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Activity of cyclic AMP phosphodiesterase and methyltransferases in leukocyte membranes from allergic patients
Clinica Chimica Acta, 143 (1984) 225-233 Elsevier
225
CCA 03004
Activity of cyclic AMP phosphodiesterase and methyltransferases in leukocyte membranes from allergic patients Annie-France Prigent a,*, Pierre Fonlupt a, Madeleine Dubois a, Georges NCmoz a, Henri Pacheco a, Yves Pacheco b, Nicole Biot b and Max Perrin-Fayolle b a Service
de Chimie Biologique, Uniit! 205 INSERM, Instiiui National des Sciences Appliquies de Lyon, 69621 Villeurbanne CGdex and b Service de Pneumologie, Hbpital Sainte - Eug&nie, Lyon (France) (Received February 14th, 1984; revision July 28th. 1984) Key wor&: Allergy; Cyclic AMP phosphodiesterase; Methyltransjerase (basal level and noradrenaline-stimulated level); Leukocytes; Membranes
Summary
Cyclic AMP metabolism and methylation of phospholipids are central events which occur at the membrane level. Since a dysfunction of cell membranes seems to characterize some allergic diseases, we investigated cyclic AMP phosphodiesterase and methyltransferase activities in leukocyte membrane fractions obtained from healthy volunteers and from allergic patients. The allergic group presented a significantly decreased methyltransferase activity compared with a control group, whereas cyclic AMP phosphodiesterase and noradrenaline (NA)-stimulated methyltransferase were found to be increased with respect to the control group. A significant correlation has been found between cyclic AMP phosphodiesterase and NA-stimulated methyltransferase with both control and allergic subjects, which suggests close relationships between these two enzymes within the cell membrane. Introduction
There is general agreement that cyclic adenosine monophosphate (CAMP) plays an important modulating role in lymphoid cell function and allergic responses. * To whom correspondence should be addressed. Abbreviations used CAMP, aderiosine 3’5’~cyclic monophosphate; PDE, cyclic phodiesterase (EC 3.1.4.17); SAM, S-adenosyl-L-methionine; ME, methykransferase.
OOOS-8981/84/$03.W
@ 1984 Ehwier
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phos-
226
Cyclic nucleotides act at several levels in the immune response such as the generation and release of mediators of anaphylaxis and the cellular responsiveness to mediators [l]. The release of these mediators is reduced or obviated by cyclic AMP or by several hormones that interact with cell surface receptors to activate adenylate cyclase [2]. A decreased /3-adrenergic response has been*proposed earlier as one of the causes of hypersensitivity of the bronchi in bronchial asthma [3]. Numerous studies of atopic leukocytes [4] or lymphocytes [S] have revealed subnormal CAMP responses to &adrenergic agonists, both in vitro and in vivo, but considerable controversy has existed over the occurrence and importance of /3-adrenoceptor desensitization in allergic patients. Whether or not allergic subjects are more prone to &receptor desensitization than healthy subjects is still an unanswered question. There are wide variations in individual susceptibility to desensitization which occur with both allergic and non-allergic subjects [Z]. Various mechanisms have been proposed to explain the refractoriness, which follows exposure of cells or isolated plasma membranes to agonists, and may be correlated with changes in the physiological responsiveness of the system. Exposure to hormones may cause a loss of binding sites. Indeed, a significant decrease in the number of receptor sites was observed in a variety of tissues from several species during the desensitization process [2]. Alternative mechanisms such as a loss of the guanine nucleotide regulatory component or a decrease in the interaction of the hormone-occupied receptor with the regulatory protein of the adenylcyclase system have also been proposed [6]. Although the alteration of one or the other component of the cyclase system is very likely to be involved in the desensitization process, other mechanisms such as an increase in cyclic nucleotide phosphodiesterase (PDE) activity may make a contribution in some tissues, e.g. pineal [7]. In this regard, discrepant reports have recently appeared. Thus, Conolly [2] was unable to demonstrate any change in phosphodiesterase activity in either bronchi or lymphocytes after desensitization by isoproterenol, whereas Chan et al [8] found a stimulation of cyclic AMP phosphodiesterase in human mononuclear leukocytes following agonist desensitization. Similarly, Grewe et al [9] observed a si~ific~tly elevated cytosolic phosphodiesterase activity in leukocytes from allergic patients as compared with leukocytes from control subjects. On the other hand, a recent attractive hypothesis, supported by in vitro experiments with cultured C, astrocytoma cells, has suggested that membrane phospholipid turnover may be involved in decreased CAMP responsiveness [lo]. Since the enzymic methylation of phospholipids alters membrane composition, symmetry and fluidity, this process is likely to have a wide spectrum of effects upon biological function and the activity of several membrane-bound enzymes [ll]. Indeed, the methylation of membrane phospholipids seems to regulate part of the P-adrenergic system and vice versa [lo-111. However, clinical studies related to an eventual alteration of phospholipid methylation in allergic diseases are few [12,13]. Furthermore, eventual relationships between methyltr~sferase and membrane-bound phosphodiesterase activities have not been studied before. The aim of the present work was to evaluate in parallel the methyltransferase (basal level and adrenergic stimulated level) and the cyclic AMP phosphodiesterase activities in the same leukocyte membrane fractions from healthy volunteers, allergic patients
227
receiving no systemic therapies.
