| Mapping the cellular mechanisms regulating atrial natriuretic peptide secretion: | ||
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Hormones, growth factors and autocrine/paracrine factors act by binding to specific receptors to initiate diverse cellular events. These factors bind to membrane-associated receptors to trigger a cascade of secondary events, including the generation of diffusible intracellular second messengers (Davis et al. 1992a). Membrane receptors are linked to an intracellular effector system via intermediate transducing proteins. A family of these membrane-bound transducing proteins has been identified, which bind guanine nucleotides (guanosine triphosphate and guanosine diphosphate, GTP and GDP), and are hence termed G-proteins (Strader et al. 1994, Gudermann et al. 1997). Several hundred G-protein-coupled receptors have been cloned (Strader et al. 1994, Gudermann et al. 1997).
The G-proteins are located on the cytoplasmic surface of the cell membrane. Each G-protein is a heterotrimer containing α-, β - and γ -subunits and they are classified based upon the amino acid sequence similarity of their α-subunits (Strader et al. 1994, Gudermann et al. 1997). Upon stimulation by ligand-receptor complex, the G-protein exchanges a bound GDP molecule for GTP, and the β - and γ -subunits then dissociate from the Gα-subunit. Both GTP-bound α-subunits and β γ dimers are signaling molecules and modulate the activity of coupled effectors, such as enzymes, ion channels, and transporters, resulting in rapid alterations of second messenger (Sternweis & Smrcka 1992, Exton 1994, Clapham 1994, Birnbaumer & Birnbaumer 1995, Gudermann et al. 1997, Dolphin 1998). There is a wide diversity of G proteins involved in signal transduction (Simon et al. 1991). Main types include Gi proteins that inhibit adenylyl cyclase and activation of potassium channels, Gs proteins that stimulate adenylyl cyclase and activate Ca2+ channels, and Gq proteins that activate phospholipase C (PLC). G-protein-regulated macromolecules known to affect cardiac cellular responses include the enzymes adenylyl cyclase, PLC, phospholipase A2 (PLA2) and guanylyl cyclase (for review see Fleming et al. 1992, Johnson & Friedman 1993). Thus, it is evident that G proteins are essential links in the cascade of biochemical events that ensue when neurotransmitters and hormones interact with receptors on myocardial cells.
The efficacy of signal transduction systems may be modified in a number of ways. These include the modulation of receptor number and/or function, desensitation of post-receptor coupling systems, and antagonistic or synergistic interactions between parallel signaling systems (Davis et al. 1992a, Böhm et al. 1997). For example, in human heart there are many receptor systems that regulate contractility and heart rate. In chronic heart failure a decrease in β 1-adrenoreceptor number and an increase in the functional activity of Gi will lead to reduced physiologic responses of the failing heart to β -adrenergic stimulation (Brodde et al. 1995).
Protein kinases regulate numerous biological processes. In the cardiac myocytes, these include the regulation of contraction, ion transport, fuel metabolism and gene expression and growth (for review see Sugden & Bogoyevich 1995). Protein kinases often participate in the transduction of a (extracellular) signal to a biological response. Binding of the regulatory molecule to its membrane receptor often changes the intracellular level of one of the second messengers, which then modulates the activity of protein kinase (Table 2). Many protein kinases require a change in phosphorylation state (phosphorylation or dephosphorylation) for activity. When the phosphorylated form of the enzyme is active, phosphorylation can be either permissive or modulatory. The majority of protein kinases transfer the γ -phosphate group from ATP to hydroxyl groups of serine/threonine residues (protein Ser/Thr kinases) or tyrosine residues (protein tyrosine kinases, PTKs) in proteins, including a change in activity or function of the substrate enzyme or protein. For many protein Ser/Thr kinases, the transduction process involves receptor activation followed by the synthesis or release of a second messenger. In contrast, the PTKs are often receptors themselves (Fantl et al. 1993) but the existence of nonreceptor PTKs is also well-established (Bolen 1993).
