Medical Physiology, 3rd Edition

Receptors Coupled to G Proteins

G protein–coupled receptors (GPCRs) constitute the largest family of receptors on the cell surface, with >1000 members either known or predicted from genome sequences. GPCRs mediate cellular responses to a diverse array of signaling molecules, such as hormones, neurotransmitters, vasoactive peptides, odorants, tastants, and other local mediators. Despite the chemical diversity of their ligands, most receptors of this class have a similar structure (Fig. 3-3). They consist of a single polypeptide chain with seven membrane-spanning α-helical segments, an extracellular N terminus that is glycosylated, a large cytoplasmic loop that is composed mainly of hydrophilic amino acids between helices 5 and 6, and a hydrophilic domain at the cytoplasmic C terminus. Most small ligands (e.g., epinephrine) bind in the plane of the membrane at a site that involves several membrane-spanning segments. In the case of larger protein ligands, a portion of the extracellular N terminus also participates in ligand binding. The 5,6-cytoplasmic loop appears to be the major site of interaction with the intracellular G protein, although the 3,4-cytoplasmic loop and the cytoplasmic C terminus also contribute to binding in some cases. Binding of the GPCR to its extracellular ligand regulates this interaction between the receptor and the G proteins, thus transmitting a signal to downstream effectors. In the next four sections of this subchapter, we discuss the general principles of how G proteins function and then consider three major second-messenger systems that G proteins trigger.

FIGURE 3-3 G protein–coupled receptor.

General Properties of G Proteins

G proteins are heterotrimers that exist in many combinations of different α, β, and γ subunits

G proteins are members of a superfamily of GTP-binding proteins. This superfamily includes the classic heterotrimeric G proteins that bind to GPCRs as well as the so-called small GTP-binding proteins, such as Ras. Both the heterotrimeric and small G proteins can hydrolyze GTP and switch between an active GTP-bound state and an inactive GDP-bound state.

Heterotrimeric G proteins are composed of three subunits, α, β, and γ. At least 16 different α subunits (~42 to 50 kDa), 5 β subunits (~33 to 35 kDa), and 11 γ subunits (~8 to 10 kDa) are present in mammalian tissue. The α subunit binds and hydrolyzes GTP and also interacts with “downstream” effector proteins such as adenylyl cyclase. Historically, the α subunits were thought to provide the principal specificity to each type of G protein, with the βγ complex functioning to anchor the trimeric complex to the membrane. However, it is now clear that the βγ complex also functions in signal transduction by interacting with effector molecules distinct from those regulated by the α subunits. Moreover, both the α and γ subunits are involved in anchoring the complex to the membrane. The α subunit is held to the membrane by either a myristyl or a palmitoyl group, whereas the γ subunit is held via a prenyl group.

The multiple α, β, and γ subunits demonstrate distinct tissue distributions and interact with different receptors and effectors (Table 3-2). Because of the potential for several hundred combinations of the known α, β, and γ subunits, G proteins are ideally suited to link a diversity of receptors to a diversity of effectors. The many classes of G proteins, in conjunction with the presence of several receptor types for a single ligand, provide a mechanism whereby a common signal can elicit the appropriate physiological response in different tissues. For example, when epinephrine binds β₁ adrenergic receptors in the heart, it stimulates adenylyl cyclase, which increases heart rate and the force of contraction. However, in the periphery, epinephrine acts on α₂ adrenergic receptors coupled to a G protein that inhibits adenylyl cyclase, thereby increasing peripheral vascular resistance and consequently increasing venous return and blood pressure.

FAMILY/SUBUNIT	% IDENTITY	TOXIN	DISTRIBUTION	RECEPTOR	EFFECTOR/ROLE
Gs (αs)
αs(s)	100	CTX	Ubiquitous	β adrenergic, TSH, glucagon, others	↑ Adenylyl cyclase
αs(l)					↑ Ca2+ channel
αolf	88	CTX	Olfactory epithelium	Odorant	↑ Adenylyl cyclase Open K+ channel
Gi (αi)
αi1	100	PTX	~Ubiquitous	M2, α2 adrenergic, others	↑ IP3, DAG, Ca2+, and AA, ↓ adenylyl cyclase
αi2	88	PTX	Ubiquitous
αi3		PTX	~Ubiquitous
αO1A	73	PTX	Brain, others	Met-enkephalin, α2 adrenergic, others
αO1B	73	PTX	Brain, others
αt1	68	PTX, CTX	Retinal rods	Rhodopsin	↑ cGMP-phosphodiesterase
αt2	68	PTX, CTX	Retinal cones	Cone opsin
αg	67	PTX, CTX	Taste buds	Taste
αz	60		Brain, adrenal, platelet	?	↓ Adenylyl cyclase
Gq
αq	100		~Ubiquitous	M1, α1 adrenergic, others	↑ PLCβ1, PLC β2, PLC β3
α11	88		~Ubiquitous
α14	79		Lung, kidney, liver
α15	57		B cell, myeloid
α16	58		T cell, myeloid	Several receptors	↑ PLCβ1, PLC β2, PLC β3
G12
α12	100		Ubiquitous
α13	67		Ubiquitous

CTX, cholera toxin; PTX, pertussis toxin.

Among the first effectors found to be sensitive to G proteins was the enzyme adenylyl cyclase. The heterotrimeric G protein known as G_s was so named because it stimulates adenylyl cyclase. A separate class of G proteins was given the name G_i because it is responsible for the ligand-dependent inhibition of adenylyl cyclase. Identification of these classes of G proteins was greatly facilitated by the observation that the αsubunits of individual G proteins are substrates for ADP ribosylation catalyzed by bacterial toxins. The toxin from Vibrio cholerae activates G_s, whereas the toxin from Bordetella pertussis inactivates the cyclase-inhibiting G_i (Box 3-1).

Infectious diarrheal disease has a multitude of causes. Cholera toxin, a secretory product of the bacterium Vibrio cholerae, is responsible in part for the devastating characteristics of cholera. The toxin is an oligomeric protein composed of one A subunit and five B subunits (AB₅). After cholera toxin enters intestinal epithelial cells, the A subunit separates from the B subunits and becomes activated by proteolytic cleavage. The resulting active A1 fragment catalyzes the ADP ribosylation of Gα_s. This ribosylation, which involves transfer of the ADP-ribose moiety from the oxidized form of nicotinamide adenine dinucleotide (NAD⁺) to the α subunit, inhibits the GTPase activity of Gα_s. As a result of this modification, Gα_s remains in its activated, GTP-bound form and can activate adenylyl cyclase. In intestinal epithelial cells, the constitutively activated Gα_s elevates levels of cAMP, which causes an increase in Cl⁻ conductance and water flow and thereby contributes to the large fluid loss characteristic of this disease.

A related bacterial product is pertussis toxin, which is also an AB₅ protein. It is produced by Bordetella pertussis, the causative agent of whooping cough. Pertussis toxin ADP-ribosylates Gα_i. This ADP-ribosylated Gα_i cannot exchange its GDP (inactive state) for GTP. Thus, α_i remains in its GDP-bound inactive state. As a result, receptor occupancy can no longer release the active α_i-GTP, so adenylyl cyclase cannot be inhibited. Thus, both cholera toxin and pertussis toxin increase the generation of cAMP.

For their work in identifying G proteins and elucidating the physiological role of these proteins, Alfred Gilman and Martin Rodbell received the 1994 Nobel Prize in Physiology or Medicine.  N3-5

G-protein activation follows a cycle

For more information about Alfred Gilman and Martin Rodbell and the work that led to their Nobel Prize, visit http://www.nobel.se/medicine/laureates/1994/index.html (accessed October 2014).

