Bone Mass In Rats Research Articles

Anabolic effect of aminoterminally truncated fibroblast growth factor 4 (FGF4) on bone

Fibroblast growth factor 4 (FGF4), a member of the FGF family, plays several important roles in bone development during embryogenesis. Systemic administration of FGF4 increases bone mass in rats, which suggests the potential therapeutic usefulness of this growth factor in treatment for osteopenia and in bone regeneration. We investigated the length of FGF4 required to exert its anabolic effects, because this information may be useful in developing new molecules to mimic the effects of FGF4. Because the active site of FGF family molecules is in the carboxylterminal region, we produced aminoterminally truncated recombinant human FGF4s (rhFGF4s) of different sizes. Human FGF4 cDNA containing almost the full length of the coding region (573 bp, 191 amino acid residues) was inserted into pUC18 vector and then deleted from the 5′ end using the ExoIII system. Each of the deleted FGF4 cDNAs was subcloned into a pET29(+) expression vector. Differently sized recombinant proteins were expressed in the BL21(DE3)pLysS Escherichia coli strain and then purified. The growth-stimulative effects on NIH3T3 cells of each recombinant protein were examined by means of MTT colorimetric assay. Full-length and the shortened recombinant proteins, which stimulated NIH3T3 cell growth, were then subcutaneously administered into male ddY mice (6 weeks old) every day for 2 weeks. Bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT). The rhFGF4 of 134 amino acid residues, the region homologous to other members of the FGF family, exerted a growth-stimulative effect on NIH3T3 cells comparable to the full-length version of FGF4; however, the shortest version, with 111 amino acid residues, showed a limited growth-stimulative effect. Systemic administration of the rhFGF4 of 134 amino acid residues increased the bone mineral density (BMD) of femurs at a dose of 0.1 mg/kg, which was comparable to that of the full-length rhFGF4. DEXA analysis, pQCT analysis, soft X-ray photos, and contact microradiographs revealed an increase in femoral trabecular bone in FGF4-treated animals; an increase in bone formation was also evident upon histomorphometric analysis. These results indicate that the region of FGF4 that is homologous to other FGF family members provides a sufficient anabolic effect in bone and that this recombinant protein is potentially useful as a therapeutic agent in bone.

Effects of potassium bicarbonate supplementation on axial and peripheral bone mass in rats on strenuous treadmill training exercise.

We administered a potassium bicarbonate supplement to rats on strenuous treadmill training in order to determine the effect on bone mass and the metabolic acidosis seen with this type of training. A sample of 45 93-day-old female Wistar rats with a mean initial weight of 267 +/- 17 g were studied. The control group (15 rats) was not exercised or given potassium bicarbonate (Ex- PB-). The experimental group (30 rats) was randomly divided into two subgroups of 15 rats each, one that exercised and did not receive potassium bicarbonate (Ex+ PB-) and one that exercised and received potassium bicarbonate (Ex+ PB+), at a dose of 0.05 mg/kg/day administered by esophageal catheter on exercise days. Training consisted of treadmill running on 5 out of 7 days for a period of 11 weeks. Running time, treadmill speed, and the percent grade were gradually increased until week 7, then maintained until rats were sacrificed at the ened of 11 weeks. The bone mineral content (BMC) and bone mineral density (BMD) of the whole right femur and 5th lumbar vertebra were measured. Femoral and vertebral length were also measured. Femur length, weight, BMC, and BMD, and femur BMC/final weight ratio, and vertebral weight, BMD, and BMC, and vertebral BMC/final weight ratio were lower in the Ex+ PB- group than in either the controls or the Ex+ PB+ group (P < 0.01-P < 0.0001); the length of the 5th lumbar vertebra did not differ between groups.

Effects of alprazolam supplementation on vertebral and femoral bone mass in rats on strenuous treadmill training exercise.

