The effect of membrane preparation and cellular maturation on human erythrocyte adenylate cyclase

We found that adenylate cyclase activity of human erythrocytes is potentially labile during isolation of their plasmalemma. Addition of 1 mM EGTA to solution used to remove hemoglobin from lysed cells protected activity. Human erythrocyte adenylate cyclase is minimally activated by catecholamines, in the absence or presence of exogenous guanyl nucleotide, but substantially by 5'-guanylyl imidodiphosphate or sodium fluoride and concentration-dependently by Mg2+ or Mn2+. Basal catalytic activity is an age-dependent component of the human erythrocyte; 5'-guanylyl imidodiphosphate- or fluoride-activated activities decline with cellular maturation proportionally to the decrease in basal activity.     

Effects of drugs on pigeon erythrocyte membrane and asymmetric control of adenylate cyclase by the lipid bilayer

In pigeon erythrocyte membrane, the β-adrenergic receptor and the enzyme adenylate cyclase can be uncoupled in two different ways depending on the type of drug used.Cationic drugs: chlorpromazine, methochlorpromazine, tetracaine, $\text{n-}\text{octylamine}$ and a neutral alcohol, octanol, abolished alprenolol receptor binding ability and in the same range of concentration of the drug, sensitized adenylate cyclase to fluoride or Gpp(NH)p stimulation. Anionic drugs: di- and trinitrophenols, indomethacin and octanoic acid did not affect the total number of β-adrenergic receptor sites and, with the exception of trinitrophenol, did not change the association constant for alprenolol but they abolished the stimulation of adenylate cyclase by isoproterenol, fluoride or Gpp(NH)p. These modifications of the adenylate cyclase system occurred in a range of drug concentration where cell shape and protection against hemolysis were also affected.As chemical composition varies widely from one drug to another, it is suggested that these effects are largely nonspecific and mediated by the lipid bilayer. They are probably related to a preferential sidedness of action of the drugs in the lipid bilayer, displaying the role of an asymmetric control of the adenylate cyclase system in the membrane by the two halves of this bilayer.     

Occurrence of creatine kinase activity in human erythrocyte membrane

Some evidences for creatine kinase activity in normal human erythrocyte membrane were presented. The creatine kinase was indicated to be a constituent of the integral proteins of erythrocyte membrane or to be tightly bound to the membrane, and was contrasted to the results obtained with adenylate kinase. Isoenzyme distribution of the erythrocyte creatine kinase by electrophoresis was identical to MM-creatine kinase from rabbit muscle.     

1-Methyladenine inhibits adenylate cyclase in starfish oocytes

Summary

The effect of 1-methyladenine (1-MeA) on adenylate cyclase (AC) basal activity and on preliminary stimulated AC activity was investigated in oocyte membrane preparations of the starfish Aphelasterias japonica. 1-MeA inhibited the membrane-bound AC activity both after its addition to intact oocytes and in cell-free experiments. GTP did not affect AC activity but it intensified the inhibitory effect of 1-MeA on AC activity. Sodium fluoride (F″) stimulated the oocyte AC (8 fold), while 1-MeA significantly reduced F″-stimulated activity. Manganese (MnCl₂, 5mM) stimulated AC (150 fold), but 1-MeA did not reduce Mn²⁺-stimulated activity. However, Mn²⁺-stimulated AC activity was inhibited by 1-MeA in the presence of MgCl₂. Forskolin stimulated AC activity (7 fold) and 1-MeA had no effect on AC. Thus, the inhibitory effect of 1-MeA on stimulated AC activity is displayed only after stimulation of the regulatory AC subunit. We suggest that 1-MeA inhibits the oocyte AC acting via inhibitory regulatory Gi protein of AC.     

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.     