In their inactive state, heterotrimeric G proteins are a complex of α, β, and γ subunits in which GDP occupies the guanine nucleotide–binding site of the α subunit. After ligand binding to the GPCR (Fig. 3-4, step 1), a conformational change in the receptor–G protein complex facilitates the release of bound GDP and simultaneous binding of GTP to the α subunit (see Fig. 3-4, step 2). This GDP-GTP exchange stimulates dissociation of the complex from the receptor (see Fig. 3-4, step 3) and causes disassembly of the trimer into a free GTP-bound α subunit and separate βγ complex (see Fig. 3-4, step 4). The GTP-bound α subunit interacts in the plane of the membrane with downstream effectors such as adenylyl cyclase and phospholipases (see Fig. 3-4, step 5), or cleavage of its myristoyl or palmitoyl group can release the α subunit from the membrane. Similarly, the βγ subunit can activate ion channels or other effectors.

FIGURE 3-4 Enzymatic cycle of heterotrimeric G proteins.

The α subunit is itself an enzyme that catalyzes the hydrolysis of GTP to GDP and inorganic phosphate (P_i). The result is an inactive α-GDP complex that dissociates from its downstream effector and reassociates with a βγ subunit (see Fig. 3-4, step 6); this reassociation terminates signaling and brings the system back to resting state (see Fig. 3-4, step 1). The βγ subunit stabilizes α-GDP and thereby substantially slows the rate of GDP-GTP exchange (see Fig. 3-4, step 2) and dampens signal transmission in the resting state.

The RGS (for “regulation of G-protein signaling”) family of proteins appears to enhance the intrinsic GTPase activity of some but not all α subunits. Investigators have identified at least 19 mammalian RGS proteins and shown that they interact with specific α subunits. RGS proteins promote GTP hydrolysis and thus the termination of signaling.

Activated α subunits couple to a variety of downstream effectors, including enzymes and ion channels

Activated α subunits can couple to a variety of enzymes. A major enzyme that acts as an effector downstream of activated α subunits is adenylyl cyclase (Fig. 3-5A), which catalyzes the conversion of ATP to cAMP. This enzyme can be either activated or inhibited by G-protein signaling, depending on whether it associates with the GTP-bound form of Gα_s (stimulatory) or Gα_i (inhibitory). Thus, different ligands—acting through different combinations of GPCRs and G proteins—can have opposing effects on the same intracellular signaling pathway.  N3-4

FIGURE 3-5 Downstream effects of activated G-protein α subunits. A, When a ligand binds to a receptor coupled to α_s, adenylyl cyclase (AC) is activated, whereas when a ligand binds to a receptor coupled to α_i, the enzyme is inhibited. The activated enzyme converts ATP to cAMP, which then can activate PKA. B, In phototransduction, a photon interacts with the receptor and activates the G protein transducin. The α_t activates phosphodiesterase (PDE), which in turn hydrolyzes cGMP; this lowers the intracellular concentrations of cGMP and therefore closes the cGMP-activated channels. C, In this example, the ligand binds to a receptor that is coupled to α_q, which activates PLC. This enzyme converts PIP₂ to IP₃ and DAG. The IP₃ leads to the release of Ca²⁺ from intracellular stores, whereas the DAG activates PKC.

G proteins can also activate enzymes that break down cyclic nucleotides. For example, the G protein called transducin contains an α_t subunit that activates the cGMP phosphodiesterase, which in turn catalyzes the breakdown of cGMP to GMP (see Fig. 3-5B). This pathway plays a key role in phototransduction in the retina (see p. 368).

G proteins can also couple to phospholipases. These enzymes catabolize phospholipids, as discussed in detail below in the section on G-protein second messengers. This superfamily of phospholipases can be grouped into phospholipases A₂, C, or D on the basis of the site at which the enzyme cleaves the phospholipid. G proteins that include the α_q subunit activate phospholipase C, which breaks phosphatidylinositol 4,5-bisphosphate into two intracellular messengers, membrane-associated diacylglycerol and cytosolic IP₃ (see Fig. 3-5C). Diacylglycerol stimulates protein kinase C, whereas IP₃ binds to a receptor on the endoplasmic reticulum (ER) membrane and triggers the release of Ca²⁺ from intracellular stores.

Some G proteins interact with ion channels. Agonists that bind to the β adrenergic receptor activate the L-type Ca²⁺ channel (see pp. 190–193) in the heart and skeletal muscle. The α subunit of the G protein G_sbinds to and directly stimulates L-type Ca²⁺ channels and also indirectly stimulates this channel via a signal-transduction cascade that involves cAMP-dependent phosphorylation of the channel.

βγ subunits can activate downstream effectors

Following activation and disassociation of the heterotrimeric G protein, βγ subunits can also interact with downstream effectors. The neurotransmitter ACh released from the vagus nerve reduces the rate and strength of heart contraction. This action in the atria of the heart is mediated by muscarinic M₂ AChRs, members of the GPCR family (see p. 341). These receptors can be activated by muscarine, an alkaloid found in certain poisonous mushrooms. Muscarinic AChRs are very different from the nicotinic AChRs discussed above, which are ligand-gated ion channels. Binding of ACh to the muscarinic M₂ receptor in the atria activates a heterotrimeric G protein, which results in the generation of both activated Gα_i as well as a free βγ subunit complex. The βγ complex then interacts with a particular class of K⁺ channels, increasing their permeability. This increase in K⁺ permeability keeps the membrane potential relatively negative and thus renders the cell more resistant to excitation. The βγ subunit complex also modulates the activity of adenylyl cyclase and phospholipase C and stimulates phospholipase A₂. Such effects of βγ can be independent of, synergize with, or antagonize the action of the α subunit. For example, studies using various isoforms of adenylyl cyclase have demonstrated that purified βγ stimulates some isoforms, inhibits others, and has no effect on still others. Different combinations of βγ isoforms may have different activities. For example, β₁γ₁ is one tenth as efficient at stimulating type II adenylyl cyclase as is β₁γ₂.

Some βγ complexes can bind to a special protein kinase called the β adrenergic receptor kinase (βARK). As a result of this interaction, βARK translocates to the plasma membrane, where it phosphorylates the ligand-receptor complex (but not the unbound receptor). This phosphorylation results in the recruitment of β-arrestin to the GPCR, which in turn mediates disassociation of the receptor-ligand complex and thus attenuates the activity of the same β adrenergic receptors that gave rise to the βγ complex in the first place. This action is an example of receptor desensitization. These phosphorylated receptors eventually undergo endocytosis, which transiently reduces the number of receptors that are available on the cell surface. This endocytosis is an important step in resensitization of the receptor system.

Small GTP-binding proteins are involved in a vast number of cellular processes

A distinct group of proteins that are structurally related to the α subunit of the heterotrimeric G proteins are the small GTP-binding proteins. More than 100 of these have been identified to date, and they have been divided into five groups: Ras, Rho, Rab, Arf, and Ran families. These 21-kDa proteins can be membrane associated (e.g., Ras) or may translocate between the membrane and the cytosol (e.g., Rho).

The three isoforms of Ras (NRas, HRas, and KRas) relay signals from the plasma membrane to the nucleus via an elaborate kinase cascade (see pp. 89–90), thereby regulating gene transcription. In some tumors, mutation of the genes encoding Ras proteins results in constitutively active Ras. These mutated genes are called oncogenes because the altered Ras gene product promotes the malignant transformation of a cell and can contribute to the development of cancer (oncogenesis). In contrast, Rho family members are primarily involved in rearrangement of the actin cytoskeleton. Rab and Arf regulate vesicle trafficking, whereas Ran regulates nucleocytoplasmic transport.

Similarly to the α subunit of heterotrimeric G proteins, the small GTP-binding proteins switch between an inactive GDP-bound form and an active GTP-bound form. Two classes of regulatory proteins modulate the activity of these small GTP-binding proteins. The first of these includes the GTPase-activating proteins (GAPs) and neurofibromin (a product of the neurofibromatosis type 1 gene). GAPs increase the rate at which small GTP-binding proteins hydrolyze bound GTP and thus result in more rapid inactivation. Counteracting the activity of GAPs are guanine nucleotide exchange factors (GEFs) such as “son of sevenless” or SOS (see p. 69), which promote the conversion of inactive Ras-GDP to active Ras-GTP. Interestingly, cAMP directly activates several GEFs, such as Epac (exchange protein activated by cAMP); this demonstrates crosstalk between a classical heterotrimeric G-protein signaling pathway and the small Ras-like G proteins.

