1452 SECTION XII I Pediatric Critical Care Pharmacology and Toxicology more than 50 members distributed in different families with those mainly involved in the transport of drugs consisting of the mul[.]
1452 S E C T I O N X I I I Pediatric Critical Care: Pharmacology and Toxicology more than 50 members distributed in different families with those mainly involved in the transport of drugs consisting of the multidrug resistance (MDR) family, the multidrug resistance protein (MRP) family, the breast cancer resistance protein, and the bilesalt export protein.14 The main function of these ABC transporters is to facilitate the efflux of drugs from the cells through apical and basal membranes P-glycoprotein (PgP) or MDR1, the most extensively studied ABC transporter, is widely distributed with a broad substrate specificity (e.g., tacrolimus, digoxin, dexamethasone, chemotherapeutic agents [etoposide, doxorubicin, vinblastine], protease inhibitors) and is subject to drug interactions through inhibition or induction It limits intestinal drug absorption, participates in drug elimination (liver and kidney), and influences drug tissue distribution (e.g., it limits drug entry into the brain) Some cancer cells also express large amounts of PgP, rendering these cancers multidrug resistant Membrane transporters of the SLC superfamily mainly involved in the transport of drugs consist of organic anion transporting polypeptide, organic cation transporter, organic cation transporters novel, organic anion transporter (OAT), organic anion/urate transporter, multidrug and toxin extrusion protein, heteromeric organic solute transporter, peptide transporter, humane equilibrative nucleoside transporter, Na1-dependent taurocholate cotransporting polypeptide, and monocarboxylate transporters The main function of the SLC family is to facilitate the entry and the elimination of drugs from the cells through apical and basal membranes.15 Receptor Type and Regulation Four families of receptors, three cell surface receptor types and one nuclear receptor, have been described (Fig 123.5) Most transmembrane signaling is accomplished by only a few molecular mechanisms, each of which has been adapted to transduce many different signals These protein families include cell surface receptors and receptors within the cell as well as enzymes and other components that generate, amplify, coordinate, and terminate post-receptor signaling G Protein–Coupled Receptors In 1994, Alfred G Gilman and Martin Rodbell were awarded the Nobel Prize in physiology or medicine for their discovery of G proteins and their role in signal transduction in cells.16 Furthermore, in 2012, Robert Lefkowitz and Brian Kobilka were awarded the Nobel Prize in chemistry for their discoveries that reveal the inner workings of the b-adrenergic receptor, which led to the seminal discovery that all G protein–coupled receptors (GPCRs) have a similar molecular structure.17 G proteins are a superfamily of propeller proteins that allow the transduction between the activated receptor (by an agonist) and different intracellular effectors such as enzymes or ion channels, relaying signals from more than 1000 receptors.18 GPCRs, also known as metabotropic receptors, are the first component of the cellular amplification cascade (Fig 123.6) The activation of target enzymes through GPCRs Channel-linked receptors (ionotropic receptors) G protein–coupled receptors (metabotropic receptors) Enzyme-linked receptors (eg, kinase-linked) Nuclear receptors Ions Ions E R G G + or – + or – R Gene transcription Protein synthesis Protein synthesis Cellular effects Cellular effects Cellular effects Seconds Milliseconds Hours Hours Muscarinic ACh receptor Nicotinic ACh receptor Epidermal growth factor receptor Steroid receptors Protein phosphorylation Cellular effects Examples Hyperpolarization or depolarization Protein phosphorylation Gene transcription Ca2+ release Time scale R/E NUCLEUS Second messengers Change in excitability R Opioid receptor α- and β-adrenergic receptors Others NMDA receptor Cytokine receptor GABAA receptor = Agonist • Fig 123.5 Four families of receptors are classically described: G protein–coupled receptors, channel- linked receptors, enzyme-linked receptors, and nuclear receptors ACh, Acetylcholine; E, enzyme; G, G protein; GABA, g-aminobutyric acid; NMDA, N-methyl-d-aspartate; R, receptor (Modified from Rang HP, Dale MM, Ritter JM, et al Rang and Dale’s Pharmacology 7th ed Philadelphia: Elsevier; 2012.) CHAPTER 123 Molecular Mechanisms of Drug Actions Amplification Amplification Amplification 1453 Amplification β-AR C α γ β GDP GPCR = Agonist Activated adenylate cyclase cAMP Protein kinase A Phosphorylated enzymes Products CELLULAR EFFECTS • Fig 123.6 Cellular amplification cascade After binding to a G protein–coupled receptor (GPCR), a li- gand (agonist) activates a target enzyme (adenylate cyclase), which synthesizes a second messenger (cAMP) The latter then activates other enzymes (protein kinases) that phosphorylate proteins and mediate specific