Tài liệu Báo cáo Y học: Receptor crosstalk Implications for cardiovascular function, disease and therapy ppt

R E V I E W A R T I C L EReceptor crosstalk Implications for cardiovascular function, disease and therapy Nduna Dzimiri Cardiovascular Pharmacology Laboratory, Biological and Medical Res

R E V I E W A R T I C L E

Receptor crosstalk

Implications for cardiovascular function, disease and therapy

Nduna Dzimiri

Cardiovascular Pharmacology Laboratory, Biological and Medical Research Department, King Faisal Specialist Hospital &Research Centre, Riyadh, Saudi Arabia

There are at least three well-defined signalling cascades

engaged directly in the physiological regulation of cardiac

circulatory function: the b1-adrenoceptors that control the

cardiac contractile apparatus,the

renin-angiotensin-aldo-sterone system involved in regulating blood pressure and the

natriuretic peptides contributing at least to the factors

determining circulating volume Apart from these pathways,

other cardiac receptor systems,particularly the a1

-adreno-ceptors,adenosine,endothelin and opioid receptors,whose

physiological role may not be immediately evident,are also

important with respect to regulating cardiovascular function

especially in disease These and the majority of other

car-diovascular receptors identified to date belong to the guanine

nucleotide binding (G) protein-coupled receptor families

that mediate signalling by coupling primarily to three G

proteins,the stimulatory (Gs),inhibitory (Gi) and Gq/11

proteins to stimulate the adenylate cyclases and

phospho-lipases,activating a small but diverse subset of effectors and

ion channels These receptor pathways are engaged in crosstalk utilizing second messengers and protein kinases as checkpoints and hubs for diverting,converging,sieving and directing the G protein-mediated messages resulting in dif-ferent signalling products Besides,the heart itself is endowed with the means to harmonize these signalling mechanisms and to fend off potentially fatal consequences of functional loss of the essential signalling pathways via compensatory reserve pathways,or by inducing some adaptive mechanisms

to be turned on,if and when required This receptor crosstalk constitutes the underlying basis for sustaining a coherently functional circulatory entity comprising mechanisms con-trolling the contractile apparatus,blood pressure and cir-culating volume,both in normal physiology and in disease Keywords: receptor crosstalk; heart; vasculature; regulatory systems; subcellular; contractile function; G-proteins; heart failure; hypertension; hypertrophy; signal transduction

I N T R O D U C T I O N

The cardiovascular circulatory function constitutes a very

sophisticated network of several highly synchronized

cir-cuits to ensure the sustention of human life by maintaining

or increasing blood supply providing oxygen and nutrients

to active tissue,and by redistributing the blood to prevent

heat loss from the body In humans,multiple cardiovascular

regulatory mechanisms have evolved to uphold this function

at three major levels: contractile apparatus,blood pressure

and circulating volume Apart from its ability to ensure a

smooth supply of nutrients to various organs,this network

also has the capacity to adapt to minor changes in vascular resistance that may influence the caliber of arterioles and other resistance vessels,and thus alter capillary hydrostatic pressure Such circulatory adjustments are effected syner-gistically by local (autoregulatory) as well as systemic mechanisms in both the heart and peripheral circulatory organs The autoregulatory mechanisms are a result of the intrinsic contractile response of smooth muscle to stretch,in combination with vasodilatation produced by metabolic changes leading to a decrease in oxygen tension,pH,and local vasoconstrictors,such as serotonin Systemic regula-tory mechanisms involve vasodilators such as the kinins,

Correspondence to N Dzimiri,Biological & Medical Research Department (MBC-03),PO Box 3354,Riyadh 11211,Saudi Arabia.

Fax: + 966 1442 7858,Tel.: + 966 1442 7870,E-mail: dzimiri@kfshrc.edu.sa

Abbreviations: A,adenosine receptor subtype; AC,adenylate cyclase; Ach,acetylcholine; ACE,angiotensin converting enzyme; ADO,adenosine receptors; ANG II,angiotensin II; ANP,atrial natriuretic peptide; ANPR,ANP receptor; AP-1,activating protein; AR,adrenoceptors; ATR, angiotensin receptor; BNP,brain natriuretic peptide; BNPR,BNP receptor; BP,blood pressure; [Ca2+],calcium channel; [Ca2+] i ,intracellular calcium; [Ca] v ,voltage-gated Ca 2+

channel; cAMP,3¢,5¢-cyclic adenosine monophosphate; cGMP,3¢,5¢-cyclic guanosine monophosphate; CNS,central nervous system; CV,cardiovascular; diacylglycerol,diacylglycerol; ET-1,endothelin-1; ETR,endothelin receptor; ETC,endothelial cells; 5-HT 4 ,5-hydroxytryptamine; I Ca ,calcium current; I K ,inward rectifying K+current; GC,guanylate cyclase; iNOS,inducible nitric oxide synthase; InsP 3 ,inositol triphosphate; IPN,isoproterenol; [K+],potassium channel; [K] ATP ,ATP-dependent K+channel; [K+] D ,delayed rectified

K + channel; MAPK,mitogen-activated protein kinase; MR,muscarinic cholinergic receptors; Na,sodium; NE,norepinephrine; NO,nitric oxide; eNOS,cardiac nitric oxide synthase; OP,opioid receptors; PE,phenylephrine; PIE,positive inotropic effect; PKC,protein kinase C; PLA 2 , phospholipase A 2 ; PLC,phospholipase C; PLD,phospholipase D; PKG,cGMP-dependent protein kinase; PPase,phosphoprotein phosphatase; PP2A,phosphoprotein phosphatase 2A; PTK,protein tyrosine kinase; PTPase,protein tyrosine phosphatase; PTX,Pertussis toxin; RAS,renin-angiotensin aldosterone system; RPIA, N 6 -phenylisopropyladenosine; VECs,vascular endothelial cells; VSM,vascular smooth muscle (Received 12 March 2002,revised 29 June 2002,accepted 14 August 2002)