Table 2. Second messenger-dependent protein kinases.
| Protein kinase | Abbreviations | Activator |
|---|---|---|
| cyclic AMP-dependent protein kinase | PKA | cyclic AMP |
| cyclic GMP-dependent protein kinase | PKG | cyclic GMP |
| Ca2+/calmodulin-dependent protein kinase | CaM kinase | 4(Ca2+)-calmodulin complex |
| Protein kinase C | PKC | Ca2+ and 1,2-diacylglycerol |
Processes that are reversibly controlled by protein phosphorylation require not only protein kinases but also protein phosphatases (PPs)(Hunter 1995). Target proteins are phosphorylated at specific sites by one or more protein kinases, and these phosphates are removed by specific PPs. The extent of phosphorylation on a particular site can be regulated by changing the activity of the protein kinases or phosphatases or both (Hunter 1995). The maximal activities of the protein kinases and phosphatases acting on a particular site must be in the controlled balance; otherwise, the protein would be either fully phosphorylated or completely dephosphorylated. Several different protein kinase or phosphatase inhibitors can be used to determine the physiological significance of the protein phosphorylation systems in various types of cells (Hidaka & Kobayashi 1992).
Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important component of several intracellular signaling pathways. Many cellular responses to the occupancy of membrane receptors include the hydrolysis of PIP2 by PLC and the subsequent generation of second messengers inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) (for review see Nishizuka 1992, Divecha & Irvine 1995). PIP2 is a precursor not only for the second messengers IP3 and DAG, but also for the putative messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3), which is produced by phosphatidylinositol 3-kinase (PI-3 kinase). PIP2 is also known to serve as a regulator of many actin-binding proteins, and as a cofactor for an isoform of phospholipase D (PLD) (for review see Lee & Rhee 1995).
All PLC enzymes identified up to now are single polypeptides that can be divided into three types (β , γ , θ )(Lee & Rhee 1995). It is generally accepted that there are at least two distinct mechanisms to link receptor occupancy to the activation of PLC isoenzymes. PLC-β isoenzymes are activated by G proteins (Sternweis & Smrcka 1992, Exton 1994), while PLC-γ isoenzymes are activated by receptor and nonreceptor PTKs (Lee & Rhee 1995). The activation mechanism of PLC-θ is not known at the present time. Cardiac myocytes predominantly express PLC-β (Hansen et al. 1995).
IP3, by mediating Ca2+ mobilization, acts synergistically with DAG to activate PKC. Activation of PKC is thought to involve the redistribution of the enzyme from a cytosolic location in resting cells to a membrane-associated site during stimulation. A rise in internal Ca2+ alone can bring about this redistribution and activation of PKC (Nishizuka 1992, Newton 1997). Isotope-labeling studies indicate that DAG is metabolized very rapidly either when it is produced endogenously or when it is added exogenously to cells (Nishizuka 1992). DAG may be phosphorylated to form phosphatidic acid, and then resynthesized back to phosphatidylinositol, or it can be hydrolyzed by DAG lipase to form arachidonic acid, which in turn can act as a second messenger. Thus, PKC is active for only a short time after the stimulation.
As mentioned earlier, in response to agonist, DAG is initially produced as a result of hydrolysis of inositol phospholipids (PIP2). This DAG production is transient and it is frequently followed by a more sustained increase in the amount of DAG. This second phase of DAG formation results from hydrolysis of phosphatidylcholine. Phosphatidylcholine is degraded by PLC to produce DAG and cholinephosphate or by PLD to produce choline and phosphatic acid, which is further dephosphorylated to DAG by phosphomonoesterase (Nishizuka 1992). In addition, a potential role of free fatty acids as second messengers has been suggested (for review see Asaoka et al. 1992, Azzi et al. 1992, Nishizuka 1992). PLA2 hydrolyzes phospholipids to liberate free fatty acids and lysophospholipids. Several cis unsaturated fatty acids, which are produced from phospholipids by the action of nonselective type of PLA2, enhance the DAG-dependent activation of PKC, and allow PKC to exhibit nearly full activity in the presence of low amounts of Ca2+. However, it is thought that the activity of PKC is increased initially by the increase in the concentrations of intracellular Ca2+ and DAG. The activity of PKC may then be sustained, even after the concentration of intracellular Ca2+ is no longer increased, if DAG and cis unsaturated fatty acids both become available (Asaoka et al. 1992, Nishizuka 1992).
