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Prolonged signaling at the parathyroid hormone receptor 1 (PTHR1) correlates with the capacity of a ligand to bind to a G protein-independent receptor conformation (R 0 ). As long-acting PTH (LA-PTH) ligands hold interest as potential treatments for hypoparathyroidism (HP), we explored the structural basis in the ligand for stable R 0 binding and prolonged cAMP signaling. A series of PTH/PTHrP hybrid analogs were synthesized and tested for actions in vitro and in vivo. Of the series, [Ala 1,3,12 ,Gln 10 ,Arg 11 ,Trp 14 ]-PTH(1-14)/PTHrP(15-36) (M-PTH/PTHrP) bound with high affinity to R 0 , induced prolonged cAMP responses in UMR106 rat osteoblast-derived cells, and induced the most prolonged increases in serum calcium (sCa) in normal rats. Daily s.c. injection of M-PTH/PTHrP into thyroparathyroidectomized (TPTX) rats, a model of HP, normalized sCa without raising urine Ca. In contrast, oral alfacalcidol, a widely used treatment for HP, normalized sCa, but induced frank hypercalciuria. M-PTH/PTHrP exhibited low solubility in aqueous solutions of neutral pH; however, replacement of Leu18, Phe22, and His26 with the less hydrophobic residues, Ala, Ala, and Lys, at those respective positions markedly improved solubility while maintaining bioactivity. Indeed, we recently showed that the resultant analog [Ala 18,22 ,Lys 26 ]-M-PTH/PTHrP or LA-PTH, effectively normalizes sCa in TPTX rats and mediates prolonged actions in monkeys. These studies provide useful information for optimizing PTH and PTHrP ligand analogs for therapeutic development. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research., (© 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.)

Background and Objectives: Fibroblast growth factor 23 (FGF23) is an independent risk factor for mortality in patients with ESRD. Before FGF23 testing can be integrated into clinical practice of ESRD, further understanding of its determinants is needed., Design, Setting, Participants, & Measurements: In a study of 67 adults undergoing peritoneal dialysis, we tested the hypothesis that longer dialysis vintage and lower residual renal function and renal phosphate clearance are associated with higher FGF23. We also compared the monthly variability of FGF23 versus parathyroid hormone (PTH) and serum phosphate., Results: In unadjusted analyses, FGF23 correlated with serum phosphate (r = 0.66, P < 0.001), residual renal function (r = -0.37, P = 0.002), dialysis vintage (r = 0.31, P = 0.01), and renal phosphate clearance (r = -0.38, P = 0.008). In adjusted analyses, absence of residual renal function and greater dialysis vintage associated with higher FGF23, independent of demographics, laboratory values, peritoneal dialysis modality and adequacy, and treatment with vitamin D analogs and phosphate binders. Urinary and dialysate FGF23 clearances were minimal. In three serial monthly measurements, within-subject variability accounted for only 10% of total FGF23 variability compared with 50% for PTH and 60% for serum phosphate., Conclusions: Increased serum phosphate, loss of residual renal function, longer dialysis vintage, and lower renal phosphate clearance are associated with elevated FGF23 levels in ESRD patients undergoing peritoneal dialysis. FGF23 may be a more stable marker of phosphate metabolism in ESRD than PTH or serum phosphate.

We examined mechanisms of down-regulation of PTH receptors and desensitization of the PTH-stimulated increase in intracellular cAMP in clonal rat osteosarcoma cells, ROS 17/2.8. ROS cells treated with 10 nM [Nle8,Nle18,Tyr34] bovine (b) PTH-(1-34) amide (NlePTH) for 3 days showed loss of specific PTH binding and PTH-stimulated cAMP accumulation to 10% of that in vehicle-treated control cells. Treatment of these cells with both 0.5 mM 8-bromo-cAMP (8-Br-cAMP) and 1 mM methylisobutylxanthine or 100 ng/ml cholera toxin for 3 days elicited no change in either of these responses. Treatment with 10 nM NlePTH for 3 days did not modify the cAMP accumulation stimulated by 30 microM forskolin or 1 micrograms/ml cholera toxin, indicating that agonist-specific desensitization of PTH-stimulated cAMP accumulation is not due to diminished activity of either the stimulatory guanyl nucleotide regulatory subunit (Gs) or the catalytic subunit of the adenylate cyclase. Treatment of ROS cells with pertussis toxin (PT; 10 ng/ml) for 12, 24, 48, and 72 h increased specific PTH binding by 21%, 28%, 35%, and 39%. The increase in PTH binding was associated with a parallel increase in PTH-stimulated cAMP accumulation and was due to an increase in the number of PTH receptors. PTH receptor affinity remained constant (apparent Kd = 0.3 nM). PT treatment of the cells partially blocked agonist-specific PTH receptor down-regulation. PT catalyzed ADP ribosylation of 41K and 39K membrane proteins, consistent with the alpha-subunits of Gi and Go, respectively. In conclusion, agonist-induced PTH receptor down-regulation in ROS 17/2.8 cells is cAMP independent and can be reversed by PT treatment. PTH receptor expression in these cells appears to be under tonic inhibitory control by mechanisms involving a PT-sensitive G protein(s).

