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Calcium, calmodulin and cAMP and the control of cellular proliferation
233
T I B S - J u l y 1982
How widespread are cyanide-resistant mitochondria in plants? The upper part of the inflorescence of the pletely sealed chambers was fully inhibited voodoo lilly (Sauromatum guttatum) and by 0.16 mM HCN. While pointing out that the flower-bearing appendix of the skunk this observation does not mean that operacabbage (Symplocarpus foetidus ) are two tion of cyanide-resistant processes, such as tissues that have had a great influence on the 'alternative electron transport pathway', the study of plant mitochondria. This is are not important for germination, the because both tissues can generate warmth authors concluded that cyanide-sensitive by respiring at a remarkable rate, and this processes are also required. respiration is unaffected by cyanide. The second, and conceivably stronger Cyanide-resistant respiration was found to jolt, has originated from the University of be associated with preparations of Arizona where Goldstein, Anderson and mitochondria from these tissues about 25 McDaniel3 convincingly demonstrated that years ago and since then similar (although the cyanide-insensitive 02 uptake measured less pronounced) cyanide-resistant respira- with mitochondria obtained from wheat tion has been identified with mitochondrial seedlings by differential centrifugation was preparations from a wide range of plant due to the oxygenation of linoleate by tissues. lipoxygenase (both of which were fortuitThe task of elucidating the biochemical ously associated with the mitochondrial mechanism of cyanide resistance has pro- preparation) and consequently had nothing ven to be formidable. To date, the fol- to do with any alternative electron transport lowing ~ have been proposed: (1) The system. Goldstein et al. found that the precyanide-resistant electron transport system paration separated on a linear Percoll denconsists of a branch from the conventional sity gradient into two bands of mitochonelectron transport system, beginning with drial activity, neither of which was resistant ubiquinone and terminating with an 'alter- to cyanide. However, one band still disnative oxidase'. It is not coupled to ATP played cyanide-resistant 02 uptake when formation, i.e. it leads mainly to production supplied with linoleate. Further purification of heat (although when NADH is the elec- of this band on a stepped Percoll density tron donor one ATP is produced, as usual, gradient resulted in one band of between NADH dehydrogenase and ubi- mitochondria that no longer possessed quinone). (2) All mitochondria with linoleate-dependent O~ uptake, and a cyanide-resistant respiration also possess second band that contained no mitochonthe conventional, cyanide-sensitive res- dria but presumably contained lipoxygenpiratory electron transport that terminates ase since it took up 02 when supplied with with cytochrome oxidase, and appear to use linoleate. this system preferentially. There is eviThis demonstration that plant mitochondence that the cyanide-resistant pathway dria are easily contaminated with lipoxybecomes operative only when the conven- genase is of special significance because it tional pathway becomes restricted (e.g. by had been shown earlier4 that lipoxygenase cyanide inhibition of cytochrome oxidase) or overwhelmed (i.e. high level of ubiquinone reduction). However, the disturbing feature of cyanide-resistant respiration is that the nature of the alternative oxidase, and the nature and number (if any) of the electron transport components between it In the early 1970s, the hypothesis that and ubiquinone have not been elucidated. cyclic AMP regulated cellular proliferation Apart from the unidentified alternative was enthusiastically supported. This came oxidase, only one further component (a about mainly tYom the observation that the flavoprotein) has been implicated. concentration of cAMP was relatively low The somewhat shaky foundations upon in transformed cells and that the addition of which the concept of cyanide-resistant res- agents that raised its concentration stopped piration is based were recently given two growthL However, the idea of cAMP further jolts. One of these came from Yu, being the 'magic bullet' against cancer fell Mitchell and Robitaille 2 (Purdue Univer- from grace, when inconsistencies in the sity), who showed that the reported ability findings invariably cropped up. of certain seeds to germinate in the presence Although these inconsistencies discourof cyanide was an artifact caused by the aged many groups, others doggedly purescape of cyanide (as HCN) from the Petri sued the involvement of cAMP in growth dishes used as germination chambers. They control and their efforts are beginning to demonstrated that germination in com- pay off in a big way. Some of the earlier
