- Email: [email protected]
Neuromethods, vol. 20 ‘intracellular messengers’
J o u r n a l of Chemical N e u r o a n a t o m y , Vol. 6:53 54 (1993)
Book Review Neuromethods, Vol. 20 'lntracellular messengers' A. A. Boulton, G. B. Baker, a n d C. W. T a y l o r (eds). H u m a n a Press, Clifton NJ. U.S.A., 1992. 580 pp., ISBN 0-89603-207-8.
This volume of the N e u r o m e t h o d s series is an extensive rexiew of current knowledge of intracellular inl\~rmation processing upon receptor activation. Although the book is initially aimed at neuroscience, its application can be easib extended to tissues other than the central nerxous system. It contains detailed methodological sections dealing with practical problems and calibration of instruments, as well as the various pitfalls that can be encountered. G proteins, ion channels, calcium homeostasis, cyclic nucleotides, inositol phosphates and protein kinase activity are also covered. One missing "second messenger" in this volume is nitric monoxide and NO synthase. The NO synthase/diaphorase system seems to play an important role in regulating the redox-state of the cell, mediating at least in part the electron transfer from various donor to acceptor molecules. Though the implications of NO synthase function in neuroscience are only beginning to emerge, this system might have been discussed within the context of cGMP. Nevertheless, throughout the book, attention is focused on how the cell processes receptor-mediated information, translated by the signal transduction system, into the individual second- and subsequent messengers, which is ultimately integrated into an adequate, exocytotic response. Furthermore, the largely biochemical body of text is extended by a molecular genetic approach into signal transduction, using various mutant strains of Drosophila. The first chapter by Milligan gives an update on G proteins, one of the most versatile signal-transduction pathwa.~s. It contains a basic evaluation on the current knowledge of subunit diversity, and functional properties of G proteins, l\~r instance in regulating receptor affinity and the subsequent channelling of information into the cell. Several sections cover the various ways of identifying the specific G protein complex, e.g. ADP-ribosylation, photoaflinity labelling, GTPase studies and the use of reconstituted systems l\~r identifying transiently expressed cloned G protein subunits. A considerable part of the chapter is devoted to the immunological characterization of the various G protein subunits and the use of antibodies, raised against specific proteins, to modify receptor affinity. Finally, functional implications of the subunit diversity is discussed, along with future possibilities using site-directed mutagenesis or chimeric constructs of cloned G proteins, as this is expected to contribute to our knowledge of how receptors ultimateb communicate with their respective intracellular effeclor SVStel'ilS
Subsequent chapters are devoted to the various types of second messengers that are known at present. The chapters by Godfrey, Taylor el al., and part of that b} Potter deal with the phosphatidylinositides, inositol phosphates, and stable analogues. Since the initial identification of inositol phosphates as second messengers by ¢~ 1993 by John Wiley and Sons Ltd
Berridge in the late seventies, this field has expanded enormously, and the inositols are found In be important regulators of cellular calcium. In addition, diacylglycerol has profound stimulatory activity by activating protein kinasc C. Godfrey focuses mainly on the current analytical methods in receptor-activated phosphotipid hydrolysis. including extraction procedures. TLC, ion-exchange and gas chromatography. Taylor and coauthors try to integrate the IP~ and cell calcium. Cell strategies in calcium sequestration are discussed, as well as thc mechanisms with which cells selectively mobilize these pools upon receptor activation. I P~-selective intracellular receptors have been implicated in calcium mobilization from intracellular stores, and such receptors have been identified recently: properties of these receptors are discussed, i.e. pharmacological characterization and purification. Finally, some current ideas on the role of receptor-activated phospholipase C, IP~ and calcium signalling are discussed. New approaches in second inessenger application into the cells by use of "caged messengers" are discussed bx Parker. Previously, micro-iontophoretic application or cell permeabilization were used to introduce active second messengers into cells: however, spatial concentrations appeared to be difficult to estimate. With "caged" compounds, inactive photolabile compounds are loaded into the cells, and subsequently activated by light flashes through the microscope. This results in instant liberation of an amount of active second messenger which can be recorded by their effects on membrane current. Imaging ofcalciurn signalling by employing fluorescent calcium-sensitive indicators is a technique that has emerged in the early eighties. Since then. various sensitive indicators have been developed, as well as the computerassisted imaging technology. Poenie and Moreton extensively discuss the principles and problems of calcium imaging. Calcium-sensitive dyes display diflerent calcium buffering capacities, binding kinetics, loading eflicienc3 and cellular distribution. These properties arc reviewed methodologically, giving guidance on a suitable indicator choice. Finally, being a quantitiative fluorographic method, methods for device calibration, image digitization, quantification and data reduction are discussed in detail, along with the various limitations of this technique. Apart from calcmm, wmous ion-selective dyes are available for on-line monitoring of local cellular changes in pH, Mg :+, Na +, K ' , and ('1 . The various techniques currently in use IBr the visualizalion of ion fluxes are discussed. One of the fimfl effector systems to which various second- and third messengers seem to converge tire the kinases, of which protein kinase C (PKC) has a prominent role. To date, several isozymes of PKC have been cloned and characterized. Activation of PKC will ultimatel\