PTH binds to specific receptors that are coupled to adenylate cyclase and activate cAMP-dependent protein kinase. Since it has been shown that PTH activates phospholipid inositol metabolism, we investigated whether PTH influences protein kinase-C (PKC) activity in rat osteosarcoma (ROS) cells 17/2.8 that contain a large number of PTH receptor. Incubation of ROS cells with PTH or phorbol 12-myristate 13-acetate (PMA) for 1-30 min caused a rapid and transient decrease in PKC activity in the cytosol, which was associated with a transient increase in PKC activity in the membrane fraction. After 1, 5, 15, and 30 min of incubation with PTH, cytosolic PKC activity decreased to 57%, 74%, 84%, and 93% of the control value, whereas membrane PKC activity increased to 156%, 122%, 111%, and 106% of the control value, respectively. After PMA treatment for 1, 5, 15, and 30 min, cytosolic PKC activity decreased by 81%, 74%, 63%, and 44%, whereas membrane-bound PKC activity increased by 83%, 44%, 28%, and 17%, respectively. The effects of PTH and PMA on PKC were dose dependent, with ED50 values of 0.3 nM PTH and 4 nM PMA. Chronic treatment of ROS cells for 3 days with PMA caused depletion of total PKC activity in cytosolic and membrane fractions to less than 10% of that in control cells. Conversely, chronic treatment of ROS cells with PTH did not deplete PKC. In addition, chronic treatment of ROS cells with PTH inhibited the responsiveness of PKC activity to subsequent acute PTH challenge, but not to acute PMA challenge, suggesting specific desensitization of this response by PTH. Activation of cytosolic PKC by diolein, phosphatidylserine, and calcium caused phosphorylation of many cytosolic proteins, including those having apparent mol wt of 39K, 35K, 33K, 25K, 19K, and 16K. Pretreatment of ROS cells with PTH resulted in a transient decrease in the phosphorylation of these cytosolic proteins by PKC. This decrease in cytosolic protein phosphorylation by treatment with PTH is temporally associated with PTH-stimulated translocation of PKC activity from the cytosol to the membranes. These data suggest a potential role for PKC in the mechanism of action of PTH in ROS cells.

We and others have recently shown that amino terminal sequences of parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP), which share a 62% homology within the first 13 residues, bind to the same receptor on ROS 17/2.8 cells. The remaining PTHrP sequence is markedly different from PTH, suggesting that receptor binding may be dependent on the first 13 amino acids of either peptide. However, since the amino acid residues 14-34 have previously been recognized as an important binding domain for PTH, conformational similarity within this portion's secondary structure of both peptides could contribute to their capacity to bind to the same receptor. To test this hypothesis, we synthesized [Tyr36,Cys38]PTHrP-(14-38) and [Tyr34]bPTH(14-34)NH2, and studied binding of both peptides to the common PTH/PTHrP receptor on ROS 17/2.8 cells. Radioiodinated, HPLC-purified [Nle8,18, Tyr34]bPTH(1-34)NH2 (NlePTH) and [Tyr36]PTHrP-(1-36)NH2 were used to functionally define receptor binding requirements. [Tyr36,Cys38]PTHrP(14-38) and [Tyr34]bPTH(14-34)NH2 competed with 125I-NlePTH for binding sites on ROS 17/2.8 cells with apparent Kds of 10 microM and 50 microM respectively. Both peptides also competed with 125I-[Tyr36]PTHrP(1-36)NH2 with apparent Kds of 30 microM and 10 microM respectively. In the same assay system, NlePTH and [Tyr36,Cys38]PTHrP(1-38)inhibited binding of either radioiodinated ligand with apparent Kds of 0.3 and 1.0 nM. These studies indicate that although [Tyr34]bPTH(14-34)NH2 and [Tyr36,Cys38]PTHrP(14-38) share virtually no sequence homology, their secondary structures must be sufficiently similar to permit binding to a common PTH/PTHrP receptor.

A method of measuring the biological activity of parathyroid hormone (PTH) in human serum that depends on the activation of its natural target enzyme, human renal cortical adenylate cyclase, is described. Optimal sensitivity ranging in different assays from 14 to 20 pg 1-34 hPTH/ml was achieved in the presence of the GTP-analogue GppNHp (10 mumol/L), 5 mmol/L MgCl2 and 1.25 mmol/L EGTA. Basal and stimulated cAMP production was reproducible within assays (c.v. below 7%, S.E.M., n = 3) and between assays (c.v. 5 to 14%, S.E.M., n = 4). The recovery of 1-34 hPTH added to individual test sera averaged 94%. The specificity of the method was established as follows: 1.) Other tested hormones, at 100 ng/ml, were ineffective; 2.) In the majority of peripheral sera from patients with hyperfunctioning parathyroid glands elevated bio-activity was detected; 3.) The circulating bio-activity fell rapidly after removal of parathyroid adenomata; 4.) Treatment with antisera for hPTH reduced the bio-activity; 5.) A PTH-antagonist inhibited the bio-activity.