Effects of chronic prednisolone treatment on bone resorption and bone composition in intact and ovariectomized rats and in ovariectomized rats receiving beta-estradiol

To examine the interactions between estrogen deficiency and glucocorticoid excess on bone metabolism the osteopenic effects of a standard dose of prednisolone (2 mg/kg BW.day) were studied in sham-ovariectomized (Sham-OVX), ovariectomized (OVX), and OVX rats given replacement beta-estradiol (OVX + E2). For 12 weeks six groups of female albino rats aged 4 months which had their skeletons labeled with 45Ca were fed matched amounts of low-calcium (0.1% Ca) hydroxyproline-free diet. The six treatment groups were: group 1, Sham-OVX; group 2, Sham-OVX + prednisolone; group 3, OVX; group 4, OVX + prednisolone; group 5, OVX + E2; group 6, OVX + E2 + prednisolone. Bone resorption was estimated by studying the urinary excretion of hydroxyproline and 45Ca. Parathyroid function was assessed indirectly from urinary cAMP excretion. Treatments did not influence parathyroid activity or serum levels of calcium or 1,25-dihydroxyvitamin D. However, ovariectomy increased bone resorption and induced osteopenia whereas prednisolone decreased bone resorption and formation and caused osteopenia. Ovariectomy increased the rate of bone resorption in prednisolone-treated rats; prednisolone lowered the rates of bone resorption and formation in OVX rats. The osteopenic effects of prednisolone and ovariectomy were additive and independent. E2 protected bone from the osteopenic effects of ovariectomy but did not affect bone loss induced by prednisolone. These results suggest prophylactic estrogen should help to avoid bone loss from estrogen deficiency in patients requiring chronic high dose glucocorticoid treatment.     

Estrogen treatment prevents osteopenia and depresses bone turnover in ovariectomized rats

Female Sprague-Dawley rats were subjected to bilateral ovariectomy (OVX) or sham surgery (control). Groups of ovariectomized (OVX) and control rats were injected daily with low, medium, or high doses of 17 beta-estradiol (10, 25, or 50 micrograms/kg BW, respectively). An additional group of OVX and control rats was injected daily with vehicle alone. All rats were killed 35 days after OVX, and their proximal tibiae were processed undecalcified for quantitative bone histomorphometry. Trabecular bone volume was markedly reduced in vehicle-treated OVX rats relative to that in control rats (12.1% vs. 26.7%). This bone loss was associated with a 2-fold increase in osteoclast surface and a 4-fold increase in osteoblast surface. The bone formation rate, studied with fluorochrome labeling, was also significantly elevated in vehicle-treated OVX rats (0.111 vs. 0.026 micron3/micron2.day). In contrast, treatment of OVX rats with the three doses of estradiol resulted in normalization of tibial trabecular bone volume and a decline in histomorphometric indices of bone resorption and formation. Our results indicate that estrogen treatment provides complete protection against osteopenia in OVX rats. The protective mechanism involves estrogenic suppression of bone turnover. These findings are consistent with the skeletal effects of estrogen therapy in postmenopausal women.     

去卵巢大鼠骨髓源性破骨样细胞增殖、分化的改变及雌激素的影响

目的　通过体外骨髓细胞培养诱导破骨样细胞 (osteoclast likecells ,OLC)的形成 ,观察去卵巢对成年大鼠OLC形成及活性的影响以及给予雌激素后的改变。　方法　 3月龄SD大鼠分为对照组、去卵巢组及雌激素替代组。术后 12周处死大鼠 ,取股骨分离骨髓细胞 ,在条件培养液中诱导其向破骨细胞分化。活体观察OLC形成情况并于培养的第 6天行细胞染色 ,以抗酒石酸酸性磷酸酶(TRAP)染色 (+)、细胞核≥ 3个的细胞为OLC ,计数各组OLC及骨陷窝。　结果　 3组中去卵巢组OLC出现早且数量〔(2 7 75± 0 92 )个 /玻片〕明显高于其它两组〔(17 93± 0 6 9)个 /玻片和 (12 81±0 6 1)个 /玻片 ,P <0 0 1〕。雌激素处理能明显抑制OLC的形成 ,但雌激素替代组的OLC仍多于对照组 (P <0 0 5 )。骨陷窝形成的变化与OLC数量改变一致。　结论　本实验结果显示大鼠去卵巢后 ,骨髓干细胞分化形成OLC数量明显增多 ,且活性增强。补充雌激素能够有效抑制OLC形成增加及其活性增强。故雌激素抑制骨吸收的机制至少部分是作用于骨髓干细胞向破骨细胞的分化。     

