Molecular aspects of the actions of cyclic nucleotides at synapses

Neurochemistry International Vol.2, pp.113-122. Pergamon Press Ltd. 1980. Printed in Great Britain.

MOLECULAR ASPECTS OF THE ACTIONS OF CYCLIC NUCLEOTIDES AT SYNAPSES

Richard Rodnight Department of Biochemistry, Institute of Psychiatry, De Crespigny Park, London, SE5 8AF, U.K.

ABSTRACT The neurotransmitters noradrenaline, dopamine, serotonin and histamine, and the neuromodulatot adenosine, activate adenylate cyclase via postsynaptic receptors. Acetylcholine and some cholinergic agonists by contrast activate guanylate cyclase via muscarinic receptors. The cyclic nucleotides are believed to modify function in the postsynaptic cell by promoting the phosphorylation of key proteins; in this respect the role of cyclic AMP is far more complex than that of cyclic GMP. In this paper evidence for these postsynaptic actions of the cyclic nucleotides is assessed from studies using cell-containing preparations (slices, etc.) from mammalian brain and sympathetic ganglia, and from cell-free preparations of synaptic plasma membranes. Despite the accumulation of extensive data the precise functional roles of cyclic nucleotides in synaptic transmission remain speculative.

KEYWORDS Cyclic AMP; cyclic GMP; protein kinases; brain slices; sympathetic ganglia; synaptic transmission.

synaptic membrane fragments;

INTRODUCTION The tremendous growth in our knowledge of the chemistry of synaptic transmission over the past two decades owes much to the pioneering work of David Nachmansohn and his many coworkers. Nachmansohn was amongst the first to recognize that electrical phenomena in excitable tissues are ultimately based on biochemical processes occurring both within the cell and at the level of the plasma membrane. Such a statement is now taken for granted, but 30 years ago the implications of the concept were only dimly perceived. We now know that the molecular events that accompany synaptic transmission are complex: in mammalian brain at least 12 substances function as central transmitters and each possesses diverse and unique actions on the postsynaptic cell, embracing not only modulation of the properties of the postsynaptic membrane, hut also numerous processes occurring in the cytosol, nucleus and other organelles. It is therefore impossible to generalise: both the electrophysiological phenomena and their molceular basis have to he considered in terms of specific neurotransmitters or neuromodulators. The aspects of this subject to be discussed in this paper concern the molecular consequences of the transmitter-induced synthesis in the postsynaptic cell of the cyclic nucleotides adenosine 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3':5'-cyclic monophosphate (cyclic GMP)° In the CNS, as in peripheral organs cyclic nucleotides function as intracellular second messengers whose synthesis is increased as a result of combination of the extracellular first messengers (hormones or neurotransmitters) with specific cell surface receptors. Only certain neurotransmitters exert all or part of their postsynaptics effects through cyclic nucleotide synthesis: these are noradrenaline, dopamine, serotonin and histamine, which combine with receptors coupled to adenylate cyclase, the enzyme synthesising cyclic AMP; and acetylcholine, acting at muscarinic receptors, which stimulates (directly or I]3