(an enzyme that is practically ubiquitous in the plant kingdom) is inhibited by both salicylhydroxamate and propyl gallate; these two substances also inhibit cyanideresistant (but not cyanide-sensitive) respiration, and they have been used as 'relatively specific' inhibitors to detect cyanide-resistant respiration. It is now clear that these inhibitors cannot be used to obtain reliable measurements of cyanideresistant respiration. In this context, it will be interesting to find what effect disulfiram has on lipoxygenase, since Grover and Laties~ at the University of California have found that this disulphide, is a powerful inhibitor of cyanide-resistant respiration by potato tuber mitochondria. Meanwhile, the conclusion of Goldstein et al. :~ that their purified wheat mitochondria 'lack demonstrable alternative oxidase respiration' leaves us wondering how many other plant mitochondria, when similarly purified, will be found to lack cyanideresistant respiration. The mitochondria from the voodoo lily and the skunk cabbage, if they can be similarly purified, would be particularly ripe for reexamination. References 1 Day, D. A., Arron.G. P. and Laties,G. G. (1980) in The Biochemistry of Plants, Vol. 2 (Davies, D. D., ed.), pp. 197-241,AcademicPress,NewYork 2 Yu, K. S.. Mitchell.C. A. and Robitaille. H. A. ( 1981) Plant Physiol. 68. 509-51 I 3 Goldstein,A. H.. Anderson,J. O. and McDaniel, R. G. ( 1981) Plant Physiol. 67. 594-596 4 Parrish, D. J. and Leol:x~ld,A C. (1978) Plant Physiol. 62,470~72 5 Grover, S. D. and Laties. G. G. (1981) Plant Physiol. 68,393-400 GRAHAMEJ. KELLY Departmentof Biochemistry,and Nutrition, University of New England.Anlfidale.Australia.
Calcium, calmodulin and cAMP and the control of cellular proliferation inconsistencies have been explained through the work of John Pawelek at Yale. Basically, he has shown that the effects of cAMP depend, to a great extent, on its absolute cellular concentration and that this 'optimal dose' varies from cell type to cell type according to the KA of Type II cAMP-dependent protein kinase (cA-PK) for cAMP 2. Jim Whitfield, AI Boynton and their colleagues in Ottawa have demonstrated that cAMP concentrations increase transiently prior to DNA synthesis in a number of cell types and that the initiation of DNA synthesis is prevented if cAMP concentrations are kept steady. These transients have been
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~aet, exogenousce~l~VJt(~tseJt r.~. .. , ~ can stimulate , In t°tbepre.treatmentofthes -~---,~nu caimodu. Ca~'_deprived . . . . vents may stvary .according n (CM) in the growth regulation syntl~esize DNA, the most tho . h . . . . Y eros. Perhaps y o f systems. Perhaps the rod-°tla vari. The response to Ca 2~ or C M is also blocked %-~-provorin 5( procluctive ~nese• are rat 1;. . . . . . • . swered questions c . . . . . . . g o f the unan,~cr .... ~-,-c~ns exogeno t~e stimulation o f versibly ~trrested at ceus the Gwhich U S b have been ewtt n a ohr ~ r mdomethacin ~:~,^ - ' an i .umDltor of D N A , synthesis by . . . . . uc synttlase which is the first proteins, such as CM ... "; usiy apphed ~ b a t i o n in low 0.02 . . . . oundary by e~zyme in the pathwa~ of prostaglandit~ rlo~oenzyme C . . . . ~* .Jype h cA-PK t s ' a n d 5 ) , Wh(~nCa;~+~Ted~edtalC;~; Olosymhesis from a r a c h a d o " , " ~"nr°'exper, mentsconvineMoreover, ~Csta;lcaid mgly show that these effects are not anifac_ rs D N A synthesis begins within 15 tmix. dins (PGA~ arachadooic PGE~ p G ~ acid • s " tual, yet there are no existinp rr, uses to a growth promoter known. ' - plato these observations odels to ex~ .' . . . .snmu~ate .~"<~' or , phospholi is, by far, one o f the most rapid re- use A~ will the m°~'~cs D N A sy~. The model has also lent itself well to heincreaseintherateofDNA thesis and this , . ~ls system is nreced...~ - synthesis indometh.o;, can also be blocked ~., .~ comparisons between normal and trans. '.ases in the c ~ , ~ - ~ u . oy transient Tho--~, . . . . ~ Jormed cells. For : P and ~.a r,~'--' ' c n t r a t l ° n s o f bo,~, __ . - - ~ c OO~rvations h a w ~-.~ - cells in ~e ~, instance, transform r.r ,, . . . . rt,.R. Th~ -" <" marion o f . ~ - ~ , ~ ,cu to the tb, , ~ neral, are refr~," ed '~'~. svnth.o;. "~ stimulation .,;.t..~. ~ ,-uuel where c.~e+ '"synchroni7.~; . . . . - " - ~ t l v e |o lOWCn2~ d d i t i o n o-~Y2 can be blocked b,, ~,-~.~M which, in turn st~n~, c o m p l e x e s be exolain S ~. n . lhis p h e n o m e n o n m i " 1 FKin~ a s . • Y pnotipase . , mutates a h . e~ ~,y aberra . . ght Ior. In appropriate 'cPeo c ' f i c cA-PK arachidonate]eleasmg arachidonate P ~ cycle~dependentsurgeTd~,m..~_ng°ftbece,l ~tself Stimulat,~ r~,,. ncentrations, whi,'~, ~.;-- • ~ converted to a pros,--~', -, t h e s i s . In addilio- e n o v o C M syn[so is bl,-~a..~o ~ , , . ~ synthesis and t,._ ~'~ ~-muiates adenyl c v c l ~ ",~g~anam thesized Tvr~ . ~'ff surge O£denovo s n . ' _ - - ~ . e u oy PKinh g'-~ luanSlent) inc---~ . ~ - ~ , causing a I~, ~, ~r~ .~ c~_pK h.. h~ "y ~* cA-PK holoenzvme" ~^~,genous tion, The cA~.~'n~ ~.e m c A M p concentra ~