PKC represents a structurally homologous group of proteins similar in size, structure and mechanism of activation. Several isoenzymes have been defined which are derived both from multiple genes and alternative splicing of a single RNA transcript (Azzi et al. 1992, Hug & Sarre 1993). PKC has been subdivided into three groups: 1) the conventional PKCs (α,β , γ ) which are regulated by Ca2+, phosphatidylserine, and DAG, 2) the novel PKCs (δ, ε, η , θ ) which are regulated by DAG and phosphatidylserine, 3) the atypical PKCs (ι, λ , ζ ) which are Ca2+ and DAG independent but are all dependent on phosphatidylserine (Sugden & Bogoyevitch 1995, Hofmann 1997, Newton 1997). At least five different isoforms of PKCs (α, β , δ, η and ζ isoforms) have been revealed in both adult and neonatal rat cardiac myocytes and four of these isoforms are Ca2+ independent subtypes (Kohout & Rogers 1993, Sugden & Bogoyevitch 1995).
PKC participates in one of the major signal transduction systems triggered by the external stimulation of cells by various ligands including hormones, neurotransmitters and growth factors (Azzi et al. 1992, Davis et al. 1992a, Nishizuka 1992). Several physiological functions have been assigned to PKC, including involment in secretion and exocytosis, modulation of ion conductance, interaction and down-regulation of receptors, smooth muscle contraction, gene expression, and cell proliferation. The connection between PKC activation and its cellular responses is mediated by proteins, which become phosphorylated. The phosphorylated residues on the target proteins are serine and threonines and PKC is known as a Ser/Thr kinase. Several substrate proteins of PKC have been identified, including receptor proteins, membrane proteins such as Ca2+-ATPase and Na+/K+-ATPase, contractile proteins such as myosin light chain, and enzymes such as myosin light chain kinase and guanylate cyclase (Azzi et al. 1992). PKC can phosphorylate and activate sarcoplasmic reticulum (SR) Ca2+ channels and it may also phosphorylate PLC to inactivate it. In addition, mitogen-activated protein kinase (MAPK) is a Ser/Thr-protein kinase, whose enzymatic activity requires phosphorylation of both threonine and tyrosine residues and it is activated by PKC (Andersson et al. 1990, Adams & Parker 1991).
In cardiac cells, stimulation of β -adrenergic receptors (β 1/β 2) on the sarcolemma by noradrenaline activates the stimulatory Gs protein by promoting the exchange of GDP for GTP. This reaction catalyzes the dissociation of the GTP-bound Gsα subunit from Gβ γ . GTP-bound Gsα then binds to and stimulates adenylyl cyclase, a membrane-bound enzyme that catalyzes the conversion of ATP to cyclic adenosine monophosphate (cAMP) (Ishikawa & Homcy 1997). So far, at least nine isoforms of adenylyl cyclase are known, and types II-VII are also detected in cardiac tissue (Ishikawa & Homcy 1997). They all share the same membrane topology; tandem repetition of a six-transmembrane domain and a large cytoplasmic domain. Biochemical characterization of the enzyme obtained from various tissues demonstrated that there were two distinct subtypes: calmodulin (CaM)-sensitive and -insensitive isoforms. Types V and VI represent the major adenylyl cyclase isoforms in the heart. These isoforms are insensitive to both CaM and Gβ γ subunits (Ishikawa & Homcy 1997).
cAMP acts as an intracellular second messenger. Severalfold increases in cAMP levels can occur within a millisecond-to-second time frame of hormonal stimulation. cAMP, in turn, activates several target molecules, primarily cyclic AMP-dependent protein kinase (protein kinase A, PKA) by dissociating its regulatory subunit from the catalytic subunit leading to a phosphorylation cascade and activation of multiple proteins (Walsh & van Patten 1994). PKA is a member of Ser/Thr kinases. PKA phosphorylates at least the L-type Ca2+ channel, phospholamban and troponin I, and causes, for example, an increase in Ca2+ entry into the cardiocytes. β -adrenergic effects on both the rate and force of cardiac contraction are mediated by PKA (for review see Sugden & Bogoyevitch 1995). The activating effects of Gsα rapidly reverse when agonist occupancy of the receptor ceases and cAMP is rapidly catabolized to 5’AMP by phosphodiesterases (for reviev see Tang & Gilman 1992, Ishikawa & Homcy 1997). PKA is then inactivated by reassociation of the catalytic subunit with the regulatory subunit and phosphorylated proteins are rapidly dephosphorylated by specific phosphatases. Participation of adenylyl cyclase and cAMP in biological processes in intact cells can be studied by treatment with permeating analogues of cAMP or with agonist that directly activate the catalytic domain of adenylyl cyclase; for example forskolin and its homologues.
