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Chapter 3

Receptors and chemical message transduction

Interpretation of studies in exercise physiology that employhormone (H) measurements or pharmacological agents that modify Hrelease or action requires an understanding of the characteristics ofreceptors (Rs) and the way they interact with Hs. Selection of anappropriate method of H measurement requires an understanding of thelimitations of available approaches. This chapter describes principal typesof receptors and transduction mechanisms that affect the functioning ofseveral key organs during exercise as well as characteristics of H-Rinteractions. Because measurement of hormones utilizes features of H-Rinteractions, methods of hormone measurement also are briefly described.Molecular biology rapidly is advancing knowledge about receptorstructure and function so that descriptions of chemical messagetransduction will have to undergo frequent revisions.

Types of chemical messenger receptors

The chemical messengers act on Rs, composite proteins locatedon cell membrane or in the interior of the cell which consist of a specificrecognition site to which H binds and an effector mechanism throughwhich the chemical message is transduced into biological action. Thereis great diversity in the structure and function of chemical messengers butthey can be classified into categories according to their location andstructural characteristics (Baulieu 1990, Mendelson 1996). By location,Rs are located in a cell’s plasma membrane or are found in the cytoplasmor in the nucleus. Water- soluble messengers such as peptide hormones,catecholamines and indoleamines have Rs embedded in the plasmamembrane, while steroid and thyroid hormones are hydrophobic lipids thatdiffuse freely through the lipophilic plasma membrane to bind tointracellular Rs and subsequently to nuclear DNA. Rs embedded inplasma membrane have (1) an extracellular domain which containshydrophilic amino acids and the amino terminal, (2) an intracellulardomain ending at the caboxy terminal, and (3) a membrane-spanninghydrophobic domain (Figure 31). The extracellular domain is frequentlyglycosylated in the course of H maturation, and carbohydrates attachedto R proteins may facilitate recognition or hormonal binding processes.Rs located in the cell interior also consist of three parts, (1) ahypervariable (HVD) hormone-specific domain at the amino terminus, (2)

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Figure 31. Structural characteristics of main categories of receptorsReceptors for water-soluble messengers are embedded in plasma

membrane and are of four kinds: (A) Rs containing an extracellulardomain ending in an amino terminus, a single membrane-spanning region,and an intracellular domain ending in a carboxy terminus; (B) Rs with asingle trans-membrane region incorporating tyrosine kinase activity in theirintracellular domain; (C) Rs with multiple membrane-spanning segmentsthat are associated with G proteins; and (D) Rs with multiple membrane-spanning segments that form ion channels. Receptors for lipophilicsteroid/thyroid hormones (E) are located in the cell interior or in thenucleus and consist of a hypervariable domain (HVD), a central DNA-

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binding domain (DBD), and one or more hormone-binding domains (HBD).________________________________________________________

a central DNA-binding domain (DBD) consisting of about 50 to 100 aminoacids and a characteristic “zinc-finger” region that binds to the hormoneresponsive element (HRE) on DNA, and (3) a hormone-binding domain(HBD) at the carboxy terminus consisting of one or more clusters ofhydrophobic amino acids to which lipophilic steroid and thyroid hormonesbind (Figure 31).

By molecular structure, membrane-spanning Rs can be categorizedinto four groups (Figure 31): (1) Rs with a single transmembrane region,(2)Rs with an intracellular tyrosine kinase effector attached to thetransmembrane region, (3) Rs with multiple membrane-spanning regionsthat are associated with guanine nucleotide-binding proteins (G proteins),and (4) Rs with multiple membrane-spanning regions that form ionchannels. Many hormones act on more than one category of receptors,and some receptors encompass more than one structural category.

Rs with a single transmembrane region (Figure 31 A) includegrowth hormone R (GH-R), prolactin R (PRL-R), nerve growth factor R(NGF-R) and some cellular nutrient receptors such as low-densitylipoprotein (LDL-R) and transferrin Rs (T-R). GH-R also exists as acirculating GH-binding protein with a truncated intracellular region, andNGF-R and T-R are dimers.

Rs with an intracellular tyrosine kinase effector (Figure 31 B)include insulin R (I-R), insulin-like growth factor-I R (IGF-I-R), epidermalgrowth factor R (EGF-R), platelet-derived growth factor R (PDGF-R), andcolony-stimulating factor-I R (CSF-I-R). Of those, I-R and IGF-I-R areheterotetramers consisting of two membrane-spanning beta units thathave tyrosine kinase activity and two extracellular alpha units attached tobeta units through cysteine cross-bridges. Some tumors overexpressEGF-R, and Rs with tyrosine kinase activity frequently participate incellular growth.

The family of Rs with multiple membrane-spanning regionsassociated with G proteins (Figure 31 C, Dohlman et al., 1987, 1991)includes Rs for many hormones and neurotransmitters. Included in thisgroup are adenosine Rs (Fredholm et al. 1994), alpha-1 and alpha-2 typeof alpha adrenergic Rs (AA-R), beta adrenergic Rs (BA-R), calcium R on

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calcitonin and PTH cells (Nemeth & Scarpa, 1987), muscarinic cholinergicRs (mACH-R), and Rs for angiotensin II , AVP, DA, endothelin (ETA andETB), LH, FSH, platelet-activating factor (PAF), serotonin (5HT-R),substance P (SP), TSH, and many others. These Rs are embedded inplasma membrane in the vicinity of G (guanine nucleotide-bindingregulatory) proteins and catalytic enzymes.

G proteins (Gilman 1989, Linder & Gilman, 1992, Spiegel et al,1993) are structurally similar but functionally diverse. They consist ofidentical beta and gamma subunits, and a functionally differentiated alphasubunit. G proteins are categorized according to their ability to affectenzyme function or ion channels. The Gs protein stimulates enzymeadenylyl cyclase (AC, Benovic et al., 1988, Strader et al., 1989) to convertthe substrate Mg2+-ATPto the second messenger cyclic AMP (cAMP)which in turn activates the enzyme cAMP-dependent protein kinase (PK)-A . Hs that activate Rs coupled to GS protein are ACTH, adenosine ( A2

Rs), alphaMSH, AVP (V2 Rs in the kidney), calcitonin, DA (D1 receptors),E (beta1,beta2, and beta3 Rs, Gilman 1987), FSH, gastrin, glucagon,gonadotropin releasing hormone (GnRH-R), histamine (H2 Rs), LH, NE(beta1, beta2 and beta3 Rs), parathyroid hormone (PTH-R), secretin, 5-HT, TSH, and VIP.

The Gi protein inhibits the activity of AC. Hormones that act on Rsassociated with Gi protein are mACH-R , adenosine (A1 Rs, Fredholm etal., 1994), bradykinin, DA (D2 Rs), NE (alpha2Rs), opioids, and SRIF.

The Gc protein stimulates the enzyme guanylyl cyclase (Garbers &Lowe, 1994) to produce cyclic guanosine monophosphate (c-GMP) andincrease the activity of the enzyme phosphodiesterase (PDE) and at timesalso open a calcium channel. PDE degrades cAMP to the inactive form 5'-AMP and activates the enzyme PK-G. Hormones that act through Gc

protein are ACH (m ACH-R) and atrial natriuretic factor (ANF).

The Gq protein (sometimes also designated as Gp protein)stimulates the enzyme phospholipase C (PL-C, Rhee & Choi, 1992) toincrease turnover of membrane phospholipids and produce secondmessengers (Berridge 1993, Exton, 1990) inositol triphosphate (IP3) anddiacyl glycerol (DG). The DG in turn activates the enzyme PK-C(Nishizuka 1992). Hs that act on Rs associated with Gq protein are foradenosine (A1-R), angiotensin II, ACH (mACH), AVP (V1 Rs in the liverand vascular smooth muscle), bombesin, CCK, DA (D2 -Rs), E (alpha1B -

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Rs, Han et al., 1987), EGF-R, GnRH, histamine (H1 -Rs), NE (alpha1 -AA-R), NGF, PDGF, PAF, 5-HT (5-HT 2-R), substance-P (SP-R),thromboxanes, TRH, and VIP. Finally, the Gk protein regulates potassiumchannels.

The last category of Rs consists of proteins with multiplemembrane-spanning regions that form ion channels (Figure 31 D). Amongthese are hormone-gated channels and ion pumps. Nicotinic ACH R(nACH-R, Stroud 1985), gamma-aminobutyric acid A R (GABAA -R), andglycine R are some of the H-gated channels. The nACH-R contains fivetransmembrane regions (Figure 31 D) of which two alpha subunitsconstitute the H recognition site. Activation of nACH-R opens a channelthat permits diffusion of Na+ and K+ across the cell membrane. Na+- K+

ATPase (Bertorello & Katz, 1995), H+- K+ ATPase, and Ca2+ ATPase areion pumps that also belong to this R family.

Mechanisms of chemical message transduction

A limited number of transduction mechanisms translates signalsfrom many hormones into biological action. Thus a considerable numberof hormones with distinct recognition sites act on the same transductionapparatus in a given cell. Biological response depends on coupling aparticular effector mechanism to a given transduction mechanism and isnot determined by either the R type or a particular transduction pathway.Thus the same R can produce opposite effects in some tissues (forexample BA-R in heart compared to smooth muscle), and Rs that oftenhave opposite effects (for example the AA-R and BA-R in smooth muscle)can have the same effect in some tissues (for example the AA-R and BA-R effects on glycogenolysis). Similarly, a transduction mechanism canhave opposite effects in different tissues (for example the cAMP pathwayin cardiac and smooth muscle). Ttransduction mechanisms that usuallyhave opposite effects (cAMP and IP3 pathways) can produce the samebiological effect in some tissues (liver glycogen).

Looking ahead, several transduction mechanisms that utilize Gs, Gi,

and Gq proteins are explained in some detail. Other transductionpathways are more briefly described, for (1) adenosine transduction , (2)sphingomyelinase, (3) growth-promoting Rs with a single trans-membraneregion, (4) Rs with tyrosine kinase activity, and (5) nitric oxide. Finally themechanism of intracellular action of steroid and thyroid Hs is reviewed.

