Which of the following hormones will bind to a membrane receptor on its target cell and stimulates the generation of second messenger ?`?

Protein Kinase C

Many lipid second messengers, including DAG, PIP3, arachidonic acid, phosphatidic acid, and lysophosphatidylcholine activate one or more of the 10 PKC isozymes expressed by vertebrate cells [Fig. 26.6]. These diverse PKC isozymes provide a selective response to various lipid second messengers. Some, but not all, PKC isozymes also require Ca2+ for activation. Sphingosine may inhibit some PKC isozymes.

Lipid second messengers activate PKC by dissociating an intramolecular pseudosubstrate sequence from the active site. Pseudosubstrates have alanine at the phosphorylation site instead of the serine found in substrates. DAG and other lipid second messengers bind C1 regions adjacent to the pseudosubstrate. DAG binding depends on phosphoglycerides, such as phosphatidylserine. Phorbol esters, pharmacological activators of PKC that promote tumor formation in laboratory experiments, bind PKC in a fashion similar to DAG. Ca2+-dependent PKC isozymes have C2 regions that mediate binding to phospholipids in the presence of Ca2+. During apoptosis, caspases cleave off this regulatory domain [see Fig. 46.12] producing constitutively active PKC isoforms.

Activated PKCs have many potential targets in cells and are implicated in the regulation of cellular activities ranging from gene expression to cell motility to the generation of lipid second messengers. PKC isozymes are selective toward certain protein substrates. The C2 regions target PKC isozymes to the plasma membrane, cytoskeleton, or nucleus.

PKCs can provide either positive or negative feedback to the signaling pathways that turn them on. PKC activates PLD and PLA2 and provides positive feedback, because those enzymes produce more DAG to sustain the activation of PKC. On the other hand, PKC provides negative feedback when it phosphorylates and inhibits both growth factor receptors and PI-PLCγ1. PKC also phosphorylates and inhibits PI-PLCβ, generating negative feedback after activation of seven-helix receptors. Negative feedback makes both of these signaling events transient.

Non-Gaseous Second Messengers

Cyclic nucleotides

Cylic AMP [cAMP] is a water-soluble second messenger found in eukaryotic and prokaryotic cells. The intracellular concentrations of cyclic AMP are approximately 0.1-1.0 μM, increasing 20-fold within seconds of activation. Cyclic AMP is rapidly and continuously catabolized to adenosine 5′ monophosphate [5′-AMP] by cytosolic and particulate formsphosphodiesterases [PDEs][Fig. 2]. PDE activity is regulated through phosphorylation by protein kinase A [PKA]and protein kinase C [PKC]Krebs [1989], Nishizuka [1989].

The levels of cellular cyclic AMP are generally regulated by changes in the activity of adenylate cyclase [AC], the enzyme responsible for the synthesis of this second messenger. This enzyme is an integral membrane effector protein that utilizes ATP as a substrate and magnesium as a cofactor to form cyclic AMP and inorganic phosphate. AC is expressed in most tissues, comprising 0.001-0.01% of the total membrane protein. Multiple isoforms of AC can be expressed in a single cell by different subcellular components.

The levels of cyclic AMP in a cell directly govern the activity of PKA and indirectly modulate metabolic and transcriptional events. Two molecules of cyclic AMP must bind to each regulatory unit of PKA for activation of this kinase. An intracellular concentration of 10 μM cyclic AMP is required for activation of PKA Siegelbaum et al [2000].

Cyclic GMP [cGMP], like cyclic AMP, is a water soluble second messenger that is rapidly and continuously catabolized to guanosine 5′ monophosphate [5′-GMP] by the magnesium-dependent PDEs including PDE1, PDE2, PDE 5, and PDE 6Siegelbaum et al [2000].

The synthesis of cyclic GMP from GTP, which requires magnesium, is catalyzed by soluble or particulate guanylyl cyclase [GC]. Upon hydrolysis of GTP by GC, cyclic GMP is formed [Fig. 2]. The soluble form of GC is calcium-sensitive is activated by increases in intracellular calcium or nitric oxide. The particulate form of GC, which is calcium-insensitive, is an integral membrane protein with a structure similar to A C. A particulate form of GC localized in retinal photoreceptor cells is regulated by light and inhibited by calcium.