circulating vasoconstrictors,such as catecholamines and

angiotensin II (Ang II),neural regulatory mechanisms,

sympathetic vasodilator systems and the vagal tone

Locally,the ability of the heart to maintain its regular

contractility and pumping rate is facilitated primarily by

postganglionic sympathetic-adrenergic nerve endings that

terminate within the myocardium and the autonomic

electrical stimulus originating in the heart Thereby,the

noradrenergic sympathetic impulses increase the cardiac

rate,contractile force and accelerate relaxation via the

b-adrenoceptors (b-ARs),while impulses in the vagal

cardiac fibres decrease heart rate via the cholinergic

pathway [1–3] Stimulation of the cardiac b-ARs activates

the stimulatory guanine nucleotide binding (Gs) protein –

adenylate cyclase (AC))3¢,5¢-cyclic adenosine

monophos-phate (cAMP) cascade,initiating the protein kinase A

(PKA)-dependent phosphorylation of several ion channels

and regulatory proteins,such as the L-type Ca2+channels,

phospholamban and myofibrillar proteins involved in the

cardiac excitation-contraction coupling and energy

meta-bolism [1,2] Regulation of the blood pressure involves

both systemic mechanisms and local angiotensin

receptor-mediated actions of Ang II on the

renin-angiotensin-aldesterone system (RAS) [4] The angiotensin receptors

(ATRs) couple to Gq/11 or Gi/o proteins to stimulate

several intracellular signalling pathways,transduced via

activation of at least five different effector systems: (a)

phospholipase C (PLC) leading to the formation of

inositol-1,4,5-triphosphate (InsP3) and diacylglycerol

(DAG); (b) voltage-dependent Ca2+channels stimulating

several downstream effectors; (c) phospholipase D

clea-ving phosphatidylcholine; (d) phospholipase A2

synthesi-zing prostaglandins and procanoids; and (e) AC inhibition

leading to a decrease in cAMP production [4]

By virtue of its nature,circulating blood volume is an

indirect product of mechanisms controlling the contractile

apparatus,blood pressure and valvular function This

function is regulated primarily by the release of natriuretic

peptides (NPs),particularly the atrial natriuretic peptide

(ANP),in response to volume expansion through

activa-tion of the hypothalamic muscarinic cholinergic neurons

by a-AR synapses [5,6] Unlike the other essential cardiac

G-protein coupled receptors,such as ARs or ATRs,the

NP receptors (NPRs) belong to the so-called type I

transmembrane single-chain receptors containing three

domains: intracellular catalytic GC,adjacent kinase-like

and extracellular ligand-binding domains They transmit

NP signalling by activating the guanylate cyclases to

produce the second messenger cGMP,leading to

cGMP-dependent protein kinase-mediated cellular actions [5]

Apart from the aforementioned pathways,the

cardiovas-cular system maintains several other receptor systems,

particularly the a1-ARs [2],adenosine (ADO)

[7],endo-thelin (ETR) [8],and opioid (OPR) [9] receptors,with yet

no fully defined cardiac physiological function,but may

contribute primarily to events associated with its adaptive

functional performance in disease The existence of this

particular group of receptors in the heart not only raises

important questions regarding the complexity of the

circulatory function,but also unequivocally points to the

fact that no cardiovascular functional entity is attributable

to a single signalling mechanism Growing evidence points

to crosstalk as a primary means by which these

mecha-nisms regulate cardiovascular circulatory function,and which is the subject of this review

C A R D I O V A S C U L A R R E C E P T O R

C R O S S T A L K S I G N A L L I N G

Crosstalk among adrenoceptor subtypes The concept of receptor cross talk has its origin in the early 1980s,when efforts were directed at explaining some of the apparently inconsistent behaviour of certain pharmacolo-gical agents,such as the a-AR and b-AR agonists Hence, among the most elaborately described crosstalk to date is that involving the cardiac AR subtypes,particularly between the b1-AR and a1-AR,in the regulation of the cardiac contractility and rhythm [10–14] This is partly attributable to the fact that,initially,differentiation among the AR subtypes has been hypothetically based on differences in the potencies of the three agonists epineph-rine,norepinephrine (NE) and isoproterenol (IPN) to the a- and b-AR subfamilies Early studies had already demonstrated that such a receptor classification could not be sustained,because of compelling evidence revealing that catecholamines not only transduce their signalling via both a- and b-AR subtypes [11],but also influence the downstream signalling components of the individual pathways in virtually the same fashion under different conditions [12] In rat neonatal cardiomyocytes for exam-ple,stimulation of a1-AR inhibits b-AR-mediated cAMP accumulation,presumably by coupling to the Giprotein, indicating that the former pathway may regulate the b-AR signalling downstream of agonist–receptor interactions [10,14] These studies led to the appreciation of the probability that both the convergence of these two pathways at the receptor–G protein–AC circuit,and their cross regulation via Gs and Gi serve to regulate mecha-nisms controlling cardiac contractile function under phy-siological conditions [10,13] However, the mode(s) by which this crosstalk is transmitted downstream of the AC appears to differ depending on the experimental setup In one study using transgenic mouse lines for example, cardiac-specific overexpression of a1B-AR was found not

to affect b-AR density or affinity to antagonists,yet the basal AC activity was increased without influencing basal cAMP levels,while IPN-stimulated AC was attenuated in association with increased Ca2+-dependent PKC-d and PKC-e as well as Ca2+-independent PKC-b2 levels in particulate cellular fractions [13] These findings led to the suggestion that overexpression of a1B-AR triggers uncoup-ling or desensitization of the b-AR by molecular crosstalk, via the PKC pathway In contrast,in another transgenic mouse model,similar overexpression of the a1B-AR was associated with significant depression of left ventricular contractility,accompanied by both attenuated basal and catecholamine-stimulated AC activity [14] Treatment of the mice with pertussis toxin (PTX) led to a reversal

of these changes,presumably pointing to an induction of coupling to PTX-sensitive Gi proteins resulting from elevated levels of a1B-AR This,together with the obser-vation of elevated GRK2 activity in these animals stimu-lated the notion that down-regulation of b-ARs may cause

an elevation in a1-AR levels It is evident that under experimental conditions,the a- and b-AR pathways

influence each other in a variety of ways,utilizing different

signalling conduits The cardiac a1-AR may influence the

b1-AR signalling via two distinct receptor-mediated

mech-anisms: via pertussis toxin (PTX)-sensitive G proteins

possibly to regulate positive inotropic function and by

enhanced a1-AR under disease conditions

Adrenoceptor crosstalk with other cardiovascular

receptors

Rapidly accumulating literature has demonstrated that,

apart from crossregulation of each other,stimulation of the

AR pathways triggers alterations in the signal transduction

of other cardiovascular systems,particularly the ATR,

ETR,muscarinic acetylcholine receptor (MR),NPR and

nitric oxide synthase (NOS) pathways [15–31] (Table 2) In

neonatal rat cardiomyocytes,the a1- and b1-AR agonists

induce ANP transcription [15,16], while Ang II stimulation

of AT1R leads to a decrease in a1A-AR mRNA levels and

stability as well as its induction of immediate early gene

c-fos expression,demonstrating that crosstalk among these

receptors occurs at the level of gene transcriptional

regula-tion [17] Furthermore,in rat-1 fibroblasts,activaregula-tion of

ET-1 receptors was found to induce a1A-AR

phosphoryla-tion,possibly involving the PKC and MAPK signalling [18],

while in human pericardial smooth muscle,it was found to

counter-regulate cAMP and MAPK signalling [20]

