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Cellular Physiology of Adrenergic Receptors
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The effects of adrenergic xenobiotics on the cell are primarily mediated through a secondary messenger system of cyclic adenosine monophosphate (cAMP). The intracellular cAMP concentration is regulated by the membrane interaction of three components: the adrenergic receptor, a “G-protein” complex, and adenyl cyclase, the enzyme that synthesizes cAMP in the cell.10,22,78,79 These receptors are described in detail in Chap. 14.
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The G protein serves as a “signal transducer” between the receptor molecule in the cell membrane and the effector enzyme, adenyl cyclase, in the cytosol. The G proteins consist of three subunits: α, β, and γ.14,52,53 The α subunit of the G protein complex binds to the adrenergic receptor in the cell membrane and to the adenyl cyclase enzyme. The G protein complex exists in several isomeric forms, depending on their interactions with the adenyl cyclase enzyme. These forms, Gs, Gi, and Gq have different functions in the regulation of cellular activity. The Gs protein complexes contain αs subunits that stimulate adenyl cyclase when “activated” by adrenergic receptor interaction. These Gs complexes are primarily responsible for the stimulatory activity of β-adrenergic agonist agents. β1- and β2-adrenergic receptors interact primarily with βs subunits in stimulatory Gs protein complexes. The αi subunits of Gs proteins inhibit the activity of adenyl cyclase. Some β2-adrenergic receptors and the α2-adrenergic receptors interact with inhibitory Gi proteins to decrease the activity of adenyl cyclase. A third form, Gq, interacts with the α1-adrenergic receptors, but does not interact directly with adenyl cyclase. Instead, the Gq interacts with phospholipase C to mediate the cell response to α1-adrenergic stimulation.
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The G protein complex is composed of the cellular receptor and the three subunits, α, β, and γ, which are involved in the cellular response to catecholamines. In the absence of a catecholamine at the receptor site, the receptor protein is bound to the β and β-γ–dimer of the G protein, and guanosine diphosphate (GDP) is bound to the α subunit. Catecholamine binding to the receptor causes a conformational change in the α subunit; GDP dissociates and guanosine triphosphate (GTP) binds to the α subunit. The α subunit (with GTP bound) then dissociates from the receptor and from the β-γ–dimer. This “activated” α subunit can now interact with adenyl cyclase or other effector enzymes. Interaction of the αs subunit with adenyl cyclase increases the activity of the enzyme resulting in a rapid increase in the intracellular cAMP (Fig. 17–1).10,14,41,52,69
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cAMP acts as a secondary messenger in the cell. cAMP interacts with protein kinase A (PKA) and other cAMP-dependent protein kinases to increase their protein phosphorylating activity.40 In the absence of cAMP, PKA is a tetramer of two regulatory and two catalytic subunits. cAMP binds to the regulatory subunits to release the active enzymatic units from the tetramer (Fig. 17–1). The activated protein kinases then transfer phosphate groups from ATP to serine (as well as to threonine and tyrosine amino acid groups) on enzymes that are involved in intracellular regulation and activities. Phosphorylation may increase or decrease the activity of specific enzymes, and specific protein kinases are highly selective in the proteins that they phosphorylate.75,76
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PKA phosphorylates a variety of cellular proteins involved in Ca2+ regulation, including the voltage-sensitive calcium channel, phospholamban, and troponin;27,28,74 these are all involved in the regulation and control of cellular muscle fiber contraction. Phosphorylation of the l-type calcium channel increases the entry of calcium ions into the cell during membrane depolarization.57 Phosphorylation of phospholamban decreases its ability to inhibit the calcium ATPase pump on the sarcoplasmic reticulum (SR). This decreased inhibition of the calcium ATPase pump increases the efficiency of Ca2+ storage in the SR. This enhances both the cellular contractility as the Ca2+ is released into the cell cytosol and the relaxation of muscle fibers as the Ca2+ is pumped back into the SR.57
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Physiologic Effects of Adrenergic Receptor Subclasses.
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The existence of two types of adrenergic receptors, α and β, was first proposed in 1948 to explain both the excitatory and the inhibitory effects of catecholamines on different smooth muscle tissue.1 The α receptor was subsequently subdivided into α1 and α2 when norepinephrine and other α-adrenergic agonists were found to inhibit the release of additional norepinephrine from neurons into the synapse. The α1-adrenergic receptors are located on postsynaptic cells outside the central nervous system, primarily on blood vessels, and mediate arteriole constriction. The “autoregulatory” α2-adrenergic receptors are primarily located on the presynaptic neuronal membrane and, when stimulated, decrease release of additional norepinephrine into the synapse. Additionally, some α2-adrenergic receptors are also found on the postsynaptic membrane in the central nervous system. Activation of these postsynaptic α2-receptors in the cardiovascular control center in the medulla and elsewhere in the central nervous system decreases sympathetic outflow from the brain. Thus, α2-adrenergic agonists generally decrease peripheral vascular resistance, decrease heart rate, and decrease blood pressure. The α1- and α2-adrenergic receptors also interact with circulating catecholamines and other sympathomimetics. The effects of sympathomimetics vary in the different organ systems due to differences in the adrenergic receptors and in the cellular responses to the receptor interactions.
