Parathyroid hormone-related protein increases DNA synthesis in proximal tubule cells by cyclic AMP- and protein kinase C-dependent pathways

The present study was performed to characterize the possible involvement of cAMP synthesis and protein kinase C (PKC) activation in the DNA synthesis-stimulating effect of parathyroid hormone-related protein (PTHrP) in proximal tubule cells. We found that DNA synthesis was stimulated by 10 microM 8BrcAMP, and 1 microM Sp-cDBIMPS, two cAMP analogs, and also by 1 microM phorbol 12-myristate 13-acetate (PMA) and 100 microM 1,2-dioctanoyl-sn-glycerol, two PKC activators, and 10 nM [Cys23] human (h)PTHrP (24-35) amide in rabbit proximal tubule cells (PTC). Both Sp-cDBIMPS and PMA, at 1 microM, also increased DNA synthesis in SV40-immortalized mouse proximal tubule cells MCT. Human PTHrP (7-34) amide [PTHrP (7-34)] dose dependently stimulated DNA synthesis in a similar manner as [34Tyr]PTHrP (1-34) amide [PTHrP (1-34)], in PTC. PMA pre-treatment for 20 h, which downregulates PKC, completely blocked the effect induced by PTHrP (7-34), but not that of PTHrP (1-34), in the latter cells. In contrast, the same PMA pre-treatment abolished the DNA synthesis stimulation by PTHrP (1-34) and PTHrP (7-34) in MCT cells, which appear to have PTH receptors mainly coupled to phospholipase C and not adenylate cyclase. Our results indicate that the stimulatory effect of PTHrP on DNA synthesis in proximal tubule cells is mediated by a cAMP- and PKC-dependent mechanism.     

Glucagon-like peptide-1-(7-36)amide increases pulmonary surfactant secretion through a cyclic adenosine 3',5'-monophosphate-dependent protein kinase mechanism in rat type II pneumocytes

Glucagon-like peptide-1 (GLP-1) receptor messenger RNA has been identified in cells considered type II pneumocytes that are involved in the synthesis and secretion of the pulmonary surfactant. In an attempt to open new insights into the control of surfactant secretion, we studied the effects of glucagon-related peptides in this process. Accordingly, type II pneumocytes were isolated from Wistar rat lungs and cultured overnight with [methyl-14C]choline, and then the basal and stimulated secretions of [14C]phosphatidylcholine were measured. GLP-1(7-36)amide stimulated phosphatidylcholine secretion in a concentration-dependent manner in the 1-100 nM range; the concentration of the peptide that produced a half-maximal response was 10 nM. Exendin-4 induced similar effects. No changes were observed when GLP-1-(1-37), GLP-2, or exendin-(9-39) was added to the medium. However, the latter reversed the stimulatory effects of GLP-1-(7-36)amide and exendin-4. A study of the mechanism through which GLP-1-(7-36)amide exerts its stimulatory effect was carried out using different agents that are well known stimulants of phosphatidylcholine secretion. GLP-1-(7-36)amide did not produce any change in the stimulatory effect observed with terbutaline or 8-bromo-cAMP, suggesting the involvement of a cAMP-dependent protein kinase in the stimulatory effect of this peptide on phosphatidylcholine secretion. It was further supported by the use of inhibitors of protein kinases and by the stimulation of cAMP production in type II pneumocytes incubated with either GLP-1-(7-36)amide or exendin-4.     

Conditionally immortalized murine bone marrow stromal cells mediate parathyroid hormone-dependent osteoclastogenesis in vitro