54
Book Review
lead to the phosphorylation of various protein substrates, thus affecting cellular processes involved with neurotransmitter release cell growth and differentiation, ion channel activity and receptor desensitization, for example. Within the cell, PKC is activated by diacyglycerol (mimicked by phorbol esters) and is sensitive to calcium-calmodulin. Methods for studying PKC, e.g., phophorylation assays, translocation studies, and the use of phorbol esters, are discussed by Burgess. In conjunction, the chapter by Morton deals with the functional implications of protein phosphorylation and dephoshorylation. In addition to PKC, other kinases like protein kinase A and G are discussed, being sensitive to cAMP, cGMP and the presence of calcium (e.g. CaM kinases). Furthermore, tyrosine kinases are discussed, as well as the undoubtedly important physiological role protein phosphatases play in the overall regulation of protein phosphorylation. The study of second messengers is a field that is largely dominated by biochemists. Nevertheless, many studies have been published using electrophysiological methods to monitor changes in electrical properties of neuronal membranes upon exposure to various second messengers; these are reviewed in two chapters. Matthews and Lamb discuss these changes in membrane potentials using the photoreceptor paradigm in rods of the toad retina. Changes in second messengers may lead to a change in ion conductancy. This chapter is an introduction into electrophysiological recordings, dealing with the various methods like patch clamp, and whole cell recording. The electrodes are often used to iontophoretically apply drugs or second messengers onto or into cells, thus leading to changes in current (monitored by voltage clamp). Moreover, rods can be activated physiologically by exposing the cell to light. Manipulation by various second messengers, activators or inhibitors of enzyme systems, may subsequently lead to an altered signal. Thus, biochemical pathways can be delineated in subsecond range, similar to calcium imaging. The second review chapter is by Minke and Selinger, describing the combination of biochemistry and electrophysiology in the photo cells (ommatidia) of various mutant Drosophila strains. In contrast with vertebrate photoreceptors, invertebrates use phosphoinositides as second messengers in cell excitation. Membrane current can be manipulated by light as well as by administration of second messengers or inhibitors into the cells. In addition, the fruit fly is a pet animal of geneticists and consequently many different mutants have been described. Some of those may incorporate mutations in genes or loci that are involved with adequate processing of visual information. In this chapter, some mutant effects
in the nina (neither inactivated nor activatedj iocub are described. The various nina genes code for proteins of which several have now been identified, for instance, ninaC encodes a myosin kinase, ninaE is an opsin-coding gene, and ninaA is necessary for correct folding and stability of rhodopsin. Changes in these genes may thus affect the sensitivity to light and the underlying biochemical pathways. Similarly, a mutant fly norpA produces no light-induced potential on the photoreceptor cells. The cloned norpA gene encodes a protein homologous to phospholipase C (PLC). Genetic recombination experiments with temperature-labile mutations in the norpA locus revealed that photo-activated norpA (i.e. PLC) is essential for the subsequent excitation of these cells. This chapter further extends the range of experimental approaches into the biochemistry of signal transduction. As the fruit fly genetics already offers a plethora of mutant flies, subsequent analysis of phototransduction for instance, may offer new paradigms. Various genes, containing those mutations, may well have a vertebrate counterpart. Finally, one of the main functions of a neuron is to communicate its current status to its neighbouring cells after information processing, by means of active secretation of neurotransmitters in an adequate manner. Therefore, strict regulation of exocytosis is mandatory, and various intracellular messenger systems converge somehow to the stimulus-secretion coupling mechanism. Burgoyne reviews these systems, e.g. calcium and calmodulin, protein kinases, phosphatases, and GTP-binding proteins and their effects on the cytoskeleton. On the whole, this volume covers the various methods used in studying intracellular messengers in the central nervous system. It is organized much the same way as the information processing in a neuron. The book starts with G proteins and more or less ends with the exocytotic process of neurotransmitters. It may serve as an introductory handbook for researchers, new in the field of second messengers, and provides a comprehensive review of current knowledge of the various systems and their complex interactions. Finally, the book introduces a molecular genetic approach to intracellular messengers, offering a valuable extension to 'classical' neurobiotogy and provides new tools in neurochemistry. E. Ronken, Ph.D.