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The β-adrenergic receptors are subclassified into three subtypes: β1, β2, and β3 (Table 17–1). The most prevalent β-adrenergic subtype in the heart is β1 (80%), although β2 (20%) and β3 (few) receptors are also present.8,17,21,61 Stimulation of the β1-adrenergic receptors increases heart rate, contractility, conduction velocity, and automaticity. The β2-adrenergic receptors primarily cause relaxation of smooth muscle with resulting bronchodilation and arteriolar dilation. The β3 receptors are located primarily on adipocytes where they play a role in lipolysis and thermogenesis.11
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The β1-, β2-, and α2-adrenergic receptors all interact with Gs proteins and stimulate the adenyl cyclase enzyme. Differences in the resultant clinical effects are primarily related to the location and number of the different receptors in different tissues and to differences in the specificity of the tissue protein kinases activated by cAMP. Stimulation of the β1-adrenergic receptor results in increased heart rate and increased contractility. β2-Adrenergic receptor stimulation causes relaxation, as opposed to contraction, of smooth muscle. Because both β-adrenergic receptor subtypes interact with stimulatory Gs proteins, their clinical effects would appear to be paradoxical. However, there are two primary reasons for their different effects when Gs complexes are stimulated by β1- or β2-adrenergic agonists. First, PKA is not a single enzyme, but a group of related isoenzymes variably expressed in different tissues.7,30,56 The actions and the substrates of the varied protein kinase isoenzymes differs between β1- and β2-adrenergic responsive tissues. Second, whereas β1-adrenergic stimulation results in cAMP-mediated effects throughout the cytoplasm, β2-adrenergic stimulation is compartmentalized within the cell. The effect of β2-adrenergic stimulation of Gs type receptors is primarily localized phosphorylation of the L-type calcium channels, increasing their activity.13,35,87,88 Additionally, some β2-adrenergic receptors are also coupled to Gi-type receptors that inhibit adenyl cyclase and prevent the diffuse cytoplasmic increases in cAMP.71,72,88 Also, β2-adrenergic receptor stimulation does not result in phosphorylation of phospholamban38 or troponins.13
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The α2-adrenergic receptor interacts with a Gi protein that has an inhibitory interaction with adenyl cyclase. Binding of α2-adrenergic agonists to the receptor inhibits (not stimulates) adenyl cyclase and decreases intracellular cAMP.
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The α1-adrenergic receptors also are associated with G proteins. However, rather than being associated with Gs proteins and adenyl cyclase, the α1-adrenergic receptors are associated with Gq proteins that are linked to phospholipase C.77 Agonist binding to the receptor activates the hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP2) to 1,2-diacylglycerol (DAG) and inositol triphosphate (IP3).25 The IP3 acts as an intracellular messenger, binds to receptors on the SR, and initiates the release of calcium ion.5 DAG activates protein kinase C, which phosphorylates slow calcium channels and other intracellular proteins, and increases the influx of calcium ion into the cell (Fig. 17–2).63,70,91
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Many xenobiotics interact with G-protein membrane receptors and alter the intracellular cAMP or Ca2+ concentration. β-Adrenergic antagonist overdose results in decreased stimulation of adenyl cyclase by Gs proteins, decreased production of cAMP, decreased activation of the cAMP-dependent kinases, and decreased Ca2+ release (Chap. 62). Similarly, by different mechanisms, calcium channel blocker overdose results in decreased cytoplasmic calcium concentration (Chap. 61).