medication
and allergic subjects
having
various non-adrenergic
Subjects Main characteristics of the groups are as follows. Comparison of cyclic AMP phosphodiesterase, methyltransferase and noradrenaline (NA)-stimulated methyltransferase activities in leukocyte membrane fractions from allergic patients and control donors: In this study, leukocytes were separated from the blood of 12 control subjects who did not exhibit clinical signs of allergy and 12 subjects with allergic asthma. Allergic asthma was defined by the following clinical criteria: bronchial hypersensitivity to carbachol; positive reactions to skin testing with a standard battery of common allergens (house dust - dermatophagoids); elevated IgE level; presence of specific IgE estimated by the RAST method. Control and allergic subjects were of both sexes and ages ranged from 20 to 40 years. In order to have a greater potency of the paired t test, controls were age and sex matched with the patients. They had not received any systemic medication (corticoids or theophylline) for at least two weeks before the experiments. Correlation between cyclic AMP phosphodiesterase and NA-stimulated methyltransferase activities in leukocyte membrane fractions: The leukocytes used in this study were from heterogeneous groups including healthy volunteers, allergic patients without systemic medication for at least two weeks, and allergic patients having various non-adrenergic therapies. In this part of the work, our aim was not to compare enzymatic activities between control and allergic subjects or to evaluate the influence of a particular therapy on these enzymatic activities, but because of the heterogeneity of the leukocyte population obtain the greatest variability among samples. Methods
Chemicals [3H]SAM (15 Ci/mmol), [8-3H]cyclic AMP (20-30 Ci/mmol) and [Ui4 Cladenosine ( 500 Ci/mmol) were supplied by the Radiochemical Centre (Amersham, UK). Snake venom (Ophiophagus hannah), bovine serum albumin and unlabelled cyclic AMP were purchased from Sigma Chemical Co. (St. Louis, MO, USA). AGlX2 resin (200-400 mesh) was from Bio-Rad Laboratories (Richmond, CA, USA). All other chemicals were reagent grade.
Purification of [‘HJSAM and isotopic dilution [ 3H]SAM was diluted isotopically with 100 parts of cold SAM. The two radioactive sources (with high and low specific activities) were chromatographed separately on silica gel plates, eluted with acetic acid and then stored at - 20 o C. Under these conditions, the radioactive source was stable for at least 8 days. By mixing the two [3H]SAM eluates with high and low specific activities, we can obtain the SAM concentration needed and the required number of disintegrations with high precision.
228
Leukocyte
membrane preparation
Leukocytes were isolated from heparinized blood by sedimentation in the presence of 5% dextran, then centrifuged and washed three times with 25 mmol/l Tris-HCl buffer, pH 7.6, and stored at - 20 *C. Since, in our previous work [13], no correlation was found between the methylase activity and the percentage of any cell type (polynuclears, lymphocytes, eosinophils, mononuclears), experiments were performed on the heterogeneous leukocyte populations without further separation of the different kinds of cells. The cells were further centrifuged at 50000 x g for 15 min and the pellet resuspended in hypotonic 5 mmol/l Tris-HCl buffer, pH 7.4, frozen and thawed. After washing by centrifugation, the membranes were resuspended in 50 mmol/l Tris-HCl buffer pH 7.4. This membrane suspension was used for the determination of the enzymic activities after appropriate dilution. Cyclic AMP phosphodiesterase
assay
Cyclic AMP phosphodiesterase activity was assayed as reported in [14] following a modified method based on the original two-step radioisotopic procedure of Thompson et al 1151with 0.25 pmol/l cyclic AMP as substrate. All the assays were performed at 30 OC, in triplicate, with enzyme dilutions adjusted to give linear reaction rates (usually less than 20-25% substrate hydrolysis). Adenosine recovery was systematically determined by means of [U-‘4C]adenosine and results corrected for these yields in each samples. Phosphodiesterase activities were expressed as pmol cyclic AMP hydrolyzed/lo6 cells per min. ~ethy~transfer~e
activity assay
Methyltransferase activity was determined by a procedure previously used [13j and derived from that described by Hirata et al (161. 0.1 pmol/l [3H]SAM (170000 dpm/assay) was used as substrate. Methyltransferase activities were expressed as pmol [ 3H]methyl group incorporated in the chloroform extractible material/lo6 cells per 30 min. NA-stimulated
methyrtransferase
assay
NA-stimulated methyltransferase activity was measured as described above, after a 5-min preincubation period of membrane fractions with or without 100 pmol/l NA. These experimental conditions were shown to give optimal stimulation in preliminary experiments performed with synapto~m~ or leukocyte preparations (P.F. unpubl, results}. Results were expressed as the ratio: NA-stimulate methylase level-basal methylase level (without NA)/basal methylase level. Results are not significantly different when activities are related to the number of cells or expressed per mg protein since the protein content of leukocytes from allergic subjects and from controls are not si~ific~tly different. However, membrane recoveries during preparation might be different in the allergic and the control groups, if variations in membrane stability occurred. In our experimental conditions, we did not find any difference between the two groups: lo6 cells always gave about 40 pg protein; so we expressed activities per lo6 cells.
229
Results
In a first set of experiments, methyltransferase, NA-stimulated methyltransferase and cyclic AMP phosphodiesterase activities were determined and compared in only two groups: control and allergic patients without systemic medication for at least two weeks. Results presented in Fig. lb showed that the methyltransferase activity, as determined by the incorporation of [ 3H]methyl group into the phospholipid fraction, was significantly lower in leukocyte preparations from allergic patients than that observed with control subjects (mean allergies: 0.115 f 0.042, mean controls: 0.153 f 0.036 in pmol [3H]methyl group incorporated per lo6 cells per 30 min, n = 12, 0.05 < (Y< 0.02) which confirms our previous results [13]. The NA-stimulated methyltransferase activity (Fig. lc) proved to be significantly higher in the same leukocyte preparations from allergic subjects than that from control donors as indicated by the marked difference of the relative increase in methyltransferase activity (basal level = 1) upon stimulation within the two populations: mean allergies: 0.477 k 0.27, mean controls: 0.255 _t 0.18, n = 12, cy< 0.01). The leukocyte membrane-bound cyclic AMP phosphodiesterase activity appeared to be higher among allergic patients as compared with the phosphodiesterase activity of control donors (Fig. la). Although the difference proved to be weakly significant due to the large individual variation within each group, this result agrees well with those reported by Grewe et al [9] for cytosolic cyclic AMP phosphodiesterase from similar leukocyte preparations. In addition, the number of NA binding sites seemed to be the same for the allergic and the control subjects (on account of the limited availability of blood samples, only 6 determinations could be performed in each group) (not shown). (a)
(C)
tb) 0.5
ii
Fig. 1. Comparison of (a) cyclic AMP phosphodiesterase (PDE), (b) methyltransferase and (c) NA-stimulated methyltransferase in leukocyte membrane fractions from A, allergic patients and T, control donors. Twelve pairs of control-allergic subjects were studied (n = 12). For a given control-allergic pair venous blood samples were collected at the same time and the subsequent leukocyte preparation was carried out simultaneously with the two samples, as previously described in 113). This procedure avoids daily or seasonal variations of the enzymic activities and/or slight variations in the experimental conditions of samples treatment. Results are expressed as described in ‘Methods’. In (b) and (c) statistical analysis of data was effected by the paired t test. b, Mean of differences 0.0376 f 0.0503, t = 2.59, n - 1 = 11 0.01 c a c 0.02. c. Mean of differences 0.2216 + 0.2389, t = 3.3, n - 1 = 11 (I c 0.01. a. Variances of the two groups (controls and allergies) were not similar, so data were analysed by the Wilcoxon t test. c = 0.941 a > 0.1.