The ability of alprazolam to diminish cortisol response and favor ovarian function could make it useful in the prevention of osteopenia in athletes in selected cases. A sample of 45 female Wistar rats, all 93 days old and with a mean initial weight of 267 +/- 17 g, were studied. Rats were exposed to a high-performance level of exercise and were divided into two groups-one group received an alprazolam supplement and one did not-and compared with controls to determine the effect of alprazolam on bone mass as measured by dual-energy X-ray absorptiometry (DKA). Exercise consisted of treadmill running on 5 out of 7 days during a period of 11 weeks. A steep grade treadmill inclination was used to stimulate high-intensity muscle activity. Final inclination was 17.5 degrees and treadmill speed was 45 cm/second. Upon completion of the experiment, all the rats were killed and the femur and 5th lumbar vertebra were dissected and cleaned. Length, weight, bone mineral content (BMC), and density (BMD) of the whole right femur and 5th lumbar vertebra were measured. In the exercise only group (no alprazolam), the length, weight, BMC, BMD, and femur BMC/final rat weight ratio of the femur, and the vertebral weight, vertebral BMD and BMC, and vertebral BMC/final rat weight ratio were lower than in the control and the exercise-alprazolam groups (P < 0.0167 - < 0.0001). Alprazolam preserves bone mass in rats exposed to intense exercise.

Effect of induced uterine retroversion on bone mass in rats

Objective: To evaluate the effect of surgical uterine retroversion on bone mass in rats. Study Design: Forty-five female Wistar rats were assigned randomly to three groups: 15 unmanipulated rats, 15 rats that underwent uterine retroversion, and 15 rats that underwent sham uterine retroversion (exposure of the uterus to air followed by closure of the abdominal cavity). Sixty days later the rats were killed and their femurs were dissected. Femurs were weighed and measured, and femoral bone mineral content (BMC) and bone mineral density (BMD) were determined by dual energy X-ray absorptiometry. Results: In the group of rats that underwent uterine retroversion, BMC, BMD, and BMC corrected for final body weight were significantly lower ( P<0.001) than in the unmanipulated control and sham uterine retroversion groups. Conclusion: Our findings indicate that uterine retroversion induced a loss of bone mass. We could not determine the mechanism of bone loss; in our opinion, these problem merits further investigations, which currently occupy our interest.

Treadmill running starting 3 months after orchidectomy restores femoral bone mass in rats.

The present study was designed to provide data on the effects on femoral bone of endurance training starting only 3 months after orchidectomy in rats. A total of 70 Wistar male rats were used at 8 weeks of age. On day 0 of the experiment, 10 rats were killed by cervical dislocation to be used as first controls. Among the 60 other animals, half was surgically castrated (CX) or sham operated (SH). On day 90, 10 CX and 10 SH were killed and used as intermediary controls (ICX and ISH). Among the other 20 CX and 20 SH, 10 within each group (CXE, SHE) were selected for treadmill running (60% maximal oxygen uptake, 1 h x day(-1), 5 days x week(-1) for 12 weeks). The 20 other rats were used as sedentary controls (CXR, SHR) and killed (as runners) on day 180. On day 90 femoral bone density (BMD) and mineral content (BMC) were lower in ICX than in ISH. On day 180 total femoral BMD was lower in CXR than in CXE. Simultaneously metaphyseal femoral BMD was lower in CXR than in CXE, SHR or SHE. Furthermore, at that time, no significant difference concerning BMD and BMC was observed between SHR and CXE. This would indicate that treadmill running starting only 3 months after orchidectomy is able to restore BMD and BMC to control values, mainly by inhibiting bone resorption (as shown by decreased urinary deoxypyridinoline excretion in CXE) without decreasing osteoblastic activity (evaluated by plasma osteocalcin concentration).

Type-1 Parathyroid Hormone (PTH)/PTH-Related Peptide (PTHrP) Receptors Activate Phospholipase C in Response to Carboxyl-Truncated Analogs of PTH(1-34)

The carboxyl(C)-truncated human (h) PTH (hPTH) analog hPTH(1–31), which activates adenylyl cyclase (AC), but not protein kinase C, in rat osteosarcoma cells, exerts an anabolic effect on rat bone in vivo similar to that of hPTH(1–34). It has been proposed, therefore, that this action of PTH(1–34) is mediated exclusively by stimulation of AC via the rat type-1 PTH/PTH-related peptide (PTHrP) receptor (PTH1R). To determine whether this selective signaling pattern also might be a property of the hPTH1R, we studied signal transduction via heterologously expressed hPTH1Rs in response to activation by hPTH(1–34), hPTH(1–31), and a C-truncated analog that does not increase rat bone mass in vivo, hPTH(1–30). In porcine LLC-PK1 cells that stably expressed recombinant hPTH1Rs, these three peptides activated AC identically (EC50 = 1–2 nm). In cells with comparable expression of rat PTH1Rs, AC activation by hPTH(1–34) and hPTH(1–31) again was identical, whereas full activation by hPTH(1–30) required higher concentrations (EC50 = 10 nmvs. 1 nm). Surprisingly, hPTH(1–31) fully stimulated phospholipase C (PLC), via both species of PTH1Rs, with potency that was similar (hPTH1Rs) or slightly reduced (rat PTH1Rs), relative to that of hPTH(1–34). hPTH(1–30), however, was 5-fold less potent than hPTH(1–34) in activating PLC via hPTH1Rs and showed weak and only partial activity via the rat PTH1R. Comparable results were obtained when human and rat PTH1Rs were transiently expressed heterologously in COS-7 cells or homologously in HEK 293 and UMR 106–01 cells, respectively. Binding affinities of these C-truncated peptides to human and rat PTH1Rs were concordant with their relative potencies in activating PLC. We conclude that hPTH(1–31) and, to a lesser extent, hPTH(1–30) can activate PLC, as well as AC, via both rat and human PTH1Rs. Accordingly, a role for PLC activation in the anabolic action of PTH in vivo cannot be excluded.

