1 Institute of Anaesthesiology, University Hospital Zurich, Switzerland. 2 Institute of Pharmacology and Toxicology, University of Zurich, Switzerland
*Corresponding author: Cardiovascular Anaesthesia Laboratory, Institute of Anaesthesiology, University Hospital Zurich, Rämistrasse 100, 8091 Zurich, Switzerland. E-mail: michael.zaugg{at}usz.ch
![]() |
Abstract |
---|
Keywords: adrenergic receptor signalling; calcium, signalling; myocardial contractility; MAPK signalling; muscle, vascular smooth muscle regulation
![]() |
Introduction |
---|
![]() |
Adrenergic receptor subtype-specific signalling via G-proteins |
---|
|
ARs couple to the heterotrimeric G-protein complex (G, Gß, G
) on the inner side of the cell membrane.3 12 60 76 94 Upon activation, the G
-subunit hydrolyses guanosine triphosphate (GTP) and dissociates from the complex, leaving the ß
-subunits as undissociable heterodimer (Fig. 1). Currently there are 20 known G
, six Gß and eleven G
subunits. When activated, all three
1-ARs interact with the pertussis toxin-insensitive G
q component, which follows the main signalling route via phospholipase C (PLC) leading to diacylglycerol (DAG), producing activation of protein kinase C (PKC) and inositol trisphosphate (IP3) for the liberation of Ca2+ from the sarcoplasmic reticulum (SR). All three
2-ARs couple to the pertussis toxin-sensitive G
i, leading to inhibition of the integral membrane protein adenylyl cyclase (AC), activation of K+-channels, and inhibition of the sarcolemmal L-type Ca2+ entry channels (DHPR). This is in contrast to the ß-ARs, which are more variable in their coupling to G-proteins (Fig. 1). The ß1-AR couples almost exclusively to G
s, inducing positive inotropy (increased contractile amplitude) and positive lusitropy (enhanced relaxation). Its canonical signalling pathways involve activation of AC and protein kinase A (PKA). ß2- and ß3-ARs are both able to signal via G
s, G
i or G
q depending on the physiological or pathophysiological conditions (different states with regard to catecholamines, inflammatory cytokines and angiotensin-II). The G
s and G
q protein families have defined main effector pathways: the AC and the PLC pathways, respectively. The G
i protein family is more amorphous and its signalling flows equally through both the G
i and the Gß
complex, affecting several different downstream signalling pathways. G
q-dependent signalling by the heterodimeric Gß
to the phosphoinositol-3 kinase (PI3K) pathway was recently established in cardiac hypertrophy.20
AC is an integral membrane protein with 12 transmembrane helices and a molecular weight of 130 kDa.25 26 65 At least nine isoforms of the AC exist (AC1AC9); AC5 is specifically expressed in cardiomyocytes, whereas AC6 occurs in heart cells other than myocytes. Additional isoforms are also expressed in the heart, but to a lesser degree. In addition to its regulation by G-proteins (stimulatory G
s and inhibitory G
i), PKC further stimulates and PKA inhibits the activity of AC isoforms. Furthermore, high Ca2+ during sustained cell activity inhibits AC5 and AC6, establishing a negative feedback loop. The fine tuning in the regulation of AC is of particular significance as its activity is rate-limiting in adrenergic signal transmission. PLC is a peripheral membrane protein at the cytoplasmic side, hydrolysing phosphatidylinositol 4,5-bisphosphate (PIP2) to DAG and IP3 (Figs 2 and 3). It has an absolute requirement for Ca2+ bound to the active site and comprises three types: PLCß (150 kDa), stimulated by G-proteins; PLC
(150 kDa), stimulated by receptor tyrosine kinases; and PLC
(84 kDa), stimulated by transglutaminase-II.29 All three types are expressed in cardiomyocytes.
|
|
![]() |
Regulation of adrenergic receptor signalling |
---|
(i) Upon agonist activation, the ß2-AR couples to the G-protein heterotrimer, mainly with its third intracellular loop between the transmembrane helices TM5 and TM6 and part of the intracellular C-terminus immediately following the TM6. It couples preferentially to Gs forming a stoichiometric 1:1 complex. However, the ß2-AR may instead also couple to G
i or G
q, activating their corresponding signalling pathways (Fig. 1). The G-protein specificity is, at least in part, determined by the type of agonist.94 This implies that the agonist-induced conformation of the receptor favours the type of G-protein with which it interacts. For instance, salbutamol and ephedrine display a much higher efficacy with G
q than G
s or G
i. On the other hand, isoproterenol is more effective than salbutamol with G
i. It was recently shown that the selective ß2-AR antagonist ICI-118,551 exerts a direct negative inotropic effect by acting as a ß2-AR agonist, directing it away from coupling to G
s towards coupling to G
i.28 Furthermore, upon stimulation by catecholamines the ß2-AR interacts much faster with G
s than with G
i and G
q. This could generate intracellular signals in a timely, ordered fashion. In general, AR signalling is terminated by phosphorylation at multiple sites in the third intracellular loop and in the C-terminal region. However, PKA-dependent phosphorylation in the third loop (serines 261 and 262) and in the proximal C-terminal region (serines 345 and 346) of the ß2-AR switches its predominant coupling from the stimulatory G
s to the inhibitory G
i, thereby inhibiting the AC and promoting activation of the mitogen activated protein kinase (MAPK) signalling cascade (see next section).12 97
(ii) Signal termination is commonly referred to as desensitization, i.e. attenuation of receptor signalling despite the continued presence of a stimulus. Desensitization may occur under physiological or pharmacological stimuli as well as under pathological conditions.12 54 72 Homologous desensitization involves phosphorylation of two adjacent serines (355 and 356) in the C-terminal region of the ß2-AR by a family of G-protein-coupled receptor kinases (GRKs), whose activation does not require the production of second messengers. The six different GRK isoforms are tissue-specific. GRK2 and GRK5 (formerly referred to as ß-ARKs; 68 and 80 kDa respectively) are expressed in cardiomyocytes and display specificity for agonist-activated receptors; non-activated receptors or antagonist-bound receptors are usually not phosphorylated by GRKs. In contrast to ß1- and ß2-AR, the ß3-AR lacks phosphorylation sites and is refractory to desensitization by GRKs or PKA. The Gß subunits seem to play a role in recruiting the cytoplasmic GRKs to the membrane environment of their receptor substrates and the membrane phospholipids required for kinase activation.12 54 Heterologous desensitization occurs in the absence of agonist occupancy and is regulated by the signalling of another receptor via an intermediary second messenger. Induction of cAMP by receptor signalling leads to phosphorylation of several intracellular serines of the ß2-AR by cAMP-dependent PKA. Thus, phosphorylation by PKA turns off signalling through the receptors normal partner G
s and, at the same time, facilitates receptor coupling to the inhibitory G
i.12 21 97 Stimulation of PKC via the PLCPIP2DAG pathway also leads to phosphorylation of the receptor and its heterologous desensitization.