Inhibition of adenylate cyclase activity of human thyroid membranes by gangliosides

Gangliosides inhibit basal, thyrotropin-induced and fluoride-induced adenylate cyclase activity of human thyroid membranes in physiological conditions. In contrast neutral glycolipids, phospholipids and neuraminic acid containing oligosaccharides show no effect. The efficacy of inhibition is more dependent upon the position of the sialic acid residues than upon their absolute number. In general gangliosides with disialyl groups are more inhibitory than those with single sialyl moieties. The inhibitory effects of the individual gangliosides on the two modes of stimulation are parallel. This parallelism suggests that the inhibitory effect is located at the postreceptor level and that the gangliosides interact directly with the adenylate cyclase system. A possible role of thyroid membrane gangliosides as suppressive cofactors of adenylate cyclase is discussed in relation to recent findings of stimulating anti-ganglioside antibodies in Graves' disease.     

Role of calmodulin in the effect of guanyl nucleotides on rat hippocampal adenylate cyclase: involvement of adenosine and opiates

Abstract: The adenylate cyclase activity of rat hippocampal plasma membranes can be stimulated by vaso-active intestinal polypeptide (VIP). Low concentrations (10⁻⁹ to 10⁻⁷M) of 5'-guanylyl-imido diphosphate (GppNHp) evoke a transient inhibition of the enzyme, which is followed by stimulation with increasing GppNHp concentrations (10⁻⁶ to 10⁻⁴M). Inclusion of ethyleneglycol - bis - (β - aminoethylether) - N,N' - tetraacetic acid (EGTA) during incubation abolishes the GppNHp inhibition while preserving GppNHp activation. The stimulation induced by GppNHp is amplified by VIP, but the inhibition is unaffected. Adenosine analogs and opiates are inhibitory ligands in the presence of GTP, and their effects can be reversed by the appropiate receptor antagonists, 3-isobutyl-1-methylxanthine and naloxone. Treatment of membranes with trypsin abolishes the GppNHp-induced inhibition without affecting the GppNHp stimulation. The inhibition induced by GppNHp is also abolished by EGTA treatment followed by washing, which coincides wtih a reduction in the adenosine- and opiate-mediated, GTP-dependent inhibition. The GppNHp inhibition can be restored in EGTA-treated but not in trypsin-treated membranes by addition of calcium-calmodulin but not by Ca²⁺ or Mg²⁺. Calcium-calmodulindepleted membranes lack calcium stimulation as well as GppNHp-induced inhibition, whereas untreated membranes and calcium-calmodulin-depleted membranes plus exogenous calcium-calmodulin showed calcium stimulation and GppNHp inhibition. These results suggest that calmodulin is involved in both Ca²⁺ stimulation and guanine nucleotide-mediated inhibition of rat hippocampal adenylate cyclase.     

Cytochemical localization of adenylate cyclase activity in the rat anterior pituitary

Summary The cytochemical localization of adenylate cyclase was studied in relation to the secretory function of the anterior pituitary glands of male rats. The reaction product of adenylate cyclase was localized on the outside of plasma membranes, but was not detected intracellularly. High activity of adenylate cyclase was detected on somatotrophs and microvilli of follicular cells, whereas no activity was found on thyrotrophs or corticotrophs. Although most of the gonadotrophs showed little or no adenylate-cyclase activity, some was detected in a small number of gonadotrophs in the central portion of the gland. In somatotrophs, activity was not detected on the plasma membranes facing perivascular spaces where exocytotic extrusion of secretory granules was frequently observed, although the remaining areas of plasma membranes of the same somatotrophs were associated with high levels of adenylate-cyclase activity. These findings indicate that the association of a high level of adenylate-cyclase activity is not directly related to the ability of the plasma membranes to fuse with secretory granule membranes.     

