Eur J Biochem 269, 4713–4730 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03181.x REVIEW ARTICLE 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-aldosterone 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-adrenoceptors, 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 cardiovascular 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 phospholipases, 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 different 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 controlling the contractile apparatus, blood pressure and circulating volume, both in normal physiology and in disease INTRODUCTION 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 synergistically 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 regulatory mechanisms involve vasodilators such as the kinins, The cardiovascular circulatory function constitutes a very sophisticated network of several highly synchronized circuits 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 Keywords: receptor crosstalk; heart; vasculature; regulatory systems; subcellular; contractile function; G-proteins; heart failure; hypertension; hypertrophy; signal transduction 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 Ca2+ 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-HT4, 5-hydroxytryptamine; ICa, calcium current; IK, inward rectifying K+ current; GC, guanylate cyclase; iNOS, inducible nitric oxide synthase; InsP3, 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; PLA2, phospholipase A2; 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, reninangiotensin aldosterone system; RPIA, N6-phenylisopropyladenosine; VECs, vascular endothelial cells; VSM, vascular smooth muscle (Received 12 March 2002, revised 29 June 2002, accepted 14 August 2002) Ó FEBS 2002 4714 N Dzimiri (Eur J Biochem 269) 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 monophosphate (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 metabolism [1,2] Regulation of the blood pressure involves both systemic mechanisms and local angiotensin receptormediated actions of Ang II on the renin-angiotensinaldesterone 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 cleaving phosphatidylcholine; (d) phospholipase A2 synthesizing 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 activation 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 cGMPdependent protein kinase-mediated cellular actions [5] Apart from the aforementioned pathways, the cardiovascular system maintains several other receptor systems, particularly the a1-ARs [2], adenosine (ADO) [7], endothelin (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 CARDIOVASCULAR RECEPTOR CROSSTALK SIGNALLING 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 pharmacological 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 epinephrine, 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 example, stimulation of a1-AR inhibits b-AR-mediated cAMP accumulation, presumably by coupling to the Gi protein, 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 mechanisms controlling cardiac contractile function under physiological 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 uncoupling 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 observation of elevated GRK2 activity in these animals stimulated the notion that down-regulation of b-ARs may cause an elevation in a1-AR levels It is evident that under experimental conditions, the a1- and b1-AR pathways Ó FEBS 2002 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 mechanisms: 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 regulation [17] Furthermore, in rat-1 fibroblasts, activation of ET-1 receptors was found to induce a1A-AR phosphorylation, 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] Growing evidence demonstrates that stimulation of these pathways 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 nonadrenergic 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-dependent 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) production [26–28] or OP2 receptors [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-ARstimulated AC activity via the Gi protein 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 neuromodulation [23] Accordingly, evoked neuromodulation Cardiovascular receptor crosstalk (Eur J Biochem 269) 4715 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 hydroxylase and dopamine b-hydroxylase 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 cGMPmediated 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 cardiomyocytes, 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 vasoactive 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 accumulation and ANP release [32], but seems to impair ANPmediated 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 Gi and Gq in addition to MAPK activation [37] The crosstalk among the vasoactive pathways occurs at two distinct levels: the 4716 N Dzimiri (Eur J Biochem 269) 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 membrane 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 Gbc plays an important role in the inhibitory synaptic transmission [3] The activation of the Na+ pump and voltage-dependent [K+] mediates smooth muscle hyperpolarization 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 knowledge about receptor crosstalk with potential relevance for the physiological regulation of circulatory function Interestingly, although this pool of interactions among cardiovascular systems appears to be congested and not very transparent, it is regulated by just a couple of G proteins, protein kinases and signalling junctions LONG-TERM AND SHORT-TERM EFFECTS OF CARDIOVASCULAR RECEPTOR CROSSTALK 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 shortterm and long-term responses may ensue 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 coordination 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 Ó FEBS 2002 cardiovascular system of at least 16 