Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of glucocorticoids on parathyroid hormone (PTH) receptors was studied using rat osteosarcoma-derived cells (ROS 17/2), which have an osteoblastic phenotype, and [125I][Nle8,Nle18,Tyr34]bovine(b)PTH-(1-34)amide as the radioligand. Treatment of cells with physiologic concentrations of hydrocortisone resulted in a time and dose-dependent increase in PTH binding. The increase in PTH binding could be observed by 10 h of exposure to hydrocortisone (2 x 10(-7) M), was maximally enhanced by 48 h, and was maintained for the subsequent 7 days of continuous exposure to the steroid. With removal of hydrocortisone, PTH receptor binding promptly returned toward control levels. The increase in PTH binding was attributed to an increase in the availability of receptor binding sites, not to altered receptor binding affinity, and was blocked by cycloheximide. PTH-stimulated adenylate cyclase was also enhanced by glucocorticoids, and a close correlation was observed between PTH binding and PTH-stimulated adenylate cyclase. However, hydrocortisone not only increased PTH binding but also enhanced the efficiency of postreceptor signaling: 5'-guanylimidodiphosphate [Gpp(NH)p]- and forskolin-stimulated adenylate cyclase activities were also increased. Thus, enhanced PTH stimulation of adenylate cyclase by glucocorticoids resulted from at least two effects--increased receptor availability and enhanced postreceptor efficiency of transmembranous signaling.
...
PMID:Glucocorticoids increase parathyroid hormone receptors in rat osteoblastic osteosarcoma cells (ROS 17/2). 285 94

PTH-like protein-(1-74) [PTHLP-(1-74)] was synthesized and purified. On the basis of chromatographic criteria and amino acid composition of the full-length peptide, direct amino acid sequencing of the N-terminus, and amino acid composition and internal sequence of proteolytic fragments of PTHLP-(1-74), the synthetic peptide appears to be of high quality and purity. Physiological comparison of PTHLP-(1-74) to [Tyr36]-PTHLP-(1-36) amide and bovine (b) PTH-(1-34) indicates that all three peptides are of equivalent potency in the fetal rat long bone and rat osteosarcoma 17/2.8 adenylate cyclase assays. However, as in earlier studies with native and N-terminal PTHLPs, PTHLP-(1-74) is considerably less potent (2%) in stimulating the canine renal cortical adenylate cyclase assay than is bPTH-(1-34). Further, PTHLP-(1-74) displayed only 12% of the activity of bPTH-(1-34) in inducing hypercalcemia when infused into rats in vivo. These studies support the possibility that subclasses of PTH receptors or varying PTH- and PTHLP-signalling transduction mechanisms may exist. In addition, they emphasize the need to precisely define the naturally occurring secretory and circulating species of this novel class of peptide hormones.
...
PMID:Synthetic parathyroid hormone-like protein-(1-74): biochemical and physiological characterization. 291 92

