Biological Activity Of Parathyroid Hormone Research Articles

The first reported case of pseudoparathryoidism type IB in Hong Kong: hypocalcemia in an adolescent boy with recurrent seizures

Pseudohypoparathyroidism (PHP) is a heterogeneous group of disorders characterized by hypocalcemia, hyperphoshatemia and inappropriately elevated serum parathyroid hormone (PTH) level due to end-organ resistance of biological activity of parathyroid hormone. It is due to epigenetic change at the GNAS locus and is divided into types Ia, Ib, Ic and II. We hereby describe the first reported case of pseudohypoparathyroidism type Ib Hong Kong.

Calcinosis Cutis in a Newborn with Transient Pseudohypoparathyroidism

Pseudohypoparathyroidism (PHP) is a heterogenous group of disorders characterized by hypocalcemia with hyperphosphatemia, increased serum concentration of parathyroid hormone (PTH), and insensitivity to the biological activity of PTH. Calcinosis cutis, the cutaneous deposition of calcium salts in the dermis, is a rare clinical symptom in infancy. The deposition of calcium in the skin may be classified as dystrophic, metastatic, idiopathic, and iatrogenic. Although a few infants with PHP and calcinosis cutis have been reported, to the authors' knowledge, the combination of neonatal transient PHP and calcinosis cutis associated with calcium treatment has not been previously reported. The authors report a newborn boy with transient PHP presenting with early hypocalcemia, hyperphosphatemia, increased PTH levels, and calcinosis cutis after intravenous treatment of calcium gluconate.

Variant of Adynamic Bone Disease in Hemodialysis Patients: Fact or Fiction?

Adynamic bone disease is a type of renal osteodystrophy characterized by low bone turnover and paucity of bone cells. It was proposed that a new type of this disease featuring high osteoclastic resorption without parathyroid hormone stimulus and designated adynamic bone disease variant occurs in hemodialysis patients. The present study is designed to evaluate the frequency and characteristics of both diseases in a large series of bone biopsy specimens. We reviewed 1,160 bone biopsy specimens from hemodialysis patients. Specimens in which adynamic bone disease was diagnosed were selected and categorized as classic or variant based on osteoclastic surface. In 218 bone biopsy specimens (18.8%), adynamic bone disease was identified, whereas the variant form was identified in 35 specimens (38.8%). Biopsy specimens categorized as the variant form were from patients who were younger and had greater phosphorus and parathyroid hormone levels. Histologically, the variant form presented greater osteoid volume, fibrosis volume, osteoid surface, osteoblast surface, and eroded surface. Similarly, values for all dynamic parameters were greater in the variant group. Osteoclastic surface correlated with phosphorus level, parathyroid hormone level, and osteoblast surface. Age and osteoblast surface were identified as independent determinants of the variant form. Adynamic bone disease variant seems to occur in younger hemodialysis patients with greater levels of parathyroid hormone, which acts on cell-covered bone surfaces. It probably is a transitional phase from low- to high-turnover status, rather than a true entity within the spectrum of renal osteodystrophy.

PTH 1-84 and PTH “7-84” in the noninvasive diagnosis of renal bone disease

Background: The intact parathyroid hormone (PTH) assay evaluates levels of serum 1-84 PTH and other N-terminally truncated PTH fragments, mainly PTH “7-84.” This PTH molecule has been found experimentally to interfere with biological activity of PTH 1-84, perhaps through its binding to the PTH receptor complex. Therefore, assuming that high levels of PTH 7-84 are a cause of bone resistance to PTH, it has been hypothesized that a decreased 1-84 to 7-84 PTH ratio caused by a relative increase in PTH 7-84 level might help in the noninvasive diagnosis of low-turnover osteodystrophy (LTO). Methods: This study was performed in 35 patients with chronic renal failure on hemodialysis therapy who underwent bone biopsy for a histological, histomorphometric, and histodynamic study. In addition, blood samples were obtained for intact PTH, 1-84 PTH, and total PTH assays. PTH 7-84 level was obtained from the difference between total and 1-84 PTH assay results. Results: Nine patients had LTO (8 patients, adynamic bone disease; 1 patient, osteomalacia), 12 patients had hyperparathyroidism (HP), and 14 patients had mixed osteodystrophy (MO). On average, 1-84 PTH levels were approximately 60% of mean values for intact PTH. The two assays were strictly correlated. Average 1-84 to 7-84 PTH ratios were 1.57 ± 0.85, 1.73 ± 1.31, and 1.95 ± 2.1 in the three histological groups (LTO, HP, and MO, respectively), with no significant difference. Conclusion: Contrary to previous expectations, results do not favor the hypothesis of a role of 7-84 PTH in bone resistance in renal osteodystrophy. The 1-84 to 7-84 PTH ratio is not a marker of LTO and is of no use in noninvasive histological diagnosis. © 2002 by the National Kidney Foundation, Inc.