Grow-ing evidence demonstrates that stimulation of these

path-ways can enhance or inhibit the release of endogenous

catecholamines often associated with the down-regulation

or desensitization of the ARs Thus,it appears that some of

this crosstalk may be a direct product of altered receptor

turnover In the vascular system and peripheral circulatory

organs,complex crosstalk regulating AR signalling often

involves synergistic actions of several pathways,or may be

an indirect product of interactions between some

nonadre-nergic pathways acting on local catecholamine release A

typical example is the interaction between the MR and

a-AR pathways in the hypothalamus,which is thought to

be responsible for the ANP release in the regulation of

circulating blood volume [6] Also,in rabbit cerebral

arteries,activation of a2-AR triggers

endothelium-depend-ent ET-1-mediated contractile response to acetylcholine

(Ach),leading to a reversal of MR effects [18,19] Crosstalk

in which nonadrenergic pathways influence b-AR include

the attenuation of IPN-stimulated cAMP accumulation by

ET-1 in human pericardial smooth muscle cells [20],

inhibition of epinephrine release and b-AR responsiveness

by adenosine [21,22] and inhibition of b-AR contractility by

either Ang II via AT1R [23–25],nitric oxide (NO)

produc-tion [26–28] or OP2receptors [29–31] Of these interactions,

probably the most exhaustively studied crosstalk is that

between b1-AR and the AT pathways,suggesting that

Ang II-mediated AT1R stimulation decreases b1-AR

responsiveness via PKC activation [24] and inhibits

b-AR-stimulated AC activity via the Giprotein in cell cultures [23]

Some studies have indicated that such AT1R activity

induces local catecholamine release from the cardiac

sympathetic neurons causing myocardial damage probably

resulting from down-regulation of the b-AR [24,25] The

regulation of NE via neuronal AT1R pathway appears to

follow two courses defined as evoked or enhanced

neuro-modulation [23] Accordingly,evoked neuroneuro-modulation

involves AT1R-mediated,antagonist-dependent rapid NE release and inhibition of K+channels,while enhanced NE neuromodulation involves the MAPK cascade ultimately leading to an increase in NE transporter,tyrosine hydroxy-lase and dopamine b-hydroxyhydroxy-lase mRNA transcription [23]

In contrast to Ang II or ET-1,adenosine has been shown to inhibit NE release from sympathetic nerve endings in rat adrenal medulla partially through its inhibitory effects on RAS pathway [21],and to exert antiadrenergic effect in rat hearts through crosstalk between its two receptor subtypes

A1- and A2a-ADO [22] The cardiac nitric oxide synthase (eNOS) pathway may contribute to a number of such mechanisms involving the crosstalk among ET,MR and

AR pathways [8,26–28] Stimulation of the eNOS may attenuate both inotropic and lustropic responses to b-AR stimulation,and appears to regulate baseline ventricular relaxation in conjunction with ANP [26,27] It was shown for example,that the inhibition of b-AR-stimulated increase

in the slow-inward Ca2+ current (ICa) and reduction in

Ca2+affinity of the contractile apparatus may be a result of

MR stimulation of the heart activating NO production

of cGMP [27] In neonatal ventricular myocytes and fibroblasts,ANP and NO were shown to synergistically attenuate the growth-promoting effects of NE by a cGMP-mediated inhibition of NE-stimulated Ca2+-influx [28] Some of the crosstalk leading to the inhibition of the b1-AR activity can be explained as resulting from a corelease of the endogenous hormones with the catecholamines in cardio-myocytes,like in the inhibition of b1-AR-stimulated AC in myocyte sarcolemma by OP2 agonists via Gi/o pathways [29,30], or the OP3 agonists in rat ventricular myocytes, devoid of the phosphoinositol pathway [31]

Crosstalk among nonadrenoceptor pathways Apart from their interaction with the ARs,activation of several G protein-linked cardiovascular pathways,notably the ATR,ETR and MR systems,can also trigger the release of,and enhance cardiovascular responses to,other vaso-active peptides such as ANP,vasopressin or aldosterone Perhaps the most comprehensively studied crosstalk to date

is that involving the ANP and RAS pathways,whereby the former is thought to exert its actions by inducing an increase

in angiotensin converting enzyme (ACE) to counteract RAS effects in regulating circulating volume However,studies so far have not delineated exactly how this may occur In sliced rat atrial tissue,Ang II induces inositol phosphate accumu-lation and ANP release [32],but seems to impair ANP-mediated inhibition of AC in the vascular smooth muscle [33] Other interesting crosstalk involving the RAS pathway includes the observation of a transcriptional regulation of ATR through inhibition of NO synthesis in rats [34] and the influence of Ang II on ET-1 synthesis and/or release apparently without influencing its circulating levels in human endothelial cells [35] These Ang II actions are probably mediated through AT1R stimulation of [Ca2+]i activity Complex interactions have also been reported involving the ET and ANP pathways In this crosstalk, ANP was reported to inhibit ET-1 in dogs with congestive heart failure [36],while ETA is thought to regulate ANP gene expression via multiple pathways involving Giand Gq

in addition to MAPK activation [37] The crosstalk among the vasoactive pathways occurs at two distinct levels: the

