Gastric Fundectomy in the Rat: Effects on Mineral and Bone Metabolism, with Emphasis on the Gastrin–Calcitonin–Parathyroid Hormone–Vitamin D Axis

In humans, gastric surgery results in in osteopenia via mechanisms that are insufficiently understood; surgery-induced changes in the hormonal axes involving the stomach, thyroid, and the parathyroids may play a role. To study this in more detail, we evaluated calcium (Ca), magnesium (Mg), and phosphorus (P) metabolism as well as physical, chemical, and histomorphometric bone parameters in rats rendered hypergastrinemic by fundectomy (FX). In independent experiments, the response to an oral Ca challenge was investigated in intact rats versus FX, and in thyroidectomized versus thyroid-intact FX rats. Sixteen weeks following FX, body weight was approximately 80% that of sham-operated controls. In urine, P excretion was elevated fivefold, the pH was significantly decreased, and cAMP excretion was elevated as compared with controls; serum parathyroid hormone (PTH), calcitonin, 25OHD, Ca, Mg, and P were normal; gastrin and 1,25(OH)₂D were elevated. On the basis of bone ash mineral content, FX rats developed significant osteopenia, and histomorphometry indicated only slightly elevated bone turnover and mineralization. Following oral Ca, thyroid-intact FX rats developed hypercalcemia, serum gastrin decreased, and calcitonin increased significantly; in thyroidectomized FX rats, calcitonin remained at baseline levels although there was a similar degree of hypercalcemia; PTH decreased during the hypercalcemic period in both groups. Serum gastrin did not correlate with calcitonin or PTH, and in multivariate regression analysis the only predictor of serum 1,25(OH)₂D was urinary phosphorus. It was concluded that in the FX rat (1) osteopenia is not caused by intestinal Ca malabsorption, vitamin D, Ca deficiency, or secondary hyperparathyroidism; (2) osteopenia may be related to PTH-independent urinary hyperexcretion of P, followed by a rise of serum 1,25(OH)₂D; (3) the existence of endocrine axes among gastrin, calcitonin, and PTH cannot be substantiated. FX osteopenia appears to be related to gastric acid abolition, and the reactive hypergastrinemia probably stabilizes the mass and turnover of bone. Received: 12 August 1997 / Accepted: 26 January 1998     

Association Study of Parathyroid Hormone Gene Polymorphism and Bone Mineral Density in Japanese Postmenopausal Women

Association of BST B1 restriction fragment length polymorphism (RFLP) of the parathyroid hormone (PTH) gene with bone mineral density (BMD) was examined in 383 healthy postmenopausal women in Japan who were unrelated. The RFLP was represented as B or b, the capital letter signifying the presence of and the small letter the absence of restriction site for BST B1. The frequency of each genotype—BB, Bb, and bb—was 82.5%, 16.7%, and 0.8%, respectively. When we statistically compared age, years after menopause, body height, and body weight between the BB genotype and the Bb genotype groups, there was no significant difference between the groups. However, the lumbar BMD and the score of BMD adjusted for age and body weight (Z score) were significantly lower in the group of genotype Bb than in the BB: 0.859 ± 0.019 g/cm² versus 0.925 ± 0.011 (mean ± SE, P= 0.01) and −0.412 ± 0.138 versus 0.067 ± 0.082 (mean ± SE, P= 0.01). In addition, the Z score of total body BMD in the Bb genotype group was lower than that in the BB group. Comparison of serum and urinary biochemical bone metabolic markers suggested that the subjects with Bb genotype might be in a relatively higher state of bone turnover than those with BB genotype. These results suggest that the polymorphism in the PTH gene would be a useful genetic marker for lower BMD and the susceptibility for osteoporosis. Received: 19 March 1998 / Accepted: 24 June 1998     

Parathyroid Hormone Modulates the Response of Osteoblast-Like Cells to Mechanical Stimulation

