Biomedical subjects

D Goltzman

Publications and source records attributed to D Goltzman.

At least 127 records · Page 7Linked to original sources

PubMed1990

Biochemical and morphological characterization of parathyroid hormone receptor binding to the rat osteosarcoma cell line UMR-106.

We have used both biochemical and morphological techniques to characterize PTH receptors on the clonal osteosarcoma cell line UMR-106, a widely used model of the osteoblast phenotype. 125I-labeled rat (r) PTH-(1-34) bound to a single class of specific saturable receptors on both whole cells and membranes prepared from UMR-106 cells in a time- and temperature-dependent manner. A decrease in PTH receptor affinity seen in the presence of guanine nucleotides demonstrated that PTH receptors on the UMR-106 cells are coupled to guanyl nucleotide-binding proteins. Although PTH is a potent stimulator of adenylate cyclase in the UMR-106 cells, comparison of PTH-stimulated adenylate cyclase and PTH binding curves indicated the presence of receptors that are not linked to the adenylate cyclase system. Our studies also demonstrated that 125I-labeled rPTH-(1-34) bound to UMR-106 cells is rapidly internalized at 22 C, whereas PTH bound at 4 C remains intact and on the cell surface. Internalization of 125I-labeled rPTH-(1-34) was associated with degradation and release of the hormone at 22 C. Three morphologically distinct cell types were identified in subconfluent cultures of UMR-106 cells. Autoradiographic analysis of 125I-labeled rPTH-(1-34) binding demonstrated differential PTH receptor expression in these cell types. The most abundant PTH binding was observed over a cell type with long cytoplasmic extensions. This cell was reminiscent of the predominant PTH target cell previously identified in the rat metaphysis in vivo, suggesting that the UMR-106 cell line may represent neoplastic transformation of the PTH target cell.

Adenylyl Cyclases↗

PubMed1990

Mechanisms of homologous and heterologous regulation of parathyroid hormone receptors in the rat osteosarcoma cell line UMR-106.

We have examined the mechanisms of homologous and heterologous regulation of PTH receptor binding and receptor-mediated adenylate cyclase activity in the osteosarcoma cell line UMR-106. Pretreatment with PTH resulted in a time- and dose-dependent decrease in PTH-stimulated adenylate cyclase which was maximal after 2 h and at a concentration of 10(-8) M rat (r)PTH-(1-34). PTH pretreatment over the same dose range also diminished receptor binding of 125I-labeled rPTH-(1-34); however, maximal loss of binding required 14 h and was greater than the loss of maximal adenylate cyclase activity. After 24 h pretreatment with rPTH-(1-34), cell surface receptors were decreased from 21,000 sites per cell to 2,700 sites per cell, and these down-regulated PTH receptors could not be detected in either vesicular or cytosolic subcellular fractions. Recovery from such homologous down-regulation appeared to require new receptor synthesis. Heterologous down-regulation of PTH receptors was demonstrated when UMR-106 cells were preincubated with prostaglandin E2 or (Bu)2cAMP. Heterologous desensitization was shown to be the result of a reversible modification of the PTH receptor which decreased binding affinity and decreased PTH-stimulated adenylate cyclase. Postreceptor components were also examined, and PTH but not prostaglandin E2 pretreatment was shown to decrease guanyl nucleotide binding (G) protein-mediated adenylate cyclase stimulation. This decrease in G protein function was associated with a loss of cholera toxin-catalyzed ADP ribosylation and was also detected by immunoblotting. These results indicate that PTH responses in osteoblastic cells are modulated by diverse mechanisms involving modifications both to the receptor and to postreceptor components of adenylate cyclase.

Adenosine Diphosphate Ribose↗

PubMed1990

Effects of passive immunization against parathyroid hormone (PTH)-like peptide and PTH in hypercalcemic tumor-bearing rats and normocalcemic controls.

