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Recent studies suggest that the uptake of immunoreactive parathyroid hormone (iPTH) displays different characteristics in liver, kidney, and bone. Using the isolated perfused canine bone, we have characterized the uptake of two synthetic analogs of PTH, bovine PTH-(3-34) [bPTH-3(3-34)] and [Nle8,Nle18,Tyr34]bPTH-(3-34) amide, which had previously been shown to inhibit PTH-stimulated adenylate cyclase activity in renal membranes. During the infusion of synthetic bPTH-(1-34) (3 ng/ml), extraction of iPTH by isolated perfused bone averaged 37 +/- 1%, and cAMP production rose from 6.2 +/- 2.0 to 21 +/- 3 pmol/min. Extraction of bPTH-(3-34) was similar (35 +/- 2%), but cAMP levels did not increase over baseline with PTH concentrations as high as 100 ng/ml. Simultaneous infusion of bPTH-(1-34) and bPTH-(3-34) at molar ratios of 1:2 led to a 50% inhibition of PTH-stimulated cAMP increases. The extraction by bone of the more potent in vitro inhibitor of renal cortical adenylate cyclase [Nle8,Nle18,Tyr34]bPTH-(3-34) NH2 (3 ng/ml) averaged 39 +/- 2%. In contrast, cAMP production rose from a baseline of 5.6 +/- 0.5 to 12.5 +/- 2.0 pmol/min, demonstrating agonist activity for the analog. These studies show that [Nle3,Nle18,Tyr34]bPTH-(3-34) NH2 has agonist properties in isolated perfused bone, and unsubstituted bPTh-(3-34) inhibits PTH-stimulated cAMP release by perfused bone.

These studies examine the metabolism of highly purified bovine parathyroid hormone [bPTH-(1--84)] by fetal rat calvaria. Enzymatically dispersed bone cells and intact (minced) calvaria were incubated with bPTH-(1--84) and the incubation medium was analyzed for degradation of PTH by polyacrylamide gel electrophoresis. Eluates of gel slices were assayed for immunoreactive PTH (iPTH) in carboxy- and amino-terminal RIAs. Both bone preparations metabolized bPTH-(1--84). The intact hormone progessively decreased with time and carboxy-terminal iPTH fragments were evident by 5 min of incubation. In the isolated cell preparations, intact hormone was completely degraded at submaximal doses of PTH (5 X 10(-9) M), as assessed by cAMP production. Degradation was incomplete in intact calvarial preparations at all doses studied. Intact calvaria were less sensitive to PTH with regard to cAMP production. No amino-terminal fragments were detected in the medium with either cell preparation. Oxidized (biologically inactive) bPTH-(1--84) was not metabolized in these systems. These findings contrast with studies in liver and kidney preparations, where oxidized bPTH has been shown to be degraded. These findings contrast with studies in liver and kidney preparations, where oxidized bPTH has been shown to be degraded. These data suggest that biological activity may be necessary for the metabolism of intact bPTH-(1--84) by bone cells and that skeletal tissue may contribute to the immunoheterogeneity of circulating PTH in the rat.