PTH recruits and activates osteoclasts to cause bone resorption. These actions of PTH are thought to be mediated indirectly via type 1 PTH/PTH-related peptide receptors (PTH1Rs) expressed by adjacent marrow stromal or osteoblastic cells, although some evidence suggests that PTH may act directly on early hematopoietic osteoclast progenitors. We have established clonal, conditionally immortalized, PTH-responsive, bone marrow stromal cell lines from mice that harbor both a transgene encoding a temperature-sensitive mutant of the simian virus 40 large T antigen and deletion of a single allele of the PTH1R gene. Of 60 stromal cell lines isolated, 45 expressed functional PTH1Rs. During coculture with normal murine spleen cells, 5 of 42 such cell lines could support formation of tartrate-resistant acid phosphatase-positive, multinucleated cells (TRAP+ MNCs) in response to 1,25-dihydroxyvitamin D3, but only 2 of these did so in response to PTH. One of these, MS1 cells, expressed numerous cytokines and proteins characteristic of the osteogenic lineage and showed increased production of interleukin-6 in response to PTH. MS1 cells supported dose-dependent induction by rat (r) PTH-(1-34) (0.1-100 nM) of TRAP+ MNCs that expressed calcitonin receptors and formed resorption lacunae on dentine slices. This effect of PTH, which required cell to cell contact between MS1 and spleen cells, was mimicked by coadministration of cAMP analog and phorbol ester but only partially by either agent alone. The carboxyl-terminal fragment rPTH-(53-84) also induced osteoclast-like cell formation, but the maximal effect was only 30% as great as that of rPTH-(1-34). Importantly, rPTH-(1-34) induced TRAP+ MNC formation even when PTH1R-/- osteoclast progenitors (from fetal liver of mice homozygous for ablation of the PTH1R gene) were cocultured with MS1 cells. We conclude that activation of PTH1Rs on cells of the osteoclast lineage is not required for PTH-(1-34)-induced osteoclast formation in the presence of appropriate PTH-responsive marrow stromal cells. MS1 cells provide a useful model for further study of PTH regulation of osteoclastogenesis.     

Residues in the membrane-spanning and extracellular loop regions of the parathyroid hormone (PTH)-2 receptor determine signaling selectivity for PTH and PTH-related peptide

The parathyroid hormone (PTH)-2 receptor displays strong ligand selectivity in that it responds fully to PTH but not at all to PTH-related peptide (PTHrP). In contrast, the PTH-1 receptor (PTH/PTHrP receptor) responds fully to both ligands. Previously it was shown that two divergent residues in PTH and PTHrP account for PTH-2 receptor selectivity; position 23 (Trp in PTH and Phe in PTHrP) determines binding selectivity and position 5 (Ile in PTH and His in PTHrP) determines signaling selectivity. To identify sites in the PTH-2 receptor involved in discriminating between His5 and Ile5, we constructed PTH-2 receptor/PTH-1 receptor chimeras, expressed them in COS-7 cells, and tested for cAMP responsiveness to [Trp23] PTHrP-(1-36), and to the nondiscriminating peptide [Ile5, Trp23]PTHrP-(1-36) (the Phe23 --> Trp modification enabled high affinity binding of each ligand to the PTH-2 receptor). The chimeras revealed that the membrane-spanning/loop region of the receptor determined His5/Ile5 signaling selectivity. Subsequent analysis of smaller cassette substitutions and then individual point mutations led to the identification of two single residues that function as major determinants of residue 5 signaling selectivity. These residues, Ile244 at the extracellular end of transmembrane helix 3, and Tyr318 at the COOH-terminal portion of extracellular loop 2, are replaced by Leu and Ile in the PTH-1 receptor, respectively. The results thus indicate a functional interaction between two residues in the core region of the PTH-2 receptor and residue 5 of the ligand.     

Transmembrane residues of the parathyroid hormone (PTH)/PTH-related peptide receptor that specifically affect binding and signaling by agonist ligands

Polar residues within the transmembrane domains (TMs) of G protein-coupled receptors have been implicated to be important determinants of receptor function. We have identified mutations at two polar sites in the TM regions of the rat parathyroid hormone (PTH)/PTH-related peptide receptor, Arg-233 in TM 2 and Gln-451 in TM 7, that caused 17-200-fold reductions in the binding affinity of the agonist peptide PTH-(1-34) without affecting the binding affinity of the antagonist/partial agonist PTH-(3-34). When mutations at the TM 2 and TM 7 sites were combined, binding affinity for PTH-(1-34) was restored to nearly that of the wild type receptor. The double mutant receptors, however, were completely defective in signaling cAMP or inositol phosphate production in response to PTH-(1-34) agonist ligand. The results demonstrate that Arg-233 and Gln-451 have important roles in determining agonist binding affinity and transmembrane signaling. Furthermore, the finding that residues in TM 2 and TM 7 are functionally linked suggests that the TM domain topology of the PTH/PTH-related peptide receptor may resemble that of receptors in the rhodopsin/beta-adrenergic receptor family, for which structural and mutagenesis data suggest interactions between TMs 2 and 7.     