230
The methyltransferase (basal level and NA-stimulated level) and the cyclic AMP phosphodiesterase activities were further studied on leukocyte preparations from three different groups of 9, 13 and 23 subjects, each group containing control donors, allergic patients and allergic subjects receiving various therapies in order to have large individual variability within each group. In all groups, venous blood samples were collected on the same day and subsequent leukocyte preparations made under the same experimental conditions for all the samples. Figure 2 shows that a good correlation exists between the cyclic AMP phosphodiesterase and the NA-stimulated methyltransferase activities. In the three different groups studied, the
. . CI: (a)
PDE
1
4
’
0.5
.
:.
)
Methyleee
,
,
0.5
1
=_Methyleee
PDE
4
0.6
1
Fig. 2. Correlations between cyclic AMP phosphodiesterase (PDE) and NA-stimulated methyltransferase (methylase) in leukocyte membrane fractions. Each symbol represents either healthy volunteer or an allergic patient. Some of the allergic subjects received no systemic medication for at least 2 wk, others received various non-adrenergic therapies. Each graph (a) (b) (c) represents a pool of homogeneous preparations obtained at the same time, in the same hospital department, and treated at the same time under the same experimental conditions (leukocyte membrane preparation, conservation and determination of enzymic activities). Results are expressed as described in ‘Methods’. The straight lines were obtained by regression analysis. a. n = 9; r = 0.919; a < 0.01 slope = 0.88; intercept, 0.02. b. n = 13; r = 0.596; 0.05 -Z a < 0.02 slope = 0.48; intercept, 0.18. c. n = 23; r = 0.622; a < 0.01 slope 0.36; intercept, 0.09.
231
correlation proves highly significant with (Y< 0.01, 0.01 < a! < 0.02 and CY< 0.01, respectively. In contrast, no correlation was found either between the cyclic AMP phosphodiesterase and the basal level of methyltransferase, or between the basal level and the NA-stimulated level of methyltransferase. This latter point must be emphasized since it suggests that the basal and the NA-stimulated methyltransferases might represent two distinct enzymic entities. Indeed, although many authors [17-191 agree that basal methylation occurs on phosphatidylethanolamine (PEA), in contrast, in the case of NA-stimulated methylation, PEA might not be the only substrate for methylation. Recent results from ours [20] and other [21] laboratories have shown that the kinetic parameters of NA-stimulated methyltransferases are quite different from those of basal methyltransferases; at the same time, it seems that NA-stimulated methylations occur not only on phospholipids [21]. Results of the present work also agree with these latter observations. Discussion Our observation, that methyltransferase activity is significantly lower in leukocyte preparations from allergic patients than that observed with control subjects, is in good agreement with our previous report [13]. On the other hand, an increased activation of methyltransferase upon adrenergic stimulation in leukocyte preparations from allergic patients as compared with controls has never been reported before. This result, however, appears to be quite logical since phospholipid methylation and plasma membrane fluidity of specialized cells were shown to closely parallel histamine release [22], and an elevated release of immediate hypersensitivity mediators following antigen exposure is indeed a central event in the pathogenesis of allergic diseases [l]. In our hands, the number of NA binding sites proves to be quite similar within both the allergic and the control groups. These results are in good agreement with those of Tashkin et al [23], who did not find any difference in the /3-adrenoceptor numbers of lymphocytes between asthmatic and normal subjects. However, the occurrence of an elevated level of phosphodiesterase activity within allergic patients might explain why, despite their normal receptor number, the lymphocytes from allergic patients showed diminished cyclic AMP responses compared with those in normal subjects [23]. The fact that highly significant correlations were obtained between the membrane-bound phosphodiesterase and adrenergic-stimulated methyltransferase activities may be of fundamental importance in the functioning of the plasma cell membrane. This result firstly suggests that the adrenergic-stimulated methyltransferase and the cyclic AMP phosphodiesterase may be closely related within the cell membrane and indicates the importance of lipids and/or phospholipids in the microenvironment of phosphodiesterase. As previously reported in astrocytoma cells [lo], the stimulation of phospholipid methylation also induces an increased degradation with generation of arachidonic acid and lysophospholipids and a concomitant loss of the adrenergic sensitivity. On the other hand, the cyclic nucleotide phosphodiesterase from various tissues and species was shown to be phospholipid and especially lysophosphohpid stimulable [24]. Thus, the stimulation of phos-
232
