Constitutive phosphodiesterase activity restricts spontaneous beating rate of cardiac pacemaker cells by suppressing local Ca2+ releases.

Spontaneous beating of rabbit sinoatrial node cells (SANCs) is controlled by cAMP-mediated, protein kinase A-dependent local subsarcolemmal ryanodine receptor Ca(2+) releases (LCRs). LCRs activated an inward Na(+)/Ca(2+) exchange current that increases the terminal diastolic depolarization rate and, therefore, the spontaneous SANC beating rate. Basal cAMP in SANCs is elevated, suggesting that cAMP degradation by phosphodiesterases (PDEs) may be low. Surprisingly, total suppression of PDE activity with a broad-spectrum PDE inhibitor, 3'-isobutylmethylxanthine (IBMX), produced a 9-fold increase in the cAMP level, doubled cAMP-mediated, protein kinase A-dependent phospholamban phosphorylation, and increased SANC firing rate by approximately 55%, indicating a high basal activity of PDEs in SANCs. A comparison of specific PDE1 to -5 inhibitors revealed that the specific PDE3 inhibitor, milrinone, accelerated spontaneous firing by approximately 47% (effects of others were minor) and increased amplitude of L-type Ca(2+) current (I(Ca,L)) by approximately 46%, indicating that PDE3 was the major constitutively active PDE in the basal state. PDE-dependent control of the spontaneous SANC firing was critically dependent on subsarcolemmal LCRs, ie, PDE inhibition increased LCR amplitude and size and decreased LCR period, leading to earlier and augmented LCR Ca(2+) release, Na(+)/Ca(2+) exchange current, and an increase in the firing rate. When ryanodine receptors were disabled by ryanodine, neither IBMX nor milrinone was able to amplify LCRs, accelerate diastolic depolarization rate, or increase the SANC firing rate, despite preserved PDE inhibition-induced augmentation of I(Ca,L) amplitude. Thus, basal constitutive PDE activation provides a novel and powerful mechanism to decrease cAMP, limit cAMP-mediated, protein kinase A-dependent increase of diastolic ryanodine receptor Ca(2+) release, and restrict the spontaneous SANC beating rate.

Muscarinic modulation of the sodium-calcium exchanger in heart failure.

BACKGROUND: The Na-Ca exchanger (NCX) is a critical calcium efflux pathway in excitable cells, but little is known regarding its autonomic regulation. METHODS AND RESULTS: We investigated beta-adrenergic receptor and muscarinic receptor regulation of the cardiac NCX in control and heart failure (HF) conditions in atrially paced pigs. NCX current in myocytes from control swine hearts was significantly increased by isoproterenol, and this response was reversed by concurrent muscarinic receptor stimulation with the addition of carbachol, demonstrating "accentuated antagonism." Okadaic acid eliminated the inhibitory effect of carbachol on isoproterenol-stimulated NCX current, indicating that muscarinic receptor regulation operates via protein phosphatase-induced dephosphorylation. However, in myocytes from atrially paced tachycardia-induced HF pigs, the NCX current was significantly larger at baseline but less responsive to isoproterenol compared with controls, whereas carbachol failed to inhibit isoproterenol-stimulated NCX current, and 8-Br-cGMP did not restore muscarinic responsiveness. Protein phosphatase type 1 dialysis significantly reduced NCX current in failing but not control cells, consistent with NCX hyperphosphorylation in HF. Protein phosphatase type 1 levels associated with NCX were significantly depressed in HF pigs compared with control, and total phosphatase activity associated with NCX was significantly decreased. CONCLUSIONS: We conclude that the NCX is autonomically modulated, but HF reduces the level and activity of associated phosphatases; defective dephosphorylation then "locks" the exchanger in a highly active state.

High basal protein kinase A-dependent phosphorylation drives rhythmic internal Ca2+ store oscillations and spontaneous beating of cardiac pacemaker cells.

