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Cyclic ADP-ribose, the ryanodine receptor and Ca2+ release
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26 Neer, M., Stovik, D. M., Daly, M., Potts, J. and Nussbaum,
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S. R. (1993) Osteoporosis ht. 3 (Suppl. l), S204-S205 Ox&d, H., Ejersted, ?., Andreassen, T. T., Tarring, 0. and Nilsson, M. H. (1993) Am. 1. Med. 95,62S-@3S Reeve,‘J. et iI. (1993) Osteopwosis ht. 3 (Suppl. I), S199-S203 Riond, J-L. (1993) Clin. Sci. 85,223-228 Rixon, R. H. et al. (1994) J Bone Miner Res. 9,1179-1189 Shen, V., Dempster, D. W., Birchman, R., Xu, R. and Lindsay, R. (1993) J. Clin. Invest. 91,2479-2487 Sogaard, C. H., Wronski, T. J., McOskar, J. E. and Mosekilde, L. (1994) Endocrinology 134,65@-657 Strein, K. (1994) in Bone Diseases and Osteoporosis: Current Treatments andFutureOpportunities(Russell,R G.,ed.),p. 131,IBCTechnicalServices Watson, P. et al. (1995) Bone 16,357-365 Whitfield, J. F., Morley, P., Ross, V., Isaacs, R. J. and Rixon, R. H. (1995) C&if. Tissue ht. 56,227~231 Whitfield, J. F. et al. C&if: Tissue ht. (in press) Wronski, T. J., Yen, C-F., Qi, H. and Dann, L. M. (1993) Endocrinology
Cyclic ADP-ribose,the ryanodine receptor and Ca2+release Rebecca Sitsapesan, Alan J. Williams
Stephen J. McGarry
In a variety of vertebrate ryanodine-sensitive
and invertebrate
tissues the
Ca*+ channel is the pathway for
Ca*+ release from intracellular for activation
and
stores. The mechanism
of the ryanodine receptor-channel
complex appears to depend both on the ryanodine receptor isoform and the cell type. In addition, a complex combination
of endogenous
compounds
channel gating. In this article,
regulates
Rebecca
Sitsapesan,
Williams
review the mechanisms
ADP-ribose
Stephen McGarry
(cADPR)-induced
by one of the recognized
ryanodine receptor-Ca*+
R. serapesan, Lectursr, S.
J. MeGany,
Postdoctoral Fellow, and
A. J. Williams, Professor, Cardiac Medlcme, National Heart and Lung Institute. lmperlal College. Dovehouse Street. London. UK SW3 6LY
3 8 6
and Alan
involved in cyclic
Ca*+ release and discuss
the likelihood that cADPR-activated mediated
intracellular
Ca*+ release is isoforms of the
channel complex.
The ryanodine receptor-Caz+ channel complex is a homotetramer, in which each monomer has a molecular weight of approximately 550 kDa (Ref. 1). Following the cloning and sequencing of the cDNA of the mammalian skeletal muscle ryanodine receptor (RY, receptor)z, two other mammalian isoforms of the protein have been identified and their amino acid sequence deduced from their cloned cDNA. The ryanodine RY, receptor, which shows 66% identity with the RY, receptor, is expressed in cardiac muscle and brain, while the RY, receptor, which shows 67% identity with the RY, receptor, is expressed in the brain and at low levels in many other tissueslJ. The sarcoplasmic reticulum (SR) membranes of bird, fish and amphibian skeletal muscles contain two isoforms of ryanodine receptor 1.45;the (Yisoform of the bullfrog receptor
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132,82%831 38 Yamamoto, N. et al. (1993) Bone Miner. 23,333-342 (1994) Guidelines for 39 United States Food and Drug Administration preclinical and clinical evaluation ofagents used in the prevention or treatment ofpostmenopausal osteoporosis 40 Kanis, J. A. (1994) Osteoporosis, Blackwell 41 Spiegel, A. M. and Weinstein, L. S. (1994) in Thp Paruthyraids, (Bilezekian, J. P. and Levine, M. A., eds), p. 231, Raven Press 42 Jouishomme, H. et al. (1992) Endocrinology 130,53-&l 43 Jouishomme, H. et al. (1994) 1. Bone Miner. Res. 9,943-949 44 Neugebauer, W., Barbier, J-R., Sung, W. L., Whitfield, J. F. and Willick, G. E. (1995) Biochemistry X,8835-8842 45 Somjen, D., Schluter, K-D., W&gender, E., Mayer, H. and Kaye, A. M. (1991) Biochem. 1.277,86%868 46 Canali‘s, E.,‘Pash, J. and Varghese, S. (1993) Crit. Rev. Euk. Gene Expression 3, 155-166 47 Rosen, C., Donahue, L. R. and Hunter, S. J. (1994) Proc. Sot. Exp. Biol. Med. 206,83-102
shows 80% identity with the RY, receptor, while the p isoform shows 86% identity with the RY, receptor’. Screening of an insect genomic library has identified an analogue of the mammalian ryanodine receptor gene that displays 45% identity with the RY, receptolh. Ryanodine disrupts Cal+ handling in cells expressing well-defined ryanodine receptor isoforms (such as mammalian skeletal and cardiac muscle7) and is known to modify Ca2+ handling in other cell types, in which information on the identity of the isoform of ryanodine receptor or the levels of expression (or both) of the receptor is either less well-defined or absent. In the latter case, the pharmacological characterization of the putative ryanodine receptor usually reflects measurements of alterations in ryanodine-sensitive Ca2+ release in intact cells or cell homogenates. Detailed characterization of the function of the ryanodine-sensitive Ca2+ channel has largely been carried out using RY, (from mammalian skeletal muscle) and RY, receptors (from mammalian cardiac muscle). Studies involving the measurement of [sH]ryanodine binding to, and Ca2+ efflux from, isolated SR membrane vesicles together with monitoring parameters of singlechannel gating and conduction following the incorporation of either intact SR membrane vesicles or isolated receptor proteins into phospholipid bilayers, have identified a number of functional characteristics that are shared by these two isofonns and also some significant differences.
Functional consequences of ligand interaction with RY, and RY, receptors A diverse array of ligands are known to modulate the function of ryanodine-sensitive Ca2+ channel@. The vast majority of these agents act at sites located on the cytosolic side of the Ca2+ channel (Fig. 1). To date, annexin VI (Ref. 9) and Ca2+ (Refs 10,ll) are the only agents thought to act at the luminal side of the channel. Although the mechanisms governing activation of RY, and RY, receptors in situ are thought to be different (mechanical coupling of RY, receptors in skeletal muscle, and Ca2+induced Ca2+release for RY, receptors in cardiac muscle), following incorporation of the receptor into planar bilayers both isoforms are regulated by cytosolic Ca2+.All ligands
0
1995, Elsevier Saence Ltd
R either will not activate channels in the absence of Ca2+, or require Ca2+ for maximum effect, suggesting that Caz+ is the ‘primary activating ligand’. The regulation of channel gating by cytosolic Ca2+ differs for RY, and RY, receptorsn. With RY, receptors, a bell-shaped Ca2+ concentration-open probability (I’,) relationship is observed; with RY, receptors, high concentrations of Caz+ tend to activate rather than inactivate. Physiological concentrations of Mg*+ (0.5-I .OmM) markedly reduce the I’, of RY, and RY, receptor-sensitive channels, and it has been suggested that this action may result from competition for cytosolic Cal+ binding sites by Mg2+ (Ref. 13). Many ‘secondary’ ligands activate both RY, and RY, receptors; however, there are often subtle differences in action, which may reflect differences in the receptor sites or, if the compound acts by a Ca2+-dependent mechanism, differences in the Ca2+ sensitivities of the isoforms. The sites which have been subjected to detailed examination, and which are most relevant to the interpretation of the action of cyclic ADP-ribose (cADPR) on ryanodine receptors, are:
Ryanodine The interaction of [jH]ryanodine with isolated membrane vesicles containing RY, and RY, receptors has identified a high-affinity binding site and possibly other low-affinity sites 1. Depending upon the experimental concentration of ryanodine used, it was found to either stimulate or inhibit Car+ efflux from isolated SR membrane vesicle+. These effects are consistent with singlechannel experiments where micromolar concentrations of ryanodine ‘lock the channel into a reduced-conductance, high-P, state (Fig. 2), and millimolar concentrations lead to irreversible channel closurers.
