TIBS 16 - MARCH1992

JOURNALCLUB

Ni-.tricoxide,

' (>IO) a versati le

.......... s e c o n d

messenger in

brain

Nitric oxide (NO) is apparently equival- ent to the endothelial-derived relaxing factor (EDRF), which stimulates soluble guanylyl cyclase (for review see Ref. I) and causes the relaxation of vascular smooth muscle. In addition to being made by endothelial cells, NO is pro- duced by macrophages and in brain tis- sue (for review see Ref. 2). It had been unclear from biochemical studies whether NO was produced in nerve cells that transmit electrical signals in the brain, by the surrounding glial cells, or only by endothelial cells within brain tissue. The enzyme responsible for NO synthesis, NO synthase (EC 1.14.23) 2, has now been shown by immuno- histochemistry to be localized within neuronal cells in specific brain regions, with highest expression in neurons of the cerebellum and olfactory bulb of adult rat brain 4.

Recent biochemical studies indicate heterogeneity in NO synthase activity between different tissues, suggesting that there are at least two isoforms. All of the isoforms convert arginine to citrulline and NO and require NADPH as a cofactor ~. The neural enzyme, like its endothelial counterpart, is constitu- tively expressed, is activated by Ca2*/calmodulin a and is selectively inhibited in a competitive fashion by N-nitro-L-arginine s. By contrast, the enzymes in macrophages, fibroblasts 7 and smooth muscle cells a appear to be similar or identical isoforms that require tetrahydrobiopterin in addition to NADPH as cofactor 7 and are induced by interferon y (Ref. 7) and tumor necrosis factor a (Ref. 9). In contrast to the brain and endothelial cell enzymes, they are inhibited by N-nitro-L-arginine much less effectively than by the simi- lar competitive inhibitors N-methyl-L- arginine and N-amino-L-arginine ~. The induction of NO synthase by tumor necrosis factor and the resulting increase of NO may be the mechanism of the severe hypotension associated with septic shock. It has been postu- lated that the differential inhibition of the two isoforms may allow for selec- tive pharmacological intervention to

brain NO synthase unaltered. This could potentially be life-saving in cases of sep- sis.

The most striking feature of NO as a second messenger is its rapid diffusi- bility across cell membranes in the absence of any carrier mechanism. This property makes NO a particularly attractive second messenger in the ner- vous system, as it allows for the signal generated in one cell to exert its effects in multiple neighboring cells, limited only by the biological half-life of the highly reactive NO (estimated to be of the order of I-5 s) I. Thus critical parameters to be determined are what substances increase NO production in neurons and, subsequently, what other messenger pathways are affected ~y the increase in NO.

The dependence of NO synthase on CaZ'/calmodulin allows for its stimu- lation through first messengers that increase intracellular Ca2÷; the most obvious of these in neurons is electrical activity. The most widely studied sys- tem in this regard is the N-methyl-D- aspartate (NMDA)-sensitive stimulation of ionotropic glutamate receptors. The resultant increase in Ca ~* can stimulate NO synthase as well as other Ca 2"- dependent enzymes. Identification of factors regulating the synthesis or turnover of NO synthase will be of great interest.

The production of NO stimulates guanylyl cyclase activity, thereby link- ing NO production to another known second messenger system. A recent immunohistological study has demon- strated high levels of cGMP in the cer- ebellum under conditions that favor NO

inhibit the production of NO in uivo 6a° Producti onn. Increased expression of by smooth muscle cells, leaving the cGMP was observed after stimulation