a, 11 b and 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 components clearly endows the heart with an enormous potential to assemble numerous signalling products to regulate intracellular Ca2+ turnover, and therefore positive inotropism, as well as vasoconstriction and vasodilatation However, despite the diversity in the multiple signalling systems engaged in the regulation of cardiovascular function, 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 accumulation 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 signalling and gene expression by coupling to both Gi and Gq proteins [36,37] In these interactions, the Gi appears to couple negatively, while the Gq may 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 NEstimulated 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 Gi protein [44], and to some extent by the bc complex of the Gq protein [45–47] Some phosphoinositide 3-kinase (PI3K) isoforms may also Ó FEBS 2002 Cardiovascular receptor crosstalk (Eur J Biochem 269) 4717 Fig 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 b1-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 Ca2+, and consequently positive inotropic effects (PIE) Alternatively, a change in the transcriptional regulation of certain genes or malfunctional signal transduction, such as PKA-mediated b1-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 Gbc-mediated crosstalk between the MAP kinases and Gi/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 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 communication 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 vasoconstrictory effects involving crosstalk between ET-1, ETB and 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 components of this complex machinery Nonetheless, cardiovascular 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 possesses 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 4718 N Dzimiri (Eur J Biochem 269) Ó FEBS 2002 Fig Regulation of the contractile apparatus through receptor crosstalk signalling Cardiovascular crosstalk engages mainly three G protein families: the stimulatory (Gs), inhibitory (Gi/o) and Gq/11 proteins, employing PLC as a central hub in regulating this crosstalk signalling In the regulation of positive inotropism, for example, the Gs-coupled b1-AR pathway constitutes the major stimulator of the contractile apparatus The Gq-coupled PLC/InsP3 (IP3) or PLC/DAG/Ca2+ pathways provide reserve pathways that can be mobilized, if and when needed All of these pathways may be inhibited by Gi/o-coupled signalling, via the muscarinic cholinergic pathway, for example, either through their inhibition AC function or via the InsP3 or DAG pathways to furnish a feedback loop in order to intermittently quench the otherwise immutable b1-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 Ca2+ channel; PLB, phospholamban; R, receptor; Ry, ryadine receptor; X, crosstalk regulatory switch; V, voltage gated Ca2+ channel 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 apparatus by their effects on PLC-b, leading to InsP3 synthesis 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 essential 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 cardiovascular 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 themselves 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 mechanism of the contractile apparatus This can be accomplished in several ways or through a combination of factors, including receptor internalization, dephosphorylation, degradation 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 b1AR-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 Ó FEBS 2002 Cardiovascular receptor crosstalk (Eur J Biochem 269) 4719 Fig 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, 2+ L-arginine; L, L-type Ca channel; V, voltage gated Ca2+ 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 receptormediated 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 probably constitute a feedback loop for controlling cAMPdependent 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 particularly the b1-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+ handling, 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 Gi in 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 contractile 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+]i transients and Ó FEBS 2002 4720 N Dzimiri (Eur J Biochem 269) Table The major human cardiac receptors and their circulatory function-related signalling products The table summarizes the circulatory functionrelated 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 a1A, a1D a2B Heart, blood vessels heart VSM, myocardial contraction Vasoconstriction Gq/11 Gi/o PLC-mediated [Ca2+]v activation [2], inhibition of cAMP accumulation [10] Ca2+-dependent [K+] activation [Ca2+]v inhibition [2] b-AR b1 b2 Myocardium Cardiac chambers PIE Heart rate control; VSM relaxation Gs Gs; Gq Gi/o AC-mediated cAMP synthesis [1,2] AC-mediated cAMP synthesis [2,55] AC inhibition [2,87] ADO A1 A2A A2B A3 Brain, heart Pacemaker VSM, brain Heart, kidney Bradycardia Vasodilatation VSM relaxation A1 modulation Gi/o Gs; G15 Gq/11? Gi3 Gq/11 AC inhibition; [K]i opening [7] Stimulation of AC and PLC-b [7] PLC/AC-mediated Ca2+ activation [7] AC inhibition [7] PLC/InsP3-mediated Ca2+ increase [7] ANG AT1R VSM Heart, aorta, kidney Heart, adrenal medulla VSM contraction Gq/11 PLC/PKC-mediated elevation of [Ca2+]i level [4] Vasodilatation; apoptosis promotion Gia2/Gia3 Ppase-stimulated MAPK activation; [K+]D opening; PTPase activation leading to T-type [Ca2+] closure [4] Vasoconstriction; PIE Vasodilatation/vasoconstriction (ETB2) Gq/11; (Gs?) Gi Gq/11 PLC/InsP3-mediated Ca2+ influx; Activation of cAMP synthesis [8] Inhibition of cAMP formation [8] PLC-mediated phosphoinositol-stimulated [Ca2+]i elevation/Ca2+ influx [8] Gi/o Gbc Gq/11 [K+]D opening; AC and [Ca2+] inhibition [3,40,45] GC AT2R ET ETA ETB VSM (blood vessel, heart) VECs, heart