cellular effects b-AR, b-Adrenergic receptor; cAMP, cyclic adenosine monophosphate GDP, guanosine diphosphate leads to the synthesis of numerous second messengers, which, in turn, activate other enzymes The intervention of the second messenger system allows for the diversity of the cellular targets (discussed later) GPCRs are complex signaling machines that participate in most physiologic and pathophysiologic processes and represent the target, directly or indirectly, of approximately 40% of all current therapeutic agents.19 Of note, pharmacologic agents have been developed for approximately only 10% of GPCRs so far Three major families of GPCRs are defined based on their amino acid sequence Family 1, the largest, includes receptors for rhodopsin, monoamines (such as b-adrenergic receptors), neuropeptides, opioids, and chemokines Family consists mainly of receptors for peptides with a large molecular weight, such as calcitonin and secretin Family has, among others, receptors for glutamate (metabotropic), GABAB, and extracellular calcium Despite these differences, the families of GPCRs share characteristic structural and functional features All GPCRs share a common serpentine structure consisting of seven transmembrane domains with three extracellular and three intracellular loops The extracellular regions are involved in ligand binding, and the intracellular regions are primarily involved in signaling.20 The latter are coupled to a heterotrimeric guanine-nucleotide–binding regulatory protein (G protein) located on the cytoplasmic portion of the cell membrane and are made of three subunits Each G protein is composed of an a-subunit that is loosely bound to a tightly associated dimer made up of b- and g-subunits The activity of a trimeric G protein is regulated by the binding and hydrolysis of guanosine triphosphate (GTP) by the a-subunit (Fig 123.7) Each of the three subunits is encoded by a separate gene selected from more than 20 a, six b, and 12 g genes, respectively The a-subunit is essential in the “receptor-effector” coupling Various a-subunits define different G protein trimers (Gs stimulatory G protein; Gi inhibitory G protein; Go,Gq5 other G proteins), each of which regulates a distinctive set of downstream signaling pathways Table 123.2 shows some examples of GPCRs with their trimeric G protein along with their target enzymes or ion channels and second messengers Some receptors act by way of more than one type of G protein trimer (e.g., m opioid receptor) Approximately 50% of the GPCRs couple to Gi/ Go proteins, approximately 25% couple to Gs, and about the same amount couple to Gq proteins.21 Gs proteins (made of assubunits) can activate adenylate cyclase and are inhibited by the cholera toxin In contrast, Gi proteins (made of ai-subunits) can inhibit adenylate cyclase and open K1 channels and are inhibited by the pertussis toxin Small G proteins are monomeric G proteins with molecular weight of 20 to 40 kDa As with heterotrimeric G proteins, their activity depends on the binding of GTP More than 100 small G proteins have been identified They are classified into different families: Ras, Rho, Rab, Rap, Ran, and ARF They are ubiquitous and play key roles in numerous cellular functions, such as cell division, proliferation, differentiation, vesicle trafficking, cytoskeletal reorganization, and gene expression.22 Channel-Linked Receptors Also known as ligand-gated ion channels or ionotropic receptors, channel-linked receptors mediate fast responses, affecting ion fluxes and membrane potential These receptors possess four distinct characteristics: (1) activation by an agonist or inactivation by an antagonist; (2) flux of ions across a central pore; (3) ion selectivity; and (4) fluctuation among open, closed, and inactivated states Broadly speaking, two types have been identified: receptors of excitatory mediators and receptors of inhibitory mediators Receptors of excitatory mediators (glutamate, aspartate, and acetylcholine), which comprise the N-methyl-d-aspartate (NMDA; Fig 123.8A) and the nicotinic acetylcholine receptors, are receptors whose activation provokes depolarization of the cell, leading to propagation of the action potential and, ultimately, secretion of a neuromediator and muscular contraction, for example These receptors are permeable to monovalent and divalent cations, mainly Na1, K1, Ca21, and magnesium (Mg21) Ketamine, a dissociative anesthetic, is a noncompetitive NMDA receptor antagonist that prevents the opening of ion channels by 1454 S E C T I O N X I I I Pediatric Critical Care: Pharmacology and Toxicology Basal activation R R β γ α γ β GDP E α Agonist binding nucleotide exchange E R GTP GDP Pi α γ β GDP E GTP Hydrolysis R β γ α GTP = Agonist Gα dissociation from Gβγ E R Target enzyme activation β γ α E GTP • Fig 123.7 Functional cycle of the G proteins The receptor (R) becomes activated after binding of an agonist Guanosine diphosphate (GDP) bound to the G protein is replaced by guanosine triphosphate (GTP), and the a-subunit of the G protein