1457CHAPTER 123 Molecular Mechanisms of Drug Actions Endogenous agonists for these receptors include steroid and thyroid hormones as well as agents such as retinoic acid and vita min D The most common[.]
CHAPTER 123 Molecular Mechanisms of Drug Actions Endogenous agonists for these receptors include steroid and thyroid hormones as well as agents such as retinoic acid and vitamin D The most commonly used drugs that target these receptors include exogenous steroids and lipid-lowering agents For many of these receptors, the corresponding hormone or vitamin has not been identified; therefore, these receptors are referred to as orphan nuclear receptors Receptor Regulation Continued exposure of a receptor to an agonist often results in a progressive loss of receptor responsiveness, with a diminished receptor-mediated response over time This is called desensitization (or tachyphylaxis) and is classified into two forms Homologous desensitization is a process in which only the activated receptor is “turned off” or desensitized, whereas heterologous (cross-) desensitization refers to processes in which the activation of one type of receptor can result in the desensitization of other types of receptors Interaction of the receptor with an antagonist prevents the occurrence of desensitization In general, desensitization occurs in three ways (Fig 123.12A): (1) inactivation or uncoupling of the receptor, which is usually the result of receptor phosphorylation and occurs within seconds to minutes of agonist exposure; (2) sequestration of the receptor in endosomes (from there, the receptor is recycled to the cell membrane); and (3) downregulation, which is characterized by receptor endocytosis and destruction in lysosomes with a net loss of receptors in the cell (at the cell membrane and within the cell) The latter develops more slowly than uncoupling, taking hours to days.27 In addition to receptor degradation, decreased synthesis of the receptor also contributes to this process Downregulation is responsible for the decreased responsiveness to prolonged exogenous catecholamine infusion frequently seen in the critical care population.28 Desensitization is usually reversible, within minutes (inactivation) to hours (sequestration/downregulation) of removal of the agonist depending on the specific receptor and cell type, concentration of the agonist, and duration of exposure to the agonist Homologous desensitization of GPCRs results from these three distinct and coordinated processes (Fig 123.12B).29 It begins within seconds of exposure to the agonist and is initiated by phosphorylation of the receptor by G protein–coupled receptor kinases (GRKs) and second messenger–dependent protein kinases (protein kinase A [PKA] and protein kinase C [PKC]) Once phosphorylated, the receptor binds with high affinity to members of the arrestin gene family, the b-arrestins The b-arrestin binding prevents the receptor–G protein interaction, leading to termination of signaling by G protein effectors (receptor inactivation or uncoupling) The receptor-bound b-arrestin can also act as an adapter protein to couple the receptor to clathrin-coated pits, inducing receptor-mediated endocytosis or sequestration Subsequently, the receptor is either recycled to the cell membrane or degraded (receptor downregulation) Resensitization of a GPCR requires its dephosphorylation and dissociation from its agonist In contrast to homologous desensitization, heterologous desensitization of GPCRs occurs when inhibition of one GPCR is induced by the activation of another GPCR One well-recognized mechanism is the phosphorylation of one GPCR by second messenger–dependent protein kinases (PKA and PKC) activated by any other GPCRs Such phosphorylation of the receptor impairs receptor–G protein coupling and leads to the inactivation of the receptor However, it is becoming increasingly clear that receptor 1457 phosphorylation is not the exclusive mediator of heterologous desensitization and that events downstream are involved.30 Similar to GPCRs, channel-linked and enzyme-linked receptors are desensitized following prolonged or repeated agonist exposure Channel-linked receptors are phosphorylated by second messenger–dependent protein kinases while tyrosine kinase receptors are internalized Upregulation refers to the increase in receptor sensitivity seen in the setting of lack of agonist stimulation or prolonged presence of a receptor antagonist This is best exemplified by a phenomenon seen when a b-adrenergic blocking agent such as propranolol is administered for a long period of time and abruptly discontinued Because a greater number of sensitized b-adrenergic receptors become available for stimulation by endogenous agonists, rebound hypertension is observed Signal Transduction Mechanisms: Intracellular Messengers and Effectors After binding of an agonist to receptors such as GPCRs or enzyme-linked receptors, the signal transduction mechanisms from the