Synthetic parathyroid hormone fragments shortened at the amino terminus stimulate glucose-6-phosphate dehydrogenase activity in the distal renal tubule.

The cytochemical bioassay, using glucose-6-phosphate dehydrogenase (G6PD) activity in the distal convoluted tubule of a guniea pig as an index, is specific and the most sensitive method of evaluating the biological activity of parathyroid hormone (PTH). Using this method, biological activities of the amino- or carboxyl-terminal PTH fragments and analogues, human (h) PTH-(3-34), [Tyr34]hPTH-(7-34)amide(NH2), [Tyr34]hPTH-(13-34)NH2, hPTH-(39-84), hPTH-(51-84), hPTH-(69-84), were tested over a concentration range of 10(-16) to 10(-13) M. In addition, the combined effect of these hormones with human or bovine PTH-(1-84) and the effect of dibutyryl (Bu)2) cAMP were also evaluated. In the 14-min time-course study, amino-terminal PTH fragments and analogues induced cyclic changes of G6PD activity with shorter cycle lengths in higher concentrations and with constant peak heights regardless of the concentrations. Human and bovine PTH-(1-84) showed the same activity on G6PD activation at 6 min. hPTH-(3-34), [Tyr34]hPTH-(7-34)NH2, and [Tyr34]hPTH-(13-34)NH2 were equipotent with PTH-(1-84) on a molar basis, and none of these analogues inhibited PTH-(1-84) even with doses up to 240 times that of PTH-(1-84). Carboxyl-terminal PTH showed no effect. (Bu)2cAMP mimicked the effect of PTH-(1-84) on G6PD activation in time course and dose response. We conclude that the amino terminus is not essential for the biological activity of PTH in the cytochemical bioassay.

Differential effects of parathyroid hormone responsive cultured human cells on biological activity of parathyroid hormone and parathyroid hormone inhibitory analogues.

We have employed parathyroid hormone (PTH) responsive human cells cultured from dermis or giant cell tumors of bone (GT) to evaluate the biological properties of a newly developed in vivo PTH inhibitor, [Tyr34]bPTH-(7-34)-amide (PTH-Inh). Short periods of incubation of cells from dermis or GT with maximal stimulatory concentrations of PTH in the presence of increasing concentrations of PTH-Inh resulted in a dose-dependent inhibition of the adenosine cyclic 3',5'-phosphate (cAMP) response (Ki = 3 X 10(-7) M and 4.2 X 10(-7) M for GT and dermal cells, respectively). In both cell cultures, PTH-Inh alone did not increase cAMP levels, and in desensitization experiments, preincubation with PTH-Inh alone did not desensitize cells to PTH. Hence, the analogue displayed no agonist properties. Unexpectedly, when PTH-Inh was incubated with dermal cells in the presence of PTH, the PTH-Inh failed to block desensitization, suggesting a loss of biological effectiveness of the inhibitor. When medium containing PTH-Inh alone was removed from dermal cells and tested for inhibition of the acute PTH response in untreated cells, there was apparent loss of inhibitory efficacy (t1/2 = 20 h). In contrast, incubation of native PTH or bPTH-(1-34) with cells did not affect the biological activity of these ligands. Unlike the dermal cells, the PTH-Inh did block desensitization to PTH in GT, and there was no loss of inhibitor efficacy when medium containing PTH-Inh was incubated with GT (48 h) and then tested in untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

A homologous biological probe for parathyroid hormone in human serum.

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.

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The structural requirement for full biological activity of parathyroid hormone (PTH), on 84-amino acid single-chain polypetide is satisfied by the 34-amino acid fragment at its NH2-terminal. The procedure to obtain a radioligand with both high specific radioactivity and bioactivity includes a gentle iodination using electrolysis or lactoperoxidase. The sulfur-free analogue [Nle8, Nle18, Tyr34] bPTH (1-34) amide, is stable during the oxidation and is also used as a radioligand. The PTH receptor in plasma membrane of chicken renal cortex is a single component with high affinity to bPTH (1-34), (Kd=7 to 10nM). Guanyl nucleotides stimulate not only PTH dependent adenylate cyclase activity but also the dissociation of 125I-bPTH (1-34) from a plasma membrane of canine renal cortex.Pseudohypoparathyroidism Type 1 (PHP-1) is an inherited disease characterized by its resistance to stimulation of adenylate cyclase by PTH. The erythrocytes of patients with PHP-1 show partially deficient activity of a guanyl nucleotide regulatory protein that is required for a functional coupling of hormone receptors and catalytic adenylate cyclase. It is also reported, however, that some of the clinically evident PHP-1 patients are possessed of quantitatively and qualitatively normal N-protein activities in erythrocytes. These findings suggest the biochemical and genetic heterogeneity of the disorder classified as PHP-1.