东菱克栓酶治疗对脑梗死患者腺苷酸环化酶影响的研究

目的研究东菱克栓酶治疗对脑梗死患者腺苷酸环化酶（AC）的影响。方法选取于2012年1月至2014年1月在本溪市中心医院治疗的脑梗死患者56例作为研究对象,采用抽签法将患者分为两组,对照组采用常规治疗,观察组在常规治疗的基础上采取东菱克栓酶治疗,利用RT-PCR技术检测两组患者治疗前后AC表达情况（AC/GPR-DH）,并观察临床疗效。结果治疗前对比分析两组患者AC/GPRDH比值,差异无统计学意义（P〉0.05）;经针对性治疗后,观察组患者的AC/GPR-DH比值上升幅度为（0.861±0.030）,较对照组（0.443±0.024）显著上升,经比较差异有统计学意义（P〈0.05）;观察组治疗总有效率（89.29%）较对照组（60.71%）高,差异有统计学意义（P〈0.05）;治疗后,观察组血脂指标浓度（TC：4.74±1.20;TG：1.06±1.04;LDL-C：3.19±1.22）下降量明显多于对照组（TC：5.25±1.15;TG：1.51±1.12;LDL-C：3.87±1.25）,差异均有统计学意义（均P〈0.05）。结论研究表明观察组治疗可明显缓解病患的并发症,具有积极意义。采用中医体质辨识理论结合解郁合欢汤治疗失眠患者具有较好的临床疗效,值得推广使用。     

Bending undulations and elasticity of the erythrocyte membrane: effects of cell shape and membrane organization

The undulatory excitations (flickering) of human and camel erythrocytes were evaluated by employing the previously used flicker spectroscopy and by local measurements of the autocorrelation function K (t) of the cell thickness fluctuations using a dynamic image processing technique. By fitting theoretical and experimental flicker spectra relative values of the bending elastic modulus K _c of the membrane and of the cytoplasmic viscosity were obtained. The effects of shape changes were monitored by simultaneous measurement of the average light intensity I ₀ passing the cells and by phase contrast microscopic observation of the cells. Evaluation of the cellular excitations in terms of the quasi-spherical model yielded values of K _c /R _inf0 ^sup3 and · R ₀ (R ₀=equivalent sphere radius) and allowed us to account (1) for volume changes, (2) for effects of surface tension and spontaneous curvature and (3) for the non-exponential decay of K (t). From the long time decay of K (t) we obtained an upper limit of the bending elastic modulus of normal cells of K _c = 2–3 · 10^–19 Nm which is an order of magnitude larger than the value found by reflection interference contrast microscopy (RICT, K _c , = 3.4 · 10^–20 Nm, Zilker et al. 1987) but considerably lower than expected for a bilayer containing 50% cholesterol (K _c = 5 · 10^–19 Nm, Duwe et al. 1989). The major part of the paper deals with long time measurements (order of hours) of variations of the apparent K _c and values of single cells (and their reversibility) caused (1) by osmotic volume changes, (2) by discocytestomatocyte transitions induced by albumin and triflouperazine, (3) by discocyte-echinocyte transitions induced by expansion of the lipid/protein bilayer (by incubation with lipid vesicles) and by ATP-depletion in physiological NaCI solution, (4), by coupling or decoupling of bilayer and cytoskeleton using wheat germ agglutinin or erythrocytes with elliptocytosis and (5) by cross-linking the cytoskeleton using diamide. These experiments showed: (1) K _c and are minimal at physiological osmolarity and temperature and well controlled over a large range of these parameters. (2) Echinocyte formation does not markedly alter the apparent membrane bending stiffness. (3) During swelling the cell may undergo a transient discocyte-stomatocyte transition. (4) Strong increases of the apparent K _c and after cup-formation or strong swelling and deflation are due to the effect of shear elasticity and surface tension. Our major conclusions are: (1) The erythrocyte membrane exhibits a shear free deformation regime which requires ATP for its maintenance. (2) Shape transitions may be caused by relative area changes either of the two monolayers of the lipid/protein bilayer (corresponding to the bilayer coupling hypothesis) or of the bilayer and the cytoskeleton where the latter mechanism appears to be more frequent. (3) The low bending stiffness and the shear free deformation regime are explained in terms of a slight excess area of the lipid bilayer leading to a pre-undulated surface profile. Freeze fracture electron microscopy studies provide direct evidence for a pre-undulated bilayer with an undulation wavelength of approximately 100 nm. Offprint requests to: E. Sackmann     

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.     