G-Protein Second Messengers: Cyclic Nucleotides

cAMP usually exerts its effect by increasing the activity of protein kinase A

Activation of G_s-coupled receptors results in the stimulation of adenylyl cyclase, which can cause [cAMP]_i to rise 5-fold in ~5 seconds (see Fig. 3-5A). This sudden rise is counteracted by cAMP breakdown to AMP by cAMP phosphodiesterase. The downstream effects of this increase in [cAMP]_i depend on the cellular microdomains in which [cAMP]_i rises as well as the specialized functions that the responding cell carries out in the organism. For example, in the adrenal cortex, ACTH stimulation of cAMP production results in the secretion of aldosterone and cortisol (see p. 1023); in the kidney, a vasopressin-induced rise in cAMP levels facilitates water reabsorption (see p. 818). Excess cAMP is also responsible for certain pathological conditions, such as cholera (see Box 3-1). Another pathological process associated with excess cAMP is McCune-Albright syndrome, characterized by a triad of (1) variable hyperfunction of multiple endocrine glands, including precocious puberty in girls; (2) bone lesions; and (3) pigmented skin lesions (café au lait spots). This disorder is caused by a somatic mutation during development that constitutively activates the G-protein α_s subunit in a mosaic pattern.

cAMP exerts many of its effects through cAMP-dependent protein kinase A (PKA). This enzyme catalyzes transfer of the terminal phosphate of ATP to specific serine or threonine residues on substrate proteins. PKA phosphorylation sites are present in a multitude of intracellular proteins, including ion channels, receptors, metabolic enzymes, and signaling pathway proteins. Phosphorylation of these sites can influence either the localization or the activity of the substrate. For example, phosphorylation of the β₂ adrenergic receptor by PKA causes receptor desensitization in neurons, whereas phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) increases its Cl⁻ channel activity.

To enhance regulation of phosphorylation events, the cell tightly controls the activity of PKA so that the enzyme can respond to subtle—and local—variations in cAMP levels. One important control mechanism is the use of regulatory subunits that constitutively inhibit PKA. In the absence of cAMP, two catalytic subunits of PKA associate with two of these regulatory subunits; the result is a heterotetrameric protein complex that has a low level of catalytic activity (Fig. 3-6). Binding of cAMP to the regulatory subunits induces a conformational change that diminishes their affinity for the catalytic subunits, and the subsequent dissociation of the complex results in activation of kinase activity. Not only can PKA activation have the short-term effects noted above, but the free catalytic subunit of PKA can also enter the nucleus, where substrate phosphorylation can activate the transcription of specific PKA-dependent genes (see p. 89). Although most cells use the same catalytic subunit, different regulatory subunits are found in different cell types.

FIGURE 3-6 Activation of PKA by cAMP.

Another mechanism that contributes to regulation of PKA is the targeting of the enzyme to specific subcellular locations. Such targeting promotes the preferential phosphorylation of substrates that are confined to precise locations within the cell. PKA targeting is achieved by the association of a PKA regulatory subunit with an A kinase anchoring protein (AKAP), which in turn binds to cytoskeletal elements or to components of cellular subcompartments. More than 35 AKAPs are known. The specificity of PKA targeting is highlighted by the observation that, in neurons, PKA is localized to postsynaptic densities through its association with AKAP79. This anchoring protein also targets calcineurin—a protein phosphatase—to the same site. This targeting of both PKA and calcineurin to the same postsynaptic site makes it possible for the cell to tightly regulate the phosphorylation state of important neuronal substrates.

The cAMP generated by adenylyl cyclase can interact with effectors other than PKA. For example, olfactory receptors (see pp. 358–359) activate a member of the G_s family called G_olf. The subsequent rise in [cAMP]_i activates a cyclic nucleotide–gated (CNG) ion channel (see Table 6-2, family No. 4). Na⁺ influx through this channel leads to membrane depolarization and the initiation of a nerve impulse.

For his work in elucidating the role played by cAMP as a second messenger in regulating glycogen metabolism (see Fig. 58-9), Earl Sutherland received the 1971 Nobel Prize in Physiology or Medicine.  N3-6 In 1992, Edmond Fischer and Edwin Krebs shared the prize for their part in demonstrating the role of protein phosphorylation in the signal-transduction process.  N3-7

For more information about Earl W. Sutherland, Jr., and the work that led to his Nobel Prize, visit http://www.nobel.se/medicine/laureates/1971/index.html (accessed October 2014).

For more information about Edmond H. Fischer and Edwin S. Krebs and the work that led to their Nobel Prize, visit http://www.nobel.se/medicine/laureates/1992/index.html (accessed October 2014).

This coordinated set of phosphorylation and dephosphorylation reactions has several physiological advantages. First, it allows a single molecule (e.g., cAMP) to regulate a range of enzymatic reactions. Second, it affords a large amplification to a small signal. The concentration of epinephrine needed to stimulate glycogenolysis in muscle is ~10⁻¹⁰ M. This subnanomolar level of hormone can raise [cAMP]_i to ~10⁻⁶ M. Thus, the catalytic cascades amplify the hormone signal 10,000-fold, which results in the liberation of enough glucose to raise blood glucose levels from ~5 to ~8 mM. Although the effects of cAMP on the synthesis and degradation of glycogen are confined to muscle and liver, a wide variety of cells use cAMP-mediated activation cascades in the response to a wide variety of hormones.

Protein phosphatases reverse the action of kinases

As discussed above, one way that the cell can terminate a cAMP signal is to use a phosphodiesterase to degrade cAMP. In this way, the subsequent steps along the signaling pathway can also be terminated. However, because the downstream effects of cAMP often involve phosphorylation of effector proteins at serine and threonine residues by kinases such as PKA, another powerful way to terminate the action of cAMP is to dephosphorylate these effector proteins. Such dephosphorylation events are mediated by enzymes called serine/threonine phosphoprotein phosphatases.

Four groups of serine/threonine phosphoprotein phosphatases (PPs) are known: 1, 2a, 2b, and 2c. These enzymes themselves are regulated by phosphorylation at their serine, threonine, and tyrosine residues. The balance between kinase and phosphatase activity plays a major role in the control of signaling events.

PP1 dephosphorylates many proteins phosphorylated by PKA, including those phosphorylated in response to epinephrine (see Fig. 58-9). Another protein, phosphoprotein phosphatase inhibitor 1 (I-1), can bind to and inhibit PP1. Interestingly, PKA phosphorylates and induces I-1 binding to PP1 (Fig. 3-7), thereby inhibiting PP1 and preserving the phosphate groups added by PKA in the first place.

FIGURE 3-7 Inactivation of PP1 by PKA.

PP2a, which is less specific than PP1, appears to be the main phosphatase responsible for reversing the action of other protein serine/threonine kinases. The Ca²⁺-dependent PP2b, also known as calcineurin, is prevalent in the brain, muscle, and immune cells and is also the pharmacological target of the immunosuppressive reagents FK-506 (tacrolimus) and cyclosporine.

The substrates for PP2c include the DNA checkpoint regulators Chk1 and Chk2, which normally sense DNA damage in the setting of organ injury and temporarily stop cell proliferation. Dephosphorylation of these kinases by PP2c inactivates them and allows the cell to re-enter the cell cycle during the repair process.

In addition to serine/threonine kinases such as PKA, a second group of kinases involved in regulating signaling pathways (discussed beginning on pp. 68–70) are tyrosine kinases that phosphorylate their substrate proteins on tyrosine residues. The enzymes that remove phosphates from these tyrosine residues—phosphotyrosine phosphatases (PTPs)—are much more variable than the serine and threonine phosphatases. The first PTP to be characterized was the cytosolic enzyme PTP1B from human placenta. PTP1B has a high degree of homology with CD45, a membrane protein that is both a receptor and a tyrosine phosphatase. The large family of PTPs can be divided into two classes: membrane-spanning receptor-like proteins such as CD45 and cytosolic tyrosine phosphatases such as PTP1B. A number of intracellular PTPs contain Src homology 2 (SH2) domains, a peptide sequence or motif that interacts with phosphorylated tyrosine groups and thus acts to recruit the phosphatase to its target substrate. Many of the PTPs are themselves regulated by phosphorylation.

cGMP exerts its effect by stimulating a nonselective cation channel in the retina

cGMP is another cyclic nucleotide that is involved in G-protein signaling events. In the outer segments of rods and cones in the visual system, the G protein does not couple to an enzyme that generates cGMP but, as noted above, couples to an enzyme that breaks it down. As discussed beginning on page 367, light activates a GPCR called rhodopsin, which activates the G protein transducin (see p. 368), which in turn activates the cGMP phosphodiesterase (see p. 368) that lowers [cGMP]_i. The fall in [cGMP]_i closes cGMP-gated nonselective cation channels that are members of the same family of CNG ion channels that cAMP activates in olfactory signaling (see pp. 358–359).