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235
TIBS -July 1 982
view, Ca 2v-deprived cells can probably be used to quantify the 'synarchic' actions of CM and cAMP, and to explore the in situ binding characteristics of CM which have recently been proposed by Rasmussen and Waisman s. Finally, the intimate involvement shown here of protein kinases in the control of cellular proliferation are tantalizingly close to the oncogene system. It seems likely that, in the next few years, these studies will progress to a deeper understanding of the events underlying the
J. P., Armato, U., Tsang, B. K. and Jones, A. (1981)Exp. Cell. Res. 135,199-211 6 MacManus, J.P., Braceland. B.M., Ri×on, R. H., Whitfield, J. F. and Morris, H. P. (1981) References FEBS Lett8. 133, 99-102 1 Buerk, R. R. (1968) .Nature (London) 219, 7 Haddox,M. K., Magun, B. E. and Russell, D. H. 1272-1274 (1980) Proc. Natl Acad. Sci. U.S.A. 77. 2 Pawalek, J. M. (1979) J. Cell. Physiol. 98, 3445-3449 619-626 8 Rasmussen,H and Waisman,D. M. Rev. Physiol. 3 Boynton, A. L. and Whitfield, J. F. Adv. (~vcl. Biochem. Pharmacol. (in press) Nucl. Res. (in press) ROBERTJ. GILLIES 4 Boynton, A. L. and Whitfield, J. F. (1981)Adv. Department of Molecular Biophysics and BioCycl. Nuc. Res. 14, 411~.t9 chemistry, Yale University, Box 6666. New Haven, 5 Boynton. A.L., Whitfield, J.F., MacManus, CT 0651 I, U.S.A.
regulation of cellular proliferation and the physiology of cellular transformation.
Platelet activating factor does more than activate platelets Biochemists who study the metabolism and function of phospholipids have rarely enjoyed the ability to attribute potent biological activity to the diverse lipid structures found in eukaryotic cells. For the most part, phospholipids have been regarded as serving structural roles in membranes or, in some cases, as precursors for more active substances such as prostaglandins. Nevertheless, the existence of hundreds of different phospholipids has enticed many workers toward the belief that some diverse new functions must lie waiting to be discovered. The recent elucidation of the structure of a phospholipid with dramatic biological potency supports this belief. When leukocytes that have been sensitized with a specific IgE antibody are challenged by antigen in vitro, they release a potent substance which causes platelets to change shape, aggregate and release their contents. The structure of this substance, originally referred to as platelet activating factor (PAF), has been identified as 1 alkyl - 2 - acetyl - sn - glycero - 3 - phosphocholine ~,2. It is clear now that the actions of this lipid are far broader than platelet activation and are believed to include involvement in anaphylactic shock, inflammation and allergic responses. Furthermore, as little as 63 nanograms of the same substance given intraveneously to hypertensive rats causes marked hypotensive responses3. After the publication of more than 60 studies related to this substance, considerably more is known about its physiological effects than about its biochemical modes of action. However, clues are beginning to emerge. The involvement of calcium is postulated because the calcium ionophore A23187 stimulates the release of PAF from various cell types. In addition, PAF itself stimulates calcium influx into rabbit platelets 4. At the 1982 meeting of the Federation of American Societies for Experimental Biology, Chilton et al. 5 reported that the addition of
ferase activity. A close correlation was observed between the amount of PAF produced by intact cells and the extent of acetyltransferase activation assayed in sonicated cells. In addition, the time course of enzyme activation coincided well with the generation of PAF by the polymorphonuclear cells. These three studies provide the groundwork for a search for agents capable of modifying PAF production in vivo. However, as with several other bioregulators, including prostaglandins, the plateletactivating lipid may play an essential beneficial role under most conditions, but in certain physiological states its contributions to thrombosis, anaphylaxis, inflammation, etc., may be life threatening. It is hoped that a better understanding of its role in the highly complex interactions between blo~d cells will provide new strategies for intervention in some of the less desirable consequences of platelet aggregation.