Dept. of Pharmacology, Faculo' of Medicine Free UniversiO', Amsterdam, The Netherlands
Book Review Neuromethods, Vol. 20 'lntracellular messengers' A. A. Boulton, G. B. Baker, a n d C. W. T a y l o r (eds). H u m a n a Press, Clifton NJ. U.S.A., 1992. 580 pp., ISBN 0-89603-207-8.
This volume of the N e u r o m e t h o d s series is an extensive rexiew of current knowledge of intracellular inl\~rmation processing upon receptor activation. Although the book is initially aimed at neuroscience, its application can be easib extended to tissues other than the central nerxous system. It contains detailed methodological sections dealing with practical problems and calibration of instruments, as well as the various pitfalls that can be encountered. G proteins, ion channels, calcium homeostasis, cyclic nucleotides, inositol phosphates and protein kinase activity are also covered. One missing "second messenger" in this volume is nitric monoxide and NO synthase. The NO synthase/diaphorase system seems to play an important role in regulating the redox-state of the cell, mediating at least in part the electron transfer from various donor to acceptor molecules. Though the implications of NO synthase function in neuroscience are only beginning to emerge, this system might have been discussed within the context of cGMP. Nevertheless, throughout the book, attention is focused on how the cell processes receptor-mediated information, translated by the signal transduction system, into the individual second- and subsequent messengers, which is ultimately integrated into an adequate, exocytotic response. Furthermore, the largely biochemical body of text is extended by a molecular genetic approach into signal transduction, using various mutant strains of Drosophila. The first chapter by Milligan gives an update on G proteins, one of the most versatile signal-transduction pathwa.~s. It contains a basic evaluation on the current knowledge of subunit diversity, and functional properties of G proteins, l\~r instance in regulating receptor affinity and the subsequent channelling of information into the cell. Several sections cover the various ways of identifying the specific G protein complex, e.g. ADP-ribosylation, photoaflinity labelling, GTPase studies and the use of reconstituted systems l\~r identifying transiently expressed cloned G protein subunits. A considerable part of the chapter is devoted to the immunological characterization of the various G protein subunits and the use of antibodies, raised against specific proteins, to modify receptor affinity. Finally, functional implications of the subunit diversity is discussed, along with future possibilities using site-directed mutagenesis or chimeric constructs of cloned G proteins, as this is expected to contribute to our knowledge of how receptors ultimateb communicate with their respective intracellular effeclor SVStel'ilS
Subsequent chapters are devoted to the various types of second messengers that are known at present. The chapters by Godfrey, Taylor el al., and part of that b} Potter deal with the phosphatidylinositides, inositol phosphates, and stable analogues. Since the initial identification of inositol phosphates as second messengers by ¢~ 1993 by John Wiley and Sons Ltd
Berridge in the late seventies, this field has expanded enormously, and the inositols are found In be important regulators of cellular calcium. In addition, diacylglycerol has profound stimulatory activity by activating protein kinasc C. Godfrey focuses mainly on the current analytical methods in receptor-activated phosphotipid hydrolysis. including extraction procedures. TLC, ion-exchange and gas chromatography. Taylor and coauthors try to integrate the IP~ and cell calcium. Cell strategies in calcium sequestration are discussed, as well as thc mechanisms with which cells selectively mobilize these pools upon receptor activation. I P~-selective intracellular receptors have been implicated in calcium mobilization from intracellular stores, and such receptors have been identified recently: properties of these receptors are discussed, i.e. pharmacological characterization and purification. Finally, some current ideas on the role of receptor-activated phospholipase C, IP~ and calcium signalling are discussed. New approaches in second inessenger application into the cells by use of "caged messengers" are discussed bx Parker. Previously, micro-iontophoretic application or cell permeabilization were used to introduce active second messengers into cells: however, spatial concentrations appeared to be difficult to estimate. With "caged" compounds, inactive photolabile compounds are loaded into the cells, and subsequently activated by light flashes through the microscope. This results in instant liberation of an amount of active second messenger which can be recorded by their effects on membrane current. Imaging ofcalciurn signalling by employing fluorescent calcium-sensitive indicators is a technique that has emerged in the early eighties. Since then. various sensitive indicators have been developed, as well as the computerassisted imaging technology. Poenie and Moreton extensively discuss the principles and problems of calcium imaging. Calcium-sensitive dyes display diflerent calcium buffering capacities, binding kinetics, loading eflicienc3 and cellular distribution. These properties arc reviewed methodologically, giving guidance on a suitable indicator choice. Finally, being a quantitiative fluorographic method, methods for device calibration, image digitization, quantification and data reduction are discussed in detail, along with the various limitations of this technique. Apart from calcmm, wmous ion-selective dyes are available for on-line monitoring of local cellular changes in pH, Mg :+, Na +, K ' , and ('1 . The various techniques currently in use IBr the visualizalion of ion fluxes are discussed. One of the fimfl effector systems to which various second- and third messengers seem to converge tire the kinases, of which protein kinase C (PKC) has a prominent role. To date, several isozymes of PKC have been cloned and characterized. Activation of PKC will ultimatel\
