- Email: [email protected]
P3. Differential metabolism of 25-hydroxyvitamin D3 by macrophages and fibroblasts cultured from synovial fluid of patients with arthritis
Abstracts from the Bone and Tooth Society Meeting, March 1990 P2. Raised CAMP levels in human placental homogenates after treatment with Forskolin, bPTH(l-34) and IBMX JMA Williams, DR Abramovich, CG Dacke,* TM Mayhew and KR Page Departments ofAnafomy, Obstetrics and Gynaecolggy and Physiology, The University, Aberdeen ‘Schoo! ofPharmacy and Biomedical Sciences, Portsmoufh Po!yfechnic, Portsmouth Parathyroid hormone (PTH) may effect placental Ca transport via a mechanism involving CAMP The effects of Forskolin and bPTH(l-34) upon tissue production of CAMP were studied using paired in vitro perfused placental lobules. Each experiment consisted of a 30 minute equilibration period followed by a 15 minute experimental period. The perfusate throughout was a Krebs Ringer (KR) solution containing 2.4mM Ca’+ and 2% w/v dextran (mol wt 60,000) except during the experimental period when KR was replaced with either KR + 10mJMIBMX + 10 ‘M Forskolin (experimental), and KR + lo-*M IBMX (control), or KR + 10-‘M IBMX + 140&l bPTH(l-34) + 0.5% w/v BSA (experimental) and KR + 10.’ IBMX + 0.5% w/v BSA (control). At minute 45 tissue samples were remoded from the lobules and microwaved for 40 seconds. All homogenates were assayed for CAMP and normalised with total protein. Perfusate samples were taken throughout the experiment and assayed for CAMP and glucose. After 15 minutes of exposure to Forskolin and IBMX there was significant (210%,?71.2%, p
by Action Research.
P3. Differential metabolism of 25-hydroxyvitamin D3 by macrophages and fibroblasts cultured from synovial fluid of patients with arthritis ME Hayes,* J Palit,t B Cooper,* D Bayley,* P Still,* J Denton,§ AJ Freernon@ and EB Mawer* Departments ofMedicine* and Rheumatology§, Uniuersit!y of Manchester Medical School, Manchester Ml3 9PT tDepartment of Rheumatology, Withington Hospital, Manchester and $fhe Rheumatic Disease Center Hope Hospital, Salford We have previously demonstrated that synovial fluid macrophages from patients with arthritis can synthesise 1,25-dihydroxyvitamin D3 (1,25-D) from 25hydroxyvitamin D3 (25-D) (Ann Rheum Dis 48,723-729). We have now investigated the effects of bacterial lipopolysaccharide (LPS), which induces 1,25-D synthesis in macrophages, and of 1,25-D itself on 25-D hydroxylation by macrophages and fibroblast-like cells (probably synoviocytes) cultured from synovial fluid.
377
Macrophages from 5 samples increased 1.25-D synthesis from 0.17-6.31 to 0.88-29.9pmoVhh110” cells (~~0.01) following 3 days exposure to LPS (0.5-lOOpg/ ml). In one of two samples also examined for the effects of 1,25-D (IOnM), 1,25-D synthesis was inhibited from 6.31kO.32 to 1.81+0.21pmol/h/106 cells (n-3, mean + SEM). Macrophage cultures were also characterised by appearance of multinucleated cells, which stained for non-specific esterase, but numbers were not 1,25-Ddependent. Fibroblast/synoviocyte cultus‘es from 3 samples synthesised 3.18,8.39 and 12.47pmol24,25dihydroxyvitamin D3 (24,25-D)/h/106 cells. In all three 1,25-D induced dose dependent increases in 24,25-D synthesis; for example 1OnM 1,25-D increiased 24,25-D synthesis to 11.12,21.49.and 44.14pmol/h1106 cells (p
P4. Binding and c&llular processing of aminoterminal parathyroi’d hormone by oppossum kidney (OK) cells RC Brown, AC Silver and JS Woodhead Department of Medical Biochemistry, Univemity qf Wales College of Medicine, Cardiff CF4 4XN, UK It is believed that the site of action of PTH in the renal proximal tubule is at the baso-lateral membrane. Apical surface binding is believed to be a non-specific process similar to other filtered low molecular weight proteins. We have examined the binding and cellular processing of “‘I labelled (8,18Nle, 34Tyr) l-34 bPTH-NH2( l-34) and (8,18Nle, 34Tyr) 3-34 bPTH-NH2(3-34) by the apical surface of confluent OK cells. Spetific, high capacity time and temperature dependent binding of 1-34 coupled to rapid degradation (solubility in 10% TCA) of l-34 was observed. Acid washing of the cell layer indicated that the receptor-ligand complex was rapidly internalised (>80% of total cell associated radiolabel was in an acid resistant compartment after 1Omin 37C). Monensin treatment was assrociated with increased intracellular uptake and reduced l-34 degradation indicating that l-34 degradaaion was associated with intracellular processing of bound l-34. In contrast, acid resistant binding and degradation of 3-34 was minimal indicating that this ligdnd was not internalised even in the presence of Forskolin. It is concluded that in OK cells apical surface binding of PTH may be of functional significance. In addition, the intact amino terminus of PTH may be a
by Action Research.
