Effect of alendronate or 8-prenylnaringenin applied as a single therapy or in combination with vibration on muscle structure and bone healing in ovariectomized rats.

Bisphosphonate alendronate (ALN), phytoestrogen 8-prenylnaringenin (8-PN) and the whole body vibration exert a favorable effect on osteoporotic bone. However, the impact of these treatments and the combination of pharmacological therapies with biomechanical stimulation on muscle and bone has not yet been explored in detail. The effect of ALN and 8-PN and their combination with the vibration (Vib) on skeletal muscle and bone healing was investigated in ovariectomized (Ovx) rats. Three-month old rats were Ovx (n = 78), or left intact (Non-Ovx; n = 12). Five weeks after Ovx, all rats were treated according to the group assignment (n = 12/13): 1) Non-Ovx; 2) Ovx; 3) Ovx + Vib; 4) Ovx + ALN; 5) Ovx + ALN + Vib; 6): Ovx + 8-PN; 7) Ovx + 8-PN + Vib. Treatments with ALN (0.58 mg/kg BW, in food), 8-PN (1.77 mg/kg BW, daily s.c. injections) and/or with vertical vibration (0.5 mm, 35 Hz, 1 g, 15 min, 2×/day, 5×/week) were conducted for ten weeks. Nine weeks after Ovx, all rats underwent bilateral tibia osteotomy with plate osteosynthesis and were sacrificed six weeks later. Vibration increased fiber size and capillary density in muscle, enlarged callus area and width, and decreased callus density in tibia, and elevated alkaline phosphatase in serum. ALN and ALN + Vib enhanced capillarization and lactate dehydrogenase activity in muscle. In tibia, ALN slowed bone healing, ALN + Vib increased callus width and density, enhanced callus formation rate and expression of osteogenic genes. 8-PN and 8-PN + Vib decreased fiber size and increased capillary density in muscle; callus density and cortical width were reduced in tibia. Vibration worsened 8-PN effect on bone healing decreasing the callus width and area. Our data suggest that Vib, ALN, 8-PN, or 8-PN + Vib do not appear to aid bone healing. ALN + Vib improved bone healing; however application is questionable since single treatments impaired bone healing. Muscle responds to the anti-osteoporosis treatments and should be included in the evaluation of the drugs.

Functional roles and efficiencies of the thioredoxin boxes of calcium-binding proteins 1 and 2 in protein folding.

The rat luminal endoplasmic-recticulum calcium-binding proteins 1 and 2 (CaBP1 and CaBP2 respectively) are members of the protein disulphide-isomerase (PDI) family. They contain two and three thioredoxin boxes (Cys-Gly-His-Cys) respectively and, like PDI, may be involved in the folding of nascent proteins. We demonstrate here that CaBP1, similar to PDI and CaBP2, can complement the lethal phenotype of the disrupted Saccharomyces cerevisiae PDI gene, provided that the natural C-terminal Lys-Asp-Glu-Leu sequence is replaced by His-Asp-Glu-Leu. Both the in vitro RNase AIII-re-activation assays and in vivo pro-(carboxypeptidase Y) processing assays using CaBP1 and CaBP2 thioredoxin (trx)-box mutants revealed that, whereas the three trx boxes in CaBP2 seem to be functionally equivalent, the first trx box of CaBP1 is significantly more active than the second trx box. Furthermore, only about 65% re-activation of denatured reduced RNase AIII could be obtained with CaBP1 or CaBP2 compared with PDI, and the yield of PDI-catalysed reactions was significantly reduced in the presence of either CaBP1 or CaBP2. In contrast with PDI, neither CaBP1 nor CaBP2 could catalyse the renaturation of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a redox-independent process, and neither protein had any effect on the PDI-catalysed refolding of GAPDH. Furthermore, although PDI can bind peptides via its b' domain, a property it shares with PDIp, the pancreas-specific PDI homologue, and although PDI can bind malfolded proteins such as 'scrambled' ribonuclease, no such interactions could be detected for CaBP2. We conclude that: (1) both CaBP2 and CaBP1 lack peptide-binding activity for GAPDH attributed to the C-terminal region of the a' domain of PDI; (2) CaBP2 lacks the general peptide-binding activity attributed to the b' domain of PDI; (3) interaction of CaBP2 with substrate (RNase AIII) is different from that of PDI and substrate; and (4) both CaBP2 and CaBP1 may promote oxidative folding by different kinetic pathways.