54
Book Review
lead to the phosphorylation of various protein substrates, thus affecting cellular processes involved with neurotransmitter release cell growth and differentiation, ion channel activity and receptor desensitization, for example. Within the cell, PKC is activated by diacyglycerol (mimicked by phorbol esters) and is sensitive to calcium-calmodulin. Methods for studying PKC, e.g., phophorylation assays, translocation studies, and the use of phorbol esters, are discussed by Burgess. In conjunction, the chapter by Morton deals with the functional implications of protein phosphorylation and dephoshorylation. In addition to PKC, other kinases like protein kinase A and G are discussed, being sensitive to cAMP, cGMP and the presence of calcium (e.g. CaM kinases). Furthermore, tyrosine kinases are discussed, as well as the undoubtedly important physiological role protein phosphatases play in the overall regulation of protein phosphorylation. The study of second messengers is a field that is largely dominated by biochemists. Nevertheless, many studies have been published using electrophysiological methods to monitor changes in electrical properties of neuronal membranes upon exposure to various second messengers; these are reviewed in two chapters. Matthews and Lamb discuss these changes in membrane potentials using the photoreceptor paradigm in rods of the toad retina. Changes in second messengers may lead to a change in ion conductancy. This chapter is an introduction into electrophysiological recordings, dealing with the various methods like patch clamp, and whole cell recording. The electrodes are often used to iontophoretically apply drugs or second messengers onto or into cells, thus leading to changes in current (monitored by voltage clamp). Moreover, rods can be activated physiologically by exposing the cell to light. Manipulation by various second messengers, activators or inhibitors of enzyme systems, may subsequently lead to an altered signal. Thus, biochemical pathways can be delineated in subsecond range, similar to calcium imaging. The second review chapter is by Minke and Selinger, describing the combination of biochemistry and electrophysiology in the photo cells (ommatidia) of various mutant Drosophila strains. In contrast with vertebrate photoreceptors, invertebrates use phosphoinositides as second messengers in cell excitation. Membrane current can be manipulated by light as well as by administration of second messengers or inhibitors into the cells. In addition, the fruit fly is a pet animal of geneticists and consequently many different mutants have been described. Some of those may incorporate mutations in genes or loci that are involved with adequate processing of visual information. In this chapter, some mutant effects
in the nina (neither inactivated nor activatedj iocub are described. The various nina genes code for proteins of which several have now been identified, for instance, ninaC encodes a myosin kinase, ninaE is an opsin-coding gene, and ninaA is necessary for correct folding and stability of rhodopsin. Changes in these genes may thus affect the sensitivity to light and the underlying biochemical pathways. Similarly, a mutant fly norpA produces no light-induced potential on the photoreceptor cells. The cloned norpA gene encodes a protein homologous to phospholipase C (PLC). Genetic recombination experiments with temperature-labile mutations in the norpA locus revealed that photo-activated norpA (i.e. PLC) is essential for the subsequent excitation of these cells. This chapter further extends the range of experimental approaches into the biochemistry of signal transduction. As the fruit fly genetics already offers a plethora of mutant flies, subsequent analysis of phototransduction for instance, may offer new paradigms. Various genes, containing those mutations, may well have a vertebrate counterpart. Finally, one of the main functions of a neuron is to communicate its current status to its neighbouring cells after information processing, by means of active secretation of neurotransmitters in an adequate manner. Therefore, strict regulation of exocytosis is mandatory, and various intracellular messenger systems converge somehow to the stimulus-secretion coupling mechanism. Burgoyne reviews these systems, e.g. calcium and calmodulin, protein kinases, phosphatases, and GTP-binding proteins and their effects on the cytoskeleton. On the whole, this volume covers the various methods used in studying intracellular messengers in the central nervous system. It is organized much the same way as the information processing in a neuron. The book starts with G proteins and more or less ends with the exocytotic process of neurotransmitters. It may serve as an introductory handbook for researchers, new in the field of second messengers, and provides a comprehensive review of current knowledge of the various systems and their complex interactions. Finally, the book introduces a molecular genetic approach to intracellular messengers, offering a valuable extension to 'classical' neurobiotogy and provides new tools in neurochemistry. E. Ronken, Ph.D.
Dept. of Pharmacology, Faculo' of Medicine Free UniversiO', Amsterdam, The Netherlands