Mechanical loading stimulates many responses in bone and osteoblasts associated with osteogenesis. Since loading and parathyroid hormone (PTH) activate similar signaling pathways in osteoblasts, we postulate that PTH can potentiate the effects of mechanical stimulation. Using an in vitro four-point bending device, we found that expression of COX-2, the inducible isoform of cyclooxygenase, was dependent on fluid forces generated across the culture plate, but not physiologic levels of strain in MC3T3-E1 osteoblast-like cells. Addition of 50 nM PTH during loading increased COX-2 expression at both subthreshold and threshold levels of fluid forces compared with either stimuli alone. We also demonstrated that application of fluid shear to MC3T3-E1 cells induced a rapid increase in [Ca²⁺]_i. Although PTH did not significantly change [Ca²⁺]_i levels, flow and PTH did produce a significantly greater [Ca²⁺]_i response and increased the number of responding cells than is found in fluid shear alone. The [Ca²⁺]_i response to these stimuli was significantly decreased when the mechanosensitive channel inhibitor, gadolinium, was present. These studies indicate that PTH increases the cellular responses of osteoblasts to mechanical loading. Furthermore, this response may be mediated by alterations in [Ca²⁺]_i by modulating the mechanosensitive channel. Received: 5 October 1999 / Accepted: 15 February 2000     

Gonadal Steroids and Bone Metabolism in Young Castrated Male Rats

At 45 days of age, 40 male Wistar rats were castrated, then randomly divided into four groups, S.C. injected for 60 days after surgery either with 17β-estradiol (E) 10 μg/kg BW/48 hours, progesterone (P) 140 μg/kg BW/48 hours, dihydrotestosterone (D) 2 μg/kg BW/48 hours, E + P + D same doses, or solvent alone (CX). Ten other rats were sham-operated (SH) and used as controls. Animals were put in balance to determine Ca and phosphorus (Pi) intestinal apparent absorption (IA Ca, IA Pi) and urinary pyridinium crosslinks excretion. Plasma was collected for measurement of intact-parathyroid hormone (PTH), calcitonin (CT), insulin-like growth factor I (IGF-I), 1,25 dihydroxyvitamin D (1,25(OH)₂D), Ca, and Pi. Orchidectomy induced marked seminal vesicles atrophy and increased plasma CT, PTH, and Ca concentrations. IA Ca was significantly higher in P rats, however, neither castration nor any other treatment had significant effects. Orchidectomy decreased femoral length, dry weight, and Ca content, whereas E or D given alone or together with P improved endochondral growth and enhanced femoral Ca content. Again, bone mineral density was lowered by orchidectomy and reestablished by both E and EPD, even above SH values, this effect being more important at the metaphyseal levels. Urinary pyridinium cross-links excretion and plasma osteocalcin concentrations were higher in the CX animals than in the controls. Although E and D given alone did reduce both biochemical turnover markers, they showed additive effect when given together (EPD). In conclusion, in the young castrated male rat, E was more efficient than D for preventing bone loss, the most important effect being induced by a combination of E + P + D. Received: 28 June 1999 / Accepted: 12 January 2000     

Bisphosphonate Maintains Parathyroid Hormone (1-34)-Induced Cortical Bone Mass and Mechanical Strength in Old Rats

This study was designed to determine the fate of new parathyroid hormone (PTH)-induced cortical bone after withdrawal of PTH treatment, and to evaluate whether subsequent treatment with a bisphosphonate would influence this. Six groups of 21-month-old rats were used: a baseline group killed at the beginning of the experiment, three groups injected with human PTH (1-34) (62 μg/kg) daily for 8 weeks (day 1-56), then one group was killed and the other two groups were injected for another 8 weeks (day 57-112) with either saline or bisphosphonate (risedronate 5 μg/kg twice a week). Two control groups were injected with vehicle for the first 8 weeks, then one group was killed and the other group injected with saline the next 8 weeks. All animals were labeled with tetracycline and calcein on day 35 and day 49 of the experiment, respectively. PTH increased periosteal (35%) and in particular endosteal mineralizing surfaces (188%), mineral appositional rates, and bone formation rates at the femur diaphysis, leading to an increase in cortical cross-sectional area of 31%. Withdrawal of PTH induced a fast and pronounced endosteal bone resorption whereas risedronate prevented this resorption. No differences were seen in apparent density of dry defatted bone and ash among the groups. PTH increased the mechanical strength of the femur diaphysis; ultimate load increased by 64% and ultimate stress by 25%. A pronounced decrease in mechanical strength and competence was found after withdrawal of PTH: ultimate load decreased by 31% and ultimate stress by 21%. Risedronate, however, prevented this decrease in mechanical strength and competence in these 2-year-old rats. Received: 13 March 1997 / Accepted: 22 August 1997     