Moderately High Consumption of Ethanol Suppresses Bone Resorption in Ovariectomized but not in Sexually Intact Adult Female Rats

Epidemiological studies suggest that moderate consumption of alcoholic beverages may be beneficial for bone in postmenopausal women. To investigate prospectively these uncontrolled obsewations, female rats were divided in four groups of 10 animals each and treated with 1) ovariectomy (OVX) and 2.5% ethanol diet (OVX-ETOH group), 2) OVX and control diet (OVX-C group), 3) sham surgery and 25% ethanol diet (SHAM-ETOH group), or 3) sham surgery and control diet (SHAM-C group). Three weeks after surgery, bone histomor-phometfy revealed that the OVX-C group, as expected, had lower trabecular bone volume and higher parameters of bone formation and resorption than the SHAM-C group (p < 0.01). Intake of ethanol did not change these parameters in the SHAM rats, but in the OVX rats it was associated with sharp reduction in parameters of bone resorption (p < 0.01) without a concomitant effect on parameters of bone formation. The cytokines are believed to contribute to accelerated bone resorption during the early postmenopausal period. Indeed, the peripheral blood monocybc cells (PBMC) from the OVX-C rats produced higher amounts of TNF-α than the PBMC from the SHAM-C rats (p < 0.05) and administration of ethanol prevented this increase in OVX rats but had no effect in SHAM rats. In summary, short-tetm intake of moderate doses of ethanol was associated with markedly different eftects in rats with and without ovarian function. Although ethanol had no significant effect on the bone tissue and TNF-α production of the SHAM rats, it was associated with markedly lower parameters of bone resorption and less TNF-α production in the OVX animals. This suggests that exposure to low-dose ethanol may protect from osteopenia following cessation of ovarian function.     

Effects of dietary sodium chloride loading on parathyroid function, 1,25-dihydroxyvitamin D, calcium balance, and bone metabolism in female rats during chronic prednisolone administration

The effects of dietary sodium chloride supplements (8 g/100 g diet) on parathyroid function, serum 1,25-dihydroxyvitamin D [1,25-(OH)2D], calcium balance, bone metabolism, and bone composition were studied in rats treated with prednisolone (2 mg/kg w X day) for 12 weeks. Animals on a low calcium diet (0.1% Ca) received the following treatments: group 1, control; group 2, NaCl; group 3, prednisolone; group 4, NaCl plus prednisolone. Parathyroid function was assessed indirectly from urinary cAMP excretion: bone resorption was estimated by studying urinary hydroxyproline excretion and mobilization of 45Ca from bone. Dietary salt loading increased the urinary excretion of calcium, 45Ca, cAMP, and hydroxyproline and raised serum 1,25-(OH)2D and net calcium absorption, but lowered calcium retention, femoral calcium, and total body calcium. Prednisolone slowed body growth and lowered net calcium absorption, calcium retention, femoral calcium, and total body calcium. Urinary calcium excretion was higher in rats receiving salt and prednisolone in combination than in animals taking salt without prednisolone, but other responses to salt and prednisolone were independent. Thus, salt and prednisolone each elicit osteopenia, and salt causes bone loss in rats receiving prednisolone. The osteopenic effect of salt is attributed to primary augmentation of urinary calcium excretion and secondary increases in PTH-medicated bone resorption. Although salt-treated rats have higher blood levels of 1,25-(OH)2D, bone loss occurs because alimentary calcium absorption is not elevated sufficiently to offset urinary calcium losses. Prednisolone lowers bone formation and net calcium absorption without lowering serum 1,25-(OH)2D values. The parathyroid-vitamin D axis remains intact in prednisolone-treated rats, as they show increases in PTH and 1,25-(OH)2D after salt treatment.     