Type-1 parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptors activate phospholipase C in response to carboxyl-truncated analogs of PTH(1-34).

The carboxyl(C)-truncated human (h) PTH (hPTH) analog hPTH(1-31), which activates adenylyl cyclase (AC), but not protein kinase C, in rat osteosarcoma cells, exerts an anabolic effect on rat bone in vivo similar to that of hPTH(1-34). It has been proposed, therefore, that this action of PTH(1-34) is mediated exclusively by stimulation of AC via the rat type-1 PTH/PTH-related peptide (PTHrP) receptor (PTH1R). To determine whether this selective signaling pattern also might be a property of the hPTH1R, we studied signal transduction via heterologously expressed hPTH1Rs in response to activation by hPTH(1-34), hPTH(1-31), and a C-truncated analog that does not increase rat bone mass in vivo, hPTH(1-30). In porcine LLC-PK1 cells that stably expressed recombinant hPTH1Rs, these three peptides activated AC identically (EC50 = 1-2 nM). In cells with comparable expression of rat PTH1Rs, AC activation by hPTH(1-34) and hPTH(1-31) again was identical, whereas full activation by hPTH(1-30) required higher concentrations (EC50 = 10 nM vs. 1 nM). Surprisingly, hPTH(1-31) fully stimulated phospholipase C (PLC), via both species of PTH1Rs, with potency that was similar (hPTH1Rs) or slightly reduced (rat PTH1Rs), relative to that of hPTH(1-34). hPTH(1-30), however, was 5-fold less potent than hPTH(1-34) in activating PLC via hPTH1Rs and showed weak and only partial activity via the rat PTH1R. Comparable results were obtained when human and rat PTH1Rs were transiently expressed heterologously in COS-7 cells or homologously in HEK 293 and UMR 106-01 cells, respectively. Binding affinities of these C-truncated peptides to human and rat PTH1Rs were concordant with their relative potencies in activating PLC. We conclude that hPTH(1-31) and, to a lesser extent, hPTH(1-30) can activate PLC, as well as AC, via both rat and human PTH1Rs. Accordingly, a role for PLC activation in the anabolic action of PTH in vivo cannot be excluded.

Glucocorticoids inhibit bone resorption by isolated rat osteoclasts by enhancing apoptosis.

We have studied the effects of glucocorticoids on the activity and viability of neonatal rat osteoclasts in vitro. In the bone slice assay, glucocorticoids caused a dose-dependent decrease in the amount of bone resorbed, which was accompanied by a parallel decrease in osteoclast number. Loss of osteoclasts was due to their death, which occurred by the process of apoptosis. Evidence for the latter was obtained by a range of techniques, including time-lapse video microscopy, acridine orange staining, DNA fragment detection and transmission electron microscopy. Immunocytochemistry revealed the presence of glucocorticoid receptors in osteoclasts, and glucocorticoid-induced cell death could be prevented by the glucocorticoid receptor antagonist, RU486. These observations suggest that glucocorticoids promote receptor-mediated apoptosis of rat osteoclasts in vitro. This finding may help to explain recent data indicating that, in sharp contrast with their effects on the human skeleton, glucocorticoids inhibit bone resorption and increase bone mass in rats in vivo.