central nervous system and cardiac humoral regulatory

mechanisms One classical example is the release of the NP

through hypothalamic MR and a1-AR crosstalk,which is

probably humorally regulated by the heart through several,

diverse feedback mechanisms [6] However,the contributory

mechanisms remain highly speculative

The role of crosstalk in cardiovascular function would be

incomplete without a brief consideration of its involvement

in the regulation of the cardiac chloride (Cl–),sodium

(Na+),K+and Ca2+channels,as they regulate the

mem-brane potential and transportation of ions and substrates,

controlling excitation and excitation-contraction coupling

of the contractile apparatus Regulation of these channels is

mediated often through interplay between Gs- and

Gi-coupled pathways For example,it has been suggested

that in cardiac myocytes, b-AR-mediated activation of the

Cl– requires the stimulation of both cAMP-dependent

PKA-mediated phosphorylation and cAMP-independent

pathways [38],and may be inhibited by a1-AR stimulation

via a PTX-insensitive G-protein [39] This crosstalk may

lead to the inhibition of b-AR-stimulated increase in

intracellular Ca2+([Ca2+]i),and/or reduction in the affinity

of the contractile apparatus to Ca2+ In the heart,

membrane-delimited activation of muscarinic K+channels

by Gbcplays an important role in the inhibitory synaptic

transmission [3] The activation of the Na+ pump and

voltage-dependent [K+] mediates smooth muscle

hyperpo-larization in the relaxation elicited by Ach,possibly through

enhancement of cGMP activity [40] Furthermore,it has

been speculated that AT1R-evoked NE neuromodulation

involves the inhibition of K+channels and stimulation of

Ca2+channels [23] In the heart,the Na+/H+exchanger

and mitochondrial K+ channels may be important in

apoptosis and ischemia/ischemic preconditioning signalling

discussed later This summary is far from being exhaustive,

and represents only a taste of the rapidly growing

know-ledge about receptor crosstalk with potential relevance for

the physiological regulation of circulatory function

Inter-estingly,although this pool of interactions among

cardio-vascular systems appears to be congested and not very

transparent,it is regulated by just a couple of G proteins,

protein kinases and signalling junctions

L O N G - T E R M A N D S H O R T - T E R M

E F F E C T S O F C A R D I O V A S C U L A R

R E C E P T O R C R O S S T A L K

In general,cardiovascular signalling may be regulated at the

level of a single functional entity such as contractile

apparatus,but more importantly so,in coordinating the

different functions into a synchronized unit In the execution

of these functions,two types of cellular responses,the

short-term and long-short-term responses may ensue Short-short-term events

include,for example,activation of Ca2+ turnover to

stimulate the contractile apparatus or vasoconstriction,

while long-term actions are essentially involved in gene

transcriptional regulation or altered expression,often as an

adaptive mechanism in disorders such as left ventricular

hypertrophy (Fig 1) Cardiovascular signalling crosstalk

mediates both short- and long-term events,and

coordina-tion of the individual contributory pathways is regulated at

various signalling junctions,particularly the G protein,AC,

PK and MAPK levels The existence in the human

cardiovascular system of at least 16 a,11 b and 5 c subunits

of the heterotrimeric G proteins [41],10 mammalian AC isoforms (ACI–ACX) [42] and multiple PKC isoenzymes exhibiting specificity and diversity in their activation of G-protein coupled receptor downstream signalling compo-nents clearly endows the heart with an enormous potential

to assemble numerous signalling products to regulate intracellular Ca2+turnover,and therefore positive inotro-pism,as well as vasoconstriction and vasodilatation However,despite the diversity in the multiple signalling systems engaged in the regulation of cardiovascular func-tion,these pathways transduce their messages by coupling primarily to three G proteins,the Gs, Gi and Gq/11 to stimulate cardiac-specific ACII and ACV or PLC isoforms, utilizing cAMP-dependent PKA,PKC-a and PKC-f to activate a small subset of downstream effectors and ion channels In particular,the Gi-coupled,PLC-mediated signalling cascades appear to occupy a central role in this crosstalk (Fig 1) This pathway mediates among other factors,the inhibition of b-AR-stimulated cAMP accumu-lation resulting from the crosstalk between the a1-AR and b-AR [10], b1-AR and OPRs [29] as well as the AT1R and ANP systems [32],although a Gi-mediated crosstalk bypassing PLC stimulation has also been proposed between b-AR and OPRs [30] The same pathway has been postulated for the ETA-induced regulation of ANP signal-ling and gene expression by coupsignal-ling to both Gi and Gq proteins [36,37] In these interactions, the Gi appears to couple negatively,while the Gqmay do so in a supportive fashion,especially in the regulation of the contractile apparatus (Fig 2) Another important regulatory pathway

is the cGMP-mediated signalling,which is thought to be involved in the crosstalk between ANPR and AT1R [37], the inhibition of ET-1 secretion by ANP [36],and the synergistic actions of NO and ANP in attenuating NE-stimulated Ca2+influx [27] Although hardly any specific physiological role in cardiovascular signalling has been clearly defined for the majority of the crosstalk among these pathways,its existence in the cardiovascular system strongly points to an orchestrated ancillary functional role in support

of the classically defined pathways

Perhaps the most challenging question at present is how crosstalk is regulated beyond the primary receptor–

G protein–second messenger circuit Although this question

is far from being answered,it can be plausibly assumed that the majority of the players have already been identified While the short-term crosstalk events appear to be mediated primarily via second messenger-dependent PKA and PKC, regulation of the long-term events probably underlies crosstalk involving both PKs and the MAPK pathways Thus,downstream of the second messengers,the PKs and the MAPKs serve as hubs for diverting,converging,sieving as well as directing signalling messages mediated by the various

G proteins coupling via the PLC pathway,for example Established crosstalk regulation by PKs includes,among others,the cross-regulation of the a1- and b-ARs [11–13,43] and attenuation of ANP-mediated inhibition of AC activity

by Ang II [33],while crosstalk at the MAPK level has been ascribed to stimulation of gene expression via different pathways [11,37] Apparently, the GPCRs stimulate the MAPK pathways mainly by coupling via Giprotein [44],and

to some extent by the bc complex of the Gqprotein [45–47] Some phosphoinositide 3-kinase (PI3K) isoforms may also

function as crosstalk mediators between nonreceptor protein

tyrosine kinase (PTK) and G-protein coupled receptor

signalling to stimulate the Ras-Raf-MAPK cascades [48,49]

