Stephen John Martin Skinner

Chondrons From Articular Cartilage (II): Analysis of the Glycosaminoglycans in the Cellular Microenvironment of Isolated Canine Chondrons

A chondron rich preparation was isolated from mature canine tibial cartilage using low-speed homogenization techniques. Proteoglycans were extracted from this preparation by exhaustive treatment with 4M guanidine-HCl. A significant proportion of the total proteoglycan, measured as uronic acid, was resistant to extraction and represented 27.9% in intact cartilage chips and 18.6% in the chondron fraction. Histochemical examination of chondrons confirmed that extraction resistant proteoglycans remained within the capsule of the chondron after 4M guanidine-HCl treatment. Electrophoretic analysis of the glycosaminoglycans extracted from intact cartilage chips and the chondron fraction showed approximately equivalent amounts of chondroitin sulphate (79.3%), keratan sulphate (16.3%) and hyaluronic acid (4.3%) present. In contrast, the extraction resistant residue in the chondron fraction was significantly enriched for hyaluronic acid (10.5%, p less than 0.05) but was depleted of chondroitin sulphate (70.9%, p less than 0.05). The major chondroitin sulphate isomer in the resistant fraction was chondroitin 6-sulphate while in the soluble fraction, the quantities of the two isomers were approximately equivalent. Comparison with previously published data suggests a role for minor collagens in the retention of proteoglycans in the cellular microenvironment.

The movement of IGF-I into the brain parenchyma after hypoxic-ischaemic injury.

The movement of peptides from CSF into the parenchyma is thought to be slow and diffusion limited. However IGF-I can reduce neuronal loss at distal sites when given centrally 2 h after hypoxic-ischaemic (HI) injury. The present study determined the distribution of [3H]IGF-I given into the lateral ventricle after unilateral HI injury in adult rats. Radioactivity in the injured cortex peaked immediately after administration then rapidly declined. Autoradiography demonstrated radioactivity in the perivascular spaces and in the corpus callosum and external capsule of the injured hemisphere. HPLC and radioimmunoassay confirmed a rise in intracerebral IGF-I levels (from 159 ± 9 to 401 ± 88 ng g−1). These data suggest that injury can enhance the movement of IGF-I into the cerebrum via the white matter tracts and perivascular spaces.

Effects of glucocorticoids and β-adrenoceptor agonists on the proliferation of airway smooth muscle

An increase in airway smooth muscle is a characteristic feature of asthma. Because beta-adrenoceptor agonists and corticosteroids are commonly used in the treatment of asthma we have studied the effects of these medicines on the growth of airway smooth muscle. These agents were incubated with bovine airway smooth muscle cells for 40 h for measurement of thymidine incorporation and 64 h for measurement of cell counts. Salbutamol inhibited thymidine incorporation (IC50 = 60 nM) and led to a reduction in cell number (IC50 = 10 nM). At 10 microM there was a 14.6 +/- 2.6% reduction in cell number. Salmeterol also inhibited the growth of the airway smooth muscle cells but the effect did not plateau at 10 microM. At this concentration there was an 89.5 +/- 3.6% reduction in thymidine incorporation and a 44.1 +/- 5.2% reduction in cell number. Cortisol and beclomethasone dipropionate were more potent than salbutamol in inhibiting thymidine incorporation with IC50 values of 5 nM and 0.2 nM respectively. Cortisol 100 nM led to a 16.6 +/- 6.5% reduction and beclomethasone dipropionate 3 nM led to a 17.8 +/- 5.8% reduction in cell number. If similar effects occur in man and in vivo, these medicines could act directly on airway smooth muscle to inhibit the development of hyperplasia.

The effects of mechanical stretching on fetal rat lung cell prostacyclin production

A model system was used to determine the effect of stretch on prostacyclin (PGI) production by organotypic fetal rat lung cultures grown on gelatin foam in vitro, measured by RIA of 6-keto-PGF1 alpha (6KF) in the culture medium. The stretching apparatus was programmable for stretch of varying frequency and duration. The effective stimuli for PGI production were: continuous pulsatile stretch greater than intermittent pulsatile stretch greater than permanent stretch (p less than 0.05). The rate of PGI production was greatest in the first 15 min of pulsatile stretch and was associated with a 70% increase in cAMP production (p less than 0.05). When the effect of magnitude of stretch was compared (15% vs 28% extension), there was a significant increase with a maximum in the 28% stretch group double that of the 15% stretch group (p less than 0.01). PGI production in response to pulsatile stretching was inhibited by indomethacin but not by pretreatment with cortisol. These results suggest that the production of PGI by lung cells may be significantly affected by the frequency and magnitude of pulsatile stretching.

Intracerebral transportation and cellular localisation of insulin-like growth factor-1 following central administration to rats with hypoxic-ischemic brain injury

Insulin-like growth factor-1 (IGF-1) has been shown to be neuroprotective when administered centrally following hypoxic-ischemic (HI) brain injury. However, the cerebral distribution and site of action of IGF-1 after intracerebroventricular (i.c.v.) administration are not known. A unilateral HI brain injury was induced in adult rats by a modified Levine method. Either 3H-IGF-1 alone, or in combination with unlabelled IGF-1, was administered into the lateral ventricle 2 h after injury. The activity of 3H-IGF-1 signal in the potentially injured cortex was compared between two treatment groups using image analysis. The regional distribution and cellular localisation of 3H-IGF-1 were examined autoradiographically in potentially injured hemispheres at 0.5 and 6 h after administration. Tritiated IGF-1 was detected predominantly in the pia mater, perivascular spaces and subcortical white matter tracts 0.5 h after administration and decreased by 6 h (p<0.05). The signals associated with the perivascular spaces and pia mater were not blocked by unlabelled IGF-1, suggesting non-saturable binding in these brain areas. IGF-1 signal was co-localised with IGF binding protein (IGFBP)-2 immunostaining in the white matter tracts where the signal was blocked by unlabelled IGF-1, suggesting competitive association. IGF-1 signal associated with neurons and glia was maximal in the cerebral cortex and less in the CA1-2 subregion of the hippocampus which were blocked by unlabelled IGF-1 (p<0.05). The signals from cortical neurons did not decrease 6 h after administration, suggesting specific and persistent binding to these cells. Our results indicate that centrally administered IGF-1 can be translocated to neurons and glia via the perivascular circulation and the ependymal cell-white matter tract pathways.