PAF to polymorphonuclear leukocytes stimulates the release and hydroxylation of arachidonic acid. No prostaglandins were detected, which is consistent with earlier data showing the effectiveness of PAF in the presence of cyclooxygenase inhibitors. At the same meeting, Shukla and Hanahan 6 reported that within 5-10 s of the addition of PAF to rabbit platelets there was a 15-20% decrease in the concentration of phosphatidylinositol in the platelet membranes. Taken together the reports suggest that PAF may act by initiating a chain of lipid-mediated membrane alterations which culminate in platelet aggregation. Sincein v i v o control of the production of PAF could have considerable pharmacological value, three groups have recently focused effort toward the enzymology of PAF synthesis. Lee et al. 7 report that normal human neutrophils possess several key References enzymes involved in the metabolism of I Demopoulos,C. A., Pinckard, R. N. and Hanahan, D. J. (1979)J. Biol. Chem. 254, 9355 PAF. Both acetyltransferase, which catalyzes the formation of PAF from inactive 2 Benveniste,J., Tence, M., Varenne,P., Bidault,J., Boullet, C. and Polonsky, J. (1979) C. R. Acad. lyso-PAF, and acetyl-hydrolase, which Sci. (D) (Paris) 289, 1037 leads to its inactivation, were present in the 3 Blank,M. L., Snyder, F., Byers,L. W., Brooks,B. human blood cells; however, acetyltransand Muirhead, E. E. (1979) Biochem. Biophys. Res. Commun, 90, 1194 ferase activity was 10-15 fold higher than that of the hydrolase. Consistent with the 4 Lee, T.-c., Malone, B., Blank. M. L. and Snyder, F. (1981) Biochem. Biophys. Res. Commun. previous reports of the involvement of cal102, 1262-1268 cium was again indicated by 2-5-fold 5 Chilton, F. H., O'Flaherty, J. T., Walsh, C. E., stimulation of both activities by A23187. Thomas, M. J., Wykle, R. L., DeChatelet, L. R. In similar studies, Ninio et al. reported and Waite. B. M. (1982)Fed. Proc. 41,2322 the preliminary characterization of an 6 Shukla, S. D. and Hanahan, D. J. (1982) Fed. Proc. 41,3757 acetyltransferase activity in extracts of murine peritoneal adherent cells s. The 7 Lee, T.-c., Malone, B., Wasserman, S. I., Fitzgerald, V. and Snyder, F. (in press) measured activity yielded a product that 8 Ninio, E., Mencia-Huerta.J. M., Heymans,F. and was active in platelet aggregation as well as Benveniste, J. (1982) Biochim. Biophys. Acta exhibiting the chromatographic and chemi710, 23-31 cal characteristics of PAF. Furthermore, 9 Alonso, F., Garcia-Gil, M., Shnchez.-Crespo,M. the enzyme activity correlated well with and Mato, J. M. (1982)J. BioL (~hem. 257. 3376 PAF releasing activity in comparisons of non-adherent vs adherent cells. J. B. OHLROGGE The third group, Alonso et al. ~, have shown that activation of human polymor- Northern Regional Research Center, Agricultural phonuclear leukocytes with zymosan Research Service, U.S. Department of Agriculture. induces a 10-fold activation of acetyltrans- Peoria, IL 61604, U.S.A.