Prevention of estrogen deficiency-related bone loss with human parathyroid hormone-(1-34): a randomized controlled trial

CONTEXT: Short-term intermittent administration of parathyroid hormone (PTH) prevents bone loss from the spine in women treated with a gonadotropin-releasing hormone (GnRH) analog. However, the effects of a longer period of PTH administration on bone mass in estrogen-deficient women, particularly on the hip and on cortical bone of the total body, are unknown. OBJECTIVE: To determine whether more prolonged PTH administration can prevent estrogen deficiency bone loss from the hip, spine, and total body in young women with endometriosis receiving GnRH analog (nafarelin acetate) therapy. DESIGN: Randomized controlled trial. SETTING: General Clinical Research Center of a tertiary care, university-affiliated hospital. PATIENTS: Forty-three women between the ages of 21 and 45 years with symptomatic endometriosis. INTERVENTION: Nafarelin alone (200 microg intranasally twice daily) or nafarelin plus human parathyroid hormone-(1-34) (hPTH-[1-34]) (40 microg subcutaneously daily). MAIN OUTCOME MEASURES: The primary end points were bone mineral density (BMD) of the anterior-posterior and lateral spine, femoral neck, trochanter, radial shaft, and total body at 12 months of treatment. RESULTS: In the women who received nafarelin alone, the mean (SEM) BMDs of the anterior-posterior spine, lateral spine, femoral neck, trochanter, and total body were 4.9% (0.6%) (P<.001), 4.9% (0.8%) (P<.001), 4.7% (1.1%) (P<.001), 4.3% (0.9%) (P<.001), and 2.0% (0.6%) (P= .003) lower than at baseline after 12 months of therapy. In contrast, coadministration of hPTH-(1-34) increased BMD of the anterior-posterior spine by 2.1% (1.1%) (P=.09) and lateral spine by 7.5% (1.9%) (P=.002) and prevented bone loss from the femoral neck, trochanter, and total body, despite severe estrogen deficiency. Radial shaft BMD did not change significantly in either group. Serum bone-specific alkaline phosphatase and osteocalcin concentrations and urinary excretion of hydroxyproline and deoxypyridinoline increased 2-fold to 3-fold during the first 6 to 9 months of therapy in the women who received nafarelin plus hPTH-(1-34) and then declined. Changes in urinary deoxypyridinolone excretion were strongly predictive (r= 0.85) of changes in spinal BMD in the women who received nafarelin plus hPTH-(1-34). CONCLUSIONS: Parathyroid hormone prevents bone loss from the proximal femur and total body and increases lumbar spinal BMD in young women with GnRH analog-induced estrogen deficiency.     

Effects of calcitonin and parathyroid hormone on calcification of primary cultures of chicken growth plate chondrocytes

Few studies have been directed toward elucidating the action of calcitonin (CT) and parathyroid hormone (PTH) on growth plate chondrocytes, cells directly involved in longitudinal bone growth and provisional calcification. In this study, primary cultures of avian growth plate chondrocytes that calcify without the supplement of beta-glycerophosphate were used to investigate the effects of synthetic human CT and 1-34 bovine PTH on (1) cell division and growth; (2) the deposition of Ca2+ and inorganic phosphate (Pi); (3) the activity of alkaline phosphatase (AP), an enzyme long associated with the mineralization process; (4) the levels of proteoglycans; and (5) the synthesis of collagens. Added continually to preconfluent cultures from day 6 until harvest, CT (1-30 nM) and PTH (0.1-1.0 nM) increased mineral deposition; the maximal increase was seen between days 18-21 at 10 nM CT (175-260%) and 0.5 nM PTH (approximately 170-280%), both p < 0.001. CT had no significant effect on cellular protein, or AP-specific activity, whereas PTH increased cellular protein, DNA, proteoglycan, and collagen content of the cultures in a dosage-dependent manner. AP activity and levels of Type II and X collagens and fibronectin in the culture medium showed a biphasic response to PTH; maximal increases were seen at 0.5 nM between days 15-18. Longer exposure (days 21-27) to PTH at higher levels (5-10 nM) caused a marked decreased in AP activity but a lesser decrease in the collagens. These results indicate that CT and PTH can act directly on chondrocytes to stimulate mineralization, but that PTH specifically stimulated cell division and synthesis of cellular and extracellular proteins by growth plate chondrocytes. The implications of these findings with regard to Ca2+ homeostasis and bone formation are discussed.     