Most studies of PTH receptor binding have been carried out with amino-terminal radioligands which only detect binding within that region of the hormone molecule. We studied the binding of electrolytically labeled intact bovine PTH [bPTH-(1-84)], and its amino-terminal fragment, bPTH-(1-34) to intact cloned rat osteosarcoma cells (ROS 17/2.8). We also measured the effects of these hormones on cell cAMP accumulation. Binding equilibrium for the two radioligands was reached by 2 h of incubation at 22 C. However, the cells had higher binding capacity (8-9% or 0.22-0.25 fmol/2 X 10(6) cells) for [125I]bPTH-(1-84) than for [125I]bPTH-(1-34) (4% or 0.11 fmol/2 X 10(6) cells). On the other hand [125I]bPTH-(1-34) bound to ROS cells with higher affinity [dissociation constant (Kd) = 19 nM] than did [125I]bPTH-(1-84) (Kd = 210 nM). Measurements of trichloroacetic acid precipitability and analysis of rebinding of previously incubated radioligand to fresh cells ruled out degradation of the tracer as an explanation for these differences. The maximum cell cAMP response to bPTH-(1-34) (Vmax = 780 +/- 32 pmol/2 X 10(6) cells X 5 min) was reached at 10(-7) M concentration with an affinity [Michaelis-Menten constant (Km)] of 3 nM. On the other hand, the Vmax with intact bPTH-(1-84) was lower (400 +/- 7 pmol/2 X 10(6) cells X 5 min), with a Km of 60 nM). Further studies with the bPTH-(1-84) tracer showed inability of hormonal fragments to compete completely for binding. At a concentration of 3 microM, bPTH-(1-84) reduced tracer binding by 82.5%, compared to 18% by bPTH-(1-34) and 10% by (Nle8,Nle18,Tyr34)bPTH-(1-34)amide, 60% by human PTH (hPTH)-(53-84), and 70% by the combination of bPTH-(1-34) and hPTH-(53-84). hPTH-(53-84) itself did not elicit a cAMP response after 5 min or 1 h of incubation nor did it significantly alter the cAMP response of the cells to bPTH-(1-84). These studies suggest that PTH binds to ROS 17/2.8 cells by sites carboxy-terminal (C-terminal) to position 34, in addition to sites within the amino-terminal portion of the hormone molecule; 72% of the binding of intact hormone to these cells was to the C-terminal 35-84 region of the PTH molecule. The significance of the C-terminal binding sites is presently unclear, but they do not appear to be coupled to adenylate cyclase. Further work is needed to determine the effects of C-terminal PTH fragments on bone cell metabolism.
...
PMID:Binding of intact parathyroid hormone to rat osteosarcoma cells: major contribution of binding sites for the carboxyl-terminal region of the hormone. 299 16

Human renal carcinoma cell line 786-0 elaborates a protein that is structurally and immunochemically distinct from parathyroid hormone (PTH) and that activates renal cortical adenylate cyclase via an interaction with the PTH receptor. Because of the high frequency of excessive bone resorption and resultant hypercalcemia in patients with malignant disease we evaluated the ability of this 786-0 cell factor to reproduce PTH action in bone-derived cells. The 786-0 factor as well as bovine PTH (BPTH) (1-34) and prostaglandin E1 produced marked increases in cyclic adenosine 3':5'-monophosphate (cAMP) accumulation in the clonal rat osteosarcoma cell line UMR-106. A competitive antagonist of PTH action, [norleucine8, norleucine18, tyrosine34] BPTH(3-34)amide, blocked the cAMP stimulation produced by 786-0 factor and BPTH(1-34) but not that produced by prostaglandin E1. In the presence of forskolin (0.1 microM) UMR-106 cells were extremely sensitive to 786-0 factor, showing significant increases in cAMP production at a concentration 10-fold less than that required to activate adenylate cyclase in renal membranes. In contrast UMR-106 cells were less sensitive to BPTH(1-34) than were renal membranes. This preferential increase in sensitivity to 786-0 factor was not seen in membranes prepared from UMR-106 cells suggesting the importance of cytosolic components. Six additional human genitourinary carcinoma cell lines were found to produce factors that increased cAMP levels in UMR-106 cells. We conclude that 786-0 factor is a potent activator of the PTH receptor-adenylate cyclase system in these bone-derived cells. These findings are consistent with the view that cancer-associated hypercalcemia may frequently be attributable to tumor secretion of proteins (such as 786-0 factor) that are distinct from PTH but are capable of activating skeletal PTH receptors.
...
PMID:Activation of the parathyroid hormone receptor-adenylate cyclase system in osteosarcoma cells by a human renal carcinoma factor. 299 59