membrane first involve the production of second messengers such as cyclic adenosine monophosphate (cAMP), cGMP, arachidonic acid and its metabolites, diacylglycerol (DAG), inositol 1,4,5-triphosphate (IP3), and Ca21 These, in turn, activate protein kinases and calcium-binding proteins, all of which result in different biological effects (Fig 123.13) Thus, the synthesis and degradation of intracellular second messengers are described first, followed by a review of the role of protein kinases and calciumbinding proteins in the transduction mechanisms Second Messengers Cyclic Adenosine Monophosphate This pathway is involved in signal transduction initiated by binding of agonists to GPCRs cAMP is synthesized from ATP after the action of adenylate cyclase, which is a transmembrane glycoprotein of the cell membrane.31 cAMP regulates many aspects of cellular function (cell division and differentiation, ion transport, and so on) by one common mechanism involving activation of protein kinases These, in turn, regulate the function of many different cellular proteins by catalyzing the phosphorylation of serine and threonine residues Phosphorylation can then either activate or inhibit target enzymes or ion channels.12 As previously mentioned, receptors coupled with Gs proteins stimulate adenylate cyclase and produce an increase in cAMP, whereas receptors coupled with Gi proteins inhibit adenylate cyclase and reduce cAMP The degradation of cAMP is catalyzed by phosphodiesterases leading to the production of 59-AMP, an inactive product Phosphodiesterases are a complex family of enzymes divided into 11 groups according to mechanism of regulation, selectivity for the substrate (cAMP or cGMP), preferential localization, and sensitivity to various inhibitors.32 In critically ill children, milrinone, used for its positive inotropic effect and vasodilating properties, is a phosphodiesterase inhibitor selective for the type III isoenzyme Cyclic Guanosine Monophosphate As discussed previously, guanylate cyclase is part of the cytosolic portion of some transmembrane receptors (membrane form) but also exists as a cytosolic enzyme (soluble form) activated by various molecules, including NO Stimulation of guanylate cyclase 1458 S E C T I O N X I I I Pediatric Critical Care: Pharmacology and Toxicology Receptor Endosome Endosome Lysosome G E G E Pi G E Pi β-Arrestin Clathrin Endosomal vesicle Endosomal vesicle GPCR GPCR Sequestration Resensitization β-Arrestin CYTOPLASM GPCR Pi Pi β-Arrestin Lysosome Receptor degradation Pi = Agonist Pi β-Arrestin β-Arrestin GRK PK B GPCR MEMBRANE GPCR Phosphorylation and Uncoupling GPCR = Agonist GPCR RECEPTOR SEQUESTRATION GPCR A RECEPTOR DOWNREGULATION RECEPTOR INACTIVATION Phosphatase Downregulation • Fig 123.12 Desensitization in response to an agonist (A) Ways in which receptors can become desen- sitized to an agonist (B) Homologous desensitization of GPCR E, G protein effectors; G, G protein; GPCR, G protein–coupled receptor; GRK, G protein–coupled receptor kinase; PK, second-messenger– dependent protein kinases (A, Modified from Alberts B, Johnson A, Lewis J, et al Molecular Biology of the Cell 4th ed London: Garland Science; 2002 B, Modified from Luttrell LM, Lefkowitz RJ The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals J Cell Sci 2002;115;455–465.) results in the accumulation of cGMP This second messenger then regulates complex signaling cascades through immediate downstream effectors, including cGMP-dependent protein kinases (e.g., protein kinase G [PKG]), cGMP-regulated phosphodiesterases (mainly types II and III), and cyclic nucleotide-gated ion channels (cells of the retina), which eventually leads to a variety of physiologic effects.33 For example, NO readily passes across the target cell membrane and activates soluble guanylate cyclase in vascular smooth muscle, resulting in increased cGMP production with the regulation of various downstream targets such as protein kinases and ion channels, which culminates in vasodilation As with cAMP, the degradation of cGMP into inactive GMP is catalyzed by phosphodiesterases In pulmonary and penile vascular smooth muscle cells, phosphodiesterase type V is responsible for the degradation of cGMP; inhibition of this enzyme results in an accumulation of cGMP in the cytosol with smooth muscle relaxation and vasodilation As such, phosphodiesterase type V inhibitors (e.g., sildenafil) are widely recognized as efficacious for the treatment of erectile dysfunction in men and have been shown CHAPTER 123 Molecular Mechanisms of Drug Actions Receptors Target enzymes Guanylate cyclase 1459 G proteins Adenylate cyclase Phospholipase C DAG lipase Second messengers cGMP cAMP IP3 DAG ↑[Ca2+]i Protein kinases Effectors PKG Enzymes, transport proteins, etc AA Prostaglandins leukotrienes Activated PKA calcium-binding PKC proteins Contractile proteins Released as local hormones Ion channels • Fig 123.13 Transduction mechanisms of membrane