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Fig.1.Modulators of the gating of the ryanodtne receptor-channel complex. Cytosolic CaZ+ IS the ‘primary activating ligand’. being effective in the absence of ail other ligands. Activation of the channel by adenine nucleotides and cafferne is regulated by both cytosolic and luminal CaZ+ Ligands acting at the adenine nucleotide site include ATP, adenosine. cyclic ADP-ribose (cADPR). ADPribose (ADPR) and P-nicotinamide-adenine dinucleotide (DNAD). Depending upon the concentration applied, ryanodme will either increase or decrease open probabrlity.
nels in bilayers by a direct action22. In contrast, phosphorylation of the RY, receptor by calmodulin-dependent protein kinase has been suggested to increase I’, (Ref. 23).
Isofornvspecific ligands Although many ligands exert similar effects on RY, and RY, receptors, some agents may be selective for certain isoforms. For example, cardiac glycosides activate RY, receptors at low nanomolar concentrations (an action that may contribute to their therapeutic effecP), but have no effect on RY, receptors. In contrast, perchlorate ions appear to activate only RY1receptors25.
ATP ATP increases the release of Ca2+ from cardiac and skeletal SR vesicles, and increases the I’, of RY, and RY, receptor-sensitive channels at physiological concentrationsPJh.17. Additionally, it has been shown that adenosine and its related analogues, 2-chloroadenosine and N-ethylcarboxamidoadenosine (NECA), activate RY, receptors by binding to ATE activation sites?.
Cafiine Caffeine activates both RY,and RY, receptorsr9J’J. In the presence of cytosolic Ca*+, micromolar concentrations of caffeine sensitize the channel to Ca2+ (Ref. 21). At millimolar concentrations, caffeine can activate the RY, receptor by a Ca2+-independent mechanism. Recently, it was shown that the sites activated by caffeine on RY, receptors are distinct from those activated by ATE (Ref. 18).
Calmodulin The effects of calmodulin on ryanodine receptor channel gating are complex. Calmodulin has been shown to inhibit Ca2+ release from membrane vesicles containing RY, and RY, receptors, and decrease the I’, of these chan-
cADPR-induced Ca2+ release in sea urchin eggs and other tissues At present, there is much interest in the possibility that cADPR may be an endogenous regulator of one or more isoforms of the ryanodine receptor. This interest stems from experiments on sea urchin eggs in which it was demonstrated that cADPR could release Ca2+ from intracellular stores via an inositol 1,4,5-trisphosphate [Ins(l,4,5)P,]-insensitive mechanism2627. Both cADI% and the enzymes responsible for the metabolism of cADPR have been detected in a variety of cell types28.29,although the mechanisms involved in regulating the intracellular concentration of cADPR have yet to be established. In addition to cADPR, ryanodine and caffeine were shown to induce Ca2+ release in sea urchin egg homogenates and potentiate the effect of cADPR (Refs 27‘30). Ca*+ release in response to cADPR exhibited desensitization to subsequent applications of cADPR, caffeine and ryanodine but not to Ins(1,4,5)P,. Based on these results it was suggested that cADPR caused release of intracellular Ca2+ in sea urchin eggs by the activation of ryanodine receptorsr7. By using [32P]8-amino-cADPR as a photoaffinity
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200 ms -
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C
Fig. 2. Ryanodine modification of the RY, receptor. A single channel is incorporated into the bilayer; CaZ+ is the permeant ion (luminal [Caz+] = 50mM) and current flows through the channel from the luminal to the cytosolic side of the bilayer. The holding potential was OmV. The dotted lines indicate the closed (C), n/anodine-modified (M) and the fully open channel levels (0). The channel was activated by ~O~.LMcytosolic Ca2+ and 1 )LM cyclic ADP-ribose. The arrow indicates the time at which irreversible ryanodine modification occurs; 1 FM ryanodine was added to the solution bathing the cytosolic side of the bilayer approximately twominutes before modification occurred. Figure modified from Ref. 45.
probe, 140 and 100kDa proteins were identified as specific binding proteins for cADPR in homogenates of sea urchin eggssi. Walseth and co-workers suggested that these proteins may be novel ryanodine receptor-channel complexes or may represent separate proteins that interact with a ryanodine receptor after binding cADPR (Ref. 31). Another possibility is that the proteins are proteolytic fragments of a ryanodine receptors*. There is evidence that cADPR causes release of intracellular Ca*+ in other cell types, including neuronal cells33,34and pancreatic acinar cells35. cADPR also increases Ca*+release from microsomal membrane preparations of hearts, brainsQ7, pancreatic l+cellsB and pituitary cellss9.In contrast, Mesziros and colleagues found no significant effect of cADPR on Caz+release from isolated skeletal SR (Ref. 36), and although Morrissette and coworkers demonstrated a cADPR-induced Ca*+ release from skeletal SR vesiclesa, they concluded that this was mediated by a non-ryanodine receptor mechanism. The bulk of the evidence discussed above indicates that cADPR releases Ca*+ from ryanodine-sensitive Ca*+ stores. Does this reflect the direct interaction of cADPR with RY,, RY, or RY, receptors; does it involve activation of the channel via an accessory protein; or does cADPR induce the release of Ca*+via a novel ryanodine-sensitive channel (with or without the involvement of an accessory protein)? While most of these possibilities are yet to be investigated, the direct actions of cADPR on RY, and RY, receptors have been studied.
Interaction of cADPR with RY, and RY, receptors Effects of cADPR on [3HI yanodine binding [aH]Ryanodine binds to RY, and RY, receptors when the channel proteins are in open or conducting states, and therefore agents that increase the I’, of these channels generally increase ryanodine binding, and agents that decrease I’, decrease ryanodine bindir@i,Q. Meszaros and colleagues reported a significant increase in [3H]ryanodine binding to cardiac SR at a free Ca*+concentration of
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0.1 pM, but they observed no effect at higher Ca*+concentrations. In skeletal SR, they observed a significant decrease in [sH]ryanodine binding at free Ca*+concentrations greater than 10 p,M, indicating that cADPR also binds to membrane vesicles containing RY, receptors and may be expected to modulate I’,. Fruen and co-workers, however, found no effect of cADPR on [sH]ryanodine binding to cardiac or skeletal SR membrane vesicles over a wide range of concentrations of free Ca*+(Ref.43). These authors concluded that cADPR has no direct effects on the functional activity of either RY, or RY, receptors. Overall, the ryanodine binding studies suggest that if cADPR does activate either RY, or RY, receptors, the effect may not be very marked. Unfortunately, there are no reports of [sH]ryanodine binding to sea urchin egg membranes. Such studies would provide important information about the properties of putative ryanodine receptors present in sea urchin eggs, and allow a comparison with the binding characteristics of RY, and RY, receptors. Effects of cADPR on single-channel gating of RY, and RY, receptors The direct effects of cADPR on RY, and RY, receptors can be observed after incorporation of the channels into planar phospholipid bilayers under voltage-clamp conditions. Unfortunately, as for the ryanodine binding studies, the reported effects of cADPR on channel gating are not entirely consistent. Neither Morrissette and colleagues40nor Fruen and coworkers43 observed an effect of cADPR on the I’, of RY, receptor-sensitive channel incorporated into artificial bilayers. However, it has since been demonstrated that cADPR does activate these channels, but that the magnitude of the effect is dependent on the luminal Ca*+concentrations; at low luminal Ca*+concentrations (<40 FM), no significant increase in P, occm+. Therefore, one explanation for the negative results could be the low luminal Ca*+concentrations (l-7 FM) usedmT43. The precursor to
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200ms Fig. 3. Comparison of the effects of cyclic ADP-ribose (cADPR) and ATP on single-channel gating. The dotted lines indicate the fully open channel level’ and the arrows indicate the closed channel level. A single channel is incorporated into the bilayer; CaZ+ is the permeant ion and current flows through the than nel from the luminal to the cytosolic side of the bilayer. The holding potential was0 mV.a: In the presence of 10 JLM cytosolic Ca*+ (left panel), 1 pM cADPR (a concentration higher than measured intracellularly) (right panel) increases the open probability of the channel (P,) from 0.021 (left) to 0.061 (right). b: In the presence of 10 PM cytosolic Cast (left panel], 2 mM ATP (physiological levels in muscle cells = 520 mM) (right panel) increases P, from 0.034 (left) to 0.927 (right). Figure modified from Ref. 45.