Use of osmotic minipumps for delivery of parathyroid hormone.

To determine if osmotic minipumps can be used for the local delivery of parathyroid hormone (PTH), we examined the bone resorbing activity of PTH in minipumps, either removed and assayed in bone organ cultures or released over rat calvaria. Biological activity of PTH was maintained for up to 6 days when the hormone solution contained serum and the minipumps and tubing were siliconized and flushed with diluent prior to use. Addition of cysteine did not enhance activity.

Parathyroid hormone bioassay using human kidney cortical cells in primary culture.

The biological activity of parathyroid hormone (PTH) has been investigated by measuring intracellular accumulation of cyclic AMP (cAMP) in human kidney cortical cultures. Enzyme dispersed cortical cells from non-invaded kidney poles of patients undergoing nephrectomy for cancer were used after 5 days of primary culture. Bovine PTH (1-84) produced a significant increase of cAMP accumulation in cultured cells at a dose (53.7 ng/ml) 10-fold lower than that found for the minimal stimulatory effect when using preparations of human purified plasma membranes. The action of bovine PTH (1-84) was very rapid, a response was detected after 5 min and a ceiling effect after 30 min. Cortical cells showed a slightly lower sensitivity to synthetic bovine PTH (1-34) (half maximal increase dose: 0.66 microgram/ml), compared to bovine PTH (1-84) (half maximal increase dose: 0.32 microgram/ml), but revealed a higher sensitivity to human PTH purified from the medium of parathyroid cell cultures (half maximal increase dose: 11.2 ng/ml). Arginine-vasopressin (AVP) also increased the cAMP accumulation of kidney cortical cultured cells, with a potency and efficacy lower than that of human 'culture' PTH, while in kidney medullary cells in primary culture AVP exerted a strong response and the effect of PTH was poor or absent. Calcitonin and glucagon were weak stimulators of kidney cortical cell cAMP accumulation.

A sensitive bioassay of parathyroid hormone in plasma.

Understanding of calcium metabolism in health and disease has been retarded by the lack of an adequately sensitive bioassay of parathyroid hormone. The problem of dissociation of bioactivity and immunoactivity, well recognized for other polypeptide hormones, is exaggerated in the case of parathyroid hormone by the disproportionately long half-time in the circulation of the immunoreactive fragments. A new method of assaying the biological activity of parathyroid hormone in plasma has been developed, based on the cytochemical methods which have yielded highly sensitive bioassays of other polypeptide hormones. It depends on the stimulation of glucose 6-phosphate dehydrogenase activity in the distal convoluted tubules of segments of guinea-pig kidney maintained in vitro, and measured by microdensitometry. The limit of sensitivity of the assay is 5 fh/ml (bPTH); the index of precision is 0.09 +/- 0.04 (mean +/- SEM; n = 11).

MEMBRANES FROM A TRANSPLANTABLE OSTEOGENIC SARCOMA RESPONSIVE TO PARATHYROID HORMONE AND PROSTAGLANDINS: REGULATION OF ADENYLATE CYCLASE AND OF HORMONE METABOLISM

Adenylate cyclase activity in particulate fractions from a transplantable rat osteogenic sarcoma was stimulated in a dose-dependent manner by prostaglandins E1 and E2 (PGE1 and PGE2) and parathyroid hormone (PTH). Prostaglandin F2alpha was active at a high concentration (3 x 10(-4) mol/l). Pretreatment of membranes with collagenase plus hyaluronidase reduced the magnitude of the PTH effect but did not affect the size of the PGE1 effect. Guanosine 5'-triphosphate and its synthetic analogue 5'-guanylylimidodiphosphate (Gpp(NH)p) activated adenylate cyclase in particulate preparations from the osteogenic sarcoma. The latter agent produced much larger effects, although the concentrations required for half-maximal enzyme activation were the same for both agonists (approximately 2 x 10(-6) mol/l). The effects of PTH and Gpp(NH)p were supra-additive at some concentrations of hormone. The effects of PGE1 and Gpp(NH)p were supra-additive at all hormone concentrations tested. Pre-incubation of membrane particles for 6 min with PTH produced an enzyme activation which was not reversed by dilution through washing; pre-incubation with PGE1 did not produce this effect. The response of membrane adenylate cyclase to Gpp(NH)p (10(-4) mol/l) was 75% greater in preparations pre-incubated with PTH than in membranes pre-incubated in buffer alone or in buffer containing PGE1. The basal rate of cyclic AMP production in the adenylate cyclase assay system decreased over a 35 min incubation period. This decrease was prevented by addition of PTH or PGE1. Addition of NaF or Gpp(NH)p produced a steady increase in the rate of production of cyclic AMP with time. Membrane preparations did not reduce the biological activity of PTH and did not degrade 125I-labelled PTH. The results demonstrate that the PTH- and PGE-responsive adenylate cyclases of the osteogenic sarcoma have distinctly different properties and that particulate preparations of the tumour do not metabolize PTH.