It is known that the secretion of PTH is often impaired in association with aluminum (Al3+) accumulation in patients with renal failure. The mechanisms involved remain ill defined. Since adenylate cyclase plays a role in the regulation of PTH secretion, these studies examine the effects of Al3+ on parathyroid adenylate cyclase. In membranes from normal bovine parathyroid glands, basal adenylate cyclase activity, in the presence of 0.2 mM ATP and 20 mM Mg2+, increased by 22% as Al3+ was raised from 0-10 microM. Higher Al3+ concentrations caused a progressive decrease in adenylate cyclase activity, reaching 68% inhibition of control activity at 2 mM Al3+. Since adenylate cyclase activation is influenced by the interaction of multiple sites within the adenylate cyclase complex, the nature of the inhibition by Al3+ was explored by examining the interaction of Al3+ with substrate ATP and with Mg2+, an allosteric activating metal ion. In the presence of 20 mM Mg2+, Al3+ concentrations of 1-2 mM resulted in noncompetitive inhibition with respect to ATP [decrease in maximum velocity (Vmax) from 4176 in the absence of Al3+ to 1106 pmol cAMP/mg protein X 15 min; Michaelis Menten constant (Km) for ATP was unchanged]. In contrast, at fixed ATP (0.2 mM), 0.5 mM resulted in competitive inhibition of adenylate cyclase with respect to Mg2+, whereas at higher Al3+ concentrations the inhibition was noncompetitive. When Mg2+ was replaced by Mn2+ (enzyme activity reflects the activity of the catalytic unit), the inhibitory effect of Al3+ on adenylate cyclase activity was abolished. These data suggest that the inhibition of parathyroid adenylate cyclase by Al3+ occurs at the level of the allosteric metal activating site. These data provide a potential mechanism for the inhibition of PTH secretion by Al3+.

Because of differing reports of an effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on parathyroid hormone (PTH) secretion, the present studies were performed in vitro using bovine parathyroid gland slices and isolated parathyroid cells. Both COOH-terminal and NH2-terminal RIAs for PTH were employed. No effect of 1,25(OH)2D3 on PTH secretion was found during a 4-h incubation of parathyroid slices in variable external calcium concentrations. The results from dual RIA measurements also showed no effect of 1,25(OH)2D3 on PTH secretion by isolated parathyroid cells during 90- to 150-min incubations with variable calcium concentrations. In addition, extensive analysis by polyacrylamide gel electrophoresis showed no effect of 1,25(OH)2D3 on cAMP generation. Whereas calcium inhibited and isoproterenol stimulated the production of cAMP, 1,25(OH)2D3 had no effect. We conclude that 1,25(OH)2D3 does not affect parathyroid gland function acutely in vitro.

Although it is well known that aluminum (Al) plays a role in the development of osteomalacia in patients with chronic renal failure, the mechanisms are not fully understood. Since the osteoblasts are the cells responsible for the formation of osteoid tissue, which is greatly affected in patients with Al-induced osteomalacia, it is possible that Al could affect the number of osteoblasts or interfere with their function. To further characterize this potential mechanism, we performed studies in isolated perfused tibiae from normal and Al-treated dogs. In this system, when PTH is added to the perfusate, cAMP, a major marker of osteoblasts, is released. The dogs were divided into two groups: control, and Al-treated (0.75 mg/kg, iv, 5 days a week for 3 months). Thereafter, the dogs were killed, and the tibiae were perfused in vitro. PTH-(1-34) (3-4 ng/ml) and 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase) were added to the perfusate. Basal cAMP secretion was the same in both groups of dogs. After PTH was added to the perfusate, cAMP increased to a peak of 188.2 +/- 30.6 pmol/min in the normal dogs vs. 113 +/- 8.15 in Al-treated dogs (P less than 0.05). Cumulative cAMP secretion over a 30-min period was 766 +/- 127.9 pmol in the normal dogs vs. 455.6 +/- 38.2 pmol in the experimental animals (P less than 0.05). The histological appearance of bone biopsies taken before and after Al administration are consistent with a suppressive effect of the cation on osteoblast function. In particular, the number of osteoblasts had decreased 8-fold (P less than 0.01) under the influence of Al, and tetracycline-based measurements of mineralization kinetics show that osteoblast-mediated calcification was dysfunctional (P less than 0.01-0.025). On the other hand, the histological features of the post Al treatment biopsies suggest that at some time during its administration, the cation stimulates osteoblastic activity. For example, new (woven) bone formation was present in two dogs, and in another, lamellar bone, deposited under the influence of Al, covered the entire trabecular surface. Moreover, Al-associated osteoid was deposited independent of prior resorptive activity, indicating that the cation promotes bone formation in the absence of prior resorption. In keeping with its trophic effect on matrix deposition, Al also led to extensive marrow fibrosis in five dogs, indicating that Al also stimulates the activity of fibroblasts, cells closely related to osteoblasts.(ABSTRACT TRUNCATED AT 400 WORDS)