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indirectly) guanylate cyclase to synthesize cyclic G ~ . There are also reports that other transmitter agents, notably noradrenaline and certain amino acids (but only at high concentrations) may stimulate guanylate cyclase in specific situations. The details of the interactions of neurotransmitters with cyclic nucleotide synthesis have been extensively discussed in the literature and will not be considered here; recent reviews include Daly (1977), Kebabian (1977), Nathanson (1977), Greengard (1978) and Rodnight (1979). The molecular consequences of the stimulation of cyclic nucleotide synthesis are, of course, the activation of specific protein kinases and the phosphorylation of acceptor proteins with either enzymic or structural functions. Protein phosphorylation is now recognized as a ubiquitous control mechanism in living organisms for modulating the functional properties of proteins. Both qualitatively and quantitatively the mammalian brain appears to make extensive use of this process for regulating its activity; and although it has proved difficult to define precise functional roles, cyclic AMP- and Ca2+-dependent protein phosphorylation appear to be of particular importance in synaptic transmission. Certainly the range of proteins phosphorylated by bound kinase activity in synaptic membranes is remarkably high compared to other cell regions and non-neuronal tissues I will first consider some of the evidence that points to functional roles for phosphorylated proteins in synaptic transmission, concentrating on acceptors phosphorylated by cyclic AMPdependent kinases, and only mentioning in passing the important and growing subject of CaL+-dependent protein phosphorylation. The study of protein phosphorylation in neural tissues has been approached in both cell-containing and cell free systems in vitro (see Williams and Rodnight, 1977, Greengard, 1978 and Rodnight, 1979 for recent accounts) and in one recent study in vivo (Berman and co-workers, 1980). Historically the "intact tissue" approach is oldest and will therefore be considered first.

EXPERIMENTAL STUDY OF PROTEIN PHOSPHORYLATION IN NEURAL TISSUES Cell-containing Systems Introductory remarks. Observations on protein phosphorylation in brain slices pre-date the cyclic nucleotide era by nearly a decade. This early work, which was initiated by P. J. Heald in 1957, is su~mnarised by Rodnight (1970; 1975): it demonstrated that brief (2-10 s) periods of depolarization increase the turnover of protein hound phosphorylserine groups in respiring slices of guinea cerebral cortex. Attempts to locate the site of the response pointed to the cell membrane fraction, but there was no indication as to whether the phenomenon was related to axonal or synaptic transmission or even to some properties of glial cells. With the discovery of cyclic AMP-dependent protein phosphorylation in the 1960's and the key observation of Kakiuchi Rail and Mcllwain (1969) that depolarization of brain slices with electrical pulses increases the cyclic AMP content of the tissue, the formulation of a definitive hypothesis became possible. This proposed that depolarization induced the release of a neurotransmitter which activated, through receptors on the postsynaptic membrane, the enzyme adenylate cyclase and that the resulting increase in cyclic AMP led to the phosphorylation of a specific protein or proteins located in the postsynaptic membrane. Concurrent research (Weller and Rodnight, 1970) demonstrated that membrane fractions from brain contained bound cyclic AMP-dependent protein kinase activity that transferred phosphate from ATP to membrane-located protein phosphorylserine groups. To investigate this hypothesis Reddingto~ and later Williams, in my laboratory, (see Williams and Rodnight, 1977) studied parameters of protein phosphorylation in slices incubated in the presence of neurotransmitters. Only noradrenaline, dopamine, serotonin and histamine, transmitters capable of activating receptor-coupled adenylate cyclase, also stimulated protein phosphorylation in the tissue. Recent investigations have therefore focussed on identifying the transmitter involved in the increase in protein phosphorylation that follows depolarization of the tissue by electrical pulses. Methodology. The procedures for preparing, incubating and stimulating slices of cerebral cortex used in thest studies are derived from the pioneering studies of Henry Mcllwain in this field (see Mcllwain, 1975); the more specific procedures for studying protein phosphorylation in intact tissues are described by Rodnight, Reddington and Gordon (1975) and Williams and Rodnight (1976). Briefly, slices of guinea pi~ cerebral are preincubated under good metabolic conditions for 30 min, labelled with [3 ~ H3PO 4 and then either electrically depolarized or exposed to low concentrations of neurotransmitters. After fixing the

