Increased thymidine incorporation into fetal rat cartilage in vitro in the presence of human somatomedin, epidermal growth factor and other growth factors

The incorporation of [3H]thymidine by rat costal cartilage in vitro was studied at different fetal and postnatal ages and the effect of partially purified human somatomedin, mouse epidermal growth factor, platelet secretion products, insulin and growth hormone on thymidine uptake by fetal cartilage was examined. Thymidine uptake in plasma-free medium was many times greater in late fetal life than after birth. The incorporation of [3H]thymidine into costal cartilage from 21-day fetuses was significantly (P less than 0.05) increased above control values in the presence of 10 micrograms somatomedin/1, and when cartilage was incubated in medium containing somatomedin and diluted human plasma there was a synergistic action. Epidermal growth factor at a concentration of 1 ng/l was a potent stimulator of thymidine uptake. Secretion products from human platelets after their aggregation by thrombin stimulated [3H]thymidine uptake at a concentration of 2% (v/v), but were inhibitory at high concentrations. High concentrations of platelet secretion products stimulated the incorporation of [35S]sulphate by cartilage. A pharmacological concentration of 10 mu. insulin/ml stimulated [3H]thymidine uptake, but not concentrations of 1 or 100 mu./ml. Growth hormone had no effect. The results showed that fetal cartilage had a greater endogenous mitogenic activity than postnatal cartilage. While somatomedins may be important in the regulation of fetal body growth, other protein growth factors also stimulate fetal skeletal tissues.     

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.     

Evidence suggesting that the direct growth-promoting effect of growth hormone on cartilage in vivo is mediated by local production of somatomedin.

We have used a catheterization system that permits chronic infusion into the arterial supply of one hindlimb of rats to study the direct effects of rat growth hormone and human somatomedin C on growth of the tibial epiphyseal cartilage plate in hypophysectomized rats. Rat growth hormone (0.4 microgram per day) or human somatomedin C (0.25, 1, or 4 micrograms per day) stimulated growth of the epiphyseal plate of the infused limb but not of that of the contralateral noninfused limb. The somatomedin C had a dose-related effect. Rabbit antiserum to human somatomedin C, but not normal rabbit serum, completely abolished the direct growth effect of the rat growth hormone when it was co-infused with the hormone. These results support the concept that growth hormone stimulates long bone growth by inducing local production of somatomedin, which in turn stimulates cell proliferation in an autocrine or paracrine fashion. However, they do not exclude the possibility that serum somatomedin may also play a role.     

Circulating growth factor studies in growth plate versus resting cartilage in vitro. II. Recognition of growth factors in rat serum

The growth-promoting effects of GH can be explained in part by the mediation of somatomedins/insulin-like growth factors (IGFs). However, large quantities of the IGFs are required to stimulate growth in vivo, and in some conditions, IGF levels may correlate poorly with GH levels and growth status. These observations suggest that other circulating factors may also be important for growth. We have examined the growth-promoting activity in rat serum, as assessed by stimulation of sulfate and/or thymidine uptake by resting and growth plate cartilage (osteochondral junction) from hypophysectomized rats in vitro. Although stimulation by a low molecular weight somatomedin fraction (approximately 5,000-12,000) accounted for about 90% of serum stimulation of sulfate uptake by resting cartilage, it explained only about 60% of stimulation of the growth plate. Growth plate and resting cartilage appeared equally insensitive to insulin, but the growth plate exhibited reduced sensitivity to inhibitor(s) in diabetic rat serum. Fractionation of normal rat serum by gel filtration at neutral pH revealed comparable stimulation of growth plate and resting cartilage by high molecular weight factors, presumably somatomedins bound to carrier proteins. After gel filtration at acid pH, both growth plate and resting cartilage responded to somatomedins with molecular weights from 5,000-12,000. However, the growth plate also responded to a 12,000-22,000 mol wt factor [Sephadex G-75; 5 X 120 cm; sulfate uptake, 68 +/- 16% above buffer (mean +/- SEM); P less than 0.01] which did not affect resting cartilage (sulfate uptake, 27 +/- 21% above buffer; P = NS). Levels of both the low and higher molecular weight factors were reduced in the serum of hypophysectomized rats. We conclude that circulating growth-promoting activity includes both the low molecular weight somatomedins and a higher molecular weight growth plate growth factor which is not recognized by resting cartilage. Use of the osteochondral junction assay system may permit elucidation of the regulation and nature of this growth factor.     

