Platelet-derived growth factor and multiplication-stimulating activity II, but not multiplication-stimulating activity III-2, stimulate [3H]thymidine and [35S]sulphate incorporation by fetal rat costal cartilage in vitro

The actions of partially purified porcine platelet-derived growth factor (PDGF) and highly purified multiplication-stimulating activity (MSA) II and MSA III-2, which are somatomedins, were investigated on the incorporation of [3H]thymidine and [35S]sulphate by fetal rat costal cartilage in vitro. This was compared with their effects in the presence of 1% fetal calf serum (FCS) on the uptake of thymidine by growth-arrested fetal rat fibroblasts. Platelet-derived growth factor at concentrations of 0.21-21 micrograms/l enhanced the incorporation of both isotopes by fetal cartilage in the presence of 1% FCS, but had an inconsistent action on thymidine uptake and no significant action on sulphate uptake in serum-free medium. Platelet-derived growth factor promoted thymidine uptake by growth-arrested, isolated fetal rat fibroblasts. Multiplication-stimulating activity II (10-100 micrograms/l) stimulated the uptake of thymidine and sulphate by fetal cartilage in medium containing 1% FCS but had no consistent action in serum-free medium, although MSA II and PDGF had a synergistic effect on thymidine uptake in the absence of serum. Multiplication-stimulating activity III-2 had no consistent action on thymidine or sulphate incorporation by fetal cartilage in either serum-free or serum-supplemented medium. However, the same preparation of MSA III-2 stimulated the uptake of [3H]thymidine into fetal rat fibroblasts with a half-maximal response at a concentration of 5-10 micrograms/l. The results identify PDGF as a possible mitogenic agent for fetal rat connective tissues in vitro and show a differential sensitivity of fetal cartilage to MSA peptides.     

Response of chondrocytes isolated from human foetal cartilage to plasma somatomedin activity

Plasma somatomedin activity enhanced the incorporation of [3H]thymidine into chondrocytes isolated from human foetal cartilage during weeks 13-21 of gestation. Human growth hormone (0.1-20 muu./ml), human placental lactogen (0.1-5 microgram/ml) and insulin (0.25-10 muu./ml) had no direct effects on the synthesis of DNA in these chondrocytes, although insulin at concentrations of 2.5-100 mu./ml increased [3H]thymidine incorporation by up to 400%.     

Stimulation of cartilage zones of the calf costochondral growth plate in vitro by growth hormone dependent rat plasma somatomedin activity

The actions of rat plasma somatomedin activity dependent on growth hormone were investigated in vitro on separated zones of cartilage from the calf costochondral junction. Plasma somatomedin maximally stimulated the uptake of[3H]thymidine into cartilage cells of the proliferating region. Cartilage deeper in the growth plate possessed the highest uptake of [35S]sulphate which was also stimulated by somatomedin. Somatomedin, therefore, appears to promote both cell replication and matrix synthesis throughout the growth plate cartilage although the two processes were greatest in different cartilage regions. Growth hormone or tri-iodothyronine did not directly alter the uptake of either isotope into the growth plate cartilage.     

Autocrine tumour growth regulation by somatomedin C: an in-vitro model

A human primary haemangiosarcoma was derived from a patient with severe hypoglycaemia. Cell line established from that tumour secreted somatomedin C in serum-free culture media. Immunoreactive somatomedin from the media eluted from Sephacryl S-200 in two peaks of 160 000 and 8000 molecular weights. Similar results were obtained when medium was acidified and chromatographed on Sephadex G-50. Binding of tracer concentrations of 125I-labelled somatomedin C to human haemangiosarcoma cells was much higher than that of 125I-labelled insulin. Half-maximal displacement of 125I-labelled somatomedin C binding occurred at an unlabelled somatomedin C concentration of 0.7 nmol/l. Insulin competed with 125I-labelled somatomedin for binding to this receptor, but 150-fold more insulin was required for half-maximal displacement. Somatomedin secreted by human haemangiosarcoma cells and purified from serum-free media strongly stimulated [methyl-3H]thymidine incorporation into the DNA of these cells. Inhibition of somatomedin C secretion by cortisol resulted in the inhibition of tumour cell proliferation but stimulation of somatomedin secretion by human GH stimulated the cell proliferation rate. It appears that production of somatomedin C in human haemangiosarcoma cells plays a part in the regulation of tumour growth by an autocrine mechanism.     