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cAMP transduction pathway

Figure 32. Transduction pathways that stimulate, inhibit, or degrade cAMPHormones that stimulate (Hs) adenylyl cyclase (AC) bind to their

receptors (Rs) and trigger an interaction between the Rs, Gs protein andAC (left). Substitution of a GTP for GDP is needed to activate the Gs

protein and lead to dissociation of its alphas

subunit. G alphas attaches to, and stimulates, AC to produce cAMP fromMg2+-ATP.Hormones that inhibit AC (Hi), bind to their receptors (Ri) and initiate atransduction event (top center) that includes dissociation of the beta-gamma subunits from the activated Gi protein and their association withthe G alphas thereby blocking the formation of cAMP. Hormones thatdegrade cAMP (Hd) engage in analogous transduction process afterbinding to their receptor (Rd). The interaction between Hd, Rd, and Gc protein results in activation of guanylyl cyclase, production of cGMP andsubsequent activation of the enzyme phosphodiesterase (PDE). PDEdegrades cAMP to inactive 5'-AMP.________________________________________________________

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H-mediated stimulation of AC (Figure 32, left) entails interactionbetween three proteins, R, Gs protein , and the enzyme AC (Benovic etal., 1988, Strader et al., 1989). In the unstimulated state, guanosinediphosphate (GDP) is bound to the alpha subunit of the Gs protein. AfterH-R binding, GDP dissociates, and a guanosine triphosphate (GTP)attaches to the alpha subunit of the Gs protein. This activation step isnecessary for the R and Gs protein to aggregate and for the alpha subunitof the GS protein to dissociate from the beta and gamma subunits. Gs-alpha-GTP then complexes with, and activates, the catalytic enzyme ACto convert Mg2+-ATP to cAMP. GTPase that is associated with theactivated Gs-alpha hydrolyzes GTP to GDP and terminates transduction ofthe endocrine message.

The cAMP acts as a second messenger that regulates the activityof cAMP-dependent PK-A. PK-A (Cohen 1992) affects a number ofenzymes involved in mobilization and utilization of metabolic fuels,muscle contraction, and endocrine and exocrine secretion throughphosphorylation of enzymes or ion-channel proteins. The cAMP alsoinfluences expression of genes for gluconeogenic enzymephosphoenolpyruvate carboxykinase (PEPCK), tyrosine aminotransferase,preprosomatostatin, VIP and cytochrome P-450 that is involved in steroidhydroxylation. Transcription effects are mediated by the cAMP response-element binding protein (CREB). CREB is activated by cAMP and bindsto the cAMP response element (CRE) on the DNA (Meyer et al. 1993).Individual steps in the Gs activation pathway elicit increased responsesand result in amplification of hormonal messages.

Hormone-mediated inhibition of AC (Figure 32, top center) alsoentails interaction between three proteins, R for the inhibitory H, Gi protein,and AC. Binding of Hs to Rs that inhibit AC causes dissociation of GDPfrom the Gi alpha subunit and substitution of GTP in the place of GDP.The inhibition of AC activity is achieved by interaction of free beta-gammasubunits from the Gi protein with Gs alpha subunits.

IP3- DG transduction pathway

The Gq transduction pathway entails the activation of PL- C (Rhee& Choi, 1992) following H-R binding (Figure 33, top). This membrane-associated enzyme then catalyzes hydrolysis of a membranephospholipid, phophatidylinositol 4,5-biphosphate (PIP2), to secondmessengers IP3 and DG (Berridge 1993, Exton 1990). IP3 binds to its R, a

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membrane-spanning protein on the ER or SR that forms a Ca2+ channel,and triggers the release of Ca2+. Calcium mediates second messengeraction by binding to specific Ca2+-binding proteins, calmodulin or troponin.After binding with Ca2+, activated calmodulin stimulates various enzymes,and activated troponin uncovers myosin binding sites on actinmyofilaments and causes skeletal muscle contraction.

Figure 33. IP3-DAG, cGMP and nitric oxide transduction pathwaysIP3 and DAG are second messengers in a transduction pathway

(top left) that involves an interaction between H,R, and Gq protein . Theresult is activation of phospholipase-C (PL-C) that converts a membranephospholipid PIP2 into two second messengers. IP3 triggers release ofCa2+ from the SR after binding to a R on SR. Ca2+ bound to proteins suchas troponin or calmodulin and these proteins then effect a number ofbiological actions. cGMP is a second messenger in a transductionpathway (right) that entails activation of guanylyl cyclase (GC) andstimulation by cGMP of enzymes such as PDE and PK-G. PDE convertscAMP into an inactive molecule. PK-G closes the L-type Ca2+ channel.The NO transduction pathway (left center) entails the activation of nitricoxide synthetase (NOS, center) by calmodulin and the control of cytosolicCa2+ release through a bifunctional enzyme (H-C). This enzyme isactivated by cGMP formed by cytoplasmic GC after NO binds to it. Onesubunit of the bifunctional enzyme (ribosylcyclase, C) can form cADP-ribose that binds to a recognition site on R-R to release Ca2+ from SRthrough its channel. The other subunit of the enzyme (hydrolase,H)closes the Ca2+ channel by hydrolyzing cADP-ribose to a non-cyclic form.

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DG, the other second messenger in the Gq transduction pathwayincreases the affinity of PK-C (Nishizuka 1992) for Ca2+. DG can also beproduced from phosphatidylcholine by activation of PL-D in response tomACH and V-1 R binding to ACH and AVP, respectively. Activated PK-Cphosphorylates enzymes and affects the expression of genes. Thegenetic effect involves activation of a DNA-binding transcription factor AP-1 that contains two proto-oncogene proteins, c-fos and c-jun, throughwhich DG stimulates cell proliferation and tumorigenesis.

cGMP and nitric oxide transduction pathways

Cyclic guanosine monophosphate (cGMP) is a second messengerthat is formed from GTP in a reaction associated with Gc protein andcatalyzed by guanylyl cyclase (GC), an enzyme found both in the plasmamembrane and in the cytoplasm (Garbers & Lowe, 1993). Its maineffector action is activation of PDE, the enzyme that inactivates cAMP(Figure 32). The membrane-bound cGMP transduction pathway (Figure33, right) blocks phosphorylation of the L-type calcium channel throughthe dephosphorylating action of protein kinase-G (PK-G, Petit-Jacques etal. 1994) after activation of cardiac mACH receptors. It is also involved inthe ANP-stimulated production of cGMP that results in vasodilatation,natriuresis and diuresis.

GC can also be activated by nitric oxide (NO), the free radicalmessenger that binds to the enzyme and thus influences a number ofphysiological processes (Knowles & Moncada, 1992) of importance inexercise. NO is produced in muscles, both smooth and skeletal, nerves,and endothelia. NO suppresses contractility in type II skeletal muscles(Kobzik et al. 1994, Reid 1996), and induces relaxation in smooth muscles(Moncada et al. 1991). Nitrergic innervation is found in the heart, bloodvessels, airway epithelia, enteric plexuses of the GI tract and in GIsphincters (Rand & Li, 1995) and VIP is often a co-transmitter. Inaddition, NO is produced in macula densa of the JGA where itparticipates in the regulation of plasma volume (Wilcox et al. 1992). NO isresponsible for vasodilatation to ACH when it acts on intraluminalendothelium (Furchgott & Zawadski, 1980). Before the recentidentification of NO as the agent responsible for endothelium-dependentvasodilatation, the putative messenger was previously known asendothelium-derived relaxation factor (EDRF).

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NO is produced (Figure 33, left) by the enzyme NO synthetase(NOS). NOS is expressed either as a constitutive enzyme that isassociated with the Ca2+ -binding protein calmodulin (Chow et al., 1992)as is the case in endothelia and nerve terminals, or as an inducibleenzyme that requires high cytoplasmic Ca2+ for activation of calmodulinas is the case in smooth muscles exposed to macrophages orlipopolysaccahride (LPS, Katusic & Cosentino, 1994). Skeletal muscleexpresses both forms of NOS (Kobzik et al., 1995). NOS is a P450cyclooxygenase (White & Marletta, 1992) consisting of a reductase and anoxygenase domain. The reductase contains flavine adenine dinucleotide(FAD) and flavine mononucleotide (FMN) which remove the electrons fromnicotinamide dinucleotide phosphate (NADPH+) and pass them on to theoxidase. The oxidase domain has an iron-containing haem group (H inFigure 33) and is associated with a H4 biopterin cofactor (BH4 in Figure33). It oxidizes L-arginine to L-citrulline and NO and reduces molecularoxygen to water (Figure 33, Abu-Soud & Stuehr, 1993). In the absence ofthe amino acid substrate and the cofactor, NOS generates superoxidesand hydrogen peroxide (Mayer et al, 1991).

NO is thought to produce vasodilatation by several signallingpathways. Among its actions that prevent smooth muscle contraction areclosure of L-type Ca2+ channels mediated by cGMP and PK-G (Figure 33,right, Ahlner et al., 1990, Furukawa et al., 1987), and closure ofsarcoplasmic or endoplasmic Ca2+ channels known also as ryanodynereceptors (R-R, Figure 33, center). NO controls R-R closure through itseffect on dissociation of a messenger cyclic adenosine diphosphateribose (cADPr) from its binding site on R-R (Lee et al. 1994). Afteractivating guanylyl cyclase, cGMP phosphorylates and activates abifunctional enzyme (H-C in Figure 33) that can either open sarcoplasmicCa2+ channels (ribosylcyclase action shown as C in Figure 33) throughsynthesis of cADP-r from nicotine adenine dinucleotide (NAD), or closethe channel (hydrolase action shown as H in Figure 33) by facilitatingcADP-r dissociation from R-R and its degradation. In the skeletal muscleand the heart, NO appears to inhibit development of tension by hydrolysisof cADP-r. NO can also cause vasodilatation by acting on enzymes thatdephosphorylate light chain of myosin (Ahlner et al., 1991, Rapoport etal., 1983). Since NO synthesis is linked to IP3-induced activation ofcalmodulin, and NO acts through the cGMP second messenger, this novelintracellular agent permits messaging across two transduction pathways.

