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From www.bloodjournal.org by guest on June 18, 2017. For personal use only. Lymphocyte II. Cyclic Membrane 3’,5’-Adenosine Monophosphatase Unstimulated Human Small Nuclear By This study enzyme phatase was S. undertaken portance ulation, Cyclic in mediating hence localization is thought may histochemical of action employed that was the pliosphodiesterase, resulting in a lead then INCREASINGLY, enzymes portaiice lead the in early to ivuiphocyte small lymphocytes I)OSSSS while if! is it that from In may the monophosphate work in indicates It where does at some other on the indicates cyclic not that AMP necessarily point in the pathway, and the be the site mediating is mean is the site this may lymphocyte including the cell nuclear membrane where cyclic AMP from intracellular effects AMP may The it and events, to serve of the be an freshly enzyme value stimulation nucleus. stimulatory is known with of the state. ascertain the cyclic nature resting the AMP) is concerned the surface to the intracellular that site localized which BEING CONCENTRATED on the membranes, as they may be of imrelated to antigen recognition, that study of (cyclic particularly This ascertain along variety the to be precipitate the prestimulation be possible to lymphocyte found This is subsulfide. The is degraded. hydrolyzed, to occur events a wide et stimulation. human chemical be ATTENTION IS with lymphocyte events, immediately associated is stimulation al.#{176} membrane, by may only degraded phosphate was membranes, action. as lead that the nuclear membrane where cyclic AMP is effective; may Shanta is enzyme destroyed. AMP technique of KENNEDY nuclear this of cyclic substrate ANIP A. at the site of enzyme sequently visualized stimenzyme AMPase, point to the site in the cell. The Cyclic the its intracell- cyclic on Lymphocyte LESLEY AND be of im- lymphocyte of the to modulate concentration, COULSON monophoshuman small AMP Located Membranes to localize cyclic 3’,5’-adenosine in unstimulated lymphocytes. that ular ALAN Enzymes. of such related data pathway cyclic they information what bio- that leads 3’,5’-adenosine as a second-echelon peptide hormones.1’2 intermediary separated systems hormone, Recent in thymocyte and from the Department of Surgery, Stanford University School of Medicine, Stanford, Calif. Submitted April 20, 1971; revised May 20, 1971; accepted May 22, 1971. This study was carried out in the Department of Pathology, Guy’s Hospital, London, England, and was supported by the University of London Laura de Saliceto Studentship, the Peel Medical Research Trust, and the British Medical Association Ernest Hart Award. ALAN S. CouLsoN, NI.A., NIB., B. Cim., PH.D., L.R.C.P., M.R.C.S.: Senior Research Fellow, Department of Surgery, Stanford University School of Medicine, Stanford, Calif.; Leverhulme Research Fellow. LE5LEY A. KENNEDY: Tissue-typing Immunologist, Departnients of Immunology and Surgery, Guy’s Hospital, London, England. BLOOD, \OL. 38, No. 4 (OCTOBER), 1971 485 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 486 COULSON lymphocyte stimulation, hence the phosphodiesterase, in this way, probably cyclic serves the it was of interest 3’,5’-AMPase, to modulate to that the attempt destroys effect AND the KENNEDY localization of cyclic AMP and thus, of cyclic AMP within cell. The histochemical Smears of AMP, and cells to this form exogenous also was verted to visualized black by The the The lead microscopically in in sulfide MATERIALS Lq;nplzocyte human described carbonyl third of Gelatine rotated allowed lymphocytes to prevent to make the slides subsequently a that medium in form later lead then the stage. The presumptive AND METHODS et al.#{176} cyclic 3’,5’- within hydrolyzed form of black site snake which staining of the by precipitated phosphate, the Shanta located radical of of containing was phosphate marked on phosphodiesterase the the at and based a medium compound liberated medium, in the latter supplied atrox.7 in was incubated degraded 5’-AMPase present employed were 5’-AMP. Crotalu9 from technique lymphocytes the venom with lead was con- could enzyme be activity. Separation small lymphocytes were separated from healthy donors using the technique by Coulson, Corner, and Coombs.8 Defibrinated blood was mixed with 100 mg iron particles ( Fine Dyestuffs and Chemicals, Manchester, England and with one its volume of 3% gelatin in phosphate-buffered saline (Batch 277, Glue and Research Association