T I B S - J u l y 1982
How widespread are cyanide-resistant mitochondria in plants? The upper part of the inflorescence of the pletely sealed chambers was fully inhibited voodoo lilly (Sauromatum guttatum) and by 0.16 mM HCN. While pointing out that the flower-bearing appendix of the skunk this observation does not mean that operacabbage (Symplocarpus foetidus ) are two tion of cyanide-resistant processes, such as tissues that have had a great influence on the 'alternative electron transport pathway', the study of plant mitochondria. This is are not important for germination, the because both tissues can generate warmth authors concluded that cyanide-sensitive by respiring at a remarkable rate, and this processes are also required. respiration is unaffected by cyanide. The second, and conceivably stronger Cyanide-resistant respiration was found to jolt, has originated from the University of be associated with preparations of Arizona where Goldstein, Anderson and mitochondria from these tissues about 25 McDaniel3 convincingly demonstrated that years ago and since then similar (although the cyanide-insensitive 02 uptake measured less pronounced) cyanide-resistant respira- with mitochondria obtained from wheat tion has been identified with mitochondrial seedlings by differential centrifugation was preparations from a wide range of plant due to the oxygenation of linoleate by tissues. lipoxygenase (both of which were fortuitThe task of elucidating the biochemical ously associated with the mitochondrial mechanism of cyanide resistance has pro- preparation) and consequently had nothing ven to be formidable. To date, the fol- to do with any alternative electron transport lowing ~ have been proposed: (1) The system. Goldstein et al. found that the precyanide-resistant electron transport system paration separated on a linear Percoll denconsists of a branch from the conventional sity gradient into two bands of mitochonelectron transport system, beginning with drial activity, neither of which was resistant ubiquinone and terminating with an 'alter- to cyanide. However, one band still disnative oxidase'. It is not coupled to ATP played cyanide-resistant 02 uptake when formation, i.e. it leads mainly to production supplied with linoleate. Further purification of heat (although when NADH is the elec- of this band on a stepped Percoll density tron donor one ATP is produced, as usual, gradient resulted in one band of between NADH dehydrogenase and ubi- mitochondria that no longer possessed quinone). (2) All mitochondria with linoleate-dependent O~ uptake, and a cyanide-resistant respiration also possess second band that contained no mitochonthe conventional, cyanide-sensitive res- dria but presumably contained lipoxygenpiratory electron transport that terminates ase since it took up 02 when supplied with with cytochrome oxidase, and appear to use linoleate. this system preferentially. There is eviThis demonstration that plant mitochondence that the cyanide-resistant pathway dria are easily contaminated with lipoxybecomes operative only when the conven- genase is of special significance because it tional pathway becomes restricted (e.g. by had been shown earlier4 that lipoxygenase cyanide inhibition of cytochrome oxidase) or overwhelmed (i.e. high level of ubiquinone reduction). However, the disturbing feature of cyanide-resistant respiration is that the nature of the alternative oxidase, and the nature and number (if any) of the electron transport components between it In the early 1970s, the hypothesis that and ubiquinone have not been elucidated. cyclic AMP regulated cellular proliferation Apart from the unidentified alternative was enthusiastically supported. This came oxidase, only one further component (a about mainly tYom the observation that the flavoprotein) has been implicated. concentration of cAMP was relatively low The somewhat shaky foundations upon in transformed cells and that the addition of which the concept of cyanide-resistant res- agents that raised its concentration stopped piration is based were recently given two growthL However, the idea of cAMP further jolts. One of these came from Yu, being the 'magic bullet' against cancer fell Mitchell and Robitaille 2 (Purdue Univer- from grace, when inconsistencies in the sity), who showed that the reported ability findings invariably cropped up. of certain seeds to germinate in the presence Although these inconsistencies discourof cyanide was an artifact caused by the aged many groups, others doggedly purescape of cyanide (as HCN) from the Petri sued the involvement of cAMP in growth dishes used as germination chambers. They control and their efforts are beginning to demonstrated that germination in com- pay off in a big way. Some of the earlier
(an enzyme that is practically ubiquitous in the plant kingdom) is inhibited by both salicylhydroxamate and propyl gallate; these two substances also inhibit cyanideresistant (but not cyanide-sensitive) respiration, and they have been used as 'relatively specific' inhibitors to detect cyanide-resistant respiration. It is now clear that these inhibitors cannot be used to obtain reliable measurements of cyanideresistant respiration. In this context, it will be interesting to find what effect disulfiram has on lipoxygenase, since Grover and Laties~ at the University of California have found that this disulphide, is a powerful inhibitor of cyanide-resistant respiration by potato tuber mitochondria. Meanwhile, the conclusion of Goldstein et al. :~ that their purified wheat mitochondria 'lack demonstrable alternative oxidase respiration' leaves us wondering how many other plant mitochondria, when similarly purified, will be found to lack cyanideresistant respiration. The mitochondria from the voodoo lily and the skunk cabbage, if they can be similarly purified, would be particularly ripe for reexamination. References 1 Day, D. A., Arron.G. P. and Laties,G. G. (1980) in The Biochemistry of Plants, Vol. 2 (Davies, D. D., ed.), pp. 197-241,AcademicPress,NewYork 2 Yu, K. S.. Mitchell.C. A. and Robitaille. H. A. ( 1981) Plant Physiol. 68. 509-51 I 3 Goldstein,A. H.. Anderson,J. O. and McDaniel, R. G. ( 1981) Plant Physiol. 67. 594-596 4 Parrish, D. J. and Leol:x~ld,A C. (1978) Plant Physiol. 62,470~72 5 Grover, S. D. and Laties. G. G. (1981) Plant Physiol. 68,393-400 GRAHAMEJ. KELLY Departmentof Biochemistry,and Nutrition, University of New England.Anlfidale.Australia.