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Adenosine transduction pathway

Adenosine is a nucleoside messenger consisting of a purine baseadenine bound to ribonucleic acid. Adenosine is released throughneurotransmission by ATP-containing S fibers or produced in thecytoplasm by progressive enzymatic degradation of ATP and ADP,nucleotide mediators of cellular metabolism. ADP is converted toadenosine by enzyme 5'-nucleotidase. Adenosine acts on A1, A2 and A3

receptors (Baraldi et al.1995, Jacobson et al. 1992). Principalnonselective antagonists are the commonly-imbibed xanthine derivativescaffeine and theophylline. Other agonists and antagonists are listed inTable 8.

A1-Rs are prevalent in adipose tissue where they inhibit lipolysis(Schwabe et al. 1973) and increase insulin sensitivity (Londos et al.,1985) ; skeletal muscle where they modulate insulin sensitivity (Challiset al.,1984,1992, Webster et al., 1996); heart where they have anti-adrenergic function (Dobson & Fenton, 1993, Headrick, 1996); respiratorysmooth muscles where they cause bronchospasm (Van Schoor et al.,1997); brain where they decrease arousal, physical activity and NEneurotransmission (Fredholm et al.1994); and anterior pituitary glandwhere they decrease GH secretion (Dorflinger & Schonbrunn, 1985).Transduction of adenosine message after binding to A1-Rs involves bothGi (Figure 32, top center) and Gq proteins (Figure 33, top, Fredholm et al.1994). Transduction through the Gi pathway produces membranehyperpolarization via opening of K+ channels and closing of voltage-dependent Ca2+ channels.

A2 Rs are found in vascular smooth muscles where they inhibitcontraction and in brain. Adenosine binding to A2-Rs is coupled to Gs

protein and activates adenylate cyclase to form cAMP (Figure 32, left).Principal biological effects (discussed also in connection with the role ofRs in vasomotor control) are dilatation of vascular smooth muscle,suppression of platelet aggregation and leukocyte adhesion (Anfossi et al.1996, Feoktisov et al., 1992), and stimulation of hepatic glycogenolysis.

Ceramide transduction pathway

The metabolism of another lipid membrane component,sphingomyelin, mediates chemical signals by the cytokine tumor-necrosisfactor- alpha (TNF-alpha). Binding of TNF-alpha to its cell membrane

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receptors activates the enzyme sphingomyelinase which hydrolyzessphingomyelin into phosphocholine and ceramide. The latter is a secondmessenger that activates a ceramide-activated protein phosphatase(CAPP). CAPP inhibits cellular proliferation and induces celldifferentiation and programmed cell death (apoptosis). VitD3 induces celldifferentiation through this pathway when it acts at membrane sitesrather than inside the nucleus (Hannun, 1994).

Anabolic-hormone transduction pathways

Among the growth-promoting Rs with a single trans-membraneregion are GH-R, PRL-R and other members of cytokine receptor family(Kitamura et al. 1994). A GH molecule binds to two GH-Rs , and thisdimer associates with one of several cytoplasmic Janus (JAK) kinases(Carter-Su et al., 1996, Figure 34). JAK2 is associated with activated GH,PRL and erythropoietin Rs, JAK1 with activated interferon receptors, andJAK 3 with interleukin-2 Rs. Although the Rs of the cytokine family lacktyrosine kinase activity that characterizes Rs of some other growth-promoting messengers, their binding to Hs stimulates JAK tophosphorylate tyrosine residues on the R and on a variety of transcriptionactivators that they recruit and stimulate. Phosphorylated moleculesinclude signal transducers and activators of transcription (STATs) thatbind to specific HREs within the regulatory regions of genes, insulin Rsubstrates (IRSs), and others. GH transduction engages other secondmessengers such as DAG, Ca2+, PK-C,mitogen-activated protein (MAP)kinases, PL-A, and phosphoinositol-3-K, but connections to thesesignalling pathways are not well understood .

There are four families of receptors that have a transmembranedomain and an intracellular tyrosine kinase domain: (1) the EGF-R family,(2) insulin/IGF-I R family, (3) PDGF R family, and (4) fibroblast growthfactor (FGF) R family. Binding of respective messengers to these Rsstimulates cell proliferation, differentiation, and migration. Transduction ofRs in these families usually includes receptor dimerization, activation oftyrosine kinase (Schlesinger 1993, Ullrich & Schlesinger, 1990), andreceptor and enzyme phosphorylation on tyrosine residues which thenenhances the capacity of the enzyme to phosphorylate other proteins.Phosphorylation takes place when proteins containing a sequence ofamino acids referred to as src homology 2 (SH2) dock and bind tophosphorylated tyrosines on the receptor. Some cellular proto-oncogeneproducts (Src for instance) that affect cellular growth contain SH2 domain

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but lack the H-binding domain.

Figure 34. Anabolic hormone transduction pathwaysGH signalling is initiated when GH dissociates from its binding

protein, binds to a membrane R and forms a dimer (top). Attachment ofJanus kinases2 (JAK2) to H-R complex triggers phosphorylation of R andof JAK2 proteins on tyrosine residues and attracts various transcriptionfactors (STATs, IRSs). These proteins bind to the H-R complex and getphosphorylated and subsequently initiate gene transcription that ofteninvolves mediation by mitogen-activated protein kinases (MAPKs). Othergrowth promoting messengers, for example PDGF, have intrinsic tyrosinekinase activity on their intracellular domain. They also have signallingpathways that include receptor dimerization, autophosphorylation andphosphorylation of other transcription mediators on tyrosine residues.Frequently the IP3-DAG second messengers or other paths ofintracellular signalling are incorporated into growth-promoting transductionprocess._______________________________________________________

Tyrosine kinases usually are components of signalling pathwaysthat interact with other transduction systems (Figure 34). For instance,PDGF binding to its R increases cytosolic calcium by utilizing the Gq

protein pathway. This takes place as a result of activation of PL-C-gammawhich binds through its SH2 domain to phosphorylated receptor and

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results in IP3 and DAG production. The autophosphorylated PDGFreceptor (Williams 1989) also activates MAP kinases (Cobb & Goldsmith,1995, Davis 1993). MAP kinases phosphorylate cellular proteins includingtranscription factor AP-1 and its constituents c-fos and c-jun.

Steroid/thyroid hormone transduction pathways

Figure 35. Steroid/thyroid hormone transduction pathwaysTransduction of the steroid or thyroid hormone message starts

when the H dissociates from its binding protein and binds to the R. Usuallytwo molecules of heat shock protein (hsp) 90 and one molecule of hsp70are bound to the R before it is occupied by the H. These molecules arereleased when H binds to its R. Dimerized H-R complex turns into atranscription factor. The DBD region of the R binds to the HRE on DNA.Other transcription factors, especially IIB, assist in the attachment of theRNA polymerase II and in initiation of gene transcription._______________________________________________________

Rs for thyroid hormones, vitD3 and vitamin A (retinoic acid) arelocated in the nucleus and are bound to the HRE on the DNA where theysuppress the expression of respective genes. Rs for glucocorticoids (G-R), mineralocorticoids (M-R), androgens (AN-R), estrogen (E-R) andprogesterone (PROG-R) are located in the cytoplasm (O’Malley 1990,

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Tsai & O’Malley, 1994) bound to two molecules of heat-shock proteins(hsp)90 and one molecule of hsp70 (Pratt 1990, 1993, Figure 35). Whenthese Rs bind with respective Hs, the H-R complex turns into atranscription factor that initiates gene expression. Rs associated with hspsrelease these proteins and form dimers (Glass 1994) which then bind tothe HRE on DNA. Other proteins known as IIA through IIF, particularly thetranscription factor (TF) IIB, bind to the TATA box on DNA and allow theattachment of RNA polymerase II and initiation of gene transcription.Other Rs that do not associate with hsps substitute their amino terminusfor the caboxy terminus, and release corepressor molecules fromattachment to TF IIB which then initiates gene transcription.

Receptors and transduction pathways that influence body functionduring exercise

There is a remarkable diversity in the types of Rs and transductionmechanisms in tissues that support physical activity. This section is anoverview of the way autonomic neurotransmitters and endocrinemessengers interact with Rs in the heart, skeletal muscle, vascularsystem, airways, and energy-storage tissues during exercise.

Transduction of chemical messages in exercising heart

Two components of the heart beat are controlled by autonomic andchemical messengers: the heart rate (chronotropic function) and heartcontractility (inotropic function). The heart syncytium has inherentrhythmicity because of the clusters of excitable cells that act aspacemakers. The sinoatrial node (SAN) is such a pacemaker that canspontaneously depolarize about 100 times per minute. The other heartpacemaker, the atrioventricular node (AVN) can discharge actionpotentials at a slower frequency . SAN and AVN become spontaneouslydepolarized because of a gradual decrease in the permeability of theirmembranes to K+ in the face of a constant permeability to Na+. Theimbalance in the magnitude of inward Na+ leak and outward K+ leaktriggers membrane depolarization, a pacemaker potential. Thisdepolarization starts in the SAN, spreads across the two atria and causesthe AVN to discharge. The depolarization then rapidly spreads across thetwo ventricles through the conducting bundle of His.

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Figure 36. Modulation of heart function by ions and chemicalmessengersA. Time course of depolarization that triggers contraction of the heartventricle.; B. Changes in permeabilities of sodium (PNa+), calcium (PCa2+),and potassium (PK+) that are responsible for the timing of ventriculardepolarization. C. The effect of sympathetic (S) neurotransmission oradrenomedullary E and of parasympathetic (PS) neurotransmission on thetiming of cardiac pacemaker potentials (the chronotropic effect). D. Theeffect of S neurotransmission or adrenomedullary E on heart contractility(the inotropic effect).________________________________________________________

The interval between successive cardiac muscle depolarizations isabout one thousand times longer than is the interval between nerve or

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skeletal muscle action potentials (Figure 36 A). This slower heart-beatrhythm is imposed by voltage-gated L-type calcium channels that openimmediately after initial and transient opening of the sodium channel. Forthe duration of time that the Ca2+ channel is open and that K+ permeabilityis reduced below resting level, the heart muscle is depolarized andrefractory to catecholamine or pacemaker stimulation (Figure 36 B).Repolarization of the heart muscle takes place when the permeability ofheart membrane to Ca2+, Na+, and K+ returns to resting level due toincreased activity of Na+- K+ pump and Ca2+ -ATPase and closure of thecation channels.