M2 Heart, brain M3 VSM, brain Decrease in heart rate, force (NIE) VSM contraction ANP BNP Kidney, myocardium Ventricle Blood volume, BP regulation BP reduction NOS eNOS VSM endothelium; Vasodilator tone in BP regulation GC cGMP-mediated NO actions [26], Ca2+/ calmodulin activation [26,28] OP OP1 Neocortex, vas differens Caudate putamen Central CV regulation Gia1/Gia3 (Goa2) Go AC inhibition [9]; inhibition of b-AR function [29,30] MR NP OP3 Central CV regulation 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 protein phosphatase (PP1) and structural restriction of PKA diffusion by its specific anchoring proteins have been proposed as candidate mechanisms underlying this compartmentalization [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 GC PLC-mediated InsP3/Ca2+, DAG/PKC activity; cAMP elevation [3,52] cGMP-dependent PKG inhibition of cardiac growth and function [5,6] cGMP-dependent PKG inhibition of VEGF synthesis and function [5,6] IK conductance activation; reduction in neuronal ICa; AC inhibition [9,80] 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 Gi signalling may be important in disease manifestation 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/o inhibits AC activity, the signalling of a1-AR, A2A-ADO, A3-ADO, M1- and M2-MR via the Gq theoretically promotes conditions for PIE due to their ability to mobilize Ca2+ (Table 2) These Ó FEBS 2002 Cardiovascular receptor crosstalk (Eur J Biochem 269) 4721 Table 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] a1-AR Gi-coupled a1B-AR stimulation b-AR agonist (IPN) Potentiates PE-mediated protein synthesis; Ca2+-dependent PKC-a-mediated early gene expression in neonatal heart [83] 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 a1B-AR overexpressing mice [13,14] Attenuates a1-AR mRNA and stimulated c-fos induction [17] Phosphorylates a1B in Rat-1 fibroblasts via PKC and PTKs [18] Synergistic with IPN, activates protein synthesis via Raf/MAPK pathways in cultured myocytes in neonatal rats [11]; Gi-coupled inhibition of b-AR-activated chloride current [39] Inhibits b1-AR stimulated cAMP accumulation [10] Inhibits cardiac b1-AR responsiveness via PKC activation and density in transgenic mouse [23–25]; Inhibits [K+]; stimulates NE release, NE transporter and [Ca2+] [23,24] Inhibits IPN-stimulated b1-AR in transgenic mouse [24], Stimulates [K+] via Gi coupling to PP2A/PLA2; stimulates NE-transporter and transcription activity [23] Inhibits pericardial cell IPN-stimulated cAMP accumulation [20] Inhibits b-AR PIE and chronotropy in transgenic mice and heart failure [26,65] and NE mitogenic effects in ventricular cells [27] Induces b1-AR hyporesponsiveness in cardiomyopathy [28] Decreased inhibition of IPN-stimulated AC activity [66] Increases IPN-stimulated AC in adult rat cardiomyocytes [66] Inhibits NE release, b1-AR contractility [76] Inhibits NE-mediated b1-AR effects in rat ventricular myocytes via a PTX-sensitive Gi/o protein [29,30] Attenuates IPN-induced cAMP accumulation in cardiac smooth muscle [20] Parasympathetic slowing of heart rate; inhibits b1-AR contractility (via Gi?) [3,28,45] Decreased AC inhibition by RPIA in adult rat myocytes [66] b1-AR Ang II via AT1R ET-1 via ETA a1-AR agonist (PE) Gi-coupled a1-AR (NE) Ang II on (neuronal) AT1R AT1R -antagonists Ang II-stimulated AT2R ET-1 NO via eNOS NO via iNOS RPIA-desensitized A1 Carbachol-stimulated M2 OP1 agonists A1 AT1R AT2R ETA ANP BNP M2 ENOS ETA-stimulated ET-1 M2 stimulation Isoproterenol-desensitized b-AR Ach-desensitized M2 A2a ANP via cGMP ET-1 NO ANP via ANPR ET-1 stimulated ETA Ang II via AT1R ANP Isoproterenol-activated b-AR NO a1-AR agonist (PE) b-AR agonists (IPN) Ang II-activated AT1R eNOS ET-1-activated ETA M1/M2 stimulation OP2 agonists ET-1-mediated activity Isoproterenol-desensitized b-AR Oxymetazoline on a2-AR ET-1 stimulated ETA, ETB M2 agonists Decreased inhibitory action of RPIA on AC [66] Counteracts A1-meditaed antiadrenergic actions [22] Inhibit ET-1 secretion following Ang II-mediated AT stimulation in porcine aorta [71]; inhibits RAS activation [70] Stimulates RAS system synergistically with Ang II [59] Up-regulation of AT1R by inhibition of NO synthesis [34] Increases ACE levels via cGMP stimulation, vasorelaxation [98] Synergistically activates Raf-1 kinase, MAPK in myocytes [8] Induces endothelial ET-1 release associated with hypertensive-induced hypertrophy [35] Counteracts ET-1 activation of AP-1 via cGMP pathway in dogs with congestive heart failure [36] Attenuation of ET-1-induced MAPK activity [21] Inhibits ET synthesis via cGMP synthesis [52] Stimulates ANP synthesis and release [6,16] Stimulates ANP transcription via Akt [15] Attenuates ANP-mediated AC inhibition via KPC in VCM [33]; stimulates ANP release via inositol phosphate/Ca2+ activation [32] Up-regulates ANP in eNOS deficient mice [26] Activates Gq-mediated Ca2+-stimulated ANP synthesis in rat myocytes [37] ANP release in response to volume expansion (via Gq) [6] Releases ANP in hypertensive patients [92] Increases BNP gene expression in porcine aorta [71] Increased AC inhibition by carbachol in rat myocytes [66] Induces endothelial ET-1-mediated contraction to Ach [19] Induce NO synthesis in endothelial cells, counterbalances NO vasodilator tone [8] Stimulates NO synthesis [28] 4722 N Dzimiri (Eur J Biochem 269) overtly antagonistic events imply that the ability to concomitantly couple to PLC via the Gi and Gq pathways 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 feedback 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 functions 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 glomerular 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 vasodilatation in regulating circulating volume, cardiovascular hemodynamics and blood pressure While the involvement of the crosstalk among these vasoactive receptor systems seems unequivocal as a physiological regulatory control for circulatory 