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.     

Converting parathyroid hormone-related peptide (PTHrP) into a potent PTH-2 receptor agonist

Most of the bone and kidney-related functions of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) are thought to be mediated by the PTH/PTHrP receptor. Recently, a homologous receptor, the PTH-2 receptor, was obtained from rat and human brain cDNA libraries. This receptor displayed the remarkable property of responding potently to PTH, but not to PTHrP. To begin to define residues involved in the ligand specificity of the PTH-2 receptor, we studied the interaction of several PTH/PTHrP hybrid ligands and other related peptide analogs with the human PTH-2 receptor. The results showed that two sites in PTH and PTHrP fully account for the different potencies that the two ligands exhibited with PTH-2 receptors; residue 5 (His in PTHrP and Ile in PTH) determined signaling capability, while residue 23 (Phe in PTHrP and Trp in PTH) determined binding affinity. By changing these two residues of PTHrP to the corresponding residues of PTH, we were able to convert PTHrP into a ligand that avidly bound to the PTH-2 receptor and fully and potently stimulated cAMP formation. Changing residue 23 alone yielded [Trp23]hPTHrP-(1-36), which was an antagonist for the PTH-2 receptor, but a full agonist for the PTH/PTHrP receptor. Residues 5 and 23 in PTH and PTHrP thus play key roles in signaling and binding interactions, respectively, with the PTH-2 receptor. Receptor-selective agonists and antagonists derived from these studies could help to identify the biological role of the PTH-2 receptor and to map specific sites of ligand-receptor interaction.     

Parathyroid hormone represses alpha 1(I) collagen promoter activity in cultured calvariae from neonatal transgenic mice

We examined the effect of PTH on the activity of alpha 1(I) collagen promoter fusion genes in cultured calvariae from transgenic mice. The parent construct, ColCAT 3.6, contains 3520 basepairs of 5' rat alpha 1(I) collagen DNA, 115 basepairs of untranslated alpha 1(I) collagen-coding DNA, and the bacterial chloramphenicol acetyltransferase reporter gene, while the 5'-deletion ColCAT 2.3 contains 2296 kilobases of rat alpha 1(I) collagen promoter sequence. Transgenic mouse lines harboring these collagen promoter fusion genes were developed using the oocyte microinjection technique, and for each construct, three different lines of mice were tested. Calvariae from 6- to 8-day-old transgenic mice were cultured for 48 h with or without bovine PTH-(1-34). ColCAT 3.6 and ColCAT 2.3 were expressed at comparable levels in calvariae and were inhibited by PTH. There were parallel decreases in the incorporation of [3H]proline into collagen and levels of the endogenous alpha 1(I) collagen mRNA and transgene mRNA. Forskolin at 10 microM mimicked the inhibitory effect of PTH on promoter activity in ColCAT 3.6 and ColCAT 2.3 calvariae. A RNase protection assay showed that the transgene was initiated correctly from the transgene promoter. These data show that PTH and cAMP can repress collagen promoter activity in calvariae from transgenic mice, suggesting that the alpha 1(I) collagen promoter may contain cis elements down-stream of -2.3 kilobases that mediate PTH and cAMP repression of collagen gene expression in bone. Cultured bone explants from transgenic mice can be used as a model to study hormonal regulation of alpha 1(I) collagen promoter constructs.     