Passive immunization using antisera raised against rat PTH-like peptide [PLP-(1-34)] and rat PTH-(1-84) was used to assess and compare the roles played by PLP and PTH in modulating mineral metabolism in hypercalcemic rats bearing the Rice-500 Leydig cell tumor and in normocalcemic control animals. After immunization, plasma calcium in the tumor-bearing animals rapidly normalized and remained within the normal range for several days, plasma phosphate rose, and urinary phosphate and cAMP fell. These changes were associated with increased longevity of the tumor-bearing rats. Reduction of plasma calcium was shown to be a function of early (5 h) neutralization of PLP bioactivity in the kidney, whereas the effect of immunoneutralization of PLP in bone appeared later (24-48 h) and was more prolonged. Immunization against PTH in normocalcemic animals resulted in a hypocalcemic episode of smaller magnitude and shorter duration than that achieved in hypercalcemic animals immunized against PLP and appeared to be unassociated with neutralization of distal tubular effects. Neutralization of proximal tubular and skeletal actions of PTH appeared to occur in a manner analogous to that seen for PLP in tumor-bearing animals, but were of shorter duration. These studies suggest that in normal animals the action of PTH in the skeleton and/or on vitamin D metabolism contributes considerably more than renal transport activity to the maintenance of normocalcemia and that compensatory mechanisms rapidly restore homeostasis. PTH appears to be the major modulator of calcium homeostasis in normal rats, with PLP playing a minor role, if any. In tumor-bearing rats, mechanisms to restore calcium levels to baseline are delayed after PLP immunoneutralization, and PLP appears to be both necessary and sufficient for the hypercalcemic state.

Animals↗

PubMed1990

Parathyroid hormone-like peptide in normal and neoplastic human endocrine tissues.

PTH-like peptide (PLP) is produced by tumors commonly associated with hypercalcemia as well as nonneoplastic tissues and several endocrine glands and tumors. To characterize the distribution of PLP in human endocrine tissues and tumors, we localized PLP in formalin-fixed paraffin-embedded material using the avidin-biotin-peroxidase technique with polyclonal antisera. Among peptide hormone-producing tissues, PLP was identified in nontumorous adenohypophysis and pituitary adenomas; medullary thyroid carcinomas; normal, hyperplastic, and adenomatous parathyroids; adrenal medulla and pheochromocytomas; normal pancreatic islets; and endocrine tumors of pancreas, gut, and lung, including small cell carcinomas. In other endocrine tissues PLP was identified in nontumorous thyroid follicular epithelium, colloid nodules, and follicular neoplasms; normal adrenal cortex, adrenocortical adenomas, and carcinomas; nontumorous testicular Leydig cells; normal ovarian granulosa and thecal cells; an ovarian thecoma and a granulosa cell tumor; placental trophoblast; and decidua. These results demonstrate that PLP is localized in many normal and neoplastic endocrine cells, including those not known to influence extracellular calcium homeostasis. The presence of PLP in a variety of endocrine tissues suggests that it may play a local physiological role in the growth or function of endocrine cells.

Adrenal Cortex Neoplasms↗

PubMed1990

Gene-encoding parathyroid hormone-like peptide: nucleotide sequence of the rat gene and comparison with the human homologue.

The single-copy gene coding for rat PTH-like peptide was isolated from a rat liver genomic DNA library. The gene spans 12 kilobases and contains four exons. Exon I encodes the 5'-noncoding region, exon II encodes the prepro region, exon III encodes the mature peptide sequence up to amino acid 139 and exon IV encodes the carboxyl-terminal two amino acids, a stop codon, and the 3'-noncoding region. Splicing of these exons yields the 1.4 kilobase mRNA which is the predominant transcript observed in the hypercalcemic rat Leydig cell tumor and several normal rat tissues. The overall exon/intron organization of the rat parathyroid hormone-like peptide (PLP) gene is similar to that of the PTH gene and emphasizes the likelihood that PLP and PTH arose from a common ancestral gene. A comparison of the single promoter region of the rat with the second promoter of the human gene indicates conserved TATA and CAAT box homologies, GC box regions (SP-1 binding sites), putative AP-2 binding sites, and a vitamin D responsive element. When compared to the seven exon human PLP gene, which uses multiple promoters and encodes three peptide isoforms, the simpler organization of the rat gene predicts, in mammals, the predominant use of a single promoter and generation of a 141-amino acid peptide as the major molecular form.

Amino Acid Sequence↗

PubMed1990

Evidence for a humoral mechanism for enhanced osteogenesis after head injury.

