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

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

Retinoic acid (RA) inhibits the increases in alkaline phosphatase (AP) and hormone-stimulated adenylate cyclase that accompany the growth of ROS 17/2.8 osteosarcoma cells in culture. The RA effects were first detected 2 days after initiation of treatment and were dose dependent, with an EC50 of 100 nM. The reduction in the hormone-responsive adenylate cyclase activity was associated with lower levels of beta-catecholamine receptors, without a change in apparent receptor affinity and with lower levels of the GTP-binding proteins Gs and Gi, visualized by NAD-dependent [32P]ADP ribosylation. The reduction in AP was correlated with a decrease in the steady state level of AP mRNA. RA had no effect on cell proliferation or saturation density. Retinoids thus inhibit the same features that are promoted by glucocorticoids in ROS 17/2.8 cells. These features seem to be subject to coordinate regulation, probably at the pretranslational level.
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PMID:Effects of retinoic acid on alkaline phosphatase messenger ribonucleic acid, catecholamine receptors, and G proteins in ROS 17/2.8 cells. 282 98

PTH receptors on two stable clonal rat osteosarcoma cell lines, ROS 17/2 and ROS 17/2.8, were characterized using an HPLC-purified, synthetic, sulfur-free, radioiodinated analog of bovine PTH, [Nle8,Nle18,Tyr,34]bovine PTH-(1-34)amide. PTH binding is specific for PTH agonists and antagonists and is dependent on the time and temperature of incubation. There is an excellent correlation between binding affinities of PTH agonists and antagonists in these intact cell systems with those in canine renal membranes. Peptides unrelated to PTH do not bind. Both ROS 17/2 and 17/2.8 have a single class of saturable, high affinity PTH binding sites that, by kinetic analysis and Scatchard analysis of saturation and competition studies, has a dissociation constant (Kd) of 0.8-1.4 nM. Bmax is approximately 36,000 and 72,000 sites per cell in ROS 17/2 and 17/2.8, respectively. A close correlation was found between the binding of PTH agonists to their receptors in ROS 17/2 cells with their relative biological potencies as measured by stimulation of adenylate cyclase in plasma membranes prepared from these cells. Prolonged treatment of ROS 17/2 and 17/2.8 cells with PTH agonists results in a dose- and time-dependent decrease of available cell-surface binding sites, without alterations in Kd. PTH antagonists do not regulate PTH receptors. Regulation of PTH receptors by PTH agonists is dependent on the dose and time of exposure to ligand over a dose range of 10(-8) to 10(-11) M. Cells exposed to agonists (greater than or equal to 10(-8) M) for 48 h show maximally decreased receptor number; continued exposure to agonists (greater than or equal to 10(-8) M) does not further decrease PTH receptor number, which remains constant at about 15% of control values. Agonist-induced down-regulation occurs with less than 10(-11) M agonists, a concentration less than 10% of the minimal dose detected by direct ligand competition. Treatment of ROS 17/2 cells with PTH agonists results in a dose- and time-dependent decrease of PTH-stimulated adenylate cyclase. This agonist-induced desensitization correlates closely with the decreased availability of PTH receptors: it is maximal in cells exposed to agonists (greater than 10(-8) M) for 48 h and also does not decrease further with continued exposure of the cells to agonist. Future studies with these stable ROS cell lines should permit detailed analysis of the biochemical mechanisms underlying homologous and heterologous regulation of PTH receptors and desensitization and sensitization of the adenylate cyclase response.
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PMID:Characterization and agonist-induced down-regulation of parathyroid hormone receptors in clonal rat osteosarcoma cells. 283 Oct 22