Frequency of food intake and natural dietary components are potent modulators of bone resorption and bone mass in rats

Prevention of low bone mass is important to reduce the incidence of osteoporotic fractures. In this report evidence is provided that feeding habits per se, that is, increased frequency of food intake as well as a diet containing soy and other raw components, decrease bone resorption and increase bone mass in growing rats. Interim results after 6 weeks indicate that food fractionation and natural dietary components are both capable of inhibiting trabecular bone loss in aged rats. These interim results indicate that the effect of both dietary interventions are additive and together are capable of nearly completely blunting the age-dependent loss of trabecular bone mineral density. These dietary manipulations are, however, only partially effective in inhibiting the strongly increased loss of trabecular bone mineral density induced by estrogen deprivation. The fact that the natural dietary components are not more effective in ovariectomized rats as compared to intact females confirms our contention that these components may not operate by mimicking the effect of estradiol. Whether bone mass in humans is also under the control of dietary habits is not known. If so, an increased frequency of meals of appropriate composition may be used to prevent osteoporosis.

Replacement therapy with nitric oxide or nitric oxide dornor in vivo, maintains the bone mass in rats after ovariectomy

Replacement therapy with nitric oxide or nitric oxide dornor in vivo, maintains the bone mass in rats after ovariectomy

Effects of low-dose long-term sodium fluoride preventive treatment on rat bone mass and biomechanical properties.

Effects of fluoride on bone strength and cortical bone mass remain controversial. We compared 9-month, low-dose sodium fluoride (NaF) treatment with estrogen replacement therapy. Female Wistar rats 4.5 months old were divided into baseline, sham-operated (sham), sham-treated with NaF at 0.5 mg NaF/kg/day in drinking water, and ovariectomy (OVX), OVX treated with NaF and with estrogen. Bone mass was measured by dual X-ray absorptiometry (DXA) in vitro. Dimensions of the first lumbar vertebral body (L1) were determined by radiogrammetry. The right femur was processed undecalcified to obtain a midshaft cross-section to determine cross-sectional moments of inertia (CSMIs). L1 compressive test and left femoral torsional test were performed. OVX induced significant bone loss in L1 and femoral midshaft. Bone mass was increased to a greater extent in NaF-treated rats than in rats receiving estrogen replacement therapy. Femoral CSMIs in OVX rats, both L1 sizes and femoral CSMIs in NaF-treated rats, were significantly increased. Estrogen treatment had the least dimension expansion. OVX significantly decreased L1 compressive variables. There was no statistical difference in compressive parameters between NaF-treated groups and controls. OVX significantly increased femoral torsional strength but NaF treatment did not. Bone fluoride content was significantly increased after treatment with NaF. No significant difference in bone mineralization degree (ash and calcium) was found between treated and control rats. The discrepancy that an increase in bone mass and geometric properties in both trabecular and cortical bones by low-dose, long-term NaF treatment did not increase vertebral strength nor proportionally improve femoral strength indicated that the bone intrinsic biomechanical properties could be changed by NaF treatment.

Effects of Low-Dose Long-Term Sodium Fluoride Preventive Treatment on Rat Bone Mass and Biomechanical Properties

Effects of Low-Dose Long-Term Sodium Fluoride Preventive Treatment on Rat Bone Mass and Biomechanical Properties

Histone H4-related osteogenic growth peptide (OGP): a novel circulating stimulator of osteoblastic activity.

It has been established that regenerating marrow induces an osteogenic response in distant skeletal sites and that this activity is mediated by factors released into the circulation by the healing tissue. In the present study we have characterized one of these factors, a 14 amino acid peptide named osteogenic growth peptide (OGP). Synthetic OGP, identical in structure to the native molecule, stimulates the proliferation and alkaline phosphatase activity of osteoblastic cells in vitro and increases bone mass in rats when injected in vivo. Immunoreactive OGP in high abundance is present physiologically in the serum, mainly in the form of an OGP-OGP binding protein complex. A marked increase in serum bound and unbound OGP accompanies the osteogenic phase of post-ablation marrow regeneration and associated systemic osteogenic response. Authentic OGP is identical to the C-terminus of histone H4 and shares a five residue motif with a T-cell receptor beta-chain V-region and the Bacillus subtilis outB locus. Since these latter proteins have not been implicated previously in the control of cell proliferation or differentiation, OGP may belong to a novel, heretofore unrecognized family of regulatory peptides. Perhaps more importantly, OGP appears to represent a new class of molecules involved in the systemic control of osteoblast proliferation and differentiation.

