal sections in vitro. The increased free cholesterol served as a positive steroidogenic factor only during the early phase of the incubation when other ACTH-induced steroidogenic factors were operative. At later times of incubation, the increased free cholesterol did not, of itself, enhance steroidogenesis unless ACTH was added. These results suggest that the ACTH-induced enhancement of intraadrenal (and mitochondrial) free cholesterol may be important in determining the amount of steroids produced in response to a given ACTH stimulus, but another ACTH-induced factor is more important in initiating the steroidogenic process. This factor (? labile protein) appears to control a metabolic process which is distal to the mitochondrial uptake of free cholesterol.     

Failure of somatostatin and beta-endorphin to affect bovine adrenal cortex cells in vitro

The main intention of this study was to examine the effect of cyclic somatostatin (SRIF) on bovine adrenal cell activity in vitro. beta-Endorphin was applied in another course of experiments. Basal aldosterone secretion when measured was found to be 0.86 +/- 0.33 ng per 10(5) cells and 1.5 h incubation time in the glomerulosa cell fractions. Cortisol production was 1.6 +/- 0.3 ng per 10(5) cells in the fasciculata/reticularis cell incubates respectively. No effect was observed when cyclic somatostatin (SRIF) was added to cell incubates of the bovine adrenal cortex. However, aldosterone secretion was increased 4.5-fold in the presence of 10(-9)M ACTH or 10(-8)M At II. Cortisol production was enhanced similarly after addition of 10(-9)M ACTH.     

Up-regulation of IGF-I receptors on IM-9 cells by IGF-II peptides

To examine a possible role for IGF-II in the regulation of IGF-I receptors we measured 125I-IGF-I binding on IM-9 cells following pre-incubation with IGF-II/IGF-I mixtures, purified MSA (a rat IGF-II-like peptide), pure IGF-I, or insulin. Whereas all preparations tested induced down-regulation of IGF-I binding after 20 hours, distinct differences were noted after six hour pre-incubation: IGF-I (100 ng/ml) and insulin (1 microgram/ml) both induced down-regulation of IGF-I binding (15 +/- 2% and 19 +/- 2% respectively). However, a mixture of IGF-II and IGF-I (100 ng/ml each) induced consistent up-regulation of IGF-I binding (16 +/- 2%) (mean +/- SE, n = 14), and a preparation enriched in IGF-II (250 ng/ml IGF-II and 75 ng/ml IGF-I) induced 20 +/- 5% (n = 3) up-regulation at six hours. Purified MSA (200 ng/ml) induced 15% up-regulation of IGF-I binding at six hours. Scatchard analysis of displacement curves showed that increased binding was due to loss of low affinity binding, with enhancement of high affinity sites. The up-regulation of IGF-I binding was unaffected by treatment with 0.1 mM cycloheximide, but was blunted by 5 microM colchicine. It is concluded that 1. IGF-II induces up-regulation of IGF-I receptors on IM-9 cells following 6 hour pre-incubation; 2. This phenomenon is not mimicked by the structurally-related peptides IGF-I or insulin; The up-regulation is due to enhanced high affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of insulin-like growth factor binding proteins (IGFBPs) secreted by bovine adrenocortical cells in primary culture: regulation by insulin-like growth factors (IGFs) and adrenocorticotropin (ACTH).

