Multiple effects of guanine nucleotides on human platelet adenylate cyclase

We report that the adenylate cyclase system in human platelets is subject to multiple regulation by guanine nucleotides. Previously it has been reported that GTP is either required for or has little effect on the response of the enzyme to prostaglandin E₁. We have found that when platelet lysates were prepared in the presence of 5 mM EDTA, GTP lowered the basal and prostaglandin E₁-stimulated adenylate cyclase activity when the enzyme was assayed in the presence of Mg²⁺. The basal and prostaglandin E₁-stimulated adenylate cyclase activities were also increased by washing, which presumably removes endogenous GTP. The analog, guanyl-5′-yl-imidodiphosphate mimics the inhibitory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase activity but it stimulates basal enzyme activity. The onset of the inhibitory effect of GTP on the adenylate cyclase system is rapid (1 min) and is maintained at a constant rate during incubation for 10 min. GTP and guanyl-5′-yl-imidodiphosphate were noncompetitive inhibitors of prostaglandin E₁. An increase in the concentration of Mg²⁺ gradually reduces the effect of GTP while having little influence on the effect of guanyl-5′-yl-imidodiphosphate. Neither the substrate concentration nor the pH (7.2–8.5) is related to the inhibitory effect of guanine nucleotides. The inhibition by nucleotides was found to show a specificity for purine nucleotides with the order of potency being guanyl-5′-yl-imidodiphosphate > dGTP > GTP > ITP > XTP > CTP > TTP. The inhibitory effect of GTP is reversible while the effect of guanyl-5′-yl-imidodiphosphate is irreversible. The GTP inhibitory effect was abolished by preparing the lysates in the presence of Ca²⁺. However, the inhibitory effect of guanyl-5′-yl-imidodiphosphate persisted. Substitution of Mn²⁺ for Mg²⁺ in the assay medium resulted in a diminution of the inhibitory effect of GTP on basal activity and converted the inhibitory effect of GTP on prostaglandin E₁-stimulated activity to a stimulatory effect. At a lower concentration of Mn²⁺ (less than 2 mM) guanyl-5′-yl-imidodiphosphate inhibited prostaglandin E₁-stimulated adenylate cyclase activity, but at a higher concentration of Mn²⁺, it caused an increase in enzyme activity exceeding that occuring in the presence of prostaglandin E₁. In the presence of Mn²⁺, dGTP mimics the effect of GTP and is 50% as effective as GTP. Our data suggest that the inhibitory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase is mainly due to its direct effect on the enzyme itself, whereas the stimulatory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase is due to enhancement of the coupling between the prostaglandin E₁ receptor and adenylate cyclase. These studies also indicate that the method of preparation of platelet lysates can profoundly alter the nature of guanine nucleotide regulation of adenylate cyclase.     

核苷对ROS17/2.8细胞株腺苷酸环化酶活性的影响

 大鼠成骨肉瘤细胞株(ROS17/2.8)系甲状旁腺素(PTH)的靶细胞。当该细胞质膜上的PTH受体与PTH结合后,可激发腺苷酸环化酶(AC)的活性。腺苷及四种不同的核苷酸(AMP、GMP、UMP、CMP)单独对AC无明显效应,但却可抑制PTH对AC的刺激作用。而鸟苷或木糖腺苷则可显著增强PTH对AC的刺激作用。提示核苷的不同代谢物在代谢调节中的多样化作用。     

Prostaglandin E1 binding and adenylate cyclase activation in normal and transformed fibroblasts

The binding of [³H]prostaglandin E₁ to membranes of clones of normal rat kidney fibroblasts (NRK cells) has been measured. Cell lines that responded to prostaglandin E₁, such as NRK and NRK transformed with Schmitt-Ruppin strain of Rous sarcoma virus (SR-NRK cells), have a high affinity prostaglandin E₁ binding site. Murine-sarcoma-virus-transformed lines of NRK cells are unresponsive to prostaglandin E₁ and have reduced prostaglandin E₁ binding. Exposure of cells to prostaglandin E₁ results both in decreases prostaglandin E₁ responsiveness and reduced prostaglandin E₁ binding.Activation of adenylate cyclase is correlated to binding of prostaglandin E₁ to receptors in both NRK and SR-NRK cell membranes. Mathematical models suggest that GTP decreases the affinity of hormone for its receptor while increasing the catalytic efficiency of adenylate cyclase, and that aggregates of occupied receptors may play an important role in the activation of adenylate cyclase.     