Calcium, calmodulin and cAMP and the control of cellular proliferation inconsistencies have been explained through the work of John Pawelek at Yale. Basically, he has shown that the effects of cAMP depend, to a great extent, on its absolute cellular concentration and that this 'optimal dose' varies from cell type to cell type according to the KA of Type II cAMP-dependent protein kinase (cA-PK) for cAMP 2. Jim Whitfield, AI Boynton and their colleagues in Ottawa have demonstrated that cAMP concentrations increase transiently prior to DNA synthesis in a number of cell types and that the initiation of DNA synthesis is prevented if cAMP concentrations are kept steady. These transients have been
234
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°n Althoogh of wellThis ge°o,al model has. been holding up in other s,,st~-_ - p e c m c , this TFp hl^~,. . lcledly such as r-+ ':~.. c ~ s stuaied by this grou c o m e by the addition ° f e.x. .o. g r,e ncan o u s 19e C MOVer- exact timing, ' - .-¢v e. r. . . .tn v;"*~. . . . . h o w e v e r , the P'
~aet, exogenousce~l~VJt(~tseJt r.~. .. , ~ can stimulate , In t°tbepre.treatmentofthes -~---,~nu caimodu. Ca~'_deprived . . . . vents may stvary .according n (CM) in the growth regulation syntl~esize DNA, the most tho . h . . . . Y eros. Perhaps y o f systems. Perhaps the rod-°tla vari. The response to Ca 2~ or C M is also blocked %-~-provorin 5( procluctive ~nese• are rat 1;. . . . . . • . swered questions c . . . . . . . g o f the unan,~cr .... ~-,-c~ns exogeno t~e stimulation o f versibly ~trrested at ceus the Gwhich U S b have been ewtt n a ohr ~ r mdomethacin ~:~,^ - ' an i .umDltor of D N A , synthesis by . . . . . uc synttlase which is the first proteins, such as CM ... "; usiy apphed ~ b a t i o n in low 0.02 . . . . oundary by e~zyme in the pathwa~ of prostaglandit~ rlo~oenzyme C . . . . ~* .Jype h cA-PK t s ' a n d 5 ) , Wh(~nCa;~+~Ted~edtalC;~; Olosymhesis from a r a c h a d o " , " ~"nr°'exper, mentsconvineMoreover, ~Csta;lcaid mgly show that these effects are not anifac_ rs D N A synthesis begins within 15 tmix. dins (PGA~ arachadooic PGE~ p G ~ acid • s " tual, yet there are no existinp rr, uses to a growth promoter known. ' - plato these observations odels to ex~ .' . . . .snmu~ate .~"<~' or , phospholi is, by far, one o f the most rapid re- use A~ will the m°~'~cs D N A sy~. The model has also lent itself well to heincreaseintherateofDNA thesis and this , . ~ls system is nreced...~ - synthesis indometh.o;, can also be blocked ~., .~ comparisons between normal and trans. '.ases in the c ~ , ~ - ~ u . oy transient Tho--~, . . . . ~ Jormed cells. For : P and ~.a r,~'--' ' c n t r a t l ° n s o f bo,~, __ . - - ~ c OO~rvations h a w ~-.~ - cells in ~e ~, instance, transform r.r ,, . . . . rt,.R. Th~ -" <" marion o f . ~ - ~ , ~ ,cu to the tb, , ~ neral, are refr~," ed '~'~. svnth.o;. "~ stimulation .,;.t..~. ~ ,-uuel where c.~e+ '"synchroni7.~; . . . . - " - ~ t l v e |o lOWCn2~ d d i t i o n o-~Y2 can be blocked b,, ~,-~.~M which, in turn st~n~, c o m p l e x e s be exolain S ~. n . lhis p h e n o m e n o n m i " 1 FKin~ a s . • Y pnotipase . , mutates a h . e~ ~,y aberra . . ght Ior. In appropriate 'cPeo c ' f i c cA-PK arachidonate]eleasmg arachidonate P ~ cycle~dependentsurgeTd~,m..~_ng°ftbece,l ~tself Stimulat,~ r~,,. ncentrations, whi,'~, ~.;-- • ~ converted to a pros,--~', -, t h e s i s . In addilio- e n o v o C M syn[so is bl,-~a..~o ~ , , . ~ synthesis and t,._ ~'~ ~-muiates adenyl c v c l ~ ",~g~anam thesized Tvr~ . ~'ff surge O£denovo s n . ' _ - - ~ . e u oy PKinh g'-~ luanSlent) inc---~ . ~ - ~ , causing a I~, ~, ~r~ .~ c~_pK h.. h~ "y ~* cA-PK holoenzvme" ~^~,genous tion, The cA~.~'n~ ~.e m c A M p concentra ~
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TIBS -July 1 982
view, Ca 2v-deprived cells can probably be used to quantify the 'synarchic' actions of CM and cAMP, and to explore the in situ binding characteristics of CM which have recently been proposed by Rasmussen and Waisman s. Finally, the intimate involvement shown here of protein kinases in the control of cellular proliferation are tantalizingly close to the oncogene system. It seems likely that, in the next few years, these studies will progress to a deeper understanding of the events underlying the