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 categories 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 metabolism 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 Ó FEBS 2002 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 overexpression 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 Gi protein 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 underscores 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 contributory 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 cardiomyopathy, 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 downregulation of b-ARs, up-regulation in a2-ARs and GRKs 2, and 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 Ó FEBS 2002 more complex and less discernible Currently, crosstalk among basically all signalling pathways regulating both vascular reactivity, such as the ETR, ATR, NOS, and contractile function such as the b-ARs, has been implicated in the development of experimental hypertension [53] However, evidence for the role of these receptor systems in human hypertension is scanty and often contradictory In recent years, attention has also focussed on the potential role of the deleterious vascular effects of endogeneous ET-1 and its crosstalk with other vasoactive hormones in hypertension Some studies have suggested that these effects can be accentuated by reduced NO generation as a result of hypertensive endothelial dysfunction [36,53] A defect in the ET-derived NO signalling presumably triggers abnormal response to Ach in hypertensive vessels, and may at least in part account for the increased vascular resistance observed in hypertension [33] On the other hand, despite persuasive evidence suggesting that ET-1 contributes to the adverse effects of hypertension-induced cardiac and vascular remodeling, as well as hypertensive renal damage [53], some studies show that its generation and responsiveness remains unaltered in hypertensive subjects In contrast, animal models of early stage heart failure suggest that the inhibition of endogenous ET-1 by ANP may play a critical role in its inhibition of RAS and maintenance of renal function, partly by counteracting neurohormone-induced vasoconstriction [36,70,71] Another potentially important crosstalk includes a complex interaction between ET, Ang II, a-AR agonists, Ca2+ and growth factors, which has been implicated in the pathogenesis of hypertension-induced vascular hypertrophy [53] The third class of cardiovascular disorders, the coronary artery diseases, is a sequel of vascular malfunctions leading to the blockade of coronary circulation resulting in inadequate supply of oxygen and nutrients to the heart itself, and therefore causing ischemia It appears that these events involve the interaction of the same vasoactive substances particularly the a-AR, ATR and ETR agonists that play important modulatory or initiator roles in the development of hypertension, arteriosclerosis and apoptosis [53,61,72,73] An imbalance in the crosstalk between ET-1 and NO has also been associated with atherosclerosis, hypoxia and ischemia [53,74] In contrast to disease manifestation, crosstalk among various other vascular signalling pathways has also been associated with ischemic preconditioning (PC), a protective mechanism against ischemic injury with immediate and delayed protective effects These mechanisms involve complex second messenger pathways comprising components such as adenosine, ADO subtypes, PKC-e, mitochondrial ATP-sensitive [K+], eNOS and paradoxical protective role of oxygen radicals [75–78] The ADO-mediated PC probably involves the activation and translocation of PKC-e to mitochondrial membranes leading to increased ATP-sensitive [K+] opening and possibly stimulation of the MAPK pathway through a yet unknown mechanism [79] In contrast, enhanced synthesis of NO by eNOS is thought to play a dual role, acting initially as the trigger and subsequently as the mediator of the condition defined as late PC [77] Other potential mechanism of PC include possible neuromodulatory crosstalk of M2-MR with the b-AR pathway through presynaptic inhibition of NE release in guinea pig hearts, without the involvement of reflex vagal activity in rat hearts, or ET-1 cross talk with the Cardiovascular receptor crosstalk (Eur J Biochem 269) 4723 NO pathway leading to cholinergic vasodilatation [75–77] In the rat myocardium, both PKC and a subpopulation of Gi/o proteins may be involved in OP1 and A1-ADOmediated ischemic preconditioning and protection against apoptosis [80] In diseases with an element of pressure overload, such as hypertension or valvular heart diseases, the heart usually responds to increased hemodynamic load by hypertrophic growth, or remodeling of myocytes and the extracellular matrix [60] The hypertrophic increase in the left ventricular mass occurs as a pathologic consequence of the disease, or as an adaptive compensatory mechanism to reduce systolic wall stress on the left ventricle as in hypertension These changes are characterized by a series of sequential gene activating events resulting in elevated protein synthesis, a more regular cell shape, and organization of the contractile proteins into sarcomeric units [81] The most important feature of left ventricular overload-induced hypertrophy is the Ôswitching onÕ of immediate early genes, particularly c-fos, c-jun and c-myc, all of which are transcription factors activated by MAPK-dependent phosphorylation [81–83] At the onset, homodimerization of Jun/Fos or Jun/Fra (collectively known as activator protein-1) allows them to induce RNA polymerase II activity, followed by the reactivation of genes such as the ANP, skeletal a-actin and b-myosin heavy chain (b-MHC) that are normally expressed during the fetal and early neonatal periods, but are quiescent in the adult myocardium [60,81] This in turn triggers the activation of some constitutively expressed structural genes, e.g cardiac a-actin