The rate of fracture-healing is accelerated and abundant callus develops in patients who have a head injury and fractures. The mechanism underlying this is unclear. We studied the possibility that increased circulating growth factors or circulating factors that stimulate local release of growth factors mediate the increased osteogenesis. Samples of serum were obtained from thirty-two subjects: patients who had a head injury alone, those who had a head injury and fractures of the lower extremities, those who had only fractures, and control subjects who had neither a head injury nor a fracture. Severe head injury was defined as that producing coma of at least three days' duration. Growth-factor activity was determined by assessing the effect of serum on the incorporation of [3H]thymidine and on cell counts in primary cultures of osteoblastic cells from the calvaria of fetal rats. Samples of serum from the two groups of patients who had a head injury had higher mitogenic activity and produced a greater increase in the number of cells than did the samples from the other two groups. The mean levels of activity were not statistically different between the first two groups or between the patients who had fractures only and the control subjects. Dilution studies showed that increased mitogenic activity in the serum from the patients who had a head injury was dose-dependent. In three patients in whom it was studied, the mitogenic activity peaked approximately thirty-seven days after the head injury was sustained.

Adult↗

PubMed1989

Characterization of the human parathyroid hormone-like peptide gene. Functional and evolutionary aspects.

The single-copy gene coding for the human parathyroid hormone-like peptide was isolated from a human placental genomic library. The gene spans 13 kilobases and contains seven exons. Exons I and II encode 5'-noncoding regions; each has its own transcription initiation site, and the two promoters are separated by over 1000 base pairs of genomic DNA. Exon III encodes the prepro-coding region, and exon IV encodes the mature peptide sequence. At the end of exon IV the splice site interrupts codon 139 of the mature peptide. Exon V, which is contiguous with exon IV, encodes a stop codon and a 3'-noncoding region. Exon VI encodes 34 additional amino acids, a stop codon, and a second 3'-noncoding region. Exon VII encodes two extra amino acids, a stop codon, and a third 3'-noncoding region. This genomic organization reveals how the multiple human parathyroid hormone-like peptide RNA transcripts, which have been observed, arise by both alternative splicing out of exons and use of multiple promoters. The mRNAs, which can potentially be formed from the primary transcript of this gene, could have one of three different carboxyl-terminal coding regions. The use of different exons to encode the different functional domains, 5'-noncoding region, pre-pro-coding region, and mature peptide region is identical to the organization of the human parathyroid hormone gene. This strongly suggests a common evolutionary origin of the two genes.

Amino Acid Sequence↗

PubMed1989

Characteristics of secondary hyperparathyroidism in vitamin D-deficient dogs.

Characteristics of secondary hyperparathyroidism were evaluated in dogs with mild vitamin D deficiency. The animals were normocalcemic with reduced concentrations of 1,25-dihydroxy vitamin D3 [1,25(OH)2D3] and elevations in parathyroid hormone (PTH) concentrations, parathyroid mass, and prepro PTH mRNA levels. Dynamic testing revealed a sigmoidal relationship between plasma calcium and PTH, although PTH concentrations were increased relative to values in vitamin D-sufficient dogs. Infusions of chelator elicited lower plasma calcium levels and greater augmentations in biologically active PTH in vitamin D-deficient than in D-sufficient animals. Induced hypercalcemia lowered both immunoreactive and bioactive hormone to stable but detectable levels. The results demonstrate the decreased capacity of vitamin D-deficient animals to defend against acute hypocalcemia, despite the presence of abundant PTH, and indicate that increased circulating PTH levels in early vitamin D deficiency is due predominantly to an augmentation in the quantity of releasable hormone. The latter appears secondary to an increase in parathyroid mass and synthetic activity regulated by 1,25(OH)2D3 per se.

Animals↗

PubMed1989

Influence of calcium and 1,25-dihydroxycholecalciferol on proliferation and proto-oncogene expression in primary cultures of bovine parathyroid cells.