signaling AA, Arachidonic acid; [Ca21]I, intracellular calcium concentration; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; DAG, diacylglycerol; IP3, inositol 1,4,5-triphosphate; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G (Modified from Rang HP, Dale MM, Ritter JM, et al Rang and Dale’s Pharmacology 7th ed Philadelphia: Elsevier; 2012.) to induce pulmonary vasodilation both in children and adults Thus, they are part of the strategies available for the treatment of pulmonary hypertension.34–36 Arachidonic Acid and Its Metabolites Arachidonic acid and its metabolites (prostaglandins and leuko trienes) are now considered intracellular messengers.37 Arachidonic acid is a component of membrane phospholipids released either in a one-step process, after phospholipase A2 (PLA2) action, or a twostep process, after phospholipase C and DAG lipase actions Arachidonic acid is then metabolized by cyclooxygenase (COX) and 5-lipoxygenase, resulting in the synthesis of prostaglandins and leukotrienes, respectively These intracellular messengers play an important role in the regulation of signal transduction implicated in pain and inflammatory responses Corticosteroids inhibit PLA2 activity, whereas nonsteroidal antiinflammatory drugs inhibit COX activity Diacylglycerol and Inositol Triphosphate Phospholipase C is the target enzyme for some GPCRs (phospholipase C-b) as well as enzyme-linked receptors such as tyrosine kinase receptors (phospholipase C-g) It splits phosphatidylinositol, a membrane-bound phospholipid, into DAG and IP3, both of which function as second messengers The most important function of DAG is to activate the membrane-bound PKC, which catalyzes the phosphorylation of a variety of intracellular proteins IP3 binds to and opens an IP3-gated Ca21 release channel on the endoplasmic reticulum membrane, which results in an increase in free intracellular Ca21 concentration ([Ca21]i) Calcium Ions [Ca21]i is critically important as a regulator of cell function An increase in [Ca21]i is the most important intracellular messenger signaling pathway known in biological systems When [Ca21]i is at its baseline value, few proteins have an affinity sufficient to bind to Ca21 Once membrane signaling occurs, an increase in [Ca21]i results, derived from either the extracellular space or from the lumen of the endoplasmic reticulum, and allows binding of proteins to Ca21 Finally, this binding can trigger contraction, secretion, a modification in metabolism regulation, or several other effects depending on the cell type involved To maintain a low resting [Ca21]i, Ca21 is permanently expulsed from the cytosol into the extracellular compartment or into the endoplasmic reticulum via a Ca21-ATPase and Na1/Ca21 exchanger Phosphorylation of Proteins Many receptor-mediated signals produce variations in the concentration of second messengers—such as cAMP, cGMP, arachidonic acid, DAG, IP3, and Ca21—as previously discussed These can then modify the activity of other proteins, mainly protein kinases and calcium-binding proteins Protein Kinases Protein kinases are enzymes located in the cytoplasm that phosphorylate proteins The main protein kinases consist of PKA, PKG, and PKC38 as well as tyrosyl protein kinases (part of tyrosine kinase receptors) They are distinguished from each other by the different intracellular second messengers involved in their 1460 S E C T I O N X I I I Pediatric Critical Care: Pharmacology and Toxicology regulation and by the selective substrates that they use They all have a binding site for Mg21-ATP (phosphate donor) and for substrate protein as well as various regulatory sites Phosphorylation of these proteins is short lived because protein phosphatases rapidly dephosphorylate proteins previously phosphorylated by protein kinases, thus, terminating the intracellular signal Calcium-Binding Proteins Calcium exerts its control in cellular function by virtue of its ability to regulate the activity of many different proteins, such as channels, transporters, and transcription factors In the majority of cases, a calcium-binding protein serves as an intermediate between Ca21 and the regulated functional protein Calcium-binding proteins represent a large group of cytosolic proteins and include the calmodulin and annexin (or lipocortin) families Multiple Drug Targets Within an Organ System: The Myocardium This section highlights the molecular mechanisms behind therapeutic agents that increase contractility and accelerate relaxation within the myocardium During cardiac excitation-contraction coupling, Ca21 is the essential second messenger (Fig 123.14).39 During the depolarization phase of the cardiac action potential, Ca21 enters the cell through voltage-gated L-type Ca21 channels Ca21 entry triggers Ca21 release from the sarcoplasmic reticulum (SR) through an SR membrane ion channel—the cardiac/isoform ryanodine receptor (RyR2) This process is known as calciuminduced Ca21 release The