Influx of extracellular Ca++ into bone cells has been postulated as an early action of PTH and other bone resorption-stimulating factors. To test this hypothesis directly, we measured the cytosolic free Ca2+ concentration ([Ca2+]i) in two hormone-responsive human (SaOS-2 and G-292) and two rat osteosarcoma cell lines (Ros 25/1 and Ros 17/2.8) and in primary cultures of bone cells from neonatal mouse calvaria using the fluorescent Ca2+ indicator Quin 2. Actions of bovine PTH-(1-34), vasoactive intestinal peptide, epidermal growth factor, prostaglandin E2, and ionomycin were studied. Medium cAMP (20 min; 37 C; 25 microM 3-isobutyl-1-methylxanthine) was quantitated by RIA. Basal [Ca2+]i was: SaOS-2, 126 +/- 8 nM; G-292, 61 +/- 6 nM; Ros 25/1, 109 +/- 15 nM; Ros 17/2.8, 363 +/- 42 nM; and primary cultures, 266 +/- 39 nM (mean +/- SE; n = 3-14). In each cell type, no acute (1 sec to 20 min) spike in [Ca2+]i was observed in response to PTH (24-120 nM), vasoactive intestinal peptide (100 nM), epidermal growth factor (17 nM), or prostaglandin E2 (2.8 microM). However, in SaOS-2 cells only, PTH reproducibly increased [Ca2+]i 10-15% above basal values beginning about 3 min after hormone addition, and this small increase returned to baseline at 15-20 min. Ionomycin (100 nM) elicited an immediate spike in [Ca2+]i to levels 2- to 4-fold above basal in all cells; the peak [Ca2+]i decayed rapidly (within 4-5 min) to baseline in G-292, Ros 25/1, and Ros 17/2.8 cells. The decay of peak [Ca2+]i in SaOS-2 was prolonged. To test for intact hormone responses in Quin 2-loaded cells, cAMP accumulation was measured. In SaOS-2 and Ros 17/2.8, both control and Quin 2-loaded cells showed similar increases in cAMP in response to PTH. Considering the limitations of the Quin 2 technique, we conclude that in the four hormone-responsive bone cell lines and primary cultures of bone cells tested, acute elevation of [Ca2+]i is not an inevitable consequence of receptor occupancy and/or adenylate cyclase activation by bone resorption-stimulating hormones.
...
PMID:Measurement of cytosolic free Ca2+ concentrations in human and rat osteosarcoma cells: actions of bone resorption-stimulating hormones. 300 4

PTH receptor-stimulating proteins may be a common mediator of humoral hypercalcemia of malignancy (HHM). Such proteins exhibit adenylate cyclase-stimulating activity (ACSA) in PTH-sensitive assays, and ACSA has been used to follow their purification. Acid/urea tumor extracts from a murine squamous carcinoma model of HHM were previously shown to have very high ACSA, which was partially, but incompletely, inhibited by the PTH antagonist Nle8,18,Tyr34-bovine PTH-(3-34) amide. ACSA from murine tumor extracts has now been further purified using solvent fractionation and reverse phase HPLC. Approximately half of the ACSA is attributable to a family of three proteins (peaks IA, IB, and IC) with properties characteristic of the PTH receptor-stimulating protein extracted from rat Leydig cell and human HHM tumors. The ACSA in these three peaks of murine tumor extract elutes in the same region as human tumor ACSA on reverse phase HPLC, has a dose-response curve parallel to that of PTH, and is fully inhibited by the PTH-(3-34) antagonist in both the renal cortical and rat osteosarcoma (ROS) adenylate cyclase assays. The remaining half of the ACSA from murine tumor extracts elutes as a single peak (peak II) at a higher acetonitrile concentration on reverse phase HPLC. In the renal cortical assay, its dose-response curve differs from that of PTH, its ACSA is not affected by the PTH-(3-34) antagonist, and it potentiates PTH- or peak I-stimulated adenylate cyclase activity. In the PTH-sensitive intact cell ROS assay, peak II exhibits no ACSA. We conclude that the potent ACSA of murine tumor acid/urea extract results in large part from amplification of the PTH-specific ACSA (peak I) by peak II. Peak II is a distinct protein, not previously reported in tumor extracts, that may act as a postreceptor step in the adenylate cyclase system.
...
PMID:Two species of adenylate cyclase-stimulating activity in a murine squamous carcinoma model of humoral hypercalcemia of malignancy. 300 44