Exposure of quiescent bovine parathyroid cells to serum or a serum substitute caused an elevation in [3H] thymidine incorporation, followed by an increase in cell number. This was preceded by a rapid transient rise in c-myc and c-fos proto-oncogene mRNA levels. Alterations in the medium calcium concentration had no effect on the growth state of quiescent parathyroid cells. In addition, varying the medium calcium concentration did not influence either the time course or the degree of induction of proto-oncogene expression or the subsequent increase in [3H] thymidine uptake or proliferation of stimulated parathyroid cells. In contrast, when 1,25-dihydroxycholecalciferol was added with serum or serum substitute to quiescent parathyroid cells, no increase in c-myc mRNA levels was observed, and the expected increase in parathyroid cell number failed to occur. The augmentation in c-fos mRNA in response to serum was not, however, altered by 1,25-dihydroxycholecalciferol. These results indicate that 1,25-dihydroxyvitamin D, but not extracellular calcium, may directly modulate parathyroid cell proliferation by altering the expression of specific replication-associated oncogenes.

Animals↗

PubMed1989

The parathyroid hormone-like peptide gene is expressed in the normal and neoplastic human endocrine pancreas.

PTH-like peptide (PLP) is produced by a number of tumors commonly associated with the development of hypercalcemia. Analysis of the expression of the PLP gene has demonstrated that a variety of non-neoplastic endocrine and nonendocrine tissues contain PLP mRNA transcripts. Using a combination of Northern blot analysis, immunohistochemistry, and RIAs, we have demonstrated that the PLP gene is expressed in normal human and rat fetal and adult islets of Langerhans. PLP gene expression was not confined to cells containing a single pancreatic islet hormone, but was found in cells of all four major endocrine subtypes. PLP mRNA transcripts were also detected in RNA prepared from isolated rat islets, and small amounts of PLP immunoreactivity were secreted by cultured rat islets. Fifteen human pancreatic endocrine tumors not associated with hypercalcemia were analyzed and PLP-immunopositive tumor cells were found in 13. These observations demonstrate that the PLP gene is expressed in the normal and neoplastic islets of Langerhans, and suggests a possible role for this peptide in the growth or function of the endocrine pancreas.

Adult↗

PubMed1989

Rat parathyroid hormone-like peptide: comparison with the human homologue and expression in malignant and normal tissue.

A rat Leydig cell tumor cDNA library was screened with a 32P-labeled genomic restriction fragment encoding human PTH-like peptide (hPLP), and three cDNA clones were isolated. The largest cDNA insert contained 1146 nucleotides. The cloned cDNA encodes a 177-amino acid protein consisting of a predicted 36-amino acid leader sequence and a 141-amino acid mature peptide in which 9 of 13 amino-terminal residues are identical to rat PTH (rPTH). Comparison of rPLP with hPLP reveals marked conservation of both the nucleotide and amino acid sequences through the prepro, amino-terminal, and midregion portions of the molecules. There is also striking conservation of the 3' noncoding regions of rPLP and hPLP mRNAs, both of which contain AU-rich repeated sequences that may affect mRNA stability. A single species of mRNA of approximately 1.4-kilobases was identified in the rat Leydig cell tumor and in normal rat stomach. Southern blot analysis is consistent with the presence of a single copy of the rPLP gene per haploid genome, and there is no major rearrangement or amplification of the rPLP gene in DNA isolated from the tumor per se. The results demonstrate the presence of a single gene transcript in a rat model of malignancy associated with hypercalcemia which encodes a peptide homologous to hPLP, document the marked interspecies sequence conservation that exists in major functional domains of the mRNAs and peptides, and show the expression of mRNA encoding rPLP in normal stomach as well as in neoplastic rat tissue.

Amino Acid Sequence↗

PubMed1988

Gene for parathyroid hormone-like peptide is on rat chromosome 2.

Parathyroid hormone-like peptide (PLP) is thought to be a mediator of hypercalcemia in both human and rodent malignancies. A rat PLP cDNA was used as a hybridization probe in Southern blot analysis of DNAs isolated from a panel of rat-mouse somatic cell hybrids. The single-copy gene for PLP was assigned to rat chromosome 2, whereas the rat parathyroid hormone (PTH) gene has previously been assigned to rat chromosome 1. Consequently, despite significant amino-terminal homology between PLP and PTH the genes encoding these peptides in the rat as well as human species have discrete chromosomal localizations.

