Journal of cyclic nucleotide and protein phosphorylation research最新文献

Incubation of dog thyroid slices with 1 mU/ml TSH resulted in enhanced intracellular and extracellular cAMP accumulation. In the absence of TSH, the intra- and extracellular cAMP concentrations remained at a constant low level. The release of cAMP from TSH-stimulated slices was inhibited by 10 microM PGA1, 1 mM probenecid or 1 mM IBMX, which are known inhibitors of cAMP escape in several tissues. Negative controls of intracellular cAMP levels are exerted in the dog thyroid by 10 microM carbamylcholine (shown to activate a Ca++- calmodulin dependent phosphodiesterase), 100 microM norepinephrine and 100 microM iodide (both inhibiting adenylate cyclase activity). The purpose of the present study was to demonstrate that these three agents do not enhance cAMP escape. The results presented here show that these agents decrease both intracellular accumulation and escape in parallel. Moreover, the escape constants obtained by numerical simulation were not greater in the presence of inhibiting concentrations of carbamylcholine, norepinephrine or iodide. Thus the inhibition by these agents of cAMP accumulation in TSH-stimulated dog thyroid slices cannot be explained by a stimulation of cAMP escape from these cells.

Isoproterenol activates adenylate cyclase indirectly via the beta-receptors. Forskolin, on the other hand, directly activates the adenylate cyclase. Both compounds can induce slow action potentials (APs) in isolated guinea pig papillary muscles, consistent with their ability to activate adenylate cyclase. Acetylcholine (ACh), 1-10 microM, depressed or abolished slow APs induced by isoproterenol or forskolin. There was no difference between the forskolin- and isoproterenol-induced slow APs with regard to their sensitivity to ACh. Similar results were obtained in cultured embryonic chick heart cells. We conclude that forskolin induces slow APs that are essentially the same as those induced by isoproterenol, and that ACh action on depressing slow APs must be either directly on the adenylate cyclase complex and/or on another step entirely (e.g., mediated through increased cGMP.

Progress curves of cyclic AMP production by human platelet lysates in the presence of 10 microM GppNp were upward curving, reaching a steady-state rate after about 20 min. Increasing concentrations of prostaglandin E1 reduced the lag time required to reach steady-state and increased the steady-state rate in a dose-dependent manner. At 0.3 microM PGE1 the progress curve was linear for at least 40 min. At higher concentrations of PGE1 the initial rate of cyclic AMP formation was linear and similar to that obtained at 1.0 microM PGE, however, the progress curve showed a downward curvature after several minutes, reaching a new steady-state rate after about 10 min. The extent of downward curvature was dose-dependent, and at 10 microM PGE1 the final steady-state rate had almost returned to that observed in the presence of GppNp alone. Analysis of initial and final steady-state rates as a function of prostaglandin concentration revealed two apparently saturable processes that were interpreted as binding to a stimulatory receptor (EC50 = 130 nM), followed by binding to a lower affinity inhibitory receptor (EC50 = 1200 nM) that showed a slow response to receptor occupancy. Qualitatively similar results were obtained with PGD2 and the stable PGI2 analog ZK36374.

Binding of 125I-iodopindolol (IPIN) to intact 1321N1 human astrocytoma cell B-adrenergic receptors was measured at 37 degrees and on ice. Control cells showed a single component of IPIN binding on ice with the same total number of receptors as measured at 37 degrees. In desensitized cells (pretreated for 20 min with 1 microM isoproterenol) approximately 40% of IPIN binding on ice exhibited kinetics similar to those observed in control cells. The remaining 60% of receptors were labelled by IPIN at a much slower rate requiring the use of very high concentrations of IPIN. Sucrose density gradient fractionation was used to separately study the labelling of plasma membrane receptors and those associated with a light vesicle fraction. Labelling by IPIN on ice of the plasma membrane receptors of control cells was rapid, labelling of the light vesicle receptors of desensitized cells was slow, and labelling of the plasma membrane receptors of desensitized cells appeared to occur with both rapid and slow components. Selective labelling of the plasma membrane receptors of intact cells thus could be obtained by incubation with IPIN on ice under selected conditions. Similar results were obtained when broken cell preparations from control and desensitized cells were used. The decreased binding of IPIN on ice to B-adrenergic receptors in the light vesicle fraction not only provides further evidence consistent with sequestration of B-adrenergic receptors during desensitization, it also provides a convenient and inexpensive means to assay the sequestration reaction.

We found specific activity of adenylate cyclase (AC) to be as high in rat skeletal muscle sarcoplasmic reticulum (SR) as in sarcolemma (SL) (39 +/- 5 pmol/mg per min and 34 +/- 5 pmol/mg per min). Detection of AC in SR could not be due to SL contamination. Activity in SR was similar in triads (heavy SR) and longitudinal reticulum (light SR), despite virtual absence of surface membrane markers in preparations of light SR. Also, AC of SR and SL may differ biochemically. In the presence of 10(-5) M 5'-guanylylimidodiphosphate, 10(-4) M isoproterenol increased SL activity 508%, crude SR 46.4%, heavy SR 68.3%, light SR only 24.3%. SR activity was 50% higher at 0.32 micromolar Ca++ than at 1 nanomolar Ca++ (p less than 0.05); higher concentrations of Ca++ noncompetively inhibited activity (Ki 0.87 micromolar). In contrast, Ca++ monophasically inhibited SL activity. Permeabilization of SR vesicles with alamethacin indicated that AC is on the cytoplasmic surface of SR; its regulation by physiological changes in cytoplasmic Ca++ could influence SR Ca++ flux.