The fact that cardiac function is regulated by diverse

signalling cascades linked via autoregulatory and systemic

regulatory mechanisms renders it mandatory for the heart

to possess an inherent machinery to integrate the

commu-nication among these individual pathways into a single

functional entity To achieve this,the heart probably

functions as an endocrine and paracrine organ [50,51] that

determines its own fate by regulating the various signalling

mechanisms through receptor crosstalk Some of these

mechanisms have their origin in the CNS (Fig 3) These

include: (a) the possible regulation of blood pressure in the

cardiovascular centres of the brain through Ach release via

cholinergic neurons [52]; (b) the negative feedback system

regulating the balance between vasodilatory and

vasocon-strictory effects involving crosstalk between ET-1,ETBand

NO [53]; (c) the regulation of both the noradrenergic and

cholinergic systems in cardioinhibitor and vasomotor

centres in the medulla oblongata [3]; and (d) the regulation

of MR by a-AR systems controlling ANP release in the

hypothalamus [6] Thus,the CNS may be intimately

involved in defining the types,sources and physiological

entities to convey defined messages at the appropriate time, using sympathetic and parasympathetic routes as links between the extracardiac and the cardiac signals

Implication of receptor crosstalk for cardiovascular physiology

Because circulatory function constitutes an integration of messages emanating from the contractile apparatus with those of the various regulators of blood pressure and circulating volume,a clear demarcation is often not possible between the mechanisms controlling the different compo-nents of this complex machinery Nonetheless,cardiovas-cular receptor systems may be broadly placed into those that regulate mainly the contractile apparatus,such as the

AR systems,and those that primarily determine the circulating blood volume,such as the ATR,ETR and NPR systems Interestingly,while the human heart posses-ses at least three defined b-AR subtypes (b1-, b2- and

b3-AR),it was traditionally believed that catecholamines preferentially elicit their inotropic,chronotropic,lusitropic and dromotropic effects via the b1-AR–Gs–AC–cAMP pathway under physiologic conditions [1,2] However, the accumulating evidence that several cardiac pathways can

Fig 1 Regulation of short and long-term signalling in normal cardiovascular physiology and disease Short-term signalling is involved in acute and instantaneous regulation functions such as cardiac contractile function Stimulation of receptors such as b 1 -AR by the catecholamines (H),for example,leads to such effects by activating the classical receptor–guanine nucleotide binding protein (G)–second messenger (AC) circuit leading to a change in the concentration of intracellular messengers such as free cytosolic Ca 2+ ,and consequently positive inotropic effects (PIE) Alternatively,

a change in the transcriptional regulation of certain genes or malfunctional signal transduction,such as PKA-mediated b 1 -AR actions,may trigger long-term cellular effect by stimulating the mitogen-activated protein kinase pathway resulting in altered protein functional expression,as in apoptosis or mitogenesis The G bc -mediated crosstalk between the MAP kinases and G i/o pathways play an important role in mediating long-term signalling changes especially in cardiac disease Inhibitory or counteractive functions are indicated as rounded arrow ends L,L-type Ca2+channel; PLB,phospholamban; Ry,ryadine receptor.

also mediate positive inotropism in vitro at least via the Gs–

AC–cAMP pathway and that multiple GPCRs can couple

to more than one G protein has cast doubt over the validity

of this paradigm Moreover,at least three potential

pathways have been delineated so far,that can lead to the

enhancement of cAMP synthesis: directly by coupling to the

Gs,or to PKC and Ca2+via Gq/11,or signalling via the bc

complex of Gi/o proteins (Table 1) Accordingly,any

signalling cascade employing members of the Gq/11 and

Gi/o protein families should also be capable of at least

indirectly regulating or modulating the contractile

appar-atus by their effects on PLC-b,leading to InsP3synthesis or

through the PKC-mediated handling of Ca2+turnover,or

even bypassing these two routes While the last alternative

symbolizes physiologically only a theoretical possibility,it

might be relevant in cardiac disease,if and when the other

two are rendered nonfunctional In contrast,although no

physiological function is currently attributed to the Gq/11/

PLC–DAG route,compelling evidence points to an

essen-tial function as an inherent supportive resource to be tapped

as required (Fig 2) Notably,all of the receptors currently

thought to be physiologically dormant in the heart,

particularly b2-AR,A2-ADO,ETA,AT1R and M1-MR

subtypes can elevate Ca2+ levels via the Gq/PLC route

(Table 1) It is therefore highly unlikely that the expression

of this contingency of Ca2+ regulators is redundant,

considering the complexity and essence of the cardiovascu-lar circulatory system On the other hand,while some of these pathways have been shown to mediate positive inotropic effects (PIE) in vitro at least [54],it does not appear desirable at all that they contribute directly to cardiac physiological regulation,but rather to avail them-selves in the event that the primary regulators of the contractile apparatus falter in disease

In contrast to enhancing PIE in normal cardiac function, receptor crosstalk may in fact be desirable,if not absolutely essential,in rhythmically quenching the b1-AR-mediated stimulation of the excitation/contraction coupling mechan-ism of the contractile apparatus This can be accomplished

in several ways or through a combination of factors, including receptor internalization,dephosphorylation,deg-radation and negative feedback loops evoking primarily the inhibition of cAMP-mediated effects In the heart,the most obvious mechanism for this action is the direct coupling of the Gia,notably Gia2,to inhibit AC signalling A number of signalling pathways,including the a1-AR, b2-AR,M2-MR,

A1- and A3-ADO have actually been found to couple to this pathway,in fashions that would lead to the inhibition of b1 -AR-stimulated AC activity (Table 2) The same route potentially mediates the negative inotropic effects (NIE) of some of these pathways,counterbalancing b1-AR positive inotropism by inhibiting Ca2+ effects For example,the

Fig 2 Regulation of the contractile apparatus through receptor crosstalk signalling Cardiovascular crosstalk engages mainly three G protein families: the stimulatory (G s ),inhibitory (G i/o ) and G q/11 proteins,employing PLC as a central hub in regulating this crosstalk signalling In the regulation of positive inotropism,for example,the G s -coupled b 1 -AR pathway constitutes the major stimulator of the contractile apparatus The

G q -coupled PLC/InsP 3 (IP 3 ) or PLC/DAG/Ca2+pathways provide reserve pathways that can be mobilized,if and when needed All of these pathways may be inhibited by G i/o -coupled signalling,via the muscarinic cholinergic pathway,for example,either through their inhibition AC function or via the InsP 3 or DAG pathways to furnish a feedback loop in order to intermittently quench the otherwise immutable b 1 -AR stimulation of the contractile apparatus Details of the individual messenger cycles have been omitted for clarity Inhibitory or counteractive functions are indicated as rounded arrow ends H,hormone (ligand); L,L-type Ca 2+ channel; PLB,phospholamban; R,receptor; Ry,ryadine receptor; X,crosstalk regulatory switch; V,voltage gated Ca 2+ channel.