Calcitonin versus Clodronate in the Prevention of Ovariectomy-Induced Osteopenia in Rats

The effects of salmon calcitonin and clodronate were compared in ovariectomised rats. Sixty female Wistar rats (∼260 g in weight) were fed the same diet and had the same living conditions. The rats were divided into the following groups: 15 rats with sham ovariectomy and no drug treatment (Sham-OVX); 45 rats with bilateral ovariectomy subdivided into 15 rats not receiving drug treatment (OVX group), 15 rats treated with subcutaneous salmon calcitonin, 2 U/kg/day every 2 days (OVX + CT group) and 15 rats treated with subcutaneous clodronate, 5 mg/kg/day every 2 days (OVX + Cl group). Sixty days after surgery, the rats were sacrificed and their femurs and fifth lumbar vertebrae were dissected and cleaned of soft tissue. Femur length, vertebral height, and bone mineral content and bone mineral density of the femur and fifth lumbar vertebra by dual-energy X-ray absorptiometry were measured. Calcitonin had a significant and stronger effect in preventing ovariectomy-induced osteopenia in the femur (OVX + CT vs OVX groups, p<0.0001); both calcitonin and clodronate had a significant effect on the fifth lumbar vertebra, which was greater in the calcitonin group (OVX + CT vs OVX + Cl groups, p<0.005). These findings indicate that calcitonin has a protective effect on both the axial (trabecular bone) and peripheral (cortical bone) skeletons, but clodronate only has a protective effect on the axial skeleton. Received: 28 May 1999 / Accepted: 29 July 1999     

Osteoblastic cells mediate osteoclastic responsiveness to parathyroid hormone

Indirect evidence suggests that cells of the osteoblastic lineage may mediate augmented osteoclastic bone resorption induced by PTH. To test this suggestion, osteoclasts were disaggregated from neonatal rat long bones and incubated on slices of human femoral cortical bone. Resorption was measured by computer-assisted morphometric and stereophotogram-metric quantification of osteoclastic excavations, identified in the scanning electron microscope after culture. We compared the effect of PTH on bone resorption by osteoclasts incubated alone with the effect of the hormone on resorption by osteoclasts cocultured with osteoblastic cells. PTH had no effect on bone resorption by osteoclasts alone, but in the presence of any of three osteoblast-containing cell populations, or in the presence of cloned, hormone-responsive osteosarcoma cells, PTH caused a 2- to 4-fold increase in osteoclastic resorption. Significant stimulation was observed at 10(-4) IU/ml PTH. None of the osteoblastic cell populations caused morphologically detectable bone resorption in the absence of osteoclasts. These results indicate that PTH acts primarily on osteoblasts, which are induced by the presence of the hormone to stimulate osteoclastic bone resorption.     

Assessment of changes due to the long-term effect of estrogen and calcium deficiency in the trabecular bone structure in rats

OBJECTIVE: To assess structural changes, especially structural anisotropy, of rat bone trabecular system 6 months after ovariectomy followed by low-calcium diet. METHOD: The study was carried out on the group of 32 female rats, half of which were ovariectomized at the age of 75 days. The animals were divided into 4 groups: one receiving a normal diet (N), another receiving a low-calcium diet (LCa), ovariectomized rats receiving a normal diet (OVX), and ovariectomized animals receiving a low-calcium diet (OVX+LCa). After 6 months the animals were killed, bone specimens were collected and cut into sections of 6 microm thickness. Digital images of the sections were analyzed using a software package enabling analysis of the transversal and longitudinal trabeculae. RESULTS: Significant changes in trabecular structure due to a low-calcium diet (trabecular bone volume loss of 19%), ovariectomy (53%) and ovariectomy combined with low-calcium diet (71%) were observed. In all the analyzed groups, the percentage loss (as compared with controls) of transversal trabeculae was more significant than the loss of longitudinal trabeculae. In the LCa group, transversal trabecular loss was 39%, longitudinal 25%, in (Ovx): 63% and 54%, respectively, and in OVX + LCa: 77% and 72%. The structural anisotropy coefficient, defined as the ratio of transversal to longitudinal trabecular surface area was 0.64 for (N), 0.50 for LCa, 0.49 for OVX, and 0.54 for OVX+LCa groups. CONCLUSIONS: The effect of ovariectomy and low-calcium diet on trabecular structure can be assessed quantitatively by means of analysis of transversal and longitudinal trabeculae associated with the main direction of strain. The degree of transversal trabecular loss is much higher than the longitudinal trabecular loss; the difference becomes smaller with the progress of bone destruction, being greatest in the LCa group, the smallest in the (OVX+ LCa) group.     