The N-terminal fragment of human hypercalcemia factor (hHCF), hHCF-(1-34)NH2, has bioactivities similar to PTH in vitro and in vivo. Because it interacts with PTH receptors and is more potent than PTH in some systems, the hHCF sequence may provide interesting leads for the design of potent and selective PTH and hHCF antagonists. Based on the antagonist activity of [Tyr34]bovine PTH-(7-34)NH2 [( Tyr34]bPTH-(7-34)NH2), we synthesized the corresponding fragment of hHCF, hHCF-(7-34)NH2 and examined its properties in vitro. In the bone-derived rat osteosarcoma cell line ROS 17/2.8, hHCF-(7-34)NH2 and [Tyr34]bPTH-(7-34)NH2 were equipotent for inhibition of radiolabeled PTH-binding. In contrast, hHCF-(7-34)NH2 was 8-fold more potent that [Tyr34]bPTH-(7-34)NH2 for inhibiting PTH-stimulated cAMP production. hHCF-(7-34)NH2 also inhibited PTH-binding and PTH-stimulated adenylate cyclase activity in bovine renal cortical membranes: hHCF-(7-34)NH2 and [Tyr34]bPTH-(7-34)NH2 were equipotent in this system. In addition, hHCF-(7-34)NH2 antagonized hHCF-(1-34)NH2 action in both systems with similar inhibition constants. However, unlike the PTH analogue, hHCF-(7-34)NH2 (8 microM) was a weak partial agonist, producing a 2.4-fold increase in cAMP (5% of the maximal response) in ROS cells. This same system also detects agonism for [Nle8, 18Tyr34]bPTH-(3-34)NH2, another PTH partial agonist/antagonist. These results demonstrate that hHCF-(7-34)NH2 interacts with PTH receptors based in large part on the region which is not homologous to PTH, and suggest the utility of the ROS 17/2.8 cell system for identifying weak agonism of PTH and hHCF analogues in vitro.
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PMID:The 7-34-fragment of human hypercalcemia factor is a partial agonist/antagonist for parathyroid hormone-stimulated cAMP production. 283 81

[Tyr36]human adenylate cyclase stimulating peptide (1-36)-NH2, an amino-terminal analog of a tumor peptide which is associated with hypercalcemia of malignancy, and [Nle8, Nle18, Tyr34]bovine parathyroid hormone (PTH)-(1-34)-NH2 both bind with similar affinities to receptors on rat osteosarcoma cells, ROS 17/2.8, when either of the peptides is used as the radioligand. Pretreatment of the cells with either peptide down-regulates available binding sites for either radioligand and desensitizes the cAMP accumulation stimulated by either peptide. Prior exposure of the cells to dexamethasone increases these responses to both peptides. Photoderivatized radioiodinated [Tyr36]human adenylate cyclase-stimulating peptide (1-36)-NH2 and [Nle8, Nle18, Tyr34]bovine PTH-(1-34)-NH2 both specifically label a Mr = 80,000 membrane protein on ROS 17/2.8 cells. The intensity of labeling this receptor band by either photoprobe is reduced by co-incubation with either peptide over the same dose range. Equivalent dose-dependent down-regulation of receptors which bind both photoprobes is also found when ROS 17/2.8 cells are preincubated with either peptide. Dexamethasone increases the intensity of receptor labeling. Our findings strongly indicate that both peptides recognize the same plasma membrane receptor on ROS 17/2.8 cells. Although the physiological function(s) of human adenylate cyclase-stimulating peptide is unknown, these results could explain why its biological actions on mineral ion metabolism so closely simulate those of PTH and raise interesting questions about the general biological and evolutionary significance of the use of the same receptor by chemically distinct peptides.
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PMID:The parathyroid hormone-like peptide associated with humoral hypercalcemia of malignancy and parathyroid hormone bind to the same receptor on the plasma membrane of ROS 17/2.8 cells. 283 57