Desensitization is an acute response involving binding of arrestin to the phosphorylated C-terminus of the ß2-AR. Arrestin, together with the heterotetrameric adapter complex AP2, delivers the receptor to clathrin-coated pits for endocytosis to endosomes or to lysosomes. In the endosomes the ß2-AR is dephosphorylated by specific protein phosphatases. This resensitized receptor then recycles back to the cell membrane. However, fusion of the endosome with lysosomes leads to ß2-AR degradation. Desensitization is not always coupled to internalization but exhibits receptor type-specificity. For instance, under agonist stimulation 5080% of the ß2-AR internalizes within a few minutes, whereas the ß1-AR does not internalize but remains at the cell surface, even in the desensitized state. The different -ARs internalize only moderately, except for the
2A-AR, which remains at the membrane. In contrast to desensitization, downregulation denotes a chronic process, during which agonist overstimulation promotes increased receptor degradation concomitant with a reduced de novo synthesis rate that does not match the loss of receptors.12 71
(iii) Recent reports on homo- and heterodimerization between receptor subtypes suggest a potential concentration of receptor complexity that could account for previously unexpected pharmacological diversity.3 2-AR and ß2-AR may form homodimers. The ß2-AR was found to form dimers on agonist activation, and the agonist-induced homodimer seems to represent the active ß2-AR species. Dimerization is established between the sixth and seventh transmembrane domains of the two receptors involved.78 Heterodimers have been found between the
2A-AR and ß1-AR, the ß1-AR and ß2-AR, and the
2C and M3-muscarinic receptors, as well as between the ß2-AR and
-opioid receptors.3 Formation of the ß1-AR and ß2-AR heterodimer inhibits the agonist-promoted internalization of the ß2-AR and its ability to activate the MAPK cascade46 (see next section).
(iv) A GPCR-associated protein may directly mediate signalling, as in the case of the G-proteins themselves. Alternatively, a GPCR-associated protein may regulate receptor signalling by controlling receptor localization and/or trafficking, e.g. by internalization. Finally, a GPCR-associated protein may act as a scaffold, physically linking the receptor to various effectors. Scaffold proteins are defined as proteins that associate with two or more partners to enhance the efficiency and/or specificity of cellular signalling pathways. The family of PKA-anchoring proteins (AKAPs) was one of the first to be recognized as scaffold proteins.30 85 87 Two AKAPs, AKAP250, also known as gravin, and AKAP79, were found to interact with the C-terminus of the ß2-AR.
![]() |
Relation of adrenergic to global cardiomyocyte signalling |
---|
G-proteins are also able to activate the monomeric GTPase Ras via different phosphorelay systems. Ras represents a master switch in transferring extracellular signals for growth and differentiation via the four MAPK cascades to the nucleus (Fig. 2).33 37 53 55 64 Among these four cascades, (i) ERKs (extracellular signal-regulated kinases) are activated by growth factors and regulate cardiac hypertrophy and apoptosis, (ii) the more recently characterized big MAP kinase-1 (BMK1, also called ERK5) pathway transmits oxidative stress signals to the cell nucleus, (iii) the four p38 MAPK isoforms (, ß,
and
) are activated by cytokines and environmental stress and are also involved in regulation of apoptosis, and (iv) the JNKs (c-Jun N-terminal kinases) are critical regulators of transcription. Specificity of AR subtype signalling may be achieved by proteins that do not have intrinsic catalytic activity but serve as adapter and anchoring proteins. By keeping the reaction partners of a particular signalling pathway in close proximity to the effector site, they form so-called signalling modules. Such a signalling module of activated PKC
with ERK has been demonstrated to be operative in mitochondria, where it induces cardioprotection.5 Another recent example is the conserved sequential MAPK cascade RafMEKERK, in which the two upstream kinases of the module, Raf and MEK, remain cytoplasmic. In resting cells, ERK is anchored to MEK, whereas upon activation it rapidly detaches and translocates to the nucleus.73 A similarly complex signalling network with redundancies, as revealed between the downstream signalling pathways of ARs (Fig. 1), can also be discerned among the MAPK cascades (Fig. 2).
![]() |
Calcium as a signal transmitter |
---|
In contrast to the low cytoplasmic Ca2+ during rest, the Ca2+ concentration outside the myocyte ranges between 1 and 2 mM, thus establishing a gradient of over 10 000-fold. This outsideinside gradient enables the Ca2+ ion to function as a signal, provided there are sufficiently sensitive Ca2+-binding signalling components in the cell.27 Inside the cardiomyocytes, the Ca2+ signal is conveyed to the target sites by the Ca2+-sensing proteins troponin-C (TnC) and calmodulin (CaM), which reversibly bind Ca2+ ions with affinities in the range of 105 to 106 M. These Ca2+ affinities are just in the range attained by Ca2+ signalling transients, taking into account the intracellular presence of around 1 mM Mg2+ ions, which are competing for the Ca2+ binding sites with an affinity 104 times lower than that of Ca2+. Some Ca2+-regulated targets have their own Ca2+ binding sites and are not dependent on TnC or CaM as an intermediate signal component.
Calcium enters the cardiomyocyte down the concentration gradient via the high voltage-activated (opening at around 20 mV) L-type Ca2+-channels of the sarcolemma (particularly accumulated in the transverse T-tubules) as often as an AP stimulates the cell. The low voltage-activated (opening between 60 and 40 mV) T-type channels are not concentrated in the T-tubules and contribute little to the Ca2+ entry from outside.6 18 34 41 They are primarily found in secretory and smooth muscle cells and in the cardiac nodal cells, where they are involved in rhythm control. The L- and T-type channels belong to a family of cell surface Ca2+-channels composed of four subunits in a 1:1 stoichiometry (1,
2, ß,
). The human
1C (Cav1.2) subunit (2221 amino acids, 249 kDa), occurring in heart (Cav1.2a) and smooth muscle (Cav1.2b sharing 93% homology), contains four domains with six transmembrane helices each (S1S6, yielding a total of 12 transmembrane helices). The S6s of each domain together form the Ca2+ pore. The four transmembrane S4 helices of 19 amino acids each contain positively charged residues at every third position, together forming the voltage sensor of the pore. The P-loop between the transmembrane helices S5 and S6 is very much conserved among the different Ca2+-channels and provides the filter selectivity for Ca2+. The ß-subunit associates with the
-subunit at the inner side of the membrane and determines the kinetics of the channel activities (opening, closing, inactivation). The ß2-subunit (73.5 kDa) is characteristic of the heart while the ß3-subunit (54.5 kDa) is more typical of smooth muscle. The Cav1.2 channel activity is enhanced by phosphorylation by PKA, PKC and CaMK, but is inhibited by Ca2+ when its cytoplasmic concentration is increased during sustained cell activity (negative feedback control).34 39 41 93 Calcium binds directly to the C-terminal intracellular part of the Cav1.2, which contains a Ca2+-binding EF-hand domain (see below), but nearby is also a binding site for CaM, which contributes to sensing Ca2+ signalling. For prolonged elevation of cytoplasmic Ca2+, ill-defined ligand-gated channels (also called store-operated channels) are thought to be involved in Ca2+ entry after the Ca2+ release from internal stores.14 22