Actions of Cyclic Nucleotides at Synapses

I15

tissue in perchloric acid the precipitated proteins are centrifugally washed, delipidated and dissolved in alkali. The alkali labile phosphate (representing the protein phosphate) is then extracted as phosphomolybdic acid and its radioactivity determined. Changes in protein phosphorylation occurring as a result of the various treatments are expressed as the radioactivity relative to the radioactivity of the acid soluble phosphate fraction of the tissue. In this way differences in the uptake of 32p by the slices are compensated. An alternative procedure employs small sections of tissue (3 x 0.33 x 0.33 mm) prepared by chopping (Mcllwain, 1975) instead of relatively large slices (e.g. Forn and Greengard, 1978). The tissue is preincubated and labelled in bulk and then transferred in equal aliquots to vessels containing the agents under investigation. No correction for variable uptake of 32p is necessary. This procedure is very satisfactory for studying the action of chemical agents on protein phosphorylation in the tissue, but is unsuitable for studies requiring depolarization by electrical pulses. Sum~narised results. The protein phosphorylation response to electrically-induced depolarization is blocked by tetrodotoxin and is maximal within 2 seconds of applying pulses (WillSams and Rodnight, 1975). Studies with tissues stimulated before and after labelling with 32p indicated that the response involved the net phosphorylation of vacant serine hydroxyl groups rather than increased turnover of protein phosphate; dephosphorylation appeared to occur within about 5 min (Williams and Rodnight, 1975). Cell fractionation studies indicated that the sensitive protein (or proteins) is located in neuronal perikarya and absent from a fraction containing glial cells and neuropil (Williams, Pavlic and Rodnight, 1974a). It therefore appears probable that the response is restricted to axosomatic inhibitory synapses. To identify the transmitter involved a pharmacological approach was used (Williams and Rodnight, 1976). Studies in which slices were depolarized in the presence of added exogenous neurotransmitters showed that whereas the protein phosphorylation responses to serotonin and histamine were additive with that to pulses, the responses to noradrenaline and pulses were non-additive. This pointed to noradrenaline as the mediator in the response to pulses and further work with adrenergic blocking agents showed conclusively that both pulses and the catecholamine act via a B-noradrenergic receptor rather than an s-receptor. Moreover in further cell fractionation studies (Williams, Pavlik and Rodnight, 1974b), the response to noradrenaline was located in the neuronal perikarya fraction, while the response to histamine and serotonin resided in the non-neuronal fraction. Finally, the inhibition of both responses by low concentrations of trifluoperazine, an inhibitor of catecholamine-stimulated adenylate cyclase, provided strong circumstantial evidence for the involvement of cyclic AMP (Williams and Rodnight, 1976). A direct link between the neurotransmitter evoked'activation of adenylate cyclase and protein phosphorylation has only been demonstrated in homogenates of striatal tissue, where the cyclase-enzyme retains sensitivity to dopamine in cell-free preparations. In such preparations of rat caudate dopamine was found to stimulate the incorporation of 32p into endogenous proteins with pharmacological characteristics similar to those observed for the dopamine sensitive adenylate cyclase system of caudate tissue (Hullihan and co-workers, 1979). Moreover, chronic depletion of striatal dopamine by stereotoxic injection of the neurotoxic drug 6-hydroxydopamine significantly increased the protein phosphorylation response to added dopamine. Discussion. The logical extension of the work is clearly the isolation, localization and characterization of the proteins phosphorylated as a result of short-term depolarization with pulses and of exposure to exogenous noradrenalin. While some progress has been made towards this goal there are serious problems (see Rodnight, 1977). Briefly, electrophoretic separation of labelled slice proteins solubilized in sodium dodecyl sulphate (SDS) on polyacrylamide gels has given equivocal results, mainly because of high background labelling. Attempts to fractionate subcellularly the labelled tissue have foundered on the problem of the lability of the protein phosphate incorporated as a result of depolarization or noradrenaline. Thus dephosphorylation occurs within seconds of homogenizing the tissue in sucrose solutions, and is presumably due to the exposure of the labile ester bonds to soluble protein phosphatases on destruction of the tissue structure. The dephosphorylation process can be completely inhibited by including millimolar copper or zinc salts in the homogenizing mddium, but this renders subsequent fractionation impossible. So far no other stabilizing agents have been found. Alternative approaches have been developed by other workers, although these are not directly addressed to the problem discussed here. Forn and Greengard (1978) focussed on the two NCI 2:~6