Isolation of a somatomedin from plasma of rats bearing growth hormone secreting tumors.

We have purified a protein which has somatomedin-like properties from the serum of Wistar-Furth rats bearing a growth hormone producing pituitary tumor (MStT/W15). Activity was measured by a placental insulin and/or somatomedin C radioreceptor assay (SmC-RRA). The serum was initially filtered through Sephadex G-150 equilibrated with 0.1 M NH4HCO3 and 0.02% NaN3. On the G-150 column, radioreceptor insulin (RRI) and radioreceptor somatomedin C (RRSm-C) activities coincided and appeared predominantly in the 160,000 mol wt range with a minor proportion in the 50,000 mol wt range. The pooled active fractions were boiled for 30 min at pH 5.5. After removing denatured protein by centrifugation, the extract was passed through G-50 Sephadex equilibrated with 1% formic acid and 0.15 M NaCl. Sixty to 90% of the SmC-RRA activity in the effluent appeared in the 9000 mol wt range. This material has an isoelectric focusing range of 8.4--9.6, similar to that described for human somatomedin C. On SDS-urea polyacrylamide gel electrophoresis only one protein band was seen. The isolated peptide (rSm) stimulated sulfate uptake in hypophysectomized rat cartilage. The potency of two preparations was variously assayed from 14.0 to 54.7 units/mg. Rat somatomedin was iodinated and purified by absorption on and elution from placental membranes. Eight to 12% of rat [125I]Sm was specifically bound by human placental membranes. Rat [125I]Sm was displaced by hSmC and rSm and human NSILA-S, partially displaced by procine proinsulin and poorly displaced by rat insulin. In preliminary studies, rat [125I]Sm was displaced from receptors on human placental membranes by sera from pituitary tumor bearing rats greater than normal rat sera greater than hypophysectomized rat sera.     

Somatomedin, growth and nutritional status

Serum somatomedins, or insulin-like growth factor(s) (IGF), originally characterized as primarily GH-dependent peptides, were found to also be dependent on insulin levels and nutritional status. Four properties characterize somatomedin peptides: their concentrations in serum are growth hormone dependent; they possess insulin actions in extraskeletal tissues; they promote the incorporation of sulfate into proteoglycans of cartilage; and they stimulate DNA synthesis and cell multiplication in certain types of cultured cells. Reduced somatomedin C levels are found in children with severe protein-energy malnutrition. Plasma concentration of growth hormone and cortisol are both elevated and there are low levels of insulin and somatomedin C. There is evidence that the ability of somatomedin C to stimulate cartilage is modulated by somatomedin inhibitor, factor that may act to limit growth in conditions of hormonal and/or nutritional deficiency. Dietary energy and protein appears to be particularly important for both generation of somatomedins and their action on growing cartilage. Measurement of somatomedins C concentration shows promise as a means for monitoring the response of malnourished patients and rats to nutrition repletion.     