Birmingham England . The blood-gelatin-iron mixture was on a blood cell suspension mixer for 30 mm at 37#{176}C,then decanted and to sediment for a further 30 mm at 37#{176}C.The supematant suspension of was diluted with an equal volume of 199 medium (Weilcome, lot M3413) the gelatin from gelling, and centrifuged to produce the pellet of cells used the smears. The time that elapsed from the initial venipuncture to the fixing of in acetone was 2 hr. The slides on which the smears were made, and which came in contact with the incubation media, were scrupulously cleaned. ) ) Fixation and Histochemistry Lymphocyte smears were air dried within a period of 5 sec and then plunged into 60% distilled water at 20#{176}C for 120 ± 5 sec. The slides were washed gently for 3 mm in tap water and then in two changes of glass distilled water for a total of 4 mm. Test slides were incubated in a medium containing the following constituents: cyclic 3’,5’-adenosine monophosphate ( 1.44 mM, Sigma, lot 60C-1620), Tns-maleate buffer (pH 7.6, 50 mM), magnesium chloride ( 10 mM), lead acetate (2 mM), and Crotalus atrox venom ( 1 mg in 10 ml, Sigma, lot 78B-1460).6 When the medium was made up in this way, the final pH was 7.30, and it had to be adjusted to 7.60 with Tris base. Incubation was continued for 90 ± 5 mm at 37#{176}C in disposable Petri dishes. At the end of incubation the pH of the medium was still 7.60. The molarity of the incubation medium was unavoidably reduced slightly by the residual dampness of the lymphocyte smear. After incubation, the slides were washed carefully by gentle agitation in five changes of glass distilled water for a total of 25 mm. Any significant curtailment of this washing led to nonspecific background staining on the slide. The next stage in the staining procedure was immersion of the slides in freshly prepared ammonium sulfide solution (1 % weight! volume in distilled water), followed by three washes in distilled water totaling 9 mm. Finally the slides were mounted in glycerol jelly. In some preliminary experiments different methods of fixation were tried, including 100% methanol and 70% ethanol is distilled water. acetone in Control With AMP every staining run the following controls was omitted; (2) exogenous 5’-nucleotidare were was employed: omitted and (1) the fluoride substrate was cyclic added to From www.bloodjournal.org by guest on June 18, 2017. For personal use only. LYMPhOCYTE inhibit MEMBRANE endogenous 5’-nucleotidase; cyclic AMPase. water was substituted ingredients of other was In omitted, 20 mM; practical (3) terms, or when fluoride addition, the solution smears cyclic AMP solution, affected. was were ammnophylline the for the cyclic AMP the medium was not sodium in 487 ENZYMES so that When substituted to preincubated in was added was omitted, the the an inhibitor 1 ml of of distilled final concentration of the exogenous 5’-nucleotidase produce 100 as a mM final sodium concentration fluoride of dissolved in 50 mM Tris buffer (pH 7.8 ) for 30 ± 3 mm at 37#{176}C.The aminophyllmne control studies involved preincubation of the slide in 100 mM ammnophyllmne B.P. ( British Pharmacopoeia) for 30 mm at 37#{176}C,followed by incubation in a medium identical to the normal test medium but with added aminophylline B.P. to a final concentration of 20 mM. Further control studies were carried out on two additional donors, including heat inactivation of the enzyme, inhibition with formahin, and variation of the pH of the incubation medium. In practical terms, heat inactivation was achieved by warming the separated lymphocytes to 60#{176}Cfor 120 ± 5 sec prior to centrifugation and fixation. Formalin treatment involved immersion of the slides for 10 mm in formahin B.P. at room temperature between the acetone fixation stage and the wash in distilled water. Variation in the pH range for the final incubation media was achieved by making up the test medium to the following different pH values: 6.0, 6.5, 6.8, 7.0, 7.6, 7.8, and 8.0. RESULTS Lymphocyte The Separation, leukocytes contained the in more lymphocytes the in the film showed and 70% compared small branes in the were smear sharply deposits closely showed the was where, of the procedure outnumbered taken on the smear the to examine Leishman was conversely, film, avoided the tail beof the lymphocytes. lymphocyte to incubation smears, with distorted density inferior yielded cells Care together; considerably fixed the after of final which were substrate. airThe treatment, and this positive