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major closely related phosphorylated proteins present in synaptic structures, and designated Proteins Ia and Ib by these workers and B3 and ~4 by ourselves (see below). Proteins Ia and Ib were extracted from incubated chopped cerebral tissue (rat) in the presence of Zn 2+ by mild acid treatment. The extracted proteins were then phosphorylated with [32 0 A T P and added cyclic AMP-dependent protein kinase. Under these conditions the amount of phosphate incorporated was a measure of the state of phosphorylation of the proteins in the intact tissue. Under basal conditions the two proteins existed in the de~hosphorylated form, but exposure of the intact tissue to cyclic AMP, 8-bromocyclic AMP or NU-monobutyryl cyclic AMP, or to a phosphodiesterase inhibitor (3-isobutyl-l-methylxanthine) increased their level of phosphorylation. Further depolarization of the tissue with veratridine or high K + was also associated with increased phosphorylation of Proteins Ia and Ib. These latter effects were dependent on external Ca 2+ being present in the medium. It is probable that these effects on protein phosphorylation are related to transmitter release rather than postsynaptic phenomena since the major location for Proteins Ia or Ib is in the limiting membrane of the synaptic vesicles (Bloom and co-workers, 1979; Ueda and co-workers, 1979). In my own laboratory attempts to replicate this work using noradrenaline as the stimulating agent were unsuccessful (H. Holmes and R. Rodnight, unpublished results), and at present it seems unlikely that Proteins Ia and Ib are the acceptors for the protein phosphorylation response to short bursts of electrical pulses and noradrenaline. In a recent report Browning and co-workers (1979) claimed to have detected a change in the phosphate acceptor function of a protein present in incubated hippocampal slices following brief repetitive electrical stimulation (I00 pulses for 1 second) of the Schaffer collateralcommissural system. These are conditions known to induce long-term potentiation of synaptic efficacy. Subsequent analysis of endogenous protein phosphorylation (see below) in a membrane fraction indicated a decrease in the acceptor function of a protein of 40000 daltons in stimulated samples which presumably reflected the induction of an increased phosphorylation state in situ. However, a serious problem in the interpretation of this work is the cellular location of the 40000 dalton phosphoprotein, which in our experience (R. Rodnight and H. Holmes, unpublished data) is a typical constituent of mitochondria rather than synaptic membranes. It is possible that the changes observed in this work on repetitive stimulation reflect calcium entry since the mitochondrial acceptor of 40000 daltons is normally associated with a Ca2+-dependent protein kinase (R. Rodnight and H. Gower, unpublished data). There is no suggestion that cyclic AMP is involved in this response. Subcellular Structures Introductory remarks. By using ~ 2 ~ A T P as donor it is possible to demonstrate in subcellular fractions of cerebral tissues the widespread occurrence of protein kinases and protein phosphatases associated with specific acceptor proteins. These protein phosphorylating systems are particularly abundant in fragments of the synaptic plasma membranes, where bound kinase activity towards integral membrane proteins is regulated in some cases by cyclic AMP and in others by Ca 2+ (with or without calmodulin). Cyclic GMP, on the other hand, has no effect on endogenous protein phosphorylation in membrane fragments, although it does stimulate the activity of a soluble protein kinase in the cerebellum (Takai and coworkers, 1975; Schlichter, Casnellie and Greengard, 1978). The characteristics of membrane located protein phosphorylating systems have been extensively studied in several laboratories over the past few years (e.g. Dunkley, Holmes and Rodnight, 1977; Ueda and Greengard, 1977; Ehrlich and co-workers, 1977; Wiegant and coworkers, 1978;DeBlas and co-workers, 1979; Zwiers and co-workers, 1979; Rubin and coworkers, 1979; Holmes and Rodnight, 1980). The subject has also been treated several times in reviews (Williams and Rodnight, 1977; Greengard, 1978; Routtenberg, 1979; Rodnight, 1979). More specific investigations include attempts to determine the subsynaptic localisation of the protein substrates for kinase action (Kelly and Cotman, 1979; Ng and Matus, 1979; Bloom and co-workers, 1979; Ueda and co-workers, 1979). At present these studies mainly serve to underline the complexity of the protein phosphorylating systems in synaptic membrane fragments, without as yet giving more than occasional clues as to their functional significance. Before considering this complexity some conmlents are needed on methodology since variations in the procedural approach used by different groups has hampered comparison of results. Methodology. The following aspects are considered here: (a) preparation of synaptic membranes; (b) labelling conditions; (c) electrophoretic fractionation and (d) quantitative