phodiesterase by lysophospholipids, generated as a consequence of increased methylations, could be involved in desensitization processes. Another important finding is the good correlation observed between each group of leukocyte preparations whatever the group of blood donors: healthy volunteers, allergic patients receiving no systemic medication and allergic subjects receiving various non-adrenergic therapies. This result supports the idea that the interrelationship between the two enzymes is not strictly linked with the allergic disease but rather traduces the membrane state characteristic of each individual. Thus, the interrelationship between adrenergicstimulated methyltransferase and cyclic nucleotide phosphodiesterase might be a good index of the physical state of the leukocyte membranes. This could explain the wide variations in individual susceptibility to desensitization within both allergic and non-allergic subjects, individuals with a low level of both enzymes being more resistant than those with elevated adrenergic-stimulated methyltransferase and phosphodiesterase. Acknowledgements This work was supported by grants from the Institut National de la Sante et de la Recherche Medicale (INSERM U. 205 and CRL 81-30-33) and by the Centre National de la Recherche Scientifique (CNRS ERA 560). References 1 Parker CW. Modulation of lymphoid cell function and allergic responses. Adv Cyclic Nucleotide Res. 1980; 12: 181-185. 2 Conolly ME. Cyclic Nucleotides, /I receptors, and bronchial asthma. Adv Cyclic Nucleotide Res. 1980; 12: 151-159. 3 Szentivanyi A. The /3 adrenergic theory of the atopic abnormality in bronchial asthma. J Allergy 1968; 42: 203-232. 4 Parker CW, Smith JW. Alterations in cyclic adenosine monophosphate metabolism in human bronchial asthma. I. Leukocyte responsiveness to /3 adrenergic agents. J Clin Invest 1973; 52: 48-59. 5 Makino S, Ikemori R, Fukuda T, Motojima S. Relationships between responsiveness of the bronchi to acetylcholine and cyclic AMP response of lymphocytes to beta-l and beta-2 adrenergic receptor stimulation in patients with asthma. Allergy 1983; 38: 37-42. 6 Iyengar R, Birbaumer L. Agonist-specific desensitization: molecular locus and possible mechanism. Adv Cyclic Nucleotide Res. 1981; 14: 93-100. 7 Oleshansky MA, Neff NH. Rat pineal adenosine cyclic 3’5 monophosphate phosphodiesterase activity. Modulation in vivo by a beta adrenergic receptor. Mol Pharmacol 1975; 11: 552-557. 8 Chan SC, Grewe SR, Stevens SR, Hanifin JM. Functional desensitization due to stimulation of cyclic AMP phosphodiesterase in human mononuclear leukocytes. J Cyclic Nucleotide Res 1982; 8: 211-224. 9 Grewe SR, Chan SC, Hanifin JM. Elevated leukocyte cyclic AMP-phosphodiesterase in atopic disease: a possible mechanism for cyclic AMP-agonist hyporesponsiveness. J Allergy Clin Immunol 1982; 70: 452-457. 10 Mallorga P, Tallman JF, Henneberry RC, Hirata F, Strittmatter WJ, Axelrod J. Mepacrine blocks &adrenergic agonist-induced desensitization in astrocytoma cells. Proc Nat1 Acad Sci USA 1980; 77: 1341-1345. 11 Strittmatter WJ, Hirata F, Axelrod J. Regulation of the /j adrenergic receptor by methylation of membrane phospholipids. Adv Cyclic Nucleotide Res 1981; 14: 83-91.
233
12 Fonlupt P, Dubois M, Gallet H, Biot N, Pacheco H. Modification de I’activitt phosphohpide-N-methyltransferase leucocytaire apres traitement chronique de sujets allergiques par la mequitazine. CR Acad Sci Paris 1983; 296: 100-1007. 13 Fonlupt P, Pacheco Y, Macovschi 0, Dubois M, Biot N, Pacheco H. Phosphatidyl-ethanolamine methylation in leukocyte membrane preparations from allergic subjects: an unexpected result. Clin Chim Acta 1984; 136: 13-18. 14 NCmoz G, Prigent AF, Pageaux JF, Pacheco H. Isoelectric-focusing patterns of cyclic nucleotide phosphodiesterase from rat heart. Biochem J 1981; 119: 113-119. 15 Thompson WJ, Brooker G, Appleman MM. Assay of cyclic nucleotide phosphodiesterase with radioactive substrates. Meth Enzymol 1974; 38: 205-212. 16 Hirata F, Viveros OH, Dihberto El, Axelrod J. Enzymatic synthesis and rapid translocation of phosphatidylchohne by two methyltransferases in erythrocyte membranes. Proc Nat1 Acad Sci USA 1978; 75: 171881721. 17 Hirata F, Axehod J. Phospholipid methylation and biological signal transmission. Science 1980; 209: 1082-1090. 18 Leprohon CE, Blusztajn JK, Wurtman RJ. Dopamine stimulation of phosphatidylchohne (lecithin) biosynthesis in rat brain neurons. Proc Nat1 Acad Sci USA 1983; 80: 2063-2066. 19 Hirata R, Strittmatter W, Axelrod J. fi-adrenergic receptor agonists increase phosphohpid methylation, membrane fluidity and /3-adrenergic receptor adenylate cyclase coupling. Proc Nat1 Acad Sci USA 1979; 76: 368-372. 20 Fonlupt P, Rey C, Pacheco H. Mise en evidence dune nouvelle methylase dans les membranes cellulaires du cerveau de rat. CR Acad Sci Paris 1984; 298: l-3. 21 Wazer DE, Cordasco DM, Segarnick DJ, Lippa AS, Meyerson LR, Benson D, Rotrosen J. Norepinephrine stimulation of phosphohpid methylation in rat cortical synaptosomes: fact or artifact? Life Sci 1983; 32: 2535-2544. 22 Axelrod J, Hirata F. Phospholipid methylation and the receptor-induced release of histamine from cells. Trends Pharmacol Sci 1982; 3: 156-158. 23 Tashkin DP, Conolly ME, Deutsch RI, Hui KK, Littner M, Scarpace P, Abrass I. Subsensitization of &adrenoceptors in airways and lymphocytes of healthy and asthmatic subjects. Am Rev Respir Dis 1982; 125: 185-193. 24 Wells JN, Hardman JG. Cyclic nucleotide phosphodiesterase. Adv Cyclic Nucleotide Res 1977; 8: 119-143.