Heart rate increases during exercise when S nerves or circulating Ebind to beta1 and beta2 (Leenen et al. 1995) adrenergic receptors. Thisactivates Gs transduction pathway and generates cAMP, the principalaction of which is to phosphorylate and open ligand-gated Na+ and Ca2+

and inward-flowing K+ channels (Figure 37). A direct consequence of theinward flow of Na+ and Ca2+ is an acceleration in the rate of pacemakerdepolarization and a shortening of the latency between depolarizations(Figure 36 C).

S nerves and E also act on beta1 and beta2 adrenergic receptorsthroughout the myocardium to increase heart contractility. An increase incontractility produces more forceful muscle contractions at any given end-diastolic pressure. Thus a catecholamine-induced increase in inotropicaction of the heart is independent of, and additive to, increased force ofcontraction due to greater end-diastolic volume (Figure 36 D). Cardiaccontractility increases as a result of three-fold action of cAMP: (1)phosphorylation of proteins in, and opening of, slow L-type of Ca2+

channels in the plasma membrane and of R-R-type of Ca2+ channels inthe SR (Witcher et al., 1991, Figure 37); (2) phosphorylation of a SRprotein phospholamban that controls active transport of calcium via Ca2+ -ATPase (Figure 37), and (3) phosphorylation of myosin in cardiacmyofibrils.

Phosphorylation of phospholamban facilitates the Ca2+-ATPaseactivity and calcium reuptake into SR for repeated release into cytoplasm(Figure 37, Langer 1997). Phosphorylation of myosin acceleratescrossbridge cycling, all of which increases cardiac contractility.Endurance training increases the affinity of the heart to beta2 adrenergicstimulation (Mazzeo et al., 1995). The heart also has alpha1A and alpha1B

adrenergic receptors (Michel et al.,1994) which modulate its inotropic

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action. Alpha1B -R exert negative inotropic action on R-Rs by reducingCa2+ release out of SR.

Figure 37. Signal transduction in the control of the heart beat.Simulation of beta1 and beta2 adrenergic Rs by NE or E triggers the cAMPtransduction process. This results in depolarization and contraction ofcardiac muscle. The principal effect of cAMP within the myocardium is toopen Na+, Ca2+ and inward-flowing K+ channels. The initial increase incytosolic Ca2+ triggers the release of additional Ca2+ from the SR. Alpha2

A-Rs (not shown) reduce heart contractility through the Gi transductionpathway. ACH does the same through three different transductionpathways: through stimulation of (1) the cGMP (Gc) pathway that results indegradation of cAMP and closure of Ca2+ channel (left) , (2) IP3-DAG (Gq)

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pathway that closes the L-type Ca2+ channel, and (3) the Gi pathway thatblocks cAMP synthesis (see Figures 32 and 33 and text for details).___________________________________________________________

At rest, in response to baroreceptor signals of increased bloodpressure, or in response to endurance training (Shi et al., 1995), PSnerves reduce the chronotropic action of the heart. ACH acts on mACHreceptors ( of M1 type in intracardiac ganglia and of M2 type in atrialmuscle , Buckley & Caulfield, 1992) in the SAN to reduce the heart rateby three different trasduction pathways (Petit-Jacques et al., 1994): (1) itinhibits the synthesis of cAMP through the Gi pathway (Figure 37, topright), (2) it hastens cAMP degradation through the Gc pathway (Figure 37,left), and (3) it blocks the opening of calcium channel through the Gq

pathway (Figure 37, right). The net effect of this action is pacemakerhyperpolarization, reduction in the rate of pacemaker depolarization andslowing of the heart rate (Figure 36 C).

In addition to the action of autonomic neurotransmitters and E,heart function in exercise is influenced by purinergic and peptidergicmessengers. Purinergic messenger adenosine acts on A1 receptors Twoneurotransmitters that are colocalized with NE in S nerves, NPY acting onprejunctional Y2 receptors and/or galanin, prolong catecholamine actionby opposing vagal neurotransmission (Potter & Ulman, 1994). Theduration of catecholamine action is thus increased and may facilitaterecovery from exercise.

A number of chemical messengers that are released duringexercise such as E, angiotensin II, AVP, IL-1, and Ca2+ trigger the releasefrom the endocardial endothelium of another cardioactive peptide,endothelin (Grossman & Morgan, 1997). The ET-1 molecular form ofendothelin binds to ETA and ETB receptors on cardiac myocytes toincrease cardiac contractility and duration of systole by paracrine route(Beyer et al., 1995, Brutsaert 1993). Commonly used ETA and ETB

agonists and antagonists are listed in Table 8. Angiotensin II also caninfluence heart function. It acts on AT1 receptors in the central nervoussystem to reset baroreflex control to higher blood pressure ranges. Byreducing vagal influence over heart function, angiotensin II increasesblood pressure without a concomittant decrease in heart rate (Reid 1996).

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Table 7: List of Commonly-Used Hormone Agonists andAntagonists

HORMONE RECEPTORTYPE

AGONIST ANTAGONIST

Adenosine A caffeine, theophylline8phenyltheophylline

A1 2chlorocyclopentadyladenosine,cyclopentadyladenosine,NECA,

DPCPX8cyclopentadyltheophylline

A2 CGS21680, CV1808 KF17837,SCH58261,CGS15943,CP66713,ZM241385

A3 APNEA, CGS21680

Adrenergic(NE,E)*

alpha1A WB4101, 5-m-uradipil,(+/-)-tamsulosin

alpha1B chloroethylclonidine

alpha1D 5-MU, BMY-7378

alpha2A oxymetazoline,octopamine, synephrine,dexmedetomidine, UK-14304

WB-4101

alpha2B chlorpromazine, ARC-239, spiroxatrine,SK&F104856, prazosin

alpha2D oxymetazoline BRL-44408

Adrenergic(E,NE)*

beta3 BRL37344, CGP12177,ispopreterenol,SR58611A,CL316243,LY79771,bucindolol, pindolol

CGP20712A, ICI 118551,bupranolol

AngiotensinII

AT1 losartan (DUP753)

AT2 PD123319

Endothelin ET SB209670

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ETA BQ-123

ETB sarafotoxin 6C

* Supplements information in table 6

Finally, nitrergic nerve terminals have been described in themyocardium (de Belder et al. 1993, Klimaschewski et al. 1992, Schmidt etal, 1992, Tanaka et al. 1993), but their function has as yet not beenelucidated.

Transduction of chemical messages in skeletal muscle

This section overviews chemical messengers delivered byneurotransmission or endocrine and paracrine routes that influence theforce of contraction, and the rate of mobilization and storage of metabolicfuels in the skeletal muscle. Skeletal muscle contraction is triggered byaction potentials in motoneurons. Coupling of neurogenic excitation andmyofilament contraction is achieved in the skeletal muscle and in theheart through an increase in cytosolic Ca2+ . In both types of muscle, initialincreases in cytoplasmic Ca2+ are caused by opening of the L-type Ca2+

channels on the plasma membrane. In the skeletal muscle, the L-typechannel (that contains a dihydropyridine or DHP receptor) is a voltagesensor (VS,Figure 38) and opens in response to muscle depolarization. Itthen causes the opening of the sarcoplasmic ryanodine Ca2+ channel orreceptor (R-R, Takeshima et al. 1989) through the mediation ofintracellular messenger, cADP-r (Thorn et al., 1994). R-R on the SR ofthe heart muscle, on the other hand, opens when Ca2+ binds to it. Ca2+

is then released through the foot-like features of the R-R (Innui et al. 1991)into the cleft next to T tubules (Figure 38, Fabiato 1985). This facilitatesCa2+ binding to troponin and formation of actin-myosin cross-bridges.

The force of the skeletal muscle contraction can be increased byBA-R stimulation delivered either through S nerve endings or circulation.E is taken up by prejunctional beta2 Rs in S nerve endings, and along withNE, can overflow out of S nerve terminals when the nerves discharge(Coppes et al., 1995). Beta2 adrenergic stimulation increases muscletension and tetanic contraction by augmenting the amount of Ca2+

released from the SR through its R-Rs Ca2+ channels (Cairns et al., 1993).

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Figure 38. Calcium channels that control muscle contractionTwo types of Ca2+ channels control the contraction of skeletal and

heart muscle: the L-type channel on the sarcolemma and the ryanodine R(R-R) on the SR. In the skeletal muscle, the L-type Ca2+ channels arevoltage sensors (VSs) that respond to sarcolemmal depolarization inducedby action potentials from motoneurons while in the heart muscle L-typechannels are opened in response to H-induced cAMP transductionprocess. The initial inflow of Ca2+ is a sufficient stimulus for the opening ofheart R-Rs while in theskeletal muscle R-Rs are opened through themediation of cADP-ribose messenger. Increase in cytosolic Ca2+ activatestroponin and facilitates muscle contraction.___________________________________________________________

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Tension-inducing action of beta2 adrenergic stimulation is greater in redthan in white fibers, and the slow-twitch red fibers have more betaadrenergic receptors (Jensen et al., 1995) and are more sensitive to BA-Rstimulation than the white fibers (Cairns & Dulhunty, 1993). BA-Rstimulation of skeletal muscle is also necessary for enzymatic andmetabolic adaptations to endurance training, increases in oxidativeenzyme activity and muscle sensitivity to insulin (Powers et al., 1995,Torgan et al., 1993). Alpha2 adrenergic stimulation can also affect theforce of muscle contraction but it does so indirectly. Maximal contraction ofthe white glycolytic, but not red oxidative, muscles attenuates the action ofS stimulation on alpha2 adrenergic Rs on vascular smooth muscles, andthe resultant vasodilatation augments the blood flow to the muscles andtheir force of contraction (Thomas et al., 1994).