For excitationcontraction coupling, Ca2+ is primarily released from intracellular stores in response to extracellular stimuli (Fig. 3). The endoplasmic reticulum (ER) in non-muscle cells and its derivative, the SR in striated muscles (cardiac and skeletal) as well as in smooth muscle, are able to accumulate Ca2+ up to a concentration of 30 mM and to store it bound to proteins with multiple low-affinity Ca2+-binding sites, such as calsequestrin and calreticulin. Calsequestrin (46.4 kDa) is found in cardiac and skeletal SR while calreticulin (45.0 kDa) is mainly present in the SR of smooth muscle and ER of non-muscle cells. Both the SR and ER contain two ligand-gated Ca2+ release channels, the ryanodine binding receptor (RYR) and the IP3 binding receptor (IP3R). The Ca2+ entering the cell on stimulation induces a far larger (5- to 20-fold) release of Ca2+ from the closely positioned intracellular SR into the cytoplasm via the RYR, a process called Ca2+-induced Ca2+ release (CICR).7 14 16
The RYR channel forms a tetramer with four equal subunits of 565 kDa each, which combine with four regulatory proteins, called FKBP12.6. FKBP, with a mass of 12.6 kDa belongs to the group of cyclophilins, which accelerate protein folding, acting as peptidyl-prolyl cis-trans isomerases or rotamases. They are inhibited by the immunosuppressor drugs FK506 (tacrolimus) and rapamycin (sirolimus), but not by cyclosporin A. Of the three isoforms, RYR1 (with a total molecular weight of around 2300 kDa) occurs in skeletal muscle, RYR2 in cardiomyocytes and RYR3 in non-muscle cells.49 88 In addition, the RYR2 serves as scaffold protein, combining with numerous key regulatory components in the junctional SR complex, including CaM, PKA, type-1 and type-2 phosphatases and, at the luminal SR surface, triadin and calsequestrin. Calcium can also be released from the SR via the IP3R channel, which is composed of four equal subunits of 313 kDa each. The IP3R channel also exists in three isoforms, with IP3R2 in cardiomyocytes. The IP3R2 is activated by IP3 produced by PLC. The rate and extent of Ca2+ liberation by IP3 is much lower than for CICR via the RYR2, and thus hardly contributes to excitationcontraction coupling in cardiomyocytes. However, intracellular Ca2+ release by IP3 is important in the slow motion of smooth muscle contraction and in fine-tuning the activity of atrial myocytes, where the SR has more IP3R2 than in the ventricular myocytes. RYR2 and IP3R2 share structural and functional similarities and have some sequence similarity in their C-terminal domains, although the latter is about half the size of the former.77 The two Ca2+ release channel types interact and are inhibited by high Ca2+ and CaM, but become activated by phosphorylation by PKA, PKC or a Ca2+-CaM-dependent protein kinase-II (CaMK-II) (Fig. 3). Phosphorylation of RYR2 induces dissociation of the FKBP regulatory protein, which inhibits the RYR2 channel when bound to it.
![]() |
Intracellular calcium handling in cardiomyocytes |
---|
The amount of AP-induced Ca2+ entry is primarily dictated by the duration of the AP and the open probability of the L-type Ca2+-channel. The Ca2+ extruded after the heart-beat must match the amount of Ca2+ that enters just before the beat, otherwise the cell would not be in steady state but would either lose or gain Ca2+. This provides a quantitative framework for the dynamic Ca2+ fluxes in the cardiomyocytes. The SERCA2a pump is one of the main players in terminating contraction and restoring resting cytoplasmic Ca2+ concentrations. It is known that during heart failure in humans, as well as in a rabbit model, the functional expression of SERCA2a is reduced and NCX1 is increased.8 32 This results in a loss of SR Ca2+ concomitant with enhanced Ca2+ extrusion, leading to a net loss of intracellular Ca2+. Consequently, less Ca2+ is available in the SR for the subsequent heart-beats, which is the central cause of systolic contractile deficit in heart failure.
The NCX1 (splice variant NCX1.1) is the cardiac isoform with 938 amino acids (104 kDa), whereas the NCX2 is preferentially expressed in the brain and NCX3 in skeletal muscle.62 70 82 100 Its topology is not yet clear, suggesting either 11 or nine transmembrane helices. Interestingly, the NCX1 may function in reverse mode during the plateau of the AP (Ca2+ influx coupled with Na+ outflux). This may be of physiological significance as the NCX1, like the L-type Ca2+-channel, is concentrated in the transverse T-tubular system and may be able to elicit Ca2+-induced Ca2+ release via the RYR2 from the SR. This would reinforce the Ca2+ release process from the RYR2 ascribed to the interaction of the L-type Ca2+-channel with the RYR2 (as mentioned above). The NCX1 can be phosphorylated and stimulated by both PKA and PKC, and is therefore under the control of ARs. PMCA and SERCA belong to a subfamily of the P-type ATPases. The sarcolemmal Ca2+ pump (PMCA) transports one Ca2+ per ATP out of the cell, while SERCA2a takes two Ca2+ per ATP up into the SR. From the isoforms of the four genes giving rise to PMCA1-PMCA4 and over 20 splice variants, PMCA1c (1249 amino acids, 138 kDa, with 10 putative transmembrane helices) seems to be the main cardiac species, but other isoforms are also expressed in cardiomyocytes. PMCA activity depends on binding CaM, and may be further stimulated by phosphorylation via PKA and/or PKC (Fig. 3).
The cardiac SR Ca2+ pump SERCA2a, together with its regulatory protein phospholamban (PLN), is the most important component linking adrenergic control to inotropy and rhythmicity.4 8 14 22 48 Three genes were identified for the SR Ca2+ pump, SERCA1, SERCA2 and SERCA3, which are spliced into several isoforms. SERCA1a is mainly expressed in fast skeletal muscle, while SERCA1b is abundant in fetal and neonatal tissues. The SERCA2 gene encodes four splice variants: SERCA2a, expressed in the heart and in slow skeletal muscle, SERCA2b, expressed in smooth muscle and with variants (types 2 and 3) in non-muscle cells and (type 4) in neuronal cells. The recently solved crystal structure of the SERCA1a pump in the Ca2+-bound state reveals (besides the 10 transmembrane helices) three large cytoplasmic domain structures constituting the nucleotide binding site, the catalytic site and the phosphorylation site.91 The cardiac SERCA2a is 997 amino acids long (110 kDa) and is under the direct control of a CaM-dependent kinase-II (CaMK-II), which enhances its transport capacity by phosphorylation of Ser38. The major regulator of SERCA2a, however, is PLN, with 52 amino acids (6.1 kDa) and one transmembrane helix (C-terminal amino acids 3252). It is predominantly expressed in ventricular cardiac muscle, but also in small amounts in slow-twitch skeletal muscle, smooth muscle and endothelial cells. As a monomer, it associates with and efficiently inhibits the SERCA2a pump, by interaction of its transmembrane helix with helices of the pump and its cytoplasmic domain with the cytoplasmic domain of the pump. Its inhibitory effect is delicately regulated by phosphorylation induced by different signalling pathways. The cytoplasmic domain is amenable to regulation by phosphorylation of Ser16 by PKA, Thr17 by CaMK-II, and Ser10 by PKC (Fig. 3). Phosphorylation to various degrees causes gradual dissociation of PLN from SERCA2a, relieving the inhibition of the Ca2+ pump and thus increasing the rate of relaxation (lusitropic effect). When not associated with SERCA2a, PLN polymerizes into pentamers.