combination of Ca21 influx and calcium-induced Ca21 release raises [Ca21]i, allowing Ca21 binding to troponin C (TnC), which permits cross-bridging between actin and myosin and, ultimately, contraction For relaxation to occur, [Ca21]i must decline, allowing Ca21 to dissociate from TnC This requires Ca21 transport out of the cytosol by four pathways: SR Ca21-ATPase (the main one, also known as SERCA), sarcolemmal Na1/Ca21 exchanger, sarcolemmal Ca21-ATPase, and mitochondrial Ca21 uniport SR Ca21-ATPase activity is modulated by phospholamban, an endogenous inhibitor Under physiologic conditions, there is no net gain or loss of cellular Ca21 with each contraction-relaxation cycle There are two main ways to pharmacologically increase the strength of cardiac contraction (see Fig 123.14)39,40: (1) by increasing the amount of [Ca21]i available for binding to TnC or (2) by increasing the sensitivity of myofilaments to Ca21 Catecholamines and phosphodiesterase type III (PDEIII) inhibitors b-Adrenergic receptor stimulation by catecholamines activates a GTP-binding protein (Gs), which stimulates adenylate cyclase to produce cAMP, whereas PDEIII inhibitors (e.g., milrinone) prevent cAMP degradation The resulting increase in cAMP activates PKA, which, in turn, phosphorylates intracellular targets, including voltage-gated L-type Ca21 channels, RyR2, phospholamban, and troponin I (TnI) Phosphorylation of voltage-gated L-type Ca21 channels enhances Ca21 Epi/Norepi ACh Sarcolemma AC α β-AR Gs α β α Gi AC γ – M2– Rec cAMP GTP Reg GTP GTP GTP cAMP ATP PKA AKAP PKA P Ca Sarcoplasmic reticulum P RyR Reg PKA Ca Ca ATP PLB P P Troponin I Ca • Fig 123.14 Cardiac Ca Myofilaments excitation-contraction coupling and molecular targets of therapeutic agents with positive inotropic and lusitropic effects (Note: Under physiologic conditions, NCX works mainly in the Ca21 extrusion mode; however, if intracellular Na1 concentration is elevated, as is the case with digoxin, it can work in the Ca21 influx mode.) AC, Adenylate cyclase; ATP, adenosine triphosphate; b-AR, badrenergic receptor; cAMP, cyclic adenosine monophosphate; Gs, stimulatory G protein; NCX, Na1/Ca21 exchanger; P, phosphorus; PDE, phosphodiesterase; PKA, protein kinase A; PLB, phospholamban; RyR2, cardiac/isoform ryanodine receptor; SR, sarcoplasmic reticulum; TnI, troponin I; TnC, troponin C CHAPTER 123 Molecular Mechanisms of Drug Actions entry into the cytosol, with a subsequent increase in calciuminduced Ca21 release from the SR and contraction (positive inotropic effect of catecholamines and PDEIII inhibitors) In contrast, phosphorylation of phospholamban activates SR Ca21-ATPase with increased Ca21 transport from the cytosol back to the SR, thus, promoting relaxation (positive lusitropic effect of catecholamines and PDEIII inhibitors) This action also contributes to the overall gain in cardiac excitationcontraction coupling by increasing the SR Ca21 content available for the next contraction However, such increased loading of the SR with Ca21 may be a key factor in the development of Ca21-mediated arrhythmias.41 The lusitropic effect of catecholamines and PDEIII inhibitors is also mediated by phosphorylation of TnI, which decreases the affinity of myofilaments for Ca21 Digoxin Digoxin enhances myocardial contractility, although modestly, by inhibiting the Na1/K1-ATPase pump with a resultant mild increase in intracellular Na1 This increase of Na1 subsequently inhibits the extrusion of Ca21 from the cytosol outside the cell by the sarcolemmal Na1/Ca21 exchanger Ca21 not extruded from the cytosol is stored in SR and allows increased release of Ca21 during the next contraction Ca21 sensitizers More recently, Ca21 sensitizers (e.g., levosimendan), a new class of inotropic agents, have been developed They improve cardiac contractility by binding to TnC and stabilizing its interaction with Ca21, which results in prolonged interaction of actin-myosin filaments One possible limitation of some Ca21 sensitizers is worsening diastolic function due to facilitation of cross-bridging at diastolic Ca21 concentrations However, this does not appear to be the case for levosimendan because its binding to TnC depends on [Ca21]i (i.e., when [Ca21]i increases during systole, it facilitates actin-myosin interaction, and when [Ca21]i decreases during diastole,, it does not) One potential beneficial effect of these agents compared with catecholamines and PDEIII inhibitors comes from the fact that they not increase [Ca21]i and, as such, have neutral effects on myocardial oxygen demand and heart rhythm.40 Promising new inotropic agents have been developed.42,43 Among these, istaroxime, a lusitropic agent, inhibits the Na1/K1ATPase pump (increased inotropy, like digoxin) and stimulates SR Ca21-ATPase (accelerated relaxation) Cardiac myosin activators (e.g., omecamtiv mecarbil) represent a new class of compounds that directly influence the cross-bridge cycle They promote actin-dependent