225
CCA 03004
Activity of cyclic AMP phosphodiesterase and methyltransferases in leukocyte membranes from allergic patients Annie-France Prigent a,*, Pierre Fonlupt a, Madeleine Dubois a, Georges NCmoz a, Henri Pacheco a, Yves Pacheco b, Nicole Biot b and Max Perrin-Fayolle b a Service
de Chimie Biologique, Uniit! 205 INSERM, Instiiui National des Sciences Appliquies de Lyon, 69621 Villeurbanne CGdex and b Service de Pneumologie, Hbpital Sainte - Eug&nie, Lyon (France) (Received February 14th, 1984; revision July 28th. 1984) Key wor&: Allergy; Cyclic AMP phosphodiesterase; Methyltransjerase (basal level and noradrenaline-stimulated level); Leukocytes; Membranes
Summary
Cyclic AMP metabolism and methylation of phospholipids are central events which occur at the membrane level. Since a dysfunction of cell membranes seems to characterize some allergic diseases, we investigated cyclic AMP phosphodiesterase and methyltransferase activities in leukocyte membrane fractions obtained from healthy volunteers and from allergic patients. The allergic group presented a significantly decreased methyltransferase activity compared with a control group, whereas cyclic AMP phosphodiesterase and noradrenaline (NA)-stimulated methyltransferase were found to be increased with respect to the control group. A significant correlation has been found between cyclic AMP phosphodiesterase and NA-stimulated methyltransferase with both control and allergic subjects, which suggests close relationships between these two enzymes within the cell membrane. Introduction
There is general agreement that cyclic adenosine monophosphate (CAMP) plays an important modulating role in lymphoid cell function and allergic responses. * To whom correspondence should be addressed. Abbreviations used CAMP, aderiosine 3’5’~cyclic monophosphate; PDE, cyclic phodiesterase (EC 3.1.4.17); SAM, S-adenosyl-L-methionine; ME, methykransferase.
OOOS-8981/84/$03.W
@ 1984 Ehwier
Science Publishas
B.V.
nucleotide
phos-
226
Cyclic nucleotides act at several levels in the immune response such as the generation and release of mediators of anaphylaxis and the cellular responsiveness to mediators [l]. The release of these mediators is reduced or obviated by cyclic AMP or by several hormones that interact with cell surface receptors to activate adenylate cyclase [2]. A decreased /3-adrenergic response has been*proposed earlier as one of the causes of hypersensitivity of the bronchi in bronchial asthma [3]. Numerous studies of atopic leukocytes [4] or lymphocytes [S] have revealed subnormal CAMP responses to &adrenergic agonists, both in vitro and in vivo, but considerable controversy has existed over the occurrence and importance of /3-adrenoceptor desensitization in allergic patients. Whether or not allergic subjects are more prone to &receptor desensitization than healthy subjects is still an unanswered question. There are wide variations in individual susceptibility to desensitization which occur with both allergic and non-allergic subjects [Z]. Various mechanisms have been proposed to explain the refractoriness, which follows exposure of cells or isolated plasma membranes to agonists, and may be correlated with changes in the physiological responsiveness of the system. Exposure to hormones may cause a loss of binding sites. Indeed, a significant decrease in the number of receptor sites was observed in a variety of tissues from several species during the desensitization process [2]. Alternative mechanisms such as a loss of the guanine nucleotide regulatory component or a decrease in the interaction of the hormone-occupied receptor with the regulatory protein of the adenylcyclase system have also been proposed [6]. Although the alteration of one or the other component of the cyclase system is very likely to be involved in the desensitization process, other mechanisms such as an increase in cyclic nucleotide phosphodiesterase (PDE) activity may make a contribution in some tissues, e.g. pineal [7]. In this regard, discrepant reports have recently appeared. Thus, Conolly [2] was unable to demonstrate any change in phosphodiesterase activity in either bronchi or lymphocytes after desensitization by isoproterenol, whereas Chan et al [8] found a stimulation of cyclic AMP phosphodiesterase in human mononuclear leukocytes following agonist desensitization. Similarly, Grewe et al [9] observed a si~ific~tly elevated cytosolic phosphodiesterase activity in leukocytes from allergic patients as compared with leukocytes from control subjects. On the other hand, a recent attractive hypothesis, supported by in vitro experiments with cultured C, astrocytoma cells, has suggested that membrane phospholipid turnover may be involved in decreased CAMP responsiveness [lo]. Since the enzymic methylation of phospholipids alters membrane composition, symmetry and fluidity, this process is likely to have a wide spectrum of effects upon biological function and the activity of several membrane-bound enzymes [ll]. Indeed, the methylation of membrane phospholipids seems to regulate part of the P-adrenergic system and vice versa [lo-111. However, clinical studies related to an eventual alteration of phospholipid methylation in allergic diseases are few [12,13]. Furthermore, eventual relationships between methyltr~sferase and membrane-bound phosphodiesterase activities have not been studied before. The aim of the present work was to evaluate in parallel the methyltransferase (basal level and adrenergic stimulated level) and the cyclic AMP phosphodiesterase activities in the same leukocyte membrane fractions from healthy volunteers, allergic patients
227
receiving no systemic therapies.