Effect of Chronic Vitamin D Infusion Upon In Vivo Glucose Disposal

We have previously demonstrated that parathyroid hormone (PTH) infusion decreases glucose disappearance rate (K_g) in vivo. Because in the rodent model used it was not possible to determine whether the PTH itself, the induced hypercalcemia, or both contributed to the glucose intolerance, we examined the effect of vitamin D infusion on insulin-mediated glucose disposal. In this model also hypercalcemia is induced but PTH levels are suppressed. Thirty male Sprague Dawley rats were continuously infused with vit D for 5 days using an Alzet miniosmotic pump, at a rate of 9.7 pmol/hour. Thirty controls were infused with the vehicle alone. On the 5th day, glucose 700 mg/kg and insulin 0.35 U/kg were given as a bolus through the left femoral vein and blood samples were obtained from the right femoral vein just prior to and at 2, 5, 10, and 20 minutes post-glucose/insulin infusion. At the end of 5 days, plasma calcium levels were higher in the vit D-infused rats than in the control rats (12.8 ± 0.1 versus 10.0 ± 0.1 mg/dL, P < 0.01) and rat PTH levels were suppressed (2.1 ± 0.1 versus 62 ± 12 pg/ml, P < 0.01). Glucose levels were higher in the vit D animals only at 5 minutes following glucose/insulin bolus (375 ± 7 versus 350 ± 6 mg/dL, P < 0.01) but at no other time. There were no differences between serum insulin levels at any time. Unlike previous findings in PTH-infused rats, K_g (measured from 2 to 20 minutes following glucose/insulin bolus) was not different between groups (4.5 ± 0.3 versus 4.7 ± 0.2, P= 0.92.) A positive correlation between serum calcium and serum glucose was found only at 5 minutes (r = 0.55, P < 0.01) and only in the vit D animals. The areas under the glucose curves approached statistically significant differences (vit D-infused 5258 ± 142 mg/dL/18 minutes versus control 4947 ± 127, P= 0.06.) Analysis of serum glucose data by two-factor analysis of variance (ANOVA) suggests that the two groups differ slightly in glucose values (P= 0.03) but have parallel K_g. In order to define whether different effects of PTH (1–34) and vit D on intracellular calcium [Ca²⁺]_i levels could partly explain the different effects of PTH and vit D infusion on glucose disposal, we investigated the effect of PTH and vit D infusions on basal and concanavalin A (con A)-stimulated changes in mononuclear [Ca⁺²]_i levels. Following 5 days of PTH, vit D, or control infusion, peripheral mononuclear cells were incubated with 50 μg/ml con A. Changes in [Ca⁺²]_i over 5 minutes were calculated by flow cytometric measurement of the calcium sensitive fluo-3 AM dye. Despite achieving significant and comparable degrees of hypercalcemia in the PTH and vit D infused animals, there were no differences in basal or con A-stimulated [Ca⁺²]_i levels from control. Consequently, we conclude that vit D-induced hypercalcemia associated with suppressed PTH levels has mild affects on glucose homeostasis but does not affect glucose disappearance rate in vivo (K_g) as does hypercalcemia induced by PTH infusion, and that neither chronic PTH infusion nor chronic vit D infusion are associated with long-standing changes in [Ca²⁺]_i levels. Received: 24 March 1998 / Accepted: 29 June 1998     

Immunohistochemical Localization of Selected Early Response Genes Expressed in Trabecular Bone of Young Rats Given hPTH 1-34

Intermittent administration of parathyroid hormone (PTH) increases trabecular bone mass in vivo by stimulating bone formation. To further characterize the cellular and molecular mediators of the anabolic response to PTH, we examined the effect of intermittent synthetic hPTH 1-34 on the expression and localization of selected early response genes, c-fos, c-jun, c-myc, and IL-6 protein, in bone tissue by immunohistochemistry. Young male Sprague-Dawley rats, 70–100 g, were injected s.c. with 8 μg/100 g PTH or vehicle control, once daily for 5 days. Femurs were harvested 1 and 24 hours after the fifth injection, then fixed, decalcified, processed for wax embedding, and sections were immunostained. Early response genes, c-fos, c-jun and IL-6, were strongly expressed in osteoblasts, osteocytes, and megakaryocytes in bones 1 hour after PTH, when compared with vehicle-treated controls or sections from rats, 24 hours after PTH injection. Osteoblasts, osteocytes, and megakaryocytes were also positive for c-myc but the differences in stain intensity between control and treated groups were marginal. Also, scattered islands of hematopoietic cells in the marrow stained intensely for IL-6 by 1 hour after PTH, but the stain intensity decreased to control level 24 hours after the last PTH injection. Scattered islands of hematopoietic cells in the bone marrow stained more strongly for c-fos than either c-jun or c-myc, but neither localization nor stain intensity were regulated by PTH at the time points examined. We conclude that during the immediate early phase of the anabolic response, PTH regulates c-fos, c-jun, and IL-6 expression in osteoblasts, osteocytes, megakaryocytes, and selected bone marrow hematopoietic cells in bone. Received: 7 July 1998 / Accepted: 10 June 1999     