Increased plasma prolactin levels in ovariectomized thyroidectomized rats treated with estrogen

It is well established that TRH exerts a stimulatory effect on the secretion of both TSH and PRL. Clinically, hyperprolactinemia is usually present in hypothyroid women, but not men. In experimental studies, results vary because of the sexes, and treatments of animals differ. The purpose of this study was to further investigate the physiological control of PRL secretion in hypothyroid female rats. Adult female Sprague-Dawley rats that were surgically ovariectomized (OVX) and/or thyroidectomized (Tx) for 2 weeks were used. Serial blood samples were collected through indwelling intraatrial catheters, and plasma PRL and TSH levels were measured by RIA. We found that OVX + Tx and polyestradiol phosphate (PEP; 0.1 mg/rat, sc)-treated rats exhibited significantly higher basal PRL and TSH levels and afternoon surge PRL levels than sham Tx rats with the same treatments. On the other hand, if OVX + Tx rats were not treated with estrogen, their plasma PRL levels were not significantly different from those in sham Tx controls. If challenged with TRH (1 microgram/rat, iv), significantly higher PRL responses were found in OVX + Tx + PEP rats than in sham Tx rats. The contents of TRH in the median eminence of Tx rats, however, were not different from those in sham Tx rats. When challenged with domperidone (10 micrograms/rat, iv), a dopamine antagonist, no difference in PRL increments was found in the two groups of animals. Treatment with CB154, a potent dopamine agonist, did not eliminate the difference in basal PRL levels between the two groups. Pretreatment with a smaller dose of domperidone (1 microgram/rat), however, enhanced the PRL-releasing effect of TRH more in Tx than in sham Tx rats. When T4 (2 or 10 micrograms/100 g BW.day for 21 days) was replaced in Tx rats starting the second day after Tx, both basal and TRH-stimulated PRL secretion were significantly decreased in a dose-dependent manner. In conclusion, the increased PRL levels in OVX + Tx + PEP rats may be due to increased responsiveness of the anterior pituitary gland to TRH, and not to a decreased responsiveness to dopamine. In addition, the elevation of plasma PRL in OVX + Tx + PEP rats is negatively correlated with plasma levels of thyroid hormone.     

Human parathyroid hormone-(1-34) increases bone mass in ovariectomized and orchidectomized rats

In intact growing rats, intermittent administration of low doses of PTH increases bone mass. As gonadal hormones are considered to be essential for normal bone growth, the anabolic effect of PTH may be mediated or modified by these hormones. The objective of this research was to determine if the anabolic effect of PTH would be altered in female ovariectomized (OVX) and male orchidectomized (ORCHX) rats. Two weeks after ovariectomy, orchidectomy, or sham operations, 5-week-old rats (eight per group) were given daily sc injections of human PTH (1-34) (8 micrograms/100 g) or vehicle. After 12 days of treatment, all rats were killed; castration was confirmed, and sera, femurs, tibias, and kidneys were collected. Calcium (Ca) and dry weight (DW) of trabecular and cortical bone of distal half-femurs were measured. Female OVX rats were osteopenic compared to their sham-operated controls, as the bone mass of distal femurs decreased while body weight increased. In PTH-treated females, total bone Ca and DW per 100 g BW increased significantly by 16% and 21%, respectively, in sham-operated rats and by 21% and 25%, respectively, in OVX rats compared to the appropriate control values. ORCHX rats were also osteopenic, as the bone mass of distal femurs was significantly decreased compared to that in sham-operated males. However, as body weight also decreased, the bone mass per unit BW was not altered. In PTH-treated males, total bone Ca and DW per 100 g BW increased significantly by 34% and 25%, respectively, in sham-operated rats by 32% and 29%, respectively, in ORCHX rats compared to their appropriate control values. Serum Ca, creatinine, and alkaline phosphatase levels were normal and comparable in all rats. We conclude that PTH increased bone mass in control, OVX, and ORCHX rats, and the anabolic response to PTH is not dependent on gonadal hormones.     