medication
and allergic subjects
having
various non-adrenergic
Subjects Main characteristics of the groups are as follows. Comparison of cyclic AMP phosphodiesterase, methyltransferase and noradrenaline (NA)-stimulated methyltransferase activities in leukocyte membrane fractions from allergic patients and control donors: In this study, leukocytes were separated from the blood of 12 control subjects who did not exhibit clinical signs of allergy and 12 subjects with allergic asthma. Allergic asthma was defined by the following clinical criteria: bronchial hypersensitivity to carbachol; positive reactions to skin testing with a standard battery of common allergens (house dust - dermatophagoids); elevated IgE level; presence of specific IgE estimated by the RAST method. Control and allergic subjects were of both sexes and ages ranged from 20 to 40 years. In order to have a greater potency of the paired t test, controls were age and sex matched with the patients. They had not received any systemic medication (corticoids or theophylline) for at least two weeks before the experiments. Correlation between cyclic AMP phosphodiesterase and NA-stimulated methyltransferase activities in leukocyte membrane fractions: The leukocytes used in this study were from heterogeneous groups including healthy volunteers, allergic patients without systemic medication for at least two weeks, and allergic patients having various non-adrenergic therapies. In this part of the work, our aim was not to compare enzymatic activities between control and allergic subjects or to evaluate the influence of a particular therapy on these enzymatic activities, but because of the heterogeneity of the leukocyte population obtain the greatest variability among samples. Methods
Chemicals [3H]SAM (15 Ci/mmol), [8-3H]cyclic AMP (20-30 Ci/mmol) and [Ui4 Cladenosine ( 500 Ci/mmol) were supplied by the Radiochemical Centre (Amersham, UK). Snake venom (Ophiophagus hannah), bovine serum albumin and unlabelled cyclic AMP were purchased from Sigma Chemical Co. (St. Louis, MO, USA). AGlX2 resin (200-400 mesh) was from Bio-Rad Laboratories (Richmond, CA, USA). All other chemicals were reagent grade.
Purification of [‘HJSAM and isotopic dilution [ 3H]SAM was diluted isotopically with 100 parts of cold SAM. The two radioactive sources (with high and low specific activities) were chromatographed separately on silica gel plates, eluted with acetic acid and then stored at - 20 o C. Under these conditions, the radioactive source was stable for at least 8 days. By mixing the two [3H]SAM eluates with high and low specific activities, we can obtain the SAM concentration needed and the required number of disintegrations with high precision.
228
Leukocyte
membrane preparation
Leukocytes were isolated from heparinized blood by sedimentation in the presence of 5% dextran, then centrifuged and washed three times with 25 mmol/l Tris-HCl buffer, pH 7.6, and stored at - 20 *C. Since, in our previous work [13], no correlation was found between the methylase activity and the percentage of any cell type (polynuclears, lymphocytes, eosinophils, mononuclears), experiments were performed on the heterogeneous leukocyte populations without further separation of the different kinds of cells. The cells were further centrifuged at 50000 x g for 15 min and the pellet resuspended in hypotonic 5 mmol/l Tris-HCl buffer, pH 7.4, frozen and thawed. After washing by centrifugation, the membranes were resuspended in 50 mmol/l Tris-HCl buffer pH 7.4. This membrane suspension was used for the determination of the enzymic activities after appropriate dilution. Cyclic AMP phosphodiesterase
assay
Cyclic AMP phosphodiesterase activity was assayed as reported in [14] following a modified method based on the original two-step radioisotopic procedure of Thompson et al 1151with 0.25 pmol/l cyclic AMP as substrate. All the assays were performed at 30 OC, in triplicate, with enzyme dilutions adjusted to give linear reaction rates (usually less than 20-25% substrate hydrolysis). Adenosine recovery was systematically determined by means of [U-‘4C]adenosine and results corrected for these yields in each samples. Phosphodiesterase activities were expressed as pmol cyclic AMP hydrolyzed/lo6 cells per min. ~ethy~transfer~e
activity assay
Methyltransferase activity was determined by a procedure previously used [13j and derived from that described by Hirata et al (161. 0.1 pmol/l [3H]SAM (170000 dpm/assay) was used as substrate. Methyltransferase activities were expressed as pmol [ 3H]methyl group incorporated in the chloroform extractible material/lo6 cells per 30 min. NA-stimulated
methyrtransferase
assay
NA-stimulated methyltransferase activity was measured as described above, after a 5-min preincubation period of membrane fractions with or without 100 pmol/l NA. These experimental conditions were shown to give optimal stimulation in preliminary experiments performed with synapto~m~ or leukocyte preparations (P.F. unpubl, results}. Results were expressed as the ratio: NA-stimulate methylase level-basal methylase level (without NA)/basal methylase level. Results are not significantly different when activities are related to the number of cells or expressed per mg protein since the protein content of leukocytes from allergic subjects and from controls are not si~ific~tly different. However, membrane recoveries during preparation might be different in the allergic and the control groups, if variations in membrane stability occurred. In our experimental conditions, we did not find any difference between the two groups: lo6 cells always gave about 40 pg protein; so we expressed activities per lo6 cells.
229
Results
In a first set of experiments, methyltransferase, NA-stimulated methyltransferase and cyclic AMP phosphodiesterase activities were determined and compared in only two groups: control and allergic patients without systemic medication for at least two weeks. Results presented in Fig. lb showed that the methyltransferase activity, as determined by the incorporation of [ 3H]methyl group into the phospholipid fraction, was significantly lower in leukocyte preparations from allergic patients than that observed with control subjects (mean allergies: 0.115 f 0.042, mean controls: 0.153 f 0.036 in pmol [3H]methyl group incorporated per lo6 cells per 30 min, n = 12, 0.05 < (Y< 0.02) which confirms our previous results [13]. The NA-stimulated methyltransferase activity (Fig. lc) proved to be significantly higher in the same leukocyte preparations from allergic subjects than that from control donors as indicated by the marked difference of the relative increase in methyltransferase activity (basal level = 1) upon stimulation within the two populations: mean allergies: 0.477 k 0.27, mean controls: 0.255 _t 0.18, n = 12, cy< 0.01). The leukocyte membrane-bound cyclic AMP phosphodiesterase activity appeared to be higher among allergic patients as compared with the phosphodiesterase activity of control donors (Fig. la). Although the difference proved to be weakly significant due to the large individual variation within each group, this result agrees well with those reported by Grewe et al [9] for cytosolic cyclic AMP phosphodiesterase from similar leukocyte preparations. In addition, the number of NA binding sites seemed to be the same for the allergic and the control subjects (on account of the limited availability of blood samples, only 6 determinations could be performed in each group) (not shown). (a)
(C)
tb) 0.5
ii
Fig. 1. Comparison of (a) cyclic AMP phosphodiesterase (PDE), (b) methyltransferase and (c) NA-stimulated methyltransferase in leukocyte membrane fractions from A, allergic patients and T, control donors. Twelve pairs of control-allergic subjects were studied (n = 12). For a given control-allergic pair venous blood samples were collected at the same time and the subsequent leukocyte preparation was carried out simultaneously with the two samples, as previously described in 113). This procedure avoids daily or seasonal variations of the enzymic activities and/or slight variations in the experimental conditions of samples treatment. Results are expressed as described in ‘Methods’. In (b) and (c) statistical analysis of data was effected by the paired t test. b, Mean of differences 0.0376 f 0.0503, t = 2.59, n - 1 = 11 0.01 c a c 0.02. c. Mean of differences 0.2216 + 0.2389, t = 3.3, n - 1 = 11 (I c 0.01. a. Variances of the two groups (controls and allergies) were not similar, so data were analysed by the Wilcoxon t test. c = 0.941 a > 0.1.