G-Protein Second Messengers: Products of Phosphoinositide Breakdown

Many messengers bind to receptors that activate phosphoinositide breakdown

Although the phosphatidylinositols (PIs) are minor constituents of cell membranes, they are largely distributed in the internal leaflet of the membrane and play an important role in signal transduction. The inositol sugar moiety of PI molecules (see Fig. 2-2A) can be phosphorylated to yield two major phosphoinositides involved in signal transduction: phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂ or PIP₂) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃ or PIP₃; see p. 69).  N3-8

Contributed by Emile Boulpaep, Walter Boron

As noted in the text, phosphatidylinositols (PIs) (see p. 10) and phosphatidylcholines (PCs) (see p. 10) can each contain a variety of acyl groups. Therefore, the phosphoinositides derived from them can also contain a variety of acyl groups.

A phosphoinositide is a PI derivative containing one, two, or three additional phosphate groups. Because there are three possible attachment sites (at sites 3, 4, or 5), there are a total of seven combinations possible.

Seven Combinations

• Three monophosphates:

• PI3P

• PI4P

• PI5P

• Three bisphosphates called PIP₂

• PI(3,4)P₂

• PI(4,5)P₂

• PI(3,5)P₂

• One trisphosphate called PIP₃

• PI(3,4,5)P₃

Certain membrane-associated receptors act though G proteins (e.g., G_q) that stimulate phospholipase C (PLC) to cleave PIP₂ into inositol 1,4,5-trisphosphate (or P₃) and diacylglycerol (DAG), as shown in Figure 3-8A. PLCs are classified into three families (β, γ, δ) that differ in their catalytic properties, cell-type–specific expression, and modes of activation. PLCβ is typically activated downstream of certain G proteins (e.g., G_q), whereas PLCγ contains an SH2 domain and is activated downstream of certain tyrosine kinases. Stimulation of PLCβ results in a rapid increase in cytosolic IP₃ levels as well as an early peak in DAG levels (see Fig. 3-8B). Both products are second messengers. The water-soluble IP₃ travels through the cytosol to stimulate Ca²⁺ release from intracellular stores (see next section). DAG remains in the plane of the membrane to activate protein kinase C, which migrates from the cytosol and binds to DAG in the membrane (see pp. 60–61).

FIGURE 3-8 Second messengers in the DAG/IP₃ pathway. ER, endoplasmic reticulum; SERCA, sarcoplasmic and endoplasmic reticulum Ca-ATPase.

Phosphatidylcholines (PCs), which—unlike PI—are an abundant phospholipid in the cell membrane, are also a source of DAG. The cell can produce DAG from PC by either of two mechanisms (see Fig. 3-8C). First, PLC can directly convert PC to phosphocholine and DAG. Second, phospholipase D (PLD), by cleaving the phosphoester bond on the other side of the phosphate, can convert PC to choline and phosphatidic acid (PA; also phospho-DAG). This PA can then be converted to DAG via PA-phosphohydrolase. Production of DAG from PC, either directly (via PLC) or indirectly (via PLD), produces the slow wave of increasing cytosolic DAG shown in Figure 3-8B. Thus, in some systems, the formation of DAG is biphasic and consists of an early peak that is transient and parallels the formation of IP₃, followed by a late phase that is slow in onset but sustained for several minutes.

IP₃ liberates Ca²⁺ from intracellular stores

As discussed on page 126, three major transport mechanisms keep free intracellular Ca²⁺ ([Ca²⁺]_i) below ~100 nM. Increases in [Ca²⁺]_i from this extremely low baseline allow Ca²⁺ to function as an important second messenger. IP₃ generated by the metabolism of membrane phospholipids travels through the cytosol and binds to the IP₃ receptor, a ligand-gated Ca²⁺ channel located in the membrane of the endoplasmic reticulum (see Fig. 3-8A). The result is a release of Ca²⁺ from intracellular stores and a rise in [Ca²⁺]_i. Indeed, it was within this system that Ca²⁺ was first identified as a messenger mediating the stimulus-response coupling of endocrine cells. The IP₃ receptor (ITPR) is a tetramer composed of subunits of ~260 kDa. At least three genes encode the subunits of the receptor. These genes are subject to alternative splicing, which further increases the potential for receptor diversity. The receptor is a substrate for phosphorylation by protein kinases A and C as well as calcium-calmodulin (Ca²⁺-CaM)–dependent protein kinases.  N3-9

Contributed by Laurie Roman

As noted in the text, the IP₃ receptor (IPTR) is a tetramer composed of subunits of ~260 kDa, and at least four different genes encode the receptor subunits. These genes are subject to alternative splicing, further increasing the potential for receptor diversity. IP₃ receptors bind their ligand with high affinity (the dissociation constant K_D = 2–10 nM) or low affinity (K_D = 40 nM). However, the extent to which these different affinities correlate with particular forms of the receptor has not been established.

Interaction of IP₃ with its receptor results in passive efflux of Ca²⁺ from the ER and thus a rapid rise in the free cytosolic Ca²⁺ concentration. The IP₃-induced changes in [Ca²⁺]_i exhibit complex temporal and spatial patterns. The rise in [Ca²⁺]_i, which can be brief or persistent, can oscillate repetitively or spread across groups of cells coupled by gap junctions. In at least some systems, the frequency of [Ca²⁺]_i oscillations seems to be physiologically important. For example, in isolated pancreatic acinar cells, graded increases in the concentration of ACh produce graded increases in the frequency—but not the magnitude—of repetitive [Ca²⁺]_i spikes. The mechanisms responsible for [Ca²⁺]_i oscillations and waves are complex. It appears that both propagation and oscillation depend on positive-feedback mechanisms, in which high [Ca²⁺]_ifacilitates Ca²⁺ release, as well as on negative-feedback mechanisms, in which high [Ca²⁺]_i inhibits further Ca²⁺ release.

Structurally related to ITPRs are the Ca²⁺-release channels known as ryanodine receptors (RYRs; see p. 230). Because cytosolic Ca²⁺ activates RYRs, these channels play an important role in elevating [Ca²⁺]_i in certain cells by a process known as calcium-induced Ca²⁺ release (CICR; see pp. 242–243)—an example of the positive feedback noted above. For example, RYRs are responsible for releasing Ca²⁺ from the sarcoplasmic reticulum of muscle and thereby switching on muscle contraction (see pp. 229–230). Moreover, cyclic ADP ribose (cADPR), the product of ADP-ribosylcyclases, increases the sensitivity of RYR to cytosolic Ca²⁺, thereby augmenting CICR.

[Ca²⁺]_i can increase as the result not only of Ca²⁺ release from intracellular stores, but also of enhanced influx through Ca²⁺ channels in the plasma membrane. By whatever mechanism, increased [Ca²⁺]_i exerts its effects by binding to cellular proteins and changing their activity, as discussed in the next two sections. Some Ca²⁺-dependent signaling events are so sensitive to Ca²⁺ that a [Ca²⁺]_i increase of as little as 100 nM can trigger a vast array of cellular responses. These responses include secretion of digestive enzymes by pancreatic acinar cells, release of insulin by β cells, contraction of vascular smooth muscle, conversion of glycogen to glucose in the liver, release of histamine by mast cells, aggregation of platelets, and DNA synthesis and cell division in fibroblasts.