The Advantage of Alfacalcidol Over Vitamin D in the Treatment of Osteoporosis

Although alfacalcidol is widely used in the treatment of osteoporosis, its mechanism of action in bone is not fully understood. Alfacalcidol stimulates intestinal calcium (Ca) absorption, increases urinary Ca excretion and serum Ca levels, and suppresses parathyroid hormone (PTH) secretion. It remains to be clarified, especially under vitamin D-replete conditions, whether alfacalcidol exerts skeletal effects solely via these Ca-related effects, whether the resultant suppression of PTH is a prerequisite for the skeletal actions of alfacalcidol, and, by inference, whether alfacalcidol has an advantage over vitamin D in the treatment of osteoporosis. To address these issues, we (1) compared the effects of alfacalcidol p.o. (0.025–0.1 μg/kg BW) vis-à-vis vitamin D₃ (50–400 μg/kg BW) on bone loss in 8-month-old, ovariectomized (OVX) rats as a function of their Ca-related effects, and (2) examined whether the skeletal effects of alfacalcidol occur independently of suppression of PTH, using parathyroidectomized (PTX) rats continuously infused with hPTH(1–34). The results indicate that (1) in OVX rats, alfacalcidol increases BMD and bone strength more effectively than vitamin D₃ at given urinary and serum Ca levels: larger doses of vitamin D₃ are required to produce a similar BMD-increasing effect, in the face of hypercalcemia and compromised bone quality; (2) at doses that maintain serum Ca below 10 mg/dl, alfacalcidol suppresses urinary deoxypyridinoline excretion more effectively than vitamin D₃; and (3) alfacalcidol is capable of increasing bone mass in PTX rats with continuous infusion of PTH, and therefore acts independently of PTH levels. It is suggested that alfacalcidol exerts bone-protective effects independently of its Ca-related effects, and is in this respect superior to vitamin D₃, and that the skeletal actions of alfacalcidol take place, at least in part, independently of suppression of PTH. Together, these results provide a rationale for the clinical utility of alfacalcidol and its advantage over vitamin D₃ in the treatment of osteoporosis.     

Changes in Geometry and Cortical Porosity in Adult, Ovary-Intact Rabbits after 5 Months Treatment with LY333334 (hPTH 1-34)

The purpose of this study was to determine if the increased cortical bone porosity induced by intermittently administered parathyroid hormone (PTH) reduces bone strength significantly. Mature ovary-intact New Zealand white rabbits were treated with once daily injections of vehicle, or PTH(1-34), LY333334, at 10 or 40 μg/kg/day for 140 days. Geometry of the femoral midshaft was measured to evaluate changes in the cross-sectional moment of inertia (CSMI). Cortical porosity was measured in the midshaft of the tibia by dividing cortical area into three zones based on equal divisions of cortical diameter: near endocortical (Zone I), near intermediate (Zone II), and near periosteal (Zone III) regions. Total cortical porosity significantly increased after PTH treatment from 1.4% in the controls to 6.3% in the higher dose group, but the location of the new porosities was not randomly distributed. In the controls, porosity of Zones I and II (both 1.7%) was almost twice as much as that of Zone III (0.9%). In the lower dose group, cortical porosity of Zone I (5.5%) and II (1.8%) was greater than in Zone III (0.9%), but these differences were not statistically significant. In the higher dose group, cortical porosity of Zone I (11.5%) and II (6.1%) significantly increased compared with Zone III (1.4%) (P < 0.0005). Histomorphometric measurements showed that bone formation rate on both periosteal and endocortical surfaces increased, resulting in increased bone area and cortical area in the higher dose group. A model was developed to evaluate the effect of the changes in geometry and porosity on CSMI in the different zones. This simulation model indicated that CSMI in the higher dose group was significantly greater than in the other two groups, despite the increased porosity. We speculate the reason to be that porosity increased near the endocortical surface, where its mechanical effect is small. This increase was more than offset by apposition of new bone on the periosteal surface. These data suggest that (1) PTH increases cortical porosity in a dose-dependent manner, primarily near endocortical surfaces; (2) because of this nonhomogeneous distribution, the mechanical effect of increased porosity is small; (3) the increased cortical porosity associated with PTH treatment is more than offset by periosteal apposition of new bone, causing an overall increase in the bending rigidity of cortical bone; and (4) these changes cannot be accurately evaluated using noninvasive methods of bone densitometry, which cannot account for the location of bone gain and bone loss. Received: 20 May 1999 / Accepted: 10 January 2000     