230
The methyltransferase (basal level and NA-stimulated level) and the cyclic AMP phosphodiesterase activities were further studied on leukocyte preparations from three different groups of 9, 13 and 23 subjects, each group containing control donors, allergic patients and allergic subjects receiving various therapies in order to have large individual variability within each group. In all groups, venous blood samples were collected on the same day and subsequent leukocyte preparations made under the same experimental conditions for all the samples. Figure 2 shows that a good correlation exists between the cyclic AMP phosphodiesterase and the NA-stimulated methyltransferase activities. In the three different groups studied, the
. . CI: (a)
PDE
1
4
’
0.5
.
:.
)
Methyleee
,
,
0.5
1
=_Methyleee
PDE
4
0.6
1
Fig. 2. Correlations between cyclic AMP phosphodiesterase (PDE) and NA-stimulated methyltransferase (methylase) in leukocyte membrane fractions. Each symbol represents either healthy volunteer or an allergic patient. Some of the allergic subjects received no systemic medication for at least 2 wk, others received various non-adrenergic therapies. Each graph (a) (b) (c) represents a pool of homogeneous preparations obtained at the same time, in the same hospital department, and treated at the same time under the same experimental conditions (leukocyte membrane preparation, conservation and determination of enzymic activities). Results are expressed as described in ‘Methods’. The straight lines were obtained by regression analysis. a. n = 9; r = 0.919; a < 0.01 slope = 0.88; intercept, 0.02. b. n = 13; r = 0.596; 0.05 -Z a < 0.02 slope = 0.48; intercept, 0.18. c. n = 23; r = 0.622; a < 0.01 slope 0.36; intercept, 0.09.
231
correlation proves highly significant with (Y< 0.01, 0.01 < a! < 0.02 and CY< 0.01, respectively. In contrast, no correlation was found either between the cyclic AMP phosphodiesterase and the basal level of methyltransferase, or between the basal level and the NA-stimulated level of methyltransferase. This latter point must be emphasized since it suggests that the basal and the NA-stimulated methyltransferases might represent two distinct enzymic entities. Indeed, although many authors [17-191 agree that basal methylation occurs on phosphatidylethanolamine (PEA), in contrast, in the case of NA-stimulated methylation, PEA might not be the only substrate for methylation. Recent results from ours [20] and other [21] laboratories have shown that the kinetic parameters of NA-stimulated methyltransferases are quite different from those of basal methyltransferases; at the same time, it seems that NA-stimulated methylations occur not only on phospholipids [21]. Results of the present work also agree with these latter observations. Discussion Our observation, that methyltransferase activity is significantly lower in leukocyte preparations from allergic patients than that observed with control subjects, is in good agreement with our previous report [13]. On the other hand, an increased activation of methyltransferase upon adrenergic stimulation in leukocyte preparations from allergic patients as compared with controls has never been reported before. This result, however, appears to be quite logical since phospholipid methylation and plasma membrane fluidity of specialized cells were shown to closely parallel histamine release [22], and an elevated release of immediate hypersensitivity mediators following antigen exposure is indeed a central event in the pathogenesis of allergic diseases [l]. In our hands, the number of NA binding sites proves to be quite similar within both the allergic and the control groups. These results are in good agreement with those of Tashkin et al [23], who did not find any difference in the /3-adrenoceptor numbers of lymphocytes between asthmatic and normal subjects. However, the occurrence of an elevated level of phosphodiesterase activity within allergic patients might explain why, despite their normal receptor number, the lymphocytes from allergic patients showed diminished cyclic AMP responses compared with those in normal subjects [23]. The fact that highly significant correlations were obtained between the membrane-bound phosphodiesterase and adrenergic-stimulated methyltransferase activities may be of fundamental importance in the functioning of the plasma cell membrane. This result firstly suggests that the adrenergic-stimulated methyltransferase and the cyclic AMP phosphodiesterase may be closely related within the cell membrane and indicates the importance of lipids and/or phospholipids in the microenvironment of phosphodiesterase. As previously reported in astrocytoma cells [lo], the stimulation of phospholipid methylation also induces an increased degradation with generation of arachidonic acid and lysophospholipids and a concomitant loss of the adrenergic sensitivity. On the other hand, the cyclic nucleotide phosphodiesterase from various tissues and species was shown to be phospholipid and especially lysophosphohpid stimulable [24]. Thus, the stimulation of phos-
232