Mobilization of metabolic fuels stored in the muscle is either directlytriggered by muscle contraction as is the case of glycogenolysis in fast-twitch white fibers (Greenhaff et al., 1991), or is stimulated by S nervesand endocrine and paracrine messengers. Stimulation of both beta2 andbeta3 adrenergic Rs increases skeletal muscle glycogenolysis, beta2 Rsat lower, and beta3 Rs at high messenger concentrations (Sillence et al.,1993), and the effect is confined to red muscle fibers (Greenhaff et al.,1991). Lipolysis, like glycogenolysis, increases in skeletal muscle withboth beta2 and beta3 stimulation (Nagase et al.,1996), with beta2 Rsresponding to lower concentrations and beta3 Rs operating at highmessenger concentrations (Sillence et al., 1993).

Messengers that affect skeletal muscle fuel synthesis areadenosine, insulin, NO and cortisol. Adenosine and NO are generated byskeletal muscle contraction. Adenosine acts on A1 Rs to reduce insulin-dependent glucose uptake in red muscle fibers (Challis et al., 1992).Insulin resistance in the muscles of genetically obese rats is reversededby administration of adenosine A1 R antagonist (Challis et al., 1984). Inthe presence of insulin and beta A-R stimulation, adenosine increasesactivity of the glycogen synthetase in red muscle fibers (Vergauwen et al.,1997). Adenosine also mediates glucocorticoid blockade of E-inducedglycogenolysis in skeletal muscle (Coderre et al., 1992).

Glucose uptake by the skeletal muscle is in part insulin-dependentand in part insulin inependent. Insulin-dependent component is mediatedby the IP3-DAG transduction pathway and phosphorylation of IRS

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(Hayashi et al., 1997, Zhou & Dohm, 1997), while the insulin-independentcomponent is induced by muscle contraction and requires increasedcytosolic Ca2+ (Hayashi et al., 1997) and the cGMP-NO pathway forsignalling (Kapur et al.,1997,Roberts et al., 1997, Young et al., 1997). NOand cGMP-dependent PDE stimulate both G uptake and cabohydratemetabolism in the absence of insulin (Young et al., 1997), and deficientrelease of NO was identified as a cause of insulin resistance in theskeletal muscle (Young & Leighton, 1998).

Alpha-adrenergic stimulation facilitates glucose uptake by themuscle particularly in the presence of FFAs (Saitoh et al., 1974) while betaadrenergic stimulation by E contributes to insulin resistance in the muscleby decreasing the activity of enzyme hexokinase (Lee et al., 1997). Unlikethe effects of insulin and NO which affect glucose uptake at the level ofmessage transduction, beta adrenergic stimulation increases intracellularglucose 6-phosphate formation through glycogenolysis and blockshexokinase activity by substrate inhibition.

Chemical messengers in vasomotor and respiratory control

Smooth muscles that surround blood vessels, respiratory airwaysand GI tract differ from cardiac and skeletal muscles in that theircontraction is initiated by diverse stimuli: autonomic neurotransmission,endocrine messengers, and metabolic and mechanical stimuli rather thandepolarizations brought about by pacemaker or motoneuron actionpotentials. These stimuli may increase or decrease smooth musclecontractions, and tension is a result of integrated stimulatory and inhibitoryinfluences. In contrast, depolarizations of cardiac and skeletal musclefibers are of all-or-none kind, and in the skeletal muscle gradation oftension is primarily achieved through motor unit recruitment.

Innervation of smooth muscles is heterogeneous. Smooth musclesare innervated both by S and PS nerves although some S fibers to bloodvessels can use ACH as a neurotransmitter (Bulbring & Burn, 1935). Snerves form a branching network of fibers along the smooth musclemembrane, and NPY and ATP are frequently colocalized with NE(Miyahara & Suzuki, 1987). Neurotransmitters are found in varicosities,serial thickenings of S nerve endings, and no specialized synapse or end-plate is seen in the adjacent smooth muscle membrane. Some sensorynerves that often co-localize SP and 5-HT or SP and CGRP can releasetransmitters centrifugally and act as effector nerves (Maggi & Meli, 1988).

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Smooth muscles are electrically coupled and allow spreading ofmembrane depolarization between cells via electrotonic and gap junctions(Burnstock & Hoyle, 1992). Depending on the location and strength of theinitiating stimulus, only segments of a smooth muscle may contract attimes. This section overviews chemical messengers that control smoothmuscle contraction and relaxation by neurotransmission and by endocrineand paracrine routes.

Chemical messengers in smooth muscle contraction areneurotransmitters NE , NPY and ACH, circulating messengers adenosine(acting on A1 Rs), angiotensin II, and AVP (acting on V1 Rs, Ekelund 1996,Marshall et al., 1993), and paracrine messengers eicosanoids, histamine,and endothelin (Figure 39, left). Constriction of arterioles by alpha1D AA-Rs and of venules by alpha1B AA-Rs (Leech & Faber, 1996) andbronchoconstriction by adenosine A1 Rs (counteracted by A1 blockeraminophylline) entails interaction with a Gq protein in the IP3- DAGtransduction pathway. PL-C catalyzes synthesis of IP3 and DAG andactivation of PK-C. PK-C increases the amplitude of the high-threshold,long-acting L current by opening Ca2+ channels on the plasma membrane.This triggers membrane depolarization and muscle contraction (Chick etal., 1996). NPY which is often co-released with NE from S nerves, alsocauses vasoconstriction, and potentiates action of NE (Dockray 1992).Vasoconstriction is also triggered by alpha2 Rs (Blaak et al., 1993,Thomaset al., 1994). Constriction of both arterioles and venules in smoothmuscles is mediated by alpha2D Rs (Leech & Faber, 1996) probablythrough the Gi transduction pathway (Figure 39). With aging, alphaadrenergic vasoconstriction to cold becomes attenuated (Frank et al.,1996).

Stimulation of the m2ACH-Rs m3ACH-R of the airway smoothmuscle (Buckley & Caulfield, 1992) precipitates bronchospasm in sensitiveindividuals (Hargreave et al., 1981). Cholinergic stimulus triggers theopening of several types of ion channels: L-type high-threshold Ca2+ ,non-selective cation, and a cloride channel, and suppressin of two typesof K+ channels (Janssen & Sims, 1992). As was the case with AA-Raction on smooth muscles, mACH-R influences ion channels through theIP3- DG transduction pathway. Various irritants can increase endothelialrelease of histamine and cause tracheal smooth-muscle constriction by acholinergic vagal reflex (Takahashi et al., 1996). Nocturnal asthmaattacks are triggered by an endogenous rhythm that superimposes adecrease in E and cortisol with an increase in ACH (Barnes 1985). In

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Figure 39. Transduction mechanisms in vasomotor controlConstrictive action of the alpha1 and alpha2 A-Rs, adenosine A1 R,

mACH cholinergic R, and ETA and ETB endothelin Rs is shown on the left,and vasodilating action of beta2 A-Rs, A2 adenosine R and NO is shownon the right. R identities are shown on the Hs for ease of presentation.Alpha1 A-R, adenosine A1 R, and ETA and ETB endothelin Rs producevasoconstriction through the IP3-DAG transduction pathway. Smoothmuscle constricts when cytosolic Ca2+ concentration increases inresponse to PK-C action on the L-type Ca2+ channels and IP3 action onR-Rs on the SR. The mACH Rs also open non-selective cation and Cl-

channels and close K+ channels. The beta2 A-R and A2 adenosine Rcause vasodilatation through the cAMP transduction pathway. The cAMP-dependent PK-A closes the L-type Ca2+ channel on the plasmamembrane, while NO closes the R-R Ca2+ channel on the SR. Alpha2 A-Rcauses vasoconstriction through the Gi transduction pathway that inhibitsformation of cAMP.___________________________________________________________

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both asthma and aging, hyperreactivity to ACH results from adownregulation of beta2 R number and affinity (Connoly et al., 1994).

Endothelin is the principal endothelium-derived vasoconstrictormessenger (Yanagisawa et al., 1988) that effects smooth-musclecontraction by a paracrine route. It binds to ETA and ETB receptors (seeTable 8 for agonists and antagonists), both of which are found in vascularsmooth muscles. Its release is elicited by TNFalpha, IL-1beta, angiotensinII, AVP, thrombin, transforming growth factor beta (TGFbeta), PDGF andshear stress, and inhibited by ANP and sodium heparin (Brooks et al.,1994). Endothelin vasoconstriction is mediated by the Gq protein andPL-C. The production of IP3 results in increased concentration of Ca2+ inthe cytoplasm. Both increased cytoplasmic Ca2+ and DAG activatemyosin light-chain kinase. Resultant phosphorylation of myosin triggerssmooth-muscle contraction (Morgan & Suematsu, 1990). Endothelinexerts multiple additional effects on cardiorespiratory function. Itinfluences heart function (see above), activates RVLM area of the braininvolved in the control of vasomotor function (Kuwaki et al. 1994) and thechemosensitive areas on the ventral surface of medulla oblongata (Kuwakiet al. 1991). Endothelin1 inhibits renin and AVP release and therebycauses increased natriuresis and diuresis, respectively. Endothelin alsostimulates secretion of ANF (Brooks et al., 1994).

Chemical messengers in smooth muscle dilatation

Relaxation of smooth muscle is triggered by several peptidesdelivered through neurotransmission, circulating messengers, and dilatorysubstances produced in the endothelium. Among the peptidergic nervevasodilators are bradykinin (Ekelund 1996), CGRP (Brain & Williams,1988, Yaoita et al., 1994), SP (Brain & Williams, 1988), and VIP (Kubotaet al., 1985).

Circulating E induces relaxation by acting on beta2 adrenergic Rs(Figure 39, right). When E binds to the smooth muscle BA-Rs, itstimulates the cAMP transduction pathway to activate PK-A. The cAMP-dependent PK-A closes Ca2+ channels on the plasma membrane and asa result reduces the amplitude of the high-threshold, long-acting L current.This prevents membrane depolarization and causes vasodilatation (Chicket al., 1996).

Of the remaining vasodilating circulating messengers, adenosine isreleased by contracting skeletal muscle, oxidative more than glycolytic

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(Mian et al. 1990). Adenosine is the messenger that maintains coronaryartery dilatation during exercise through its action on the KATP channel(Dunker et al., 1995). Adenosine binds to A2 -Rs and causesvasodilatation by activating the cAMP transduction pathway as was thecase with E after binding to BA-Rs (Figure 39). In addition, arteriolardilatation by insulin is mediated by adenosine receptor and membranehyperpolarization by way of ATP-sensitive potassium channels (McKay &Hester, 1996). Endurance training increases vascular sensitivity todilatory messengers by reducing the action of alpha2 ARs (Delp 1995).