Omeprazole-induced hypergastrinemia: role of gastric acidity

The increase in gastrin caused by the gastric proton pump inhibitor, omeprazole, is presumably related to inhibition of gastric acid secretion (GAS). We investigated omeprazole's effect on gastrin release by studying two doses of omeprazole which produced marked acid suppression. Six gastric fistula dogs received omeprazole, 3 mumole/kg, daily for 20 days and, after a rest interval of 2 months, omeprazole, 10 mumole/kg again for 20 days. Both doses of omeprazole increased gastrin levels and produced a decrease in GAS which was still significant (P less than 0.05) 3 days postfinal dose. The increase in the integrated gastrin response by omeprazole, 10 mumole, was greater than by omeprazole, 3 mumole. Omeprazole, 10 mumole, also reduced GAS and gastric acidity, and increased gastric pH more consistently than omeprazole, 3 mumole. The magnitude of the gastrin response corresponded with the degree of acid inhibition and pH increase. Therefore, the data support the hypothesis that the hypergastrinemia caused by omeprazole is dependent on gastric pH and GAS suppression.     

Bone Mineral in Pre- and Postmenopausal Women with Insulin-Dependent and Non-insulin-Dependent Diabetes Mellitus

We have recently shown that bone radiogrammetric dimensions are associated with vitamin D receptor gene polymorphism. Since parathyroid hormone (PTH) plays a central role in maintaining calcium homeostasis and in bone remodeling, we investigated whether bone radiogrammetric dimensions are associated with a PTH gene polymorphism in 91 healthy Caucasian women, who were premenopausal at entry into the study. These women had assessments of bone by radiogrammetry every five years for a median period of 20 years (range 4–27). DNA was extracted from white blood cells. A segment of the PTH gene with a polymorphism at a BstBI restriction site was amplified by polymerase chain reaction. Diameter, cortical thickness and cross-sectional area at standard sites of the metacarpals, radius and femur were measured with radiogrammetry. Higher metacarpal diameter and cross-sectional cortical area, and a slower decrease in radial cortical area with age, were associated with the absence of the BstBI restriction site of the PTH gene. PTH gene polymorphism accounts for about 7–9% of the total variances of bone dimensional variables. These findings suggest that the dimensions of long bones are influenced by allelic variations in the PTH gene or in genes nearby. Received: 29 January 1998 / Accepted: 3 August 1998     

Parathyroid Hormone Secretory Response to EDTA-Induced Hypocalcemia in Black and White Premenopausal Women

One consistent racial difference in mineral homeostasis is increased efficiency of renal calcium conservation in blacks which could account, in part, for differences in bone density and fracture risk. Since parathyroid hormone (PTH) is the major regulator of calcium homeostasis, we investigated its secretion in black and white women in response to hypocalcemia. Two hour EDTA infusions (50 mg/kg) were performed in 34 premenopausal women (17 black, 17 white). Blood was sampled at 30-minute intervals during the infusion, at 60-minute intervals for 3 more hours, and at 24 hours. Serum ionized calcium decreased identically in both groups with a nadir at 2 hours and returned to baseline within 24 hours. Serum 1-84 PTH levels rose similarly in both groups with a peak PTH level that was slightly higher in black women, and on average, slightly earlier than that in white women. Serum PTH levels remained elevated in both groups at 24 hours with no overall group differences in PTH response. In black, but not white women, serum 25OHD levels correlated negatively with both basal PTH and peak PTH level, achieved with infusion. Serum 1,25(OH)₂D levels rose and osteocalcin levels decreased, with no group differences. We conclude that overall, premenopausal black women show no clear differences in PTH secretory activity to an EDTA-induced hypocalcemic stimulus. Basal vitamin D status appeared to be a determinant of the degree of the PTH response in black women, with the peak PTH level being inversely correlated with levels of 25OHD. Since we have previously shown that the skeleton contributes less to acute calcium needs in blacks than in whites, the lack of a racial difference in PTH secretory responsivity suggests that calcium homeostasis is more likely maintained in blacks through greater PTH sensitivity at extraskeletal sites, such as the kidney. Received: 31 August 1998 / Accepted: 12 March 1999     