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assessment of the radioactivity of the labelled protein. these differ from the published procedures referred to.

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(a) There are a variety of methods available for the preparation of synaptic plasma membrane fractions all of which start with a synaptosomal fraction. We use the procedure of Jones and Matus (1974) which yields a preparation consisting of about 80 per cent of fragments of the pre- and postsynaptic membranes. Further purification is unnecessary (and may lead to loss of phosphorylating activity) because subcellular studies have shown that the contaminating structures (mitochondria, myelin fragments, axolemmal membranes and glial membranes) are (a) relatively low in protein phosphorylating systems and (b) contain virtually no phosphate acceptors co~mnon with those found in synaptic membranes. Many of the phosphorylating systems present in the membrane fractions are labile and most workers only use fresh preparations. However, loss of activity can be largely prevented by storage at a concentration of 5mg of protein/ml in 50 per cent (v/v) glycerol at -25°C. Evidently prolonged storage in the frozen state leads to irreversible change in the molecular structure of the complex of kinase and substrate. (b) Labelling conditions vary considerably between different groups. We use a basic medium consisting of 30mM-TrisHCl, pH 7.4, ImM-MgSO 4 and 2 0 ~ M - ~ 2 p ] A T P with a final protein concentration of 1.25mg/ml. Under these conditions (see Dunkley and co-workers, 1977; Holmes and Rodnight, 1980) approximately 25 per cent of the ATP is consumed (mainly by ATPase action) in the i0 s standard incubation time. The K m for the cyclic AMP-dependent systems is around 12~M (Rodnight, 1975, but see Uno, Ueda and Greengard, 1977). Many workers use a higher concentration of Mg 2+ (e.g. iOmM) and with concentrations of ATP less than 25~M this does result in enhanced phosphorylatingz+ activity. However, wlth° IOOBM-ATP very little difference between I and lOmM-Mg was seen (R. Rodnight, unpublished data). A trial of several buffers indicated none more satisfactory than TrisHCl. HEPES buffer neutralised with Na + or K + is unsatisfactory as both these ions inhibit protein kinase activity: the cyclic A ~ systems are particularly sensitive to Na + with appreciable inhibition being evident at IOmM-concentration. Therefore the use of 5OmM-sodium acetate buffer by several groups is particularly unfortunate. The reaction is typically stopped with the solution of SDS and mercaptoethanol recommended originally by Laemmli (1970) which solubilizes the membrane protein. There is no advantage in heating or boiling the mixture: no perceptible proteolysis occurs within 24 hours of adding the SDS mixture and boiling leads to a loss of resolution of some polypeptides. (c) Conditions used in this laboratory for fractionating the labelled proteins on polyacrylamide gels are given in Holmes and Rodnight (1980). Exponential gradient gels give excellent separation except in the region occupied by proteins in the molecular weight range 40000-60000 daltons. For these proteins iO per cent or 12 per cent single concentration gels are superior. It is now widely recognised that molecular weight determinations based on the comparison of migration rates with those of known standards are at the best only approximate, but it is not always realised that the ionic composition of the incubating medium may affect the rate of migration. For example, certain proteins labelled in the presence of IOmM-Mg 2+ migrate more slowly than when labelled in media containing ImM-Mg 2+ . (d) Most workers use radioautography to assess the labelling of the protein bands. The gel (stained with dye to mark the protein bands) is dried under vacuum and then exposed to "no-screen" X-ray paper (Kodak NS-2T is satisfactory) under conditions of complete darkness. (X-ray papers containing screen are unsatisfactory.) Further details are given in Dunkley and co-workers (1977). The developed film is scanned in a suitable densitometer to give a profile of labelled proteins (see Fig I.). If standard conditions of labelling, exposure and development are used, and providing the peaks are symmetrical and background radiation is reasonably low, the ~ a k heights may be taken as a quantitative measure in arbitrary units of the amount of ~ P incorporated. Moreover it is possible to standardise peak heights against cpm of 32p by applying various volumes of a solution of the latter to a dried gel with a microsyringe to give bands of radioactivity of approximately the same dimensions as the labelled protein bands. By scanning these 'standard' bands the relationship between peak height and cpm can be plotted graphically. From the specific radioactivity of the E32~ ATP used in the labelling reaction a measure of the phosphate incorporated in molar terms can be obtained. This procedure is more convenient and as accurate as the more conventional method of cutting out bands from the gel and counting radioactivity in suitable equipment.