Local, rhythmic, subsarcolemmal Ca2+ releases (LCRs) from the sarcoplasmic reticulum (SR) during diastolic depolarization in sinoatrial nodal cells (SANC) occur even in the basal state and activate an inward Na(+)-Ca2+ exchanger current that affects spontaneous beating. Why SANC can generate spontaneous LCRs under basal conditions, whereas ventricular cells cannot, has not previously been explained. Here we show that a high basal cAMP level of isolated rabbit SANC and its attendant increase in protein kinase A (PKA)-dependent phosphorylation are obligatory for the occurrence of spontaneous, basal LCRs and for spontaneous beating. Gradations in basal PKA activity, indexed by gradations in phospholamban phosphorylation effected by a specific PKA inhibitory peptide were highly correlated with concomitant gradations in LCR spatiotemporal synchronization and phase, as well as beating rate. Higher levels of basal PKA inhibition abolish LCRs and spontaneous beating ceases. Stimulation of beta-adrenergic receptors extends the range of PKA-dependent control of LCRs and beating rate beyond that in the basal state. The link between SR Ca2+ cycling and beating rate is also present in vivo, as the regulation of beating rate by local beta-adrenergic receptor stimulation of the sinoatrial node in intact dogs is markedly blunted when SR Ca2+ cycling is disrupted by ryanodine. Thus, PKA-dependent phosphorylation of proteins that regulate cell Ca2+ balance and spontaneous SR Ca2+ cycling, ie, phospholamban and L-type Ca2+ channels (and likely others not measured in this study), controls the phase and size of LCRs and the resultant Na(+)-Ca2+ exchanger current and is crucial for both basal and reserve cardiac pacemaker function.

Sodium/calcium exchanger expression in the mouse and rat olfactory systems.

Sodium/calcium (Na(+)/Ca(2+)) exchangers are membrane transport systems that regulate Ca(2+)-homeostasis in many eukaryotic cells. In olfactory and vomeronasal sensory neurons ligand-induced olfactory signal transduction is associated with influx and elevation of intracellular Ca(2+), [Ca(2+)](i). While much effort has been devoted to the characterization of Ca(2+)-related excitation and adaptation events of olfactory chemosensory neurons (OSNs), much less is known about mechanisms that return [Ca(2+)](i) to the resting state. To identify proteins participating in the poststimulus Ca(2+)-clearance of mouse OSNs, we analyzed the expression of three potassium (K(+))-independent (NCX1, 2, 3) and three K(+)-dependent (NCKX1, 2, 3) Na(+)/Ca(2+) exchangers. In situ hybridization showed that mRNAs of all six Na(+)/Ca(2+) exchangers coexist in neurons of the olfactory and vomeronasal systems, and that some are already detectable in the embryo. Of these, NCX1 and NCKX1 represent the most and least abundant mRNAs, respectively. Moreover, immunohistochemistry revealed that the NCX1, 2, and 3 proteins are expressed in nearly all neurons of the olfactory epithelium, the vomeronasal organ, the septal organ of Masera, and the Grueneberg ganglion. These three exchanger proteins display different expression profiles in dendrites, knobs, and plasma membranes of OSNs and in sustentacular cells. Furthermore, we show that NCX1 mRNA in rat olfactory mucosa is expressed as 8 alternative splice variants. This is the first comprehensive analysis of Na(+)/Ca(2+) exchanger expression in the mammalian olfactory system. Our results suggest that Ca(2+)-extrusion by OSNs utilizes multiple different Na(+)/Ca(2+) exchangers and that different subtypes are targeted to different subcellular compartments.

The regulation of the Na/Ca exchanger and plasmalemmal Ca2+ ATPase by other proteins.

Na/Ca exchanger (NCX) and plasma membrane Ca2+ ATPase are the Ca2+ efflux mechanisms known in mammalian cells. NCX is the main transporter to efflux intracellular Ca2+ in the heart. NCX protein contains nine putative transmembrane domains and a large intracellular loop joining two sets of the transmembrane domains. The intracellular loop regulates the activity of the NCX by interacting with other proteins and nonprotein factors, such as ions, PIP2. Several proteins that are associated with NCX have been identified recently. Similarly, plasmalemmal Ca2+ ATPase (PMCA) has 10 putative transmembrane domains, and the C-terminal intracellular region inhibits transporter activity. There are several proteins associated with PMCA, and the roles of the associated proteins of PMCA vary from specific localization to involving PMCA in signal transduction. Elucidation of structural and functional roles played by these associated proteins of NCX and PMCA will provide opportunities to develop drugs of potential therapeutic value.

Phosphorylation and other conundrums of Na/Ca exchanger, NCX1.