M2-MR-mediated activation of NO-stimulated cGMP

synthesis is thought to contribute to various cardiac

functions including NIE,an abbreviation of contraction,

and enhancement of diastolic relaxation [27] The NIE may

serve as a physiological role of intermittently quenching

the otherwise immutable b1-AR-mediated excitation of

the contractile apparatus It has been suggested that an

elevation of Ca2+ entry via L-type Ca2+ channels in

response to b-AR stimulation,rather than its release from

intracellular stores,may mediate its inhibition of ACV and

ACVI and act as a negative regulator of the

receptor-mediated AC activity [56] Therefore,some of the cardiac

signalling circuits involving Ca2+-dependent inhibition of

these AC isoforms,or inhibition of G-protein coupled

receptor activity via PKA-mediated phosphorylation

prob-ably constitute a feedback loop for controlling

cAMP-dependent increases in [Ca2+]i Thus,the dual regulatory

control of the cardiac AC activity by the Gs and Gi is

probably designated to provide means for filtering

parti-cularly the b-AR signalling in the regulation of the

contractile apparatus,employing the PKs as feedback regulatory switches,or for redirecting signalling messages

to meet certain requirements (Fig 2)

Apart from this function,crosstalk between b1- and

b2-AR,involving switching of the b2-AR from Gs to Gi coupling appears also to provide the heart with the ability to cope with various situations,and to mediate actions that differentiate these two AR subtypes on cardiac Ca2+ hand-ling,contractility,cAMP accumulation,PKA-mediated protein phosphorylation,and in modulating noncontractile cellular processes [54,55] Thus, the dual coupling of the

b2-AR to Gs and Giin the heart is thought to result in compartmentalization of the Gs-stimulated cAMP signal, selectively affecting plasma membrane effectors such as L-type Ca2+ channels,and bypassing cytoplasmic target proteins such as phospholamban and myofilament con-tractile proteins [55] Accordingly,Gi-dependent functional compartmentalization of the b2-AR-directed cAMP/PKA signalling to the sarcolemmal microdomain dissociates the receptor-induced augmentation of [Ca2+] transients and

Fig 3 Crosstalk in the regulation of cardiovascular circulatory function The regulation of the blood pressure and circulating blood volume is maintained through crosstalk of various signalling pathways,some of which are controlled in the cardiovascular control centres in the central nervous system Receptor such as AT1R and ET1R stimulate vasoconstriction employing primarily the PLC pathway,whereas the nitric oxide synthase and NP pathways regulation cell/blood volume by causing smooth cell relaxation In the CNS crosstalk between the cholinergic and the a-AR among others,has been implicated in the release of ANP,or regulation of nitric oxide pathways leading to feedback pathways for the regulation of the blood pressure and volume Inhibitory or counteractive functions are indicated as rounded arrow ends Ad,adenosine; Arg,

L -arginine; L,L-type Ca 2+ channel; V,voltage gated Ca 2+ channel.

contractility from cAMP production and PKA-dependent

cytoplasmic protein phosphorylation,apparently allowing

cAMP to perform selective functions during b-AR subtype

stimulation [55] Pathways involving the activation of type 1

protein phosphatase (PP1) and structural restriction of

PKA diffusion by its specific anchoring proteins have been

proposed as candidate mechanisms underlying this

com-partmentalization [57] Besides,the switching from Gs- to

Gi-mediated PKA coupling of G-protein coupled receptor

pathways is thought to mediate mitogenesis via the Sos

pathway [43] Activation of Src (or closely related PTKs),

and subsequently Tyr phosphorylation of adapter or

scaffold proteins,leads to the recruitment of guanine

nucleotide exchange factors,such as the Grb2–mSos

complex to the plasma membrane,followed by sequential

activation of Ras/Raf pathway to regulate gene expression

essential for proliferation [43–47] (Fig 1) This process, traditionally conceived as an escape route for G-protein coupled receptors from unabated stimulation by their agonists,has recently gained some recognition as a normal physiological process to regenerate receptors following desensitization Other crosstalk involving interaction of Gs and Gisignalling may be important in disease manifesta-tion These are discussed below

An interesting feature of cardiac signalling is the fact that coupling of receptor subtypes within the same family via different G proteins often generates opposed signalling products For example,while coupling of a2-AR,M2-MR,

A1- and A3-ADO to PLC via the Gi/oinhibits AC activity, the signalling of a1-AR,A2A-ADO,A3-ADO,M1- and

M2-MR via the Gqtheoretically promotes conditions for PIE due to their ability to mobilize Ca2+(Table 2) These

Table 1 The major human cardiac receptors and their circulatory related signalling products The table summarizes the circulatory function-related signalling pathways and products of important cardiovascular receptor subtypes in the various organs The relevant literature has been quoted in the main text.

Family Type Localization CV function Coupler Signalling mechanism [References]

a-AR a 1A ,

a 1D

Heart,blood

vessels

VSM,myocardial contraction

G q/11 PLC-mediated [Ca2+] v activation [2],inhibition

of cAMP accumulation [10]

a 2B heart Vasoconstriction G i/o Ca 2+ -dependent [K + ] activation

[Ca2+] v inhibition [2]

b-AR b 1 Myocardium PIE G s AC-mediated cAMP synthesis [1,2]

b 2 Cardiac chambers Heart rate control;

VSM relaxation

G s ; G q

G i/o

AC-mediated cAMP synthesis [2,55]

AC inhibition [2,87]

ADO A 1 Brain,heart Bradycardia G i/o AC inhibition; [K] i opening [7]

A 2A Pacemaker Vasodilatation G s ; G 15 Stimulation of AC and PLC-b [7]

A 2B VSM,brain VSM relaxation G q/11 ? PLC/AC-mediated Ca2+activation [7]

A 3 Heart,kidney A 1 modulation G i3

G q/11

AC inhibition [7]

PLC/InsP 3 -mediated Ca2+increase [7]

ANG AT1R VSM Heart,

aorta,kidney

VSM contraction G q/11 PLC/PKC-mediated elevation of [Ca 2+ ] i level [4]

AT2R Heart,adrenal

medulla

Vasodilatation;

apoptosis promotion

G ia2 /G ia3 Ppase-stimulated MAPK activation; [K+] D opening;

PTPase activation leading to T-type [Ca2+] closure [4]

ET ET A VSM (blood

vessel,heart)

Vasoconstriction; PIE G q/11 ;

(G s ?)