To further increase the complexity, sarcolipin (SLN), which is a homologue of PLN with 31 amino acids (3.8 kDa), forming just one transmembrane helix and lacking most of the cytoplasmic portion present in PLN, was discovered recently.4 48 It is highly expressed in human fast-twitch skeletal muscle and in small amounts predominantly in atrial muscle. SLN inhibits the SERCA1a (fast skeletal muscle) and SERCA2a (cardiomyocytes) by lowering the Ca2+-binding affinity and slowing the ATPase turnover rate. In addition, SLN is able to induce a superinhibitory effect apparently by binding to PLN and thus preventing PLN from polymerizing into pentamers. A small amount of SLN may be sufficiently potent to shift the equilibrium of pentameric PLN towards the monomer, inhibiting SERCA2a. In view of the decisive power of the SR-SERCA2a pump in regulating contractility, both SERCA2a and PLN represent potential targets for new therapeutic approaches.
![]() |
Adrenergic fine-tuning of contractility |
---|
|
![]() |
FrankStarling and negative feedback mechanisms |
---|
In general, the positive inotropic and lusitropic effects are mainly elicited by ß1- and ß2-AR stimulation and mediated through cAMP-dependent PKA phosphorylation of PLN, TnI, the L-type Ca2+-channel and RYR2. The degree of positive inotropy is directly related to the amount of cellular Ca2+, which steadily increases with time under these conditions. Without safety precautions, it would end in cardiac arrest in systolic contracture. This is, however, normally not observed (except with digitalis glycoside overdoses), as the cardiac phosphodiesterase (PDE) isoforms degrade cAMP, thus keeping the overall ß1-AR stimulation at bay. At least four types of PDE isoforms (PDE1PDE4, varying in size from 61 to 124 kDa), with over 20 splice variants, most of them membrane-associated, have been identified in cardiomyocytes.25 35 52 83 Via cyclic nucleotide metabolism, PDEs are also involved in the regulation of the L-type Ca2+-channels. The activity of PDE1 depends on the association of the four Ca2+ bound form of CaM, which increases concomitantly with the increase in cytoplasmic Ca2+; PDE2 is stimulated by cAMP; PDE3 is inhibited by cAMP; and PDE4 is insensitive to cAMP. The PDEs are operative as homodimers and contribute decisively to the contractile responsiveness. The fact that PDE activity reduces inotropy by degradation of cAMP prompted the development of PDE inhibitors, such as amrinone, milrinone and enoximone (bipyridines inhibit PDE enzymes, as do methylxanthines), which are in clinical use for the acute treatment of congestive heart failure. Despite its complexity, the PDE system provides a powerful negative feedback control system of great physiological significance (Fig. 3).
In conclusion, although Ca2+ is traditionally described as a second messenger that is liberated from intracellular stores (SR), Ca2+ entering the cell may activate a number of processes acting directly as a first messenger.14 In particular, it amplifies its own signalling capacity by the Ca2+-induced Ca2+ release from the SR via the RYR2, thus also acting as a second messenger. Furthermore, the second messenger IP3, liberated by PLC from PIP2 (as mentioned above), provokes the release of Ca2+ via the IP3R of the SR, acting then as a third messenger. In addition, adrenergic signalling pathways exert their effects (balancing between positive or negative inotropy and lusitropy) almost exclusively by modulating cytoplasmic Ca2+ concentrations and signalling transients in amplitude and frequency, thus adding yet another step to the Ca2+ signalling cascade. Consequently, Ca2+ may well be viewed as operating at the same time as a first, second and third messenger. In the healthy heart, the combined signalling circuits coordinate contractility and energy production in a concerted way. The cellular and molecular basis explains how adrenergic stimulation induces positive inotropy together with positive lusitropy (faster relaxation) under increased haemodynamic load. This combination of stronger but shorter contraction twitches makes it possible to accommodate more beats per time interval, thus increasing cardiac output under increased workload. However, upon transition from compensated haemodynamics to overt heart failure or during ischaemia/reperfusion injuries, the integrated signalling network may become unbalanced and dysfunctional, eventually ending in collapse.32 49 71 79 99
![]() |
Adrenergic regulation of heart rate |
---|
![]() |
Regulation of vascular smooth muscle contraction |
---|
The actin filaments do not contain troponin, yet smooth muscle contraction is still regulated by Ca2+, albeit in an indirect way.38 50 57 86 The onoff switch for contraction depends on the phosphorylation of smooth muscle myosin RLC at Ser19 by the Ca2+-CaM-dependent smooth muscle MLCK isoform and dephosphorylation by a myosin light chain phosphatase (MLCP) (Fig. 5). The extent of RLC phosphorylation determines shortening velocity and tension development in smooth muscle. A second phosphorylation of RLC at the adjacent Thr18 has only a small additional positive effect on ATPase activity. Alternatively, the RLC can also be phosphorylated by PKC, CaMK-II and other protein kinases. In addition, contraction and relaxation of smooth muscle may also be induced by hormones and cell mediators, bypassing the canonical Ca2+ signalling pathways (see below).
|
It was first thought that regulation of smooth and non-muscle contractility resides primarily in the activatory process of RLC phosphorylation, followed by a more or less constant rate of dephosphorylation by a constitutively active MLCP (thought to represent a housekeeping enzyme). More recently it was recognized that the MLCP also is regulated by phosphorylation.84 86 MLCP is a holoenzyme consisting of three subunits: a catalytic subunit of 37 kDa (PP1c), a large myosin binding subunit (MBS) of around 130 kDa (also known as myosin phosphatase targeting subunit, MYPT), and a small 20 kDa subunit (M20) of unknown function. The PP1c is a member of the type-1 protein serinethreonine phosphatase family. The MBS exists in two splice variants, M130 and M133. MBS binds the relatively non-specific phosphatase PP1c near its N-terminus and confers substrate specificity by forming a complex with myosin-II. MBS preferentially associated with myosin-II when the RLC is phosphorylated and ready for dephosphorylation. M20 binds to the N-terminal portion of MBS. Inhibition of MLCP by phosphorylation at Thr655 in M130 (amino acid numbering for the human species) or Thr696 in M133 by Rho-kinase-II (ROK-II) leads to sustained vasoconstriction without concomitant lowering of the cytoplasmic Ca2+ concentration (Fig. 5). Phosphorylation of MBS induces dissociation from myosin-II and from the catalytic PP1c subunit. Additional phosphorylation sites for several different types of kinases have also been shown for MBS in vitro, but their physiological significance has not yet been established. Dephosphorylation of MBS proceeds slowly and does not allow fast relaxation of the contractile apparatus.
However, another small molecular weight (16.7 kDa) protein, CPI-17, is a potent reversible inhibitor of MLCP.84 86 The inhibitory activity of CPI-17 is increased more than 1000-fold by phosphorylation at Ser38 by PKC, PKA, PKG or ROK, and also quickly reversed upon dephosphorylation by the protein serinethreonine phosphatases PP2A, PP2B and PP2C, but not by type-1 protein phosphatases. Consequently, CPI-17 is considered to act in concert with myosin RLC phosphorylation by MLCK as the onoff switch of smooth muscle contraction.