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Summarised Results. As illustrated in Figure I synaptic membrane fragments contain at least 7 proteins (or polypeptides) which are distinctly phosphorylated by endogenous cyclic AMPdependent protein kinase activity and an equal number of acceptors phosphorylated by kinase activity independent of the nucleotide. Addition of Ca 2+ and calmodulin to the incubation medium stimulates the phosphorylation of several of these peaks and reveals others (data not shown; see Schulman and Greengard, 1978; Holmes and Rodnight, 1980). These include the peaks designated ~3' Y6 and an acceptor of 50000 daltons not evident in Figure I. Also Huttner and Greengard (1979) have shown that the acceptors of 79000 and 86000 (83 and B4or Protein I) are associated with a Ca 2+ plus calmodulin-dependent protein kinase that transfers phosphate to sites distinct from those phosphorylated by cyclic AMP-dependent protein kiaase activity. (Peak designation in this paper follows our previous practice of dividing the profile into 4 regions - O, ~, 8, and y - and numbering the peaks within these regions.) c0~o

oJ~. . . .

Qo~-

o Iol 3b

5

3

u~u~

Mol Wt x 10-3

I!, p 3

23Yt5 *- cAMP

Fig. I. Densitometric scan of a radioautograph of 32p-labelled synaptic membrane fragments made from rat cerebral cortex and fractionated on an exponential gradient polyacrylamide gel (6-14%). The blocked out areas indicate the stimulation given by the inclusion of 50~M-cyclic AMP in the incubation medium. The medium also contained ImM-EGTA, added to chelate endogenous Ca 2+. This was necessary because Ca 2+ inhibits cyclic AMPdependent protein kinase activity. However, endogenous Ca 2+ also activates the Ca2+-dependent kinase that phosphorylates the 47000 dalton aeceptor; therefore EGTA inhibits the phosphorylation of this acceptor and the cross hatched area above this peak indicates the extra phosphorylation of this acceptor observed in the absence of EGTA. ATP concn, was 2OpM.