Blueberry consumption prevents loss of collagen in bone matrix and inhibits senescence pathways in osteoblastic cells

Ovariectomy (OVX)-induced bone loss has been linked to increased bone turnover and higher bone matrix collagen degradation as the result of osteoclast activation. However, the role of degraded collagen matrix in the fate of resident bone-forming cells is unclear. In this report, we show that OVX-induced bone loss is associated with profound decreases in collagen 1 and Sirt1. This was accompanied by increases in expression and activity of the senescence marker collagenase and expression of p16/p21 in bone. Feeding a diet supplemented with blueberries (BB) to pre-pubertal rats throughout development or only prior to puberty [postnatal day 21 (PND21) to PND34] prevents OVX-induced effects on expression of these molecules at PND68. In order to provide more evidence and gain a better understanding on the association between bone collagen matrix and resident bone cell fate, in vitro studies on the cellular senescence pathway using primary calvarial cells and three cell lines (ST2 cells, OB6, and MLO-Y4) were conducted. We found that senescence was inhibited by collagen in a dose–response manner. Treatment of cells with serum from OVX rats accelerated osteoblastic cell senescence pathways, but serum from BB-fed OVX rats had no effect. In the presence of low collagen or treatment with OVX rat serum, ST2 cells exhibited higher potential to differentiate into adipocytes. Finally, we demonstrated that bone cell senescence is associated with decreased Sirt1 expression and activated p53, p16, and p21. These results suggest that (1) a significant prevention of OVX-induced bone cell senescence from adult rats can occur after only 14 days consumption of a BB-containing diet immediately prior to puberty, and (2) the molecular mechanisms underlying this effect involves, at least in part, prevention of collagen degradation.     

Calcium metabolism of intact and thyroparathyroidectomized rats fed a bicarbonate enriched diet

Rats fed a bicarbonate-enriched diet become alkalotic during their feeding period (ca. 10 h/day). Alkalosis produced a significant reduction in the plasmatic concentration of ionized calcium and increased parathyroid hormone secretion confirmed by concurrent hypophosphatemia and increased urinary excretion of cAMP. Three weeks of treatment produced, however, a significant reduction of bone Ca resorption. The increased endogenous fecal excretion of calcium counteracted the enhancement of true calcium absorption. No significant change in the skeletal calcium mass was observed. Thyroparathyroidectomized rats fed the alkaline diet reduced further their rate of bone Ca resorption and increased significantly their skeletal calcium mass.     

Melatonin effect on bone metabolism in rats treated with methylprednisolone

The present study was undertaken to examine the effect of melatonin (25 microg/mL of drinking water, about 500 microg/day) on a 10-wk long treatment of male rats with methylprednisolone (5 mg/kg s.c., 5 days/wk). Bone densitometry and mechanical properties, calcemia, phosphatemia and serum bone alkaline phosphatase activity and C-telopeptide fragments of collagen type I (CTX) were measured. Both melatonin and methylprednisolone decreased significantly body weight (BW) and the combination of both treatments resulted in the lowest BW values found. Consequently, all results were analyzed with BW as a covariate. Densitometrically, methylprednisolone augmented bone mineral content (BMC), bone area (BA) and bone mineral density (BMD) in the entire skeleton, BMC in cortical bone, and BMC and BMD in trabecular bone. Melatonin increased BMC and BA in whole skeleton and BMC and BMD in trabecular bone. For BMC and BA of whole skeleton, BMC of cortical bone, and BMC and BMD of trabecular bone, the combination of glucocorticoids and melatonin resulted in the highest values observed. Femoral weight of rats receiving methylprednisolone or melatonin increased significantly and both treatments summated to achieve the greatest effect. In femoral biomechanical testing, methylprednisolone augmented ultimate load and work to failure significantly. Rats receiving the combined treatment of methylprednisolone and melatonin showed the highest values of work to failure. The circulating levels of CTX, an index of bone resorption, decreased after methylprednisolone or melatonin, both treatments summating to achieve the lowest CTX values found. Serum calcium increased after methylprednisolone and serum phosphorus decreased after treatment with methylprednisolone or melatonin while serum bone alkaline phosphatase levels remained unchanged. The results are compatible with the view that low doses of methylprednisolone or melatonin decrease bone resorption and have a bone-protecting effect.     