The same mechanisms that normally keep [Ca²⁺]_i at extremely low levels (see p. 126) are also responsible for reversing the increases in [Ca²⁺]_i that occur during signaling events. Increases in [Ca²⁺]_i activate an ATP-fueled Ca pump (SERCA; see p. 118) that begins pumping Ca²⁺ back into the ER. In addition, a Ca pump (see p. 118) and Na-Ca exchanger (see pp. 123–124) at the plasma membrane extrude excess Ca²⁺from the cell. These processes are much slower than Ca²⁺ release, so [Ca²⁺]_i remains high until IP₃ is dephosphorylated, terminating Ca²⁺ release via ITPR and thereby allowing the transporters to restore [Ca²⁺]_i to basal levels.

Calcium activates calmodulin-dependent protein kinases

How does an increase in [Ca²⁺]_i lead to downstream responses in the signal-transduction cascade? The effects of changes in [Ca²⁺]_i are mediated by Ca²⁺-binding proteins, the most important of which is calmodulin (CaM). CaM is a high-affinity cytoplasmic Ca²⁺-binding protein of 148 amino acids. Each molecule of CaM cooperatively binds four calcium ions. Ca²⁺ binding induces a major conformational change in CaM that allows it to bind to other proteins (Fig. 3-9). Although CaM does not have intrinsic enzymatic activity, it forms a complex with a number of enzymes and thereby confers a Ca²⁺ dependence on their activity. For example, binding of the Ca²⁺-CaM complex activates the enzyme that degrades cAMP, cAMP phosphodiesterase.

FIGURE 3-9 CaM. After four intracellular Ca²⁺ ions bind to CaM, the Ca²⁺-CaM complex can bind to and activate another protein. In this example, the activated protein is a Ca²⁺-CaM–dependent kinase.

Many of the effects of CaM occur as the Ca²⁺-CaM complex binds to and activates a family of Ca²⁺-CaM–dependent kinases known as CaM kinases (CaMKs). These kinases phosphorylate specific serine and threonine residues of a variety of proteins. An important CaMK in smooth-muscle cells is myosin light-chain kinase (MLCK) (see p. 247). Another CaMK is glycogen phosphorylase kinase (PK), which plays a role in glycogen degradation (see p. 1182).

MLCK, PK, and some other CaMKs have a rather narrow substrate specificity. The ubiquitous CaM kinase II (CaMKII), on the other hand, has a broad substrate specificity. Especially high levels of this multifunctional enzyme are present at the synaptic terminals of neurons. One of the actions of CaMKII is to phosphorylate and thereby activate the rate-limiting enzyme (tyrosine hydroxylase; see Fig. 13-8) in the synthesis of catecholamine neurotransmitters. CaMKII can also phosphorylate itself, which allows it to remain active in the absence of Ca²⁺.

DAGs and Ca²⁺ activate protein kinase C

As noted above, hydrolysis of PIP₂ by PLC yields not only the IP₃ that leads to Ca²⁺ release from internal stores but also DAG (see Fig. 3-8A). The most important function of DAG is to activate protein kinase C (PKC), an intracellular serine/threonine kinase. In mammals, the PKC family comprises at least 10 members that differ in their tissue and cellular localization. This family is further subdivided into three groups that all require membrane-associated phosphatidylserine but have different requirements for Ca²⁺ and DAG. The classical PKC family members PKCα, PKCβ, and PKCγ require both DAG and Ca²⁺ for activation, whereas the novel PKCs (such as PKCδ, PKCε, and PKCη) require DAG but are independent of Ca²⁺, and the atypical PKCs (PKCζ and PKCλ) appear to be independent of both DAG and Ca²⁺. As a consequence, the signals generated by the PKC pathway depend on the isoforms of the enzyme that a cell expresses as well as on the levels of Ca²⁺ and DAG at specific locations at the cell membrane. Moreover, proteins such as receptor for activated C-kinase (RACK) and receptor for inactivated C-kinase (RICK) can target specific PKC isoforms to specific cellular compartments.

In its basal state, PKCα is an inactive, soluble cytosolic protein. When a GPCR activates PLC, both DAG (generated in the inner leaflet of the plasma membrane) and Ca²⁺ (released in response to IP₃) bind to the PKC regulatory domain; this results in translocation of PKCα to the membrane and activation of the PKC kinase domain. Even though the initial Ca²⁺ signal is transient, PKCα activation can be sustained, resulting in activation of physiological responses, such as proliferation and differentiation. Elevated levels of active PKCα are maintained by a slow wave of elevated DAG (see Fig. 3-8B), which is due to the hydrolysis of PC by PLC and PLD.

Physiological stimulation of the classical and novel PKCs by DAG can be mimicked by the exogenous application of a class of tumor promoters called phorbol esters. These plant products bind to the regulatory domain of PKCs and thus specifically activate them even in the absence of DAG.

Among the major substrates of PKC are the myristoylated, alanine-rich C-kinase substrate proteins, known as MARCKS proteins. These acidic proteins contain consensus sites for PKC phosphorylation as well as CaM- and actin-binding sites. MARCKS proteins cross-link actin filaments and thus appear to play a role in translating extracellular signals into actin plasticity and changes in cell shape. Unphosphorylated MARCKS proteins are associated with the plasma membrane, and they cross-link actin. Phosphorylation of the MARCKS proteins causes them to translocate into the cytosol, where they are no longer able to cross-link actin. Thus, mitogenic growth factors that activate PKC may produce morphological changes and anchorage-independent cell proliferation in part by modifying the activity of MARCKS proteins.

PKC can also directly or indirectly modulate transcription factors and thereby enhance the transcription of specific genes (see p. 86). Such genomic actions of PKC explain why phorbol esters are tumor promoters.

G-Protein Second Messengers: Arachidonic Acid Metabolites

In addition to DAG, other hydrolysis products of membrane phospholipids can act as signaling molecules.  N3-10 The best characterized of these hydrolysis products is arachidonic acid (AA), which is attached by an ester bond to the second carbon of the glycerol backbone of membrane phospholipids (Fig. 3-10). Phospholipase A₂ initiates the cellular actions of AA by releasing this fatty acid from glycerol-based phospholipids.  N3-11 A series of enzymes subsequently convert AA into a family of biologically active metabolites that are collectively called eicosanoids (from the Greek eikosi [20]) because, like AA, they all have 20 carbon atoms. Three major pathways can convert AA into these eicosanoids (Fig. 3-11). In the first pathway, cyclooxygenase (COX) enzymes produce thromboxanes (TXs), prostaglandins (PGs), and prostacyclins. In the second pathway, 5-lipoxygenase enzymes produce leukotrienes (LTs) and some hydroxyeicosatetraenoic acid (HETE) compounds. In the third pathway, the epoxygenase enzymes, which are members of the cytochrome P-450 class, produce other HETE compounds as well as cis-epoxyeicosatrienoic acid (EET) compounds. These three enzymes catalyze the stereospecific insertion of molecular O₂ into various positions in AA. The cyclooxygenases, lipoxygenases, and epoxygenases are selectively distributed in different cell types, which further increases the complexity of eicosanoid biology. Eicosanoids have powerful biological activities, including effects on allergic and inflammatory processes, platelet aggregation, vascular smooth muscle, and gastric acid secretion.

The nomenclature of the eicosanoids is not as arcane as it might first appear. The numerical subscript 2 (as in PGH₂) or 4 (as in LTA₄) refers to the number of double bonds in the eicosanoid backbone. For example, AA has four double bonds, as do the leukotrienes.

For the cyclooxygenase metabolites, the letter (A to I) immediately preceding the 2 refers to the structure of the 5-carbon ring that is formed about halfway along the 20-carbon chain of the eicosanoid. For the leukotrienes, the letters A and B that immediately precede the 4 refer to differences in the eicosanoid backbone. For the cysteinyl leukotrienes, the letter C refers to the full glutathione conjugate (see Fig. 46-8). Removal of glutamate from LTC₄ yields LTD₄, and removal of glycine from LTD₄ yields LTE₄, leaving behind only cysteine.