Presence of endothelial relaxing factors has been recognized since1980 (Furchgott & Zawadski, 1980), and since then, NO and adenosinehave been identified as endothelium-derived vasodilators withindependent mechanism of action that was already described (Luscher &Dohi, 1992, Matsunaga 1996).

Endocrine message transduction in the control of metabolic fuelduring exercise

Principal metabolic fuels, glucose and free fatty acids, aremobilized during exercise from their storage depots and metabolized toprovide energy for muscle contraction. The roles of ANS in this processwas discussed in chapter 1 and of individual chemical messengers will bedescribed in chapter 5. This section briefly reviewes H-R interactions thatmediate (1) hepatic glucose output, (2) free fatty acid release from theadipose tissue, (3) fuel utilization during exercise, and (4) messenger-mediated glucose uptake and resynthesis of glycogen and triglycerideduring recovery from exercise.

Glucose release by the liver is made possible by two metabolicpathways of glucose formation: glycogenolysis or enzymatic degradationof the inert glucose polymer glycogen, and biosynthesis of glucose fromthe breakdown products of protein, triglyceride, and carbohydratemetabolism. Both processes are controlled by the sympathoadrenal andseveral endocrine and paracrine messengers. Principal controllers ofhepatic glycogenolysis are glucagon and sympathoadrenalcatecholamines with a quantitatively less important contributions fromadenosine ( Van Stapel et al, 1991), angiotensin, V1 receptors of AVP (Aliet al., 1989), ATP (Keppens 1993), EGF (Grau et al., 1996), andeicosanoid PG D2 produced by Kuppfer cells in the liver (Casteleijn etal.,1988).

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Glycogenolysis is increased by stimulation of both alpha1 and betaadrenergic receptors at rest and during exercise (Coker et al., 1997,Figure 40). NE binding to alpha1 adrenergic receptors, and AVP acting onV1 receptors in liver, transduce their message through the Gq signallingpathway. Stimulation of PL-C brings about IP3 synthesis and Ca2+

release from SR. Calmodulin that is activated by increased Ca2+

concentration, catayzes phosphorylation of phosphorylase kinase.Activated phosphorylase kinase phosphorylates the enzymephosphorylase which then detaches a glucose residue from glucogen andphosphorylates it in position 1.

E acting on beta1 and beta2 A-Rs, adenosine acting on A2-R, andglucagon stimulate glycogenolysis by the cAMP transduction pathway(Figure 40, top). Although both E and glucagon activate AC and shareone type of the Gs protein, glucagon transduction pathway utilizes another,small type of Gs protein (Yagami 1995). Phosphorylation of phosphorylasekinase is carried out by cAMP-dependent PK-A, and the remainder of theenzymatic cascade is the same as in IP3 -DAG transduction pathway.There are gender (Moriyama et al., 1997) and age (Van Ermen &Fraeyman, 1994) differences in the relative participation of the twopathways in hepatic glucose production. In the female, alpha adrenergiccontrol of glycogenolysis predominates and is converted to the c-AMP-mediated pathway that is prevalent in the males in the absence of adrenalglucocorticosteroids (Moriyama et al., 1997). Gluconeogenic enzymesPEPCK and pyruvate caboxylase (PC) are stimulated by S stimulation ofalpha1 A-Rs and by glucagon, GH, and cortisol.

Lipolysis, hydrolysis of a triglyceride to glycerol and free fatty acidsis stimulated by sympathoadrenal catecholamines and by messengersbeta lipotropin, glucagon, glucocorticoids , GH , prolactin, and secretin.Action of all of the above chemical messengers except for glucocorticoidsand GH is through the cAMP transduction pathway (Figure 40, bottom).CAMP activates cAMP-dependent PK which in turn phosphorylates andactivates triglycerol hydrolase called hormone-sensitive lipase (HSL). HSLhydrolyzes storage triglycerides into free fatty acids (FFAs) and glycerol.Stimulation of lipolysis by GH and glucocorticoids is delayed one to twohours because these hormones act through transcription and synthesis ofHSL. Mechanisms by which the growth-promoting protein Hs and steroidHs influence gene transcription was already described. Finally, hronic GHadministration acts by antagonizing the antilipolytic action of adenosine

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(Doris et al.,1996).

LegendFigure 40. Chemical signalling in mobilization of metabolic fuels

Mobilization of glucose from liver glycogen (glycogenolysis) isshown above the mobilization of free fatty acids (FFAs) out of atriglyceride (TG) molecule (lipolysis).Both alpha1 and beta A-Rs and A2 adenosine Rs facilitate glycogenolysis.The alpha1

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A-Rs stimulate formation of IP3 , increase in the concentration of cytoslicCa2+ and activation of calmodulin. Calmodulin catalyzes activation of theenzyme phosphorylase phosphatase (PP) by phosphorylation. PP in turnactivates glycogen phosphorylase again by phosphorylation, andphosphorylase cleaves glucose-1P from the glycogen molecule.Glycogenolysis in response to activation of beta A-Rs and A2 adenosineRs is achieved through the cAMP transduction pathway. CAMP-dependent PK-A activates PP by phosphorylation , and the rest of theglycogenolytic cascade is the same as after stimulation of alpha1 A-Rs.Lipolysis is stimulated when beta A-Rs initiate the cAMP transductionreaction and activate cAMP-dependent PK-A. PK-A activates hormone-sensitive lipase (HSL) by phosphorylating it, and HSL hydrolyzes TG toFFAs and glycerol. The adenosine A1 Rs and insulin are antilipolytic. TheA1 R antilipolytic action is by Gi and Gq transduction pathways resulting ininhibition of cAMP synthesis and dephosphorylation of HSL by PK-C,respectively. Insulin antilipolytic action (not shown) is mediated throughthe cGMP transduction pathway.___________________________________________________________

Lipolysis in adipose tissue entails adrenergic stimulation of beta1,beta2, and beta3 Rs (Emorine et al., 1989). Although there is somedisagreement regarding the expression of beta3 Rs in humans, this R typeis activated at high stimulus intensities (Atgie et al., 1997) and isfunctional in human intra-abdominal fat (Hoffstedt et al.,1995). Differencesin the sensitivity of beta Rs (Wahrenberg et al., 1992) and of antilipolyticalpha2 Rs (Hellstrom 1997) in the two genders (Lonnqvist et al., 1997) andto hormonal stimulation were found to cause differential fat loss inexercising or sedentary humans. Stimulation of beta3 Rs increases energyexpenditure by uncoupling oxidation from phosphorylation of ADP in thebrown adipose tissue (Nagase et al. 1996) and beta1 stimulation increasesthe basal metabolic rate (Lamont et al., 1997,Tremblay et al., 1992).

Alpha2 adrenergic Rs block lipolysis through the Gi transductionpathway (Figure 40, Tarkovacs 1994), and alpha2 R blockade increasesbeta stimulation by NE and lipolysis (Lafontan et al.,1992). Differentialdistribution and sensitivities of alpha2 and beta1 and beta2 adrenergicreceptors in different fat depots may be responsible for regionaldifferences in lipolysis and fat accumulation (Hellmer et al.,1992).Likewise, differential S activation of alpha-adrenergic Rs in different fatdepots may contribute to regional differences in fat deposition and lipolysis(Robidoux et al., 1995). The alpha2A Rs in the

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intraabdominal fat are less sensitive to the lipolytic action of E than are thesubcutaneous adipocytes (Vikman et al., 1996), while the same receptortype is more sensitive in gluteal than in abdominal subcutaneous fat(Galitzky et al., 1993).

In addition to catecholamines acting on alpha2 Rs, other hormoneswith antilipolytic action are adenosine, insulin, insulin-like growth factors,oxytocin,GIP, and glucocorticoids which along with insulin can down-regulate beta3 Rs (Langin et al., 1995). Adenosine inhibits lipolysis byacting on A1 -Rs that are coupled to both Gi and Gq transductionpathways. The former inhibits formation of cAMP, and the latter blocksHSL phosphorylation via increased activity of PK-C. A1 -Rs displayweaker coupling to the Gi protein than alpha2 Rs (Larrouy et al., 1994).Abnormal A1 adenosine R function has been proposed as one of thecauses of obesity (La Noue & Martin, 1994).

Glycogen synthesis and lipogenesis in the liver and adiposetissue are largely under the control of insulin. Insulin has a multiplicity ofmetabolic and biosynthetic actions, and their molecular mechanisms areincompletely understood. With the exception of stimulation ofcarbohydrate metabolism, most insulin actions are anabolic. Insulinstimulates glucose uptake and glycogen synthesis and inhibitsglycogenolysis and gluconeogenesis . It stimulates triglyceride andprotein synthesis and inhibits lipolysis and protein degradation.

A model of insulin transduction mechanism that best fits theavailable observations, includes both the IP3-DAG and cGMPtransduction pathways (Figure 33, Manganiello et al.,1996). Insulinactivates its receptor and triggers phosphorylation of the instrinsic tyrosinekinase. Activated receptor then phosphorylates other enzymes includingGC, PKs and phosphatases. Activation by insulin of dephosphorylatingphosphatases is one way that insulin inactivates phosphorylase and HSL.The inhibitory effects of insulin on glycogenolysis and lipolysis are alsomediated through the activation of PDE by the second messenger cGMP.PDE reduces intracellular concentration of cAMP and of cAMP-dependentPK-A. The same inhibitory effects of insulin are also reported to involve IP3

and PK-C.

Biosynthetic actions of insulin include phosphorylation oftranscription factors and kinases that belong to the growth-factor signallingcascades (Manganiello et al., 1996, Figure 34). Thus insulin- stimulated

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MAPKs phosphorylate and activate ribosomal S6 kinases that are involvedin growth and differentiation, and protein phosphatases whichdephosphorylate and stimulate glycogen synthetase and HSL. Becauseinsulin transduction mechanisms combine features of IP3-DAG andcGMP pathways (as illustrated in Figure 33), NO could be the connectinglink in insulin signalling. At this point however, NO is known to only beinvolved in the glucose-uptake aspect of insulin action (Kapur, et al.,1997,Young & Leighton, 1998).