PLC/InsP 3 -mediated Ca 2+ influx;

Activation of cAMP synthesis [8]

ET B VECs,heart

Vasodilatation/vasocon-striction (ET B2 )

G i

G q/11

Inhibition of cAMP formation [8]

PLC-mediated phosphoinositol-stimulated [Ca 2+ ] i elevation/Ca2+influx [8]

MR M 2 Heart,brain Decrease in heart

rate,force (NIE)

G i/o

G bc

[K + ] D opening; AC and [Ca 2+ ] inhibition [3,40,45]

M 3 VSM,brain VSM contraction G q/11 PLC-mediated InsP 3 /Ca2+,DAG/PKC activity;

cAMP elevation [3,52]

NP ANP Kidney,

myocardium

Blood volume,

BP regulation

GC cGMP-dependent PKG inhibition of cardiac growth

and function [5,6]

BNP Ventricle BP reduction GC cGMP-dependent PKG inhibition of VEGF synthesis

and function [5,6]

NOS eNOS VSM endothelium; Vasodilator tone

in BP regulation

GC cGMP-mediated NO actions [26],Ca2+/

calmodulin activation [26,28]

OP OP 1 Neocortex,

vas differens

Central CV regulation G ia1 /G ia3

(G oa2 )

AC inhibition [9]; inhibition of b-AR function [29,30]

OP 3 Caudate putamen Central CV regulation G o I K conductance activation; reduction in

neuronal I Ca ; AC inhibition [9,80]

Table 2 Circulatory function-related effects of cardiovascular receptor crosstalk The table shows signalling products of crosstalk interaction of cardiac receptors on other G-protein coupled receptor receptor system Details of the crosstalk activity are given in the cited references in the main text.

Receptor Interactive agonist/receptor Cardiovascular signalling product [References]

a 1 -AR G i -coupled a 1B -AR

stimulation

Potentiates PE-mediated protein synthesis; Ca2+-dependent PKC-a-mediated early gene expression in neonatal heart [83]

b-AR agonist (IPN) Synergistic with PE,activates protein synthesis via Raf/MAPK pathway in neonatal

rats myocytes [11];

PKC-mediated depression of b-AR response to IPN in a 1B -AR overexpressing mice [13,14] Ang II via AT1R Attenuates a 1 -AR mRNA and stimulated c-fos induction [17]

ET-1 via ET A Phosphorylates a 1B in Rat-1 fibroblasts via PKC and PTKs [18]

b 1 -AR a 1 -AR agonist (PE) Synergistic with IPN,activates protein synthesis via Raf/MAPK pathways in

cultured myocytes in neonatal rats [11];

G i -coupled inhibition of b-AR-activated chloride current [39]

G i -coupled a 1 -AR (NE) Inhibits b 1 -AR stimulated cAMP accumulation [10]

Ang II on

(neuronal) AT1R

Inhibits cardiac b 1 -AR responsiveness via PKC activation and density in transgenic mouse [23–25]; Inhibits [K+]; stimulates NE release,NE transporter and [Ca2+] [23,24]

AT1R -antagonists

Ang II-stimulated AT2R

Inhibits IPN-stimulated b 1 -AR in transgenic mouse [24], Stimulates [K + ] via G i coupling to PP2A/PLA 2 ; stimulates NE-transporter and transcription activity [23]

ET-1 Inhibits pericardial cell IPN-stimulated cAMP accumulation [20]

NO via eNOS Inhibits b-AR PIE and chronotropy in transgenic mice and heart failure [26,65]

and NE mitogenic effects in ventricular cells [27]

NO via iNOS Induces b 1 -AR hyporesponsiveness in cardiomyopathy [28]

RPIA-desensitized A 1 Decreased inhibition of IPN-stimulated AC activity [66]

Carbachol-stimulated M 2 Increases IPN-stimulated AC in adult rat cardiomyocytes [66] Inhibits

NE release, b 1 -AR contractility [76]

OP 1 agonists Inhibits NE-mediated b 1 -AR effects in rat ventricular myocytes

via a PTX-sensitive G i/o protein [29,30]

ET A -stimulated ET-1 Attenuates IPN-induced cAMP accumulation in cardiac smooth muscle [20]

M 2 stimulation Parasympathetic slowing of heart rate; inhibits b 1 -AR contractility (via G i ?) [3,28,45]

A 1 Isoproterenol-desensitized

b-AR

Decreased AC inhibition by RPIA in adult rat myocytes [66]

Ach-desensitized M 2 Decreased inhibitory action of RPIA on AC [66]

A 2a Counteracts A 1 -meditaed antiadrenergic actions [22]

AT1R ANP via cGMP Inhibit ET-1 secretion following Ang II-mediated AT stimulation

in porcine aorta [71]; inhibits RAS activation [70]

ET-1 Stimulates RAS system synergistically with Ang II [59]

NO Up-regulation of AT1R by inhibition of NO synthesis [34]

AT2R ANP via ANPR Increases ACE levels via cGMP stimulation,vasorelaxation [98]

ET-1 stimulated ET A Synergistically activates Raf-1 kinase,MAPK in myocytes [8]

ET A Ang II via AT1R Induces endothelial ET-1 release associated with hypertensive-induced hypertrophy [35].

ANP Counteracts ET-1 activation of AP-1 via cGMP pathway in dogs

with congestive heart failure [36].

Isoproterenol-activated

b-AR

Attenuation of ET-1-induced MAPK activity [21]

NO Inhibits ET synthesis via cGMP synthesis [52]

ANP a 1 -AR agonist (PE) Stimulates ANP synthesis and release [6,16]

b-AR agonists (IPN) Stimulates ANP transcription via Akt [15]

Ang II-activated AT1R Attenuates ANP-mediated AC inhibition via KPC in VCM [33];

stimulates ANP release via inositol phosphate/Ca 2+ activation [32]

eNOS Up-regulates ANP in eNOS deficient mice [26]

ET-1-activated ET A Activates G q -mediated Ca2+-stimulated ANP synthesis in rat myocytes [37]

M 1 /M 2 stimulation ANP release in response to volume expansion (via G q ) [6]

OP 2 agonists Releases ANP in hypertensive patients [92]

BNP ET-1-mediated activity Increases BNP gene expression in porcine aorta [71]

M 2

Isoproterenol-desen-sitized b-AR

Increased AC inhibition by carbachol in rat myocytes [66]