The interaction between glucocorticoids (GC) and PTH has been suggested to play a role in the pathogenesis of GC-induced osteopenia. The present studies were designed to examine the effect of acute (5-h) or chronic (4-week) GC administration in vivo on 1) cAMP release by the isolated perfused dog tibia before (basal) and after the addition of synthetic bovine PTH-(1-34) [syn bPTH-(1-34)] (stimulated) to the perfusate in vitro, in the presence or absence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX; 1 mM), and 2) the percent arteriovenous difference of immunoreactive PTH across bone. Acute administration of 6 mg/kg methylprednisolone (MP) did not affect the basal release of cAMP from bone (6.9 +/- 1.6 pmol/min in control vs. 6.1 +/- 1.2 pmol/min in MP-treated animals); however, syn bPTH-(1-34) stimulated release of cAMP was higher in the MP-treated animals (45 +/- 8.1 pmol/min) than in controls (26.8 +/- 3.0 pmol/min). When IBMX was added to the perfusate, basal cAMP release was not different in control and MP-treated bone (17.2 +/- 2.1 pmol/min in control vs. 19.1 +/- 1.9 pmol/min in MP-treated bone), and syn bPTH-(1-34)-stimulated release of cAMP was equivalent in both groups. In contrast, chronic prednisone therapy lead to a decrease in both basal and PTH-stimulated release of cAMP from bone (3.1 +/- 0.4 and 6.9 +/- 1.6 pmol/min for basal, and 13.1 +/- 1.7 and 26.8 +/- 3.0 pmol/min for stimulated values, respectively). However, the percent changes from the basal levels were not different in the two groups. These results were correlated with histological studies of rib biopsies obtained from these animals, which showed evidence of osteopenia and decreased bone turnover. Neither acute nor chronic GC administration had any effect on arterio-venous differences for PTH across the bone. Thus, these studies demonstrate that 1) acute administration of MP enhances the response of bone to PTH, an effect that is not apparent in the presence of the phosphodiesterase inhibitor IBMX; and 2) chronic prednisone therapy decreased basal and PTH-stimulated cAMP release, an effect that correlated with histological evidence of decreased bone turnover.

In the present investigation, sodium fluoride (NaF) was employed to explore the role of guanine nucleotide-binding proteins (G-proteins), protein kinase-C, or cytosolic calcium [( Ca]i) in the regulation of cytosolic pH [( pH]i) in dispersed bovine parathyroid cells, using the pH-sensitive fluorescent dye BCECF. When cells acidified by nigericin in Na-free medium were resuspended in Na-containing buffer, [pH]i returned to basal levels. This recovery was blocked by continued removal of Na+ or the addition of amiloride. NaF (10 mM) increased [32P]phosphate incorporation into phosphatidylinositol bisphosphate, suggesting an increase in phosphatidylinositol bisphosphate turnover. NaF caused an initial acidification, followed by an alkaline recovery in a dose-dependent manner (1-10 mM). Amiloride blocked the NaF-induced alkaline recovery. The protein kinase-C activator phorbol 12-myristate 13-acetate (10(-7) M) caused cytosolic alkalinization, while the protein kinase-C inhibitor H7 (6 x 10(-5) M) significantly inhibited the NaF-induced alkaline recovery. Pertussis toxin (1 microgram/ml) did not affect the NaF-induced changes in [pH]i. Removal of extracellular Ca2+ with EGTA blocked the NaF-induced increase in [Ca]i and alkaline recovery. Ionomycin (5 x 10(-7) M) caused cytosolic alkalinization, but pretreatment with EGTA inhibited the ionomycin-induced cytosolic alkalinization. The present studies clearly demonstrated the presence of an amiloride-sensitive Na+/H+ exchanger in parathyroid cells. Our findings suggest that the NaF-induced cytosolic alkaline recovery was via two complementing pathways: 1) activation of protein kinase-C, followed by stimulation of a Na+/H+ exchanger, and 2) existence of extracellular calcium and/or an increase in [Ca]i.