Expression of the parathyroid hormone receptor and correlation with other osteoblastic parameters in fetal rat osteoblasts

Primary fetal rat calvarial cell cultures were examined for the expression of different osteoblastic parameters at the single cell level and in the whole population. The presence of the parathyroid hormone (PTH) receptor was studied by employing receptor autoradiography. After 3 days of culture, 10% of the cells expressed the PTH receptor. Immunolocalization of osteocalcin in 3-day-old cell cultures was found to be strongly correlated with the presence of the PTH receptor. Alkaline phosphatase (APase) localization in 3-day-old cultures correlated with only 69% of the PTH receptor expressing cells. Our results show that in 3-day-old rat calvarial cell cultures, only about 10% of the cells show markers of osteoblastic differentiation. The presence of the PTH receptor is strongly correlated with the presence of osteocalcin, but less with the presence of APase, indicating that it is the mature osteoblast that expresses the PTH receptor. After 7 days of culture, most receptor labeling, APase, and osteocalcin expression was found in multilayered areas of cells (nodules). Received: 28 February 1995 / Accepted: 27 July 1995     

Lack of Relationship Between BsmI Vitamin D Receptor Polymorphism and Primary Hyperparathyroidism in a Spanish Female Population

The role of vitamin D receptor (VDR) gene polymorphisms in the pathogenesis of hyperparathyroidism is uncertain. Controversial results have been reported. The aim of this study was to explore the relevance of BsmI VDR gene polymorphism in patients with primary hyperparathyroidism (PHPT) due to adenomas. For this purpose, 36 postmenopausal patients with PHPT, mean age 64 years, were compared with a normal age-matched female population (n = 81). BsmI polymorphism distribution in PHPT group was as follows: Bb 50% (18/36); BB 22% (8/36); bb 28% (10/36). In the control group, the distribution was Bb 49% (40/81); BB 16% (13/81); bb 35% (28/81). No statistical differences were found between the two groups. In the PHPT group, no statistical associations were found between different allelic distribution and age, creatinine, hematocrit, phosphorus, alkaline phosphatase (ALP), total calcium, serum parathyroid hormone (PTH), or gland weight. By contrast, levels of serum calcium and iPTH values positively correlated with the PT weight (r = 0.421 and 0.599, respectively, P= 0.0001). Our data suggest that at least in this group, BsmI VDR gene polymorphism appears to be of minor relevance in clinical presentation and possibly, tumorigenesis in PHPT due to adenomas. Received: 21 April 1998 / Accepted: 12 March 1999     

Parathyroid Hormone (1-34) Receptor-Binding and Second-Messenger Response in Rat Incisor Odontoblasts

Even though indirect evidence indicates that PTH exerts an anabolic effect on dentinogenesis, the existence of PTH receptors and any second-messenger response in odontoblasts have not been demonstrated. The aim of this study was to investigate whether rat incisor odontoblasts express PTH receptors, and to identify which second messenger pathway the hormone may activate. Odontoblasts were dissected from rat incisors. Amino-terminal (1-34) fragment rat PTH [rPTH(1-34)] conjugated to fluorescein isothiocyanate visualized receptor sites on the cell surface. Upon incubation of odontoblasts with rPTH(1-34), cAMP formation was increased. However, no fluctuations in intracellular calcium activity were observed upon rPTH(1-34) stimulation when using Fura-2 as a Ca²⁺ probe. In long-time incubations, stimulation with PTH(1-34) upregulated APase activity. The results demonstrate that rPTH(1-34) evokes an anabolic response in dentinogenically active odontoblasts, and that this may be mediated through the protein kinase A/cAMP pathway, whereas no indications for Ca²⁺ as a second messenger were evident. Received: 12 March 1997 / Accepted: 26 June 1997     

Divergent effects of forskolin on 3′,5′ cyclic adenosine monophosphate production and parathyroid hormone secretion