PKGs constitute a second group of cyclic nucleotide-dependent protein Ser/Thr kinases (Francis & Corbin 1994, Sugden & Bogoyevitch 1995). They are involved in the mediation of the effects of NO and natriuretic peptides which activate guanylyl cyclase (Chinkers & Garbers 1991, Maack 1992, Anand-Srivastava & Trachte 1993, Anderson et al. 1994), thereby increasing intracellular concentration of cGMP. The increased cGMP activates PKG, which in turn phosphorylates a number of smooth muscle proteins, including the myosin light chain leading further to the relaxation of smooth muscle and vasodilatation.
Ca2+ acts as an intracellular second messenger of large varieties in species and is involved in cellular processes ranging from contraction and secretion to gene expression (Gnegy 1993). Cells contain a number of Ca2+-binding proteins, and in most cell types, the major Ca2+-binding protein is CaM. CaM is a ubiquitous, multifunctional Ca2+ -binding protein that binds four Ca2+ ions (Gnegy 1993). This complex of 4(Ca2+)-CaM activates downstream targets and acts as Ca2+-dependent regulator of cyclic nucleotide metabolism, Ca2+-transport, protein phosphorylation-dephosphorylation cascades, ion transport, cytoskeletal function, and cell proliferation. CaM activates isoenzymes of enzymes such as adenylyl cyclase, cyclic nucleotide phosphodiesterase, Ca2+-ATPase, Mg2+-ATPase, calcineurin, NOS, and several protein kinases (for review see Gnegy 1993).
CaM also activates a class of proteins called Ca2+/CaM-dependent protein kinases (CaM kinases). These kinases are a structurally related group of enzymes that constitute a subfamily within the larger family of protein Ser/Thr kinases (Hanks & Quinn 1991). Currently recognized members of the CaM kinases are phosphorylase kinase, myosin light chain kinase and CaM kinase Ia/Ib, II, III and IV (Schulman 1993, Braun & Schulman 1995). CaM kinase II is a multifunctional protein kinase which is activated by CaM-mediated autophosphorylation, thus, CaM-binding removes the restraint of an autoinhibitory domain on autophosphorylation (Schulman 1993, Braun & Schulman 1995). CaM kinase II is also present in the heart (Mayer et al. 1994, Sugden & Bogoyevitch 1995, Braun & Schulman 1995). CaM kinase II has the ability to phosphorylate and alter the function of a variety of substrates including cardiac Ca2+-ATPase, IP3 receptor, NOS, caldesmon, myosin light chain kinase, phospholamban, PLA2, cardiac ryanodine receptor (RYR) and troponin I (Braun & Schulman 1995). This indicates that CaM kinase II has a central role in the regulation of cardiac exitation-contraction coupling, ion handling, and vascular smooth muscle contraction.
Characteristic feature of many receptors is that they have intrinsic PTK activity and belong to the receptor protein tyrosine kinase (RPTK) family (Fantl et al. 1993, van der Geer & Hunter 1994). Such receptors are coupled to the regulation of many cellular programs, such as growth and differentiation. Most known ligands for RPTKs are soluble proteins such as growth factors, but membrane-bound proteins as well as extracellular matrix proteins may also activate RPTKs (Fantl et al. 1993, van der Geer & Hunter 1994). Ligand binding to the extracellular domain of RPTK is followed by receptor dimerization, stimulation of PTK activity and autophosphorylation. This leads to downstream activation of a number of common signaling molecules such as PLC-γ , PI-3 kinase, GTPase-activating protein, pp60-src, p21ras, raf-1 kinase and MAPKs, and S6 ribosomal kinases (Carpenter 1992, Fantl et al. 1993, van der Geer & Hunter 1994). Another class of PTKs is nonreceptor PTKs which represent a collection of cellular enzymes that are grouped together because of their lack of extracellular sequences (Bolen 1993). A number of nonreceptor PTKs have been found to be associated with other cell surface proteins and shown to be capable of facilitating cell surface initiated signal transduction much like the class of RPTKs.