Porins interact with macrophage membranes and inhibit their phagocyting activity. We have tested the porin effect on a biologically relevant membrane-bound enzymic activity, the adenylate cyclase system, which appears to be stimulated both in the presence of Mn2+ and Mg2+ or Mg2+ + Gpp(NH)p. Moreover, for mice macrophages incubated in the presence of porins, there is an increase in the intracellular cAMP content after 5 min of incubation, with a maximum after 15 min of incubation. The results shown suggest that the porin effects on the adenylate cyclase can represent the molecular basis of the porin-dependent inhibition of the macrophages phagocytosis. Our point of view, which proposes a cAMP role in inhibiting the phagocyting activity in macrophages, is supported also by the results of the experiments carried out in the presence of both dibutyryl-cAMP or aminophylline. The phagocyting activity is inhibited in all cases and independently of the bacteria to be phagocyted.

Xenopus oocytes are stimulated to undergo meiotic cell division in response to several types of mitogenic stimuli. Agents that reduce cAMP levels induce cell division in oocytes, and this occurs due to inhibition of adenylate cyclase with progesterone as well as by activation of phosphodiesterase with insulin. Phorbol esters and microinjected protein kinase C also promote cell division, implicating phospholipid breakdown as another signalling pathway competent to induce proliferation in this system. A third signalling pathway is via the tyrosine kinase activity of the insulin receptor. A proximal activation of a ribosomal protein S6 kinase by insulin has provided insight into the regulation of this pathway. All three of these signal transduction pathways lead to the activation of a cytoplasmic protein able to induce nuclear breakdown, chromosome condensation and spindle formation in vivo and in vitro. This protein, known as maturation-promoting factor, is associated with changes in protein phosphorylation on both serine and tyrosine residues. These results support a model in which signal transduction by different pathways activates a common cell cycle control element that regulates the G2----M transition via changes in protein phosphorylation.

Strips of bovine mesenteric arteries, brought to sustained contraction by addition of 3.0 microM phenylephrine, relaxed during exposure to shortwave light. The action spectrum of the photorelaxation was characterized by most effectiveness of relaxation in the range of 360 to 400 nm; on the contrary, shortwave light of 280 to 300 nm increased the tension. The photo-induced relaxation was accompanied by an increase in the cGMP level, measured 30 sec after the onset of radiation, and the action spectrum of the increase in cGMP seemed to coincide fairly well with the action spectrum of relaxation. Both the increase in cGMP and relaxation in response to increasing light intensity at 400 nm fitted the rectangular hyperbolic equation. The K values (the intensity which gave half maximal response) for cGMP increase and relaxation, respectively, obtained by non-linear least square regression analysis, were found to assume very close values (1.3 mW/cm2 for cGMP increase and 1.5 mW/cm2 for relaxation). The action spectrum of the crude soluble guanylate cyclase (GC) activity displayed a peak around 400 nm. Our results suggest that there is a close association between photo-induced increase in cGMP and relaxation, probably as a result of interaction between shortwave light and the heme moiety of GC.

Addition of N6-(L-2-phenylisopropyl)-adenosine (PIA) to cultured FRTL-5 rat thyroid cells led to a concentration-dependent inhibition of TSH-stimulated cAMP formation. Half-maximal inhibition was attained with approximately 0.5 nM PIA. Forskolin and cholera toxin-stimulated cAMP production were also inhibited by PIA. 3-Isobutyl-methylxanthine inhibited the effect of PIA. These results are consistent with the presence of inhibitory adenosine receptors (Ri). Ri-sites were further demonstrated by the binding of 3H-cyclohexyl-adenosine to FRTL-5 plasma membranes. High (Kd = 0.50 +/- 0.07 nM) and low affinity (Kd = 5.95 +/- 2.33 nM) binding sites were observed. Pretreatment of FRTL-5 cells with pertussis, but not cholera, toxin effectively antagonized the inhibitory effects of PIA on cAMP production. ADP-ribosylation of FRTL-5 membranes with [32P]-NAD in the presence of cholera or pertussis toxin specifically labeled a 45,000 and 41,000 Mr species, respectively, which correspond to the alpha subunit of the stimulatory and inhibitory guanine nucleotide regulatory proteins. These results demonstrate that PIA inhibits TSH-stimulated cAMP production via Ri-sites on FRTL-5 thyroid cells. PIA appears to exert its inhibitory effects through the inhibitory guanine nucleotide regulatory protein.

Eukaryotic protein synthesis initiation factor 2, eIF-2, was purified from either hemin-supplemented (translationally active) or hemin-deficient (translationally inactive) rabbit reticulocyte lysate under conditions chosen and demonstrated to preserve the in situ phosphorylation state. Direct analysis of the phosphate content of the alpha-subunit of eIF-2 was determined by chemical analysis of the isolated alpha-subunit or by a combination of vertical slab gel isoelectric focusing and immunoblotting. These results were compared with those obtained from an indirect analysis utilising the incorporation of [gamma-32P]ATP into eIF-2 by the heme-sensitive eIF-2 alpha-kinase. All three analyses demonstrate that the phosphorylation site specific for the heme-sensitive kinase is unoccupied in translationally active lysate and 25-30% occupied in translationally inhibited lysate. In addition, both direct analyses support the existence of a second phosphorylation site on the alpha-subunit, not regulated by hemin and distinct from that phosphorylated by the heme-sensitive kinase. Different reticulocyte lysate batches vary with respect to the activity of the kinase responsible for phosphorylation of the second site. Further investigations demonstrated that this kinase is a membrane-associated protein.