J. P., Armato, U., Tsang, B. K. and Jones, A. (1981)Exp. Cell. Res. 135,199-211 6 MacManus, J.P., Braceland. B.M., Ri×on, R. H., Whitfield, J. F. and Morris, H. P. (1981) References FEBS Lett8. 133, 99-102 1 Buerk, R. R. (1968) .Nature (London) 219, 7 Haddox,M. K., Magun, B. E. and Russell, D. H. 1272-1274 (1980) Proc. Natl Acad. Sci. U.S.A. 77. 2 Pawalek, J. M. (1979) J. Cell. Physiol. 98, 3445-3449 619-626 8 Rasmussen,H and Waisman,D. M. Rev. Physiol. 3 Boynton, A. L. and Whitfield, J. F. Adv. (~vcl. Biochem. Pharmacol. (in press) Nucl. Res. (in press) ROBERTJ. GILLIES 4 Boynton, A. L. and Whitfield, J. F. (1981)Adv. Department of Molecular Biophysics and BioCycl. Nuc. Res. 14, 411~.t9 chemistry, Yale University, Box 6666. New Haven, 5 Boynton. A.L., Whitfield, J.F., MacManus, CT 0651 I, U.S.A.
regulation of cellular proliferation and the physiology of cellular transformation.
Platelet activating factor does more than activate platelets Biochemists who study the metabolism and function of phospholipids have rarely enjoyed the ability to attribute potent biological activity to the diverse lipid structures found in eukaryotic cells. For the most part, phospholipids have been regarded as serving structural roles in membranes or, in some cases, as precursors for more active substances such as prostaglandins. Nevertheless, the existence of hundreds of different phospholipids has enticed many workers toward the belief that some diverse new functions must lie waiting to be discovered. The recent elucidation of the structure of a phospholipid with dramatic biological potency supports this belief. When leukocytes that have been sensitized with a specific IgE antibody are challenged by antigen in vitro, they release a potent substance which causes platelets to change shape, aggregate and release their contents. The structure of this substance, originally referred to as platelet activating factor (PAF), has been identified as 1 alkyl - 2 - acetyl - sn - glycero - 3 - phosphocholine ~,2. It is clear now that the actions of this lipid are far broader than platelet activation and are believed to include involvement in anaphylactic shock, inflammation and allergic responses. Furthermore, as little as 63 nanograms of the same substance given intraveneously to hypertensive rats causes marked hypotensive responses3. After the publication of more than 60 studies related to this substance, considerably more is known about its physiological effects than about its biochemical modes of action. However, clues are beginning to emerge. The involvement of calcium is postulated because the calcium ionophore A23187 stimulates the release of PAF from various cell types. In addition, PAF itself stimulates calcium influx into rabbit platelets 4. At the 1982 meeting of the Federation of American Societies for Experimental Biology, Chilton et al. 5 reported that the addition of
ferase activity. A close correlation was observed between the amount of PAF produced by intact cells and the extent of acetyltransferase activation assayed in sonicated cells. In addition, the time course of enzyme activation coincided well with the generation of PAF by the polymorphonuclear cells. These three studies provide the groundwork for a search for agents capable of modifying PAF production in vivo. However, as with several other bioregulators, including prostaglandins, the plateletactivating lipid may play an essential beneficial role under most conditions, but in certain physiological states its contributions to thrombosis, anaphylaxis, inflammation, etc., may be life threatening. It is hoped that a better understanding of its role in the highly complex interactions between blo~d cells will provide new strategies for intervention in some of the less desirable consequences of platelet aggregation.