cADPR, p-nicotinamide-adenine dinucleotide @NAD+), and the metabolite ADP-ribose (ADPR), were also shown to activate the RY, receptor-channel complex at micromolar concentrations@. Mesziros and colleagues detected an increase in the I’, of RY, receptor-sensitive channel only at a sub-activating cytosolic Caz+ concentration (lOn~)x, while others have demonstrated that cADPR has no effect on I’, over a range of cytosolic Ca2+ concentrations@. In contrast, it has recently been demonstrated that cADPR increases I’, in a Ca2+-dependent mannel”‘5; significant increases in I’, were obtained only in the presence of activating cytosolic Ca2+ concentrations. The discrepancies in the results from different laboratories may result either from species differences or from variations in the luminal Ca2+ concentrations for, as is the case with the RY, receptors, the gating of the RY, receptor-channel complex appears to be regulated by luminal Ca2+ (Ref. 10). The concentrations of cADPR required to activate the RY, receptor-channel complex (21 p~)36,45 are much higher than the tissue levels measured to date (approximately 200-68Onr@s. Even these high concentrations of cADPR are not as effective at activating RY, receptors as physiological concentrations of ATP (Fig. 3). There is evidence that cADPR and ATP may compete for the same binding sites on RY, receptors45, and it has also been demonstrated that in the presence of physiological levels
of ATP and Mg2+, cADPR is unable to activate the RY, receptorAanne1 complex&. Moreover, as for RY, receptors, pNAD+ and ADPR cause significant increases in I’,,(Ref. 45). The mechanisms involved in the activation of RY, and RY, receptors by cADPR appear to be very simila+,4s. The results indicate that while cADPR may be a pharmacological ligand for the adenine nucleotide sites on these ryanodine receptors, it is unlikely to act as a physiological regulator of these ryanodine receptor isoforms by binding directly to the channels.
Pharmacological profiles of cADPR-induced Ca2+ release and ryanodine receptor-channel gating On balance, the data presented appear to indicate that RY, and RY, receptors do not contain distinct, high-affinity, binding sites for cADPR; rather cADPR, PNAD+ and ADPR can increase channel opening via an interaction with the adenine nucleotide sites on the protein. This is clearly at odds with the action of cADPR in sea urchin eggs. However, this is not the only discrepancy between the characteristics of cADPR-induced Ca2+ release in sea urchin eggs and the established properties of RY, and R.Y, receptors. Points of particular interest include: l Single-channel studies with RY, or RY, receptors indicate that in the presence of activating cytosolic Caz+ and in the absence of other ligands, cADPR is ineffective at concentrations less than 1 FM (Refs 44, 45). In contrast,
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20 no cADPR can activate Ca2+ release in sea urchin eggs, and binding studies yield a K, = 171~ (Refs 27, 46). l Single-channel studies with RY, or RY, receptors”+” have demonstrated that PNAD+ and ADPR can increase channel I’@whereas in homogenates of sea urchin eggs ADPR has no effect and PNAD+ releases intracellular Ca2+ only after a lag-time of approximately four minutes (presumably after conversion of PNAD+ to cADPR)26. l Experiments reported by Graeff and co-workers indicate that ATP does not activate Ca2+ release in homogenates of sea urchin eggs47. In contrast, Ca2+-flux measurements in membrane vesicles containing RY, and RY, receptors demonstrate significant ATP-induced Caz+ release, and ATP can fully activate RY, and RY, receptorsensitive channels incorporated into bilayersisJ6J7 (Fig. 3). l Calmodulin decreases the I’, of RY, and RY, receptorgated channels, whereas calmodulin potentiates the effect of cADPR in homogenates of sea urchin eggs in a similar manner to caffeine and Sr2+ (Refs 22, 48). l In membrane preparations containing RY, and RY, receptors, Ca2+ is effective at releasing Ca2+, and can activate these channels reconstituted into bilayers in the absence of other activating ligands. However, in sea urchin eggs, Ca2+-induced Ca2+release is more difficult to evoke27,49.In homogenates of sea urchin eggs, it appears that Ca*+ alone is not a very effective activator of intracellular Caz+ release27 and requires a second ligand (such as caffeine) to cause marked release. l The literature contains no reports of the detection of RY,, RY, or RY, receptors in sea urchin eggs. However, a 380 kDa protein has been detected in the cortex of sea urchin eggs using an antibody raised to the RY, receptorw.
this complex in turn binds and activates the ryanodine receptor31. If the accessory binding protein does not incorporate into a planar bilayer along with the ryanodine receptor, this mechanism could explain why a specific activation of the ryanodine receptor is not seen in singlechannel studies using nanomolar concentrations of cADPR. However, it is still necessary to suggest that, in addition to increasing I’, the binding of the cADPRaccessory protein complex brings about a very marked alteration of the pharmacological profile of the ryanodine receptor-channel complex (ATP becomes ineffective, calmodulin increases rather than decreases I’,). The addition of purified sea urchin accessory proteins to reconstituted RY, and RY, receptor-channel complexes would be an important experiment. (3) cADPR induces Caz+release through a specific interaction with a ryanodine receptor that is not a RY, or RY, receptor; again this may involve the binding of cADPR to an accessory binding protein. As outlined, there is some variation in the degree of identity amongst the isoforms of ryanodine receptor that have been examined. Within the group of receptors on which detailed information on channel function exists, there are differences between isoforms, sometimes in terms of the degree of activation pro duced by a given concentration of ligand, and in some cases ligands are specific for a particular isoform. The identification of the isoform(s) of ryanodine recep tor expressed in sea urchin eggs and the characterization of the properties of the receptor and ion channel may help to resolve these uncertainties.
Concluding remarks cADPR induces the release of Ca2+ from ryanodinesensitive, Ins(l,4,5)P,-insensitive, intracellular stores in sea urchin eggs and other cell types, and therefore it is not unreasonable to suggest that cADPR-induced release of Ca2+might be mediated by a ryanodine-sensitive channel. The direct demonstration of the activation of RY, receptors by cADPR (Ref. 36), and the initial experiments suggesting a lack of effect of cADPR on RY, receptors27,40, have led to general acceptance of the proposal that cADPR-induced Ca2+ release is mediated by the RY, receptor. However, it is clear from the arguments discussed that there are significant discrepancies between the pharmacological profile of cADPR-induced Ca2+ release and that of the RY, receptor. There are a number of hypothetical mechanisms for cADPR-induced Ca2+ release via a ryanodine-sensitive channel. (1) cADPR binds to a distinct site on the ryanodine receptor-channel complex and increases I’,. The singlechannel data described indicate that this mechanism is unlikely to be involved if the receptor in question is either the RY, or RY, receptor. (2) cADPR binds to a separate receptor protein (the 100 or 140 kDa proteins identified in sea urchin egg), and