![]() |
Phasic contraction and vascular tone |
---|
The Rho protein family comprises a group of small monomeric GTPases (around 20 kDa) that function as pivotal regulators in cell motility, cell proliferation and apoptosis, involving the actin cytoskeleton and the microtubule network. RhoA with bound GDP is kept in the cytoplasm by association with GDI (GDP dissociation inhibitor). The activity of RhoA is governed by the two factors GEF (GTP exchange factor) and GAP (GTPase activating protein). Agonist binding to surface receptors induces the receptor-coupled Gq to activate GEF, which in turn promotes dissociation of GDI from RhoA and its exchange of GDP for GTP (Fig. 5). RhoA with bound GTP associates with ROK, releasing its autoinhibitory peptide loop and the activated ROK then inhibits the MLCP by phosphorylation of the MBS subunit. Subsequently, the myosin RLC remains partially phosphorylated, maintaining vascular tone. ROK is a protein serinethreonine kinase of around 160 kDa with two isoforms: ROK-I (ROKß) and ROK-II (ROK
). ROK-II is the main species in muscles and brain. Besides the activation by RhoA, its activity can be modulated further by autophosphorylation. In addition to inhibition of the MLCP (by phosphorylation of MBS), ROK-II is also able to phosphorylate the myosin RLC and thus contributes in two ways to the maintenance of vascular tone.
Another mechanism contributing to the vascular tone is the integrin-linked kinase system (Fig. 5).36 89 Integrins are a family of cell adhesion receptors composed of an -subunit (several types of 140210 kDa) and a ß-subunit (several types of 90130 kDa), which are involved in establishing cell-to-cell and cell-to-extracellular matrix (ECM) contacts. Integrins are potential force-transduction proteins because they span the surface membrane and link the ECM to the underlying actin cytoskeleton in specialized focal contact regions. Integrins activate signalling cascades similar to those activated by growth factor receptors (Figs 1 and 2), but unlike these, integrins possess no intrinsic tyrosine kinase activity and must therefore signal via cytoplasmic kinases (Fig. 5). Such an integrin-linked serine-threonine kinase (ILK) has recently been shown to phosphorylate the MBS of MLCP, CPI-17, as well as the myosin RLC in a Ca2+-independent manner. Induction of Ca2+-independent contraction by RLC phosphorylation by ILK becomes apparent only when the MLCP is inhibited. Whether additional tyrosine kinases upstream of ILK are involved in this signalling pathway is not known at present.
Further fine-tuning of smooth muscle contraction is brought about by regulatory mechanisms at the actin filaments.50 58 95 The regulatory protein caldesmon (93.3 kDa) is complexed to actin, tropomyosin and a Ca2+-binding protein (CaBP). In the absence of Ca2+, caldesmon restricts the interaction of the myosin heads with actin, but when Ca2+ is bound to CaBP this inhibition is relieved. CaBP has the same molecular weight as CaM (16.7 kDa) and is believed to represent a covalently modified form of CaM by phosphorylation at Thr79 and Ser81. An additional basic protein, calponin, of 33 kDa, predominantly found in smooth muscle, binds to filamentous actin and CaM. Its role is not clear yet, but it seems to be involved in 1-agonist-induced signal transduction. In particular, it may facilitate the PKC-ERK1/2 signalling pathway, leading to phosphorylation of caldesmon, which also relieves the actomyosin inhibition of the latter.
![]() |
Intracellular calcium handling in smooth muscle cells |
---|
The smooth muscle L-type Ca2+-channel mainly serves to fill and maintain the Ca2+ content of the SR (Fig. 5). As in myocytes, the Ca2+-channel is activated by PKA phosphorylation induced by ß-AR stimulation, increasing cAMP. PKA is recruited to the membrane for Ca2+-channel phosphorylation by the PKA-anchoring protein AKAP. At high concentrations of cAMP, the Ca2+-channel stimulation by PKA may, however, be attenuated for the following reasons. PKG is an important mediator of vascular relaxation induced by the endothelial derived relaxing factor nitric oxide (NO), which stimulates the cytoplasmic guanylyl cyclase (GC) by interaction with its haem group and produces cGMP.2,47 cGMP activates PKG, which lowers intracellular Ca2+ by inhibition of the L-type Ca2+-channel, activates the BKCa (see below), and also activates the MLCP (Fig. 5). Whether inhibition of the Ca2+-channel is achieved by direct phosphorylation of the Cav1.2b subunit or by activation of a protein phosphatase is not clear at present. To complicate matters further, neither cAMP nor cGMP displays absolute specificity for its respective kinase. Thus, some of the actions of cAMP at high concentrations may be due to stimulation of both PKA (channel activation) as well as of PKG (channel inhibition).
One of the most salient features of the caveolae arrangement for the regulation of Ca2+ entry into the cell rests on the close juxtaposition with the SR.10 15 47 81 Calcium entry locally activates the RYR release channel of the SR and thus triggers so-called Ca2+ sparks, which in turn activate the BKCa channel in the caveolar vicinity. The BKCa consists of the channel-forming -subunit (125 kDa) and a small regulatory ß-subunit (21.5 kDa). The ß-subunit is selectively expressed in smooth muscle, localizes to the intracellular side of the channel, and markedly increases the Ca2+-sensitivity of the channel complex. PKA and PKG seem to stimulate whereas PKC may inhibit the BKCa channel activity. The BKCa is activated either by elevation of the intracellular Ca2+ or by diminishing the membrane potential. Together with the voltage-dependent K+-channels, it is mostly responsible for the membrane or resting potential. The caveolar Ca2+ pathway over the short distances from the L-type Ca2+-channel to the SR and from the RYR of the SR back to the sarcolemmal BKCa represents a feedback loop where Ca2+ entry leads immediately to a more negative resting potential. This arises by activation of the potassium outflow through the BKCa shielding the cell from undue Ca2+ entry. The BKCa seems not to be present in the myocyte surface membrane.
As mentioned above for the integrin-ILK signalling for phosphorylation of myosin RLC and MLCP, integrin-dependent signalling involving a tyrosine phosphorylation cascade can also activate the L-type Ca2+-channel. This signalling pathway may translate mechanical forces of the ECM-linked integrins directly to an increase in Ca2+ entry, leading to an increase in vascular tone.
![]() |
Hallmarks of smooth muscle versus myocardial regulation |
---|
|
![]() |
Interplay between calcium and adrenergic receptor subtype-specific signalling |
---|
|
An interesting aspect concerns the signalling differences between ß1- and ß2-AR. Although both ß1-ARs and ß2-ARs increase contractile amplitude and hasten relaxation in ventricular myocytes, several striking differences with respect to signal transduction downstream from the ß-ARs have been revealed. While ß1-AR signalling strictly adheres to the canonical cAMPPKA pathway, the ß2-AR adopts a much more flexible approach by being able to signal via Gs, G
i, G
q and/or Gß
depending on spatiotemporal relations in the receptor microenvironment (Fig. 1). It was demonstrated that apoptotic myocyte cell death is dissociated from ß2-AR and selectively mediated by ß1-AR in isolated ventricular myocytes and in intact hearts in vivo.96 98 99 The possible activation of different G-proteins by ß2-ARs may result in distinct downstream signalling pathways. The chronic catecholamine-dependent positive inotropy of ß2-AR is supported by an increased Ca2+ influx via the L-type Ca2+-channel without cardiotoxic consequences. This is achieved by locally restricted signalling to the Ca2+-channel located close by in the membrane, and bypasses the ß1-typical effects on PLN and the contractile proteins. On the other hand, coupling to G
i/Gß
activates several cytoprotective mechanisms (counteracting in part the G
s-mediated routes), such as activation of ATP-dependent K+-channels in the sarcolemma and in the inner mitochondrial membrane, mediated by different PKC isoforms, and activation of the protein kinase B (PKB, also called Akt). In the latter case, signalling proceeds via the PI3K and PI3K-dependent kinase-1 (PDK1) pathway (Fig. 1). PKB activation leads to increased expression of the antiapoptotic protein Bcl-2 and inhibition of proapoptotic factors, such as caspase-9 and Fas-ligand expression.