Cellular and molecular correlates of the decline in responsiveness of cartilage and thymus to growth hormone in aged animals

The responses in vitro of cartilage to somatomedin and of thymocytes to growth hormone have been studied in tissues derived from rats of various ages. The basal and somatomedin stimulated incorporation of radioactive proline into proteins and of sulphate into mucopolysaccharides diminishes markedly with age. Chondrocytes per unit area in cartilage of old rats are about 4- to 5-fold reduced in number as compared to those in cartilage from 1-day old rats. The organ size and yield of thymocytes are reduced in aged rats. Besides the fall in number of cells, the average metabolic activity of cells as measured by uridine incorporation into RNA in vitro is also diminished. There is a progressive decline in stimulation by growth hormone of uridine incorporation in isolated thymocytes with age. Immunocytochemical studies reveal the location of the hormone along the membrane. The amount of the hormone bound by thymocytes, estimated by immunoenzymatic methods is 3- to 9-fold lower in thymocytes from 14-months old rats as compared to similar preparations from 4-week old rats. Thymocytes from both young and old rats are composed of subpopulations, one of which binds growth hormone. The proportion of the hormone binding cells is higher in thymocytes from young rats as compared to those from aged animals.     

Characterization of a specific somatomedin-c receptor on isolated bovine growth plate chondrocytes

The interaction of somatomedin (Sm) with growth plate chondrocytes (GPCs) is believed to be the primary stimulus of skeletal growth. Using techniques designed to disrupt as little as possible the phenotypic characteristics of GPCs, we have been able to obtain 3-4 x 10(8) viable cells from the major physes of one newborn calf. The availability of these cells plus essentially pure Sm-C/insulin-like growth factor I, the most GH-dependent Sm, has now made possible detailed studies of the interaction of this radiolabeled peptide with the GPC receptor and of the subsequent processing of this hormone by these cells. The enzymatic methods required to free GPCs from their matrix led to loss of receptors, followed by rapid receptor regeneration by de novo synthesis in suspension cultures. Binding of [125I]iodo-Sm-C to GPCs was time dependent and saturable, with optima at 15 C and pH 7.8. At 37 C, binding peaked at 90 min and declined thereafter. Multiplication-stimulating activity, insulin, and nerve growth factor were less potent than unlabeled Sm-C in competition with [125I]iodo-Sm-C for its receptor. Human GH, epidermal growth factor, and fibroblast growth factor failed to show competition even at 10(-6) M. Analysis of the fate of [125I]iodo-Sm-C bound to GPCs at 37 C provided evidence that this hormone is internalized and extruded from the cell in a partially degraded form. Scatchard analysis of [125I]iodo-Sm-C binding to GPCs and to chondrocytes isolated from articular cartilage revealed similar Ka values, but reproducibly 2-6 times more receptors on growth plate than on articular chondrocytes.     

Incorporation of [35S]sulphate into mucopolysaccharide by teleost cartilage in vitro: the influence of mammalian growth hormone, teleost plasma, and mammalian plasma.

Branchial cartilages from two species of teleost fish, Salmo gairdneri and Tilapia andersoni, were found to be capable of synthesizing mucopolysaccharide for up to 4 days in vitro. Synthesis of mucopolysaccharide, the cartilage matrix, was measured by monitoring the amount of bound [³⁵S]sulphate incorporated by the fish cartilage units. The binding of ³⁵S appeared to be increased by the addition of bovine growth hormone to the organ culture medium. Plasma from Tilapia did not contain stimulatory somatomedin-like activity, when added to either Tilapia or rat cartilage cultures. However, mammalian serum and plasma, known to be rich in somatomedin, markedly stimulated the binding of ³⁵S by Tilapia cartilage units in vitro. The degree of stimulation of ³⁵S binding in fish cartilage by mammalian serum was greater and more consistent than that observed with bovine growth hormone. The possibility of a direct effect of growth hormone on skeletal growth in teleost fish is discussed.     

Steroid hormone effects on sonatomedin. I. Somatomedin action in vitro.