The influence of 1,25-dihydroxyvitamin D-3 on the cAMP response to parathyroid hormone was studied in the osteoblast-like rat osteosarcoma cells ROS 17/2.8. The stimulation by parathyroid hormone of cAMP production in intact cells and of adenylate cyclase activity in isolated plasma membranes was attenuated by 1,25-dihydroxyvitamin D-3 treatment. This was associated with a reduction of the stimulatory guanine nucleotide regulatory protein, as demonstrated by a lower response to NaF and guanosine 5'-[beta, gamma-imido]triphosphate, and by a lower activity of solubilized plasma membrane extracts in the reconstitution assay. 1,25-dihydroxyvitamin D-3 blunted also the cAMP response to parathyroid hormone in cells incubated with the glucocorticoid dexamethasone, where a higher activity of the adenylate cyclase catalytic unit was observed. Thus, the two steroids appear to affect distinct levels of the adenylate cyclase system. Furthermore, the two hormones also showed an antagonistic effect upon the production of osteocalcin, an osteoblast-specific extracellular matrix protein. The release of this non-collagenous matrix protein by ROS 17/2.8 cells was increased by 1,25-dihydroxyvitamin D-3 and decreased by dexamethasone.
...
PMID:Heterologous desensitization by 1,25-dihydroxyvitamin D-3 of cyclic AMP response to parathyroid hormone in osteoblast-like cells and the role of the stimulatory guanine nucleotide regulatory protein. 301 22

Tumour extracts from two patients with humoral hypercalcaemia of malignancy contained material which stimulated adenylate cyclase in chick renal membranes and in rat osteosarcoma cells. Adenylate cyclase-stimulating activity in each system was inhibited by a specific parathyroid hormone (PTH) antagonist. Studies in two HPLC systems suggested that the adenylate cyclase-stimulating factors extracted from these tumours differed from each other and from synthetic human parathyroid hormone 1-34. The presence of similar PTH-like adenylate cyclase stimulating material(s) in oncogenic osteomalacia suggests that adenylate cyclase stimulating factor(s) may not be the direct or the sole cause of hypercalcaemia.
...
PMID:Humoral hypercalcaemia of malignancy: report of two further patients with biochemical studies on tumour extracts. 301 3

This study examines the osteoblastic properties of the established human osteosarcoma cell line Saos-2. Saos-2 cells inoculated into diffusion chambers, which were implanted i.p. into nude mice, produced mineralized matrix in 4 of 6 chambers at 8 weeks. In 5 of 6 chambers there was a strong positive alkaline phosphatase reaction. In culture the alkaline phosphatase levels increased with time and cell density, reaching very high levels at confluence: 4-7 mumol/mg protein/min. The cells show a sensitive adenylate cyclase response to parathyroid hormone, 50% effective dose = 2.8 nM, which increases with cell density and is further raised by dexamethasone treatment. They also exhibit typical binding of 1-25-dihydroxyvitamin D3 to 3.2S receptor protein with an apparent Kd of 0.21 nM; the numbers of sites per cell were 3,300 at 50,000 cells/cm2 and 1,800 at 280,000 cells/cm2. The presence of osteonectin was visualized with a monoclonal antibody which revealed a reticular pattern on the cell surface. Osteonectin was also detected in the medium by Western blots, migrating at around Mr 40,000 in nonreduced gels and Mr 44,000 in reduced gels. The Saos-2 cells thus possess several osteoblastic features and could be useful as a permanent line of human osteoblast-like cells and as a source of bone-related molecules.
...
PMID:Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties. 304 Feb 34

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.
...
PMID:Recombinant human parathyroid hormone synthesized in Escherichia coli. Purification and characterization. 327 65


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---