phodiesterase by lysophospholipids, generated as a consequence of increased methylations, could be involved in desensitization processes. Another important finding is the good correlation observed between each group of leukocyte preparations whatever the group of blood donors: healthy volunteers, allergic patients receiving no systemic medication and allergic subjects receiving various non-adrenergic therapies. This result supports the idea that the interrelationship between the two enzymes is not strictly linked with the allergic disease but rather traduces the membrane state characteristic of each individual. Thus, the interrelationship between adrenergicstimulated methyltransferase and cyclic nucleotide phosphodiesterase might be a good index of the physical state of the leukocyte membranes. This could explain the wide variations in individual susceptibility to desensitization within both allergic and non-allergic subjects, individuals with a low level of both enzymes being more resistant than those with elevated adrenergic-stimulated methyltransferase and phosphodiesterase. Acknowledgements This work was supported by grants from the Institut National de la Sante et de la Recherche Medicale (INSERM U. 205 and CRL 81-30-33) and by the Centre National de la Recherche Scientifique (CNRS ERA 560). References 1 Parker CW. Modulation of lymphoid cell function and allergic responses. Adv Cyclic Nucleotide Res. 1980; 12: 181-185. 2 Conolly ME. Cyclic Nucleotides, /I receptors, and bronchial asthma. Adv Cyclic Nucleotide Res. 1980; 12: 151-159. 3 Szentivanyi A. The /3 adrenergic theory of the atopic abnormality in bronchial asthma. J Allergy 1968; 42: 203-232. 4 Parker CW, Smith JW. Alterations in cyclic adenosine monophosphate metabolism in human bronchial asthma. I. Leukocyte responsiveness to /3 adrenergic agents. J Clin Invest 1973; 52: 48-59. 5 Makino S, Ikemori R, Fukuda T, Motojima S. Relationships between responsiveness of the bronchi to acetylcholine and cyclic AMP response of lymphocytes to beta-l and beta-2 adrenergic receptor stimulation in patients with asthma. Allergy 1983; 38: 37-42. 6 Iyengar R, Birbaumer L. Agonist-specific desensitization: molecular locus and possible mechanism. Adv Cyclic Nucleotide Res. 1981; 14: 93-100. 7 Oleshansky MA, Neff NH. Rat pineal adenosine cyclic 3’5 monophosphate phosphodiesterase activity. Modulation in vivo by a beta adrenergic receptor. Mol Pharmacol 1975; 11: 552-557. 8 Chan SC, Grewe SR, Stevens SR, Hanifin JM. Functional desensitization due to stimulation of cyclic AMP phosphodiesterase in human mononuclear leukocytes. J Cyclic Nucleotide Res 1982; 8: 211-224. 9 Grewe SR, Chan SC, Hanifin JM. Elevated leukocyte cyclic AMP-phosphodiesterase in atopic disease: a possible mechanism for cyclic AMP-agonist hyporesponsiveness. J Allergy Clin Immunol 1982; 70: 452-457. 10 Mallorga P, Tallman JF, Henneberry RC, Hirata F, Strittmatter WJ, Axelrod J. Mepacrine blocks &adrenergic agonist-induced desensitization in astrocytoma cells. Proc Nat1 Acad Sci USA 1980; 77: 1341-1345. 11 Strittmatter WJ, Hirata F, Axelrod J. Regulation of the /j adrenergic receptor by methylation of membrane phospholipids. Adv Cyclic Nucleotide Res 1981; 14: 83-91.
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12 Fonlupt P, Dubois M, Gallet H, Biot N, Pacheco H. Modification de I’activitt phosphohpide-N-methyltransferase leucocytaire apres traitement chronique de sujets allergiques par la mequitazine. CR Acad Sci Paris 1983; 296: 100-1007. 13 Fonlupt P, Pacheco Y, Macovschi 0, Dubois M, Biot N, Pacheco H. Phosphatidyl-ethanolamine methylation in leukocyte membrane preparations from allergic subjects: an unexpected result. Clin Chim Acta 1984; 136: 13-18. 14 NCmoz G, Prigent AF, Pageaux JF, Pacheco H. Isoelectric-focusing patterns of cyclic nucleotide phosphodiesterase from rat heart. Biochem J 1981; 119: 113-119. 15 Thompson WJ, Brooker G, Appleman MM. Assay of cyclic nucleotide phosphodiesterase with radioactive substrates. Meth Enzymol 1974; 38: 205-212. 16 Hirata F, Viveros OH, Dihberto El, Axelrod J. Enzymatic synthesis and rapid translocation of phosphatidylchohne by two methyltransferases in erythrocyte membranes. Proc Nat1 Acad Sci USA 1978; 75: 171881721. 17 Hirata F, Axehod J. Phospholipid methylation and biological signal transmission. Science 1980; 209: 1082-1090. 18 Leprohon CE, Blusztajn JK, Wurtman RJ. Dopamine stimulation of phosphatidylchohne (lecithin) biosynthesis in rat brain neurons. Proc Nat1 Acad Sci USA 1983; 80: 2063-2066. 19 Hirata R, Strittmatter W, Axelrod J. fi-adrenergic receptor agonists increase phosphohpid methylation, membrane fluidity and /3-adrenergic receptor adenylate cyclase coupling. Proc Nat1 Acad Sci USA 1979; 76: 368-372. 20 Fonlupt P, Rey C, Pacheco H. Mise en evidence dune nouvelle methylase dans les membranes cellulaires du cerveau de rat. CR Acad Sci Paris 1984; 298: l-3. 21 Wazer DE, Cordasco DM, Segarnick DJ, Lippa AS, Meyerson LR, Benson D, Rotrosen J. Norepinephrine stimulation of phosphohpid methylation in rat cortical synaptosomes: fact or artifact? Life Sci 1983; 32: 2535-2544. 22 Axelrod J, Hirata F. Phospholipid methylation and the receptor-induced release of histamine from cells. Trends Pharmacol Sci 1982; 3: 156-158. 23 Tashkin DP, Conolly ME, Deutsch RI, Hui KK, Littner M, Scarpace P, Abrass I. Subsensitization of &adrenoceptors in airways and lymphocytes of healthy and asthmatic subjects. Am Rev Respir Dis 1982; 125: 185-193. 24 Wells JN, Hardman JG. Cyclic nucleotide phosphodiesterase. Adv Cyclic Nucleotide Res 1977; 8: 119-143.