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.     

Subcellular distribution of axonally transported adenylate cyclase: effect of nerve constriction and comparison with acetylcholinesterase

Abstract: Despite several studies indicating that cyclic nucleotides and their associated enzymes are present in peripheral nerves, their role in neuronal function remains unknown. One possible role is that of a modulating influence in the processes associated with axonal growth and maintenance, and in axonal regeneration. This study has used the frog sciatic nerve as a preparation for investigating the subcellular distribution of neuronai adenylate cyclase activity in normal and crush-injured nerves. The experiments have focused primarily on the axonal transport of adenylate cyclase activity and its subcellular redistribution at the site of constriction. The adenylate cyclase activity measurements were also compared with similar measurements of acetylcholinesterase distribution. Adenylate cyclase activity in normal sciatic nerves increased in the segment proximal to a nerve constriction over time, but did not increase distal to the constriction. Subcellular fractionation of the accumulating activity indicated that the majority of axonally transported enzyme was associated with microsomal organelles; however, an additional transported component was found in the nuclear/mitochondrial fraction. The transport velocities of these two components were different. The microsomal activity appeared to be transported with Group I proteins, while the nuclear/mitochondrial activity was transported with Group II. Rapidly transported Group I proteins have been suggested to be destined principally for the axolemma or the agranular reticu-lum, and the more slowly transported Group II proteins to be associated with intracellular organelles, including synaptic structures. Thus, axonally transported adenylate cyclase activity may have more than one functional role in peripheral nerve. The association of both adenylate cyclase and Protein I, an endogenous substrate for cyclic AMP, with Group II transport offers the intriguing possibility of a structural correspondence. Adenylate cyclase activity in Group I, however, did not appear to be transported with organelles which also contained acetylcholinesterase. The two enzymes, in terms of both velocity of transport and susceptibility to retrograde transport, were handled differently by the neuron. The subcellular distribution of adenylate cyclase activity in an isolated nerve segment was also found to change over time. Microsomal activity decreased, while nuclear/mitochondrial activity transiently increased and then also decreased. This may offer some indication of the morphological location of adenylate cyclase and its potential involvement in Wallerian degeneration and nerve regeneration, particularly in view of recent reports concerning the importance of local injury-induced changes to the initiation of nerve regeneration. We have proposed a dynamic association between axonal calcium and cyclic AMP concentration, which provides a method for membrane renewal or degradation in the intact axon and may offer a molecular basis for the structural reorganization occurring in the proximal segment of an injured nerve.     

Effects of proteolysis on the actions of monovalent cations and 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine on platelet adenylate cyclase

NaCl stimulated the adenylate cyclase activities of human and rabbit platelet particulate fractions prepared in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetate, but inhibited the activities of particulate fractions proteolysed by endogenous Ca2+-activated protease or treatment with alpha-chymotrypsin. Studies with other monovalent cations showed that LiCl had weak effects similar to those of NaCl, whereas KCl inhibited the enzyme in both proteolysed and non-proteolysed preparations. The results suggest that NaCl exerts stimulatory and inhibitory effects through different sites. NaCl potentiated and proteolysis greatly reduced the inhibition of platelet adenylate cyclase by 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (platelet-activating factor).     

Inhibition by somatostatin of the vasopressin-stimulated adenylate cyclase in a kidney-derived line of cells grown in defined medium

LLC-PK1L cells, a kidney-derived cell line grown in defined medium, possess a vasopressin-sensitive adenylate cyclase. Somatostatin was able to inhibit the vasopressin-induced increase in adenylate cyclase activity, without affecting the basal enzyme activity. This inhibition was competitive. No effect of somatostatin could be detected on [3H]vasopressin binding suggesting an interaction of somatostatin with the vasopressin-sensitive system distal to the hormone-receptor interaction. At variance with N6-L-2-phenylisopropyladenosine (PIA), GTP did not potentiate the inhibition by somatostatin. The inhibition of the vasopressin stimulation by somatostatin and that by PIA were additive. Changing the composition of the cell growth medium increased the number of vasopressin receptors per cell. Cells with a high number of vasopressin receptors were less sensitive to inhibition by somatostatin. Such results suggested that somatostatin and vasopressin receptors and/or the inhibitory (Ni) and stimulatory (Ns) regulatory transducing components are regulated by different mechanisms.     