The effect of prostaglandins (PG) on free cytosolic calcium concentrations [( Ca2+]i) and cAMP levels was studied in the osteosarcoma cell line UMR-106. PGF2 alpha and PGE2, but not 6-keto-PGF1 alpha, induced an increase in [Ca2+]i which was mainly due to Ca2+ release from intracellular stores. The EC50 for PGF2 alpha was approximately 7 nM, whereas that for PGE2 was approximately 1.8 microM. Maximal doses of PGF2 alpha increased [Ca2+]i to higher levels than PGE2. Both active PGs also stimulated phosphatidylinositol turnover in UMR-106 cells. The effects of the two PGs were independent of each other and appear to involve separate receptors for each PG. PGE2 was a very potent stimulator of cAMP production and increased cAMP by approximately 80-fold with an EC50 of 0.073 microM. PGF2 alpha was a very poor stimulator of cAMP production; 25 microM PGF2 alpha increased cAMP by 5-fold. The increase in cellular cAMP levels activated a plasma membrane Ca2+ channel which resulted in a secondary, slow increase in [Ca2+]i. High concentrations of both PGs (10-50 microM) inhibited this channel independent of their effect on cAMP levels. Pretreatment of the cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate inhibited the PG-mediated increase in phosphatidylinositol turnover and the increase in [Ca2+]i. However, pretreatment with 12-O-tetradecanoyl-13-acetate had no effect on the PGE2-mediated increase in cAMP. The latter finding, together with the dose responses for PGE2-mediated increases in [Ca2+]i and cAMP levels, suggests the presence of two subclasses of PGE2 receptors: one coupled to adenylate cyclase and the other to phospholipase C. With respect to osteoblast function, the cAMP signaling system is antiproliferative, whereas the Ca2+ messenger system, although having no proliferative effect by itself, tempers cAMP's antiproliferative effect.
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PMID:Relationship of cAMP and calcium messenger systems in prostaglandin-stimulated UMR-106 cells. 283 4

A tumor-derived protein with a spectrum of biologic activities remarkably similar to that of parathyroid hormone (PTH) has recently been purified and its sequence deduced from cloned cDNA. This PTH-like protein (PLP) has substantial sequence homology with PTH only in the amino-terminal 1-13 region and shows little similarity to other regions of PTH thought to be important for binding to receptors. In the present study, we compared the actions of two synthetic PLP peptides, PLP-(1-34)amide and [Tyr36]PLP-(1-36)amide, with those of bovine parathyroid hormone (bPTH)-(1-34) on receptors and adenylate cyclase in bone cells and in renal membranes. Synthetic PLP peptides were potent activators of adenylate cyclase in canine renal membranes (EC50 = 3.0 nM) and in UMR-106 osteosarcoma cells (EC50 = 0.05 nM). Bovine PTH-(1-34) was 6-fold more potent than the PLP peptides in renal membranes, but was 2-fold less potent in UMR-106 cells. A competitive PTH receptor antagonist, [Tyr34]bPTH-(7-34)amide, rapidly and fully inhibited adenylate cyclase stimulation by the PLP peptides as well as bPTH-(1-34). Competitive binding experiments with 125I-labeled PLP peptides revealed the presence of high affinity PLP receptors in UMR-106 cells IC50 = 3-4 nM) and in renal membranes (IC50 = 0.3 nM). There was no evidence of heterogeneity of PLP receptors. Bovine PTH-(1-34) was equipotent with the PLP peptides in binding to PLP receptors. Likewise, PLP peptides and bPTH-(1-34) were equipotent in competing with 125I-bPTH-(1-34) for binding to PTH receptors in renal membranes. Photoaffinity cross-linking experiments revealed that PTH and PLP peptides both interact with a major 85-kDa and minor 55- and 130-kDa components of canine renal membranes. We conclude that PTH and PLP activate adenylate cyclase by binding to common receptors in bone and kidney. The results further imply that subtle differences exist between PTH and PLP peptides in their ability to induce receptor-adenylate cyclase coupling.
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PMID:Synthetic peptides comprising the amino-terminal sequence of a parathyroid hormone-like protein from human malignancies. Binding to parathyroid hormone receptors and activation of adenylate cyclase in bone cells and kidney. 284 1