Cyclic GMP activates protein kinase G [PKG] Pilz and Casteel [2003], a single polypeptide containing regulatory and a catalytic domains, with the latter being similar in sequence to PK A. Cyclic GMP binds to the regulatory subunit of PKG.

Inositol lipids

The phosphatidyl inositol [PI] signaling pathway involves phospholipase C [PLC], an enzyme that cleaves membrane phospholipids Berridge [1989]. There are 20 different isoforms of PLC that regulate the activities of kinases, esterases, and phospholipases. The endogenous substrate for PLC is phosphatidylinositol 4,5-bisphosphate [PIP2], with hydrolysis of the phosphodiester bond linking the glycerol backbone to the polar head group to form diacylglycerol [DAG] and inositol 1,4,5-trisphosphate [IP3] [Fig. 3].

DAG and IP3 are second messengers that can act independently or in unison. DAG activates protein kinase C and IP3 binds to a receptor on the endoplasmic reticulum to release calcium from intracellular stores. The beta isozymes of PLC are regulated by G-proteins [G-alphaq/11 and G-betagamma] Berridge [1989], Gilman [1989].

The arachidonic acid signaling cascade involves phospholipase A2 [PLA2], which cleaves arachidonic acid from the second position of the phospholipid backbone Siegelbaum et al [2000]. Once arachidonic acid is released, it is metabolized to the prostaglandins, thromboxanes, and leukotrienes, all of which may act as second messengers and can directly affect numerous cellular elements, including ion channels [Fig. 4]. Phospholipase A2 is present in both particulate and soluble forms.

Calcium

Calcium is involved in numerous signaling pathways. The levels of intracellular calcium are tightly regulated by voltage-dependent channels, IP3-gated channels, and ryanodine receptors.

Calcium activates numerous kinases, as well as other enzymes including phospholipase A2 leading to the formation of cytochrome P450- and cyclooxygenase-dependent metabolites such as epoxides and prostaglandins. Calcium may also regulate calcium-activated potassium channelsresulting Wu [2003] in hyperpolarization of smooth muscle membrane. Calcium can bind to specific calcium binding proteins such as calmodulin [eukaryotic cells] or troponin C [skeletal muscle], initiating other cellular activities such as stimulation of constitutive nitric oxide synthase[Fig. 5].

The Second Messengers

Second messengers refer to small intracellular molecules that are produced after the first messenger [hormone or neurotransmitter]-dependent receptor activation. Second messengers are intended to activate intracellular signaling pathways that amplify the signal and culminate with the activation or inhibition of transcription factors, inducing a cellular response.

The chemical nature of the second messenger is diverse: cyclic nucleotides, lipid derivatives and small active compounds, and some ions. The most studied second messengers are cyclic 3′5′-adenosine monophosphate [AMP] or cyclic guanosine monophosphate [GMP], calcium, DAG, IP3, and reactive oxygen and nitrogen species [ROS, NOS]. In the following sections, we emphasize on the general characteristics and mechanisms of action of some of them.

Second messenger–independent protein Ser/Thr kinases

Although the second messenger–dependent protein kinases were identified first as playing an important role in neuronal function, we now know that many second messenger–independent protein Ser/Thr kinases regulate numerous fundamental neuronal functions. Second messenger–independent protein Ser/Thr kinases generally act downstream of second messenger-induced pathways or protein Tyr kinase signaling [Figure 25-2A]. Their activity is commonly controlled by regulatory phosphorylation and/or association with regulatory cofactors. Second messenger–independent protein Ser/Thr kinases contribute to the transduction of signals via the phosphorylation of specific phosphoproteins and feedback onto upstream protein kinases and phosphatases.