Anabolic Effect of Human Synthetic Parathyroid Hormone-(1-34) Depends on Growth Hormone*

We tested whether GH is required for the anabolic effect of PTH on bone, using sham-operated (sham) and hypophysectomized (HX) young male rats. HX rats were supplemented daily with 3% sucrose water, T4, and corticosterone. Rats received vehicle or human PTH-(1-34) at 8 micrograms/100 g, sc, once daily, alone or in combination with rat or ovine GH at 0.2 mg/100 g, sc, twice daily or 12 micrograms ovine GH/100 g.day by continuous sc infusion. After 12 days, rats were sedated, and blood, femurs, and tibias were removed. Femur trabecular and cortical bone calcium (Ca), dry weight (DW), and hydroxyproline were measured. PTH increased bone Ca, DW, and hydroxyproline in shams by approximately 30%, but consistently failed to induce an anabolic response in HX rats. GH alone stimulated systemic growth in HX rats and increased their bone Ca and DW by 2-fold. The anabolic effect of PTH was restored in HX rats given both PTH and GH. Total bone mass in these rats was approximately 20% more (P less than 0.05) than the bone mass of rats given GH alone. When food was restricted in shams to limit systemic growth, PTH still induced an increase in bone mass. We conclude that GH or GH-dependent factors, such as insulin-like growth factor-I, which increases in PTH-treated bones in vitro, are required for the anabolic response of bone to PTH in vivo.

Effects of low doses of strontium on bone quality and quantity in rats

Strontium (Sr) has been shown to increase bone mass when given at low doses. In this study, the diets of rats containing 0.50% calcium were supplemented with Sr (0.19 and 0.40% of SrCl 2 orally) for periods of four and eight weeks. Long bones and vertebrae were studied by density fractionation and each fraction was analyzed chemically. X-ray diffraction was used to determine crystal size. Static and dynamic histomorphometric parameters of bone formation were also measured. We found a shift towards lower density in the mineralization profiles of Sr supplemented rats (0.40%), as well as a decrease in bone crystal size at the larger dose of Sr. The CO 3 content and the Ca Sr ratio of the bone decreased with increasing Sr content. We found an increase in the vertebral trabecular bone volume together with an increase in osteoid volume in Sr supplemented rats. This study shows that Sr at the larger dose induces bone hypomineralization as well as an increase in bone mass in rats fed a relatively low calcium diet.

Comparison of the anabolic effects of synthetic parathyroid hormone-related protein (PTHrP) 1-34 and PTH 1-34 on bone in rats.

The objective of this study was to determine whether intermittent synthetic human PTH-related protein (PTHrP 1-34) will mimic the anabolic effect of PTH and increase bone mass in rats. Dose response experiments were done on young, male Sprague-Dawley rats given sc vehicle, human (h) PTH (1-34) at 8 micrograms/100 g or PTHrP (1-34) at 1-32 micrograms/100 g daily for 12 days or 26 days. On the last day, 3 h after injections, rats were killed and serum, femurs, and tibias harvested. Trabecular and cortical bone of distal half femurs were analyzed for calcium (Ca) and hydroxyproline content and dry weight. Tibia metaphyseal bone was analyzed using conventional histomorphometry techniques. Our results showed that low doses of PTHrP (1-34) did not increase bone mass or bone forming surfaces. After 12 days, PTH, at 8 micrograms/100 g, increased trabecular Ca, dry weight, and hydroxyproline by approximately 19%, 36%, and 53%, respectively, while the bone mass of PTHrP-treated rats was comparable to vehicle-treated rats. PTHrP at a higher dose of 32 micrograms/100 g, increased trabecular bone mass by 30-37%, compared to the 43-48% increase induced by PTH at 8 micrograms/100 g after 12 days. When treatment was extended to 26 days, PTHrP, at 16 micrograms/100 g, increased trabecular bone mass by 24-36%, respectively, compared to the 43-61% increase induced by PTH at 8 micrograms/100 g. Unlike PTH, which increased cortical bone mass by 15-25%, PTHrP increased cortical bone mass only at the highest dose tested, 32 micrograms/100 g. Bone forming surfaces but not bone apposition rate were increased by PTH and PTHrP while resorption measures remained comparable to control values. Although serum Ca and Pi remained in the physiological range for all rats, the values for PTHrP-treated rats were consistently higher. In conclusion, PTHrP (1-34) was less potent and less effective than PTH (1-34) in inducing an anabolic response in bone in vivo.