There is evidence that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] affects phospholipid metabolism of intestine, kidney, and bone. There are no such studies concerning the parathyroid gland, which is also a target tissue for 1,25-(OH)2D3. In this investigation we examined the effect of 1,25-(OH)2D3 on the incorporation of radiolabeled choline, inositol, serine, and ethanolamine into the phospholipids of cultured bovine parathyroid cells. Treatment with 10(-8) M 1,25-(OH)2D3 for 48 h caused a significant decrease in [14C]choline incorporation, although there were no differences in the incorporation of radiolabeled inositol, serine, or ethanolamine. Time-course and dose-response evaluations of the 1,25-(OH)2D3 effect revealed that the decrease in [14C]choline incorporation was seen within 12 h of incubation and occurred with as little as 10(-9) M, respectively. In contrast, neither 10(-7) M 25-hydroxyvitamin D3 nor 10(-7) M 24,25-(OH)2D3 caused significant changes in [14C]choline incorporation. When 5 X 10(-6) M cycloheximide was added to the medium, the inhibitory effect of 1,25-(OH)2D3 on [14C]choline incorporation was completely abolished. A significant decrease in phosphatidylcholine content was observed after treatment with 10(-8) M 1,25-(OH)2D3 for 96 h. 1,25-(OH)2D3 did not cause a dramatic change in the fatty acid composition of the phosphatidylcholine. The present studies demonstrate that in parathyroid cells 1,25-(OH)2D3 causes a decrease in [14C]choline incorporation, which could be due to a decrease in the synthesis of phosphatidylcholine or increased degradation. This effect is specific for 1,25-(OH)2D3 and requires new protein synthesis.

The present studies investigate the effects of glucocorticoids on the function of the parathyroid glands using primary cultures of bovine parathyroid cells. Treatment of parathyroid cell cultures with dexamethasone for 48 h caused a dose-dependent stimulation of PTH secretion. The minimal concentration of dexamethasone required for a significant stimulation of PTH secretion was 0.1 nM. The stimulatory effect of dexamethasone on the secretion of PTH was found within 12 h of treatment with 100 nM dexamethasone. The steroids deoxycorticosterone and cortexolone, which do not have glucocorticoid activity were without effect of PTH secretion. Since glucocorticoids may modulate the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in other tissues, additional studies were performed to evaluate the interactions of glucocorticoids and 1,25-(OH)2D3. Addition of 1,25-(OH)2D3 to parathyroid cell cultures for 48 h significantly suppressed PTH secretion. In the presence of dexamethasone, however, 1,25-(OH)2D3 also significantly decreased PTH secretion, although it did not reduce PTH secretion to control levels. The treatment of parathyroid cell cultures with 100 nM dexamethasone did not affect the parathyroid cell content of 1,25-(OH)2D3 receptors. In summary, these studies indicate that glucocorticoids significantly increase the secretion of PTH in vitro. This stimulatory effect can be inhibited by 1,25-(OH)2D3. The parathyroid gland is an additional site of physiological antagonism of glucocorticoids and 1,25-(OH)2D3.