M. and Sorrentino, V. (1995) J Cell Biol. 128,893-904 Olivares, E. B. et al. (1991) Biophys. J. 59,1153-1163 O’Brien, J., Valdivia, H. H. and Block, B. A. (1995) Biophys. 1. 68,471-482 Takeshima, H. et al. (1994) FEBS Mt. 337,81-87 Sutko, J. L., Ito, K. and Kenyon, J. L. (1985) FASEB J. 44,2984-2988 Coronado, R., Morrissette, J., Sukhareva, M. and Vaughan, D. M. (1994) Am. J. Physiol. 266, C1485-Cl504 Diaz-Munoz, M., Hamilton, S. L., Kaetzel, M. A., Hazarika, I? and Dedman, J. R. (1990) J. BioI. Chem. 265,15894-15899 Sitsapesan, R. and Williams, A. J. (1994) J. Membr. Biol. 137, 215-226 Sitsapesan, R. and Williams, A. J. (1995) J. Membr. Biol. 146, 133-144 Chu, A., Fill, M., Stefani, E. and Entman, M. L. (1993) 1. Membr. Biol. 135,49-59 Smith, J. S., Coronado, R. and Meissner, G. (1986) J. Gen. Physiol. 88,573-588 Meissner, G. (1986) J. Biol. Chem. 261,6300-6306 Lai, F. A., Misra, M., Xu, I., Smith, H. A. and Meissner, G. (1989) J. Biof. Chem. 264,1677&16785 Meissner, G. and Henderson, J. S. (1987) J. Biol. Chem. 262, 30653073 Rousseau, E., Smith, J. S., Henderson, J. S. and Meissner, G. (1986) Biophys. J. 50,1009-1014 McGarry, S. J. and Williams, A. J. (1994) J. Membr. Biol. 137,169-177 Rousseau, E., LaDine, J., Liu, Q-Y. and Meissner, G. (1988) Arch Biochem. Biuphys. 267,75-86 Rousseau, E. and Meissner, G. (1989) Am. J. Physiol. 256, H328-H333 Sitsapesan, R. and Williams, A. J. (1990) 1. Physiol. 423,425-439 Smith, J. S., Rousseau, E. and Meissner, G. (1989) Circ. Res. 61, 352-359
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Selected references 1 Ogawa, Y. (1994) Crit. Rev. Biochem. Mol. Biol. 29,229-274 2 Take&ma, H. et al. (1989) Nature 339,439-445 3 Giannini, G., Conti, A., Mammarella, S., Scrobogna, 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
R 23 Witcher, D. R., Kovacs, R. J., Schulman, H., Cefali, D. C. and Jones, L. R. (1991)J. Biol. Chem. 266,11144-11152 24 McGarry, S. J. and Williams, A. J. (1993) Br. J. Pharmacol. 108, 1043-1050 25 Fruen, B. R., Mickelson, J. R., Roghair, T. J., Cheng, H-L. and Louis, C. F. (1994) Am. J. Physiol. 266, C1729-Cl735 26 Clapper, D. L., Walseth, T. F., Dargie, l’. J. and Lee, H. C. (1987) J. Biol. Chem. 262,9561-9568 27 Galione. A.. Lee. H. C. and Busa. W. B. (1991) Science 253.1143-1146 28 Walseth, T. F., Aarhus, R., Zeleznikar, R. J., Jr and Lee, H. C. (1991) Biochim. Biophys. Acta 1094,1X3-120 29 Lee, H. C., Galione, A. and Walseth, T. F. (1994) Vitam. Horm. 48,199-257 30 Buck, W. R., Rakow, T. L. and Shen, S. S. (1992) Exp. Cell Res. 202,59-66 31 Walseth, T. F., Aarhus, R., Kerr, I. A. and Lee, H. C. (1993) J. Biol Chem. 268,26686-26691 32 Galione, A. and White, A. (1994) Trends Cell Biol. 4,431-436 33 Currie, K. I’. M., Swarm, K., Galione, A. and Scott, R. H. (1992) Mol. Biol. Cell 3, 1415-1425 34 Hua, S-Y. et al. (1994) Neuron 12,7073-1079 35 Thorn, P., Gerasimenko, 0. and Petersen, 0. H. (1994) EMBO J. 13,2038-2043 36 Meszaros, L. G., Bak, J. and Chu, A. (1993) Nature 364,76-79 37 White, A. M., Watson, S. I’. and Galione, A. (1993) FEBS Lett. 318,259-263 H. (1993) Science 38 Takasawa. S.. Nata. K.. Yonekura. H. andokamoto. ~ 259,370-37i 39 Koshiyama, H., Lee, H. C. and Tashjian, A. H., Jr (1991) J. Biol. Chem. 26616985-16988 40 Morrissette, J., Heisermann, G., Cleary, J., Ruoho, A. and Coronado, R. I
Central 5-HT, receptors Richard M. Eglen, Erik H. F. Wong, Aline Dumuis and Joiil Bockaert Activation of the 5-HT, receptor mediates widespread effects in central and peripheral nervous systems. Recent developments, such as the identification of novel, selective agonists and antagonists, receptor,
as well the cloning of the
have provided insights into the physiological
role of the receptor. colleagues
In this article, Richard Eglen and
assess the emerging evidence relating to the
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(1993) FEBS Left. 330,270-274 41 Holmberg, S. R. M. and Williams, A. J. (1990) Biochim. Biuphys. Acta 1022,187-193 42 Chu, A., Diaz-Munoz, M., Hawkes, M. J., Brush, K. and Hamilton, S. L. (1990) Mol. Pharmacol. 37,735-741 43 Fruen, 8. R., Mickelson, J. R., Shomer, N. H., Velez, P. and Louis, C. F. (1994) FEBS Lett. 352,123-126 44 Sitsapesan, R. and Williams, A. J. (1995) Am. 1. Physiol. 268, C1235-Cl240 45 Sitsapesan, R., McGarry, S. J. and Williams, A. J. (1994) Crrc. Res. 75,596-600 46 Lee, H. C. (1991) J. Bial. Chem. 266,2276-2281 47 Graeff, R. M., Podein, R. J., Aarhus, R. and Lee, H. C. (1995) Biochem. Biophys. Res. Commun. 206,78&791 48 Lee, H. C., Aarhus, R., Graeff, R., Gurnack, M. E. and Walseth, T. F. (1994) Nature 370,307-309 49 Rakow, T. L. and Shen, S. S. (1990) Proc. NatI Acad. SCI. USA 87,9285-9289 50 McPherson, S. M., McPherson, P. S., Mathews, L., Campbell, K. P. and Longo, F. J. (1992) J, Cell Biol. 116, 1111-1121
Editor’s note Ryanodine receptors are often assigned the nomenclature ‘RyRl’ etc. The nomenclature used in this article is in accordance with the criteria for receptor nomenclature established by the IUPHAR Committee for Receptor Nomenclature and Drug Classification (Pharmacol. Rev. in press).
Acknowlsdgemml The authors are grateful
:P,:‘,~,~~~$:,,,,, $“p,lLlll
and SB204070, these compounds lack selectivity toward the 5-I-IT, receptor. Furthermore, esters such as SDZ205557, RS23597, GR113808 or SB204070 may have limited activity in vivo due to hydrolysis. Together, these properties have precluded extensive pharmacological characterizations of central roles of the 5-HT, receptor in vivo. Several agonists and antagonists (Box 1) have recently been reported to have both enhanced 5-HT, selectivity and in vim stability. The use of these compounds has helped to elucidate the function of 5-HT, receptors in the brain, and several lines of evidence suggest a role for the receptor in cognitive processing7. Other functions of 5-HT, activation may involve modulation of dopamine transmissions and anxiolysisg.
function of the 5-HT., receptor in the brain. The cerebral distribution of the receptor,
along with neurochemical
and electrophysiological
data, suggests a role in cognition.
The role of the receptor
in modulation of dopamine
transmission
and anxiolysis is also addressed.
Definition of 5-HT, receptors is based on pharmacological, biochemical and, most recently, structural standpointsiJ. The receptor is activated by indoles (for example, 5-HT, 5methoxytryptamine), benzamides [for example, (s)-zacopride, cisapride, renzapride], benzimidazolones (for example, BIMUI, BIMU8) and Wnaphthalimides [for example, (s)-RS56532]*J (Fig. 1). Antagonists for the receptor include benzoates (for example, SDZ205557, RS23597), indoles (for example, tropisetron, GR113808, GR125487)‘,“5 and benzodioxanes (for example, SB204070)6 (Fig. 2). Except for GR113808, GR125487
0
1995,
Elsevier Science Ltd
Distribution of 5-HT, receptors The development of specific radioligands, such as [aH]GR113808 (Ref. 10) or [1zI]SB207710 (Ref. ll), for the 5HT, receptor has enabled mapping of the central distribution of the receptorQ(Figs 3, 4). Radioligand binding and autoradiography studies have been performed in brain tissue from raW2J3, mousei*, guinea-pigr0J2,13, pig’s, calP6, monkey13 and humani4J6J7 (Table 1). In all species studied so far, the highest receptor densities (100-400 fmol mg protein-i) are found in the limbic system. 5-HT, receptors are also found in components of other neuronal pathways, including the corticostriatal-tectal pathway and the septo-hippocampalhabenul&nterpeduncular pathway. The location of the 5-HT, receptor in the limbic system (that is, islands of Calleja, olfactory tubercle, frontal cortex, fundus striatus, ventral pallidurn, septal region,
TiPS - November
1995 (Vol. 16)