A number of PTKs have been shown to be activated by neuropeptides and mechanical forces. Bombesin, vasopressin and ET-1 have been shown to stimulate tyrosine phosphorylation (Zachary et al. 1992, Koide et al. 1992). In Dictyostelium cells tyrosine phosphorylation of actin correlates with changes in cell shape (Howard et al. 1993). Focal adhesion kinase (FAK) is a cytoplasmic PTK, specifically located at the focal adhesion, which has been shown to be phosphorylated and activated by a number of growth factors and integrin-dependent cell adhesion. Stretching of mesangial cells has been shown to stimulate FAK (Hamasaki et al. 1995). In fetal lung cell mechanical stretch caused activation of Src, nonreceptor type of PTKs, within 5 min (Liu et al. 1996). Since cardiac myocytes are also targets for the action of growth factors (Schneider & Parker 1990), PTKs may have some functional role in the regulation of cardiac cells.
MAPKs are a group of important mediators that transduce extracellular signals to intracellular responses. At least three different MAPK isoforms have recently been described in mammalian cells, including extracellular signal-regulated kinase (ERK), p38 kinase and stretch-activated protein kinase (SAPK), also known as c-Jun N-terminal protein kinase (Bokemeyer et al. 1996, Force et al. 1996). Recent developments have extensively characterized the ERK cascade, which is the best studied MAPK signaling cascade. ERK1/ERK2 are the forms of MAPKs expressed in the heart (Sugden & Bogoyevitch 1995). In addition, SAPK is a new subgroup of MAPKs also present in cardiac cells (Kyriakis et al. 1994, Force et al. 1996) activated in response to extracellular stimuli binding both to tyrosine kinase receptors or G-protein-coupled receptors. A common feature of all MAPK isoforms is the requirement for phosphorylation of both threonine and tyrosine regulatory sites by a specific upstream protein kinase for activation (Anderson et al. 1990). The ERK family member of MAPKs is activated by MEK (MAPK kinase / ERK kinase). In response to extracellular stimuli MAPKs regulate the transcriptional activity of several transcription factors via phosphorylation of either stimulatory or inhibitory regulatory sites. PTK receptors, G-protein coupled receptors and cytokine receptors were shown to be capable of activating the ERK cascade (for review see Bokemeyer et al. 1996). Ang II, ET-1 and fibroblast growth factor have been shown to be potent stimulators of ERK in cardiac myocytes (Sadoshima et al. 1993, Bogoyevitch et al. 1994, Lazou et al. 1994).
Ionized calcium is the most common signal transduction element in cells (for review see Davis et al. 1992a, Petersen et al. 1994, Clapham 1995). Changes in intracellular Ca2+ concentration can control specialized functions like excitability, contraction, exocytosis, metabolism and gene expression. In most cells, elevations in intracellular Ca2+ arise from Ca2+ entry via Ca2+ channels in the surface membrane, or Ca2+ discharge from internal stores, or both. In the beating heart Ca2+ homeostasis uses 20-25% of total energy output of myocytes (Langer 1992). Homeostasis is maintained by Ca2+ pumps in the sarcolemma and the SR, and by the sarcolemmal Na+-Ca2+ exchanger. Unlike many other second-messenger molecules, Ca2+ cannot be metabolized, so cells tightly regulate intracellular levels through numerous binding and specialized extrusion proteins.
Normal intracellular Ca2+ levels are 10,000-fold lower than the concentration found extracellularly (100 nM vs. 2 mM). At least two mechanisms exist to pump Ca2+ out of the cell, one involving a Ca2+-ATPase, and another a Na+/Ca2+ exchanger, which couples the cellular uptake of Na+ to Ca2+ extrusion. Of these, the Na+/Ca2+ exchanger has the largest transport capacity (Doohan & Rasmussen 1993). In cells Ca2+ is stored to the SR and mitochondria. Ca2+ pumps in the SR membrane (smooth endoplasmic reticulum calcium pumps, SERCA pumps) use ATP to pump Ca2+ ions into the SR, where they are sequestered by buffer molecules (Lytton & Nikam 1992, Clapham 1995). Ca2+ ion can also be removed to the extracellular space by plasma membrane Ca2+ pumps (PMCA pumps). Both SERCA and PMCA pumps are ATPases. The SERCA pumps are products of three different genes. SERCA2 pumps are expressed in cardiac (Grover & Khan 1992) and slow-twitch skeletal muscle, while SERCA1 pumps are expressed in fast-twitch skeletal muscle (Lytton & Nigam 1992, Clapham 1995). Intracellular Ca2+-binding proteins can be classified as trigger or buffer proteins (see Lytton & Nikam 1992, Clapham 1995). Trigger proteins, e.g. CaM, change their conformation upon binding Ca2+ and modulate effector molecules such as enzymes and ion channels. Ca2+ storage in SR is achieved by the low-affinity, high-capacity Ca2+-binding protein, e.g. in cardiac cells it is called calsequestrin.
