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Biomedical subjects

G A Rodan

Publications and source records attributed to G A Rodan.

At least 181 records · Page 10Linked to original sources
PubMed1981

Parathyroid hormone and isoproterenol stimulation of adenylate cyclase in rat osteosarcoma clonal cells. Hormone competition and site heterogeneity.

A clonal cell line from rat osteosarcoma was found to possess parathyroid hormone and isoproterenol sensitive adenylate cyclase. This study examines the relationship between the two hormones and triphosphoguanine nucleotide with respect to enzyme activation. Concentration-dependence curves, analyzed by computer-aided curve fitting, revealed: (1) in the presence of 5 microM GTP there were two apparent affinities for parathyroid hormone (Km 9 and 89 nM) and isoproterenol (Km 72 and 340 nM; (2) and two affinities for guanosine-5' (beta, gamma-imido)triphosphate (Km 0.25 and 1.3 microM); (3) hormones and guanine nucleotides reciprocally shifted each other's concentration dependence curve to the high affinity sites; (4) parathyroid hormone and isoproterenol interacting with high affinity sites competed for the same adenylate cyclase; (5) parathyroid hormone and isoproterenol, acting on low affinity sites had additive effects and also stimulated adenylate cyclase in the absence of added guanine nucleotides. The findings are consistent with (i) competition of parathyroid hormone and isoproterenol for the activation of the high (hormone) affinity complex containing: receptors, nucleotide subunit, triphosphoguanine nucleotide, catalytic unit (ii) the apparent presence of receptor-nucleotide sub-unit GDP-catalytic unit complexes with low hormone affinity which are stimulated by parathyroid hormone and isoproterenol separately.

Adenylyl Cyclases↗
PubMed1981

The role of Mg2+ in hormone stimulation of rat osteosarcoma adenylate cyclase.

1. Mg2+ concentration dependence of adenylate cyclase activity, in a rat osteosarcoma cell line (ROS 2/3), exhibits two apparent affinities with Km values of approx. 2 mM and 10 mM. 2. Only one Mg2+ affinity with a Km value of around 1 mM was apparent at saturating concentrations of: (i) guanosine-5'-(beta, gamma-imido)triphosphate; (ii) parathyroid hormone and GTP; and (iii) (-)-isoproterenol and GTP. 3. Conversely, at saturating concentrations of Mg2+ (40 mM) only high hormone concentrations, acting on low affinity sites, stimulated adenylate cyclase. 4. At saturating concentrations of guanosine-5'-(beta, gamma-imido)triphosphate, hormone stimulation decreased with increasing Mg2+ concentrations and none was seen at 40 mM Mg2+. The findings suggest that hormone stimulation of adenylate cyclase is associated with Mg2+ activation of a 'high hormone affinity' responsive state dependent on triphosphoguanine nucleotide. The hormone effect on Mg2+ affinity fully accounts for hormone stimulation of adenylate cyclase at physiologically relevant concentrations.

Adenylyl Cyclases↗
PubMed1981

Cytochemical evidence for the segregation of adenylate cyclase, Ca2+-, Mg2+-ATPase, K+-dependent p-nitrophenyl phosphatase in separate membrane compartments in human platelets.

The blood platelet has three morphologically distinct membrane systems. In addition to the plasma membrane the platelet has an 'open canalicular system' (surface-connected intracytoplasmic membrane system) and a microsome-like 'dense tubular system'. The open canalicular and dense tubular systems have been implicated in Ca2+ transport, cyclic nucleotide (cAMP) synthesis and prostaglandin and thromboxane synthesis. Precise definition of the function of the different membrane systems requires analysis of their unique chemical activities. Broken cell preparations are used to advantage for such studies. However, clean separation and definition of the origin and composition of the membrane fractions has been difficult because well-defined marker enzymes for the various membrane systems have not been conclusively established. Platelets were fixed for 5 min in 1% paraformaldehyde-0.2% glutaraldehyde and assayed for K+-dependent p-nitrophenyl phosphatase, Ca2+-, Mg2+-ATPase and adenylate cyclase K+-dependent p-nitrophenyl phosphatase was localized only at the plasma membrane while Ca2+-, Mg2+-ATPase and adenylate cyclase were found relatively segregated to the open canalicular and dense tubular systems. The segregation of these enzymes to separate membrane compartments may have significant implications with regard to understanding platelet function.

4-Nitrophenylphosphatase↗
PubMed1980

Osteosarcoma cytosol factor promotes parathyroid hormone stimulation of adenylate cyclase independent of GTP.

The adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1)-stimulating factor from rat osteosarcoma cytosol was purified 600-fold by ion-exchange chromatography. The factor has an apparent Mr of 20000, is cold-labile, but retains activity at -20 degrees C in 10% glycerol. The factor enhanced parathyroid hormone stimulation of adenylate cyclase and restored hormone responsiveness to membranes washed with 0.5 M NaCl. These 'GTP-like' effects were not inhibited by 100 microM GDP-beta-S, which completely abolished the GTP enhancement of both basal and hormone-stimulated adenylate cyclase. Adenylate cyclase activity in the presence of the stimulating factor was linear with time, and showed hyperbolic dependence on factor concentration. The factor also linearized (in double reciprocal plots) the downward-concave Mg2+-dependence of adenylate cyclase, increasing the apparent affinity of the enzyme for Mg2+. The presence of the factor in two clonal osteosarcoma cell lines correlated with parathyroid hormone-stimulatable adenylate cyclase. Factor stimulation was absent while GTP stimulation was retained in the hormone-nonresponsive clone. Factor and hormone sensitivity were restored by in vivo passage. This factor thus may represent a guanyl nucleotide-independent path for cellular regulation of hormone response.

Adenylyl Cyclases↗
PubMed1980

Comparison of bone and osteosarcoma adenylate cyclase. Partial purification of membranes and kinetic properties of enzyme.

The purpose of this study was to compare the adenylate cyclase of a tumour (rat osteosarcoma) growing in vivo with that of fast-growing embryonic bone. In the tumour the enzyme activity per total protein or DNA (under the same assay conditions) was 6--10-fold lower than in embryonic bone. To characterize this difference, we examined the kinetic properties of the enzyme in partially purified plasma membranes from the two tissues. A purification procedure based on differential centrifugation and discontinuous-sucrose-gradient centrifugation yielded a 10-fold increase in the specific activities of adenylate cyclase and 5'-nucleotidase in bone. The same procedure yielded an enriched membrane preparation from the tumour, but, relative to 5'-nucleotidase, a loss of 30% in adenylate cyclase occurred, which could not be recovered from another fraction. Kinetic analysis revealed that the lower adenylate cyclase activity in the tumour was due to a decrease in Vmax.. There was no significant difference in Ks (approx. 0.15 mM), and in the Km for GTP and p[NH]ppG. There were marked differences, however, in the extent of stimulation by p[NH]ppG, GTP and hormone, which was greater in tumour, and in the K1 for adenosine inhibition, which was 140 microM in bone and 500 microM in tumour. Under maximum stimulatory conditions, the enzyme activity in the tumour approached that in bone. The kinetic differences between bone and tumour enzyme were decreased by detergent solubilization, suggesting that the membrane environment plays a role in the generation of the observed differences.

Adenylyl Cyclases↗
PubMed1980

Comparison of bone and osteosarcoma adenylate cyclase. Effects of Mg2+, Ca2+, ATP4- and HATP3- in the assay mixture.

The effects of Mg(2+) and Ca(2+) on bone and osteosarcoma adenylate cyclase were investigated. The concentrations of the cations and other ionic species in the assay mixture were calculated by solving the simultaneous equations describing the relevant ionic interactions (multiple equilibria). We re-examined the effects of HATP(3-) and ATP(4-) on enzyme activity and found that (i) the concentration of the minor ATP species is less than 1% of that of MgATP(2-), and their ratio to MgATP(2-) is constant if Mg(2+) and H(+) concentrations are unchanged; (ii) Mg(2+) addition decreased the ratio of the minor species to MgATP(2-) and increased the enzyme activity, but no meaningful kinetic model could attribute this effect of HATP(3-) or ATP(4-). On the other hand, kinetic analysis of Mg(2+) effects showed: (i) stimulation via two metal sites, separate from the catalytic (MgATP(2-)) site, with apparent K(m) values of approximately 1 and 8mm; (ii) that the low affinity increased towards the higher one when the enzyme activity rose as a result of increased substrate or guanine nucleotide concentrations, this effect being less pronounced in tumour; (iii) conversely, that two apparent affinities for MgATP(2-) merged into one at high Mg(2+) concentration; (iv) kinetically, that this relationship is of the mixed con-competitive type, which is consistent with a role for Mg(2+) as a requisite activator, and binding occurring in non-ordered sequence. Analysis of the Ca(2+) effects showed: (i) competition with Mg(2+) at the metal site (K(i) 20mum for bone and 40mum for tumour); (ii) that relative to the substrate the inhibition was uncompetitive, i.e. velocity decreased and affinity increased proportionally, which is consistent with Ca(2+) binding after substrate binding. These findings support the existence of interacting enzyme complexes, losing co-operativity at increased enzyme activity. They also indicate a potential physiological role for Ca(2+) in enzyme regulation and point to quantitative differences between bone and tumour with regard to these properties.

Adenosine Triphosphate↗
PubMed1980

Parathyroid hormone-responsive clonal cell lines from rat osteosarcoma.