Animals↗

PubMed1988

Recombinant human parathyroid hormone synthesized in Escherichia coli. Purification and characterization.

Recombinant human parathyroid hormone (hPTH) was expressed in Escherichia coli harboring a plasmid containing a synthetic human parathyroid hormone gene under the control of the E. coli lac promoter. Three major forms of the hormone were isolated by acid extraction and purified to homogeneity by high performance liquid chromatography. By amino acid analysis and NH2-terminal sequencing, these were identified as hPTH-(1-84), formyl-methionyl-hPTH-(1-84), and hPTH-(8-84). The recombinant hPTH-(1-84) was immunologically indistinguishable from a World Health Organization standard of extracted native hPTH-(1-84). Recombinant hPTH-(1-84) was also bioactive in renal and skeletal adenylate cyclase assays. In the skeletal bioassay performed in UMR 108 osteosarcoma cells its activity was identical to that of an hPTH-(1-84) standard. In this bioassay, formyl-methionyl-hPTH-(1-84) had 10% of the activity of hPTH-(1-84) and hPTH-(8-84) was inactive. The results demonstrate the importance of isolating hPTH-(1-84) from other recombinant forms and metabolites to achieve full hormonal bioactivity and indicate that purified recombinant hPTH-(1-84) can thereby be obtained which should be a useful source of hormone for both basic and clinical studies.

Adenylyl Cyclases↗

PubMed1988

Kenny syndrome: description of additional abnormalities and molecular studies.

An 18-month-old girl presented with clinical manifestations of Kenny syndrome including growth retardation, ophthalmologic abnormalities, a persistent open anterior fontanel, dysmorphic facies, anemia, radiologic skeletal abnormalities, and severe hypoparathyroidism. Analysis of restriction patterns of DNA with human parathyroid hormone (PTH) DNA probes revealed no gross abnormalities of the PTH gene that could contribute to the hypoparathyroidism. In addition to the previously described characteristics of the syndrome, hypoplastic nails, persistent neutropenia, abnormal T cell function and neonatal liver disease all occurred and may be additional manifestations of Kenny syndrome, requiring diagnostic or therapeutic consideration.

Abnormalities, Multiple↗

PubMed1988

Parathyroid hormone desensitization in renal membranes of vitamin D-deficient rats is associated with a postreceptor defect.

We examined the characteristics of PTH resistance in vitamin D-deficient rats employing renal membranes in vitro. Homologous desensitization was characterized by diminished PTH-stimulated adenylate cyclase activity and was associated with a reduction in PTH-binding capacity, but not affinity. Heterologous desensitization was also seen, as manifested by decreased calcitonin (CT)-stimulated adenylate cyclase activity with normal CT receptor binding. The reduced capacity of the nonhormonal effectors NaF and guanylylimidodiphosphate to stimulate adenylate cyclase indicated a postreceptor defect at the level of the guanyl nucleotide-binding protein (G protein), whereas a normal forskolin response was consistent with a fully functional catalytic component. The G protein deficiency was confirmed by demonstrating that the addition of extracts of vitamin D-sufficient membranes to preparations of vitamin D-deficient membranes restored the normal responses to NaF and guanylylimidodiphosphate. In addition, cholera toxin- and pertussis toxin-catalyzed labeling of vitamin D-deficient renal membranes with [32P]NAD revealed a decrease in both the stimulatory and inhibitory binding proteins. Experiments with testicular membranes in vitro indicated that the adenylate cyclase abnormality was absent in tissue lacking PTH receptors. The results suggest that a major contribution to PTH resistance in vitamin D-deficient animals is a postreceptor defect at the level of the G proteins and that this defect is manifest only in tissue expressing the PTH receptor.

Adenosine Diphosphate Ribose↗

PubMed1988

In vivo distribution of parathyroid hormone receptors in bone: evidence that a predominant osseous target cell is not the mature osteoblast.