Second messenger pathways link an extracellular signal [ie, first messenger] that activates a receptor to an intracellular response such as a change in membrane potential or gene expression. Despite the diverse range of neurotransmitter receptors, many components of receptor-activated second messenger pathways overlap and take part in complex, seemingly indiscriminate signaling networks. However, interactions are in fact highly specific, with specificity conferred by [1] the location of receptors within a particular brain area, cell type, and subcellular space; [2] the synergistic interaction among receptors; and [3] the interaction with anchoring and scaffolding proteins. Kinases and phosphatases anchored in close vicinity to receptors modulate the activity of neighboring substrate proteins. Duration of stimuli and second messenger activity, together with the subcellular location of second messengers, allows for additional outcome specificity. Thus, although many signaling pathways share common effectors, a relatively small number of substrates can create a diverse range of responses. Here we will examine the conventional second messenger pathways of the basal ganglia [BG] represented by 3′-5′-cyclic adenosine monophosphate, phosphoinositide metabolism, calcium, and receptor tyrosine kinases, and the main receptors of the BG associated with them. We will provide examples of interactions between these pathways and discuss novel insights into intracellular signaling mechanisms.

I. Introduction

Second messengers are vital elements of intracellular signaling pathways that can be activated by receptor-ligand interaction at postsynaptic as well as presynaptic membranes. The nuclei of the basal ganglia participate in the modulation of a diverse range of behaviors, emotions, and motor functions in humans. Second messenger pathways in the basal ganglia have been studied extensively and many components of these pathways are targets of pharmaceutical therapies for neurological illnesses. This chapter will introduce the main second messenger pathways that participate in neurotransmission in the basal ganglia and will identify the key molecules that are needed for normal basal ganglia function.

Despite a limited number of molecules that function as second messengers, second messenger pathways show a high degree of specificity in linking particular receptors to cellular responses. For example, Ca2+ is a ubiquitous signaling molecule used by various G-protein coupled receptors, ionotropic receptors and ion channels, yet there is a high degree of specificity contingent on the source, location and timing of Ca2+ influx into the cytoplasm. Although we will focus here on common themes of signaling pathways, it is important to keep in mind that neurons have the ability to navigate these pathways to very selective targets. These targets are usually proteins that, through covalent modifications such as phosphorylation, alter their enzymatic activity, affect membrane permeability or mediate vesicle fusion. The initial, rapid effects of signal transduction pathways are not dependent on protein synthesis, but can be followed by long-lasting responses that involve the activation of transcription factors, induction of gene expression and protein synthesis, and ultimately rearrangements of synapses.

In this chapter we will highlight the major second messenger pathways that are involved in basal ganglia neurotransmission [see Chapter 1 for an overview of basal ganglia circuits]. As discussed in other chapters in this book, different receptor types co-exist in any given neuron in any particular brain region [see Chapter 4]. These receptors can activate multiple second messenger pathways simultaneously in opposing or synergistic fashion. Thus, cells can integrate information from multiple brain areas. The dynamic integration of various signals is of interest for therapeutic approaches that target disorders of basal ganglia nuclei.

Second-messenger systems

Second messengers are small intracellular molecules that mediate the effects of first messengers, i.e., neurotransmitters and hormones. Some of the important second messengers in the nervous system are cAMP, cyclic guanosine monophosphate [cGMP], diacylglycerol [DAG], inositol trisphosphate [IP3], and Ca2 + ions. Formation of cAMP and cGMP is catalyzed by the enzymes adenylyl cyclase and guanylyl cyclase, respectively. In retinal cells, cGMP levels are regulated by rhodopsin-induced stimulation of phosphodiesterase [PDE], the enzyme that degrades cGMP. DAG and IP3 are second messengers that are jointly liberated from the membrane lipid phosphatidylinositol bisphosphate [PIP2] by a PIP2-specific phospholipase C. The final second messenger, Ca2 +, can enter the neuronal cytoplasm from several different sources. Thus, cytoplasmic Ca2 + concentrations can be elevated either by influx through NMDA receptor channels and/or voltage-gated Ca2 + channels, or by release from intracellular storage compartments such as the smooth endoplasmic reticulum.