仙灵骨葆对去势大鼠骨量和生物力学性能的影响

目的探讨仙灵骨葆对骨质疏松(OP)大鼠骨量、骨代谢和生物力学性能的影响。方法 3月龄雌性SD大鼠24只分为3组,每组8只:正常对照组(N)、卵巢切除组(OVX)、卵巢切除+仙灵骨葆治疗组(XLGB)。除N组外,其余两组行卵巢切除术,6 w后XLGB组给予药物干预:250 mg.kg-1.d-1,OVX组给予等量生理盐水,8 w后处死所有大鼠。留取尿液、血清检测血PINP值、尿DPYD/Cr、NTX/Cr值。取左侧股骨行骨密度测定,取左侧胫骨制备硬组织不脱钙切片,备行骨组织形态计量学检测,取右侧股骨行三点弯曲试验,检测其最大载荷。结果 OVX组血PINP、尿DPYD/Cr、尿NTX/Cr值显著高于N组,XLGB能显著降低血PINP、尿DPYD/Cr、尿NTX/Cr值,但仍显著高于N组。OVX组股骨全长及近、中、远三段骨密度均显著低于N组,XLGB组近、远端骨密度显著高于OVX组。BV/TV在OVX组显著低于N组,XLGB组显著高于OVX组;OVX、XLGB组骨吸收指标Oc.N、Er.Pm均显著高于N组,XLGB组Oc.N、Er.Pm显著低于OVX组,BFR/BV显著高于OVX组。最大载荷三组之间无显著差别。结论仙灵骨葆灌胃可抑制卵巢切除大鼠骨量丢失,其机制与促进骨形成、抑制骨吸收,降低骨转换水平,进而维持骨量及微观结构有关。     

Salmon calcitonin prevents cyclosporin-A-induced high turnover bone loss

Cyclosporin-A (CsA) has greatly influenced the outcome of organ transplantation and has also been effective in the treatment of many autoimmune diseases. Unfortunately, it has deleterious effects on bone remodelling, causing a high turnover bone loss, with bone resorption exceeding bone formation. Salmon calcitonin (SCtn) has been shown to inhibit bone resorption in high turnover states such as Paget's disease and postmenopausal osteoporosis. In an attempt to attenuate the high turnover bone remodelling caused by CsA alone, we studied the bone mineral effects of CsA in combination with SCtn in male Sprague-Dawley rats. Group A (n = 20) received vehicle as control, group B (n = 20) received CsA (15 mg/kg BW) by daily gavage and SCtn vehicle sc, group C (n = 20) received SCtn (1.3 IU/kg BW) daily sc and CsA vehicle, and group D (n = 20) received a combination of CsA and Ctn daily, as described above. Rats were bled weekly for determination of circulating biochemical bone parameters. Eight rats from each group were killed on day 14 (short term), and the remaining rats were killed on day 28 (long term). Tibiae were removed for bone histomorphometry after death, which revealed a reduction of trabecular bone volume and an increase in osteoclast number induced by CsA alone. These changes were significantly attenuated by the combination of CsA and SCtn to resemble the histomorphometry of the control group. The inhibition of osteoclast number by SCtn is the most plausible mechanism by which the combination therapy attenuates the high turnover bone loss induced by CsA alone.     