Hormonal regulation of somatomedin secretion by fetal rat hepatocytes in primary culture

To determine which hormones might regulate somatomedin secretion in the fetus, we measured somatomedin levels in conditioned medium from primary cultures of fetal rat hepatocytes. We employed a bioassay [( 3H]thymidine incorporation into DNA of chick embryo fibroblasts), a displacement assay [competition for binding of radiolabeled multiplication-stimulating activity (rat insulin-like growth factor II) to the somatomedin-binding protein] for total somatomedin, and the RIA for somatomedin-C. Epidermal growth factor and dexamethasone were the most active hormones tested; total somatomedin levels were 2-3 times above control levels. Rat GH was much less stimulatory. Human placental lactogen, glucagon, and insulin had little or no effect. Stimulation of somatomedin secretion by both epidermal growth factor and dexamethasone was time and dose dependent. The maximal response occurred at 48 h at a concentration of about 1 X 10(-7) M of either hormone. In the bioassay, stimulation by epidermal growth factor, but not dexamethasone, was detected. The steroid enhanced the secretion of an inhibitor that completely masked the mitogenic activity of the increased somatomedin levels. The somatomedin secreted by fetal hepatocytes exhibited immunological cross-reactivity with human somatomedin-C, but the levels were 500-fold less than those measured by our displacement assay. This suggests that the predominant fetal rat somatomedin is not somatomedin-C. We conclude that epidermal growth factor and dexamethasone, but not GH or placental lactogen, stimulated the secretion by fetal hepatocytes of a somatomedin which resembled multiplication-stimulating activity.     

Regulation of amino acid uptake and deoxyribonucleic acid synthesis in isolated human fetal fibroblasts and myoblasts: effect of human placental lactogen, somatomedin-C, multiplication-stimulating activity, and insulin

We compared the abilities of human placental lactogen (hPL), somatomedin-C/insulin-like growth factor I (SM-C/IGF-I), multiplication-stimulating activity (MSA), and insulin to induce a rapid anabolic event, the uptake of the nonmetabolizable amino acid [3H]alpha-aminoisobutyric acid ([3H] AIB) or the more long term action of increasing [3H]thymidine incorporation, as a measure of DNA synthesis, in isolated human fetal fibroblasts and myoblasts. Myoblasts were derived from skeletal muscle and fibroblasts from skin explants removed from human fetuses delivered between 12 and 19 weeks gestation after prostaglandin-induced abortion. Each of the four peptides caused a dose-dependent increase in [3H]AIB uptake by both fibroblasts and myoblasts, with mean half-maximal concentrations (ED50) ranging from 0.9-1.9 nM. The concentration of each peptide required to stimulate [3H]thymidine uptake was significantly greater, with the exception of insulin, which was inactive. For myoblast cultures, the mean ED50 values were: hPL, 7.9 nM; SM-C/IGF-I, 2.0 nM; and MSA, 2.2 nM. For fibroblast cultures, the mean ED50 values were: hPL, 2.3 nM; SM-C/IGF-I, 3.3 nM; and MSA, 4.3 nM. Insulin did not stimulate [3H]thymidine incorporation into either cell type at concentrations up to 6.9 nM. Incubation in the presence of monoclonal antibody against SM-C/IGF-I abolished the ability of SM-C/IGF-I to stimulate either [3H]thymidine or [3H]AIB uptake into fetal fibroblasts. The antibody substantially inhibited the incorporation of [3H]thymidine by these cells in response to hPL, but was less effective in blocking hPL-stimulated [3H]AIB uptake. It did not inhibit the uptake of either radioisotope in response to MSA or [3H]AIB uptake in response to insulin. The actions of SM-C/IGF-I and hPL on thymidine incorporation were additive at submaximal concentrations, but not so at maximal individual concentrations. Their actions on AIB uptake were additive at both submaximal and maximal concentrations. The results suggest that hPL as well as the SMs may contribute to the growth stimulus in human fetal connective tissues. Since incubation with SM-C/IGF-I antibody reduced the mitogenic response of fetal cells to hPL, the actions on DNA synthesis may be partially mediated by local release of SM. However, the similar ED50 values with which these peptides stimulated [3H]AIB uptake during a short incubation, and their additive effects at maximal individual concentrations, suggest that hPL may also have direct actions.     