Effects of PTH-rP(107-111) and PTH-rP(7-34) on adult cardiomyocytes

We investigated whether parathyroid hormone-related peptide (PTH-rP), recently found expressed in the heart, exerts growth and contractile effects on adult cardiomyocytes from rat hearts. Synthetic PTH-rP peptides were used covering either a protein kinase C (PKC)-activating domain [PTH-rP(107-111)], or an adenylate cyclase activating domain [PTH-rP(1-34) and PTH-rP(7-34)]. PTH-rP(107-111) (1 micro M) increased creatine kinase BB activity (CK-BB), a CK isoform re-expressed during cardiac hypertrophy, within 24 h by 62+/-12%. This induction was abolished in the presence of the mitogen-activated-protein (MAP)-kinase-kinase inhibitor PD 98059. PTH-rP(107-111) activated p42-MAP-kinase within 15 min, increased protein synthesis (19+/- 4%), total protein mass (19+/-5%), cell volume (45+/-7%), and cross-sectional area (38+/-9%) of cardiomyocytes. Activation of p42-MAP-kinase and increase in protein synthesis were abolished in presence of bisindolylmaleimide, a PKC inhibitor. PTH- rP(107-111) did not directly influence contractile activity but reduced the contractile response to isoprenaline. In contrast, PTH-rP(1-34) and PTH-rP(7-34) induced spontaneous contractile activity in 3-day-old cultures. This induction was abolished in presence of Rp-cAMPS, a protein kinase A inhibitor, indicating an involvement of cAMP in this response. PTH-rP(1-34) also increased the cellular accumulation of cAMP. It is concluded that PTH-rP exert direct effects on adult cardiomyocytes by activating either PKC via a functional domain covered by amino acids 107-111 or by activation of cAMP-dependent protein kinase via a functional domain covered by amino acids 7-34. Since these parts of PTH-rP have either no homology [PTH-rP(107-111)] or only a limited structural similarity [PTH-rP(7-34)] to parathyroid hormone, these activities of PTH-rP have to be clearly distinguished from those described for parathyroid hormone.     

Role of glycosylation in expression and function of the human parathyroid hormone/parathyroid hormone-related protein receptor

Parathyroid hormone (PTH) regulates calcium metabolism through a specific G protein-coupled, seven-transmembrane helix-containing receptor. This receptor also binds and is activated by PTH-related protein (PTHrP). The human (h) PTH/PTHrP receptor is a membrane glycoprotein with an apparent molecular weight of approximately 85000 which contains four putative N-glycosylation sites. To elucidate the functional role of receptor glycosylation, if any, we studied hormone binding and signal transduction in human embryonic kidney cells transfected with hPTH/PTHrP receptor (HEK-293/C-21). These cells stably express 300000-400000 receptors per cell. Inhibition of N-glycosylation with an optimized concentration of tunicamycin yielded completely nonglycosylated hPTH/PTHrP receptor (approximately 60 kDa). This receptor form is fully functional; it maintains nanomolar binding affinity for PTH- and PTHrP-derived agonists and antagonists. PTH and PTHrP agonists stimulate cyclic AMP accumulation and increases in cytosolic calcium levels. In addition, the highly potent benzophenone (pBz2)-containing PTH-derived radioligand [Nle8,18,Lys13(epsilon-pBz2),L-2-Nal23,Tyr34 3-125I)]bPTH(1-34)NH2 can photoaffinity cross-link specifically to the nonglycosylated receptor. The molecular weight (approximately 60000) of the band representing the photo-cross-linked, nonglycosylated receptor (obtained from the tunicamycin-treated HEK-293/C-21 cells) was similar to that of the deglycosylated photo-cross-linked receptor (obtained by enzymatic treatment with Endoglycosidase-F/N-glycosidase-F). Our findings indicate that glycosylation of the hPTH/PTHrP receptor is not essential for its effective expression on the plasma membrane or for the binding of ligands known to interact with the native receptor. The nonglycosylated hPTH/PTHrP receptor remains fully functional with regard to both of its known signal transduction pathways: cAMP-protein kinase A and phospholipase C-cytosolic calcium.     