R. M. Eglen.
DIrector. lnst~fute of Pharmacology. Syntex (USAI. 3401 H~llww
Avenue. Palo
Alto, CA 94304. USA. E. Ii. F. WOIUJ. Head of CNS Elmlogy. Pharmacia SpA, Ne~~ano. MaIan, Italy. A Dumuis. Director of Research. and J. Bockash Head, Centre National de la Recherche Sclentiflque Laboratory (IJPA 90231. Rue de la Cardonllle. 34094 Montpell~er France
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26 Neer, M., Stovik, D. M., Daly, M., Potts, J. and Nussbaum,
27 28 29 30 31 32 33 34 35 36 37
S. R. (1993) Osteoporosis ht. 3 (Suppl. l), S204-S205 Ox&d, H., Ejersted, ?., Andreassen, T. T., Tarring, 0. and Nilsson, M. H. (1993) Am. 1. Med. 95,62S-@3S Reeve,‘J. et iI. (1993) Osteopwosis ht. 3 (Suppl. I), S199-S203 Riond, J-L. (1993) Clin. Sci. 85,223-228 Rixon, R. H. et al. (1994) J Bone Miner Res. 9,1179-1189 Shen, V., Dempster, D. W., Birchman, R., Xu, R. and Lindsay, R. (1993) J. Clin. Invest. 91,2479-2487 Sogaard, C. H., Wronski, T. J., McOskar, J. E. and Mosekilde, L. (1994) Endocrinology 134,65@-657 Strein, K. (1994) in Bone Diseases and Osteoporosis: Current Treatments andFutureOpportunities(Russell,R G.,ed.),p. 131,IBCTechnicalServices Watson, P. et al. (1995) Bone 16,357-365 Whitfield, J. F., Morley, P., Ross, V., Isaacs, R. J. and Rixon, R. H. (1995) C&if. Tissue ht. 56,227~231 Whitfield, J. F. et al. C&if: Tissue ht. (in press) Wronski, T. J., Yen, C-F., Qi, H. and Dann, L. M. (1993) Endocrinology
Cyclic ADP-ribose,the ryanodine receptor and Ca2+release Rebecca Sitsapesan, Alan J. Williams
Stephen J. McGarry
In a variety of vertebrate ryanodine-sensitive
and invertebrate
tissues the
Ca*+ channel is the pathway for
Ca*+ release from intracellular for activation
and
stores. The mechanism
of the ryanodine receptor-channel
complex appears to depend both on the ryanodine receptor isoform and the cell type. In addition, a complex combination
of endogenous
compounds
channel gating. In this article,
regulates
Rebecca
Sitsapesan,
Williams
review the mechanisms
ADP-ribose
Stephen McGarry
(cADPR)-induced
by one of the recognized
ryanodine receptor-Ca*+
R. serapesan, Lectursr, S.
J. MeGany,
Postdoctoral Fellow, and
A. J. Williams, Professor, Cardiac Medlcme, National Heart and Lung Institute. lmperlal College. Dovehouse Street. London. UK SW3 6LY
3 8 6
and Alan
involved in cyclic
Ca*+ release and discuss
the likelihood that cADPR-activated mediated
intracellular
Ca*+ release is isoforms of the
channel complex.
The ryanodine receptor-Caz+ channel complex is a homotetramer, in which each monomer has a molecular weight of approximately 550 kDa (Ref. 1). Following the cloning and sequencing of the cDNA of the mammalian skeletal muscle ryanodine receptor (RY, receptor)z, two other mammalian isoforms of the protein have been identified and their amino acid sequence deduced from their cloned cDNA. The ryanodine RY, receptor, which shows 66% identity with the RY, receptor, is expressed in cardiac muscle and brain, while the RY, receptor, which shows 67% identity with the RY, receptor, is expressed in the brain and at low levels in many other tissueslJ. The sarcoplasmic reticulum (SR) membranes of bird, fish and amphibian skeletal muscles contain two isoforms of ryanodine receptor 1.45;the (Yisoform of the bullfrog receptor
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132,82%831 38 Yamamoto, N. et al. (1993) Bone Miner. 23,333-342 (1994) Guidelines for 39 United States Food and Drug Administration preclinical and clinical evaluation ofagents used in the prevention or treatment ofpostmenopausal osteoporosis 40 Kanis, J. A. (1994) Osteoporosis, Blackwell 41 Spiegel, A. M. and Weinstein, L. S. (1994) in Thp Paruthyraids, (Bilezekian, J. P. and Levine, M. A., eds), p. 231, Raven Press 42 Jouishomme, H. et al. (1992) Endocrinology 130,53-&l 43 Jouishomme, H. et al. (1994) 1. Bone Miner. Res. 9,943-949 44 Neugebauer, W., Barbier, J-R., Sung, W. L., Whitfield, J. F. and Willick, G. E. (1995) Biochemistry X,8835-8842 45 Somjen, D., Schluter, K-D., W&gender, E., Mayer, H. and Kaye, A. M. (1991) Biochem. 1.277,86%868 46 Canali‘s, E.,‘Pash, J. and Varghese, S. (1993) Crit. Rev. Euk. Gene Expression 3, 155-166 47 Rosen, C., Donahue, L. R. and Hunter, S. J. (1994) Proc. Sot. Exp. Biol. Med. 206,83-102
shows 80% identity with the RY, receptor, while the p isoform shows 86% identity with the RY, receptor’. Screening of an insect genomic library has identified an analogue of the mammalian ryanodine receptor gene that displays 45% identity with the RY, receptolh. Ryanodine disrupts Cal+ handling in cells expressing well-defined ryanodine receptor isoforms (such as mammalian skeletal and cardiac muscle7) and is known to modify Ca2+ handling in other cell types, in which information on the identity of the isoform of ryanodine receptor or the levels of expression (or both) of the receptor is either less well-defined or absent. In the latter case, the pharmacological characterization of the putative ryanodine receptor usually reflects measurements of alterations in ryanodine-sensitive Ca2+ release in intact cells or cell homogenates. Detailed characterization of the function of the ryanodine-sensitive Ca2+ channel has largely been carried out using RY, (from mammalian skeletal muscle) and RY, receptors (from mammalian cardiac muscle). Studies involving the measurement of [sH]ryanodine binding to, and Ca2+ efflux from, isolated SR membrane vesicles together with monitoring parameters of singlechannel gating and conduction following the incorporation of either intact SR membrane vesicles or isolated receptor proteins into phospholipid bilayers, have identified a number of functional characteristics that are shared by these two isofonns and also some significant differences.
Functional consequences of ligand interaction with RY, and RY, receptors A diverse array of ligands are known to modulate the function of ryanodine-sensitive Ca2+ channel@. The vast majority of these agents act at sites located on the cytosolic side of the Ca2+ channel (Fig. 1). To date, annexin VI (Ref. 9) and Ca2+ (Refs 10,ll) are the only agents thought to act at the luminal side of the channel. Although the mechanisms governing activation of RY, and RY, receptors in situ are thought to be different (mechanical coupling of RY, receptors in skeletal muscle, and Ca2+induced Ca2+release for RY, receptors in cardiac muscle), following incorporation of the receptor into planar bilayers both isoforms are regulated by cytosolic Ca2+.All ligands
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R either will not activate channels in the absence of Ca2+, or require Ca2+ for maximum effect, suggesting that Caz+ is the ‘primary activating ligand’. The regulation of channel gating by cytosolic Ca2+ differs for RY, and RY, receptorsn. With RY, receptors, a bell-shaped Ca2+ concentration-open probability (I’,) relationship is observed; with RY, receptors, high concentrations of Caz+ tend to activate rather than inactivate. Physiological concentrations of Mg*+ (0.5-I .OmM) markedly reduce the I’, of RY, and RY, receptor-sensitive channels, and it has been suggested that this action may result from competition for cytosolic Cal+ binding sites by Mg2+ (Ref. 13). Many ‘secondary’ ligands activate both RY, and RY, receptors; however, there are often subtle differences in action, which may reflect differences in the receptor sites or, if the compound acts by a Ca2+-dependent mechanism, differences in the Ca2+ sensitivities of the isoforms. The sites which have been subjected to detailed examination, and which are most relevant to the interpretation of the action of cyclic ADP-ribose (cADPR) on ryanodine receptors, are:
Ryanodine The interaction of [jH]ryanodine with isolated membrane vesicles containing RY, and RY, receptors has identified a high-affinity binding site and possibly other low-affinity sites 1. Depending upon the experimental concentration of ryanodine used, it was found to either stimulate or inhibit Car+ efflux from isolated SR membrane vesicle+. These effects are consistent with singlechannel experiments where micromolar concentrations of ryanodine ‘lock the channel into a reduced-conductance, high-P, state (Fig. 2), and millimolar concentrations lead to irreversible channel closurers.