Increased somatomedin and cartilage metabolic activity in rabbit fetuses injected with insulin in utero

Summary The effect of insulin injection in fetal rabbits on plasma somatomedin activity and cartilage metabolism was investigated. One fetus in each of 12 litters was injected with 1 unit of insulin zinc suspension subcutaneously on day 27 of gestation and a control fetus was injected with the same volume of 0.154 mol/l saline. The litter was delivered by caesarean section on day 29 and each fetus identified. Plasma somatomedin activity was determined by fetal rabbit cartilage bioassay. Costal cartilage from individual fetuses was incubated in medium containing [³H]thymidine or [³⁵S]sulphate as indicators of cell replication and matrix synthesis respectively. Individual values for somatomedin activity or cartilage isotope uptake were ranked within a litter. In each case the rank in the litter of the insulin-injected fetus, but not the saline-injected fetus, was significantly higher than the mean rank of the litter. Insulin did not stimulate cartilage metabolism in vitro.     

Somatomedin and analogues of cyclic AMP increase the number of cells synthesizing DNA in cartilage from hypophysectomized rats

The effects of somatomedin and certain nucleotides on nuclear labelling of cartilage cells with [3H]thymidine were determined by autoradiography. Segments of costal cartilage from hypophysectomized rats were incubated for 24 h in a basal medium with or without additions and then pulsed for 2 h with [3H]thymidine in the basal medium. Both somatomedin (0.1 U/ml) and Bt2cAMP (10(-4)M) increased the number of labelled nuclei, and the combined effects were more than additive. A parallelism between the effects of these agents on nuclear labelling and their effects on total thymidine incorporation into DNA was demonstrated. The 8-bromated derivative of cAMP (10(-4)M) also enhanced chondrocyte nuclear labelling, but neither 8-Br-5'-AMP (10(-4)7) nor 8-Br-cGMP (10(-4)M) exhibited actions of the cAMP analogues. It is concluded that in cartilage obtained from hypophysectomized rats and incubated under the specified conditions (1) both somatomedin and cAMP analogues increase the number of cells synthesizing DNA as well as total thymidine incorporation into DNA, (2) the effects of the hormone and cyclic nucleotide in combination are synergistic, and (3) the increased incorporation of labelled thymidine into DNA reflects increased DNA synthesis and not merely an alteration of the specific activity of the intracellular thymidine nucleotide pool.     

Metabolic effects of growth factors and polycyclic aromatic hydrocarbons on cultured human placental cells of early and late gestation