Bone mass measured by photonabsorptiometry and radiogrammetry in Wistar rats. Age and sex variations.

Using radiogrammetric and photonabsorptiometric methods, we determined changes in bone mass in Wistar rats as a function of age and sex. In the four age groups studied (10-, 18-, 30-, and 78-week-old rats), males displayed greater bone mass than females. With increasing age, bone mass also increases up to the 78th week. However, the rate of this increase was slower in the 78-week age group. Whereas the length and periosteal diameter of the bones studied were increased in old age groups, the endosteal diameter decreased in females and increased in males. The results show that, unlike human bone, rat bone mass persistently increases with aging, and female rats continue to gain bone at the endosteal site. Furthermore, these findings demonstrate that radiogrammetric morphometry and photonabsorptiometry may be used as an experimental tool to investigate bone mass changes in small animals.

Increased trabecular bone mass in rats treated with human synthetic parathyroid hormone

Although PTH inhibits bone collagen synthesis acutely, prolonged administration of PTH increases trabecular bone mass. The objectives of this study were to develop a simple method to quantitate the anabolic response of cortical and trabecular bone, to determine if the trabecular bone response is at the expense of cortical bone, and to correlate the bone response with changes in serum calcium or phosphate. Subcutaneous injections of 2 to 16 μg/100 g body weight hPTH(1–34) were given daily to weanling rats for 12 days. The trabecular and cortical bone were manually separated from the distal femur, and calcium, hydroxyproline, and extracted dry weight were measured. Growth and renal function were not impaired. Serum calcium (range: 8.9–9.5 mg%) and serum phosphate (range: 7.7–8.5 mg%) did not differ significantly from control serum calcium, 9.1 mg%, or serum phosphate, 8.1 mg%. A dose-related anabolic response was observed in calcium, hydroxyproline, and dry weight of trabecular bone. At the highest dose of hPTH for 12 days, these values were increased above control by 26%, 33%, and 26%, respectively ( P < .01). While cortical bone values increased over 12 days, only the dry weight increase was significant ( P < .01). Our method showed an increase in trabecular bone mass after daily subcutaneous injections of hPTH (1–34) at doses that were neither toxic nor hypercalcemic. Although the absolute change of cortical bone mass was greater than that of trabecular bone, it was more variable and not statistically significant. Nevertheless, these results indicate that trabecular bone mass did not increase at the expense of cortical bone.

Influence of liver enzyme induction caused by diphenylhydantoin and diazepam on bone mass of rats.

25 albino rats of Wistar strain were used for the study. They were divided into three groups. Animals of the first group (n = 10) were treated with Diphenylhydantoin in a daily dose of 70 mg/kg for three months. Animals of the second group (n = 10) were treated with Diazepam in a daily dose of 10 mg/kg for 3 months. Animals of the third group (n = 5) were treated with 5% acacia oil for the same period of time. Food and water consumption together with motor activity were measured. As indices of enzyme induction, D-Glucaric acid and liver weight were determined. Also the following bone indices were determined: femur Ca/femur weight, femur Ca/body weight, humerus Ca/humerus weight, humerus Ca/body weight, femur specific weight, humerus specific weight. Statistically significant alterations of bone mass were found.

Immobilization and bone mass in rats. Effects of parathyroidectomy and acetazolamide.

The effects of parathyroidectomy and acetazolamide on the changes in bone mass which occur following immobilization were studied in male and female rats. The forelimb was immobilized by sectioning the right brachial nerve plexus. At 10 to 14 days following denervation, both humeri were removed. The dry weight of the bones was determined and the difference between the weight of the bones from the intact and denervated, limbs was used at an index of the denervation response. Animals were either parathyroidectomired or placed on a diet containing 0.5% acetazolamide sodium at the time of denervation. Parathyroidectomy inhibited the development of the denervation response to the extent of 45%, 30%, and 85% in three experiments. Acetazolamide, incorporated as the sodium salt in laboratory chow at a concentration of 0.5%, also blocked the development of the denervation response. In three experiments, treatment with the drug for 10 to 14 days inhibited the response 42%, 47%, and 64% respectively. Under conditions of pair-feeding 0.5% acetazolamide incorporated in the diet caused a slight but significant hypercalcemia. It is concluded that parathyroidectomy and acetazolamide administration, both of which are known to inhibit bone resorption, are effective in significantly reducting the development of the bone mass changes, seen after denervation of the limb in the rat.