dissociates from the bg-subunit complex The a-subunit/GTP complex binds to the target enzyme (E) or ion channel, whereas the bg-subunit complex stimulates several other downstream effectors The GTPase activity of the a-subunit is increased when the target enzyme or ion channel is bound, leading to hydrolysis of the bound GTP to GDP, whereupon the a-subunit reunites with the bg-subunit complex and the agonist dissociates from the receptor Examples of G Protein–Coupled Receptors With TABLE 123.2 Their Trimeric G Protein, Target Enzyme, or Ion Channel, and Second Messengers GPCR G Protein Target Enzyme/Ion Channel Second Messengers b1- and b2-Adrenergic D1 H2 V2 Gs h Adenylate cyclase h cAMP a2-Adrenergic M2,4 m Opioid AT1 Gi g Adenylate cyclase g cAMP a1-Adrenergic M1,3,5 ETA,B AT1 H1 V1 Gq/11 h Phospholipase C m-Opioid Gi/o Opens K1 channels m-Opioid Go Closes voltage-dependent Ca21 channels h IP3, DAG, [Ca21]i g [Ca21]i AT, Angiotensin; Ca21, calcium; [Ca21]i intracellular calcium concentration; cAMP, cyclic adenosine monophosphate; D, dopaminergic; DAG, diacylglycerol; ET, endothelin; Gi, inhibitory G protein; Go, other G proteins; GPCR, G protein–coupled receptor, Gs, stimulatory G protein; H, histamine; IP3, inositol 1,4,5-triphosphate; K1, potassium; M, muscarinic; V, vasopressin glutamate In addition, the potential neuroprotective effects of ketamine appear to be mediated via NMDA receptor blockade Receptors of inhibitory mediators—the activation of which provokes hyperpolarization of the cell and therefore decreases cellular excitability—are a group that includes GABAA (Fig 123.8B) and glycine receptors These ligand-gated ion channels are selective for anions such as chloride (Cl2) or phosphorus (PO432) GABA is the major inhibitory neurotransmitter in the central nervous system; drugs that potentiate GABAergic inhibition in the brain, such as benzodiazepines and barbiturates, result in sedation and hypnosis.7 Benzodiazepine agonists enhance Cl2 ion conductance induced by GABA by increasing the frequency of channel-opening events, whereas barbiturates seem to so by increasing the duration of the GABA-gated channel openings Both classes of agents bind to sites on the GABAA molecule that are different from each other as well as from the GABA receptor site Enzyme-Linked Receptors Enzyme-linked receptors have an extracellular ligand-binding domain linked to an intracellular domain that possesses an intrinsic catalytic activity This large and heterogeneous group of membrane receptors can be divided into four subfamilies according to their catalytic activity (tyrosine kinase, guanylate cyclase, tyrosine phosphatase, and serine/threonine kinase) Cytosolic enzymes presenting an activity similar to that of enzymelinked receptors are also considered to belong to this family of receptors (e.g., soluble guanylate cyclase receptors activated by nitric oxide [NO]) Tyrosine kinase receptors include receptors for neurotrophin,23 growth factors (epidermal growth factor, platelet-derived growth factor), and insulin along with many other trophic hormones These receptors shift from an inactive monomeric state to an active dimeric state upon agonist binding (dimerization) This is followed by autophosphorylation of the intracellular domain of each receptor and binding of SH2-domain proteins that are themselves phosphorylated Depending on the receptor subtype, SH2-domain proteins allow the phosphorylated receptor to activate other functional proteins, which eventually results in stimulation of gene transcription, or are enzymes such as phospholipases, leading to the formation of second messengers (discussed later) One important pathway involved in the transduction mechanisms of tyrosine kinase receptors includes the Ras/Raf/MAP kinase pathway, which is important in cell division, growth, and differentiation (Fig 123.9) CHAPTER 123 Molecular Mechanisms of Drug Actions GABAA receptor NMDA receptor Receptor site NMDA antagonists Glutamate Modulatory site Glycine antagonists Na+ Ca2+ – Receptor site GABA antagonists – + CI– – + Glycine GABA Modulatory sites Benzodiazepine agonists + – + – Mg2+ Barbiturates + – A Benzodiazepine inverse agonists + – Benzodiazepine antagonists Steroids Channelblocking drugs (eg, ketamine) K+ B • Fig 123.8 Two important members of the channel-linked receptors, the NMDA receptor (A) and the GABAA receptor (B) The main sites of drug action on these receptors are shown GABA, g-aminobutyric acid; NMDA, N-methyl-d-aspartate Growth factor Conformation change Tyrosine Phosphorylation Dimerization autophosphorylation of Grb2 Receptor domain Transmembrane α helix MEMBRANE Tyrosine kinase domain P P P P Ras GDP P Grb2 Activation of RAS GDP/GTP exchange Ras GTP Tyrosine residue Activation Raf Phosphorylation Grb2 Binding of SH2-domain protein (Grb2) Mek KINASE CASCADE Phosphorylation Map kinase Phosphorylation Various transcription factors NUCLEUS Gene transcription • Fig 123.9 Functioning of kinase-linked receptors The main steps are dimerization of the receptor, au- tophosphorylation, and phosphorylation of targeted proteins The growth factor pathway is shown with the kinase cascade involving the successive phosphorylation of many enzymes (Raf, Mek, Map kinase), eventually leading to gene transcription GDP, Guanosine diphosphate; GTP, guanosine triphosphate (Modified from Rang HP, Dale MM, Ritter JM, et al Rang and Dale’s Pharmacology 7th ed Philadelphia: Elsevier; 2012.) 