Expression of cation exchanger NHE and anion exchanger AE isoforms in primary human bone-derived osteoblasts.

The authors used isoform-specific antibodies against cation (NHE) and anion (AE) exchange isoforms in order to establish their specific expression and localization in dispersed human bone-derived cells. Immunocytochemical preparations of permeabilized osteoblasts probed with polyclonal antibodies were optically analysed by conventional immunofluorescence and con-focal laser scanning microscopy. These techniques demonstrated the abundant presence of epitopes of the cation exchangers NHE1 and NHE3 and the anion exchanger AE2 in these cells. The NHE1 and NHE3 isoform proteins were predominantly located in subplasmalemmal and nucleoplasmic vesicles. The AE2 isoform was densely localized to a subcellular location characteristic of the Golgi complex. The molecular identity of the AE and NHE isoforms was investigated by RT-PCR that confirmed the presence of NHE1 and NHE3 transcripts in addition to NHE4. RT-PCR and diagnostic restriction analysis of amplified AE cDNA established preferential AE2 expression. Since AE2 has been shown to act as a sulfate transporter at low pH, it is possible that it performs this function in the osteoblast Golgi complex where sulfation reactions occur post-translationally on numerous extracellular matrix macromolecules prior to secretion and mineralization. The Na(+)/H(+)exchanger proteins are regulated by mitogenic and non-mitogenic stimuli in the osseus environment and are involved in the large fluxes of ions and protons that necessarily occur during bone formation and resorption and thus play an important role in intracellular ion homeostasis in osteoblasts.

Type 1 collagen synthesis by skin fibroblasts from 17 patients with osteogenesis imperfecta type III.

The aim of this study was to look for osteogenesis imperfecta (O.I.) specific features in collagen synthesised by skin fibroblast cultures obtained from patients with severe progressive deforming O.I. type III. Results from 17 O.I. type III cultures were contrasted with results from 6 relatives, 3 unrelated controls, 6 O.I. type II, 7 O.I. type IV and 7 O.I. type I cultures. Biosynthesised radiolabelled collagen types I and III were extracted and separated by gel electrophoresis as intact alpha chains or as cyanogen bromide digested peptides. Various abnormalities of type I collagen synthesis were detected in cultures from 13/17 O.I. type III patients. In conclusion, synthesised collagen abnormalities were detected in cells from most O.I. type III patients studied and were O.I.-specific, not O.I. type III-specific at the individual level. However, the frequency of detection of these features was partially specific to the O.I. type III phenotype.

Type I collagen biosynthesis by skin fibroblasts from patients with idiopathic juvenile osteoporosis.

1. Skin fibroblast lines were cultured from nine patients who had the features of idiopathic juvenile osteoporosis, six relatives, five unrelated control subjects and three unrelated patients with osteogenesis imperfecta type I. Some patients with idiopathic juvenile osteoporosis were adults whose previous osteoporosis was in remission. Two patients with idiopathic juvenile osteoporosis were siblings and one patient with idiopathic juvenile osteoporosis had a daughter with severe osteogenesis imperfecta (type III). 2. The ratio of type III to type I collagen, synthesized by fibroblasts, was increased in two of the patients with osteogenesis imperfecta type I and in the daughter with osteogenesis imperfecta type III, but was normal in all the other patients with idiopathic juvenile osteoporosis and the other relatives. 3. Radiolabelled collagen was digested by cyanogen bromide and separated on SDS-PAGE. Unreduced collagen peptides migrated normally, except those from both the two siblings with idiopathic juvenile osteoporosis. In these two lines, abnormal migration suggested the presence of collagen I mutations. 4. The secretion of synthesized collagen by these two idiopathic juvenile osteoporosis lines and two others was reduced to only 43-45% as compared with a line from a 13-year-old control subject, which was defined as 100%. The three osteogenesis imperfecta type I lines secreted 18-37%, the other five idiopathic juvenile osteoporosis lines secreted 57-75%, the relatives (including the daughter with severe osteogenesis imperfecta) secreted 49-115% and the controls secreted 69-102%.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Plasma somatomedin activity and diabetic retinopathy.

Somatomedin activity was measured by a rabbit chondrocyte bioassay in plasma of adult patients with insulin-dependent diabetes of 11-28 years duration. Mean somatomedin activity in patients with background or proliferative retinopathy, assessed by ophthalmoscopy or fluorescein angiography, was 1.42 +/- 0.65 U/ml (n = 20) which was significantly greater than in age- and weight-matched control subjects (1.05 +/- 0.22 U/ml, n = 9). In contrast, somatomedin activity was not raised in patients who had no retinopathy (1.16 +/- 0.33 U/ml, n = 10). Diabetic patients with retinopathy also showed the greatest differences between repeat samples suggesting wider fluctuations in plasma somatomedin levels. Plasma growth hormone and glucose measured either simultaneously with somatomedin or during a 12-24 h profile were not different when diabetics with or without retinopathy were compared. The somatomedins have mitogenic actions in vivo on a variety of connective tissues. The possibility that this may extend to retinal vessels should be further evaluated.