For 5-HPETE and 5-HETE, the fifth carbon atom (counting the carboxyl group as number 1) is derivatized with a hydroperoxy or hydroxy group, respectively.

FIGURE 3-10 Release of AA from membrane phospholipids by PLA₂. AA is esterified to membrane phospholipids at the second carbon of the glycerol backbone. PLA₂ cleaves the phospholipid at the indicated position and releases AA as well as a lysophospholipid.

FIGURE 3-11 AA signaling pathways. In the direct pathway, an agonist binds to a receptor that activates PLA₂, which releases AA from a membrane phospholipid (see Fig. 3-10). In one of three indirect pathways, an agonist binds to a different receptor that activates PLC and thereby leads to the formation of DAG and IP₃, as in Figure 3-8; DAG lipase then releases the AA from DAG. In a second indirect pathway, the IP₃ releases Ca²⁺ from internal stores, which leads to the activation of PLA₂ (see the direct pathway). In a third indirect pathway (not shown), MAPK stimulates PLA₂. Regardless of its source, the AA may follow any of three pathways to form a wide array of eicosanoids. The cyclooxygenase pathway produces thromboxanes (TXA₂ and TXB₂), prostacyclin (i.e., PGI₂), and prostaglandins. The 5-lipoxygenase pathway produces 5-HETE and the leukotrienes. The epoxygenase pathway leads to the production of other HETEs and EETs. AACoA, arachidonic-Acid–coenzyme A; ASA, acetylsalicylic acid.

Contributed by Ed Moczydlowski

Although it is not a member of the arachidonic acid (AA) family, platelet-activating factor (PAF) is an important lipid signaling molecule. PAF is an ether lipid that the cell synthesizes either de novo or by remodeling of a membrane-bound precursor. PAF occurs in a wide variety of organisms and mediates many biological activities. In mammals, PAF is a potent inducer of platelet aggregation and stimulates the chemotaxis and degranulation of neutrophils, thereby facilitating the release of LTB₄ and 5-HETE. PAF is involved in several aspects of allergic reactions; for example, it stimulates histamine release and enhances the secretion of immunoglobulin E, immunoglobulin A, and tumor necrosis factor. Endothelial cells are also an important target of PAF; PAF causes a negative shift of V_m in these cells by activating Ca²⁺-dependent K⁺ channels. PAF also enhances vascular permeability and the adhesion of neutrophils and platelets to endothelial cells.

PAF exerts its effects by binding to a specific receptor on the plasma membrane. A major consequence of PAF binding to its GPCR is formation of IP₃ and stimulation of a group of MAPKs. PAF acetylhydrolase terminates the action of this signaling lipid.

Contributed by Laurie Roman

Phospholipase A₂ (PLA₂) catalyzes the hydrolytic cleavage of glycerol-based phospholipids (see Fig. 2-2A–C) at the second carbon of the glycerol backbone, yielding AA and a lysophospholipid (see Fig. 3-10). Some of the cytosolic PLA₂ enzymes require Ca²⁺ for activity. In addition, raising [Ca²⁺]_i from the physiological level of ~100 nM to ~300 nM facilitates the association of cytoplasmic PLA₂ with cell membranes, where the PLA₂ can be activated by specific G proteins.

Phospholipase A₂ is the primary enzyme responsible for releasing AA

The first step in the phospholipase A₂ (PLA₂) signal-transduction cascade is binding of an extracellular agonist to a membrane receptor (see Fig. 3-11). These receptors include those for serotonin (5-HT₂receptors), glutamate (mGLUR1 receptors), fibroblast growth factor-β, interferon-α (IFN-α), IFN-β, and IFN-γ. Once the receptor is occupied by its agonist, it can activate a G protein that belongs to the G_i/G_ofamily. The mechanism by which this activated G protein stimulates PLA₂ is not well understood. It does not appear that a G-protein α subunit is involved. The G-protein βγ dimer may stimulate PLA₂ either directly or via mitogen-activated protein kinases (MAPKs) (see p. 69), which phosphorylates PLA₂ at a serine residue. The result is rapid hydrolysis of phospholipids that contain AA.

In contrast to the direct pathway just mentioned, agonists acting on other receptors may promote AA release indirectly. First, a ligand may bind to a receptor coupled to PLC, which would lead to the release of DAG (see Fig. 3-11). As noted above, DAG lipase can cleave DAG to yield AA and a monoacylglycerol (MAG). Agonists that act via this pathway include dopamine (D2 receptors), adenosine (A₁ receptors), norepinephrine (α₂ adrenergic receptors), and serotonin (5-HT₁ receptors). Second, any agonist that raises [Ca²⁺]_i can promote AA formation because Ca²⁺ can stimulate some cytosolic forms of PLA₂. Third, any signal-transduction pathway that activates MAPK can also enhance AA release because MAPK phosphorylates PLA₂.

Cyclooxygenases, lipoxygenases, and epoxygenases mediate the formation of biologically active eicosanoids

Once it is released from the membrane, AA can diffuse out of the cell, be reincorporated into membrane phospholipids, or be metabolized (see Fig. 3-11).

In the first pathway of AA metabolism (see Fig. 3-11), cyclooxygenases  N3-12 catalyze the stepwise conversion of AA into the intermediates prostaglandin G₂ (PGG₂) and prostaglandin H₂ (PGH₂). PGH₂ is the precursor of the other prostaglandins, the prostacyclins and the thromboxanes. As noted in Box 3-3, cyclooxygenase exists in two predominant isoforms, cyclooxygenase 1 (COX-1) and COX-2, as well as the COX-1b spice variant of COX-1. In many cells, COX-1 is expressed in a constitutive fashion, whereas COX-2 levels can be induced by specific stimuli. For example, in monocytes stimulated by inflammatory agents such as interleukin-1β (IL-1β), only levels of COX-2 increase. These observations have led to the concept that expression of COX-1 is important for homeostatic prostaglandin functions such as platelet aggregation and regulation of vascular tone, whereas upregulation of COX-2 is primarily important for mediating prostaglandin-dependent inflammatory responses. However, as selective inhibitors of COX-2 have become available, it has become clear that this is an oversimplification.

Cyclooxygenase is a bifunctional enzyme that first oxidizes AA to PGG₂ through its cyclooxygenase activity and then peroxidizes this compound to PGH₂. X-ray crystallographic studies of COX-1 reveal that the sites for the two enzymatic activities (i.e., cyclooxygenase and peroxidase) are adjacent but spatially distinct. The cyclooxygenase site is a long hydrophobic channel. Aspirin (acetylsalicylic acid) irreversibly inhibits COX-1 by acetylating a serine residue at the top of this channel. Several of the other NSAIDs interact, via their carboxyl groups, with other amino acids in the same region.

COX-1 activation plays an important role in intravascular thrombosis because it leads to TXA₂ synthesis by platelets. Inhibition of this process by low-dose aspirin is a mainstay for prevention of coronary thrombosis in patients with atherosclerotic coronary artery disease. However, COX-1 activation is also important for producing the cytoprotective prostanoids PGE₂ (a prostaglandin) and PGI₂ (a prostacyclin) in the gastric mucosa. It is the loss of these compounds that can lead to the unwanted side effect of gastrointestinal bleeding after long-term aspirin use. N3-15

Contributed by Emile Boulpaep, Walter Boron

Both COX-1 and COX-2 appear to be required for production of PGE₂ in the renal glomerulus, a process that is important in maintaining normal glomerular perfusion in the event of decreased renal blood flow. Thus, another risk of cyclooxygenase inhibitors is diminished renal function in patients with heart failure or volume depletion.

Similar to the nonselective cyclooxygenase inhibitors, COX-2 inhibitors have been shown to decrease renal perfusion and increase the risk of hemodynamic acute renal failure in susceptible individuals.

Reference

Schnermann J, Chou C-L, Ma T, et al. Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice. Proc Natl Acad Sci U S A. 1998;95:9660–9664.