© 1991,Elsevier Science Publishers Ltd,(UK) 0376-5067/91/$02.00

with NMDA, kainic acid or sodium nitro- prusside, all of which directly or in- directly lead to increased production of NO. Moreover, although NMDA and kainic acid stimulate neuronal NO pro- duction, increased cGMP immunoreac- tivity was observed in glial structures. This cGMP increase was inhibited by inhibitors of NO synthase, scavengers of NO or reduction in intracellular Ca 2÷, suggesting that NO serves as an inter- cellular mediator in the cerebellum by modulating cGMP levels. The roles of soluble guanylyl cyclase are varied. In cerebeUar glia it has been postulated that cGMP normalizes the ionic en- vironment by regulating ion-channel gating n. Other studies have suggested that cGMP inhibits the release of neurotransmitters t2. Another activity of cGMP was demonstrated in an un- related study, in which cGMP stimulated by NO activation of guanylyl cyclase was shown to block gap junction conductivity in retinal cells TM. Effects of NO that are independent of guanylyl cyclase have also been observed. An NO-stimulated ADP-ribosyltransferase activity ms has been reported to be present in a number of tissues, includ- Lng brain ~4. In addition, NO decreases cytosulk: tree calcium by a cGMP-inde- pendent mechanism ~.

The expression of NO synthase and generation of NO may have widespread effects during brain development and in adult brain function. For example, it has been hypothesized ~7 that NO plays an important role in axonal segregation during development, as a molecular mechanism by which concurrently fir- ing axons could communicate with one another. Nerve terminals that simul- taneously release neurotransmitters tend to segregate into groups (e.g. the barrel-field or eye-specific stripes in the cortex). These authors suggest that the high concentration of NO resulting from neurotransmitter release in a localized area may be necessary for axonal segre- gation during development. In adult ani- mals, similar mechanisms may account for synaptic plasticity ~7. For example, recent paPers bare indicated that a sequence of events involving NO syn- thase and guanylyl cyclase occurs to

81

TIBS 16-MARCH1991

effect long term depression of synapses in the cerebellum ~8'~. It is possible to measure NO directly 2° and it has now been shown that blocking the formation of NO prevents long term depression of synapses in cerebellar slice cultures2L

Taken together, all of these studies suggest a role for NO in modulating multiple messenger pathways in devel- oping and adult brains. NO promises to be an extensive and versatile messen- ger in brain development and function- ing, given the importance of activity- dependent mechanisms in the estab- lishment of brain connectivity during development specifically mediated through NMDA receptors. In addition, it is possible that NO modulates the effi- cacy with which certain neurons com- municate at their synapses (i.e. synap- tic plasticity) ~9 and it may affect the release of neurotransmitters ~2. More extensive studies of the mechanisms affected by the generation of NO should therefore deepen our understanding both of nervous system development

and of the functioning of the adult ner- vous system.

Acknowledgement The author wishes to thank Dr

Joseph Gaily for helpful discussions.

References 1 Ignarro, L. J. (1990) Hypertension 16,

477-483 2 Marietta, M. A. (1989) Trends Biochem. Sci.

14, 488-492 3 Bredt, D. S. and Snyder, S. H. (1990) Proc. Natl

Acad. Sci. USA 87,682--685 4 Bredt, D. S., Hwang, P. M. and Snyder, S. H.

(1990) Nature 347, 768 5 Moncada, S., Palmer, R. M. J. and Higgs, E. A.

(1989) Biochem. PharmacoL 38, 1709-1715 6 Lambert, L. E., Whitten, J. P., Baron, B. M.,

Cheng, H. S., Doherty, N. S. and McDonald, I. A. (1991) Life ScL 48, 69-75

7 Werner-Felmayer, G., Werner, E. R., Fuchs, D., Hausen, A., Reibnegger, G. and Wachter, H. (1990) J. Exp. Med. 172,1599-1607

8 Knowles, R. G., Salter, M., Brooks, S. L. and Moncada, S. (1990) Biochem. Biophys. Res. Commun. 172, 1042-1048

9 Marsden, P. A. and Ballermann, B. J. (1990) J. Exp. Med. 172, 1843-1852

10 Kilbourn, R. G., Jubran, A., Gross, 5. S., Grifflth, O. W., Levi, R., Adams, J. and Lodata, R. F.

Regulatory d

,phosphorylation of chloroplast

antenna proteins ..