The metabolic effects of epidermal growth factor (EGF), insulin, insulin-like growth factor-I (IGF-I), and IGF-II were determined on human placental cells in monolayer culture obtained from early gestation (less than 20 weeks) and late gestation (38-42 weeks). Parameters studied were uptake of aminoisobutyric acid (AIB), uptake of 3-O-methylglucose and [3H]thymidine incorporation into cell protein. Since benzo[alpha]pyrene (BP) inhibits EGF binding and autophosphorylation in cultured human placental cells, particularly in early gestation, we also studied the effect of benzo[alpha]pyrene and other polycyclic aromatic hydrocarbons (PAHs) on EGF-mediated AIB uptake. The metabolic effects of EGF, insulin, and the IGFs in cultured human placental cells varied with gestational age and the growth factor studied. All three classes of growth factors stimulated AIB uptake in both early and late gestation at concentrations from 10-100 micrograms/L, well within a physiological range. However, insulin stimulation of AIB uptake was maximal at a high concentration (200 micrograms/L) in both early and late gestation cells, suggesting an action via type 1 IGF receptors rather than via insulin receptors. EGF stimulated 3-O-methylglucose uptake only in term placental cells. No significant stimulation of [3H]thymidine incorporation by any of the growth factors tested was seen with either early or late gestation cells. The effect of PAHs on AIB uptake by cultured placental cells was variable. BP alone stimulated AIB uptake by both very early and late gestation cells and enhanced EGF-stimulated AIB uptake. alpha-naphthoflavone alone inhibited AIB uptake at all gestational ages and inhibited EGF-stimulated AIB uptake. beta-Naphthoflavone and 3-methylcholanthrene minimally inhibited AIB uptake by early gestation cells and did not modify EGF-stimulated uptake at any gestational period. Our prior results demonstrated that BP more significantly inhibited EGF than IGF or insulin receptor binding as well as autophosphorylation in early gestation placenta, and that BP was the most potent of the PAHs tested. Thus, the direct effect of the PAHs on placental EGF receptors and amino acid transport may indicate altered function of EGF in the regulation of placental growth in smoking mothers that is developmentally regulated.     

Action of multiplication-stimulating activity on [3H]thymidine incorporation in rabbit and human fetal chondrocytes in vitro

The mitogenic action of multiplication-stimulating activity (MSA) on normal mammalian chondrocytes has been examined. Addition of MSA (NIH, PkII-MSA, 2.5-500 ng/ml or Collaborative Research, CR-MSA, 50-250 ng/ml) to primary suspensions of chondrocytes prepared by enzymic digestion of costal and articular cartilage of rabbits (356-481 g body wt) resulted in a dose-dependent increase in [3H]thymidine incorporation into the trichloroacetic acid-precipitated cell contents. CR-MSA (50-250 ng/ml) also had a significant stimulatory effect on [3H]thymidine incorporation into human fetal chondrocytes (22 weeks of gestation) prepared by enzymic digestion. When PkII-MSA was added in the presence of 1.25% of a standard adult or cord plasma to either rabbit or human fetal (18 weeks) chondrocytes, the increase in [3H]thymidine incorporation appeared to be synergistic. The mitogenic action of MSA can thus be demonstrated on primary suspensions of mammalian chondrocytes. The action of MSA on human chondrocytes has not previously been reported.     

Response of isoalted rabbit articular and epiphyseal chondrocytes to rat liver somatomedin.

Isolated rat liver, when perfused with medium containing bovine growth homone produced somatomedin-like activity (liver somatomedin). Liver somatomedin is useful in studies of the hormonal control of the cartilage plate in vitro, since unlike serum it is not contaminated with other hormones or growth factors (apart from growth hormone). Chondrocytes isolated from various regions of the growth cartiage responded differently to liver s-omatomedin; proliferative chondrocytes, like those isolated from the articular cartilage, showed increased [3H]thymidine uptake in response to liver somatomedin, whereas hypertrophic chondrocytes did not respond. It is suggested that there is a reduction in the response to somatomedin by growth plate chondrocytes as they pass from the proliferative to the hypertrophic state. Thyroxine, thought to be involved in thr processes of hypertrophy and new bone formation, did not directly affect [3H]thymidine uptake by proliferative chondrocytes, but inhibited stimulation of both their activity by liver somatomedin. Mesurement of [3H]thymidine uptake by isolated articular chondrocytes may provide a useful assay for both liver and serum somatomedin. The graded response of chondrocytes to increasing concentrations of liver somatomedin paralleled the response to increasing levels of serum somatomedin.     

Angiotensin stimulation of bovine adrenocortical cell growth.

