STIMULATION OF CREATINE-KINASE ACTIVITY IN RAT SKELETAL TISSUE IN-VIVO AND IN-VITRO BY PROTEASE-RESISTANT VARIANTS OF PARATHYROID-HORMONE FRAGMENTS

We have reported that mid-region fragments of human parathyroid hormon e (hPTH), exemplified by hPTH-(28-48), stimulated [H-3]thymidine incor poration into DNA and increased the specific activity of the brain-typ e isoenzyme of creatine kinase (CK) in both skeletal-derived cell cult ures (ROS 17/2.8 cells) and immature rat epiphyseal cartilage and diap hyseal bone, without stimulating cyclic AMP synthesis which is a prere quisite for bone resorption. In the present study, substitution of ami no acids in hPTH-(28-48), which resulted in increased resistance to pr oteolysis, produced variants that stimulated skeletal systems at two o rders of magnitude lower concentration than the wild-type fragment. We modified hPTH-(28-48) at Leu-37 by replacement with Met, Thr or Val. Under conditions in which 20 % of the native hPTH-(28-48) resisted pro teolysis by cathepsin D for 6 h, approx. 40% of the L37V mutant and 70 % of the L37T mutant remained intact. Substitution of Met for Phe-34 in addition to Thr for Leu-37, or the substitution of Met for Phe-34 a lone, produced 100 %-resistant fragments. These variants at residue 34 caused maximal stimulation of CK in ROS 17/2.8 cells at 0.24 nM compa red with 24 nM for hPTH-(28-48). The double mutant stimulated CK activ ity significantly in immature rats, at a minimum dose of 12.5 ng/rat, and caused maximal stimulation al 125 ng/rat, a 10-fold lower dose tha n for hPTH-(28-48). The effect of the double mutant lasted up to 24 h which differs from the stimulation by hPTH-(28-48) in which CR specifi c activity returns to the control level at 24 h. This same dose also s ignificantly stimulated CK activity in gonadectomized rats. These resu lts show the advantage of using protease-resistant mid-region variants of hPTH-(28-48) to stimulate bone cells, in terms of lower doses and longer duration of effectiveness, both in vitro and in vivo.