Summary Forskolin, a diterpene which directly stimulates adenylate cyclase, markedly stimulated cAMP production in intact rat parathyroid glands and dispersed cells from hyperplastic and adenomatous human parathyroid tissues. Stimulation of cAMP production in human parathyroid adenomas occurred as early as 2 min and continued for at least 2 h; furthermore, a dose-response relationship was observed, with a maximal 80-fold cAMP response occurring at 100 μM forskolin. When PTH secretion by rat or human parathyroid tissues was studied at low (0.5 mM) and high (2.5 mM) extracellular Ca²⁺ in either the presence or absence of forskolin, no significant stimulation by forskolin was observed at 15 min, 1 h, and 2 h. When 10 human parathyroid specimens were studied with varying concentrations of forskolin at 1 mM Ca²⁺, 6 failed to show stimulation of PTH secretion and 4 showed modest but detectable increases in PTH that did not appear dose-related. We conclude that (1) at low and high Ca²⁺ levels, marked stimulation of cAMP production by forskolin can occur without a corresponding increase in PTH secretion; (2) inhibition of PTH secretion by high extracellular Ca²⁺ levels continues unchanged despite stimulation of cAMP production by forskolin; and (3) at intermediate Ca²⁺ levels (1.0 mM), PTH secretion is affected either minimally or not at all by forskolin in human hyperparathyroid tissue preparations. The marked stimulation of parathyroid adenylate cyclase by forskolin without concomitant increases in PTH secretion in the majority of tissues suggests that the level of cAMP production is not a primary or sufficient determinant of hormone secretion.     

Administration of a Glucocorticoid With Depot Effect Counteracts the Stimulating Effect of Growth Hormone on Cancellous and Cortical Bone of the Vertebral Body in Rats

Our earlier studies have shown that growth hormone administration could not counteract decreased longitudinal growth and cortical osteopenia of rat femora induced by a glucocorticoid with depot effect. In the present study we examined the effects of glucocorticoid on vertebral bone as well as the effect of growth hormone on vertebral bone in young growing animals also given glucocorticoid injections. Five groups of female rats (3 1/2 months) were treated for 80 days as follows: (1) saline, (2) prednisolone: Delcortol 5 mg/kg/day, (3) growth hormone: 5 mg/kg/day, (4) prednisolone and growth hormone, (5) food restriction. Vertebral dimensions, histomorphometry, and mechanical competence of the vertebral bone were examined. Growth hormone administration increased body weight, vertebral height, cross-sectional area, and volume. The compressive strength of the L₄-corpus cylinder was also increased due to an increase in cancellous bone volume and an increase in the area of cortical bone surrounding the vertebral body. Glucocorticoid administration decreased body weight, height, and volume of the intact vertebrae. Histological examination revealed that glucocorticoid administration decreased the area of cortical bone surrounding the vertebral body but had no effect on the cancellous bone volume. No effect of glucocorticoid administration on mechanical strength of the L₄ corpus cylinder could be detected. In agreement with our findings in cortical bone, we found no effect of growth hormone on vertebral bone when given to animals also receiving glucocorticoid injections. Growth hormone increases longitudinal growth, cortical and cancellous bone mass, and mechanical competence of the vertebral body. Glucocorticoid administration decreases longitudinal growth of the vertebrae and cortical bone mass without affecting cancellous bone mass of the vertebral body. Despite this, administration of a glucocorticoid with depot effect totally inhibits the effect of growth hormone on vertebral bone. Received: 12 March 1997 / Accepted: 14 November 1997     

Parathyroid Hormone,Prostaglandin E2, and 1,25-Dihydroxyvitamin D3 Decrease the Level of Na+-Ca2+ Exchange Protein in Osteoblastic Cells

We previously described Na⁺-Ca²⁺ exchange in osteoblastic rat osteosarcoma cells (UMR-106) and demonstrated that Na⁺-dependent Ca²⁺ transport was inhibited by 24-hour treatment of cells with parathyroid hormone (PTH), prostaglandin E₂ (PGE₂), or 1,25(OH)₂D₃. To determine whether this inhibition of Na⁺-Ca²⁺ exchange is at the level of exchanger protein synthesis we have examined exchanger protein levels using immunoblot analysis. UMR-106 cells were treated for 24 hours with or without PTH, PGE₂, or 1,25(OH)₂D₃. Plasma membrane fractions (7500 g) were obtained and proteins were separated by SDS-PAGE, transferred to nylon membranes, and immunoblotted with a polyclonal antibody to the canine cardiac Na⁺-Ca²⁺ exchanger. In rat cardiac membranes, we detected 125 and 75 kD bands, similar to findings for the canine exchanger. In the osteoblastic UMR cell membranes, a specific band was detected at 90 kD that decreased 65% after treatment of cells with PTH. Inhibition by PTH was dose dependent, was maximal with 10⁻⁷ M PTH, and required 16–24 hour treatment time. Similar inhibition was observed after a 24 hour treatment with 10⁻⁶ M PGE₂ or 10⁻⁸ M 1,25(OH)₂D₃. These results demonstrate the presence of a specific protein in UMR cells that cross-reacts with antibody directed against the cardiac Na⁺-Ca²⁺ exchanger. Thus, the previously reported inhibition of Na⁺-Ca²⁺ exchange activity by calcemic agents in osteoblasts appears to be due to regulation of exchanger protein levels in these osteoblastic cells. Received: 5 February 1996 / Accepted: 18 October 1996     