The Na+/Ca2+ exchanger (NCX) is an important Ca2+ transport mechanism in virtually all cells in the body. There are three genes that control the expression of NCX in mammals. There are at least 16 alternatively spliced isoforms of NCX1 that target muscle and nerve and other tissues. Here we briefly discuss three remarkable regulatory issues or "conundrums" that involve the most prevalently expressed gene, NCX1. (1) How is NCX1 regulated by phosphorylation? We suggest that the macromolecular complex of NCX1 plays a critical role in the regulation of NCX. The role of the macromolecular complex and evidence supporting its existence and functional importance is presented. (2) Can there be transport block of a single "mode" of NCX1 transport by drugs or therapeutic agents? The simple answer is "no." A brief explanation is provided. (3) How can NCX1 knockout mice live? The answer is "by other compensatory regulatory mechanisms." These conundrums highlight important features in NCX1 and lay the foundation for new experiments to elucidate function and regulation of NCX1 and provide a context for investigations that seek to understand novel therapeutic agents.

The integration of spontaneous intracellular Ca2+ cycling and surface membrane ion channel activation entrains normal automaticity in cells of the heart's pacemaker.

Although the ensemble of voltage- and time-dependent rhythms of surface membrane ion channels, the membrane "Clock", is the immediate cause of a sinoatrial nodal cell (SANC) action potential (AP), it does not necessarily follow that this ion channel ensemble is the formal cause of spontaneous, rhythmic APs. SANC also generates intracellular oscillatory spontaneous Ca(2+) releases that ignite excitation (SCaRIE) of the surface membrane via Na(+)/Ca(2+) exchanger activation. The idea that a rhythmic intracellular Ca(2+) Clock might keep time for normal automaticity of SANC, however, has not been assimilated into mainstream pacemaker dogma. Recent experimental evidence, derived from simultaneous, confocal imaging of submembrane Ca(2+) and membrane potential of SANC, and supported by numerical modeling, indicates that normal automaticity of SANC is entrained and stabilized by the tight integration of the SR Ca(2+) Clock that generates rhythmic SCaRIE, and the surface membrane Clock that responds to SCaRIE to immediately produce APs of an adequate shape. Thus, tightly controlled, rhythmic SCaRIE does not merely fine tune SANC AP firing, but is the formal cause of the basal and reserve rhythms, insuring pacemaker stability by rhythmically integrating multiple Ca(2+)-dependent functions, and effects normal automaticity by rhythmic ignition of the surface membrane Clock.

Protein kinase A hyperphosphorylation increases basal current but decreases beta-adrenergic responsiveness of the sarcolemmal Na+-Ca2+ exchanger in failing pig myocytes.

The sodium-calcium exchanger (NCX) protein is the major cardiac calcium extrusion mechanism and is upregulated in heart failure (HF). NCX expression level and functional activity as regulated by beta-adrenergic receptor (beta-AR) stimulation in swine with and without tachycardia-induced heart failure were studied. The Ni2+-sensitive NCX current was measured in myocytes from HF and control animals in the basal state or in the presence of isoproterenol, forskolin, 8-Br-cAMP, okadaic acid, or protein phosphatase type 1. Western blot analysis revealed a significant increase in both the 120-kDa (29%) and 80-kDa (69%) fragments in HF (P<0.05 versus control). Despite this modest increase in protein, the basal peak outward NCX current was increased almost 5-fold in HF (P<0.05 versus control). Stimulation with isoproterenol, however, increased the control currents to a significantly greater extent than HF (500% increase in control versus 100% increase in HF, P<0.01); peak stimulated current was not different in HF and control. This reduction in responsiveness to beta-AR stimulation was refractory to forskolin, 8-Br-cAMP, or okadaic acid stimulation. In vitro protein kinase A back-phosphorylation revealed higher phosphorylation capacity of NCX protein in control versus HF, consistent with increased phosphorylation in vivo (hyperphosphorylation) in HF. Protein phosphatase type 1 exposure resulted in a significant reduction (73%) in peak basal current in HF (compared with no significant difference in controls), confirming that the increased basal NCX current in HF is predominantly attributable to hyperphosphorylation. NCX expression and activity are thus increased in HF, although beta-AR responsiveness is decreased because of NCX hyperphosphorylation.

Ca(2+) -stimulated basal adenylyl cyclase activity localization in membrane lipid microdomains of cardiac sinoatrial nodal pacemaker cells.