S-100b protein regulates the activity of skeletal muscle adenylate cyclase in vitro

We have investigated the effect of the b isoform of S-100 proteins on adenylate cyclase activity of rat skeletal muscle. S-100b inhibits the adenylate cyclase activity in the presence of Mg²⁺ (5.0–50 mM), while it activates the same enzyme in the presence of Ca²⁺ (0.1–1.0 mM) dose-dependently in both cases. S-100b counteracts the stimulatory effect of NaF on adenylate cyclase in the presence of Mg²⁺ and the inhibitory effect of RMI 12330 A in the presence of Ca²⁺.     

Solubilization of membrane-bound adenylate cyclase of isolated rat adrenal cortex cells

The membrane-bound adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) of isolated rat adrenal cortex cells can be rendered soluble using 0.02 M Lubrol 12A9. The solubilized enzyme can be filtered through Millipore filters with pores 0.22 μm in diameter. Using gel filtration, on Sephadex G-200, adenylate cyclase activity was eluted with a distribution coefficient of 0.139, whereas on Sephadex G-100 the activity was eluted in the excluded volume. Half-maximum activation of the postulated guanyl nucleotide regulator site of adenylate was achieved with 5′-guanylyl-imidodiphosphate at a concentration of 1 · 10^?6 M. In contrast, however, using intact isolated rat adrenal cortex cells the guanyl nucleotide regulator site could not be stimulated by 5′-guanylyl-imidodiphosphate.     

Partial purification and properties of the cytoplasmic factors modulating adenylate cyclase activity in rat lung plasma membranes

The adult rat lung supernatant contains some factors which markedly enhance adenylate cyclase activity in membranes (Nijjar, M.S. (1979) Biochim. Biophys. Acta 584, 43–50). These factors were separated into two less active components (peaks 1 and 2) by DEAE-cellulose chromatography. However, their recombination restored the full activation of adenylate cyclase. Further purification and characterization of these factors revealed that the activation in peak 1 contained two proteins of low (14 500) and high (65 000) molecular weight whereas the activator in peak 2 contained only one protein of 65 000. The kinetics of adenylate cyclase activation revealed that both the K_m and V values were affected. The data also demonstrate that calmodulin was not involved in the cytoplasmic activation of adenylate cyclase in rat lungs.     

Adenylate cyclase activity during growth and encystment of Acanthamoeba castellanii

The adenylate cyclase activity of Acanthamoeba castellanii (Neff) was studied in extracts prepared after breaking cells in the Potter-Elvehjem homogenizer. The adenylate cyclase activity of cells is low during the exponential growth phase, but then rises 2–4-fold during the stationary phase to a peak, roughly at the time that cyst forms are detectable in the culture. A 2–4-fold activity rise to a peak also occurs 4–8 h after late log cells are transferred to a non-nutrient encystment medium, a time which is shortly before numbers of cyst forms can be detected in the culture. The pattern of activity observed when stationary phase cells are transferred to encystment medium is complex and depends in part on whether the cultures have exhibited the peak of cyclase activity and have begun to initiate cyst formation prior to the transfer. Within the usual time frame after transfer to encystment medium, early logarithmic phase cells do not exhibit a 2–4-fold rise of cyclase activity and they do not encyst. The results suggest a relationship between encystment and the pattern of rise and fall in cyclase specific activity. Fractionation of the homogenate of trophozoites indicated that adenylate cyclase activity was associated with the particulate microsomal fraction.     

Alamethicin or detergent permeabilization of the cell membrane as a tool for adenylate cyclase determination: Application to the study of hormone responsiveness in lymphocytes

Treatment of mouse lymphocytes with very low concentrations of alamethicin or Lubrol PX induces spontaneous permeabilization of the plasma membrane to ATP and allows determination of adenylate cyclase activity in whole cells. The permeabilized cells retain responsiveness to hormones (isoproterenol, adenosine analogs) and to fluoride. The main advantage of this new method is that it does not require any homogenization step, and thus adenylate cyclase activities can be accurately and reproducibly measured with very low amounts of cells. It should be especially useful for the study of purified lymphocyte subpopulations.     