Activation of the endothelial nitric oxide synthase (eNOS), which is constitutively expressed in cardiomyocytes, greatly depends on ß3-AR signalling and leads to activation of the cGMP-dependent PKG.2 47 90 NO may also reach the myocytes from extracellular sources, such as the endothelial cells. Thus, NO operates in both autocrine and paracrine fashions. The NOGCcGMPPKG pathway also exerts a negative inotropic effect in the myocytes by reducing Ca2+ influx through L-type Ca2+-channels. This negative inotropic effect of the ß3-AR acts under normal conditions as a negative counter-regulation. In contrast, in the failing heart, where ß3-ARs are upregulated with concomitant downregulation of ß1-ARs, this negative inotropic effect may aggravate the deterioration of cardiac function.
![]() |
Conclusions |
---|
![]() |
Acknowledgements |
---|
![]() |
References |
---|
2 Andrews KL, Triggle CR, Ellis A. NO and the vasculature: where does it come from and what does it do? Heart Fail Rev 2002; 7: 42345[CrossRef][Medline]
3 Angers S, Salahpour A, Bouvier M. Dimerization: an emerging concept for G-protein-coupled receptor ontogeny and function. Annu Rev Pharmacol Toxicol 2002; 42: 40935[CrossRef][ISI][Medline]
4 Asahi M, Nakayama H, Tada M, Otsu K. Regulation of sarco(endo)plasmic reticulum Ca2+ adenosine triphosphatase by phospholamban and sarcolipin: implication for cardiac hypertrophy and failure. Trends Cardiovasc Med 2003; 13: 1527[CrossRef][ISI][Medline]
5 Baines CP, Zhang J, Wang GW, et al. Mitochondrial PKC and MAPK form signaling modules in the murine heart. Enhanced mitochondrial PKC
-MAPK interactions and differential MAPK activation in PKC
-induced cardioprotection. Circ Res 2002; 90: 3907
6 Barreiro G, Guimaraes CRW, de Alencastro RB. A molecular dynamics study of an L-type calcium channels model. Protein Eng 2002; 15: 10922[CrossRef][ISI][Medline]
7 Berridge MJ. Elementary and global aspects of calcium signalling. J Exp Biol 1997; 200: 3159
8 Bers DM. Cardiac excitation-contraction coupling. Nature 2002; 415: 198205[CrossRef][ISI][Medline]
9 Biel M, Ludwig A, Zong X, Hofmann F. Hyperpolarization-activated cation channels: a multigene family. Rev Physiol Biochem Pharmacol 1999; 136: 16581[ISI][Medline]
10 Bowles DK, Wamhoff BR. Coronary smooth muscle adaptation to exercise: does it play a role in cardioprotection? Acta Physiol Scand 2003; 178: 11721[CrossRef][ISI][Medline]
11 Brodde OE, Michel MC. Adrenergic and muscarinic receptors in the human heart. Pharmacol Rev 1999; 51: 65189
12 Bünemann M, Lee KB, Pals-Rylaarsdam R, Roseberry AG, Hosey MM. Desensitization of G-protein-coupled receptors in the cardiovascular system. Annu Rev Physiol 1999; 61: 16992[CrossRef][ISI][Medline]
13 Canaves JM, Taylor SS. Classification and phylogenetic analysis of the cAMP-dependent protein kinase regulatory subunit family. J Mol Evol 2002; 54: 1729[ISI][Medline]
14 Carafoli E. Calcium signaling: a tale for all seasons. Proc Natl Acad Sci USA 2002; 99: 111522
15 Cox DH, Aldrich RW. Role of the ß1 subunit in large-conductance Ca2+-activated K+ channel gating energetics. Mechanisms of enhanced Ca2+ sensitivity. J Gen Physiol 2000; 116: 41132
16 da Silva CP, Guse AH. Intracellular Ca2+ release mechanisms: multiple pathways having multiple functions within the same cell type? Biochim Biophys Acta 2000; 1498: 12233[CrossRef][ISI][Medline]
17 Eckert RE, Karsten AJ, Utz J, Ziegler M. Regulation of renal artery smooth muscle tone by 1-adrenoceptors: role of voltage-gated calcium channels and intracellular calcium stores. Urol Res 2000; 28: 1227[CrossRef][ISI][Medline]
18 Ertel SI, Ertel EA. Low-voltage-activated T-type Ca2+ channels. Trends Pharmacol Sci 1997; 18: 3742[CrossRef][ISI][Medline]
19 Esler M, Lambert G, Brunner-La Rocca HP, Vaddadi G, Kaye D. Sympathetic nerve activity and neurotransmitter release in humans: translation from pathophysiology into clinical practice. Acta Physiol Scand 2003; 177: 27584[CrossRef][ISI][Medline]
20 Esposito G, Rapacciuolo A, Naga Prasad SV, Rockman HA. Cardiac hypertrophy: role of G protein-coupled receptors. J Cardiac Fail 2002; 8 (Suppl. 6): S40914[CrossRef][ISI][Medline]
21 Ferguson SSG. Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 2001; 53: 124
22 Frank KF, Bölck B, Erdmann E, Schwinger RHG. Sarcoplasmic reticulum Ca2+-ATPase modulates cardiac contraction and relaxation. Cardiovasc Res 2003; 57: 207[CrossRef][ISI][Medline]
23 Fukuda N, Sasaki D, Ishiwata S, Kurihara S. Length dependence of tension generation in rat skinned cardiac muscle. Circ Res 2001; 104: 163945
24 Gavras I, Manolis AJ, Gavras H. The 2-adrenergic receptors in hypertension and heart failure: experimental and clinical studies. J Hypertens 2001; 19: 211524[CrossRef][ISI][Medline]
25 Georget M, Mateo P, Vandecasteele G, et al. Cyclic AMP compartmentation due to increased cAMP-phosphodiesterase activity in transgenic mice with a cardiac-directed expression of the human adenylyl cyclase type-8 (AC8). FASEB J 2003; 17: 138091
26 Goaillard JM, Vincent P, Fischmeister R. Simultaneous measurements of intracellular cAMP and L-type Ca2+ current in single frog ventricular myocytes. J Physiol (London) 2001; 530: 7991
27 Gomes AV, Potter JD, Szczesna-Cordary D. The role of troponins in muscle contraction. IUBMB Life 2002; 54: 32333[ISI][Medline]
28 Gong H, Sun H, Koch WJ, et al. Specific ß2-AR blocker ICI 118,551 actively decreases contraction through a Gi-coupled form of the ß2-AR in myocytes from failing human heart. Circulation 2002; 105: 2497503
29 Grobler JA, Hurley JH. Catalysis by phospholipase C1 requires that Ca2+ bind to the catalytic domain, but not the C2 domain. Biochemistry 1998; 37: 50208[CrossRef][ISI][Medline]