Recent studies have shown that oxidation of bovine PTH-(1-34) [bPTH-(1-34)] with H2O2 abolished the vascular effects of PTH in rats and dogs, but the hypercalcemic effect of the oxidized PTH was preserved in the Japanese quail in vivo. These observations seem at variance with previous studies from our laboratory in the isolated perfused canine tibia preparation in which no uptake of immunoreactive PTH or stimulation of cAMP release was demonstrated during infusion of oxidized bPTH-(1-34). The present studies examine the skeletal and renal effects of oxidized PTH-(1-34) in rats and dogs in vivo. Oxidation of PTH with H2O2 reduced its activation of adenylate cyclase by 95% in dog renal cortical membrane. Awake normal dogs were studied before and during the infusion of bPTH-(1-34) or oxidized PTH-(1-34) (4 U/kg X h). With active PTH, ionized Ca+2 rose in each dog (range, 0.7-1.5 mg/dl), while with oxidized PTH, Ca+2 remained within 0.1 mg/dl of the baseline values. Fractional excretion of PO4 rose from 1.58 +/- 0.6% to 29.5 +/- 2.5% with active PTH and from 1.4 +/- 0.4% to 5.7 +/- 1% with oxidized PTH. The latter did not differ from the value in vehicle-infused dogs. Further studies were performed in 30 acutely parathyroidectomized rats. Plasma Ca+2 rose from 8.2 +/- 0.1 to 9.0 +/- 0.3 mg/dl with active PTH (20 micrograms/kg), fell to 7.7 +/- 0.2 with oxidized PTH, and fell to 7.3 +/- 0.3 mg/dl with vehicle. In parathyroid-intact rats plasma Ca+2 increased by 0.9 mg/dl whether given active PTH, oxidized PTH, or vehicle. We conclude that oxidation of bPTH-(1-34) results in loss of both the renal and skeletal effects of PTH in vivo in rats and dogs.

Vitamin A toxicity has been associated with alterations in mineral metabolism and may result in osteopenia, fractures, deformities, and growth arrest. The pathogenesis of the bone lesions that occur in vitamin A toxicity is, however, ill defined and was examined in the present study. The administration of pharmacological doses of vitamin A to growing male rats resulted in weakness and spontaneous fractures. Undecalcified bone histology of vitamin A toxic animals was characterized by increased bone resorption, osteoclastosis, a paucity of trabecular surfaces covered with osteoid, and lesions which appear to be pathognomonic of hypervitaminosis A. The serum calcium and magnesium levels of vitamin A-toxic animals were unremarkable, but serum phosphate levels were significantly higher than control values. Urinary hydroxyproline excretion reflected bone histology and was significantly increased in experimental rats. Circulating levels of the potent bone resorbers, PTH, 1,25-dihydroxyvitamin D, and 25-hydroxyvitamin D, were, however, comparable in vitamin A-toxic and control animals, suggesting a possible direct effect of vitamin A on bone. Subsequently, the effects of vitamin A (retinol) on in vitro collagen synthesis (incorporation of [3H]proline into collagen) and bone resorption (45Ca release from bone) were examined using a fetal rat calvarial culture. Retinol added to the culture medium for 20-24 h in concentrations ranging from 0.5-10 micrograms/ml selectively inhibited collagen synthesis in a dose-dependent fashion. Higher concentrations of retinol were toxic and resulted in a general inhibition of protein synthesis. Bone resorption was stimulated by 0.5 and 2.5 micrograms/ml retinol. We conclude that vitamin A toxicity in rats causes bone lesions, the genesis of which can be explained, at least in part, by a direct effect of the vitamin on skeletal tissue.

Rats were semistarved over a 7-week period, resulting in a loss of 28.2 +/- 1.6% (SEM) of their initial body weights, while ad libitum fed controls gained 15.1 +/- 1.8% (SEM). Bone loss occurred and skeletal turnover was markedly reduced in the semistarved rats, as evidenced by a paucity of osteoid and osteoclasts, failure of the bone to assume a tetracycline label, and reduced urinary hydroxyproline excretion. Despite these changes, there were no alterations of serum or bone alkaline phosphatase activity with semistarvation, and analysis of tibial mineral content revealed reductions only in magnesium and sodium. The malnourished animals, however, were hypercalciuric and hypophosphatemic. Semistarvation had no effect on circulating levels of immunoreactive parathyroid hormone or 25-hydroxyvitamin D, but did result in reduced serum levels of corticosterone, insulin, and 1,25-dihydroxyvitamin D. Therefore, it appears that the effects of semistarvation on the rat skeleton are osteoporotic rather than osteomalacic, and that the defect is the consequence of reduced bone turnover. The contribution which the abnormalities of bone-regulating hormones play in the genesis of this skeletal lesion remains to be determined.