Oxymetazoline on a 2 -AR Induces endothelial ET-1-mediated contraction to Ach [19]

ENOS ET-1 stimulated ET A ,ET B Induce NO synthesis in endothelial cells,counterbalances NO vasodilator tone [8]

M 2 agonists Stimulates NO synthesis [28].

overtly antagonistic events imply that the ability to

concomitantly couple to PLC via the Giand Gqpathways

furnishes at least an auxiliary intraregulatory mechanism

that can be mobilized to switch from one pathway to

another to regulate the cardiac contractile apparatus The

observation that crosstalk between Gi and Gq-coupled

receptors is mediated by the Gbc subunits [58] similarly

underscores the significance of crosstalk at the level of

proteins regulating second messenger function in cardiac

inotropy,and strongly corroborates the probability of the

reserve resources of both PIE and NIE being regulated by

the same components upstream of the second messengers

Therefore,these manifestations point to PLCs as a nucleus

for sorting and channelling signals that determine auxiliary

cardiac circulatory function,providing a regulatory

feed-back route for harnessing the b1-AR stimulation of the

contractile apparatus (Fig 2)

Apart from serving as a negative feedback in regulating

inotropism,crosstalk is also likely to be required to a greater

extent in the regulation of blood pressure and circulating

volume than in contractile function under physiological

conditions,especially considering the fact that these

func-tions are controlled by vasoactive autocoids with directly

opposing effects on the vascular system These interactions

are likely to be dominant in the vascular beds,but are

apparently also in the kidney,where ET-1 and Ang-II cause

vasoconstriction,decreasing renal blood flow,and

glomer-ular filtration rate,while bradykinin and ANP cause

vasodilation and increase glomerular capillary permeability

[50,52,59] Such crosstalk may primarily be designed to finely

tune the balance between vasoconstriction and

vasodilata-tion in regulating circulating volume,cardiovascular

hemo-dynamics and blood pressure While the involvement of the

crosstalk among these vasoactive receptor systems seems

unequivocal as a physiological regulatory control for

circu-latory function,very little is known to date with respect to the

level at which the different pathways communicate with each

other Nonetheless,it seems clear that both divergence and

convergence in G-protein coupled receptor pathways at the

various junctions serve to furnish the heart with the means

to respond appropriately to intercellular and intracellular

signals to meet the individual environmental requirements

Implications of receptor crosstalk for cardiac

adaptation to diseases

Cardiovascular diseases fall broadly into three main

cate-gories The first group comprises pathologies such as dilated

cardiomyopathy that directly affect the heart muscle,

gravely compromising cardiac contractile function In these

diseases,the heart may transverse various phases,such as

left ventricular hypertrophy,prior to reaching end-stage

heart failure,a state in which the heart cannot meet its

fundamental functional demands without some form of

assistance This complex process involves alterations in the

myocyte structure and function,abnormalities in Ca2+

homeostasis,excitation-contraction coupling and changes

in the cytoskeletal architecture,partly resulting in apoptotic

cell death [60,61] It appears that adaptive energy

metabo-lism lends itself as the first line of defense to protect cardiac

function from collapse in disorders affecting the contractile

apparatus The major myocardial energy substrate probably

switches from fatty acids to glucose,associated with

substantial down-regulation of the fatty acid utilization enzymes [62],under the control of a yet unidentified gene regulatory program The transition from compensated to decompensated heart failure is associated with overexpres-sion of neurohormones and peptides,such as Ang II,ET,

NE and proinflammatory cytokines At receptor level,the paradigm is that the b1-ARs are down-regulated,and the

b2-ARs uncoupled,accompanied by an up-regulation in the a-AR [1], b-AR-specific receptor kinase 2/3 (GRK2/3) and Giprotein levels [63] While the mechanisms underlying increases in a1-AR levels through down-regulation of

b1-AR are yet to be clarified,the fact that such elevations are manifest in human disease implies that the origin of such crosstalk is embedded in the mechanisms leading to cardiac disease manifestation Recently,attention has focused on the role of signalling pathways,such as b1-AR in the manifestation of heart failure Some studies have suggested for example,that PKA-mediated b1-AR signalling may induce apoptosis [61] and alterations in this pathway are an underlying cause of cardiac toxicity Apparently,in adult mouse cardiomyocytes,the apoptosis induced by the

Gs-mediated signalling or other assaulting factors can be counteracted through a process involving the coupling of the b2-AR to Gi, Gbc,PI3K and the serine-threonine kinase Akt-glycogen synthase kinase 3b pathway,which is thought

to mediate survival mechanisms [61] (Fig 1) This under-scores the notion that the cardiomyocyte is equipped with reserve mechanisms to counter potentially detrimental effects resulting from malfunctional signal transduction Malfunctional crosstalk is certainly an important contribu-tory component of the progression of cardiac disease to heart failure Such crosstalk between b-ARs and,among others,the ATR,ANPR,ETR,ADO or OPR subtypes has been implicated in the manifestation of heart failure through

a variety of mechanisms [12,31,36,64,65] In experimental right-sided congestive heart failure for example,crosstalk between myocardial OPRs and b-ARs has been associated with changes in the regulation of cardiac Ca2+metabolism and contractility in response to stress [64],while in cardio-myopathy, b-AR hyporesponsiveness has been attributed to excessive NO production mediated by the eNOS [27,65] Crosstalk in which ANP inhibits ET-1 secretion [36] and ET-1 conversely stimulates ANP up-regulation [37] has also been implicated as a cause of chronic congestive heart failure Furthermore,under desensitization conditions, cardiac G-protein-coupled receptor may be engaged in complex crosstalk in which the activation of any one of them may induce desensitization of the other,employing a pathway involving AC,PKC and PTKs [66] It remains to

be ascertained however,whether such manifestations are of any practical significance for human disease conditions The second category of cardiac diseases comprises disorders,such as left ventricular dysfunction from valvular heart diseases or hypertension that may be triggered as a result of a defect in the global circulatory function Naturally,these diseases often involve both local and global malfunctions in cardiovascular regulatory pathways as in valvular heart diseases,in which a concomitant down-regulation of b-ARs,up-down-regulation in a2-ARs and GRKs 2,

3 and 5 occur both in the myocardium and peripheral blood circulation [67–69] Furthermore,in disorders underlying environmental interactions with genetic factors,such as hypertension,the role of receptor crosstalk becomes even