Inflammatory stimuli induce COX-2 in a number of cell types, and it is inhibition of COX-2 that provides the anti-inflammatory actions of high-dose aspirin (a weak COX-2 inhibitor) and other nonselective cyclooxygenase inhibitors such as ibuprofen. Because the two enzymes are only 60% homologous, pharmaceutical companies have now generated compounds that specifically inhibit COX-2, such as celecoxib. COX-2 inhibitors work well as anti-inflammatory agents and have a reduced likelihood of causing gastrointestinal bleeding because they do not inhibit COX-1–dependent prostacyclin production. COX-2 inhibitors have been reported to increase the risk of thrombotic cardiovascular events when they are taken for long periods.

Contributed by Laurie Roman

Cyclooxygenase catalyzes the stepwise conversion of AA into the intermediates PGG₂ and PGH₂. Thus, this enzyme is also referred to as prostaglandin H synthetase (PGHS). As noted in Box 3-3, it is the same enzyme that catalyzes both reactions. Cyclooxygenase exists in three isoforms, COX-1 (a transcript of 2.8 kilobases [kb]), COX-2 (a 4.1-kb transcript), and COX-3 (a splice variant of COX-1 that is also known as COX-1b).

In the second pathway of AA metabolism, 5-lipoxygenase initiates the conversion of AA into biologically active leukotrienes. For example, in myeloid cells, 5-lipoxygenase converts AA to 5-hydroperoxyeicosatetraenoic acid (5-HPETE),  N3-13 which is short-lived and rapidly degraded by a peroxidase to the corresponding alcohol 5-HETE. Alternatively, a dehydrase can convert 5-HPETE to an unstable epoxide, leukotriene A₄ (LTA₄), which can be either further metabolized by LTA₄ hydrolase to LTB₄ or coupled (“conjugated”) by LTC₄ synthase to the tripeptide glutathione (see p. 955). This conjugation—via the cysteine residue of glutathione—yields LTC₄. Enzymes sequentially remove portions of the glutathione moiety to produce LTD₄ and LTE₄. LTC₄, LTD₄, and LTE₄ are the “cysteinyl” leukotrienes; they participate in allergic and inflammatory responses and make up the mixture previously described as the slow-reacting substance of anaphylaxis.

Contributed by Emile Boulpaep, Walter Boron

5-HPETE = 5-S-hydroperoxy-6-8-trans-11,14-cis-eicosatetraenoic acid

5-HETE = 5-hydroxyeicosatetraenoic acid

EET = cis-epoxyeicosatrienoic acid

The third pathway of AA metabolism begins with the transformation of AA by epoxygenase (a cytochrome P-450 oxidase).  N3-14 Molecular O₂ is a substrate in this reaction. The epoxygenase pathway converts AA into two major products, HETEs and EETs. Members of both groups display a diverse array of biological activities. Moreover, the cells of different tissues (e.g., liver, kidney, eye, and pituitary) use different biosynthetic pathways to generate different epoxygenase products.

Contributed by Emile Boulpaep, Walter Boron

As shown in Figure 3-11, one pathway of arachidonic-acid (AA) metabolism begins with the transformation of AA by epoxygenase (a cytochrome P-450 oxidase) to two major products: HETEs and EETs. Epoxygenase requires molecular oxygen (i.e., it is an oxidase) and has several required cofactors, including cytochrome P-450 reductase, NADPH/NADP⁺ (reduced/oxidized forms of nicotinamide adenine dinucleotide phosphate), or NADH/NAD⁺ (reduced/oxidized forms of nicotinamide adenine dinucleotide).

Prostaglandins, prostacyclins, and thromboxanes (cyclooxygenase products) are vasoactive, regulate platelet action, and modulate ion transport  N3-16

The metabolism of PGH₂ to generate selected prostanoid derivatives is cell specific. For example, platelets convert PGH₂ to thromboxane A₂ (TXA₂), a short-lived compound that can aggregate platelets, bring about the platelet release reaction, and constrict small blood vessels. In contrast, endothelial cells convert PGH₂ to prostacyclin I₂ (PGI₂), which inhibits platelet aggregation and dilates blood vessels. Many cell types convert PGH₂ to prostaglandins. Acting locally in a paracrine or autocrine fashion, prostaglandins are involved in such processes as platelet aggregation, airway constriction, renin release, and inflammation. N3-16 Prostaglandin synthesis has also been implicated in the pathophysiological mechanism of cardiovascular disease, cancer, and inflammatory diseases. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, acetaminophen, ibuprofen, indomethacin, and naproxen directly target cyclooxygenase. NSAID inhibition of cyclooxygenase is a useful tool in the treatment of inflammation and fever and, at least in the case of aspirin, in the prevention of heart disease.

Contributed by Laurie Roman

The prostanoids may participate in regulation of the Na-K pump, which plays a central role in salt and water transport in the kidney and the maintenance of ion gradients in all cell types. For example, the inhibition of the Na-K pump produced by IL-1 appears to be mediated by the formation of PGE₂. Indeed, IL-1 stimulates the formation of PGE₂, and application of exogenous PGE₂ inhibits Na-K pump activity directly. Moreover, cyclooxygenase blockers prevent the Na-K pump inhibition induced by IL-1. This action on the Na-K pump is not limited to the kidney; AA metabolites also inhibit the pump in the brain.

Prostaglandins also are vasoactive and are important in the regulation of renal blood flow.

The diverse cellular responses to prostanoids are mediated by a family of G protein–coupled prostanoid receptors. This family currently has nine proposed members, including receptors for thromboxane/PGH₂ (TP), PGI₂ (IP), PGE₂ (EP₁ to EP₄), PGD₂ (DP and CRTH2), and PGF_2α (FP). These prostanoid receptors signal via G_q, G_i, or G_s, depending on cell type. These in turn regulate intracellular adenylyl cyclase (see p. 53) and phospholipases (see p. 58).

The leukotrienes (5-lipoxygenase products) play a major role in inflammatory responses

Many lipoxygenase metabolites of AA have a role in allergic and inflammatory diseases (Table 3-3).  N3-17 LTB₄ is produced by inflammatory cells such as neutrophils and macrophages. The cysteinyl leukotrienes including LTC₄ and LTE₄ are synthesized by mast cells, basophils, and eosinophils, cells that are commonly associated with allergic inflammatory responses such as asthma and urticaria.

Since the original description of the slow-reacting substance of anaphylaxis, which is generated during antigenic challenge of a sensitized lung, leukotrienes have been presumed to play a part in allergic disease of the airways (see Table 3-3). The involvement of cells (mast cells, basophils, and eosinophils) that produce cysteinyl leukotrienes (LTC₄ through LTF₄) in these pathobiological processes supports this concept. In addition, the levels of LTC₄, LTD₄, and LTE₄ are increased in lavage fluid from the nares of patients with allergic rhinitis after the application of specific antigens to the nasal airways. Introducing LTC₄ or LTD₄ into the airways as an aerosol (nebulizer concentration of only 10 µM) causes maximal expiratory airflow (a rough measure of airway resistance; see p. 602) to decline by ~30%. This bronchoconstrictor effect is 1000-fold more potent than that of histamine, the “reference” agonist. Leukotrienes affect both large and small airways; histamine affects relatively smaller airways. Activation of the cysLT1 receptor in mast cells and eosinophils results in the chemotaxis of these cells to sites of inflammation. Because antagonists of the cysLT1 receptor (e.g., montelukast sodium) can partially block these bronchoconstrictive and proinflammatory effects, these agents are useful in the treatment of allergen-induced asthma and rhinitis.

In addition to being involved in allergic disease, several of the leukotrienes are associated with other inflammatory disorders. Synovial fluid from patients with rheumatoid arthritis contains 5-lipoxygenase products. Another example is the skin disease psoriasis. In patients with active psoriasis, LTB₄, LTC₄, and LTD₄ have been recovered from skin chambers overlying abraded lesions. Leukotrienes also appear to be involved in inflammatory bowel disease. LTB₄ and other leukotrienes are generated and released in vitro from intestinal mucosa obtained from patients with ulcerative colitis or Crohn disease.