1455 1456 S E C T I O N X I I I Pediatric Critical Care: Pharmacology and Toxicology Unlike tyrosine kinase receptors, cytokine receptors not usually possess intrinsic kinase activity; instead, they associate with cytosolic Janus kinases (JAKs) After dimerization of the receptors, which occurs after binding of the cytokine, JAKs phosphorylate tyrosine residues on the receptor, which then result in the binding of another set of proteins called signal transducers and activators of transcription (STATs) The bound STATs are themselves then phosphorylated by the JAKs and dimerize and dissociate to migrate in the nucleus and activate gene expression to regulate diverse biological processes controlling the synthesis and release of many inflammatory mediators, growth, development, and homeostasis Guanylate cyclase–linked receptors are unique because they synthesize their own second messengers upon agonist binding The natriuretic peptide receptors—including atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide receptors—belong to this family (Fig 123.10) The extracellular NH2terminal constitutes the binding domain There is a short transmembrane segment whose role is to anchor the receptor protein to the membrane The intracellular domain is made of two different entities: (1) a protein kinase homology domain whose function is to control and relay receptor activation to the catalytic domain and (2) a guanylate cyclase catalytic domain, also known as particulate guanylate cyclase, involved in the synthesis of cyclic guanosine monophosphate (cGMP) from GTP.24 In addition to this particulate guanylate cyclase (the membrane form of the enzyme), an intracellular soluble form exists It is a heterodimer consisting of a- and b-subunits, both of which are necessary for enzyme activity, and is expressed in most tissues, though not uniformly.25 It is activated by intermediate substances derived from the biosynthesis of eicosanoids (prostaglandins and leukotrienes) and by NO and NO donors such as sodium nitroprusside and nitroglycerin (discussed later) Guanylate cyclases and cGMP-mediated signaling cascades play a central role in the regulation of diverse pathophysiologic processes, including vascular smooth muscle motility, intestinal fluid and electrolyte homeostasis, and retinal phototransduction.26 Nuclear Receptors Nuclear receptors belong to a family of functionally and structurally related proteins They regulate gene expression and are not associated with a membrane Their principal mechanism of action is shown in Fig 123.11 The agonist, which must be lipid soluble, diffuses into the cell and binds to the nuclear receptor located either in the cytosol or nucleus The complex agonist-activated receptor then binds on high-affinity sites on DNA, hormone response element (HRE), situated on the promoter region of genes, whose transcription can then be induced or suppressed Because gene transcription is at their origin, these effects are slow to develop ANPCR Extracellular domain Transmembrane segment protein Kinase domain homology Guanylate cyclase catalytic domain Carboxyl terminal • Fig 123.10 Molecular structure of the natriuretic peptide receptors Left, Atrial natriuretic peptide–C receptor (ANPCR) is a clearance receptor that does not possess the kinase and guanylate cyclase domains It plays a role in the catabolism of natriuretic peptides Right, Typical particulate guanylate cyclase receptor (pGC; ANP-A or -B receptor) is shown with its extracellular dimeric protein-binding domain The intracellular domain consists of a protein kinase homology domain and a guanylate cyclase catalytic domain Target cell DNA Receptor receptor NUCLEUS HRE Activated receptor Repression Receptor Induction mRNA Cellular effects pGC Amino terminal Proteins + or – CYTOSOL = Ligand • Fig 123.11 Activation and action of nuclear receptors, located either in the cytosol (e.g., steroid receptors) or in the nucleus (e.g., vitamin D receptor) DNA, deoxyribonucleic acid; HRE, hormone response element; mRNA, messenger ribonucleic acid ... ligand-binding domain linked to an intracellular domain that possesses an intrinsic catalytic activity This large and heterogeneous group of membrane receptors can be divided into four subfamilies according... presenting an activity similar to that of enzymelinked receptors are also considered to belong to this family of receptors (e.g., soluble guanylate cyclase receptors activated by nitric oxide [NO])... from an inactive monomeric state to an active dimeric state upon agonist binding (dimerization) This is followed by autophosphorylation of the intracellular domain of each receptor and binding