Factors controlling proliferation of adrenocortical cells have been studied in monolayer cultures of bovine adrenocortical cells. Angiotensin II stimulated cell proliferation and [3H]thymidine incorporation into DNA with a half-maximal effective concentration of 0.96 +/- 0.27 nM. Similar sensitivity to angiotensin III with reduced sensitivity to angiotensin I and tetradecapeptide renin substrate was observed. Although sensitivity to angiotensin II was equivalent to that for fibroblast growth factor (1.5 nM half-maximal effective concentration), maximal effects of angiotensin were less than for fibroblast growth factor and serum. High concentrations of insulin (1-10 micrometer) also stimulated [3H]thymidine incorporation into DNA and cell proliferation. [Sar1,Ile5,Ile8]Angiotensin II, a competitive antagonist of angiotensin II, blocked angiotensin II stimulation of DNA synthesis but did not affect fibroblast growth factor and insulin stimulation of DNA synthesis. Corticotropin (ACTH) blocked the stimulatory effects of both angiotensin II and fibroblast growth factor. The dose-response curves for angiotensin II stimulation of steroidogenesis were similar to those for stimulation of [3H]thymidine incorporation into DNA. Among the seven cell types examined, only adrenocortical cells responded to angiotension II with stimulation of DNA synthesis.     

Identification of a receptor for somatomedin-like polypeptides in human fibroblasts.

We have demonstrated a specific receptor for somatomedin-like growth polypeptides in human fibroblasts in culture using the closely related polypeptide, multiplication stimulating activity (MSA), as the radioligand. Polypeptides purified from human plasma, somatomedin A and acid soluble nonsuppressible insulin-like activity (NSILA-s), competed potently for 125I-MSA binding, as did unlabeled MSA. Although insulin and proinsulin also strongly inhibited MSA binding, the properties of the growth peptide receptor differed from those of the human fibroblasts insulin receptor. Somatomedin A, NSILA-s, MSA, insulin and proinsulin all stimulated the incorporation of [3H]thymidine into DNA in human fibroblasts. We propose that these polypeptides induce DNA synthesis through their interaction with the growth peptide receptor.     

Acromegaloid patients with type A insulin resistance: parallel defects in insulin and insulin-like growth factor-I receptors and biological responses in cultured fibroblasts

A subset of patients with the syndrome of acanthosis nigricans and insulin resistance type A is characterized by acromegaloid features in addition to hyperinsulinemia, hyperandrogenemia, and an inherent defect in insulin receptor function. It has been proposed that the acromegaloid features result from the interaction of insulin at concentrations encountered in vivo, with a functionally intact insulin-like growth factor-I (IGF-I) receptor closely related to the insulin receptor. We investigated this possibility by examining binding and hormone-stimulated [14C]glucose uptake, [3H]thymidine uptake, and receptor autophosphorylation by both insulin and IGF-I in cultured fibroblasts from two affected patients. In comparison to normal fibroblasts, [125I]insulin binding, insulin-stimulated [14C]glucose, and [3H]thymidine uptake, and insulin-stimulated autophosphorylation were each reduced by approximately 50-60% of the absolute values in controls. In contrast to expectation, each of these apparent defects in insulin binding and action were mirrored by a parallel decrease in IGF-I binding and action. Thus, [125I]IGF binding was approximately 50%, IGF-I stimulated [3H]thymidine uptake was approximately 40% and 60% of the control value, and IGF-I-stimulated receptor autophosphorylation was reduced by 40%. Incubation of fibroblasts with insulin at 25 ng/mL reduced subsequent binding of [125I]IGF-I by approximately 20% and did not enhance maximal stimulation of [3H]thymidine incorporation. We conclude that in some patients with acanthosis nigricans and acromegaloid features, IGF-I receptors of cultured fibroblasts may share the inherent defects of insulin receptor function. These in vitro data do not explain the acromegaloid features observed in vivo, suggesting that acromegaloid features are mediated by other mechanisms.     