phosphate release, moving the cross-bridge into its strongly bound force-producing state As a consequence, more cross-bridges are activated per unit of time, and the contractile force increases Although still under investigation in adults, these novel approaches to improving cardiac function provide the hope that such agents may soon be available 1461 signaling pathways may partially account for therapeutic failure and toxicity Most genetic variations involve single-nucleotide polymorphisms (SNPs)—that is, the exchange of a single nucleotide in the DNA sequence Small insertions and deletions, variable-number tandem repeats, gene deletions, and gene duplications can also take place Depending on where SNPs occur, they can result in no change in the protein amino acid sequence (silent polymorphism or synonymous SNP) or in a change in the coded amino acid sequence (nonsynonymous SNP) that can have no functional consequence or can result in altered protein function The latter can have significant clinical or therapeutic implications In addition, given that genes often present many SNPs, it has become increasingly recognized that single SNPs fail to predict drug responses, whereas combinations of SNPs on a given chromosome (specific haplotype) are clinically more significant and can better determine drug effects.45 Genetic Polymorphisms and Drug Disposition Many major enzymes involved in phase I and phase II drug metabolism have known polymorphisms leading to phenotypic differences (i.e., clinically significant alteration in drug-metabolizing enzyme activities).44 For a specific polymorphic drug-metabolizing enzyme, homozygous individuals for the wild-type allele exhibit normal enzymatic activity (extensive metabolizers), heterozygous individuals may have reduced enzymatic activity (heterozygous extensive/intermediate metabolizers), and homozygous individuals with the mutant allele have low enzymatic activity (poor metabolizers) For some enzymes (e.g., CYP2D6), individuals have ultrarapid metabolism as a result of gene duplications of functional alleles (ultrarapid metabolizers) The clinical consequences of such polymorphisms may be fourfold: (1) poor metabolizers can have an enhanced drug effect, either therapeutic or toxic, resulting from higher plasma concentrations of a given drug; (2) poor metabolizers can experience a diminished drug effect resulting from the inability of a prodrug to be converted into the active metabolite due to low enzymatic activity; (3) ultrarapid metabolizers can experience diminished drug effect resulting from markedly lower plasma concentrations of a given drug; and (4) ultrarapid metabolizers can experience enhanced drug effect from an excessive conversion of a prodrug into the active metabolite as a result of supranormal enzymatic activity Codeine is a good example of the clinical impact of CYP2D6 polymorphism CYP2D6 catalyzes the biotransformation of codeine into morphine, the active compound CYP2D6 poor metabolizers are at increased risk of experiencing inadequate analgesia as a failure to convert codeine into morphine, whereas CYP2D6 ultrarapid metabolizers may be at increased risk for opioid-related adverse effects as a result of increased formation of morphine from codeine.46,47 Drug Response and Genetic Polymorphisms Genetic Polymorphisms, Drug Targets, and Signaling Mechanisms “The right dose of the right drug to the right person” is one of the goals of pharmacogenomics and personalized medicine It is estimated that genetic factors can account for 20% to 95% of variability in drug disposition and effects.44 Although drug response variation is often multifactorial (even more so in critically ill children), genetic polymorphisms occurring in genes that encode drug-metabolizing enzymes, drug targets (e.g., receptors, enzymes), drug transporters, or proteins involved in Genetic polymorphisms in signaling mechanisms involving GPCRs, transporters, and enzymes have also been identified and can influence drug response.48–50 Mutations in G proteins have also been shown to cause certain diseases.51 As an example, there has been considerable progress in cardiovascular pharmacogenetics in adults, for whom the mainstay of treatment comprises b-blockers, angiotensin-converting enzyme inhibitors, aldosterone antagonists, and diuretics The most studied genetic variants relating to ... Drug Targets Within an Organ System: The Myocardium This section highlights the molecular mechanisms behind therapeutic agents that increase contractility and accelerate relaxation within the myocardium... muscle cells, phosphodiesterase type V is responsible for the degradation of cGMP; inhibition of this enzyme results in an accumulation of cGMP in the cytosol with smooth muscle relaxation and... or from the lumen of the endoplasmic reticulum, and allows binding of proteins to Ca21 Finally, this binding can trigger contraction, secretion, a modification in metabolism regulation, or several