The effects of cortisol, estradiol, and testosterone on somatomedin action on cartilage incubated in vitro have been examined. The addition of hormones in the absence of serum had no effect on the incorporation of sulfate by cartilage from hypophysectomized rats, embryonic chicks, or normal young pigs. Normal human serum provided a source of somatomedin which stimulated the incorporation of sulfate by cartilage in a dose-response relationship; the potency of serum with and without added steroid hormone was determined after formal parallel-line analysis. Moderately supraphysiologic levels of cortisol, 17 beta-estradiol, and testosterone generally had little effect on somatomedin action in the test systems. Very high levels of serum cortisol (100-1000 mug/100 ml) inhibited somatomedin action on pig cartilage, but had little effect on rat or chick cartilage. A 20 ng/100 ml increase in serum estradiol had no effect on somatomedin action on chick cartilage, but appeared to enhance somatomedin action on pig cartilage. A 5 mug/100 ml increase in serum testosterone did not affect somatomedin action on either chick or pig cartilage. These studies suggest that the alteration of somatomedin action is not a major mechanism in the effect of steroid hormones on growth. In addition, since modest increases in serum levels of cortisol, estradiol, and testosterone had little effect on somatomedin action in our assay systems, these systems should be satisfactory for the study of hormone effects on somatomedin generation.     

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%.     

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.     

Homocysteic acid: An examination of its possible growth hormone-like activity

Hypophysectomized rats were injected intraperitoneally for 4 days with various doses of homocysteic acid or growth hormone. The effects of these compounds on epiphyseal cartilage thickness and circulating somatomedin activity levels were evaluated in an attempt to repeat the results of Clpath, Smith, and McCully², who reported that this compound had growth hormone-like activity. DNA polymerase activity in livers of animals treated with growth hormone or with 10 mg/day of homocysteic acid was also measured. Using larger number of animals and including higher doses of homocysteic acid than those previously employed, we did not observe an increase of epiphyseal cartilage thickness in homocysteic acid treated hypophysectomized rats. Growth hormone significantly increased cartilage thickness. DNA polymerase levels in homocysteic acid treated hypophysectomized rats were not substantially increased although a larger, dose-dependent increase was observed with pGH and hGH. Neither homocysteic acid nor GH increased circulating somatomedin activity under the conditions used in this investigation. These observations demonstrate that homocysteic acid was not a substance with growth hormone-like activity in our hands and cast doubt on its possible future usefulness as a substitute for GH in clinical situations.     

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.     

The effects of insulin and growth hormone on the release of somatomedin by the isolated rat liver.

Livers from hypophysectomized (hypox) rats were perfused with oxygenated Waymouth's medium in a system which permitted continuous recirculation for separate 30 minute periods after which fresh medium was supplied. In most experiments 6 changes of medium were carried out over a 3 hour period. The somatomedin activity of each perfusate was determined by measuring its ability to stimulate sulfate uptake in hypox rat cartilage in vitro. For comparison between experiments the results are expressed as the per cent stimulation of sulfate uptake by the perfusate compared with the unperfused buffer. Without hormonal additions there was a progressive fall in the release of somatomedin activity during the 6 periods of study. When compared with the results without hormone, the addition of 1000 muU/ml of insulin per ml of medium during the 2nd to 6th period led to a significant increase in perfusate somatomedin activity at all periods. The addition of 100 muU/ml of insulin was without significant effect. The possible inter-relationship between insulin and growth hormone in the regulation of somatomedin release was studied with a dose of bGH of 250 ng/ml which had previously been shown to be insufficient by itsel to stimulate somatomedin release. When added to a medium containing 1000 muU/ml of insulin, this dose of bGH did not significantly stimulate somatomedin release beyond that obtained with insulin alone. However, when 250 ng/ml was added to a medium containing 100 muU/ml insulin, a significant stimulation of somatomedin release was observed while the addition of each hormone separately was without significant effect. These results support the hypothesis that insulin shares with GH the regulation of somatomedin release by the liver. Differences in insulin concentration may explain some clinical situations in which somatomedin concentrations cannot be correlated with GH levels.     

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.     

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.     

