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J T Potts

Publications and source records attributed to J T Potts.

At least 109 records · Page 6Linked to original sources
PubMed1987

Highly sensitive two-site immunoradiometric assay of parathyrin, and its clinical utility in evaluating patients with hypercalcemia.

We have developed a highly sensitive, two-site immunoradiometric assay (IRMA) for human parathyrin (PTH) that is specific for the intact, secreted, biologically active 84-amino-acid peptide. This assay has several technical advantages: it does not detect even high concentrations of inactive carboxyl-terminal fragments, results are available within 24 h, and the detection limit for intact hormone is low (1 ng/L). The assay readily measures concentrations of PTH in all healthy subjects and distinguishes these values from low or undetectable PTH values observed in clinical situations in which PTH secretion is expected to be suppressed. We found complete separation of results from 37 patients with surgically proven hyperparathyroidism and those from 23 patients with hypercalcemia associated with malignancy, the latter having PTH values at or below the lower limits of normal for this assay. The sensitivity, specificity, and rapid turnaround time of this two-site IRMA should advance the laboratory evaluation of patients with disorders of calcium metabolism.

Humans↗
PubMed1986

Restoration of spinal bone in osteoporotic men by treatment with human parathyroid hormone (1-34) and 1,25-dihydroxyvitamin D.

Daily subcutaneous injection of a synthetic human parathyroid hormone fragment, combined with daily ingestion of 1,25(OH)2 vitamin D, significantly increased trabecular bone density in the spine (p less than .01), and improved intestinal calcium and phosphorus absorption and total body retention of dietary calcium and phosphorus in middle-aged men with idiopathic osteoporosis. The increases in spinal bone mineral were marked and progressive during a year of treatment. These results indicate that increasing intestinal absorption of dietary calcium while simultaneously stimulating new bone formation with small doses of parathyroid hormone can restore spinal bone in osteoporotic men.

Adult↗
PubMed1986

Inhibition of the in vivo parathyroid hormone-mediated calcemic response in rats by a synthetic hormone antagonist.

The parathyroid hormone (PTH) analog, [Tyr34]bovine PTH-(7-34)-amide, can inhibit the PTH-mediated elevation of plasma calcium in thyroparathyroidectomized rats in vivo. The analog is devoid of PTH-like agonist activity in this system. Repeated doses of analog inhibit the animal's calcemic response to PTH. The elevation in serum calcium levels mediated by PTH in this assay reflects PTH action (calcium mobilization) on bone. Earlier studies demonstrated antagonist properties of the analog in a renal-based assay; PTH-stimulated increases in urinary phosphate and cyclic AMP excretion were completely inhibited by the synthetic analog. Along with previous studies, this report indicates that [Tyr34]bovine PTH-(7-34)-amide is an effective in vivo antagonist for several major parameters of PTH action in both kidney and bone.

Animals↗
PubMed1985

Evaluation of an in vitro parathyroid hormone antagonist in vivo in dogs.

A potent competitive inhibitor of PTH-stimulated biological responses in vitro, [Nle8,Nle18,Tyr34] bovine PTH (bPTH)-(3-34)amide, was evaluated in vivo in dogs. These studies confirm observations in vitro, suggesting that positions 1 and 2 of the peptide are critical to its biological activity. However, unlike the results from studies in vitro, this PTH analog is a weak agonist with effects on parathyroid target tissues that produce hypercalcemia and phosphaturia and increase urinary cAMP excretion. Assessed by these three parameters of hormonal action in vivo, the estimated potency of this analog is less than 1% of that of the intact hormone. In addition, PTH-induced biological responses were not inhibited by relatively large doses of the bPTH-(3-34) analog. These results emphasize the need for a systemic, integrated approach, combining chemical with biological studies, to design effective inhibitors of hormonal action in vivo. Although the rationale for introducing particular modifications into the peptide structure is most frequently based on bioassays performed in vitro, the success of the strategy chosen must rely, ultimately, upon the demonstration of specific biological properties in vivo.

Animals↗
PubMed1985

Rat parathyroid hormone-(1-34) fragment: renal adenylate cyclase activity and receptor binding properties in vitro.