(1990) Biochem. Biophys. Res. Commun. 172, 1132-1138

11 DeVente, J., Bol, J. G. J. M., Berkelmans, H. S., Schipper, J. and Steinbusch, H. M. W. (1990) Eur. J. Neurosci. 2, 845--862

12 Yonehara, N., Matsuda, T., Saito, K., Ishida, H. and Yoshida, H. (1980) Brain Res. 182, 137-144

13 Myachi, E., Murakami, M. and Nakaki, T. (1990) NeuroReport 1,107-110

14 Brune, B. and Lapetina, E. G. (1989) J. BioL Chem. 264, 8455-8458

15 Brune, B. and Lapetina, E. G. (1990) Arch. Biochem. Biophys. 279, 286-290

16 Garg, U. C. and Hassid, A. (1991) J. Biol. Chem. 266, 9-12

17 Gaily, J. A., Montague, P. R., Reeke, G. N., Jr and Edelman, G. M. (1990) Proc. Natl Acad. Sci. USA 87, 3547-3551

18 Crepel, F. and Jaillard, D. (1990) NeuroReport 1, 133-136

19 Ito, M. and Karachot, L. (1990) NeuroReport 1, 129-132

20 Shibuki, K. (1990) NeuroscL Res. 9, 69-76 21 Shibuki, K. and Okada, D. (1991) Nature 349,

326-328 22 Greenberg, S., Diecke, F. P. J., Peevy, K. and

Tanaka, T. P. (1989) Eur. J. Pharm. 162, 67-80

KATHRYN L. CROSSIN The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.

Noncyclic electron flow from H20 to NADP ÷ during oxygenic photosynthesis requires two light-dependent steps, carried out by the reaction centers of photosystems I and If, respectivelyL To maximize the efficiency of noncyclic electron transport the rates of the photosystem I-driven and photosystem ll-driven processes must be balanced. The rate-limiting step of noncyclic elec- tron flow is the photosystem i-driven oxidation of the plastoquinol that is generated by the photosystem If-driven reduction of plastoquinoneL Thus, oxy- genic phototrophs need to increase the rate of the photosystem l-driven oxi- dation of plastoquinol when reduced plastoquinone accumulates. This is accomplished by a regulatory mechan- ism 2-4 that increases energy transfer to photosystem I from antenna pigments otherwise associated with photosystem II. This regulatory mechanism is often spoken of in terms of a transition from a state of low energy transfer from photo- system II to photosystem I (State I) to a state of high energy transfer (State 2). in chloroplasts of higher plants and algae, the transition from State 1 to State 2 appears to involve the physical movement of one or more of the pro-

teins that bind antenna pigments from a region of the thylakoid membrane close to the photosystem II reaction center to a region near the photosystem I reac- tion center 4. This set of 25--27 kDa antenna proteins, which are collectively referred to as LHC II, are phosphoryl- ated during the State l-State 2 tran- sition 2-s. Phosphorylation of amino acid residues of the membrane-bound LHC II that face the soluble strom@ involves transfer of the 7-phosphoryl residue of ATP and is catalysed by a specific mem- brane-associated kinase, or perhaps by a set of kinases 7-n. The transition from State 2 to State I is associated with the dephosphorylation of LHC II, catalysed by a phosphatase 2,3.

It has been recognized for some time that the LHC II kinase is controlled by the oxidation state of a chloroplast component 2.3. The results of oxidation-

reduction titrations and other exper- iments suggested that this component might be the plastoquinone pool itself, with reduction of the pool leading to an activation of the kinase 3,za'z6. How- ever, recent evidence suggests the cytochrome b{ complex is instead the regulator of LHC II kinase zT-'2. These new results, described below, provide the first evidence that the cytochrome bJ complex may piay a role in the regu- lation of energy distribution, in addition to its previously docunmnted role u3 in catalysing the electron flow from plasto- quinol to plastocyanin that links the two light reactions of noncyclic elec- tron flow.

Important evidence for the possible role of the cytochrome bef complex in activating the LHC II kinase came from studies with a mutant of Lemna per- pusilla that lacks the cytochrome b{ complex ~7. This mutant shows appar- ently normal membrane ultrastructure and contains normal photosystem I and II reaction centers, plastoquinone pool and the normal complement of LHC II peptides but shows no phosphorylation of LHC II peptides, either in the light or in the presence of the reductant duro- quinol, in the dark. By contrast, phos-

82 © 1991, Elsevier Science Publishers Ltd,(lIK) 0376-5067/91/$02.00