The alteration of membrane proteins in human erythrocyte membranes induced by quinolinic acid,an endogenous neurotoxin Correlation of effect with structure

Quinolinic acid (2,3-pyridinedicarboxylic acid), an endogenous metabolite of l-tryptophan, reportedly via the kynurenine pathway, has been previously shown to possess neurotoxic properties when injected into rat striatum (Schwarcz R., Whetsell, W.O., Jr. and Mangano R.M. (1983) Science 219, 316–318) and to alter the physical state of human erythrocyte membrane proteins, as judged by ESR spectroscopy (Farmer, B.T., II and Butterfield, D.A. (1984) Life Sci. 35, 501–509). Both the morphologic and ESR studies employed nicotinic acid as one comparative control and found that the effect of quinolinic acid is significantly different from that of nicotinic acid. In the present study, we report that the effects of several structural analogues and positional isomers of quinolinic acid on the ESR parameter associated with the physical state of membrane proteins in human erythrocyte membranes suggest the following conclusions concerning the structure-effect relationship of quinolinic acid: The alteration in the conformation of membrane proteins: (1) requires the presence of two carboxylic acid groups; (2) is independent of their relationship to one another on the pyridine ring; (3) is slightly dependent on the presence of the pyridine nitrogen atom but is independent of the positional relationship of the two carboxylic acid moieties to the heteroatom; and (4) seems to depend upon the presence of restricted internal motion derived from the aromaticity in these compounds.     

The sphingomyelin pools in the outer and inner layer of the human erythrocyte membrane are composed of different molecular species

Analyses of the fatty acid composition of the outer and inner pools of sphingomyelin in the human erythrocyte membrane revealed significant differences in molecular species composition of these two pools. The sphingomyelin in the inner monolayer, representing 15–20% of the total sphingomyelin content of this membrane, is characterized by a relatively high content (73%) of fatty acids, which have less than 20 carbon atoms, whereas these account for only 31% of the total fatty acids in the sphingomyelin in the outer leaflet. On the other hand, the ratio saturated/unsaturated fatty acids in the two pools is similar. Significant differences are also observed for the fatty acid composition of the sphingomyelin in human serum when compared to that in the outer monolayer of the corresponding red cell. These results are interpreted to indicate an (almost) complete absence of transbilayer movements of sphingomyelin molecules in the human erythrocyte membrane, whereas an exchange of this phospholipid between the red cell membrane and serum is either virtually absent, or affects only a minor fraction of the sphingomyelin in the outer membrane layer.     

The human erythrocyte membrane skeleton may be an ionic gel

Biochemical and biophysical observations indicate that the erythrocyte membrane skeleton is composed of a swollen network of long, flexible and ionizable macromolecules located at the cytoplasmic surface of the fluid membrane lipid bilayer. We have analyzed the mechanochemical properties of the erythrocyte membrane assuming that the membrane skeleton constitutes an ionic gel (swollen ionic elastomer). Using recently established statistical thermodynamic theory for such gels, our analysis yields mathematical expressions for the mechanochemical properties of erythrocyte membranes that incorporate membrane molecular parameters to an extent not achieved previously. The erythrocyte membrane elastic shear modulus and maximum elastic extension ratio predicted by our membrane model are in quantitative agreement with reported values for these parameters. The gel theory predicts further that the membrane skeleton modulus of area compression, K _G, may be small as well as large relative to the membrane elastic shear modulus, G, depending on the environmental conditions. Our analysis shows that the ratio between these two parameters affects both the geometry and the stability of the favoured cell shapes.