The transduction of electrical signals to cellular function is initiated by the opening of voltage-gated Ca2+ channels in response to depolarization of the surface membrane. Ca2+ penetrates the plasma membrane through the Ca2+-channels which could be voltage-, receptor-, second messenger- or mechanically operated or tonically active (Tsien & Tsien 1990, Spedding & Paoletti 1992, Balke & Gold 1992). L-type voltage-operated Ca2+ channels are high-voltage activated channels (Miller 1992) and they are the major pathway for voltage-gated Ca2+ entry involved in the activation of contraction in heart and smooth muscle, and in the control of transmitter release from endocrine cells. T-type Ca2+ channels are low-voltage activated Ca2+ channels and the most obvious function of T-type channels is to support pacemaker activity (Balke & Gold, 1992). N-type and P-type voltage-operated Ca2+ channels are found in neurons and second messenger-operated Ca2+ channels in neutrophils, platelets and lymphocytes. Receptor-operated Ca2+ channels open in direct response to the binding of an external ligand and tonically active Ca2+ channels help to set resting Ca2+ level in muscle cells. The mechanically operated channels include stretch-activated and stretch-inactivated ion channels (Tsien & Tsien 1990, Spedding & Paoletti 1992).
IP3 and ryanodine receptors represent the two principal intracellular Ca2+ channels responsible for mobilizing stored Ca2+ (see Taylor & Marshall 1992, Berridge 1993, Tsunoda 1993, Ehrlich et al. 1994). Some cells either have ryanodine-sensitive Ca2+ stores or IP3-sensitive stores, whereas e.g. atrial cells contain both. In response to many stimuli both IP3 and DAG are formed by the hydrolysis of an inositol lipid precursor stored in the plasma membrane. This IP3 acts as an intracellular second messenger by binding to the specialized tetrameric IP3 receptor that spans the endoplasmic reticular membrane and by triggering release of Ca2+ from the endoplasmic reticulum. IP3 is the only known physiological activator of the IP3 receptor, and Ca2+ is the only known physiological inhibitor since high cytoplasmic Ca2+ concentration decreases the binding of IP3 to its own receptor (Ehrlich et al. 1994).
RYR is the other major intracellular Ca2+ channel and the RYRs identified in cardiac cells are called RYR2 (for review see Coronado et al. 1994, Meissner 1994). The plant alkaloid ryanodine opens the channels at nanomolar concentrations but closes them at micromolar doses. SR Ca2+ stores can be depleted by ryanodine. The activity of the RYR channel is strongly enhanced by adenine nucleotides, caffeine and Ca2+ itself and inhibited by procaine (Tsien & Tsien 1990, Coronado et al. 1994). RYR, when activated by Ca2+-release agents, induces a release of Ca2+ from the primary Ca2+-binding protein of SR, calsequestrin. Excitation-contraction coupling is initiated when depolarization permits Ca2+ to enter the sarcoplasm through voltage-dependent Ca2+ channels in the sarcolemma, releasing a large quantity of Ca2+ from the SR (Callewaert 1992). This is called Ca2+-induced Ca2+ release. Ca2+ interacts with troponin C and produces a conformational change in the regulatory complex of muscle filament, which moves tropomyosin away and allows actin and myosin to form cross-bridges and thus initiate contraction (Valdeolmillos et al. 1989). Relaxation occurs when Ca2+ dissociates from the contractile apparatus and is resequestered (Callewaert 1992, Barry & Bridge 1993). In cardiac cells RYRs may be up- or downregulated by the phosphorylation of protein kinases such as CaM kinase, PKA and PKC (Coronado et al. 1994).