and myosin light chain type 2, encoding the signalling products contributory to the hypertrophic structural transformations [60] Although the initiators of these sequelae of events remain unclear, several biochemical and mechanical stimuli, including the a1-AR and b1-AR agonists, Ang II, ET, tumor-promoting phorbol esters, growth factors and mechanical stress are capable of triggering this transcriptional cascade linked in a temporal sequence by a variety of mechanisms [12,71,82] Stimulation of these pathways induces cardiac hypertrophy by promoting individually or synergistically protein synthesis and immediate early gene expression via the MAPK pathways [12,81,82] Besides, complex interactions between ET, ANG II, a-AR agonists, Ca2+ and growth factors as well as transcriptional regulation of G-protein-coupled receptors by other pathways are also common features of experimental cellular hypertrophy [83,84] These interactions are primarily products of their activating PKs to turn on the Ras-GTP complex and its downstream effectors, raf-1 and MAPKs [81,82] possibly involving the PLC-b signalling via PKC-e in crosstalk with phospholipase D [84] As per current notion, cardiac hypertrophy may result from stimulation of the Gq-coupled receptors, such as AT1R, in cardiac myocytes triggering rapid Tyr phosphorylation of Shc protein and its association with the adapter protein Grb2 and mSos-1, a GEF of p21ras [84] to promote the translocation of mSos-1 to the membrane, in order to interact with MAPK or other pathways Mechanical stress may also lead to cardiac hypertrophy by triggering the transcriptional activation of genes contributing to compensatory responses via the stressregulated p38MAPK pathway [85] The need for the heart to maintain a tightly controlled, reliable signalling machinery becomes even more important in disease in order to fend off potentially fatal consequences 4724 N Dzimiri (Eur J Biochem 269) of a defect or total loss of the contractile function to enable it sustain its essential task The heart can fulfill this by either maintaining some reserve pathway(s) that can always be tapped in need, or by inducing novel adaptive signalling schemes One of the most intriguing manifestations of contractile function is the fact that the heart muscle tissue concurrently maintains b2-AR and a1-AR, both of which have been regarded to be physiologically dormant, yet they can be stimulated by the same pharmacological agents leading to similar or counteractive signalling products as those of b1-AR activation [12,13,53,54] The observation of a causal relationship between the attenuation in b1-AR and/ or uncoupling of b2-AR with an up-regulation in a1-AR in both experimental heart failure and in human disease strongly argues for the existence of a1-AR as a purposeful and productive resource for adaptive regulation of contractility in response to cardiac disease Although there is hardly any direct evidence for such a mechanism at present, the logical explanation for this association is the existence of a signalling switch to turn on the a-AR in response to a malfunctioning b1-AR pathway Moreover, the overwhelming notion seems to suggest that stimulation of b2-AR is devoid of the toxic effects associated with stimulation of the b1-AR subtype in the progression of cardiac disease to endstage [54,86,87] Put together, it appears that selective activation of cardiac b2-AR in presence of a defunct b1-AR pathway may provide catecholamine-dependent inotropic support without cardiotoxic consequences, which might have beneficial effects in the failing heart [54,58] Basically, in order to compensate for a defunct b1-AR, the mobilization of Ca2+ required for the PIE is attainable most probably by a mechanism that bypasses the Gs-AC pathway, and signalling via PLC is one important option for providing such an alternative mechanism Similar pathways may be utilized by the b2-AR, which has been shown to be capable of bypassing the L-type Ca2+ channel in its modulation of cellular contraction [87] Alone, the diversity in the mode by which the b-AR pathways regulate Ca2+ metabolism is already evident at the G protein coupling circuit At this junction, coupling to at least the Gq can mediate PIE as an alternative to that attained via the Gs proteins As discussed above, the Gq-coupled activation of myocardial a1-ARs, ETRs and ATRs results in the accelerated production of InsP3 and DAG from phosphoinositol hydrolysis, both of which directly or indirectly lead to an elevation of [Ca2+]i and consequently, cardiac inotropy regulation in vitro at least (Fig 2) Therefore, the coexistence of these potent PIE providers in the heart, without displaying any evident involvement in cardiac physiologic function, is a clear lead for their potential as resources that can be availed, if required to support a defunct or ailing contractile apparatus A mechanism can be envisaged in which an inefficient b1-AR signalling itself may utilize the Gq protein as a switch to turn such reserve pathways on and off in cardiac disease in case of demand The existence of various isoforms and subtypes of major G-protein coupled receptor downstream signalling components furnishes the heart with a sizeable resource of alternative signalling combinations in cardiac disease At least four stimulatory Gsa gene splice variants and two Gia subunits are involved in the regulation of the cardiac AC signalling [88] In the heart, the Gsa is abundantly expressed, while only the Gia2 subtype is predominant, and probably Ó FEBS 2002 constitutes a primary mediator in the inhibition of the b1-AR-stimulated AC activity through various routes [88] In cardiac myocytes where AC V and AC VI are most abundant, inhibition of their catalytic activity results primarily from the activation of L-type Ca2+ channels [89,90] Incidentally, this is the same group of ACs that has Gsa as their activator, the Gia and Ca2+ as inhibitors, while the bc complex