30 Hall RA, Lefkowitz RJ. Regulation of G-protein-coupled receptor signaling by scaffold proteins. Circ Res 2002; 91: 67280
31 Han JL, Zhang YY, Lu ZZ, Mao JM, Chen MZ, Han QD. Functional 1-adrenergic receptor subtypes in human right gastroepiploic artery. Acta Pharmacol Sin 2003; 24: 32731[ISI][Medline]
32 Hasenfuss G, Schillinger W, Preuss M, et al. Relationship between Na+-Ca2+-exchanger protein levels and diastolic function of failing human myocardium. Circulation 1999; 99: 6418
33 Hefti MA, Harder BA, Eppenberger HM, Schaub MC. Signaling pathways in cardiac hypertrophy. J Mol Cell Cardiol 1997; 29: 287392[CrossRef][ISI][Medline]
34 Hering S, Berjukow S, Sokolov S, et al. Molecular determinants of inactivation in voltage-gated Ca2+ channels. J Physiol (London) 2000; 528: 23749
35 Houslay MD, Adams DR. PDE4 cAMP phosphodiesterase: modular enzymes that orchestrate signalling cross-talk, desensitization and compartmentalization. Biochem J 2003; 370: 118[CrossRef][ISI][Medline]
36 Ingber DE. Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology. Circ Res 2002; 91: 87787
37 Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002; 298: 19112
38 Kamm KE, Stull JT. Dedicated myosin light chain kinases with diverse cellular functions. J Biol Chem 2001; 276: 452730
39 Kamp TJ, Hell JW. Regulation of cardiac L-type calcium channels by protein kinase A and protein kinase C. Circ Res 2002; 87: 1095102[ISI]
40 Kaupp UB, Seifert R. Molecular diversity of pacemaker ion channels. Annu Rev Physiol 2001; 63: 23557[CrossRef][ISI][Medline]
41 Keef KD, Hume JR, Zhong J. Regulation of cardiac and smooth muscle Ca2+ channels (Cav1.2a,b) by protein kinases. Am J Physiol 2001; 281: C174356[ISI]
42 Koch WJ. Gene transfer of ß-adrenergic signaling components for heart failure. J Cardiac Fail 2002; 8 (Suppl. 6): S52631[CrossRef][ISI][Medline]
43 Koshimizu T, Tanoue A, Hirasawa A, Yamauchi J, Tsujimoto G. Recent advances in 1-adrenoceptor pharmacology. Pharmacol Ther 2003; 98: 23544[CrossRef][ISI][Medline]
44 Krebs J. Calmodulin-dependent protein kinases. In: Carafoli E, Krebs J, eds. Calcium Homeostasis. Berlin: Springer, 2000; 201123
45 Kristensen B, Birkelund S, Jorgensen PL. Trafficking of Na, K-ATPase fused to enhanced green fluorescent protein is mediated by protein kinase A or C. J Membr Biol 2003; 191: 2536[CrossRef][ISI][Medline]
46 Lavoie C, Mercier JF, Salahpour A, et al. ß1/ß2-adrenergic receptor heterodimerization regulates ß2-adrenergic receptor internalization and ERK signaling efficacy. J Biol Chem 2002; 277: 3540210
47 Lincoln TM, Dey N, Sellak H. cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression. J Appl Physiol 2001; 91: 142130
48 MacLennan DH, Kranias EG. Phospholamban: a crucial regulator of cardiac contractility. Nat Rev Cell Biol 2003; 4: 56677[CrossRef][ISI]
49 Marks AR. Cardiac intracellular calcium release channels. Role in heart failure. Circ Res 2000; 87: 811
50 Marston S, Burton D, Copeland O, et al. Structural interactions between actin, tropomyosin, caldesmon and calcium binding protein and the regulation of smooth muscle thin filaments. Acta Physiol Scand 1998; 164: 40114[CrossRef][ISI][Medline]
51 Marston SB, Redwood CS. Modulation of thin filament activation by breakdown or isoform switching of thin filament proteins. Physiological and pathological implications. Circ Res 2003; 93: 11708
52 Maurice DH, Palmer D, Tilley DG, et al. Cyclic nucleotide phosphodiesterase activity, expression, and targeting in cells of the cardiovascular system. Mol Pharmacol 2003; 64: 53346
53 Michel MC, Li Y, Heusch G. Mitogen-activated protein kinases in the heart. Naunyn Schmiedebergs Arch Pharmacol 2001; 363: 24566[CrossRef][ISI][Medline]
54 Michelotti GA, Price DT, Schwinn DA. Alpha1-adrenergic receptor regulation: basic science and clinical implications. Pharmacol Ther 2000; 88: 281309[CrossRef][ISI][Medline]
55 Molkentin JD, Dorn GW. Cytoplasmic signaling pathways that regulate cardiac hypertrophy. Annu Rev Physiol 2001; 63: 391426[CrossRef][ISI][Medline]
56 Moosmang S, Stieber J, Zong X, Biel M, Hofmann F, Ludwig A. Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissues. Eur J Biochem 2001; 268: 164652
57 Morano I. Tuning smooth muscle contraction by molecular motors. J Mol Med 2003; 81: 4817[CrossRef][ISI][Medline]
58 Murphy RA. What is special about smooth muscle? The significance of covalent crossbridge regulation. FASEB J 1994; 8: 31118
59 Nakayama S, Kawasaki H, Kretsinger R. Evolution of EF-hand proteins. In: Carafoli E, Krebs J, eds. Calcium Homeostasis. Springer, 2000; 2958
60 Neves S, Ram PT, Iyengar R. G-protein pathways. Science 2002; 296: 16369
61 Newton AC. Regulation of the ABC kinases by phosphorylation: protein kinase-C as a paradigm. Biochem J 2003; 370: 36171[CrossRef][ISI][Medline]
62 Nicoll DA, Ottolia M, Lu L, Lu Y, Philipson KD. A new topological model of the cardiac sarcolemmal N+-Ca2+ exchanger. J Biol Chem 1999; 274: 9107
63 Oldenburg O, Qin Q, Krieg T, et al. Bradykinin induces mitochondrial ROS generation via NO, cGMP, PKG, and mitoKATP channel opening and leads to cardioprotection. Am J Physiol 2004; 286: H46876[ISI]
64 Owuor ED, Kong ANT. Antioxidant and oxidant regulated signal transduction pathways. Biochem Pharmacol 2002; 64: 76570[CrossRef][ISI][Medline]
65 Patel TB, Du Z, Pierre S, Cartin L, Scholich K. Molecular biological approaches to unravel adenylyl cyclase signaling and function. Gene 2001; 269: 1325[CrossRef][ISI][Medline]
66 Perry SV. Troponin T: genetics, properties and function. J Muscle Res Cell Motil 1998; 19: 575602[CrossRef][ISI][Medline]