Parathyroid hormone 7-84 induces hypocalcemia and inhibits the parathyroid hormone 1-84 secretory response to hypocalcemia in rats with intact parathyroid glands

Biologic effects of large C-terminal parathyroid hormone (PTH) fragments, opposite to those of N-terminal PTH, have been demonstrated. C-terminal PTH fragments are co-secreted with N-terminal PTH from the parathyroids. The aim of our study was to examine whether C-terminal PTH 7-84 regulates secretion of PTH 1-84 and affects the expression of genes of relevance for parathyroid function, PTH, calcium-sensing receptor (CaR), PTH type 1 receptor (PTHR1), and PTH-related peptide (PTHrP) genes in rat parathyroid glands. PTH 7-84 induced a significant decrease in plasma Ca2+ in rats with intact parathyroid glands. Despite the reduction of plasma Ca2+, no stimulation of PTH 1-84 secretion took place. Furthermore, the PTH 1-84 secretory response to EGTA-induced acute and severe hypocalcemia was significantly inhibited by PTH 7-84. During recovery from hypocalcemia, plasma Ca2+ levels were significantly lower in the PTH 7-84-treated group, as compared with the vehicle group, and at the same time plasma PTH 1-84 levels were significantly suppressed. The expression of PTH, CaR, PTHR1, and PTHrP genes in the rat parathyroid glands was not affected by PTH 7-84. The peripheral metabolism of PTH 1-84 was not affected by PTH 7-84. PTH 7-84 did not cross-react with the rat bioactive PTH 1-84 assay. In normal rats with intact parathyroid glands, PTH 7-84 inhibited the PTH 1-84 secretory response to hypocalcemia and induced a significant decrease in plasma Ca2+. These effects of PTH 7-84 on PTH 1-84 secretion and on plasma Ca2+ levels were not associated with significant changes in PTH, PTHR1, CaR, and PTHrP gene expressions in the rat parathyroid glands. It is hypothesized that PTH 7-84 regulates PTH secretion via an autocrine/paracrine regulatory mechanism.     

Prednisolone Induces an Increase in Serum Calcium Concentration: Possible Involvement of the Kidney, the Bone, and the Intestine

To evaluate the early effect of glucocorticoids on calcium metabolism, 15 subjects aged 22–58 years (5 males, 10 females) with chronic glomerulonephritis were orally treated with 40 mg daily of prednisolone. Five of these subjects were diagnosed with nephrotic syndrome and none had a serum creatinine concentration of more than 1.4 mg/dl. Serum specimens and 24-hour urine specimens were obtained just before and 24 hours after a single oral dose of prednisolone. Serum calcium, ionized calcium, phosphate, intact parathyroid hormone (PTH), intact osteocalcin and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), and urinary excretion of calcium, phosphate, and deoxypyridinoline were measured. Both serum calcium and ionized calcium concentrations were significantly increased from 4.39 ± 0.10 to 4.47 ± 0.09 mEq/liter (P= 0.037) and from 2.48 ± 0.04 to 2.55 ± 0.04 mEq/liter (P= 0.002), respectively, 24 hours following a single oral dose of prednisolone. Serum intact PTH concentration slightly decreased, but the difference was not significant by statistical analysis. Serum intact osteocalcin concentration was markedly suppressed. In contrast, no significant changes were observed in urinary excretion of deoxypyridinoline. Serum 1,25(OH)₂D₃ concentration measured in five patients was significantly increased. No significant changes in urinary excretion of calcium was observed in the face of these findings. It thus follows that a single oral dose of prednisolone administration increases serum calcium and ionized calcium concentrations, possibly mediated by suppressed bone formation, increased intestinal absorption of calcium, and impaired urinary excretion of calcium. Received: 19 February 1998 / Accepted: 12 March 1999