In previous studies, we have shown that insulin-like growth factor II (IGF-II) stimulates basal as well as ACTH-induced cortisol secretion from bovine adrenocortical cells more potently than IGF-I [1]. The steroidogenic effect of both IGFs is mediated through interaction with the IGF-I receptor, and modified by locally produced IGF-binding proteins (IGFBPs). In the present study, we therefore characterized the IGFBPs secreted by bovine adrenocortical cells in primary culture, and investigated the effect of corticotropin (ACTH) and recombinant human IGF-I and IGF-II on the regulation of IGFBP synthesis. By Western ligand blotting, four different molecular forms of IGF-binding proteins were identified in conditioned medium of bovine adrenocortical cells with apparent molecular weights of 39-44 kDa, 34 kDA, 29-31 kDa, and 24 kDa. In accordance to their electrophoretic mobility, glycosylation status and binding affinity, these bands were identified by immunoprecipitation and immunoblotting as IGFBP-3, IGFBP-2, IGFBP-1, and deglycosilated IGFBP-4, respectively. Quantification of the specific bands by gamma counting revealed that, in unstimulated cells, IGFBP-3 accounts for approximately half of the detected IGFBP activity, followed by IGFBP-1, IGFBP-2 and IGFBP-4. ACTH treatment predominantly increased the abundance of IGFBP-1 and to a lesser extent IGFBP-3 in a time and dose-dependent fashion. In contrast, IGF-I or IGF-II (6.5 nM) preferentially induced the accumulation of IGFBP-3 (1.9-fold) and to a lesser extent of IGFBP-4, but did not show any effect on IGFBP-1. When ACTH and IGFs were combined, an additive stimulatory effect on the accumulation of IGFBP-3 and IGFBP-4 was observed. In contrast to their different steroidogenic potency, no significant difference in the stimulatory effect of IGF-I and IGF-II on IGFBP secretion was found. In conclusion, bovine adrenocortical cells synthesize IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4, and their secretion is regulated differentially by ACTH and IGFs. These results, together with earlier findings, suggest that IGF-binding proteins play a modulatory role in the regulation of differentiated adrenocortical functions. Therefore, bovine adult adrenocortical cells provide a useful tissue culture model in which the complex interactions between two IGF-ligands, at least four IGF binding proteins and two IGF-receptors can be evaluated.     

Effects of glucagon and glucagon-like peptide-1 on glucocorticoid secretion of dispersed rat adrenocortical cells.

The effects of glucagon and glucagon-like peptide-1 (GLP-1) on the secretory activity of rat adrenocortical cells have been investigated in vitro. Neither hormones affected basal or agonist-stimulated aldosterone secretion of dispersed rat zona glomerulosa cells or basal corticosterone production of zona fasciculata-reticularis (inner) cells. In contrast, glucagon and GLP-1 partially (40%) inhibited ACTH (10(-9) M)-enhanced corticosterone secretion of inner cells, maximal effective concentration being 10(-7) M. The effect of 10(-7) M glucagon or GPL-1 was suppressed by 10(-6) M Des-His1-[Glu9]-glucagon amide (glucagon-A) and exendin-4(3-39) (GPL-1-A), which are selective antagonists of glucagon and GLP-1 receptors, respectively. Glucagon and GLP-1 (10(-7) M) decreased by about 45-50% cyclic-AMP production by dispersed inner adrenocortical cells in response to ACTH (10(-9) M), but not to the adenylate cyclase activator forskolin (10(-5) M). Again this effect was blocked by 10(-6) M glucagon-A or GLP-1-A. The exposure of dispersed inner cells to 10(-7) M glucagon plus GLP-1 completely suppressed corticosterone response to ACTH (10(-9) M). However, they only partially inhibited (by about 65-70%) both corticosterone response to forskolin (10(-5) M) or dibutyryl-cyclic-AMP (10(-5) M) and ACTH (10(-9) M)-enhanced cyclic-AMP production. Quantitative HPLC showed that 10(-7) M glucagon or GLP-1 did not affect ACTH-stimulated pregnenolone production, evoked a slight rise in progesterone and 11-deoxycorticosterone release, and markedly reduced (by about 55%) corticosterone secretion of dispersed inner adrenocortical cells. In light of these findings the following conclusion are drawn: (i) glucagon and GLP-1, via the activation of specific receptors, inhibit glucocorticoid response of rat adrenal cortex to ACTH; and (ii) the mechanism underlying the effect of glucagon and GLP-1 is probably two-fold, and involves both the inhibition of the ACTH-induced activation of adenylate cyclase and the impairment of the late steps of glucocorticoid synthesis.     