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Fig.1.Modulators of the gating of the ryanodtne receptor-channel complex. Cytosolic CaZ+ IS the ‘primary activating ligand’. being effective in the absence of ail other ligands. Activation of the channel by adenine nucleotides and cafferne is regulated by both cytosolic and luminal CaZ+ Ligands acting at the adenine nucleotide site include ATP, adenosine. cyclic ADP-ribose (cADPR). ADPribose (ADPR) and P-nicotinamide-adenine dinucleotide (DNAD). Depending upon the concentration applied, ryanodme will either increase or decrease open probabrlity.
nels in bilayers by a direct action22. In contrast, phosphorylation of the RY, receptor by calmodulin-dependent protein kinase has been suggested to increase I’, (Ref. 23).
Isofornvspecific ligands Although many ligands exert similar effects on RY, and RY, receptors, some agents may be selective for certain isoforms. For example, cardiac glycosides activate RY, receptors at low nanomolar concentrations (an action that may contribute to their therapeutic effecP), but have no effect on RY, receptors. In contrast, perchlorate ions appear to activate only RY1receptors25.
ATP ATP increases the release of Ca2+ from cardiac and skeletal SR vesicles, and increases the I’, of RY, and RY, receptor-sensitive channels at physiological concentrationsPJh.17. Additionally, it has been shown that adenosine and its related analogues, 2-chloroadenosine and N-ethylcarboxamidoadenosine (NECA), activate RY, receptors by binding to ATE activation sites?.
Cafiine Caffeine activates both RY,and RY, receptorsr9J’J. In the presence of cytosolic Ca*+, micromolar concentrations of caffeine sensitize the channel to Ca2+ (Ref. 21). At millimolar concentrations, caffeine can activate the RY, receptor by a Ca2+-independent mechanism. Recently, it was shown that the sites activated by caffeine on RY, receptors are distinct from those activated by ATE (Ref. 18).
Calmodulin The effects of calmodulin on ryanodine receptor channel gating are complex. Calmodulin has been shown to inhibit Ca2+ release from membrane vesicles containing RY, and RY, receptors, and decrease the I’, of these chan-
cADPR-induced Ca2+ release in sea urchin eggs and other tissues At present, there is much interest in the possibility that cADPR may be an endogenous regulator of one or more isoforms of the ryanodine receptor. This interest stems from experiments on sea urchin eggs in which it was demonstrated that cADPR could release Ca2+ from intracellular stores via an inositol 1,4,5-trisphosphate [Ins(l,4,5)P,]-insensitive mechanism2627. Both cADI% and the enzymes responsible for the metabolism of cADPR have been detected in a variety of cell types28.29,although the mechanisms involved in regulating the intracellular concentration of cADPR have yet to be established. In addition to cADPR, ryanodine and caffeine were shown to induce Ca2+ release in sea urchin egg homogenates and potentiate the effect of cADPR (Refs 27‘30). Ca*+ release in response to cADPR exhibited desensitization to subsequent applications of cADPR, caffeine and ryanodine but not to Ins(1,4,5)P,. Based on these results it was suggested that cADPR caused release of intracellular Ca2+ in sea urchin eggs by the activation of ryanodine receptorsr7. By using [32P]8-amino-cADPR as a photoaffinity
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-
-
-
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-
-
C
Fig. 2. Ryanodine modification of the RY, receptor. A single channel is incorporated into the bilayer; CaZ+ is the permeant ion (luminal [Caz+] = 50mM) and current flows through the channel from the luminal to the cytosolic side of the bilayer. The holding potential was OmV. The dotted lines indicate the closed (C), n/anodine-modified (M) and the fully open channel levels (0). The channel was activated by ~O~.LMcytosolic Ca2+ and 1 )LM cyclic ADP-ribose. The arrow indicates the time at which irreversible ryanodine modification occurs; 1 FM ryanodine was added to the solution bathing the cytosolic side of the bilayer approximately twominutes before modification occurred. Figure modified from Ref. 45.
probe, 140 and 100kDa proteins were identified as specific binding proteins for cADPR in homogenates of sea urchin eggssi. Walseth and co-workers suggested that these proteins may be novel ryanodine receptor-channel complexes or may represent separate proteins that interact with a ryanodine receptor after binding cADPR (Ref. 31). Another possibility is that the proteins are proteolytic fragments of a ryanodine receptors*. There is evidence that cADPR causes release of intracellular Ca*+ in other cell types, including neuronal cells33,34and pancreatic acinar cells35. cADPR also increases Ca*+release from microsomal membrane preparations of hearts, brainsQ7, pancreatic l+cellsB and pituitary cellss9.In contrast, Mesziros and colleagues found no significant effect of cADPR on Caz+release from isolated skeletal SR (Ref. 36), and although Morrissette and coworkers demonstrated a cADPR-induced Ca*+ release from skeletal SR vesiclesa, they concluded that this was mediated by a non-ryanodine receptor mechanism. The bulk of the evidence discussed above indicates that cADPR releases Ca*+ from ryanodine-sensitive Ca*+ stores. Does this reflect the direct interaction of cADPR with RY,, RY, or RY, receptors; does it involve activation of the channel via an accessory protein; or does cADPR induce the release of Ca*+via a novel ryanodine-sensitive channel (with or without the involvement of an accessory protein)? While most of these possibilities are yet to be investigated, the direct actions of cADPR on RY, and RY, receptors have been studied.
Interaction of cADPR with RY, and RY, receptors Effects of cADPR on [3HI yanodine binding [aH]Ryanodine binds to RY, and RY, receptors when the channel proteins are in open or conducting states, and therefore agents that increase the I’, of these channels generally increase ryanodine binding, and agents that decrease I’, decrease ryanodine bindir@i,Q. Meszaros and colleagues reported a significant increase in [3H]ryanodine binding to cardiac SR at a free Ca*+concentration of
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0.1 pM, but they observed no effect at higher Ca*+concentrations. In skeletal SR, they observed a significant decrease in [sH]ryanodine binding at free Ca*+concentrations greater than 10 p,M, indicating that cADPR also binds to membrane vesicles containing RY, receptors and may be expected to modulate I’,. Fruen and co-workers, however, found no effect of cADPR on [sH]ryanodine binding to cardiac or skeletal SR membrane vesicles over a wide range of concentrations of free Ca*+(Ref.43). These authors concluded that cADPR has no direct effects on the functional activity of either RY, or RY, receptors. Overall, the ryanodine binding studies suggest that if cADPR does activate either RY, or RY, receptors, the effect may not be very marked. Unfortunately, there are no reports of [sH]ryanodine binding to sea urchin egg membranes. Such studies would provide important information about the properties of putative ryanodine receptors present in sea urchin eggs, and allow a comparison with the binding characteristics of RY, and RY, receptors. Effects of cADPR on single-channel gating of RY, and RY, receptors The direct effects of cADPR on RY, and RY, receptors can be observed after incorporation of the channels into planar phospholipid bilayers under voltage-clamp conditions. Unfortunately, as for the ryanodine binding studies, the reported effects of cADPR on channel gating are not entirely consistent. Neither Morrissette and colleagues40nor Fruen and coworkers43 observed an effect of cADPR on the I’, of RY, receptor-sensitive channel incorporated into artificial bilayers. However, it has since been demonstrated that cADPR does activate these channels, but that the magnitude of the effect is dependent on the luminal Ca*+concentrations; at low luminal Ca*+concentrations (<40 FM), no significant increase in P, occm+. Therefore, one explanation for the negative results could be the low luminal Ca*+concentrations (l-7 FM) usedmT43. The precursor to
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200ms Fig. 3. Comparison of the effects of cyclic ADP-ribose (cADPR) and ATP on single-channel gating. The dotted lines indicate the fully open channel level’ and the arrows indicate the closed channel level. A single channel is incorporated into the bilayer; CaZ+ is the permeant ion and current flows through the than nel from the luminal to the cytosolic side of the bilayer. The holding potential was0 mV.a: In the presence of 10 JLM cytosolic Ca*+ (left panel), 1 pM cADPR (a concentration higher than measured intracellularly) (right panel) increases the open probability of the channel (P,) from 0.021 (left) to 0.061 (right). b: In the presence of 10 PM cytosolic Cast (left panel], 2 mM ATP (physiological levels in muscle cells = 520 mM) (right panel) increases P, from 0.034 (left) to 0.927 (right). Figure modified from Ref. 45.