staining. histochemical Methanol preparations slides. AMPase found of all with prior in Cyclic was lymphocytes product was and no black and similarly staining smear head sedimentation red smears. The of the were done in acetone final of the closely spreading acetone of the Positive tail too reduction to the Localization the classical. swollen marked ethanol was the although 6: 1 in the crowded trials fixed appeared there roughly before excessive Preliminary dried but not cells ratio were from lymphocytes, appeared cells Morphology and supernatant 99% just morphology cause the than lymphocytes their Fixation, to be 10 of the defined were localized different on the donors nuclear membrane employed. The with no extension into the nucleus seen elsewhere in the cell (Fig. 1). examined and no staining. were consistently negative. in the final reaction or cytoplasm, The cell mem- The erythrocytes Controls In was all added, the controls the where lymphocyte cyclic nuclear AMP was membranes omitted were or where unstained. 18 control experiments where exogenous 5’-nucleotidase fluoride added, the nuclear membrane appeared slightly plasm very slightly gray. On the other 16 occasions and was gray during aminophylline In two out of omitted and and the cytothe prelimi- From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 488 COULSON AND KENNEDY 1 - - .4 . 1: o ‘is #{149} - 2 I A 90 Fig. 1.-Freshly separated small lymphocytes from different donors showing localization of cyclic 3’,5’-AMPase on nuclear membranes. Smears of lymphocytes were incubated in a medium containing cyclic AMP, exogenous 5’-nucleotidase and lead ions; lead phosphate deposited at the site of enzyme activity is converted to black lead sulfide by ammonium sulfide treatment. X 1750. In Fig. 1A the two small black granules to the right of the lymphocyte and the two small circles below it are artifacts. The eight erythrocytes discernible in the picture are unstained. nary trials, the Treatment tion of either the and membrane formahin enzyme 8.0 6.8 nuclear with or activity. 7.0, below the density there was and was of no unstained. heat When inactivation the pH of the staining nuclear resulted in complete the incubation medium was considerably membrane inhibiwas reduced; at pH staining. DIscussIoN The inhibitory I)ility partures viev AMP. the an of the at sulfide deposit 3’,5’-AMP the iron left high magnifications against sulfide the recognizable. Sutherland’ has the second stage at a site distant procedure employed, the related out, the from the not AMP first occur, 5’-nucleotidase, cyclic 3’,5’- of and the known of Stage. sharply final the lead cyclic Confusion residual any it the investigated carbonyl because has lymphoma is cells of of shape of NK/Ly the cyclic of degradation traces characteristic zone of the de- because persistently diffuseness and did enzyme, perinuclear pointed of product separation A in the case of the to support meaningfulness was lack suscepti- and employed the in the This reaction of technique product the cells strongly degradation questioned reaction membrane. of the medium the However, final occurring lead from demonstrated1#{176} As argues incubation has and heating histochemical he product. nuclear substrate between the the the the aminophylline prior initiates and final experiments, localized of process criticized and to both pH procedure, localization clearly optimal Pearse#{176} has these formaldehyde procedure enzymatic a multistep in of staining from that is effect of the with particles the was previously been cells. to play the role From www.bloodjournal.org by guest on June 18, 2017. For personal use only. LYMPhOCYTE of a MEMBRANE secondary situations. to be the intracellular The the result of of degrades does not hormone-like intracellular synthesis that 489 ENZYMES content a balance cyclic it. and point of within the lymphocyte. is at the cell membrane brane the and nucleus The and glutinin-stimulated of stimulate cyclic that indicate thyroid Small cyclic AMP an earlier nucleoside observation17 diphosphatase spectively with of early cyclic to the noted on and to nuclear best membrane for membrane be to as will with mediate the the playing site any suggestive. suppress but of Studies growth for the AMP described absence where is merely cyclic 3’,5’-AMP the site in Various phytohemag- addition that some phytohemagglutinin, rat thymic their will lymphocytes mitogenic hormone.1214 paucity of of part effect of enzyme para- content,16 of enzyme present, particularly