Effect of elastase on aortic smooth muscle cell proliferation

We studied the effects of elastase on [3H]thymidine incorporation into aortic smooth muscle cells. When elastase was added to cultured aortic smooth muscle cells, [3H]thymidine incorporation was inhibited in a dose-dependent manner in the presence of fetal bovine serum. Elastase also inhibited this incorporation in cells treated with epidermal growth factor. Epidermal growth factor (50 ng/ml) stimulated thymidine incorporation, without elastase, but with the addition of 20 units/ml of elastase the incorporation was inhibited 70%. The incorporation of thymidine into cells treated with 50 ng/ml epidermal growth factor was also inhibited 50% by a low concentration of elastase (5 units/ml). These inhibitory effects on thymidine incorporation were also observed in cells stimulated with platelet-derived growth factor. Platelet-derived growth factor (20 units/ml) markedly stimulated thymidine incorporation into cells, and elastase inhibited its activity in a dose-dependent manner. These results suggest that elastase has the potential to prevent the development of atherosclerosis by inhibiting smooth muscle proliferation.     

Monoclonal antibody to the type I insulin-like growth factor (IGF-I) receptor blocks IGF-I receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-I and insulin in human skin fibroblasts.

Insulin and insulin-like growth factor type I (IGF-I) stimulate an overlapping spectrum of biological responses in human skin fibroblasts. Although insulin and IGF-I are known to stimulate the incorporation of [3H]thymidine into DNA in these cells, the identity of the receptor(s) that mediates this effect has not been fully clarified. The mouse anti-human IGF-I receptor antibody alpha IR-3 binds with specificity to IGF-I but not to insulin receptors in human placental membranes; it also specifically inhibits the binding of 125I-labeled IGF-I but not 125I-labeled insulin to suspensions of human skin fibroblasts in a dose-dependent manner. alpha IR-3 competitively inhibits IGF-I-mediated stimulation of [3H]thymidine incorporation into DNA. This inhibition is dependent on the concentration of alpha IR-3 and in the presence of a fixed antibody concentration can be partially overcome by high concentrations of IGF-I. In contrast, at concentrations of less than 1 microgram/ml, the effect of insulin to stimulate [3H]thymidine incorporation is not inhibited by alpha IR-3. However, the incremental effects of higher concentrations (greater than 1 microgram/ml) of insulin on [3H]thymidine incorporation are inhibited by alpha IR-3. alpha IR-3 is a highly specific antagonist of IGF-I receptor-mediated mitogenesis in human skin fibroblasts. By using this antibody, it is shown directly that insulin can act through the IGF-I receptor to stimulate DNA synthesis but can also activate this effect through the insulin receptor itself.     

Dexamethasone and 8-bromo-cyclic AMP depress the incorporation of [3H]thymidine into mouse condylar cartilage by different pathways

We examined whether cyclic AMP (cAMP) affects the incorporation of [3H]thymidine into cartilage cells and, if so, whether this action could be related to the inhibitory effect of glucocorticoid hormones on the growth of ossifying cartilage. Incorporation of [3H]thymidine into trichloroacetic acid-precipitable material by mouse cartilage was measured concomitantly with the concentration of cAMP. Dexamethasone (1 mumol/l) significantly (P less than 0.05) depressed the incorporation of [3H]thymidine. The cAMP analogue 8-bromo-cAMP (0.01-1 mmol/l) also depressed the incorporation of the radionucleotide in a dose-dependent fashion. When various concentrations of 8-bromo-cAMP were added with dexamethasone (1 mumol/l), no apparent changes took place compared with the effect of dexamethasone alone. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2-1 mmol/l) elicited an inhibitory effect on [3H]thymidine incorporation and a stimulatory influence on cartilage cAMP concentrations. Dexamethasone, at doses (0.01-1 mumol/l) causing significant inhibition of [3H]thymidine incorporation, failed to increase cartilage levels of cAMP. It seems, therefore, that the depressive effect of dexamethasone on [3H]thymidine incorporation in condylar cartilage is not mediated through an increase of cAMP in the tissue.     

Actions of insulin and hydrocortisone on macromolecular synthesis in primary epithelial cell cultures from mouse mammary glands.