PAF to polymorphonuclear leukocytes stimulates the release and hydroxylation of arachidonic acid. No prostaglandins were detected, which is consistent with earlier data showing the effectiveness of PAF in the presence of cyclooxygenase inhibitors. At the same meeting, Shukla and Hanahan 6 reported that within 5-10 s of the addition of PAF to rabbit platelets there was a 15-20% decrease in the concentration of phosphatidylinositol in the platelet membranes. Taken together the reports suggest that PAF may act by initiating a chain of lipid-mediated membrane alterations which culminate in platelet aggregation. Sincein v i v o control of the production of PAF could have considerable pharmacological value, three groups have recently focused effort toward the enzymology of PAF synthesis. Lee et al. 7 report that normal human neutrophils possess several key References enzymes involved in the metabolism of I Demopoulos,C. A., Pinckard, R. N. and Hanahan, D. J. (1979)J. Biol. Chem. 254, 9355 PAF. Both acetyltransferase, which catalyzes the formation of PAF from inactive 2 Benveniste,J., Tence, M., Varenne,P., Bidault,J., Boullet, C. and Polonsky, J. (1979) C. R. Acad. lyso-PAF, and acetyl-hydrolase, which Sci. (D) (Paris) 289, 1037 leads to its inactivation, were present in the 3 Blank,M. L., Snyder, F., Byers,L. W., Brooks,B. human blood cells; however, acetyltransand Muirhead, E. E. (1979) Biochem. Biophys. Res. Commun, 90, 1194 ferase activity was 10-15 fold higher than that of the hydrolase. Consistent with the 4 Lee, T.-c., Malone, B., Blank. M. L. and Snyder, F. (1981) Biochem. Biophys. Res. Commun. previous reports of the involvement of cal102, 1262-1268 cium was again indicated by 2-5-fold 5 Chilton, F. H., O'Flaherty, J. T., Walsh, C. E., stimulation of both activities by A23187. Thomas, M. J., Wykle, R. L., DeChatelet, L. R. In similar studies, Ninio et al. reported and Waite. B. M. (1982)Fed. Proc. 41,2322 the preliminary characterization of an 6 Shukla, S. D. and Hanahan, D. J. (1982) Fed. Proc. 41,3757 acetyltransferase activity in extracts of murine peritoneal adherent cells s. The 7 Lee, T.-c., Malone, B., Wasserman, S. I., Fitzgerald, V. and Snyder, F. (in press) measured activity yielded a product that 8 Ninio, E., Mencia-Huerta.J. M., Heymans,F. and was active in platelet aggregation as well as Benveniste, J. (1982) Biochim. Biophys. Acta exhibiting the chromatographic and chemi710, 23-31 cal characteristics of PAF. Furthermore, 9 Alonso, F., Garcia-Gil, M., Shnchez.-Crespo,M. the enzyme activity correlated well with and Mato, J. M. (1982)J. BioL (~hem. 257. 3376 PAF releasing activity in comparisons of non-adherent vs adherent cells. J. B. OHLROGGE The third group, Alonso et al. ~, have shown that activation of human polymor- Northern Regional Research Center, Agricultural phonuclear leukocytes with zymosan Research Service, U.S. Department of Agriculture. induces a 10-fold activation of acetyltrans- Peoria, IL 61604, U.S.A.