that associated with important role in early triggering events following thus be of immunologic significance. In this context the increase in lymphocyte during transformation may synthesis AMP ascribe in and however, the small amount membranes, may play an antigen recognition,16 and case nuclear evidence appears bradykinin, are the effects AMP transformation. hormone, lymphocytes that 3’,5’-AMPase AMP mem- transformation, AMP, lymphocyte to of appears cyclase cyclic as the variety It is possible that the site where cyclic or some other site between the cell cyclic lymphocyte concentrations AMPase wide moment adenyl enzyme structure the that a any cyclic can found at enzyme cyclic available activation have in AMP modulator latter that presently lymphocyte investigators5 the the to the is effective is effective degradation. in small cyclic between AMP, Localization necessarily substance of and its them release modulator a definite membrane-associated be correlated of lymphokines.18’1#{176} enzyme place cyclic in the M. B., retro- However, AMPase, in the it is still lymphocyte too stimulation pathway. REFERENCES 1. role Sutherland, of cyclic E. ANIP. W.: JAMA On the biological 214:1281, 1970. 2. Robison, G. A., Butcher, R. W., and Sutherland, E. W.: Cyclic AMP. Ann. Rev. Biochem. 37:149, 1968. 3. MacNianus, J. P., and Whitfield, J. F.: Stimulation of DNA synthesis and mitotic activity of thymic lymphocytes by cyclic adenosine 3’,5’-monophosphate. Exp. Cell Res. 58:188, 1969. 4. Hirschhorn, R., Grossman, J., and Weissniann, C.: Effect of cyclic 3’,S’-adenosine monophosphate and theophyllmne on lym- phocyte transformation. Proc. Soc. Exp. Biol. Med. 133:1361, 1970. 5. Rigby, P. C., and Ryan, W. L.: The effect of cyclic AMP and related compounds on human lymphocyte transformation (HLT) stimulated by phytohemagglutinmn (PHA). Rev. Eur. Etud. Clin. Biol. 15:774, 1970. 6. Shanta, T. R., Woods, W. D., Waitz- man, and chemmcal method 3’,S’-nucleotide chemie 7:177, Bourne, C. H.: for localization phospho-diesterase. Histo- of cyclic Histo- 1966. 7. Richards, G. M., Do Vair, C., and Laskowski, M., Sr.: Comparison of the levels of phosphodiesterase, monophosphatases Biochemistry endonuclease, mn several (Wash.) 4:501, snake and venoms. 1965. 8. Coulson, A. S., Cumer, B. W., and Coombs, R. R. A.: Macrophage-like properties of some guinea-pig transformed cells. mt. Arch. Allerg. 32:264, 1967. 9. Pearse, A. C. E.: Histochemistry, Theoretical and Applied. London, Churchill, 1968, p. 526. 10. Kurnatowski, A., and Willighagen, R. C.: Cytochemistry of the NK!Ly lymphoma. Nature (London) 198:1211, 1963. 11. Butcher, R. W., and Sutherland, E. W.: Adenosine 3’,5’-phosphate in biological From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 490 COULSON materials. I. Purification and properties of cyclic 3’,5’-nucleotide phosphodiesterase and use of this enzyme to characterize adenosine 3’,5’-phosphate in human urine. J. Biol. Chein. 237:1244, 1962. 12. Whitfield, J. F., NlacManus, J. P., and Rixon, R. H.: The possible mediation by cyclic ANIP of parathyroid hormone-induced stimulation of mitotic activity and deoxyribonucleic acid synthesis in rat thymic lymphocytes. J. Cell. Physiol. 75:213, 1970. 13. -, -, and Gillan, D. J.: Cyclic AMP mediation of bradykinmn-mnduced stimulation of mitotic activity and DNA synthesis in thymocytes. Proc. Soc. Exp. Biol. Med. 133: 1270, 1970. 14. MacManus, J. P., and Whitfield, J. F.: Mediation of the mitogenic action of growth hormone by adenosine 3’,5’-monophosphate (Cyclic 132:409, Proc. AMP). AND Soc. Exp. KENNEDY Biol. Med. 1969. 15. Brittinger, C., Hirschhorn, R., Douglas, S. D., and Weissmann, C.: Studies on lysosomes. XI. Characterization of a hydrol- rich fraction ase Cell Biol. from 16. Coulson, J. Theor. The -: lymphocytes Quart. 18. Golgi and J. Exp. -, applications Guy Hosp. and lymphocytes. A. S.: Recognition in lymphocytes. 17. human J. 1968. 37:394, Biol. apparatus in transformed Physiol. Inman, of Rep. 50:271, pathway 25:127, 1969. in small blast cells. 1965. D. R.: Current clmnmcal tissue culture. press. lymphocyte In 19. David, J.: Mediators of munity liberated by lymphocytes. Practice 6:79, 1971. cellular imHospital From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 1971 38: 485-490 Lymphocyte Membrane Enzymes. II. Cyclic 3',5'-Adenosine Monophosphatase Located on Unstimulated Human Small Lymphocyte Nuclear Membranes ALAN S. COULSON and LESLEY A. 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