The Estimation of Elastin in Fetal Tissues by Radioimmunoassay of Isodesmosine

A radioimmunoassay was developed for the determination of isodesmosine as the tetraacetyl derivative. Isodesmosine tetraacetate conjugated with bovine albumin was injected into rabbits which developed useful titers of antibodies after five months. The radioligand for the assay was prepared by acetylating isodesmosine with [3H] acetic anhydride. The bound ligand was separated from free ligand by coprecipitation with human gamma-globulin in 46% saturated ammonium sulfate solution. The sensitivity of the assay was 2 ng isodesmosine. The antiserum was specific for isodesmosine tetraacetate and only desmosine tetraacetate gave appreciable cross-reactivity (4%). The assay was found to be suitable for the accurate estimation of elastin in small samples (5 mg dry weight) of rat and ovine fetal lung tissue and for elastin degradation products in amniotic fluid (0.5 ml).

Effects of Corticosteroids, Prostaglandin E2, and Beta-Agonists on Adenylate Cyclase Activity in Fetal Rat Lung Fibroblasts and Type II Epithelial Cells

The effect of glucocorticoids on the response of adenylate cyclase in fetal rat lung fibroblast and Type II epithelial cell cultures to beta-agonists and prostaglandin E2 (PGE2) was investigated. There was significant stimulation of cyclic AMP synthesis by adrenalin in both fibroblasts and Type II cells, which was potentiated in a dose-dependent manner by cortisol. Stimulation of adenylate cyclase by PGE2 (10-1000 nM) was demonstrated in fibroblasts but not in Type II cells. The response to PGE2 was stimulated by pretreatment with cortisol only in fibroblasts (p less than .01), and no latent response to PGE2 was evident in Type II cells after cortisol treatment. These experiments suggest that both cell types isolated from late gestation fetal lung contain active beta-agonist and glucocorticoid receptors that synergize in raising intracellular cyclic AMP, which has multiple effects, including surfactant secretion from Type II cells. Since the adenylate cyclase response to PGE2 and its enhancement by glucocorticoids occurred only in fibroblasts, it is concluded that the reported effects of PGE2 on surfactant release are not mediated via raised intracellular cyclic AMP in Type II cells.

Interaction between prostacyclin and contrisol in fetal lung cells: Effects on cAMP production

Glucocorticoids secreted by the fetal adrenal, or administered for therapeutic reasons, stimulate fetal lung maturation in the human and other species. Prostacyclin, produced within the lung may be another agent with maturational effects. In this investigation we have demonstrated that glucocorticoids interact with lung cells and increase their response to a prostacyclin analogue (Iloprost, PGIp). This agent stimulates adenylate cyclase activity in fetal lung fibroblasts, fetal lung epithelial cells and in neonatal vascular smooth muscle cells. The cAMP response to PGIp in fibroblasts and epithelial cells occurred in the range 3nM-1 microM. When fibroblasts were pretreated with cortisol before PGIp, cAMP was increased 2-3 fold (p less than 0.01). There was a similar increase in cAMP after cortisol pretreatment in response to PGIp by fetal lung epithelial cells, but not with smooth muscle cells. The action of cortisol was blocked by an inhibitor of RNA synthesis (Actinomycin D) but not by an inhibitor of DNA synthesis (5-fluorodeoxy-uridine). Additional experiments with cholera and pertussis toxins, and with forskolin suggest that cortisol principally increases the quantity or activity of the adenylate cyclase sub-unit in fetal lung fibroblasts and, in doing so, increases the cAMP response to PGIp.

Transferrin Stimulates Proteoglycan Accumulation by Fetal Lung Cells in Culture

The role of transferrin in growth and the formation of extracellular matrix was investigated by comparing its effects on proteoglycan metabolism and cell proliferation in primary cultures of fetal rat lung fibroblasts and Type II epithelial cells. Transferrin specifically stimulated the accumulation of dermatan/chondroitin sulfate proteoglycans associated with the cells and matrix in a dose-dependent manner (0-200 micrograms/ml, r = .850 in fibroblasts and r = .810 in Type II cells). This effect was not due to increased synthesis since there was a corresponding decrease in proteoglycans and their degradation products released into the medium. The effect is probably mediated via an action on the proteoglycan core protein, since there was no effect of transferrin on enzyme activity promoting glycosaminoglycan synthesis on the synthetic initiator beta-D-xyloside. The effect of transferrin on proteoglycan distribution was not a secondary effect caused by changes in collagen synthesis and was not linked to cell proliferation or the concentration of Fe3+ ions in the culture medium.

The adenylate cyclase response to parathyroid hormone in fetal lung fibroblasts is enhanced by cortisol

Parathyroid hormone (PTH, <10−8 M) stimulated adenylate cyclase in fibroblasts, but not epithelial cells, isolated from fetal rat lung. In contrast to osteosarcoma cells (UMR 106), the response of fibroblasts to PTH was increased by pretreatment with cortisol (< 10−8–10−7 M).