Characteristics of hormone receptor interactions

Hormone-receptor binding can be distinguished from other types ofchemical bonds by its specificity, reversibility, affinity, and saturability.Specificity refers to capacity of receptors to bind a particular hormoneoften present in circulation at very low concentrations. Specificity ofhormone-receptor interactions depends to a large extent on specificstructural, chemical, and electrical complementarity between them.Hormones with highest specificity have greater affinity for a particularreceptor type than other molecules. Specificity is also determined by thetype of transduction mechanism associated with a receptor in a giventissue. For instance, the same hormone may influence synthesis ofdifferent proteins in different tissues, and here the transductionmechanism leads to expression of different genes. In addition, G-R, M-R,PROG-R, and AN-R bind to the same HRE on DNA. In such situationsmessage specificity is achieved through coupling of receptors witheffector machinery in different tissues mediating only certain effects ofhormone but not others. This association is created during cellulardifferentiation when only some of several genes susceptible to hormonalcontrol located in Dnase I-sensitive regions of DNA get expressed. Most ofcellular genes are Dnase I resistant and do not get expressed duringcellular differentiation but remain tightly packaged with histone proteins innucleosomes.

Reversibility refers to the property of hormones and receptors

to form reversible non-covalent bonds described by the Michaelis-Menten equation: ka

[H] + [R] = [HR] kd

The interaction between H and R follows the law of mass of action and

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depends on the concentrations of the two reactants. At equilibrium, onecan determine association (KA) and dissociation constants (KD) from thefollowing relationships:

KA= ka/kd= [HR]/[H][R] KD=kd/ka=[H][R]/[HR]

where KA is given in M-1 and KD in M at equilibrium, and ka is theassociation rate constant in M-1 sec-1and kd is dissociation rate constant insec-1. These binding relationships are often used to determine a totalnumber of receptor sites (N) and H-R affinity. Total number of receptorscan be calculated from [R] + [HR], while the total amount of hormone isderived from [H] (that is, free hormone or F) + [HR] (that is, boundhormone or B). The amount of hormone bound at equilibrium can beestablished from:

[B]=[N][F]/KD + [F].

The graphic representation of the Michaelis-Menten equation(Figure 41) can be used to determine saturability of H-R binding. Saturability refers to the presence of finite number of receptors in anygiven cell that determines maximal binding capacity. Saturability isillustrated in the levelling-off of H-R binding at high free H concentrationsat which N or maximal binding is attained. In contrast, non-specific bindinghas a direct linear relationship to hormone concentration, is not saturable,and needs to be subtracted from the total binding to yield specific binding.Another frequently used method to determine N is Scatchard analysis(Figure 41). It is derived from the relationship:

[B]/[F]=KA([N]-[B]).

N is found as the intercept of the straight line calculated from [B]/[F] as afunction of [B], and affinity ( KD) from the negative slope, -KA.

The affinity or the strength of H-R association is also revealed bythe Michaelis-Menten equation and Scatchard analysis. The affinity isrepresented by KA or the reciprocal of KD. The KD is operationallydefined as the hormone concentration at which H-R binding is one half ofmaximal, and in the Scatchard plot from the slope, -KA.

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Figure 41. Characteristics of H-R bindingA. H-R binding at equilibrium is shown as a function of the concentrationof free H (F).Maximal binding (N or Bmax) is a measure of saturability of the equilibriumreaction and of the total number of available Rs. KD (or KA) is F at whichhalf-maximal binding is achieved and is used as a measure of affinity ofthe H-R interaction. B. Non-specific binding (BNS) is proportional to F , isnon-saturable, and needs to be subtracted from total binding (BT) to obtainspecific H-R binding (HS). C. Scatchard plot presents the change in the

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proportion of bound (H-R) over F as a function of change in bound Hconcentration (B). The intercept of the curve with the abscissa gives the N,and the slope reveals the affinity of H-R interaction. D. When fullbiological response can be elicited by less than N (50% of available Rs inthis example), then the binding of the H with spare Rs increases thesensitivity of the biological response. Binding of H with 100%, 75%, and50% (but not with 25%) of the available Rs (or with 50%, 25% and 0% ofspare Rs, respectively) will produce a full biological response. As fewerspare Rs are involved in H-R interaction, higher concentrations of F areneeded to elicit the full response. The rightward shift of the H-R curves asspare Rs decline illustrates the concomitant reduction in responsesensitivity (E).__________________________________________________________

Factors that affect receptor sensitivity

The biological response of a target cell to a hormone is determinedby additional factors such concentration of hormone, concentration ofreceptors, timing and duration of receptor stimulation, and cumulativeinfluence of competing or modulating chemical messengers. In someinstances, the response of a target is proportional to hormoneconcentration and the number of receptor sites that are occupied. In sucha situation the binding and biological response curves are superimposableand maximal response is achieved at maximal receptor binding. Moreoften, however, maximal biological response is achieved atconcentrations that are considerably lower than those required to occupyall of the receptors. Thus if binding of 5% of receptors is needed formaximal biological response, the remaining 95% of unoccupied receptorsare referred to as spare receptors.

The number of spare receptors is a measure of receptor sensitivityto hormone. For instance, thyroid hormones increase cardiac sensitivity toadrenergic stimulation by inducing synthesis of additional spare betaadrenergic receptors. When the number of spare receptors decreases dueto functional changes or presence of competitive chemical messengers, agreater number of receptors will be needed to achieve the same biologicalresponse (Figure 41). As the number of spare receptors progressivelydecreases, the KD increases. Thus with a reduction in spare receptors,both the sensitivity and the affinity of H-R binding decreases whilereceptor number (N) is unchanged.

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A decrease in available receptors can result from receptorphosphorylation by a PK or by sequestration of receptors from the cellsurface in response to exposure to the hormone. The H-R complexescluster in areas of membrane called coated pits because of coating by aprotein clathrin. These H-R complexes are internalized within coatedvesicles and degraded by lysosomal enzymes. Such hormonally-inducedreduction in available receptors is referred to as homologous down-regulation or desensitization. Desensitization is a decrease inresponsiveness of a cell to a constant level and high concentration of ahormone upon prolonged exposure. It can result both from reduction inreceptor number or from uncoupling of receptors from some component ofits transduction system.

The timing of a hormone stimulus may affect receptor sensitivityand the magnitude of biological response. Most peptide hormones displayan intermittent pattern of spontaneous secretion which secures optimalbiological response. Intermittent hormone secretion followed by hormonedisappearance due to degradation allows recovery of receptor sensitivityafter homologous down-regulation. Prolonged exposure to a highinvariant concentration of a normally pulsatile hormone results in a loss ofresponsiveness and can be used to block this hormone’s biologicalfunction.

In non-competitive binding, modulating messengers can change Nwithout altering the affinity of H-R interactions. In heterologousdesensitization, one agonist reduces the responsiveness of a cell to otheragonists acting through different receptors. Finally, genetic alterations inreceptor structure or transduction machinery can also alter the sensitivityand responsiveness of H-R interactions.

There is a considerable degree of plasticity in H-R interactions.Receptor numbers and receptor types may change with age or as a resultof metabolic and endocrine influences. In addition, the number and type ofchemical messengers delivered to the receptor by endocrine, paracrine,autocrine or neurotransmission routes may also change with age or due tometabolic and endocrine alterations. Changes in sensitivity to hormoneshave been observed in response to physical training or variouspathological states, and will be discussed in that context throughout thebook.

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Methods of hormone measurement

Selection of the appropriate method of H measurement (Baulieu1990, Griffin 1996) depends on the type of question asked, secretory andbinding characteristics of the H, acceptable level of invasiveness incollection of H sample, and physiological condition of the subject donatingthe sample (Table 8). Bioassays (BAs), radioimmunoassays (RIAs), andimmunometric assays (IMAs) provide information on H concentration inbody fluids and tissue extracts. Receptor-binding assays (RBAs) canasses N and H-R affinity, while solution hybridization assay (SHA)measures H synthesis through assessment of the concentration of specificmRNA in nucleic-acid extracts. For localization of Hs in tissues and cells,immunohistochemical (IHC) and immynocytochemical (ICC) approachesare used, while in situ hybridization (ISH) reveals sites of hormonesynthesis. The principles and limitations of these methods of Hmeasurement will be outlined and strategies for overcoming limitations ofthese approaches discussed.

Table 8

Principal Methods of Hormone Measurement

ASSAYTYPE

PRINCIPLE ASSESSES: R LABEL

BA Specificbiological action

H concentration organism, organ, tissue based on biological response

in vitro BA Specificbiological action

H concentration tissue, cells based on biological response

RIA Competitivebinding

H concentration body fluids, tissueextracts

125I-H, 3H-H

IMA, RMA,FMA, EMA

Specific binding H concentration body fluids, tissueextracts

125I-, 3H-Ab fluorophor-Abenzyme-Ab

SHA Specifichybridization

H mRNAconcentration

tissue extracts 36S-mRNA, 3P-mRNA

RRA Competitivebinding

N, H-R affinity cells, membranes,receptors

125I-H, 3H-H

IHC, ICC Specific binding H location tissue slices 125I-, 3H-H fluorophor-Henzyme-H

ISH Specific binding H location tissue slices 36S-mRNA,3P-mRNA

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BAsBAs utilize some measurable feature of the animal’s physiology for

determination of hormone concentration. An example of a BA is thehypophysectomized-rat tibia test in which the increase in the width ofepiphyseal growth plate is used to measure growth-promoting action ofGH. A special advantage of BAs is that they directly measure biologicalaction of a H, while their lack of sensitivity is a disadvantage. In vitro BAsare more recent developments. They are performed with cells or cellfragments and provide a more sensitive measure of endocrine biologicalaction.