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Glucagon receptors, which are similar to the β-adrenergic receptors, are coupled to Gs proteins and stimulate adenyl cyclase activity.2,23,38,84,89 The ability of glucagon to increase cAMP is further enhanced by its inhibitory activity on phosphodiesterase (preventing cAMP breakdown).18,48 Phosphodiesterase inhibitors, such as amrinone, milrinone, and enoximone, exert at least some of their inotropic activity by preventing the degradation of cAMP and enhancing calcium cycling.42,81,85 In a canine model of propranolol poisoning, amrinone significantly increased inotropy, stroke volume, and cardiac output.42
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Intracellular Calcium, Calcium Channels, and Myocyte Contractility
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The contraction and relaxation cycle of the myocyte is controlled by the flux of Ca2+ into and out of the SR into the cytoplasm of the cell.15,39,66 Only a small proportion of the Ca2+ involved in myofibril contraction actually enters the cell through the exterior cell membrane during the action potential and membrane depolarization. The majority of the calcium is actually released from the SR of the cell invaginations of the myocyte membrane known as T-tubules place L-type calcium channels in close approximation to calcium release channels (ryanodine receptors {RyR}) on the sarcoplasmic reticulum. The local increase in Ca2+ concentration that follows the opening of a single L-type calcium channel triggers the opening of the associated RyR channels resulting in a large release of Ca2+ from the SR.12 Myocytes contain tens of thousands of couplons, clusters of L-type calcium channels and RyR channels. The Ca2+ released from one couplon is not sufficient to trigger firing of neighboring couplons. Therefore, myocyte contraction requires synchronized release of Ca2+ from numerous couplons throughout the myocyte. The cell membrane depolarization synchronizes opening of L-type channels and subsequent Ca2+ release from the sarcoplasmic reticulum.16,57 This phenomenon of calcium-induced calcium release results in a rapid increase in the intracellular Ca2+ concentration and initiates a rapid myosin and actin interaction.15
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At the conclusion of cellular contraction, SR-associated Ca2+-adenosine triphosphatase (ATPase) pumps return the cytosolic Ca2+-ATPase into the SR. This SR associated Ca2+-ATPase pump is regulated by phospholamban, a cellular protein. When phospholamban is bound to the Ca2+-ATPase pump, the activity of the pump is decreased and less Ca2+-ATPase is stored in the SR. Phosphorylation of phospholamban decreases its affinity for binding to the Ca2+-ATPase pump. Dissociation of the phosphorylated phospholamban increases the activity of the Ca2+-ATPase pump. β-Adrenergic stimulation increases protein kinase activity and leads to phosphorylation of phospholamban, dissociation of the phosphorylated phospholamban from the pump, and an increase in the total SR Ca2+ stores.19,20 The increased activity of the SR associated Ca2+-ATPase pump enhances the contractility and increases the rate of relaxation of the myocytes.
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Cellular contraction occurs when myosin filaments interact with the actin-tropomyosin helix. A complex of troponins T, I, and C binds to the actin helix near the myosin binding site and act as regulators of the interaction. Troponin T binds the regulatory complex to the actin helix, troponin I prevents myosin from accessing its binding site on the actin helix, and troponin C acts as a Ca2+ trigger to initiate contraction. When the intracellular Ca2+ concentration increases, 4 molecules of Ca2+ bind to troponin C and a conformational shift occurs in the troponin complex. Troponin I shifts away and the myosin-binding site is exposed. Myosin then binds to the exposed site and myofibril contraction occurs (Fig. 17–3).31,32,67,68
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Calcium transport through the cellular membrane ion channels is critical for normal cardiac muscle function and contractility and for maintenance of vascular smooth muscle tone. The physiologic response to calcium channel blockers and to xenobiotics that interact with the α- or β-adrenergic receptors is mediated through changes in the intracellular Ca2+. Calcium channel blockers in current clinical use primarily block the L-type calcium channel, although their specificity differs for the calcium channels on the vascular smooth muscle cells versus on the myocardial cells. This results in variable effects of the different calcium channel blockers on the vascular tone and peripheral vascular resistance, and on the contractility and electrical activity of the myocardial cells. Additionally, certain calcium channel blockers interact with the neuronal calcium channels, such as the P/Q type, and are used to treat neurologic disorders such as migraine headache.
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Patients poisoned by calcium channel blockers have less Ca2+ entry into the cell during cardiac membrane depolarization. Administration of exogenous Ca2+ increases the concentration gradient across the cell membrane, enhances flow through available Ca2+ channels, and restores the triggered response of the RyR-2 channels to release Ca2+ from the sarcoplasmic reticulum (Antidotes in Depth: A29). Because β-adrenergic antagonists have negative effects on Ca2+ handling by the SR, similar effects occur in the myocyte affected by β-adrenergic antagonists.
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In the vascular smooth muscle, the cytosolic Ca2+ concentration maintains the basal contraction of the vascular muscle. Any decrease of Ca2+ influx results in relaxation and arterial vasodilation.55 Any influx of calcium binds calmodulin, and the resulting complex stimulates myosin light-chain kinase activity.47 The myosin light-chain kinase phosphorylates myosin. The phosphorylated myosin has increased activity for binding to actin, which causes contraction (Fig. 61–1).6,32