Second messengers vary in their range of action within a cell. For example, Ca2 + has a very short range of action due to factors such as cytoplasmic buffering and sequestration by internal storage sites. In contrast, IP3 and particularly cAMP seem to diffuse considerably longer distances before being metabolized. This is important in light of the fact that some mechanisms of plasticity may require the coordinated action of several second messengers. Therefore, if Ca2 + levels are locally elevated in a dendritic spine [perhaps due to the activation of NMDA receptor channels] but adenylyl cyclase-coupled receptors are not present in that spine, cAMP generated at another location in the dendrite might be able to reach the spine and interact with the Ca2 + to produce an appropriate postsynaptic response [Kasai & Petersen, 1994].

Second messengers generally operate through activation of protein kinases. These are enzymes that modify the functioning of various target proteins through the addition of phosphate groups to specific amino-acid residues [i.e., through phosphorylation]. The electrically charged phosphate groups alter the conformation of the affected proteins, thereby influencing their biological activity. In the case of an enzyme, for example, its catalytic activity might be either increased or reduced in the phosphorylated state. Phosphorylated proteins are returned to their original state by enzymes called phosphatases. This process terminates the cellular effects of second messengers by reversing the phosphorylation-induced changes in protein function.

Each second messenger is associated with a particular type of protein kinase. For example, cAMP activates cAMP-dependent protein kinase [also called protein kinase A; PKA], whereas cGMP similarly functions via cGMP-dependent kinase. Ca2 + first binds to a receptor protein called calmodulin, and then this complex can activate Ca2 +/calmodulin-dependent protein kinase. DAG, one of the second messengers produced from PIP2 hydrolysis, remains in the membrane and activates protein kinase C [PKC]. The other messenger, IP3, is liberated into the cytoplasm. It subsequently binds to a receptor on endoplasmic reticulum membranes and opens a channel for Ca2 + release from the endoplasmic reticulum into the cytoplasm [here we may consider Ca2 + to be a third messenger in the biochemical cascade]. The phospholipase C/PIP2 system [sometimes termed the phosphoinositide second-messenger system] can therefore activate a variety of Ca2 +-dependent mechanisms. One of these actually turns out to be protein kinase C, because this enzyme is activated by Ca2 + as well as DAG.

An interesting feature of the type II Ca2 +/calmodulin-dependent protein kinase [CaMK II] enables it to act as a kind of "molecular switch." When activated, this enzyme can engage in autophosphorylation, i.e., phosphorylation of itself. This "turns on the switch" by causing the kinase to become temporarily independent of Ca2 +. Eventually, the switch is turned off when the kinase becomes dephosphorylated. Some investigators have hypothesized that conversion of CaMK II and possibly other kinases to a stimulation-independent state may play a role in long-term potentiation or other forms of neuronal plasticity [Bliss & Collingridge, 1993; also see Frey, this volume].

Second-messenger-induced protein phosphorylation alters numerous functions related to synaptic transmission [Figure 6]. Among the neuronal proteins subject to phosphorylation are the neurotransmitter-synthesizing enzymes tyrosine hydroxylase and tryptophan hydroxylase, a number of different transmitter receptors, voltage-gated ion channels, synaptic-vesicle proteins, cytoskeletal proteins, and nuclear proteins involved in gene regulation. Second-messenger-induced phosphorylation thus plays a critical role in virtually all aspects of neuronal signaling. Different populations of neurons may exhibit distinctive patterns of phosphorylated proteins, depending on the transmitter inputs and receptor subtypes found in each population, the G proteins and second messengers activated by those receptors, and the available substrates for the kinases that are stimulated.

1 Introduction

Second messengers mediate an enormous spectrum of cellular responses to external stimuli, ranging from the regulation of cell proliferation and metabolism to cell death. Notably, different signals might arise within one cell at the same time, rendering it absolutely necessary for the cell to achieve signal specificity [Zaccolo and Pozzan, 2003; Zaccolo et al., 2002]. Clearly, a vast proportion of this specificity is due to amplitude, pattern [e.g., oscillations], and subcellular compartmentalization of signals. In particular, the latter is completely cell-type dependent and can usually not be reconstituted in vitro in a reliable manner. Therefore, the physiological roles of second messenger signaling can often only be understood when studied in the living tissue or in the intact organism.