Hyperparasitism of intrasnail stages of Fasciola hepatica by a mosquito microsporidian parasite

Laboratory studies were conducted to investigate the infective behavior of Nosema algerae spores when ingested by Fasciola hepatica free of F. hepatica-infected snails (Lymnaea cubensis). Among the F. hepatica-infected snails exposed to N. algerae, 38.09% harbored microsporidia-infected F. hepatica rediae. The F. hepatica-free snails exposed to N. algerae as well as the controls did not become infected. Light microscopical studies of Giemsa-stained microsporidia distinguished this organism from microsporidia previously described in trematode larvae. Based on the infectivity studies and morphological data, it was concluded that N. algerae, a mosquito pathogen, was transmitted to intrasnail stages of F. hepatica.     

Phylogenetic Characteristics of Fasciola hepatica Isolated from a Korean Patient

Fascioliasis is a parasitic infection caused by liver flukes. Although several cases have been reported in Korea, phylogenetic analysis of isolates is lacking. In this study, a 66-year-old woman with right upper quadrant (RUQ) abdominal pain was diagnosed as fascioliasis involving abdominal muscle by imaging study. She received praziquantel treatment, but symptoms were not improved. Lateral movement of the abscess lesion was followed. Trematode parasite was surgically removed from the patient’s rectus abdominis muscle. The fluke was identified as Fasciola hepatica based on sequence analysis of 18S rDNA. To determine the phylogenetic position of this Fasciola strain (named Korean Fasciola 1; KF1), the cox1 gene (273 bp) was analyzed and compared with the genes of 17 F. hepatica strains isolated from cows, sheep, goats, and humans from various countries. Phylogenetic analysis showed that KF1 was closely related with the isolates from China goat.     

Isolation and characterization of microsatellite markers in the liver fluke (Fasciola hepatica)

Six microsatellite markers were isolated from Fasciola hepatica, a re‐emerging parasite that causes important veterinary and public health problems. In a sample of 52 liver flukes from a region of hyperendemicity (Bolivian Altiplano), five microsatellite were polymorphic. Our results showed that liver flukes present important genetic variability, suggesting a preferential outcrossing reproduction mode for this hermaphroditic parasite.     

Lateral mobility of β-receptors involved in adenylate cyclase activation

Cationized ferritin was found to inhibit the lateral mobility of intramembrane proteins in turkey erythrocyte membranes and the activation of adenylate cyclase by the (?)-epinephrine-bound β-adrenergic receptor. It was observed that cationized ferritin has only a small direct effect on the β-receptor and on the adenylate cyclase moiety. It is concluded that the cationized ferritin-induced inhibition of the hormone-dependent cyclase activity results from the inhibition of the lateral mobility of the receptor and therefore a decrease in the bimolecular rate of interaction between the receptor and the enzyme.     

Regulation of rat lung adenylate cyclase by cytoplasmic factor(s) during development

Basal adenylate cyclase activity in rat lung homogenate was low prenatally but increased several-fold after birth and remained elevated to maturity. The results also demostrate the appearance of some factors(s) in the lung cytoplasm at a certain age which markedly activated adenylate cyclase. During late gestation and early neonatal life, when the cytoplasmic factor(s) was low or absent, basal adenylate cyclase activity was low and norepinephrine and NaF produced maximum activation of the enzyme. However, when the cytoplasmic factor(s) appeared in the adult lungs, basal adenylate cyclase activity was elevated and both norepinephrine and NaF produced little or no activation of the enzyme. These data suggest a role for the cytoplasmic factor(s) in regulating rat lung adenylate cyclase.The cytoplasmic factor(s) appeared to be a protein since it was inactivated by trypsin digestion and by heating to 75°C. Activation of adenylate cyclase was not due to small ions or other low molecular weight components of the cytoplasm as dialysis of the supernatant did not alter its activation of adenylate cyclase. The cytoplasmic factor(s) did not appear to be either GTP or calcium-dependent regulator of cyclic AMP phosphodiesterase as these did not activate the rat lung adenylate cyclase.     

Further purification and partial characterization of the rat lung cytoplasmic factors modulating adenylate cyclase activity in plasma membrane

Summary The adult rat lung cytoplasm contains some factors which markedly stimulate adenylate cyclase activity in plasma membranes (Nijjar, M. S. Biochim. Biophys. Acta 584:43–50, 1979). Adenylate cyclase activator (ACA) was purified from rat lungs by DEAE-cellulose chromatography, preparative isoelectric focusing and by repeated high-performance liquid chromatography on a Sepharogel TSK 2000SW column. The final preparation showed about 200 fold purification in ACA activity over the original lung supernatant, and appeared to be homogeneous on the basis of its migration into a single band on SDS-polyacrylamide gel electrophoresis, and co-elution of ACA activity with protein from a gel exclusion column. ACA is an acidic (pl 4.8 ± 0.1), heat labile, monomeric protein of 40000 ± 2000 dalton molecular weight, and does not resemble calmodulin.     