Monolayer cultures of mammary gland epithelial cells were prepared from the abdominal glands of midpregnancy mice. After collagenase digestion of mammary tissue and separation by differential centrifugation, the isolated epithelial cells were cultured in Eagle's Minimal Essential Medium supplemented with 10% fetal bovine serum and insulin (6 micrograms/ml). Six days later, when the cultures were in log growth and nearly confluent, the effects of insulin and/or hydrocortisone on the rates of RNA, DNA, and protein synthesis were determined in a serum-free medium. At physiological concentrations, insulin enhanced the rates of uptake and incorporation of [3H]uridine into RNA, of [3H]thymidine into DNA, and of [3H]leucine into protein. Hydrocortisone was shown to be biphasic with regard to concentration in attenuating or augmenting insulin's effects on macromolecular synthesis.     

Repeatedly passed FRTL-5 rat thyroid cells can develop insulin and insulin-like growth factor-I-sensitive cyclooxygenase and prostaglandin E2 isomerase-like activities together with altered basal and thyrotropin-responsive thymidine incorporation into DNA

Repeatedly passed or aged rat FRTL-5 thyroid cells develop a high level of basal [3H]thymidine incorporation into DNA and a reduced response to TSH in medium containing 5% serum and insulin (5H medium). The basal [3H]thymidine incorporation into DNA of aged cells can exceed the TSH-induced increase in earlier passages of the same cell line (fresh cells) and the TSH response decreases from more than 10-fold above basal in fresh cells to less than 2-fold in aged cells. This change is not associated with a loss of the diploid karyotype, a change in basal cAMP levels, or a change in dependence on TSH for cell growth. Attenuation of the TSH response in the [3H]thymidine incorporation assay is more evident than the reduced effect of TSH on cAMP levels or iodide transport; moreover, the TSH effect on cAMP levels does not correlate with that on [3H] thymidine incorporation as a function of hormone concentration. The high basal activity in [3H]thymidine incorporation into DNA in aged cells is due to an increased responsiveness to insulin, insulin-like growth factor-I (IGF-I), or serum. Thus, removal of serum and insulin from the medium eliminates the high basal [3H]thymidine incorporation into DNA, and this activity is restored by insulin or IGF-I in a concentration-dependent manner. The increased responsiveness of aged cells to insulin or IGF-I is inhibited by indomethacin or hydrocortisone and is associated with insulin or IGF-I, but not TSH, stimulation of cyclooxygenase and prostaglandin E2 (PGE2) isomerase-like activity. Fresh cells, in contrast, require TSH plus insulin or IGF-I to increase these activities. Increased responsiveness of cyclooxygenase activity to insulin or IGF-I in aged cells reflects at least in part an increase in cyclooxygenase mRNA levels. We suggest that insulin/IGF-I stimulation of PGE2 production leads to the high basal thymidine incorporation into DNA in aged cells maintained in TSH-depleted (5H) medium; the reduced stimulation by TSH of cAMP content or iodide uptake may reflect PG inhibition (negative feedback regulation) of cAMP production.     

Insulin-like growth factor (IGF)-II and insulin, but not IGF-I, are mitogenic for fetal rat adrenal cells in vitro

The agents controlling growth of the fetal adrenal gland are poorly defined. The purpose of the present study was to determine the factors required to promote proliferation of adrenal cells obtained from fetal rats at 20 days of gestation and grown in monolayer cell culture under serum-free conditions. Insulin stimulated [3H] thymidine incorporation into DNA at all concentrations (0.01-10 mg/l) tested. Rat insulin-like growth factor (IGF)-II in the presence of insulin promoted a dose-dependent increase in mitogenic activity, with a half-maximal concentration of approximately 1 microgram/l; IGF-II had no effect in the absence of insulin. There was no significant effect of IGF-I on mitogenic activity in the presence or absence of insulin. Adrenal cell DNA synthesis was not stimulated by ACTH, several ACTH-related peptides, a variety of known growth factors, several steroids, or conditioned medium from fetal adrenal cells. We conclude that in the presence of insulin, IGF-II is a specific growth factor for the fetal rat adrenal. We also suggest that rat fetal adrenal cells, in common with other epithelial cell types under serum-free culture conditions, may respond to this progression factor without an obligatory requirement for an initial exposure to competence factors.