Hormonal control of the replication of human fetal fibroblasts: role of somatomedin C/insulin-like growth factor I

Sparse cultures of fetal and postnatal human fibroblasts were equivalent in their responsiveness to the mitogenic action of somatomedin C/insulin-like growth factor I (SM-C/IGF-I). At both developmental stages, the addition of SM-C/IGF-I (100 ng/ml) increased cell number at day 3 1.4-fold in serum-free medium and 2-fold in the presence of 0.25% human hypopituitary serum. Furthermore, dose-response curves indicated that there was no difference in the sensitivity of fetal and postnatal fibroblasts to the growth-promoting effects of SM-C/IGF-I, with a half-maximal response occurring at 6 ng/ml SM-C/IGF-I. This biological action of SM-C/IGF-I correlated with SM-C/IGF-I binding to fetal and postnatal fibroblast monolayers. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) also stimulated replication of fetal and postnatal fibroblasts. The mitogenic effects of SM-C/IGF-I, EGF, and PDGF were additive. Dexamethasone, which alone had no effect, was synergistic with SM-C/IGF-I in stimulating replication of postnatal fibroblasts. The combination of SM-C/IGF-I (100 ng/ml), dexamethasone (10(-7) M), EGF (10 ng/ml), and PDGF (5 ng/ml) had the same mitogenic effectiveness as 10% calf serum (CS) in postnatal cells. In marked contrast, there was no mitogenic interaction between SM-C/IGF-I and dexamethasone in fetal fibroblasts. In fetal cells, SM-C/IGF-I + EGF + PDGF +/- dexamethasone could only account for 50% of the activity of 10% CS. Moreover, fetal cells were 50-100% more responsive than postnatal cells to the proliferative effect of serum.     

Insulin-like growth factor binding protein-3 mediates IGF-I action in a bovine mammary epithelial cell line independent of an IGF interaction

IGF-I is mitogenic for the bovine mammary epithelial cell line MAC-T. In addition, IGF-I specifically upregulates IGFBP-3 synthesis in these cells. To investigate this effect on cell growth and IGF-I responsiveness, cell lines were developed that constitutively express IGFBP-3. MAC-T cells transfected with IGFBP-3 (+BP3) or vector alone (Mock) grew similarly over 7 days in 10 or 1% fetal calf serum. Basal DNA synthesis was lower (70%) in +BP3 cells compared to Mock cells. However, DNA synthesis was increased by IGF-I (1-50 ng/ml) relative to untreated controls to a greater extent in +BP3 cells compared to Mock cells. IGF-I (20 ng/ml) increased DNA synthesis 11- and threefold in +BP3 and Mock cells, respectively. Additionally, +BP3 cells were more sensitive to the lower concentrations of IGF-I (1-5 ng/ml). In contrast, preincubation of Mock cells with exogenous IGFBP-3 did not enhance responsiveness or sensitivity to IGF-I. Basal DNA synthesis was unaffected by either an IGF neutralizing antibody or exogenous IGFBP3, indicating the differences observed between +BP3 and Mock cells were not attributable to sequestration of endogenous IGF-I by IGFBP-3. There were no differences between +BP3 and Mock cells in IGF-I receptor number or affinity. DNA synthesis was also increased in +BP3 cells, compared to controls, in response to 5 microg/ml insulin and 2.5 ng/ml Long R(3)IGF-I, indicating that the potentiated response did not require an interaction with IGFBP-3. These results suggest that IGF-I regulation of IGFBP-3 represents a regulatory loop, the function of which is to increase IGF-I bioactivity, using a mechanism that does require an IGF-I-IGFBP-3 interaction.     

Modulation of IgM secretion and H chain mRNA expression in CH12.LX.C4.5F5 B cells by adrenocorticotropic hormone