Pharmacological characterization of a simple behavioral response mediated selectively by central adenosine A1 receptors, using in vivo and in vitro techniques

Mouse embryonic carcinoma P19 cell aggregates treated with retinoic acid (RA) sequentially differentiate into neurons and astrocytes, whereas attached cells develop a mesodermal phenotype. The expression of calcitonin (CT) and PTH/PTH-related protein (PTHrP) receptors was investigated in embryonic cells, and during neural and mesodermal differentiation. In embryonic P19 cells, specific binding of [125I]salmon (s) CT(1-32) ([125I]sCT(1-32)) was 56 fmol/mg protein, and of [125I]chicken (ch) [Tyr36]PTHrP(1-36) amide ([125I]chPTHrP(1-36)) < 0.5 fmol/mg protein. Correspondingly, cAMP was maximally stimulated 47-fold by sCT(1-32) (EC50 0.05 nM) and 3-fold by chPTHrP(1-36) (EC50 1.3 nM). Receptor autoradiography revealed specific binding of [125I]sCT(1-32) to the undifferentiated P19 cells, but not to RA induced neurons and astrocytes. At the same time, [125I]sCT(1-32) binding and cAMP accumulation by sCT were gradually decreased. But, specific binding of [125I]chPTHrP(1-36) was raised at least 6-fold compared with embryonic cells to 3 fmol/mg protein, in parallel with a 10-fold higher maximal cAMP accumulation. A similar, but delayed suppression of CT and stimulation of PTH/PTHrP receptor expression was observed during mesodermal cell differentiation. The results indicate that CT receptors are associated with undifferentiated P19 cells, whereas PTH/PTHrP receptors are expressed in RA induced neural and mesodermal cells.     

Parathyroid hormone-induced dopamine turnover in the rat medial basal hypothalamus

The parathyroid hormone (PTH) gene is expressed and translated in the rat hypothalamus, and the possibility that PTH may modulate neural activity was therefore examined in anesthetized rats. Intracerebroventricular (ICV) injections of 1.0 or 10.0 micrograms rat, human, or bovine PTH(1-34) was followed 60 min later by increased concentrations of DOPAC (dihydroxyacetic acid) and the DOPAC:dopamine (DA) ratio in the medial basal hypothalamus (MBH), but not in other (brainstem, cerebral cortex, cerebellum) regions of the brain. Tissue concentrations of norepinephrine and serotonin were unchanged by ICV PTH administration, although MBH concentrations of 5-hydroxyindolacetic acid (5-HIAA) were increased following PTH administration. An increase in MBH DA turnover (as indicated by an increased DOPAC:DA ratio) was also induced by the ICV injection of 10 micrograms PTH-related protein [PTHrP(1-34)]. Pretreatment with the receptor antagonists PTH(7-34) or PTHrP(7-34) completely blocked the subsequent DOPAC response to ICV PTH or PTHrP, respectively. The DOPAC concentrations in hypothalamic extracellular fluid (ECF), sampled by microdialysis, were also increased within 20 min of PTH(1-34) perfusion, in the absence of changes in the ECF concentrations of 5-HIAA. These results demonstrate that PTH and PTH-like peptides specifically increase DA turnover in the rat MBH and suggest novel roles for these hormones in neural regulation.     

Parathyroid hormone-related protein-(1-36) is biologically active when administered subcutaneously to humans