67 Perry SV. Troponin-I: inhibitor or facilitator. Mol Cell Biochem 1999; 190: 932[CrossRef][ISI][Medline]
68 Peyton PJ, Myles PS, Silbert BS, et al. Perioperative epidural analgesia and outcome after major abdominal surgery in high risk patients. Anesth Analg 2003; 96: 54854
69 Philipp M, Brede M, Hein L. Physiological significance of 2-adrenergic receptor subtype diversity: one receptor is not enough. Am J Physiol 2002; 283: R28795[ISI]
70 Pogwizd SM, Schlotthauer K, Li L, Yuan W, Bers DM. Arrhythmogenesis and contractile dysfunction in heart failure. Roles of sodium-calcium exchange, inward rectifier potassium current, and residual ß-adrenergic responsiveness. Circ Res 2001; 88: 115967
71 Port JD, Bristow MR. Altered beta-adrenergic receptor gene regulation and signaling in chronic heart failure. J Mol Cell Cardiol 2001; 33: 887905[CrossRef][ISI][Medline]
72 Post SR, Hammond HK, Insel PA. ß-adrenergic receptors and receptor signaling in heart failure. Annu Rev Pharmacol Toxicol 1999; 39: 34360[CrossRef][ISI][Medline]
73 Pouyssegur J, Volmat V, Lenormand P. Fidelity and spatio-temporal control in MAPK kinase (ERKs) signalling. Biochem Pharmacol 2002; 64: 75563[CrossRef][ISI][Medline]
74 Riemann B, Schäfers M, Law MP, Wichter T, Schober O. Radioligands for imaging myocardial - and ß-adrenoceptors. Nuklearmedizin 2003; 1: 49
75 Riento K, Ridley AJ. Rocks: multifunctional kinases in cell behaviour. Nat Rev Cell Biol 2003; 4: 44656[CrossRef][ISI]
76 Rockman HA, Koch WJ, Lefkowitz RJ. Seven-transmembrane-spanning receptors and heart function. Nature 2002; 414: 20612
77 Saimi Y, Kung C. Calmodulin as an ion channel subunit. Annu Rev Physiol 2002; 64: 289311[CrossRef][ISI][Medline]
78 Salahpour A, Angers S, Bouvier M. Functional significance of oligomerization of G-protein-coupled receptors. Trends Endocrinol Metab 2000; 11: 1638[CrossRef][ISI][Medline]
79 Schaub MC, Hefti MA, Zuellig RA, Morano I. Modulation of contractility in human cardiac hypertrophy by myosin essential light chain isoforms. Cardiovasc Res 1998; 37: 381404[CrossRef][ISI][Medline]
80 Scholz J, Tonner PH. 2-Adrenoceptor agonists in anaesthesia: a new paradigm. Curr Opin Anesthesiol 2000; 13: 43742[CrossRef]
81 Schubert R, Nelson MT. Protein kinases: tuners of the BKCa channel in smooth muscle. Trends Pharmacol Sci 2001; 22: 50512[CrossRef][ISI][Medline]
82 Schulze DH, Muqhal M, Lederer WJ, Ruknudin AM. Sodium/calcium exchanger (NCX1) macromolecular complex. J Biol Chem 2003; 278: 2884955
83 Shakur Y, Fong M, Hensley J, et al. Comparison of the effects of Cilostazol and milrinone on cAMP-PDE activity, intracellular cAMP and calcium in the heart. Cardiovasc Drug Ther 2002; 16: 41727[CrossRef][ISI][Medline]
84 Shin HM, Je HD, Gallant C, et al. Differential association and localization of myosin phosphatase subunits during agonist-induced signal transduction in smooth muscle. Circ Res 2002; 90: 54653
85 Skalhegg B, Tasken K. Specificity in the cAMP/PKA signaling pathway. Differential expression, regulation, and subcellular localization of subunits of PKA. Front Biosci 2000; 5: 67893
86 Somlyo AP, Somlyo AV. Ca2+ sensitivity of smooth muscle and non-muscle myosin-II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 2003; 83: 132558
87 Steiberg SF, Brunton LL. Compartmentation of G protein-coupled signaling pathways in cardiac myocytes. Annu Rev Pharmacol Toxicol 2001; 41: 75173[CrossRef][ISI][Medline]
88 Striggow F, Ehrlich BE. Ligand-gated calcium channels inside and out. Curr Opin Cell Biol 1996; 8: 4905[CrossRef][ISI][Medline]
89 Swärd K, Mita M, Wilson DP, Deng JT, Susnjar M, Walsh MP. The role of RhoA and Rho-associated kinase in vascular smooth muscle contraction. Curr Hypertension Reports 2003; 5: 6672[ISI]
90 Tavernier G, Toumaniantz G, Erfanian M, et al. ß3-Adrenergic stimulation produces a decrease of cardiac contractility ex vivo in mice overexpressing the human ß3-adrenergic receptor. Cardiovasc Res 2003; 59: 28896[CrossRef][ISI][Medline]
91 Toyoshima C, Asahi M, Sugita Y, Khanna R, Tsuda T, MacLennan DH. Modeling of the inhibitory interaction of phospholamban with the Ca2+ ATPase. Proc Natl Acad Sci USA 2003; 100: 46772
92 Turnbull L, McCloskey DT, OConnell T, Simpson PC, Baker AJ. 1-adrenergic receptor response in
1AB-AR knockout mouse hearts suggest the presence of
1D-AR. Am J Physiol 2003; 284: H11049[ISI]
93 Walsh KB, Cheng Q. Intracellular Ca2+ regulates responsiveness of cardiac L-type Ca2+ current to protein kinase-A: role of calmodulin. Am J Physiol 2004; 286: H18694[ISI]
94 Wenzel-Seifert K, Seifert R. Molecular analysis of ß2-adrenoceptor coupling to Gs-, Gi, and Gq-proteins. Mol Pharmacol 2000; 58: 95466
95 Wier WG, Morgan KG. Alpha1-adrenergic signaling mechanisms in contraction of resistance arteries. Rev Physiol Biochem Pharmacol 2004; 150: 91139
96 Xiang Y, Kobilka BK. Myocyte adrenoceptor signaling pathways. Science 2003; 300: 15302
97 Zamah AM, Delahunty M, Luttrell LM, Lefkowitz RJ. Protein kinase A-mediated phosphorylation of the ß2-adrenergic receptor regulates its coupling to Gs and Gi. J Biol Chem 2002; 277: 3124956
98 Zaugg M, Schaub MC, Pasch T, Spahn DR. Modulation of ß-adrenergic receptor subtype activities in perioperative medicine: mechanisms and sites of action. Br J Anaesth 2002; 88: 10123
99 Zaugg M, Schaub MC. Signaling and cellular mechanisms in cardiac protection by ischemic and pharmacological preconditioning. J Muscle Res Cell Motil 2003; 24: 21949[CrossRef][ISI][Medline]
100 Zylinska L, Kawecka I, Lachowicz L, Szemraj J. The isoform- and location-dependence of the functioning of the plasma membrane calcium pump. Cell Mol Biol Lett 2002; 7: 103745[ISI][Medline]