cADPR, p-nicotinamide-adenine dinucleotide @NAD+), and the metabolite ADP-ribose (ADPR), were also shown to activate the RY, receptor-channel complex at micromolar concentrations@. Mesziros and colleagues detected an increase in the I’, of RY, receptor-sensitive channel only at a sub-activating cytosolic Caz+ concentration (lOn~)x, while others have demonstrated that cADPR has no effect on I’, over a range of cytosolic Ca2+ concentrations@. In contrast, it has recently been demonstrated that cADPR increases I’, in a Ca2+-dependent mannel”‘5; significant increases in I’, were obtained only in the presence of activating cytosolic Ca2+ concentrations. The discrepancies in the results from different laboratories may result either from species differences or from variations in the luminal Ca2+ concentrations for, as is the case with the RY, receptors, the gating of the RY, receptor-channel complex appears to be regulated by luminal Ca2+ (Ref. 10). The concentrations of cADPR required to activate the RY, receptor-channel complex (21 p~)36,45 are much higher than the tissue levels measured to date (approximately 200-68Onr@s. Even these high concentrations of cADPR are not as effective at activating RY, receptors as physiological concentrations of ATP (Fig. 3). There is evidence that cADPR and ATP may compete for the same binding sites on RY, receptors45, and it has also been demonstrated that in the presence of physiological levels
of ATP and Mg2+, cADPR is unable to activate the RY, receptorAanne1 complex&. Moreover, as for RY, receptors, pNAD+ and ADPR cause significant increases in I’,,(Ref. 45). The mechanisms involved in the activation of RY, and RY, receptors by cADPR appear to be very simila+,4s. The results indicate that while cADPR may be a pharmacological ligand for the adenine nucleotide sites on these ryanodine receptors, it is unlikely to act as a physiological regulator of these ryanodine receptor isoforms by binding directly to the channels.
Pharmacological profiles of cADPR-induced Ca2+ release and ryanodine receptor-channel gating On balance, the data presented appear to indicate that RY, and RY, receptors do not contain distinct, high-affinity, binding sites for cADPR; rather cADPR, PNAD+ and ADPR can increase channel opening via an interaction with the adenine nucleotide sites on the protein. This is clearly at odds with the action of cADPR in sea urchin eggs. However, this is not the only discrepancy between the characteristics of cADPR-induced Ca2+ release in sea urchin eggs and the established properties of RY, and R.Y, receptors. Points of particular interest include: l Single-channel studies with RY, or RY, receptors indicate that in the presence of activating cytosolic Caz+ and in the absence of other ligands, cADPR is ineffective at concentrations less than 1 FM (Refs 44, 45). In contrast,
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20 no cADPR can activate Ca2+ release in sea urchin eggs, and binding studies yield a K, = 171~ (Refs 27, 46). l Single-channel studies with RY, or RY, receptors”+” have demonstrated that PNAD+ and ADPR can increase channel I’@whereas in homogenates of sea urchin eggs ADPR has no effect and PNAD+ releases intracellular Ca2+ only after a lag-time of approximately four minutes (presumably after conversion of PNAD+ to cADPR)26. l Experiments reported by Graeff and co-workers indicate that ATP does not activate Ca2+ release in homogenates of sea urchin eggs47. In contrast, Ca2+-flux measurements in membrane vesicles containing RY, and RY, receptors demonstrate significant ATP-induced Caz+ release, and ATP can fully activate RY, and RY, receptorsensitive channels incorporated into bilayersisJ6J7 (Fig. 3). l Calmodulin decreases the I’, of RY, and RY, receptorgated channels, whereas calmodulin potentiates the effect of cADPR in homogenates of sea urchin eggs in a similar manner to caffeine and Sr2+ (Refs 22, 48). l In membrane preparations containing RY, and RY, receptors, Ca2+ is effective at releasing Ca2+, and can activate these channels reconstituted into bilayers in the absence of other activating ligands. However, in sea urchin eggs, Ca2+-induced Ca2+release is more difficult to evoke27,49.In homogenates of sea urchin eggs, it appears that Ca*+ alone is not a very effective activator of intracellular Caz+ release27 and requires a second ligand (such as caffeine) to cause marked release. l The literature contains no reports of the detection of RY,, RY, or RY, receptors in sea urchin eggs. However, a 380 kDa protein has been detected in the cortex of sea urchin eggs using an antibody raised to the RY, receptorw.
this complex in turn binds and activates the ryanodine receptor31. If the accessory binding protein does not incorporate into a planar bilayer along with the ryanodine receptor, this mechanism could explain why a specific activation of the ryanodine receptor is not seen in singlechannel studies using nanomolar concentrations of cADPR. However, it is still necessary to suggest that, in addition to increasing I’, the binding of the cADPRaccessory protein complex brings about a very marked alteration of the pharmacological profile of the ryanodine receptor-channel complex (ATP becomes ineffective, calmodulin increases rather than decreases I’,). The addition of purified sea urchin accessory proteins to reconstituted RY, and RY, receptor-channel complexes would be an important experiment. (3) cADPR induces Caz+release through a specific interaction with a ryanodine receptor that is not a RY, or RY, receptor; again this may involve the binding of cADPR to an accessory binding protein. As outlined, there is some variation in the degree of identity amongst the isoforms of ryanodine receptor that have been examined. Within the group of receptors on which detailed information on channel function exists, there are differences between isoforms, sometimes in terms of the degree of activation pro duced by a given concentration of ligand, and in some cases ligands are specific for a particular isoform. The identification of the isoform(s) of ryanodine recep tor expressed in sea urchin eggs and the characterization of the properties of the receptor and ion channel may help to resolve these uncertainties.
Concluding remarks cADPR induces the release of Ca2+ from ryanodinesensitive, Ins(l,4,5)P,-insensitive, intracellular stores in sea urchin eggs and other cell types, and therefore it is not unreasonable to suggest that cADPR-induced release of Ca2+might be mediated by a ryanodine-sensitive channel. The direct demonstration of the activation of RY, receptors by cADPR (Ref. 36), and the initial experiments suggesting a lack of effect of cADPR on RY, receptors27,40, have led to general acceptance of the proposal that cADPR-induced Ca2+ release is mediated by the RY, receptor. However, it is clear from the arguments discussed that there are significant discrepancies between the pharmacological profile of cADPR-induced Ca2+ release and that of the RY, receptor. There are a number of hypothetical mechanisms for cADPR-induced Ca2+ release via a ryanodine-sensitive channel. (1) cADPR binds to a distinct site on the ryanodine receptor-channel complex and increases I’,. The singlechannel data described indicate that this mechanism is unlikely to be involved if the receptor in question is either the RY, or RY, receptor. (2) cADPR binds to a separate receptor protein (the 100 or 140 kDa proteins identified in sea urchin egg), and