Spontaneous, rhythmic subsarcolemmal local Ca(2+) releases driven by cAMP-mediated, protein kinase A (PKA)-dependent phosphorylation are crucial for normal pacemaker function of sinoatrial nodal cells (SANC). Because local Ca(2+) releases occur beneath the cell surface membrane, near to where adenylyl cyclases (ACs) reside, we hypothesized that the dual Ca(2+) and cAMP/PKA regulatory components of automaticity are coupled via Ca(2+) activation of AC activity within membrane microdomains. Here we show by quantitative reverse transcriptase PCR that SANC express Ca(2+)-activated AC isoforms 1 and 8, in addition to AC type 2, 5, and 6 transcripts. Immunolabeling of cell fractions, isolated by sucrose gradient ultracentrifugation, confirmed that ACs localize to membrane lipid microdomains. AC activity within these lipid microdomains is activated by Ca(2+) over the entire physiological Ca(2+) range. In intact SANC, the high basal AC activity produces a high level of cAMP that is further elevated by phosphodiesterase inhibition. cAMP and cAMP-mediated PKA-dependent activation of ion channels and Ca(2+) cycling proteins drive sarcoplasmic reticulum Ca(2+) releases, which, in turn, activate ACs. This feed forward "fail safe" system, kept in check by a high basal phosphodiesterase activity, is central to the generation of normal rhythmic, spontaneous action potentials by pacemaker cells.

Sodium/calcium exchanger (NCX1) macromolecular complex.

The sodium-calcium exchanger, NCX1, is a ubiquitously expressed membrane protein essential in calcium homeostasis for many cells including those in mammalian heart and brain. The function of NCX1 depends on subcellular ("local") factors, the phosphorylation state of NCX1, and the subcellular location of NCX1 within the cell. Here we investigate the molecular organization of NCX1 within the cardiac myocyte. We show that NCX1 is dynamically phosphorylated by protein kinase A (PKA)-dependent phosphorylation in vitro. We also provide evidence that the regulation of this phosphorylation is attributed to the existence of an NCX1 macromolecular complex. Specifically, we show that the macromolecular complex includes both the catalytic and regulatory subunits of PKA. However, only the RI regulatory subunit is found in this macromolecular complex, not RII. Other critical regulatory enzymes are also associated with NCX1, including protein kinase C (PKC) and two serine/threonine protein phosphatases, PP1 and PP2A. Importantly, the protein kinase A-anchoring protein, mAKAP, is found and its presence in the macromolecular complex suggests that these regulatory enzymes are coordinately positioned to regulate NCX1 as has been found in diverse cells for a number of channel proteins. Dual immunocytochemical staining showed the colocalization of NCX1 protein with mAKAP and PKA-RI proteins in cardiomyocytes. Finally, leucine/isoleucine zipper motifs have been identified as possible sites of interaction. Our finding of an NCX1 macromolecular complex in heart suggests how NCX1 regulation is achieved in heart and other cells. The existence of the NCX1 macromolecular complex may also provide an explanation for recent controversial findings.

Characterization of Cah, a calcium-binding and heat-extractable autotransporter protein of enterohaemorrhagic Escherichia coli.

We have identified and characterized a protein of enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7 that shares homology with antigen 43 and AIDA-I of E. coli. The gene encoding this protein consists of a 2850 bp open reading frame and was named cah for calcium binding antigen 43 homologue. The prototype EHEC strain EDL933 possesses identical duplicate copies of cah (cah1 and cah2), which showed 100% identity at the nucleotide level. We showed that E. coli K-12 containing the recombinant cah gene produced two proteins, an approximately 80 kDa outer membrane protein and a 43.0 kDa heat-extractable protein. The Cah protein contains a predicted 52-amino-acid extended signal sequence found in several autotransporter proteins, and N-terminal sequencing data indicated that the 43.0 kDa passenger protein was derived from cleavage of the signal sequence from alanine at position 53. Phenotypes such as autoaggregation and change in bacterial shape were observed when a recombinant plasmid containing the cah gene was introduced into a laboratory E. coli strain, and these phenotypes were eliminated upon mutation of the cah gene. The passenger domain contains six domains found in calcium-binding proteins, and the recombinant Cah passenger protein bound 45Ca2+. In E. coli O157:H7, Cah is a heat-extractable protein, the expression of which is induced in minimal essential media and under divalent ion-depleting conditions; it also participates in the formation of biofilms. Our results provide insight into the expression, secretion and preliminary features of the calcium-binding Cah autotransporter protein of EHEC O157:H7.