appears not to partake in their signalling [89] It has been argued that the Gsa and Goa not inhibit certain ACs directly, because this effect would be antagonistic to those of their analogous Gbc subunits [89] In addition, individual AC isoforms particularly the AC I, AC III and AC VIII can also be regulated by Ca2+ directly, Ca2+/CaM mechanism, or through phosphorylation processes via PKA [90,91] These Ca2+ effects present an opportunity to cross-regulate receptor-stimulated AC activity without the involvement of Gs protein, PKC or calmodulin, and can be utilized by those pathways bypassing or devoid of the Gs-mediated systems These are but a handful of potential interregulatory mechanisms between AC, Ca2+ and G proteins, which may be engaged in the crosstalk among different receptor subtypes in the management of PIE in heart disease In addition, some receptor proteins, such as ANPR, have been shown to undergo transcriptional and post-translational regulation by stimulators of other signalling systems in the progression of particularly left ventricular hypertrophy and hypertension [15–18,92] Such modulation of one cardiac gene by the expression of another strongly suggests that regulators of both transcription and post-translation mediate various forms of adaptive crosstalk in response to cardiac disease A further example is the down-regulation of medium-chain acyl-CoA dehydrogenase (MCAD) transcription in the hypertrophied but lacking in the nonfailing ventricle, immediately following the manifestation of pressure overload [62], also indicative of a compensatory control at the translational or post-translational level Thus, there exists a huge contingency of potential regulators of cardiac receptor signalling at various stages of signal generation to furnish the heart with the capability to adapt itself to altered functional conditions in the most appropriate fashion in disease Implications of receptor crosstalk for pharmacological treatment of cardiac disease Traditionally, the search for the most appropriate therapeutic management of heart failure have focused mainly on reducing the inotropic load to improve or maintain sufficient left ventricular function, typically by blocking the b-AR activity For quite a long time, the underlying cardioprotective mechanism of this approach was associated with the ability of such b-AR blockage to prevent the deleterious consequences of cardiac sympathetic stimulation However, despite the fact that b-AR antagonist(b-blocker-) induced reduction in heart rate may essentially contribute to benefits, such as improving myocardial energy balance, by generating a less negative force-frequency relationship [93], there is no reason to believe that these agents are more effective than one exerting NIE in slowing down the cardiac pumping rate Moreover, b-blocker therapy has been associated with several untoward effects, such as augmenting plasma ANP, BNP and cGMP levels Ó FEBS 2002 [94] It may also provoke other secondary effects, such as atrial 5-hydroxytryptamine (5-HT4) receptor hyperresponsiveness or enhanced serotonin-evoked increases in cAMP levels, as a result of modified crosstalk between the 5-HT4, b-AR and MRs [95] More importantly, neither heart failure nor cardiac hypertrophy constitutes a disease, but both simply indicate the existence of advanced or end-stage cardiac disease It is not even certain whether or not either of them can directly influence the functional expression of cardiac signalling pathways For this reason, it has become increasingly apparent that understanding of the actual underlying cause of the disease is a prerequisite for both prevention and effective therapeutic management of cardiovascular diseases Until these mechanisms are elucidated, therapeutic strategies will continue to be directed mainly at containing these end-stage manifestations rather than combating the underlying cause of the disease It is nonetheless now evident that in the presence of a primary disorder of myocardial contractility and/or extraordinary pressure on the heart, ventricular performance depends on several compensatory mechanisms These mechanisms include among others, the Frank-Starling phenomenon, reflex release of cardiac hormones such as the ANP, vagal innervation of the atria, and sustention of both the parasympathetic and sympathetic functions in heart failure [96] It is also well established that neurohumoral activation of the heart in form of increased sympathetic, RAS, vasopressin or ANP activity may contribute to the transition from left ventricular dysfunction to clinical heart failure, and is an independent predictor of poor prognosis [97] Thus, while these compensatory mechanisms provide circulatory support in patients with acute heart failure, neurohumoral activation over an extended period of time might be harmful to patients with chronic congestive heart failure, as several neurohumoral factors may be inappropriately activated [96] Moreover, in providing inotropic support, agonists of different pathways affect the contractile function in various ways, a fact that may be important in the treatment of heart failure For example, selective partial b1-AR agonists and phosphodiesterase III inhibitors are thought to cause relatively limited accumulation of cAMP for a given PIE than does isoprenaline, while stimulation of a-AR or ETRs can enhance myocardial contractility by enhancing the Ca2+ responsiveness of the myofilaments [54] It is also believed that b-AR stimulation results in a proportionally large increase in [Ca2+]i transients compared to other PIE providers, due to additional cAMP-dependent Ca2+-desensitizing effects on the myofibrils [98] This differential regulation of Ca2+ by these receptors might provide a useful therapeutic strategy, in cases where offloading or reduction in the inotropic load may be necessary As a result, new therapeutic concepts are emerging to inhibit the potentially deleterious