All of these major phosphate acceptors are typical of synaptic structures.except for Y3 . (54000 daltons) and possibility 03b (195000) which appears to correspond zn molecular wezght to a cytosolic phosphoprotein. Thus the major acceptors are absent in mitochondria, axolemmal membranes, myelin fragments and glial membranes. However, it is important to emphasise that the pattern of 32p-labelled protein visualised on radioautographs depends upon many variables. These include (a) the concentration of ATP used for labelling, (b) the incubation time and (c) the exposure time for radioautography. Variable (a) reflects differing affinities of different kinases for ATP; thus for the major cyclic AMP-dependent systems transferring to acceptors of 79000 and 86000 the Km for ATP may be as low as I~M (Uno and co-workers, 1977). Our own estimates for the cyclic AMP systems are rather higher (about 12~M), but we agree that the use of an ATP concentration of less than 5~M greatly increases the relative labelling of ~3 and B4 (Protein I). In contrast the Ca2+ and Ca 2+ plus calmodulin-dependent kinases have Km values greater than 50~M-ATP (R.Rodnight and H°Gower, unpublished data) and the use of relatively high concentrations of ATP proportionally increases the labelling of corresponding acceptors. Incubation time is important partly because after exhaustion of the ATP (mainly by ATPase action) variable dephosphorylation occurs and partly because the acceptors have different capacities. Finally prolonged exposure times reveal many minor phosphorylated proteins which are invisible under standard conditions.

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Preparations of synaptic membrane fragments made from any region of the mammalian brain are It is thereclearly derived from synapses utilising a range of different neurotransmitters. fore relevant to ask whether the observed pattern of phosphorylated proteins in cerebral corSome approach to this question may be made by comparing the tex is typical of all synapses. phosphorylation pattern between membrane preparations made from different regions of the brain, in the expectation that variations in transmitter specific neuronal pathways will be As an illustration of this approach Figure reflected in different phosphorylation patterns. 2 compares the pattern of high molecular weight phosphorylated proteins in synaptic membrane preparations made from cerebral cortex and cerebellum.

Cerebral cortex

Fig. 2. Pattern of phosphorylated proteins in the range 79000 - 200000 daltons in synaptic membrane fragments from cerebral cortex and cerebellum of the rat. Labelling conditions were as given in Fig. 1, the blocked out areasindicating the extent of stimulation by cyclic AMP. (R.Rodnight and H.Holmes, unpublished data.)

The evident difference between these patterns could indicate either that the cerebellum lacks synaptic plasma membranes containing acceptors of 138000, 162000 and 198000 daltons or that they do not accept phosphate in the assay system. Further work along these lines, particularly if combined with lesioning techniques designed to destroy specific pathways, would be justified. Discussion. Clues as to the physiological function of the phosphorylated proteins typical of synaptic structures will come from knowledge of their precise localisation in the synaptic complex. As mentioned above remarkable progress towards this end has been achieved for the 79000 and 86000 dalton substrates by Greengard'sgroup. Both these proteins and their associated kinase have been extracted from brain, purified and characterised (Ueda and Greengard, 1977; Uno and co-workers, 1977); they are basic, elongated molecules with a high content of glycine and proline, suggesting that the polymeric unit is a structural protein similar to collagen. By careful subfractionation of synaptosomal components (Ueda and co-workers, 1979) and by immunocytochemical methods (Bloom and co-workers, 1979) this acceptor protein has been localised in two sites within synapses: in or on the limiting membrane of synaptic vesicles and to a lesser extent in the postsynaptic membrane, particularly within submembranous material known as the 'postsynaptic densities'. The immunocytochemical data are particularly interesting in that they show that the distribution of the protein is restricted to certain synapses, although no clues as to which neurotransmitters these synapses utilise has yet emerged. In further work this group have also shown that Protein I also occurs in specific synapses in the peripheral nervous system (DeCamilli and co-workers, 1979). Presumably the high content of this acceptor in synaptic plamsa membrane fragments reflects its presence in the postsynaptic membrane and to some extent contamination of the preparation by synaptic vesicles.

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Little is known of the location of the other acceptor proteins illustrated in Fig.l, except that their concentration tends to be higher in preparations of junctional complexes (Ng and Matus, 1979; DeBlas and co-workers, 1979; Kelly and co-workers, 1979). Since these preparations are representative of the pre- and postsynaptic junction no conclusions are possible about specific functions.