RIAsRIAs utilize the principle of reversibility of H-R interactions

described by Michaelis-Menten equation (discussed in the preceedingsection) to measure H concentration at H and a specific antibody (Ab)dilutions that are well below saturation. Because H-R interactions in a RIAtake place at low Ab concentration of about 10-5 g/L, prolonged incubation(hours to days) is needed to reach equilibrium, and assay sensitivity(measured by half-maximal binding) is increased as Ab concentration isreduced. Amounts of all reactants in a RIA are kept constant with theexception of H in reference preparation (or standard) which issystematically varied, and in unknowns (or samples). Radioactively-labelled H allows detection of bound hormone. Peptide and protein Hs areusually labelled with 125I on tyrosine or histidine residues (which need tobe attached to small molecules that lack them) or with 3H. For bestbinding, only monoiodinated H is used in a RIA and is separated out of theof iodination complex by chromatography. Steroid and thyroid hormonesare tagged with 3H on one or more molecular sites to achieve thenecessary level of specific activity.

The specificity in a RIA is imparted by the Ab that consists of IgGsdeveloped to a partially-purified (polyclonal Abs) or a highly-purified(monoclonal Abs) H preparations. Polyclonal Abs are a mix ofheterogeneous IgG molecules that may bind to different parts of Hmolecule, while monoclonal Abs have uniform structure and bindingproperties. Abs develop to peptides with a molecular weight greater than1000 Da. Smaller Hs need to be complexed to large proteins to increasetheir antigenicity. Abs will cross-react with more than one H if they theybind to parts of molecule that has primary structure shared by these Hs.

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Because of such Ab cross-reactivity, it is often impossible to distinguishbetween some Hs and their biologically-inert precursors or derivatives (forexample beta-LPH from beta endorphin) or between Hs that havestructural homologies (LH and FSH).

The amount of H can be estimated with a RIA from the comparisonof competitive inhibition of binding of radioactively labelled H by a knownamount of nonradioactive H in standards. The amount of H in samples isthen extrapolated from the binding curve in which the percent of bindingsuppression (B/B0) is plotted as a function of free H (or standard)concentration. The curvilinear plot is converted to a straight line by usinga log-logit or other type of mathematical transformation, but Hmeasurements are invalid at the low and high ends of the binding curve.To distinguish H-R complex containing variable proportion of labelled Hfrom unbound radioactive H, the dilute H-R complex is separated bycentrifugation after incubation with the second Ab against the gammaglobulins from the animal species that provided the first Ab. Additionalmethods of H-R separation are, precipitation of HR-complex bypolyethylene glycol (PEG) or after attachment to a solid-phase (assay-tube coating), and precipitation of free H by activated charcoal (steroid Hassays).

The advantage of a RIA method is in its capacity to detect Hs atvery low concentrations (10-12 M). The disadvantage of the method is thatit can not measure H concentrations outside the range of the standardcurve and it may measure H isoforms, precursors or degradation productsthat have little if any biological activity. The latter limitation can beovercome by repeating measurements after H dilution, and the latter bycomparison of RIA with parallel in-vitro BA measurements.

IMAsImmunometric assays utilize the specificity of H-R binding for

assessment of H concentration. Two Abs with binding sites to differentparts of H molecule are used at saturating concentrations. One Ab istagged with either a radioisotope, a chemical that imparts to the Ab thecapacity for phosphorescence or chemiluminescence, or an enzyme, andthe second Ab is coupled to a solid phase (test-tube,microtiter-platecoating, or synthetic beads). During a very brief incubation (minutes tohours), H creates a “sandwich” by connecting two Abs.

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IMAs can be more sensitive than RIAs as is the case withchemiluminescent GH assay and have made it possible to measure someHs (TSH, PTH) that were difficult to measure with RIAs. Additionaladvantages of IMAs are their capacity to measure Hs throughout theentire range of concentrations, lack of intereference by endogenous HAbs, and sensitivity of H-R interaction that is independent of Abconcentration. The disadvantage of IMAs is that they require high Abconcentrations.

SHAsSolution hybridization or nuclease-protection assay is used to

assess H synthesis by measurement of the messenger ribonucleic acid(mRNA) for a given hormone (Durnam & Palmiter, 1983). This methoduses high incubation temperatures to produce hybrids between non-radioactive antisense copies of mRNA (standards) and radioactive sensecopies of mRNA of a given H. Only the specific sense and antisensecopies of a particular H mRNAs with complementary base-pair sequencieswill hybridize, and the remaining nucleic acids in the tissue extract willremain single-stranded and vulnerable to enzyme RNAse that is used toseparate radioactive hybrids from unhybridized mRNA.

RBAsReceptor-binding assays, like RIAs, take advantage of competitive

suppression of binding of labelled H with Rs on cells or cell membranes bynonradioactive H. Radioreceptor assays (RRAs) are performed atdifferent concentrations of Rs to determine H concentration at whichmaximal binding occurs (N), and at different concentrations of free H, todetermine H-R affinity.

IHC and ICC methodsImmunohistochemical and immunocytochemical methods utilize the

specificity of H-R binding to localize Hs within the body. To that end, Hsare labelled with isotopes, chemicals that convey to them the capacity forfluorescence or chemiluminescence, or enzymes, and appropriateprocedures are then used for microscopic visualization of the location oflabelled hormone in tissue slices.

ISH methodIn situ hybridization method is a hybridization procedure that allows

microscopic visualization of H mRNA. The location of newly-synthesized H

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can be determined after incubation of tissue slices with radioactive mRNAfor a particular H under the conditions that favor its hybridization withcomplementary RNA that is present in the tissue.

Limitations of H measurement methods and strategies that overcomethem

Methods of H measurements often provide limited or misleadinginformation when characteristics of H secretion and binding are notconsidered. Individual measurements are valid indicators of concentrationfor those Hs that maintain constant concentration over extended periodsof time. Hormones that display pulsatile secretory pattern require frequentsequential sampling to adequately define variations in their concentration.H concentration is influenced by the rates of H release and degradation.H degradation can be estimated from the arterio-venous differences in Hconcentration across organs (kidney, liver) where degradation occurs andfrom the rate of decay of H pulses assessed at frequent intervals andsubjected to Cluster or deconvolution analyses (Veldhuis 1992, Veldhuis &Johnson, 1988).

H- binding proteins and endogenous Abs against circulating Hs canconfound H measurements and complicate the interpretation of RIAresults. Where endogenous Abs are present, exogenous Abs will bind toresidual free H, and reduced binding of radioactive H will bemisinterpreted as high H concentration. Where endogenous Abs aresuspected, RIA should be run without the addition of exogenous Ab andprecipitated with ammonium sulfate. Ammonium precipitation is also usedfor assessment of bioactive steroid Hs which circulate bound to specificproteins. Although less than 5% of testosterone and cortisol circulate freeof binding protein, additional H dissociates from its binding protein withinthe capillaries and can also be assessed through ammonium precipitation.For assessment of biologically active IGF-I concentration, its bindingproteins are usually removed with acid precipitation.

Concentration of H in systemic circulation does not provideinformation about H concentration in vascular beds into which the H is firstreleased. Yet collection of samples from hypothalamo-hypophyseal orhepatic portal vessels and cerebrospinal fluid is even more invasive thanblood collection from systemic circulation. Invasive procedures can beavoided when H concentration in the saliva or urine bears a predictable

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relationship with concentrations of circulating H. Urinary H measurementsare carried out when information about total H secretion during anextended period of time is sought. For example 24-h urinaryhydroxycorticoids or free cortisol are a valid measure of dailyglucocorticoid output provided creatinine clearance is also measured toassess glomerular filtration rate.

Figure 42. Dynamic tests of hypothalamo-pitutary-adrenal functionThe functional integrity of the hypothalamic-pituitary-adrenal (HPA)

axis is assessed by measurement of two or more of its Hs in response tostimulation or inhibition of synthesis or release of one of its Hs. A showsthe spontaneous operation of the HPA axis where the hypothalamic CRFrelease triggers pituitary ACTH release, and ACTH triggers adrenocorticalcortisol secretion with the appropriate biological effects. Cortisol exerts anegative feedback over CRF and ACTH synthesis and release to maintainits concentration in the blood within appropriate range. B is a test ofpituitary corticotrop competence to respond to exogenous CRFadministration with increased ACTH and cortisol secretion (shown as darkarrows on the right). C is a test of expected adrenocortical response toACTH administration. D is a dexamethasone suppression test that is usedin diagnosis of depression. Administration of exogenous corticosteroidanalog (DEX) produces increased negative feedback and is expected tosuppress CRF and ACTH secretion in non-depressed individuals.Metapyrone blocks corticosteroid synthesis (E) and RU486 blockscorticosteroid action at its Rs(F). Both diminish the negative feedback andare expected to increase CRF and ACTH secretion.________________________________________________________

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An additional limitation of individual H measurements is that theyyield ambiguous results without the information about their secretagogues.This can be avoided by dynamic tests in which H measurements arecarried out after administration of eliciting stimuli (stimulation tests) or ofHs that provide negative feedback (suppression tests). Stimulation tests(Figure 42, CRF and ACTH tests) are usually done by administeringtrophic or releasing Hs (Htro) and by measuring the magnitude of secretoryresponse of the target H (Htar). Four patterns of H concentrations canidentify abnormalities in endocrine secretion. Low response of Htar to ahigh concentration of Htro identifies target gland failure. High response ofHtar to a low concentration of Htro identifies autonomous oversecretion ofHtar. When both the Htro and Htar are low, and administration of exogenousHtro yields normal response, deficiency is in Htro secretion. An example ofthis is hyposecretion of GnRH and LH in exercise-induced amenorrheawhere normal LH is elicited after administration of exogenous GnRH (DeSouza & Metzger, 1991). When both the Htro and Htar are high, there maybe autonomous oversecretion of Htro, insensitivity of Htro to negativefeedback by Htar or tissue resistance to the actions of Htar. An example ofthe last possibility is tissue resistance to insulin action that is associatedwith high plasma concentrations of insulin (Htar) and glucose (equivalent toHtro).

Suppression tests are usually done by administering excess of Htar

negative feedback (Figure 42:dexamethasone test), suppression of Htar

synthesis (Figure 42: metapyrone test), or blockade of H Rs in targettissues (Figure 42:RU486 test). Supplementary endocrine and metabolic information is often needed for proper interpretation of dynamicH tests as metabolic abnormalities usually affect tissue sensitivity tohormones, and endocrine abnormalities affect secretion and action ofother Hs.

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