PTH-related protein (PTHrP) is responsible for most cases of humoral hypercalcemia of malignancy (HHM). It mimics the actions of PTH as a result of its structural homology with PTH and its ability to bind to and signal via the PTH/PTHrP receptor in bone and kidney. PTHrP-(1-36) appears to be one of several secretory forms of PTHrP. This peptide has been administered iv to normal volunteers previously and has been shown to produce effects that are qualitatively and quantitatively the same as those produced by PTH-(1-34). To determine whether PTHrP-(1-36) could be used sc in humans as a diagnostic reagent for elucidating the differences between HHM and hyperparathyroidism, we performed a 12-h dose-finding study examining whether sc PTHrP-(1-36) could elicit effects on mineral homeostasis. PTHrP-(1-36) administered sc in three doses (0.82, 1.64, and 3.28 micrograms/kg) to 21 normal women produced increases in circulating PTHrP-(1-36), reductions in serum phosphorus and the renal phosphorus threshold, increments in fractional calcium excretion and nephrogenous cAMP excretion, and increases in plasma 1,25-dihydroxyvitamin D. These changes were highly significant in statistical terms and were observed at doses that had no effect on serum calcium or endogenous PTH. These studies demonstrate the feasibility of using PTHrP-(1-36) as a diagnostic probe for future studies aimed at elucidating the differing pathophysiologies of HHM and hyperparathyroidism.     

Parathyroid hormone (1-34) increased total body bone mass in aged female rats

Daily subcutaneous administration of bovine parathyroid hormone (PTH)(1-34) stimulates bone formation and increases bone mass in rat tibiae, femora and lumbar spine. However, the effects of PTH on the whole body bone mineral content and density determined by dual energy x-ray absortiometry (DEXA) have not been previously reported in rats. Eighteen-month-old intact female rats were subcutaneously injected daily with 0, 40, 80 or 160 micrograms/kg/day of bovine PTH (1-34) for either 15 or 60 days. Whole body DEXA was performed at 1 day before autopsy, and bone area, bone mineral content (BMC) and bone mineral density (BMD) of the total body were determined. Total femoral, tibial and lumbar spine BMD was also determined ex vivo. Cancellous bone histomorphometry was performed on sections of double-labeled proximal tibial metaphyses. Whole body bone mineral content and density were significantly increased by 60 days, but not by 15 days, of PTH treatment at all dose groups compared with vehicle controls. Lumbar vertebral and total femoral BMD was significantly increased at all doses of PTH by 15 days of administration and further increased by 60 days. All doses of PTH increased trabecular bone area in proximal tibial metaphyses by 15 days and further increased by 60 days. All doses of PTH increased trabecular bone area in proximal tibial metaphyses by 15 days and further increased by 60 days. In proximal tibial cancellous bone, dose-dependent increases in percent labeled perimeter, mineral apposition rate and bone formation rate-bone volume referent were found between 40 and 160 micrograms/kg of PTH treatment by 15 days, and no further increases were found by 60 days. Our results showed that in aged female rats, bovine PTH(1-34) increased bone formation and total body bone mass.     

Effects of endothelin-1 on Ca2+ signaling and secretion in parathyroid cells

It has been previously reported that parathyroid cells express endothelin (ET) receptors and secrete ET-1 in an extracellular Ca2+ concentration ([Ca2+]e)-dependent manner. Here, we examined the effects of ET-1 on intracellular signaling and parathyroid hormone (PTH) secretion in dispersed bovine parathyroid (bPT) cells, which comprise several cell types including epithelial and endothelial cells, in two cell lines, the rat parathyroid epithelial (PT-r) and the bovine parathyroid endothelial (BPE-1) cells. An RNA-polymerase chain reaction analysis revealed that both ETA and ETB receptors are expressed in bovine parathyroid tissue and BPE-1 cells, and only the ETA receptor is expressed in PT-r cells. PT-r cells also expressed an inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) receptor, and ionomycin induced an increase in the intracellular Ca2+ concentrations ([Ca2+]i) in a Ca(2+)-deficient medium, indicating the presence of an operative intracellular Ca2+ pool in these cells. In cells bathed in 1 mM [Ca2+]e, ET-1 induced a rapid and transient increase in the Ins(1,4,5)P3 production, which was associated with a similar profile of increase in [Ca2+]i and with a peak response of about 800 nM. No changes in the profile of [Ca2+]i responses were observed in ET-1-stimulated cells in the presence of Ca2+ channel blockers, or in Ca(2+)-deficient medium, indicating that Ca2+ mobilization was not associated with Ca2+ entry. Furthermore, a sustained stimulation with ET-1 induced a decrease in [Ca2+]i below the prestimulatory level in a large population of cells, and the percentage of the cell population that shows the sustained decrease of [Ca2+]i increased in higher ET-1 concentrations. [Ca2+]i in PT-r cells was also controlled by a [Ca2+]e-dependent mechanism that changed [Ca2+]i from 28 to 506 nM in a 0.1-3 mM concentration range with an EC50 of 1.2 mM, which is comparable to that reported for bPT cells. In the same range of [Ca2+]e, PTH secretion from bPT cells was inhibited with an IC50 of 1 mM, and ET-1 increased PTH release in a dose-dependent manner but without affecting the IC50 for the [Ca2+]e-dependent inhibition. Thus, the parathyroid epithelial cells appear to respond to ET-1 in a unique way, and the ET autocrine system can be regarded as a possible mechanism to modulate the sensitivity of [Ca2+]e-dependent PTH release.     