CYCLIC NUCLEOTIDES, PROTEIN PHOSPHORYLATION AND MEMBRANE POLARISATION A variety of evidence has been adduced to support the hypothesis that cyclic nucleotides mediate the polarisation state of the postsynaptic membrane (see Greengard, 1978; Rodnight, 1979). Implicit in this hypothesis is the suggestion that cyclic nucleotides generated by transmitter-evoked stimulation of cyclases, activate contiguous protein kinases which regulate by phosphorylation membrane protein(s) concerned in the control of ion permeability or transport. It is highly unlikely that such processes could account for the very rapid bioelectrogenesis that follows the combination of transmitters such as glutamate and yaminobutyric acid with their receptors, but the potential changes induced by some amine transmitters have a longer latency and could conceivably arise from an enzymic process. Evidence for this concept has been sought in iontophoretic studies on central neurones and in sympathetic ganglia. The evidence is conflicting and has recently tended to discount earlier claims (e.g. Libet, 1979; Brown, Caufield and Kirby, 1979). In many central neurones iontophorectically applied cyclic AMP produces a depression of cell firing which mimicks the depression induced by noradrenaline (Phillis, 1977; Stone and Taylor, 1977). Most of the depressant action of cyclic AMP, however, is probably due to activation of adenosine receptors, although a small component may by generated by cyclic AMP transported intracellularly (Stone and Taylor, 1977). In contrast, iontophorectically applied cyclic GMP usually increases cell firing in a manner similar to the action of acetylcholine at muscarinic receptors (Stone and Taylor, 1977). Here also there are problems, however; notably the failure of phosphodiesterase inhibitors to potentiate the muscarinic effects of acetylcholine. The hypothesis has also been intensively investigated in sympathetic ganglia, particularly by Greengard (see Greengard, 1978 for review). It was originally proposed that the slow inhibitory postsynaptic potential (s-IPSP) generated in response to preganglionic stimulation was mediated through the release of dopamine from interneurones and the subsequent stimulation of a dopamine-senstive adenylate cyclase in the postganglionic cell. However, while it is clear that the s-IPSP is mediated by dopamine and that in some species at least dopamine stimulates cyclic AMP production in ganglia, there is now compelling evidence that these two events are separately mediated (Libet, 1979; Brown and co-workers, 1979). However, dopamine does have another action in sympathetic ganglia that appears to be mediated by cyclic AMP (Libet, 1979). Thus Libet has shown that exposure of ganglia to dopamine or cyclic AMP potentiates over a period of hours the generation of the slow excitatory postsynaptic potential in response to cholinergic agonists. Morever, this process is disrupted by cyclic GMP. It seems inherently more likely that such longterm modulations by neurotransmitters will be found to involve cyclic nucleotides and protein phosphorylation than the more immediate changes in cell polarisation.

REFERENCES Berman, R.F., J.P.Hullihan, W.J. Kinnier and J.E.Wilson (1980). Phosphorylation of synaptic membranes. J.Neurochem., 34, 431-437. Bloom, F.E., T.Ueda, E.Battenberg and PoGreengard (1979). Immunocytochemical localisation in synapses of Protein I, an endogenous substrate for protein kinases in mammalian brain. Proc, Natn. Acad. Sci. (USA), 76, 5982-5986. Brown, D.A., M.P.Caulfield and P.J~-Kirby (1979). Relation between catecholamine-induced cyclic AMP changes and hyperpolarisation in isolated rat sympathetic ganglia. J.Physiol., 290, 441-451. Browning, M., T.Dunwiddie, W.Bennett, W.Gispen and G.Lynch (1979). Synaptic phosphoproteins: specific changes after repetitive stimulation of the hippocampal slice. Science, 203, 60-62. Daly, J.W. (1977). The formation and degradation and function of cyclic nucleotides in the nervous system. Int. Rev. Neurobiol., 20, 105-168. DeBlas, A.L.D., Y-J.Wang, R.Sorensen and H.R.Mahler (1979). Protein phosphorylation in synaptic membranes regulated by adenosine 3',5'-momophosphate; regional and subcellular

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