Effect of hormonal and neuronal agents on adenylate cyclase from smooth muscle of the gastric antrum

Smooth muscle adenylate cyclase of a membrane preparation of canine gastric antrum has been characterized, and the effect of hormonal and neuronal agents examined. The enzyme is active in the presence of Mg²⁺ or Mn²⁺, but is inhibited by Ca²⁺. The K_m is 0.5 mM ATP, similar to the K_m of skeletal muscle adenylate cyclase. The enzyme is activated by isoproterenol but not norepinephrine, consistent with a β₂-catecholamine receptor-adenylate cyclase interaction. Secretin activates the enzyme in concentrations as low as 1 · 10^?11 M, while glucagon was effective only at 1 · 10^?6 M. Prostaglandin E₁ and E₂ have a biphasic effect with activation of adenylate cyclase at 1 · 10^?5 M and a small but significant inhibition of enzyme activity at 1 · 10^?11 M.     

Determination of adenylate cyclase activity in a variety of organisms: Evidence against the occurrence of the enzyme in higher plants

Adenylate cyclase activity was examined in a variety of organisms using a highly sensitive assay. Activity was found in a blue-green alga, four green algae, two cellular slime molds, a fungus and moss protonemata. Fern prothalli and fronds gave variable results. No activity was detected in any of the higher plant tissues tested. The results throw further doubt on the existence of adenosine 3:5-cyclic monophosphate in higher plants.Abbreviation cAMP adenosine 3:5-cyclic monophosphate     

Protective role of adenylate cyclase in the context of a live pertussis vaccine candidate

Despite high vaccination coverage, pertussis remains an important respiratory infectious disease and the least-controlled vaccine-preventable infectious disease in children. Natural infection with Bordetella pertussis is known to induce strong and long-lasting immunity that wanes later than vaccine-mediated immunity. Therefore, a live attenuated B. pertussis vaccine, named BPZE1, has been developed and has recently completed a phase I clinical trial in adult human volunteers. In this study, we investigated the contribution of adenylate cyclase (CyaA) in BPZE1-mediated protection against pertussis. A CyaA-deficient BPZE1 mutant was thus constructed. Absence of CyaA did not compromise the adherence properties of the bacteria onto mammalian cells. However, the CyaA-deficient mutant displayed a slight impairment in the ability to survive within macrophages compared to the parental BPZE1 strain. In vivo, whereas the protective efficacy of the CyaA-deficient mutant was comparable to the parental strain at a vaccine dose of 5 × 10⁵ colony forming units (CFU), it was significantly impaired at a vaccine dose of 5 × 10³ CFU. This impairment correlated with impaired lung colonization ability, and impaired IFN-γ production in the animal immunized with the CyaA-deficient BPZE1 mutant while the pertussis-specific antibody profile and Th17 response were comparable to those observed in BPZE1-immunized mice. Our findings thus support a role of CyaA in BPZE1-mediated protection through induction of cellular mediated immunity.     

Catecholamine-induced stimulation of adenylate cyclase in parallel with a change in tubulin assembly in ehrlich ascites tumor cells

In vitro incubation of Ehrlich ascites tumor cells in the presence of norepinephrine induced desensitization of adenylate cyclase to the later norepinephrine stimulation. Such a desensitization was not accompanied by a decrease in the number of receptor sites. Formation of actin filaments from actin monomers was not changed in the desensitized cells, whereas polymerization of tubulin was significantly increased. The increase in the polymerization was dependent on the concentration of norepinephrine.     

Isolation of plasma membranes from rat lungs. Effect of age on the subcellular distribution of adenylate cyclase activity

A simple and rapid method of isolating plasma membranes from rat lungs is described. The method involves homogenization of tissue in isotonic sucrose-buffered medium followed by differential and sucrose density gradient centrifugation. Plasma membranes obtained by this procedure were essentially free from other subcellular contamination. Plasma membranes isolated from 2-day-old rat lungs showed 6 to 7-fold purification of adenylate cyclase and 5′-nucleotidase activities compared to the original homogenate In contrast, plasma membranes from 35-day-old rat lungs showed no purification of adenylate cyclase activity although 5′-nucleotidase activity showed similar enrichment. These results suggest that adenylate cyclase activity is not a reliable marker for plasma membranes from adult rat lungs.