M. and Sorrentino, V. (1995) J Cell Biol. 128,893-904 Olivares, E. B. et al. (1991) Biophys. J. 59,1153-1163 O’Brien, J., Valdivia, H. H. and Block, B. A. (1995) Biophys. 1. 68,471-482 Takeshima, H. et al. (1994) FEBS Mt. 337,81-87 Sutko, J. L., Ito, K. and Kenyon, J. L. (1985) FASEB J. 44,2984-2988 Coronado, R., Morrissette, J., Sukhareva, M. and Vaughan, D. M. (1994) Am. J. Physiol. 266, C1485-Cl504 Diaz-Munoz, M., Hamilton, S. L., Kaetzel, M. A., Hazarika, I? and Dedman, J. R. (1990) J. BioI. Chem. 265,15894-15899 Sitsapesan, R. and Williams, A. J. (1994) J. Membr. Biol. 137, 215-226 Sitsapesan, R. and Williams, A. J. (1995) J. Membr. Biol. 146, 133-144 Chu, A., Fill, M., Stefani, E. and Entman, M. L. (1993) 1. Membr. Biol. 135,49-59 Smith, J. S., Coronado, R. and Meissner, G. (1986) J. Gen. Physiol. 88,573-588 Meissner, G. (1986) J. Biol. Chem. 261,6300-6306 Lai, F. A., Misra, M., Xu, I., Smith, H. A. and Meissner, G. (1989) J. Biof. Chem. 264,1677&16785 Meissner, G. and Henderson, J. S. (1987) J. Biol. Chem. 262, 30653073 Rousseau, E., Smith, J. S., Henderson, J. S. and Meissner, G. (1986) Biophys. J. 50,1009-1014 McGarry, S. J. and Williams, A. J. (1994) J. Membr. Biol. 137,169-177 Rousseau, E., LaDine, J., Liu, Q-Y. and Meissner, G. (1988) Arch Biochem. Biuphys. 267,75-86 Rousseau, E. and Meissner, G. (1989) Am. J. Physiol. 256, H328-H333 Sitsapesan, R. and Williams, A. J. (1990) 1. Physiol. 423,425-439 Smith, J. S., Rousseau, E. and Meissner, G. (1989) Circ. Res. 61, 352-359
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Selected references 1 Ogawa, Y. (1994) Crit. Rev. Biochem. Mol. Biol. 29,229-274 2 Take&ma, H. et al. (1989) Nature 339,439-445 3 Giannini, G., Conti, A., Mammarella, S., Scrobogna, 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
R 23 Witcher, D. R., Kovacs, R. J., Schulman, H., Cefali, D. C. and Jones, L. R. (1991)J. Biol. Chem. 266,11144-11152 24 McGarry, S. J. and Williams, A. J. (1993) Br. J. Pharmacol. 108, 1043-1050 25 Fruen, B. R., Mickelson, J. R., Roghair, T. J., Cheng, H-L. and Louis, C. F. (1994) Am. J. Physiol. 266, C1729-Cl735 26 Clapper, D. L., Walseth, T. F., Dargie, l’. J. and Lee, H. C. (1987) J. Biol. Chem. 262,9561-9568 27 Galione. A.. Lee. H. C. and Busa. W. B. (1991) Science 253.1143-1146 28 Walseth, T. F., Aarhus, R., Zeleznikar, R. J., Jr and Lee, H. C. (1991) Biochim. Biophys. Acta 1094,1X3-120 29 Lee, H. C., Galione, A. and Walseth, T. F. (1994) Vitam. Horm. 48,199-257 30 Buck, W. R., Rakow, T. L. and Shen, S. S. (1992) Exp. Cell Res. 202,59-66 31 Walseth, T. F., Aarhus, R., Kerr, I. A. and Lee, H. C. (1993) J. Biol Chem. 268,26686-26691 32 Galione, A. and White, A. (1994) Trends Cell Biol. 4,431-436 33 Currie, K. I’. M., Swarm, K., Galione, A. and Scott, R. H. (1992) Mol. Biol. Cell 3, 1415-1425 34 Hua, S-Y. et al. (1994) Neuron 12,7073-1079 35 Thorn, P., Gerasimenko, 0. and Petersen, 0. H. (1994) EMBO J. 13,2038-2043 36 Meszaros, L. G., Bak, J. and Chu, A. (1993) Nature 364,76-79 37 White, A. M., Watson, S. I’. and Galione, A. (1993) FEBS Lett. 318,259-263 H. (1993) Science 38 Takasawa. S.. Nata. K.. Yonekura. H. andokamoto. ~ 259,370-37i 39 Koshiyama, H., Lee, H. C. and Tashjian, A. H., Jr (1991) J. Biol. Chem. 26616985-16988 40 Morrissette, J., Heisermann, G., Cleary, J., Ruoho, A. and Coronado, R. I
Central 5-HT, receptors Richard M. Eglen, Erik H. F. Wong, Aline Dumuis and Joiil Bockaert Activation of the 5-HT, receptor mediates widespread effects in central and peripheral nervous systems. Recent developments, such as the identification of novel, selective agonists and antagonists, receptor,
as well the cloning of the
have provided insights into the physiological
role of the receptor. colleagues
In this article, Richard Eglen and
assess the emerging evidence relating to the
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(1993) FEBS Left. 330,270-274 41 Holmberg, S. R. M. and Williams, A. J. (1990) Biochim. Biuphys. Acta 1022,187-193 42 Chu, A., Diaz-Munoz, M., Hawkes, M. J., Brush, K. and Hamilton, S. L. (1990) Mol. Pharmacol. 37,735-741 43 Fruen, 8. R., Mickelson, J. R., Shomer, N. H., Velez, P. and Louis, C. F. (1994) FEBS Lett. 352,123-126 44 Sitsapesan, R. and Williams, A. J. (1995) Am. 1. Physiol. 268, C1235-Cl240 45 Sitsapesan, R., McGarry, S. J. and Williams, A. J. (1994) Crrc. Res. 75,596-600 46 Lee, H. C. (1991) J. Bial. Chem. 266,2276-2281 47 Graeff, R. M., Podein, R. J., Aarhus, R. and Lee, H. C. (1995) Biochem. Biophys. Res. Commun. 206,78&791 48 Lee, H. C., Aarhus, R., Graeff, R., Gurnack, M. E. and Walseth, T. F. (1994) Nature 370,307-309 49 Rakow, T. L. and Shen, S. S. (1990) Proc. NatI Acad. SCI. USA 87,9285-9289 50 McPherson, S. M., McPherson, P. S., Mathews, L., Campbell, K. P. and Longo, F. J. (1992) J, Cell Biol. 116, 1111-1121
Editor’s note Ryanodine receptors are often assigned the nomenclature ‘RyRl’ etc. The nomenclature used in this article is in accordance with the criteria for receptor nomenclature established by the IUPHAR Committee for Receptor Nomenclature and Drug Classification (Pharmacol. Rev. in press).
Acknowlsdgemml The authors are grateful
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and SB204070, these compounds lack selectivity toward the 5-I-IT, receptor. Furthermore, esters such as SDZ205557, RS23597, GR113808 or SB204070 may have limited activity in vivo due to hydrolysis. Together, these properties have precluded extensive pharmacological characterizations of central roles of the 5-HT, receptor in vivo. Several agonists and antagonists (Box 1) have recently been reported to have both enhanced 5-HT, selectivity and in vim stability. The use of these compounds has helped to elucidate the function of 5-HT, receptors in the brain, and several lines of evidence suggest a role for the receptor in cognitive processing7. Other functions of 5-HT, activation may involve modulation of dopamine transmissions and anxiolysisg.
function of the 5-HT., receptor in the brain. The cerebral distribution of the receptor,
along with neurochemical
and electrophysiological
data, suggests a role in cognition.
The role of the receptor
in modulation of dopamine
transmission
and anxiolysis is also addressed.
Definition of 5-HT, receptors is based on pharmacological, biochemical and, most recently, structural standpointsiJ. The receptor is activated by indoles (for example, 5-HT, 5methoxytryptamine), benzamides [for example, (s)-zacopride, cisapride, renzapride], benzimidazolones (for example, BIMUI, BIMU8) and Wnaphthalimides [for example, (s)-RS56532]*J (Fig. 1). Antagonists for the receptor include benzoates (for example, SDZ205557, RS23597), indoles (for example, tropisetron, GR113808, GR125487)‘,“5 and benzodioxanes (for example, SB204070)6 (Fig. 2). Except for GR113808, GR125487
0
1995,
Elsevier Science Ltd
Distribution of 5-HT, receptors The development of specific radioligands, such as [aH]GR113808 (Ref. 10) or [1zI]SB207710 (Ref. ll), for the 5HT, receptor has enabled mapping of the central distribution of the receptorQ(Figs 3, 4). Radioligand binding and autoradiography studies have been performed in brain tissue from raW2J3, mousei*, guinea-pigr0J2,13, pig’s, calP6, monkey13 and humani4J6J7 (Table 1). In all species studied so far, the highest receptor densities (100-400 fmol mg protein-i) are found in the limbic system. 5-HT, receptors are also found in components of other neuronal pathways, including the corticostriatal-tectal pathway and the septo-hippocampalhabenul&nterpeduncular pathway. The location of the 5-HT, receptor in the limbic system (that is, islands of Calleja, olfactory tubercle, frontal cortex, fundus striatus, ventral pallidurn, septal region,
TiPS - November
1995 (Vol. 16)
R. M. Eglen.
DIrector. lnst~fute of Pharmacology. Syntex (USAI. 3401 H~llww
Avenue. Palo
Alto, CA 94304. USA. E. Ii. F. WOIUJ. Head of CNS Elmlogy. Pharmacia SpA, Ne~~ano. MaIan, Italy. A Dumuis. Director of Research. and J. Bockash Head, Centre National de la Recherche Sclentiflque Laboratory (IJPA 90231. Rue de la Cardonllle. 34094 Montpell~er France
391