The rat PTH molecule contains five sequence differences from either the bovine or the human hormone within the biologically active 1-34 region. A synthetic rat 1-34 peptide was tested for activity by in vitro activation of canine and rat renal adenylate cyclase and binding to canine renal membrane receptors. The mean potency of 21,400 Medical Research Council units/mg in the canine adenylate cyclase system and 24,900 in the rat system was 8- to 10-fold higher than human 1-34 and 2- to 4-fold greater than bovine 1-34. These values represent the highest potency we have observed to date for a PTH preparation by these assay systems. In contrast, receptor binding of the rat fragment was comparable to that of bovine and human 1-34. Half-maximal inhibition of radioligand binding occurred at 1.7- 2.0 X 10(-9) M with all synthetic hormones. Hence, the amino acid substitutions in rat 1-34 appear to affect the cyclase-activating sequence domain without increasing avidity for the receptor. Analogs combining the rat sequence with modifications known to enhance receptor binding and/or retard enzymatic degradation offer a promising approach to the preparation of still more potent parathyroid agonists.

Adenylyl Cyclases↗
PubMed1984

Gene encoding parathyroid hormone. Nucleotide sequence of the rat gene and deduced amino acid sequence of rat preproparathyroid hormone.

The nucleotide sequence of the rat parathyroid hormone (PTH) gene was established from a 14.5-kilobase pair fragment of rat liver DNA cloned in bacteriophage Charon 4A. The transcriptional unit of the gene of 2.5 kilobase pairs is interrupted by two introns (1600 and 111 base pairs). Blot hybridization of restriction enzyme digests of rat spleen DNA using 32P-labeled fragments of the cloned PTH gene suggests that the gene is unique and present in a single copy in the genome. A promoter sequence (Goldberg-Hogness or TATA box) is situated 28 base pairs upstream from the point of initiation of transcription which was found by S1 nuclease mapping and by oligonucleotide-primed reverse transcription of rat PTH mRNA. The gene is flanked on its 5' side by repetitive DNA and contains a different, more abundant repetitive DNA on its 3' side. The mRNA encoded by the parathyroid hormone gene consists of 800 +/- 50 nucleotides as determined by electrophoresis on agarose gels. The 5' untranslated region of the mRNA contains three AUG triplets. Only one AUG triplet initiates biosynthesis of preproparathyroid hormone (prepro-PTH). The 3' untranslated segment of the mRNA contains two AATAAA sequences characteristic of polyadenylation signals. The mRNA encodes prepro-PTH, a precursor of PTH of 115 amino acids. When the amino acid sequence of the rat precursor is compared with the analagous bovine and human precursors, it becomes evident that the hormone sequences are highly conserved in two regions of known function near the NH2 terminus and in a third region near the carboxyl terminus whose biologic function, if any, has not yet been defined.

Amino Acid Sequence↗
PubMed1984

Photoaffinity labeling of parathyroid hormone receptors: comparison of receptors across species and target tissues and after desensitization to hormone.

Cells derived from human giant cell tumors of bone and fibroblasts derived from human neonatal foreskin respond to parathyroid hormone (PTH) by increasing the intracellular and extracellular levels of adenosine cyclic 3',5'-phosphate (cAMP). Using photoaffinity labeling methods, we examined these cells for the presence of a PTH receptor or a binding subunit of a receptor complex. A previously designed biologically active and photolabile radioligand analogue of PTH was reacted with these intact cells. After photolysis, the cells were extracted, and the proteins were denatured, reduced, and separated by electrophoresis on sodium dodecyl sulfate (Na-DodSO4)-polyacrylamide gels followed by autoradiography. A single membrane component, Mr 70 000, was labeled specifically in intact cells cultured from skeletal and dermal tissue. By mixing, in pairs, photolabeled proteins from (a) intact human cells derived from giant cell tumors of bone, (b) intact human fibroblasts, and (c) canine renal cortical membranes, the receptors (or their binding subunits) for PTH were compared directly and found to be identical in terms of molecular size (as determined by the migration position on NaDod-SO4-polyacrylamide gels) across species (dog and human) and target tissue (bone, skin, and kidney). Preincubation of cells cultured from giant cell tumors of bone with PTH resulted in loss of the PTH-induced cAMP response (desensitization). Preincubation with PTH was accompanied by a marked decrease in photoaffinity labeling of the PTH binding component and suggests that the loss of hormone response in cells preincubated with PTH was related to a decrease in the number or availability of PTH receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1984

Protamine: a powerful in vivo inhibitor of bone resorption.

Protamine is shown to be a powerful disrupter of calcium homeostasis, acutely inducing a severe hypocalcemia in both rabbits and rats. The magnitude of its effect correlates with bone turnover. Protamine does not significantly alter the renal excretion of calcium, and is effective whether or not there is calcium present in the gut. Protamine causes a significant fall in the specific activity of 45Ca in the blood in animals whose bone has been prelabeled with 45Ca. These data suggest that protamine induces hypocalcemia by blocking calcium efflux from bone. Further work seems indicated to define the biochemical mechanism of this action.