Bone effects of hexarelin,a GH-releasing peptide,in female rats: influence of estrogen milieu

OBJECTIVE: The present study was performed to evaluate the potential influence of the estrogen milieu in modulating the effects of GH/IGF stimulation by a GH-releasing peptide, hexarelin (HEXA), on bone metabolism and mineral density in middle-aged female rats. METHODS: HEXA was administered for 60 days (50 microg/kg s.c. twice a day) to intact and ovariectomized (OVX) 11-month-old female rats and changes in bone parameters were evaluated with respect to those of the same rats under baseline conditions and with those of control rats (intact and OVX) administered isovolumetric amounts of physiological saline. Serum total alkaline phosphatase (ALP) and urinary deoxypyridinoline (Dpd) were measured before and at various times during HEXA treatment. Bone mineral content (BMC) and density of lumbar vertebrae and femoral mid-diaphyses were measured by dual energy X-ray absorptiometry before and after treatment. In all groups, serum IGF-I levels were determined before and during treatment and the GH secretory response to HEXA was assessed at the end of the experiment. RESULTS: In intact rats, HEXA did not modify Dpd urinary excretion, induced a trend toward an increase of serum ALP activity and significantly increased BMC (+6.5%) and bone area (+4.1%) only at lumbar vertebrae. In OVX rats, HEXA did not modify the OVX-induced increase in bone turnover markers (Dpd and ALP) and did not affect the OVX-induced vertebral bone loss, but significantly increased BMC (+7.2%) and bone area (+5.3%) at femoral mid-diaphyses. HEXA significantly increased serum IGF-I levels at day 14, but not at day 60, in both intact and OVX rats, whereas the GH secretory response to HEXA was higher in the former than in the latter. CONCLUSIONS: Overall, the present data demonstrate that chronic HEXA treatment increases BMC and bone area at lumbar vertebrae in intact rats and at femoral diaphyses in OVX rats. The different sensitivity to HEXA of the skeletal districts examined is related to the estrogen milieu and may reflect a complex interplay between estrogens and GH/IGF function.     

The effects of gonadal steroids on the content of substance P in the rat anterior pituitary

The effects of gonadectomy and of the administration of gonadal steroids on the content of substance P in the anterior pituitary (AP-SP) were studied in adult rats. The effect of gonadal status on the AP-SP content of thyroidectomized (TX) rats was also studied. We have confirmed that the AP-SP content in adult males is higher than that in adult females. Ovariectomy (OVX) caused an increase in AP-SP content which was apparent 6 days after surgery. Estradiol (E2; 2 micrograms/rat daily) administered for 13 days beginning the day after OVX prevented the increase in AP-SP content induced by OVX. Orchiectomy of adult rats had no effect on AP-SP content 14 and 45 days after surgery. E2 administered to adult female rats for 13 days caused a reduction in the AP-SP content, whereas dihydrotestosterone (0.2 mg/rat daily for 13 days) caused an increase that was even more pronounced in TX rats. E2 administration to TX adult female rats caused a significant decrease in the AP-SP content both when treatment was begun on the day after surgery or 2 weeks later. Administration of T4 (1.5 and 25 micrograms/100 g BW daily for 7 days) to rats made hypothyroid by thyroidectomy 2 weeks earlier abolished the increase in AP-SP content seen in TX animals. Neither E2 nor dihydrotestosterone had an effect on the substance P content of any of the brain regions examined. The AP-SP content of pregnant or lactating rats was not different from that of age-matched controls. The content of substance P in the AP and median eminence did not vary significantly throughout the estrous cycle. The data indicate that AP-SP content is dependent on the gonadal status of the animal and that gonadal steroids interact with thyroid hormones in the regulation of substance P turnover in the AP.     