consequences of the compensatory mechanisms and to indirectly influence signalling pathways using agonists/antagonists of different receptor systems A classical example is the introduction of ACE inhibitors in the treatment of congestive heart failure, based on the notion that pharmacological interventions with the myocardial RAS significantly causes reversal of local sympathetic neuroeffector defects [96,97,99] It is thought that the administration of ACE inhibitors increases cardiac and peripheral b2-AR levels as well as improving prognosis and Cardiovascular receptor crosstalk (Eur J Biochem 269) 4725 cardiac function [100–102] This prompted the notion that the ACE inhibitors prevent b-AR down-regulation or alternatively increase b-AR up-regulation by inhibiting the Ang II-mediated catecholamine release via AT1R stimulation in heart failure Unfortunately, the numerous endeavors to explain these therapeutic approaches based on the sympathetic actions of these signalling pathways have yielded no tangible mechanistic explanation for the rationale behind these strategies Some studies have also postulated that the antihypertensive actions of ACE inhibitors, AT1R and b2-AR antagonists are partly due to their ability to modulate the expression and function of other vascular receptor systems, such as the NPs and bradykinin [70,103,104] The interchangeability of the inhibition of the b-AR and the RAS pathways in the treatment of both heart failure and hypertension intuitively demonstrates the essence of their crosstalk in controlling circulatory function Apart from these interactions, crosstalk amongst AR subtypes, and with other pathways such as the NP or ADO, also lends itself attractive as therapeutic target in heart failure The crosstalk among AR subtypes may prove to be the therapy of choice in those disorders exhibiting b-AR down-regulation in association with a-AR upregulation, as these processes rapidly revert to normal following the correction of the disease [105] We recently demonstrated the therapeutic potential of this approach by successfully administering phentolamine as a last resort in containing persistent infundibular obstruction unresponsive to classical therapeutic regiments, possibly as a result of tenaciously elevated a-AR activity, following successful balloon valvuloplasty of severe pulmonary stenosis [106] The influence of the AR agonists on the NP pathways may also be important in the treatment of heart failure, as b-blockers can modulate crosstalk between these signalling pathways, even in the absence of other variables such as hypertension or left ventricular hypertrophy Studies in animal disease models have provided important leads for the relevance of crosstalk in therapeutic management of heart failure, hypertension and ischemic heart diseases One of the most well studied pathways is crosstalk between A2A-ADO and A1-ADO [22], which is thought to be the basis for the proadrenergic actions of the former In rats treated with an A1-ADO antagonist, prejunctional a2-ARs become supersensitive to their selective agonists accompanied by an increase in plasma renin Furthermore, captopril prevents the development of hypertension and morphological changes in the arteries, and prejunctional b-AR-mediated NE release is enhanced, thus altering the adrenergic regulation of the cardiac function [23] These observations suggest that selectively targeting A1- or A2A-ADO may be useful either to protect the failing heart from chronic, excessive adrenergic stimulation or to potentiate the inotropic response to such stimulation when clinically indicated The antiadrenergic properties of A1-ADO may also offer protection against injury from myocardial ischemia and reperfusion [22] In contrast, crosstalk between A1- and A2A-ADO suggests a therapeutic benefit of A2A-antagonists in situations where enhanced antiadrenergic response is desirable, such as acute myocardial ischemia [22] This lends the possibility to potentiate the anti-ischemic effects of A1-ADO through selective manipulation of the A2A-ADO The clinical implications of the proadrenergic actions of A2A-ADO are not limited to 4726 N Dzimiri (Eur J Biochem 269) Ó FEBS 2002 Fig Potential therapeutic targets involving crosstalk signalling in cardiovascular disease The changes in progression of cardiac disease to end-stage heart failure, such as cardiac hypertrophy, are often a results of long-term signalling effects The mechanisms underlie partly the change in intracellular messengers triggering the activation of transcription factors (TFs) and their translocation into the nucleus, where they bind to their target promoters A constructive interaction of the TF with the promoter induces transcription of an appropriate gene and consequently, synthesis of the corresponding mRNA, whose message is translated into the corresponding protein In this process, crosstalk can be regulated at several hotspots, leading to diverse signalling messages These hotspots lend themselves useful therapeutic targets for the future The figure shows some examples of such potential therapeutic targets at some of these hotspots H, hormone (ligand); E, enzyme, G, G protein; RTK, receptor tyrosine kinase myocardial ischemia and heart failure, but may also be important in the treatment of cardiac arrhythmias, whereby A2A-selective antagonists may enhance the actions of adenosine in terminating supraventricular arrhythmias Furthermore, both MR expression and cardiac cellular responsiveness to muscarinic stimuli have been shown to increase significantly in hypoxic myocytes exposed to epinephrine, while in contrast, MR agonists may reduce NE overflow in animal in situ heart preparations [77] These observations also point to potential pharmacological strategies in the prevention of myocardial ischemic injury by influencing the cholinergic system The fact that crosstalk takes place at several junctions of the signalling pathways bespeaks