Animals↗
PubMed1984

Secretion of human parathyroid hormone from rat pituitary cells infected with a recombinant retrovirus encoding preproparathyroid hormone.

In order to study the functions of precursors to secreted proteins, we expressed cloned DNA encoding human preproparathyroid hormone (preproPTH) in rat pituitary cells. We first constructed a recombinant plasmid containing human preproPTH cDNA and retroviral control signals. This recombinant plasmid was transfected into psi-2 cells, a packaging cell line that produces Moloney murine leukemia viral particles containing no retroviral RNA. The transfected psi-2 cells generated helper-free recombinant retrovirus encoding preproPTH, and this recombinant retrovirus was used to infect GH4 rat pituitary cells. Clonal lines of the infected GH4 cells contained copies of the recombinant provirus stably integrated via the long terminal repeats, and the expected RNA transcripts of proviral DNA accumulated in the cytoplasm, although no infectious virus was produced. The infected cells synthesized and processed preproPTH appropriately and secreted PTH in response to thyrotropin-releasing hormone, a secretagogue for GH4 cells. Use of recombinant retrovirus permits the introduction of DNA encoding normal and mutant secreted proteins into a number of cell types specialized for secretion. Analysis of the fate of the resultant proteins will help define the specific molecular interactions involved in transmembrane transport and processing of precursor proteins.

Amino Acid Sequence↗
PubMed1984

Renal 1,25-dihydroxyvitamin D, phosphaturic, and cyclic-AMP responses to intravenous synthetic human parathyroid hormone-(1-34) administration in normal subjects.

The exogenous administration of bovine parathyroid hormone or parathyroid extract has been used to differentiate states of parathyroid hormone resistance and parathyroid gland secretory failure, and in recent years to test renal 1,25-dihydroxyvitamin D (1,25-(OH)2-D) secretion. We evaluated the effect of synthetic human parathyroid hormone (hPTH-(1-34] administration on the renal 1,25-(OH)2-D, phosphaturic and cyclic-AMP responses in eleven normal young adults. The intravenous administration of 200 units of hPTH-(1-34) over 10 min produced a 1.3-5.4 fold increase (P less than 0.01) in renal phosphate clearance and a 19-75 fold increase (P less than 0.0001) in urinary cyclic-AMP excretion. Serum 1,25-(OH)2-D levels showed a small and insignificant change at 2.5 h and a significant (P less than 0.05) but small (21 +/- 24 pmol/l) increase at 7 h after the first injection. In eight subjects a second injection of hPTH-(1-34) was given at 7 h. In these individuals serum 1,25-(OH)2-D levels at 24 h were 40 +/- 14 pmol/l (44%) higher than baseline (P less than 0.01), but were variable over the 24 h period. The present study shows that hPTH-(1-34) produces renal phosphaturic and cyclic-AMP responses in normals similar to those produced by bovine PTH preparations. However, the serum 1,25-(OH)2-D response to one or two intravenous injections of hPTH-(1-34) is small, variable, and inconsistent and, therefore, will not provide a consistent way of stimulating renal 1,25-(OH)2-D secretion.

Adult↗
PubMed1983

A parathyroid hormone inhibitor in vivo: design and biological evaluation of a hormone analog.

A synthetic analog of bovine parathyroid hormone (bPTH), [tyrosine-34] bPTH-(7-34)NH2, was found to inhibit parathyroid hormone action in vivo. When the analog and parathyroid hormone were infused simultaneously to rats at a molar ratio of 200 to 1, the analog inhibited the excretion of urinary phosphate and adenosine 3',5'-monophosphate. When infused alone at the same dose rate, the analog was devoid of agonist activity. The compound was prepared by following design principles developed for inhibitors of parathyroid hormone, and is believed to be the first antagonist of parathyroid hormone that is effective in vivo.

Amino Acid Sequence↗
PubMed1983

Nucleotide sequence of the human parathyroid hormone gene.

From a lambda phage gene library we have isolated phage containing the gene encoding human preproparathyroid hormone. The phage were isolated by using both the plaque-hybridization technique and the in vivo recombination-selection technique. The human preproparathyroid hormone gene contains two intervening sequences that separate the gene into a 5' noncoding domain, a "prepro" sequence domain, and a domain containing the parathyroid hormone sequence and the 3' noncoding region. The gene is approximately 4,200 base pairs long. Restriction endonuclease analysis of human leukocyte DNA shows that the haploid human genome contains one copy of the preproparathyroid hormone gene. A 14-base-pair sequence of alternating purines and pyrimidines that has the potential of adopting the Z-DNA conformation lies 134 base pairs upstream from the presumed site of initiation of transcription.