Several clonal cell lines from a transplantable rat osteosarcoma, selected on the basis of parathyroid hormone (PTH)-sensitive adenylate cyclase, were established in culture. Bovine PTH-(1-84) (0.1 microM) stimulation of adenylate cyclase varied among clones from 8-fold to none. The level of PTH response was a stable property of each clonal line that was retained through numerous passages in vitro (nearly 3 yr in the oldest clone). Highly PTH-responsive lines had a cuboidal-eliptoid morphology and differed from the nonresponsive lines, which had a more fibroblastic appearance. PTH responsiveness correlated with several properties, presumably associated with the osteoblastic phenotype: elevated alkaline phosphatase activity, synthesis of the gamma-carboxyglutamic acid-containing bone protein, and production of mineralized tumors in host rats. PTH (1.0 nM; 24 h) reduced the alkaline phosphatase activity by 40% when tested in a responsive clone. The acid phosphatase activity of the various cell lines was uniformly low. These osteosarcoma-derived cell lines which are stable in vitro thus seem to reflect the phenotypic heterogeneity observed in the tumor in situ. They could be useful in studies of phenotypic expression, PTH action, and a possible relationship between the two.

1-Carboxyglutamic Acid↗
PubMed1979

Adenylate cyclase and ATPase activities in red cell membranes of patients and genetic carriers of Duchenne muscular dystrophy.

Basal adenylate cyclase activity was increased in red cell ghosts from both patients with Duchenne muscular dystrophy and their mothers when the activities were compared to proper age-matched controls. The activity of ATPase measured in the presence of Na+, K+, and Mg2+ was not found to be different in erythrocyte ghosts from Duchenne dystrophic patients, age-matched controls, or the mothers of Duchenne patients, and ouabain inhibited ATPase in ghosts to the same extent in all membrane preparations.

Adenosine Triphosphatases↗
PubMed1979

Adenylate cyclase in muscular dystrophy.

The purpose of this study was to determine whether the previously reported differences in adenylate cyclase activity between the sarcolemma of normal and dystrophic chick muscles are also found in the SR, to search for a possible relationship between the adenylate cyclase changes and the pathophysiology of dystrophy, and to investigate whether the findings can be extended to Duchenne human muscular dystrophy by studying the adenylate cyclase and ATPase activities of erythrocyte ghosts from DMD patients and carriers. Microsomes were separated by standard techniques from the pectoralis muscles of normal and dystrophic ckeckens of various ages. The microsomal yields were significantly larger in dystrophic muscles. Adenylate cyclase activities in dystrophic microsomes were higher than those in matched controls and increased with the progression of the disease. The ratio between the two rose from one at 2 weeks of age to nine at about 9--10 weeks. Kinetic analyses showed that the ks for MgATP2- was about 40 microM (at 3 mM Mg2+ and 0.3 mM Ca2+) both in normal and dystrophic microsomes, that calcium caused umcompetitive inhibition of the enzyme (Ki = 0.2 mM), that the effect of calcium was noncooperative (Hill coefficient, nH = 1), that calcium did not affect the cooperativity for MgATP2-, and that magnesium competitively removed the calcium inhibition and caused additional, cooperative stimulation of the enzymatic activity (ka = 1.5 mM; NH =2). The major difference between normal and dystrophic adenylate cyclase was a higher enzymatic velocity in the latter, suggesting a larger amount of enzyme. We investigated whether altered cAMP levels may effect calcium accumulation. Calcium uptake measured (in the presence of oxalate) at several ages revealed no difference between normal and dystrophic chickens. The extent of calcium binding was also similar, although the kd for Ca2+ was lower in dystrophic microsomes. Binding was enhanced in the presence of exogenous protein kinase, but the responses of normal and dystrophic tissues were similar. We concluded that the elevation of adenylate cyclase in dystrophy was not related to microsomal calcium accumultion. Ivestigation of the localization of microsomal adenylate cyclase supported this view. Separation of calcium-loaded microsomes on a discontinuous sucrose gradient into four fractions demonstrated that adenylate cyclase activity, measured in the presence of Lubrol-PX and EGTA, was inversely related to calcium-accumulating activity. Na+, K+-ATPase comigrated with adenylate cyclase. Highest specific activities were found in the lightest fraction. These observations were confirmed by histochemical studies. The reaction product from adenylate cyclase activity was present predominantly in the terminal cisternae of the SR. In the context of the literature, our findings suggest that the rises in adenylate cyclase and Na+, K+-ATPase in avian dystrophy are compensatory changes, elicited by a defect in ECC at the calcium release step...

Adenylyl Cyclases↗
PubMed1978

DNA synthesis in cartilage cells is stimulated by oscillating electric fields.

External oscillating electric fields (1166 volts per centimeter, 5 hertz) enhanced the incorporation of [3H] thymidine into the DNA of chondrocytes isolated from the proliferative layer of embryonic (16 days) chick epiphysis. Verapamil or tetrodotoxin at 10(-6)M concentrations completely blocked the electric field effect. Tetracaine reduced the incorporation of [3H] thymidine in both control and electrically stimulated cells. The findings support the hypothesis that Na and Ca2 fluxes generated by the electrical perturbation trigger DNA synthesis in these cells.

Animals↗