Influence of aluminum on the regulation of PTH- and 1,25(OH)2D3-dependent pathways in the rat osteosarcoma cell line ROS 17/2.8

The role of hormonal status in the development of aluminum (Al)-dependent renal osteodystrophy, which is characterized by reduced bone matrix deposition, still remains largely unknown. To address this question, we used the osteoblast-like osteosarcoma cell line ROS 17/2.8 to evaluate the role of Al on parathyroid hormone (PTH)- and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent activities in these cells. Al (1 microM) caused an inhibition of basal and 1,25(OH)2D3-induced alkaline phosphatase, but only at low doses (< 1 nM) of the steroid. Al partly inhibited basal osteocalcin (OC) secretion in ROS cells (p < 0.001), and the dose-dependent increase in 1,25(OH)2D3-induced OC release by these cells was also reduced by 1 microM Al at low concentrations of the steroid (< or = 1 nM), whereas high doses of 1,25(OH)2D3 (> or = 5 nM) totally prevented the inhibiting effects of Al. Al also had strong inhibitory actions on PTH-dependent cAMP production by ROS cells over the concentration range tested (0.5-50 nM). This inhibitory action of Al was also observed for PTH-related peptide- (PTHrp, 50 nM) but not for Isoproterenol-dependent (100 nM) cAMP formation. To evaluate more fully the mechanism of this inhibition of cAMP formation, we investigated the effect of Al on toxin-modulated, G protein-dependent regulation of cAMP formation and on the activation of adenylate cyclase by Forskolin. Cholera toxin (CT, 10 micrograms/ml), applied to cells for 4 h prior to PTH challenge, enhanced cAMP production about 2-fold above PTH alone (p < 0.001), a process that was further stimulated by Al. Pertussis toxin (PT, 1 microgram/ml, 4 h) did not modify basal PTH-dependent cAMP formation by ROS cells. However, PT treatment prevented the inhibitory effect of Al on cAMP formation by these cells (p < 0.025). The stimulation of adenylate cyclase by Forskolin (0.1 and 1 microM), which bypasses G protein regulation, was not modified by Al, indicating that Al does not affect adenylate cyclase directly. Northern blot analysis of PTH receptor mRNA levels showed that Al did not modify PTH receptor message in ROS cells. Likewise, Western blot analyses of G protein subunits showed that Al did not significantly alter Gs alpha subunit levels, in accordance with the results obtained for cAMP-dependent formation in response to CT. In contrast, Gi alpha-1 and Gi alpha-2 subunits were decreased by Al treatment, consistent with PT-restricted increases in cAMP formation in Al-treated ROS cells. Taken together, these results suggest that Al has multiple actions in osteoblast-like ROS cells. The effects of Al are modulated by hormonal control of the pathways investigated. Al affects 1,25(OH)2D3-regulated functions only when this steroid is low. Al has large inhibitory effects on PTH- and PTHrp-dependent cAMP formation. This last feature is related to the ability of Al to alter the G protein transducing pathway for PTH/PTHrp-dependent formation of cAMP since it does not affect adenylate cyclase activity directly and does not affect the PTH receptor message level. Thus, Al has stronger deleterious effects in osteoblast-like cells with an already compromised 1,25(OH)2D3 status and can modulate specifically PTH/PTHrp-mediated cAMP formation at the postreceptor level.