Since the discovery of green fluorescent protein [GFP], numerous fluorescent, genetically encoded sensors able to monitor second messengers in living cells were created and new ones are continuously developed [Pozzan et al., 2003; Whitaker, 2010; Zhang et al., 2002]. In combination with adequate approaches, genetically modified organisms, and molecular or electronic biosensors measuring additional biophysical parameters, the use of GFP-based sensors can now deliver invaluable insights into many second messenger-dependent physiological processes in intact tissue. In most cases, probes for second messenger dynamics exploit Förster resonance energy transfer [FRET], usually between cyan and yellow fluorescent proteins [FPs], to transform conformational changes in the sensor molecule into measurable changes in their fluorescence spectra. The investigation of such probes in living samples has been enabled by multiphoton imaging, which allows excitation of short-wavelength dyes deep in tissue. However, the merit of tissue penetration of multiphoton light is dampened by its broad excitation range, which makes the discrimination of more than two fluorescent probes difficult. However, since dyes with longer wavelengths can efficiently be visualized even with standard confocal microscopy deep in tissue, multimodal imaging, using multiphoton together with single-photon confocal microscopy is useful to combine the visualization of second messenger signals with other readouts.

Transgenic techniques have rendered possible the introduction of genetically encoded probes into mice and a few other model organisms. However, at least in mammals, these methods cannot cope with the speed of novel sensors development and the progress in biomedical research. Therefore, faster approaches to introduce molecular sensors into target tissues are needed. While for some organs viral vectors might be ideal, electroporation-mediated gene transfer has proven to be an efficient means in skeletal muscle.

This chapter first describes the steps from genetic manipulation of skeletal muscle to the multimodal imaging and data analysis of second messenger signaling and other factors in this tissue in living mice. Then, we introduce adaptations of this paradigm to other tissues, namely, cardiac muscle and neuronal tissue.

Second Messengers and Their Functions

Most second messengers activate enzymatic processes, and are thus part of biochemical cascades that serve to amplify the responses to the first messenger. A number of protein kinases are the targets of second messengers, although protein phosphatases can also be activated by second messengers. As a result, a large variety of proteins can be functionally modified following the activation of metabotropic receptors. These include cytoskeletal proteins, neurotransmitter receptors, and ionic channels as well as transcription factors that regulate the expression of specific genes. Thus, the properties of ionotropic receptors can be regulated by phosphorylation reactions triggered by activation of a number of metabotropic receptors. In particular, channel kinetics, desensitization parameters, and even localization of a particular receptor can be modified as a result of the previous activation of metabotropic receptors for the same or a different neurotransmitter in its vicinity. As discussed later, some of these biochemical cascades have properties of biochemical switches, and have been postulated to play important roles in synaptic plasticity processes. In any event, second messenger-mediated cascades are highly regulated, and provide for rich interactions between receptors for several neurotransmitters.

VC4 Second Messengers and Transduction Pathways

Second messengers and internal biochemical events following mechanical or chemical stimuli have been the subject of study in various protists. In the case of the Pantophobiac mutants of Paramecium, for example, it was found that normal behavior could be restored by microinjection of wild-type calmodulin [CaM]. This led to the discovery that these mutants were specific point mutations with amino acid substitutions at specific CaM sites. Further studies showed that the Ca2+-CaM complex regulates calcium-dependent Na+ channels by direct interaction and is also required for the functioning of K+ channels. Work on responses to acetate and biotin in mutants with different defects in ion conductances, however, indicates that these two stimuli operate via different ion channels [Bell et al., 2007, Valentine et al., 2008].

Progress at this level has been greatest with the cellular slime mold Dictyostelium discoideum. In this organism, there appear to be several chemosensory transduction pathways, similar in general to those found in animal cells, such as leukocytes, but differing in some aspects [Van Haastert and Veltman, 2007]. In both cases, the presence of multiple pathways introduces a complexity of potential response mechanisms and behavior [Iglesias and Devreotes, 2008].