Ovariectomy augments hypertension in aging female Dahl salt-sensitive rats

The ovariectomized (OVX) Dahl salt-sensitive (DS) rat fed a low-salt diet is a model of postmenopausal hypertension. In addition to estrogen loss, aging can also contribute to postmenopausal hypertension. We hypothesized that: (1) female DS rats on a low-salt diet become hypertensive with age; (2) ovariectomy accelerates age-dependent hypertension in the DS rat caused by estrogen depletion; and (3) this hypertension correlates with increased type 1 angiotensin receptor (AT1R) number (Bmax). Blood pressure was monitored by telemetry from 3 to 12 months and AT1R Bmax was determined by Scatchard analysis in glomeruli and adrenal cortex. Three groups of DS rats were studied: intact, OVX, and 17beta-estradiol-replaced OVX (OVX+E). In intact rats, aging to 12 months resulted in hypertension (159+/-6 mm Hg) and an 82% decrease in estrogen. Blood pressure in OVX was significantly higher than OVX+E through 12 months of age (173+/-4 versus 150+/-8 mm Hg). At 4 months, OVX increased AT1R Bmax compared with intact and OVX+E in both glomeruli and adrenal cortex. Aging also increased AT1R Bmax in these tissues in intact rats. In summary, female DS rats fed a low-salt diet have hypertension develop with age, that is accelerated by OVX and attenuated by estrogen replacement. Concurrently, AT1Rs are upregulated by age and OVX, which is prevented by estrogen replacement. This study suggests that an increased activity of the renin angiotensin system contributes to the development of hypertension, and estrogen protects against this process.     

Increased dietary calcium lowers blood pressure in the spontaneously hypertensive rat

The effect of increased dietary calcium on the development of hypertension in spontaneously hypertensive (SH) rats was investigated by feeding lab chow fortified with calcium carbonate (2.5% calcium, hCa) beginning at 4 wk of age. A control SH group was fed regular lab chow (1.2% calcium, rCa). Two groups of age-matched Wistar-Kyoto (WKY) rats were treated in parallel. Systolic blood pressure (BP) was measured weekly until the age of 18 wk using a tail cuff method. The hCa diet significantly attenuated the time course of hypertension in SH rats even though both SH groups eventually developed hypertension. The hCa also lowered BP in WKYs, but to a lesser extent. Urine output (24-hr volumes) was not affected by hCa, but in both SH and WKY groups fed the hCa diet, the excretion of Na+, K+ and Ca++ was markedly elevated at 11, 15, and 19 wk of age. Urine osmolality was also elevated. Plasma Na+, Ca++ and osmolality were not significantly altered by the diet in either SH or WKY rats; plasma potassium was significantly lower in the SH group fed the hCa diet than in the group given rCa. The hCa diet did not significantly affect the body or heart, kidney, adrenal, or thymus weights. The results suggest that hCa diet may attenuate genetic hypertension by inducing an osmotic diuresis.     

Osteoprotegerin inhibits artery calcification induced by warfarin and by vitamin D

The present experiments were carried out to test the hypothesis that arterial calcification is linked to bone resorption by determining whether the selective inhibition of bone resorption with osteoprotegerin will inhibit arterial calcification. In the first test, arterial calcification was induced by treating 22-day-old male rats with warfarin, a procedure that inhibits the gamma-carboxylation of matrix Gla protein and causes extensive calcification of the arterial media. Compared with rats treated for 1 week with warfarin alone, rats treated with warfarin plus osteoprotegerin at a dose of 1 mg/kg per day had dramatically reduced alizarin red staining for calcification in the aorta and in the carotid, hepatic, mesenteric, renal, and femoral arteries, and they had 90% lower levels of calcium and phosphate in the abdominal aorta (P<0.001) and in tracheal ring cartilage (P<0.01). More rapid arterial calcification was induced by treating 49-day-old male rats with toxic doses of vitamin D. Treatment for 96 hours with vitamin D caused widespread alizarin red staining for calcification in the aorta and the femoral, mesenteric, hepatic, renal, and carotid arteries, and osteoprotegerin completely prevented calcification in each of these arteries and reduced the levels of calcium and phosphate in the abdominal aorta to control levels (P<0.001). Treatment with vitamin D also caused extensive calcification in the lungs, trachea, kidneys, stomach, and small intestine, and treatment with osteoprotegerin reduced or prevented calcification in each of these sites. Measurement of serum levels of cross-linked N-teleopeptides showed that osteoprotegerin dramatically reduced bone resorption activity in each of these experiments (P<0.001). Therefore, we conclude that doses of osteoprotegerin that inhibit bone resorption are able to potently inhibit the calcification of arteries that is induced by warfarin treatment and by vitamin D treatment. These results support the hypothesis that arterial calcification is linked to bone resorption.