While the stimulatory effect of parathyroid hormone (PTH) on osteoblast-like cell adenylate cyclase is well known, the effect of PTH on cytosolic calcium ion ([Ca2+]i) mobilization is controversial, one group finding no effect but others reporting various increases. We investigated the effects on [Ca2+]i of synthetic rat PTH fragment 1-34 (rPTH(1-34)) and two bovine PTH analogues that inhibit PTH's stimulation of adenylate cyclase (bovine 8,18Nle, 34Tyr-PTH(3-34) and 34Tyr-PTH(7-34]. [Ca2+]i was measured before, during, and after exposure to PTH analogues in perifused, attached osteoblast-like rat osteosarcoma cells (ROS 17/2.8) that had been scrape-loaded with the luminescent photoprotein aequorin. Resting [Ca2+]i was 0.094 +/- 0.056 microM (mean +/- S.D., n = 103) and rose in a time- and dose-specific way after exposure to rPTH(1-34). At 10(-10) M rPTH(1-34), [Ca2+]i rose 100% within 30 s to a plateau; higher concentrations of PTH yielded increasing initial peaks of [Ca2+]i followed by lower plateaus. At 10(-6) M, the initial peak was 5-fold basal, or 0.64 +/- 0.07 microM. Both analogues of PTH were at least partial agonists for [Ca2+]i mobilization and did not reduce peak [Ca2+]i when co-perifused with rPTH(1-34). However, the analogues did reduce significantly rPTH(1-34)-induced cAMP accumulation and did not increase cAMP accumulation by themselves. Thus, rPTH(1-34) strongly mobilizes [Ca2+]i in ROS 17/2.8 cells, at near-physiologic concentrations. Failure of the PTH analogues to block the effect of PTH on [Ca2+]i while inhibiting the effect on cAMP accumulation suggests separate pathways for PTH activation of adenylate cyclase and mobilization of calcium.
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PMID:Differential effects of parathyroid hormone and its analogues on cytosolic calcium ion and cAMP levels in cultured rat osteoblast-like cells. 284 23

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.
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PMID:Influence of the amino-terminus on in vitro and in vivo biological activity of synthetic parathyroid hormone-like peptides of malignancy. 284 83

Binding of parathyroid hormone (PTH) to circulating bovine lymphocytes was studied using, as the radioligand, a synthetic sulfur-free analog of bovine PTH, [Nle8,Nle18,Tyr34]bPTH-(1-34)amide, which was labeled to high specific activity with 125I and was purified by reverse-phase high-performance liquid chromatography. Binding of PTH to lymphocytes satisfies several criteria indicative of a specific interaction between the hormone and its receptor. Specific binding is saturable at 3.3 fmoles of radioligand bound per 10(7) cells, occurs more rapidly at 37 degrees C than at lower temperatures, and reaches equilibrium within 2 hr at 15 degrees C. Inhibition of specific binding occurs with intact PTH, with biologically active PTH analog or fragment, and with synthetic PTH antagonists, but not with biologically inactive PTH fragments, or peptide hormones unrelated to PTH antagonists, but not with biologically inactive PTH fragments, or peptide hormones receptors on lymphocytes and those previously reported with receptors in canine renal membranes, and on rat osteosarcoma cells. The dissociation constant (Kd) is approximately 10(-9) M, as calculated from the association and dissociation rate constants. This correlates very closely both with the apparent Kd, as estimated from Scatchard analysis of radioligand saturation and competition studies, and with previously reported Kd of PTH receptors in canine renal membranes and on intact rat osteosarcoma and opossum kidney cells. In addition, the relative binding affinity of intact hormone and a synthetic PTH agonist to to receptors on lymphocytes correlates closely with the relative biologic potency of these peptides in stimulating adenylate cyclase in membranes from these cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Properties of parathyroid hormone receptors on circulating bovine lymphocytes. 285 Jul 27

We identified the subunits of the stimulatory and inhibitory guanine nucleotide binding proteins (Gs and Gi, respectively) associated with adenylate cyclase in rat osteosarcoma (ROS) cells. Pertussis toxin catalyzed ADP-ribosylation of Gi alpha in ROS cells increased agonist (PTH and isoproterenol)-stimulated, but not basal, cAMP production. The effect of pertussis toxin was dose and time dependent, and slowly reversible (T 1/2 approximately 30 h) during continued culture without toxin. Pertussis toxin treatment of ROS cell lines (17/2.8 and 24/l) with markedly different agonist responsiveness increased agonist-stimulated cAMP production in proportion to the response without toxin treatment. Pertussis toxin treatment further increased cAMP response to PTH in dexamethasone treated cells. We conclude that ROS cells contain functional Gi which modulates agonist-stimulated cAMP formation. Alterations in ROS cAMP responsiveness caused by steroids, and the reduced responsiveness of the 24/1 cell line, however, are unlikely to be due to changes in Gi.
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PMID:The inhibitory guanine nucleotide regulatory protein modulates agonist-stimulated cAMP production in rat osteosarcoma cells. 285 47


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