the prospects of various potential strategies for the treatment of cardiovascular diseases in future Also, the indication that hypertrophic adaptive mechanisms are regulated at the transcriptional level renders the components of these processes valuable as potential therapeutic targets in the prevention of, for example, cardiac hypertrophy Future strategies will therefore have to focus on preventing or suppressing the initiators of injury or disease, rather than heart failure or cardiac hypertrophy emanating from these diseases Such efforts are likely to concentrate primarily on developing agents exerting their influence at four major signalling junctions: (a) receptor–G protein–effector circuit; (b) activators of tran- scription factors (TFs); (c) regulators of transcription; and (d) regulators of protein translational function (Fig 4) While the underlying basis for these crosstalk mechanisms remains largely unknown, it is nonetheless bound to occupy an increasingly central position in these therapeutic approaches in the foreseeable future SUMMARY AND PERSPECTIVES The above discussion clearly demonstrates the fact that cardiovascular receptor signalling is a maze comprising several interlocked receptor pathways that are tightly coordinated and perfectly synchronized to ensure harmony, integrity and continuity of this vital function throughout life The signalling pathways involved in the regulation of this function are extremely diverse and range from direct programs, such as the AR and AT pathways, to multistep ones, such as the PKA and Ras/MAPK pathways The most important question remains how the various pathways communicate with one another to execute this noble function Currently available data is concordant with the notion that cardiovascular signal transduction systems converge at certain checkpoints, probably under the humoral control of the heart itself A malfunction or alteration in the transduction of any one of these signalling pathways may positively or adversely affect Ó FEBS 2002 the signalling of another in their regulation of this function Our present knowledge also strongly testifies that the heart is indeed furnished with various yet unknown candidates to protect its circulatory function, if and whenever needed The major limitation in our endeavors to comprehend these concepts is the fact that this knowledge has been derived from studies mainly in isolated organs or cells It goes without saying that any alteration in a signalling pathway or the removal of any component from its physiological environment may alter the function of other components, or even whole systems, and so also signal transduction mechanisms, leading to new signalling products altogether Thus, while this information is very valuable, caution is nonetheless called for in interpreting its relevance to cardiovascular physiological signalling Despite this and the fact that currently there are no appropriate noninvasive procedures to verify some of these important notions, our current understanding of receptor crosstalk in cardiovascular function presents a milestone in our endeavors to comprehend the modus operandi of this complex machinery It is certainly just a matter of time before the literature is flooded with valuable pieces of this maze, which may ultimately enhance our understanding of the regulation of these signalling pathways, as a whole Two important issues are likely to dictate the focus of research in this field in the near future The first is with regard to the mechanisms coordinating the arrays of signalling messages from various sources to elicit such a perfectly synchronized physiological function Given the fragility and vital nature of the circulatory function and the potential contribution of receptor crosstalk in its maintenance, it is evident why evolution has had to endow the heart with the ability to control its own fate However, while the place of the heart as an endocrine organ seems apparent, its ability to mobilize any of the receptor systems as compensatory measure and/or adaptive mechanism(s) at its disposal remains to be elucidated Therefore, future studies are also to be directed at providing some answers to the important questions regarding the mode by which the heart may regulate these mechanisms under normal physiological and pathophysiological states It is obviously impossible to predict how long it will take to overcome these formidable tasks Nonetheless, the current pace of the advancement in our knowledge on the subject justifies the great expectation and hope to succeed in employing this knowledge to combat this major killer disease worldwide sooner rather than later ACKNOWLEDGEMENTS The preparation of this review and the work of the author cited herein were supported by the Royal Cardiovascular Research Grant of the King Faisal Specialist Hospital and Research Centre I would also like to thank Dr Abdelilah Aboussekhra for his useful suggestions and Paul Muiya for his assistance in the preparation of the manuscript Cardiovascular receptor crosstalk (Eur J Biochem 269) 4727 10 11 12 13 14 15 16 REFERENCES Dzimiri, N (1999) Regulation of b-adrenoceptor signaling in cardiac function and disease Pharmacol Rev 51, 465–502 Bylund, D.B., Bond, R.A., Clarke, D.E., Eikelberg, D.C., Hieble, J.P., Langer, S.Z., Lefkowitz, R.J., Minneman, K.P., Molinoff, P.B., Ruffolo, R.R., Strossberg, A.D & Trendelenburg, U.G 17 (1998) 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J Cell Biol 142, 523–535 86 Zhang, S.J., Cheng, H., Zhou, Y. Y., Wang,... interlocked receptor pathways that are tightly coordinated and perfectly synchronized to ensure harmony, integrity and continuity of this vital function throughout life The signalling pathways involved... omitted for clarity Inhibitory or counteractive functions are indicated as rounded arrow ends H, hormone (ligand); L, L-type Ca2+ channel; PLB, phospholamban; R, receptor; Ry, ryadine receptor; X, crosstalk