Metabolism and biological activity of parathyroid hormone in renal cortical membranes.

Recent studies from several laboratories have documented the presence of fragments of parathyroid hormone in blood or peripheral tissues or in both. Inasmuch as amino-terminal fragments are known to be biologically active, it has been suggested that fragments, rather than the intact polypeptide of 84 amino acids, might be the active molecular species in tissue fluids. Accordingly, the metabolism of native bovine parathyroid hormone, bPTH-(1-84), was studied in purified renal cortical membranes from several species and correlated with hormonal stimulation of adenylyl cyclase in these membranes in vitro. Analysis of whole incubation mixtures or membrane-bound hormone by gel electrophoresis and gel chromatography after incubation of [3H]bPTH-(1-84) or 125-I-labeled bPTH-(1-84) or unlabeled biologically active bPTH-(1-84) with purified canine renal cortical membranes revealed no evidence of proteolysis, and yet the uncleaved hormone readily stimulated adenylyl cyclase. Kinetic studies of hormone-stimulated adenylyl cyclase activity revealed no difference in rate of onset of activity between bPTH-(1-84) And the active synthetic amino-terminal tetratriacontapeptide bPTH-(1-34), and hence there was no evidence of precursor-product relationship between the native hormone and an active amino-terminal fragment. The results suggest, insofar as the activity detected in these membranes reflects the biological response of the hormone in vivo, that the native hormone is indeed biologically active at the receptor level directly without the requirement for cleavage into active fragments.

Analysis of the requirements for parathyroid hormone action in renal membranes with the use of inhibiting analogues.

Two synthetic analogues of bovine parathyroid hormone (PTH) with NH2-terminal modifications, PTH-(3-34) and [desamino-Ala-1]PTH-(1-34), were found to lack agonist activity but to demonstrate antagonist properties when tested in the rat renal cortical adenylyl cyclase assay in vitro against the native hormone or against PTH-(1-34), the active synthetic NH2-terminal tetratriacontapeptide. The inhibition exhibited by these analogues was proportional in degree to the dose of inhibitor, abolished by oxidation of the analogue, reversible by addition of an excess of active hormone, and specific for parathyroid hormone-stimulated renal adenylyl cyclase. No inhibition of basal or sodium fluoride-stimulated renal adenylyl cyclase could be demonstrated. Two other synthetic bovine analogues, PTH-(13-34) and PTH-(1-26), were devoid of agonist and antagonist properties. The over-all results suggest that the requirements for receptor binding of parathyroid hormone are rather broad. Conformational factors or binding interactions involving specific residues, or both seem to require the entire sequence from residue 3 to residue 27 for receptor binding to occur. A dichotomy between receptor binding and adenylyl cyclase activation was demonstrated only by alterations or deletions involving the first 2 NH2-terminal residues of the hormone and emphasizes the importance of these residues in eliciting the biological activity of parathyroid hormone. The two antagonists, [desamino-Ala-1]PTH-(1-34) and PTH-(3-34), should be useful in further analysis of the initial steps in hormone action.

The isolation and partial purification of a non-parathyroid hormone calcemic fraction from bovine parathyroid glands.

The isolation and partial purification of a new calcemic material found in bovine parathyroid glands is described. This material, designated calcemic fraction-A, is different from parathyroid hormone but possesses on a weight basis approximately one-half to one-third the biological activity of parathyroid hormone in both in vivo and in vitro test systems. The biosynthesis of calcemic fraction-A, studied in an in vitro incubation system, was inhibited by high concentrations of calcium in the incubation medium. Analysis of calcemic fraction-A by radioimmunoassay indicated it to be immunoreactive with anti-parathyroid hormone antiserum in a fashion identical to authentic parathyroid hormone but with lower potency. Based on gel filtration it appears to be larger in size than parathyroid hormone. (Endocrinology 89: 1440, 1971)

Effect of Oxidation and Reduction upon the Biological Activity of Parathyroid Hormone

Parathyroid hormone loses biological activity upon oxidation with hydrogen peroxide. Part or all of this lost activity can be regained by subsequent reduction with cysteine. The extent and reversibility of this oxidation is dependent upon pH.