DISEASE	EVIDENCE
Asthma	Bronchoconstriction from inhaled LTE4; identification of LTC4, LTD4, and LTE4 in serum or urine or both
Psoriasis	Detection of LTB4 and LTE2 in fluids from psoriatic lesions
Adult respiratory distress syndrome (ARDS)	Elevated levels of LTB4 in plasma
Allergic rhinitis	Elevated levels of LTB4 in nasal fluids
Gout	Detection of LTB4 in joint fluid
Rheumatoid arthritis	Elevated levels of LTB4 in joint fluids and serum
Inflammatory bowel disease (ulcerative colitis and Crohn disease)	Identification of LTB4 in gastrointestinal fluids and LTE4 in urine

Contributed by Laurie Roman

LTC₄, LTD₄, LTE₄, and LTF₄ are often referred to as the “cysteinyl leukotrienes” or sometimes as the “peptidyl leukotrienes.” As summarized in Figure 3-11, the enzyme glutathione-S-transferase (GST) conjugates LTA₄, which is unstable, to the sulfhydryl group of the cysteine in glutathione (glutathione, also abbreviated GSH, is the branched tripeptide Glu-Cys-Gly) to produce LTC₄. (See page 955 to learn how the liver uses GSH for conjugation reactions.) The enzyme γ-glutamyl transferase clips off the glutamate residue of LTC₄ to produce LTD₄ (which is conjugated to -Cys-Gly). A dipeptidase clips the dipeptide bond between Cys and Gly to release the terminal Gly as well as LTE₄ (which is conjugated to only the -Cys).

Leukotrienes have multiple effects on the vascular endothelium during inflammation. Various regulatory processes may interact at the level of the small blood vessels to increase the margination (i.e., the attachment to the vessel wall) of subgroups of leukocytes, increase the permeability at the postcapillary venule, and evoke diapedesis (i.e., the migration of the cell through the endothelium) of the adherent leukocytes to create a focus of interstitial inflammation. Each of these steps can be affected by leukotrienes as well as other agents.

The infiltration of leukocytes begins when the cells adhere to the endothelium of the postcapillary venule. Mediators that can increase the adhesiveness of leukocytes include LTB₄ and several of the cysteinyl leukotrienes. Increased vascular permeability, influenced by the pulling apart of adjacent endothelial cells, can occur in response to LTC₄, LTD₄, and LTE₄. After adherent leukocytes accumulate—and the size of the interendothelial cell pores increases—a stimulus for diapedesis produces an influx of leukocytes into the interstitial space. Once in the interstitial space, the leukocytes come under the influence of LTB₄, a potent chemotactic factor (i.e., chemical attractant) for neutrophils (a type of white blood cell that phagocytoses invading organisms) and less so for eosinophils (another type of white blood cell). LTB₄ is also chemokinetic (i.e., speeds up chemotaxis) for eosinophils.

In the lungs, the cysteinyl leukotrienes appear to stimulate the secretion of mucus by the bronchial mucosa. Nanomolar concentrations of LTC₄ and LTD₄ stimulate the contraction of the smooth muscles of bronchi as well as smaller airways.

Both LTB₄ (generated by a hydrolase from the unstable LTA₄) and the cysteinyl leukotrienes (i.e., LTC₄, LTD₄, and LTE₄) act as growth or differentiation factors for a number of cell types in vitro. LTB₄stimulates myelopoiesis (formation of white blood cells) in human bone marrow, whereas LTC₄ and LTD₄ stimulate the proliferation of glomerular epithelial cells in the kidney. Picomolar concentrations of LTB₄ stimulate the differentiation of a particular type of T lymphocytes referred to as competent suppressor or CD8⁺ lymphocytes. Additional immunological regulatory functions that may be subserved by LTB₄ include the stimulation of IFN-γ and IL-2 production by T cells.

The cysteinyl leukotriene receptors cysLT1 and cysLT2 are GPCRs found on airway smooth-muscle cells as well as on eosinophils, mast cells, and lymphocytes. CysLT1, which couples to both pertussis toxin–sensitive and pertussis toxin–insensitive G proteins, mediates phospholipase-dependent increases in [Ca²⁺]_i. In the airways, these events produce a potent bronchoconstriction, whereas activation of the receptor in mast cells and eosinophils causes release of the proinflammatory cytokines histamine and tumor necrosis factor-alpha (TNF-α).

In addition to playing a role in the inflammatory response, the lipoxygenase metabolites can also influence the activity of many ion channels, either directly or by regulating protein kinases. For example, in synaptic nerve endings, lipoxygenase metabolites decrease the excitability of cells by activating K⁺ channels. Lipoxygenase products may also regulate secretion. In pancreatic islet cells, free AA generated in response to glucose appears to be part of a negative-feedback loop that prevents excess insulin secretion by inhibiting CaM kinase II.

The HETEs and EETs (epoxygenase products) tend to enhance Ca²⁺ release from intracellular stores and to enhance cell proliferation

The epoxygenase pathway leads to the production of HETEs other than 5-HETE as well as EETs. HETEs and EETs have been implicated in a wide variety of processes, some of which are summarized in Table 3-4. For example, in stimulated mononuclear leukocytes, HETEs enhance Ca²⁺ release from intracellular stores and promote cell proliferation. In smooth-muscle cells, HETEs increase proliferation and migration; these AA metabolites may be one of the primary factors involved in the formation of atherosclerotic plaque. In blood vessels, HETEs can be potent vasoconstrictors. EETs enhance the release of Ca²⁺ from intracellular stores, increase Na-H exchange, and stimulate cell proliferation. In blood vessels, EETs primarily induce vasodilation and angiogenesis, although they have vasoconstrictive properties in the smaller pulmonary blood vessels.

	CELL/TISSUE	ACTION
HETEs	Stimulated mononuclear leukocytes	↑ Cell proliferation ↑ Ca2+ release from intracellular stores ↓TNFα production
	β cells of pancreatic islets	Implicated in the destruction of these cells in type 1 (juvenile-onset) diabetes mellitus
	Endothelial cells	↓ Release of fibrinolytic factors ↓ Binding of antithrombin
	Vascular smooth-muscle cells	↑ Cell proliferation ↑ Migration Formation of atherosclerotic plaque?
	Blood vessels	Potent vasoconstrictors “Myogenic” vasoconstrictive response of renal and cerebral arteries
EETs	Cells, general	↑ Ca2+ release from intracellular stores  ↑ Na-H exchange ↑ Cell proliferation ↓ Cyclooxygenase activity
	Endocrine cells	↓ Release of somatostatin, insulin, glucagon
	Toad bladder	↓ Vasopressin-stimulated H2O permeability ↓ Renin release
	Blood vessels	Vasodilation Angiogenesis
	Endothelium Platelets	↑ Tumor cell adhesion ↓ Aggregation

Degradation of the eicosanoids terminates their activity

Inactivation of the products of eicosanoids is an important mechanism for terminating their biological action. In the case of COX products, the enzyme 15-hydroxyprostaglandin dehydrogenase catalyzes the initial reactions that convert biologically active prostaglandins into their inactive 15-keto metabolites. This enzyme also appears to be active in the catabolism of thromboxanes.

As far as the 5-lipoxygenase products are concerned, the specificity and cellular distribution of the enzymes that metabolize leukotrienes parallel the diversity of the enzymes involved in their synthesis. For example, 20-hydrolase-LTB₄, a member of the P-450 family, catalyzes the ω-oxidation of LTB₄, thereby terminating its biological activity. LTC₄ is metabolized through two pathways. One oxidizes the LTC₄. The other pathway first removes the glutamic acid residue of the conjugated glutathione, which yields LTD₄, and then removes the glycine residue, which yields LTE₄; the latter is readily excreted into the urine.

The metabolic breakdown of the HETE and EET products of epoxygenase (cytochrome P-450) is rapid and complex. The predominant pathway of inactivation appears to be hydrolysis via soluble epoxide hydrolase to form dihydroxyeicosatrienoic acids (DHETs), which themselves can induce biological responses, such as vasodilation. Once formed, DHETs can be excreted intact in the urine or can form conjugates with reduced glutathione (GSH). In addition, both EETs and DHETs can undergo β-oxidation to form epoxy fatty acids or can be metabolized by cyclooxygenase to generate various prostaglandin analogs.