The murine B cell line CH12.LX.C4.5F5 (CH12 (5F5) expresses adrenocorticotropin (ACTH) receptors, which can modulate IgM secretion by these cells. Interestingly, the response to ACTH was concentration dependent, inducing IgM secretion at subnanomolar amounts and suppressing secretion at micromolar amounts. With the use of an enzyme-linking immunospot assay it was possible to demonstrate that the ACTH-induced increase in IgM secretion by CH12 (5F5) cells was caused at least in part by an increase in the number of cells secreting IgM. CH12 (5F5) cells activated with suboptimal concentrations of LPS demonstrated a similar biphasic response. ACTH at concentrations of 10(-13) to 10(-9) M augmented IgM secretion in LPS-activated cells as much as sixfold, whereas 10(-6) M ACTH slightly decreased LPS-induced IgM secretion. At the mRNA level, subnanomolar concentrations of ACTH increased microH chain mRNA expression up to twofold in unstimulated or LPS-stimulated CH12 (5F5) cells. Taken together, these studies show that physiologically relevant concentrations of ACTH can interact directly with receptors on these B lymphocytes to enhance IgM secretion and microH chain mRNA expression. Although ACTH does increase intracellular cAMP levels in CH12 (5F5) B cells, it is unlikely that the induction of this second messenger pathway is by itself responsible for the ACTH induced B cell differentiation. The concentration of ACTH necessary to stimulate significant intracellular cAMP increases was 10- to 100-fold higher than that required to increase IgM secretion. Furthermore, CH12 (5F5) cells treated with varying concentrations of 8-bromo cAMP or cholera toxin were inhibited in their ability to secrete IgM. These results strongly suggest that the enhancing effects of ACTH on CH12 (5F5) IgM secretion are via mechanisms independent of those mediated by cAMP.     

Effect of IGF-I on pig oocyte maturation,fertilization, and early embryonic development in vitro,and on granulosa and cumulus cell biosynthetic activity

Porcine granulosa cells have been shown previously to both secrete and respond to insulin-like growth factor-I (IGF-I), suggesting an autocrine function of this peptide in the follicle. The present work was undertaken to determine possible effects of IGF-I on in vitro maturation, in vitro fertilization, and early embryonic development in culture. Granulosa and cumulus cell proliferation and differentiation based on ³H-thymidine uptake and progesterone production, respectively, were also assessed. The results showed that the cleavage rate of oocytes was markedly stimulated in a dose-dependent manner by the addition of IGF-I to the oocyte maturation medium (P < 0.05). Embryo development beyond the 8-cell stage was improved by IGF-I, reaching a maximum of 22% at 200 ng/ml IGF-I. Treatment with IGF-I after fertilization increased the percentage of total oocyte cleavage (P < 0.05) to approximately 52%, 43%, and 57% at, respectively, 25, 50, and 100 ng/ml IGF-I. ³H-thymidine incorporation by granulosa cells was significantly increased in cultures treated with FSH (3-fold) or IGF-I (6-fold) compared to the control. For the cumulus cells, FSH caused a similar increase (3-fold) in ³H-thymidine incorporation while IGF-I stimulated a 15-fold increase. Progesterone production by the granulosa cells was increased to the same extent by treatment with FSH or IGF-I (4.7 and 5.1-fold, respectively). However, for the cumulus cells, while FSH caused a marked 16-fold increase in progesterone production, IGF-I caused only a marginal increase of 2.5-fold. These results indicate a beneficial effect of IGF-I on in vitro porcine oocyte maturation and pre-implantation embryo development, suggesting a physiological role for IGF-I in vivo. The in vivo effect of IGF-I may be indirect via autocrine stimulation of cumulus and/or granulosa cells resulting in enhanced oocyte maturation and fertilization. © 1994 Wiley-Liss, Inc.     

Specificity of insulin-like growth factor binding to type-II IGF receptors in rabbit mammary gland and hypophysectomized rat liver

We have reevaluated IGF binding specificity to membrane receptors in rabbit mammary gland (RMG) and hypophysectomized rat liver (HRL) using recombinant DNA-derived and synthetic analogues of human IGF-I and highly purified IGF-II. SDS-PAGE demonstrated that [125I]IGF-I bound to type-I IGF receptors in RMG; this binding was inhibited in a similar fashion by the IGF-I analogues (IC50 = 10 ng/ml) and to a lesser extent by IGF-II (IC50 = 60 ng/ml). [125I]IGF-II bound to type-II IGF receptors in both RMG and HRL. The IC50 for IGF-II was 9 and 3 ng/ml with RMG and HRL, respectively. At a dose as high as 1 microgram/ml, IGF-I analogues inhibited less than 20% of [125I]IGF-II binding. These results suggest that IGF-I has little or no affinity for type-II IGF receptors.