Previous studies in vitro and in vivo have demonstrated the presence of receptor sites for PTH on cells of the osteoblast phenotype. Nevertheless, it is unclear whether the diverse functions of this hormone in bone can all be attributed to its interaction with a single cell type. In this study, we have used a radioautographic method to examine the competitive binding of 125I-labeled rat PTH-(1-34) to the long bones of rats in vivo. Our studies confirm the presence of competitive binding to mature osteoblasts and the absence of significant competitive binding to multinucleated osteoclasts. However, by light and electron microscopic radioautographic analysis, the majority of specific competitive PTH binding was present over a cell in the intertrabecular space of the metaphyseal region, which was distinct from the mature osteoblast. This large mononuclear cell with multiple cytoplasmic extensions appeared to interface with both the bone matrix and the microvascular osseous circulation and may provide an additional target to mediate hormonal effects on the skeleton.

Animals↗

PubMed1988

Influence of the amino-terminus on in vitro and in vivo biological activity of synthetic parathyroid hormone-like peptides of malignancy.

We compared the bioactivities of a synthetic truncated NH2-terminal fragment of the human (h) PTH-like peptide (PLP) associated with malignancies [hPLP-(3-34)], an intact NH2-terminal fragment [hPLP-(1-34)], and an NH2-terminal fragment of PTH [hPTH-(1-34)]. Although hPLP-(1-34) was less potent than hPTH-(1-34) in stimulating adenylate cyclase in rat renal membranes, hPLP-(1-34) and hPTH-(1-34) were equipotent in stimulating adenylate cyclase in OK renal cells as well as in UMR 108 osteosarcoma cells in vitro. In osteosarcoma cells, each of these peptides could desensitize adenylate cyclase responses to itself and to the other peptide, but could not reduce stimulation by prostaglandin E2. Renal membranes of vitamin D-deficient rats with secondary hyperparathyroidism had a reduced PLP-stimulated as well as PTH-stimulated adenylate cyclase response. The truncated analog hPLP-(3-34) was only a weak partial agonist and an antagonist in vitro, produced equivalent inhibition of hPLP-(1-34) and hPTH-(1-34) in renal and osseous cells, and could not desensitize agonist responses. In thyroparathyroidectomized rats in vivo, hPLP-(1-34) and hPTH-(1-34) increased cAMP excretion, enhanced phosphaturia, maintained plasma calcium, and reduced calciuria. Equimolar concentrations of hPLP-(3-34) produced no increases above control levels; however, high concentrations of this peptide mimicked PTH actions on renal and plasma ion handling while modestly augmenting cAMP excretion. These results demonstrate the importance of the first two residues of PLP for bioactivity, indicate that PLP and PTH interact at common receptor sites in vivo as well as in vitro, suggest that PLP may not be less potent than PTH in renal target cells, and indicate that the net result of interaction of these peptides with their common receptor in target tissues may reflect both activation and desensitization of receptor-mediated events.

Adenylyl Cyclases↗

PubMed1988

Does the maternal kidney contribute to the increased circulating 1,25-dihydroxyvitamin D concentrations during pregnancy?

The observation that during pregnancy the circulating 1,25-dihydroxyvitamin D, 1,25(OH)2D, concentrations are higher than in the nonpregnant state as well as recent evidence showing that, in vitro, the placenta and/or decidua are sites of 1,25(OH)2D synthesis has led to the general belief that the increased circulating 1,25(OH)2D concentrations originate from the placenta and/or decidua during pregnancy. The observation of a patient with end-stage renal disease who became pregnant after 10 years of chronic hemodialysis treatment has revealed that, despite delivery of a viable infant who had a normal development for gestational age and of normal serum 25-hydroxyvitamin D levels, her serum 1,25(OH)2D3 concentrations were only 10-15 pg/ml following 25 weeks of gestation. These 1,25(OH)2D3 concentrations are far lower than those usually encountered in normal women at the end of the 2nd trimester of pregnancy. It is felt that an important contribution of the placenta and/or of the decidua to the synthesis of the hormone should have led to higher 1,25(OH)2D3 concentrations than those observed in this patient. These observations, along with evidence from the literature, prompted us to reappraise the hypothesis on the origin of the circulating maternal 1,25(OH)2D3 during pregnancy and to postulate that the kidney might be more important than previously thought to the synthesis of the hormone during pregnancy.

Alkaline Phosphatase↗