Amino Acid Sequence↗
PubMed1983

A multiresponse parathyroid hormone assay: an inhibitor has agonist properties in vivo.

Vitamin D-deficient rats subjected to thyroparathyroidectomy (TPTX) were used to evaluate in vivo the biological properties of native bovine parathyroid hormone (bPTH) and chemically synthesized fragments and analogues of the hormone on several parameters of hormone action: calcium and phosphorus fluxes, generation of cyclic adenosine 3',5'-monophosphate (cAMP), and the metabolism of 25-hydroxyvitamin D3 [25(OH)D3]. Vitamin D-deficient rats, after TPTX or sham operation, were intravenously infused with a nutrient containing 7.5 mM CaCl2 for 30 h. During the last 7 h, PTH or one of its analogues was infused intravenously at rates between 0.04 and 20 nmol/h. One hour after the start of the peptide infusion, tritiated 25(OH)D3 was injected. Urine was collected hourly for phosphate and cAMP determinations and, at the end of the experiment, blood was obtained to determine the relative accumulation of tritiated 1,25-dihydroxyvitamin D3 ([3H]1,25(OH)2D3). Infusion of bPTH-(1--84), bPTH-(1--34), human (h)PTH-(1--34), or [Nle8, Nle18, Tyr34]bPTH-(1--34) amide was accompanied by a comparable dose-dependent decrease in plasma phosphate and a dose-dependent increase in plasma calcium and [3H]-1,25(OH)2D3, and urinary excretion of phosphate and cAMP. An evaluation of [Nle8, Nle18, Tyr34]bPTH-(3--34) amide, a potent inhibitor of PTH action in vitro in the renal adenylate cyclase assay, revealed that the analogue possessed weak agonist properties in vivo. The analogue increased excretion of both cAMP and phosphate in the urine, decreased plasma phosphate levels, and increased the accumulation of [3H]-1,25(OH)2D3 in the plasma. This multiparameter model system should aid in the elucidation of the in vivo biological effects of PTH and its analogues.

Animals↗
PubMed1983

Circulating bovine lymphocytes contain receptors for parathyroid hormone.

No cell type practicably obtainable in vivo, such as blood cells, is known to contain parathyroid hormone (PTH) receptors; this deficiency has hampered investigation of receptor regulation. Second, PTH in vivo is among the potent stimulators of osteoclastic activity, although no direct hormonal effects on these cells have been identified. Several lines of evidence suggest that cells of the immune system may mediate PTH effects on osteoclasts. We, therefore, studied bovine blood cells for the presence of PTH receptors and PTH-stimulated adenylate cyclase. Using an analogue of bovine PTH, (125)I-labeled [Nle(8),Nle(18),Tyr(34)]bPTH-(1--34)amide, we found PTH-specific binding to intact, nonadherent mononuclear cells (lymphocytes) and PTH-stimulated adenylate cyclase in plasma membranes prepared from these cells, and not with cells or membranes from other blood cells. Lymphocytes may serve to study the effects of physiologic and pathologic perturbations on PTH-receptor function in vivo. Exploration of PTH-related lymphocyte responses may help define the relation between cells of the immune system and osteoclastic bone resorption.

Adenylyl Cyclases↗
PubMed1983

Parathyroid hormone messenger ribonucleic acid: effects of calcium on cellular regulation in vitro.

The effects of changes in the concentration of extracellular calcium the principal regulatory of PTH secretion, on the cellular levels of the mRNA encoding prepro-PTH, a precursor of PTH, were studied in bovine parathyroid slices in vitro using a sensitive radiodensitometric cDNA hybridization assay and mRNA translation in a wheat germ cell-free system. Although lowering the calcium from 5 to 0.5 mM during 5- to 70-h incubations stimulated the secretion of PTH 4- to 8-fold and pro-PTH synthesis 1.4-fold, the cellular levels of total hybridizable PTH mRNA did not change. However, a 2- to 3-fold increase in PTH mRNA, which could be hybridized to oligo-dT-cellulose [poly(A)-rich], was observed in the slices incubated in 5 mM calcium (suppressed slices). The poly(A)-rich PTH mRNA constituted 30% of the total PTH mRNA in suppressed gland slices and 10% of the total PTH mRNA in stimulated gland slices. PTH mRNA did not decay measurably when RNA synthesis in the gland slices was inhibited with actinomycin D and alpha-amanitin for 6 h. Both the stability and the lack of perturbation of the cellular levels of total of total hybridizable PTH mRNA by changes in the concentration of extracellular calcium suggest that the short term regulation of PTH biosynthesis involves posttranscriptional mechanisms.

Animals↗