P3. Differential metabolism of 25-hydroxyvitamin D3 by macrophages and fibroblasts cultured from synovial fluid of patients with arthritis ME Hayes,* J Palit,t B Cooper,* D Bayley,* P Still,* J Denton,§ AJ Freernon@ and EB Mawer* Departments ofMedicine* and Rheumatology§, Uniuersit!y of Manchester Medical School, Manchester Ml3 9PT tDepartment of Rheumatology, Withington Hospital, Manchester and $fhe Rheumatic Disease Center Hope Hospital, Salford We have previously demonstrated that synovial fluid macrophages from patients with arthritis can synthesise 1,25-dihydroxyvitamin D3 (1,25-D) from 25hydroxyvitamin D3 (25-D) (Ann Rheum Dis 48,723-729). We have now investigated the effects of bacterial lipopolysaccharide (LPS), which induces 1,25-D synthesis in macrophages, and of 1,25-D itself on 25-D hydroxylation by macrophages and fibroblast-like cells (probably synoviocytes) cultured from synovial fluid.
377
Macrophages from 5 samples increased 1.25-D synthesis from 0.17-6.31 to 0.88-29.9pmoVhh110” cells (~~0.01) following 3 days exposure to LPS (0.5-lOOpg/ ml). In one of two samples also examined for the effects of 1,25-D (IOnM), 1,25-D synthesis was inhibited from 6.31kO.32 to 1.81+0.21pmol/h/106 cells (n-3, mean + SEM). Macrophage cultures were also characterised by appearance of multinucleated cells, which stained for non-specific esterase, but numbers were not 1,25-Ddependent. Fibroblast/synoviocyte cultus‘es from 3 samples synthesised 3.18,8.39 and 12.47pmol24,25dihydroxyvitamin D3 (24,25-D)/h/106 cells. In all three 1,25-D induced dose dependent increases in 24,25-D synthesis; for example 1OnM 1,25-D increiased 24,25-D synthesis to 11.12,21.49.and 44.14pmol/h1106 cells (p
P4. Binding and c&llular processing of aminoterminal parathyroi’d hormone by oppossum kidney (OK) cells RC Brown, AC Silver and JS Woodhead Department of Medical Biochemistry, Univemity qf Wales College of Medicine, Cardiff CF4 4XN, UK It is believed that the site of action of PTH in the renal proximal tubule is at the baso-lateral membrane. Apical surface binding is believed to be a non-specific process similar to other filtered low molecular weight proteins. We have examined the binding and cellular processing of “‘I labelled (8,18Nle, 34Tyr) l-34 bPTH-NH2( l-34) and (8,18Nle, 34Tyr) 3-34 bPTH-NH2(3-34) by the apical surface of confluent OK cells. Spetific, high capacity time and temperature dependent binding of 1-34 coupled to rapid degradation (solubility in 10% TCA) of l-34 was observed. Acid washing of the cell layer indicated that the receptor-ligand complex was rapidly internalised (>80% of total cell associated radiolabel was in an acid resistant compartment after 1Omin 37C). Monensin treatment was assrociated with increased intracellular uptake and reduced l-34 degradation indicating that l-34 degradaaion was associated with intracellular processing of bound l-34. In contrast, acid resistant binding and degradation of 3-34 was minimal indicating that this ligdnd was not internalised even in the presence of Forskolin. It is concluded that in OK cells apical surface binding of PTH may be of functional significance. In addition, the intact amino terminus of PTH may be a