The effects of vitamin D metabolites on the plasma and matrix vesicle membranes of growth and resting cartilage cells in vitro

This study compares the effects of vitamins 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-(OH)2D3 on populations of chondrocytes at different developmental stages. Confluent third passage chondrocytes derived from the resting zone and adjacent growth region of rat costochondral cartilage were cultured in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum and increasing concentrations of hormone. After determination of cell number, matrix vesicles and plasma membranes were isolated by differential centrifugation. The effects of hormone on alkaline phosphatase, 5'-nucleotidase, ouabain-sensitive Na+/K+-ATPase, and phospholipid composition were dependent on vitamin D metabolite and were cell specific. Growth cartilage chondrocytes responded primarily to 1,25-(OH)2D3, whereas resting zone cells responded primarily to 24,25-(OH)2D3. 1,25-(OH)2D3 inhibited growth cartilage cell number at pharmacological concentrations and had no effect on resting cartilage cell number. In contrast, 24,25-(OH)2D3 appeared to stimulate resting cartilage cell number at physiological concentrations and inhibit these cells at pharmacological doses, but had no effect on growth cartilage chondrocytes. These data were supported by [3H]thymidine incorporation studies. 1,25-(OH)2D3 stimulated alkaline phosphatase, 5'-nucleotidase activity, and Na+/K+-ATPase activity in the matrix vesicles of growth cartilage cells. 1,25-(OH)2D3 also stimulated Na+/K+-ATPase activity in the matrix vesicles and plasma membranes of resting zone cells. Incubation with 24,25-(OH)2D3 stimulated alkaline phosphatase, 5'-nucleotidase, and Na+/K+-ATPase in the matrix vesicles produced by resting zone cells. In addition, 24,25-(OH)2D3 stimulated Na+/K+-ATPase activity in the plasma membranes of resting zone cells as well as in both matrix vesicles and plasma membranes of growth cartilage cells.     

Glucocorticoid effects on somatomedins and somatomedin inhibitors

Glucocorticoid excess may be associated with poor growth despite normal levels of GH and adequate nutrition. Steroid-induced growth failure could be mediated by defective generation and/or action of somatomedins. To probe potential mechanisms, we examined the effect of corticosteroid administration on net somatomedin activity, immunoreactive somatomedin-C, and separated biologically active somatomedins and somatomedin inhibitors. Twelve children receiving alternate day steroid therapy had circulating somatomedin activity measured by porcine cartilage bioassay. Somatomedin activity fell 6 h after steroids [from 1.02 +/- 0.09 (+/- SEM) to 0.35 +/- 0.07 U/ml; P less than 0.001] and then rose toward normal. No significant change in somatomedin activity occurred during the day off therapy. Further studies were conducted in normal subjects given a single 60-mg dose of prednisone. Six hours after prednisone, somatomedin activity (rat cartilage bioassay) decreased by 46% (P less than 0.01), yet somatomedin-C did not change. To pursue this discrepancy, serum was fractionated on Sephadex G-50, pH 2.4, and separated somatomedin and somatomedin inhibitory bioactivity was measured. Biologically active somatomedins (Kav, 0.50-0.63) were comparable before and after prednisone treatment, as was inhibitory activity found at Kav 0.13-0.25. In contrast, somatomedin inhibitory activity at Kav 0.25-0.38 doubled (111 +/- 8% inhibition of somatomedin action vs. 54 +/- 11%; P less than 0.005) after prednisone therapy. The somatomedin inhibitor in these fractions blunted serum stimulation of sulfate, thymidine, and uridine uptake by test cartilage. These inhibitory effects could not be attributed to direct steroid action, as levels were less than 2 micrograms/dl in inhibitory fractions and addition of cortisol and prednisolone to the bioassay system failed to decrease somatomedin activity. We conclude that glucocorticoid administration is followed by an increase in circulating somatomedin inhibitors. Such inhibitors may explain the steroid-induced fall in net somatomedin activity and contribute to impaired growth.