Derek S. Damron

Sphingosylphosphorylcholine is a ligand for ovarian cancer G-protein-coupled receptor 1.

Sphingosylphosphorylcholine (SPC) is a bioactive lipid that acts as an intracellular and extracellular signalling molecule in numerous biological processes. Many of the cellular actions of SPC are believed to be mediated by the activation of unidentified G-protein-coupled receptors. Here we show that SPC is a high-affinity ligand for an orphan receptor, ovarian cancer G-protein-coupled receptor 1 (OGR1). In OGR1-transfected cells, SPC binds to OGR1 with high affinity (Kd = 33.3 nM) and high specificity and transiently increases intracellular calcium. The specific binding of SPC to OGR1 also activates p42/44 mitogen-activated protein kinases (MAP kinases) and inhibits cell proliferation. In addition, SPC causes internalization of OGR1 in a structurally specific manner.

β1-Adrenergic Receptor Autoantibodies Mediate Dilated Cardiomyopathy by Agonistically Inducing Cardiomyocyte Apoptosis

Background— Antibodies to the &bgr;1-adrenergic receptor (&bgr;1AR) are detected in a substantial number of patients with idiopathic dilated cardiomyopathy (DCM). The mechanism whereby these autoantibodies exert their pathogenic effect is unknown. Here, we define a causal mechanism whereby &bgr;1AR-specific autoantibodies mediate noninflammatory cardiomyocyte cell death during murine DCM. Methods and Results— We used the &bgr;1AR protein as an immunogen in SWXJ mice and generated a polyclonal battery of autoantibodies that showed selective binding to the &bgr;1AR. After transfer into naive male hosts, &bgr;1AR antibodies elicited fulminant DCM at high frequency. DCM was attenuated after immunoadsorption of &bgr;1AR IgG before transfer and by selective pharmacological antagonism of host &bgr;1AR but not &bgr;2AR. We found that &bgr;1AR autoantibodies shifted the &bgr;1AR into the agonist-coupled high-affinity state and activated the canonical cAMP-dependent protein kinase A signaling pathway in cardiomyocytes. These events led to functional alterations in intracellular calcium handling and contractile function. Sustained agonism by &bgr;1AR autoantibodies elicited caspase-3 activation, cardiomyocyte apoptosis, and DCM in vivo, and these processes were prevented by in vivo treatment with the pan-caspase inhibitor Z-VAD-FMK. Conclusions— Our data show how &bgr;1AR-specific autoantibodies elicit DCM by agonistically inducing cardiomyocyte apoptosis.

Lysophosphatidylcholine Inhibits Endothelial Cell Migration by Increasing Intracellular Calcium and Activating Calpain

Objective—Endothelial cell (EC) migration, essential for reestablishing arterial integrity after vascular injury, is inhibited by oxidized LDL (oxLDL) and lysophosphatidylcholine (lysoPC) that are present in the arterial wall. We tested the hypothesis that a mechanism responsible for lysoPC-induced inhibition is increased intracellular free calcium concentration ([Ca2+]i). Methods and Results—LysoPC, at concentrations that inhibit in vitro EC migration to 35% of control, increased [Ca2+]i levels 3-fold. These effects of lysoPC were concentration dependent and reversible. LysoPC induced Ca2+ influx within 10 minutes, and [Ca2+]i remained elevated for 2 hours. The calcium ionophore A23187 also increased [Ca2+]i and inhibited EC migration. Chelators of intracellular Ca2+ (BAPTA/AM and EGTA/AM) and nonvoltage-sensitive channel blockers (lanthanum chloride and gadolinium chloride) blunted the lysoPC-induced [Ca2+]i rise and partially preserved EC migration. After lysoPC treatment, calpain, a calcium-dependent cysteine protease, was activated, and cytoskeletal changes occurred. Calpain inhibitors (calpastatin, MDL28170, and calpeptin) added before lysoPC prevented cytoskeletal protein cleavage and preserved EC migration at 60% of control levels. Conclusions—LysoPC increases [Ca2+]i. In turn, activating calpains that can alter the cytoskeleton are activated and EC migration is inhibited.

Arachidonic Acid–Dependent Phosphorylation of Troponin I and Myosin Light Chain 2 in Cardiac Myocytes

Recent evidence has suggested that arachidonic acid (AA) may be an important signaling molecule in cardiac excitation-contraction coupling. We previously showed that AA and endothelin-1 (ET) inhibit distinct K+ channels via protein kinase C-dependent pathways in rat ventricular myocytes. In addition, we demonstrated that Ca2+ transients in populations of fura 2-loaded myocytes were potentiated by AA and ET via activation of protein kinase C. In this study, we have used suspensions of [32P]orthophosphate (32Pi)-labeled rat ventricular myocytes to study the effects of AA and ET at the level of the myofilaments. After a 10-minute incubation of the labeled cells with phorbol 12-myristate 13-acetate (PMA), AA, or ET in the presence or absence of the protein kinase C inhibitor calphostin C, the myofibrillar proteins were separated by PAGE. Measurement of unloaded cell shortening using video edge detection in single electrically stimulated myocytes was also used to assess the effects of AA and ET on myocyte contractility. Incubation with either PMA, AA, or ET resulted in similar increases in 32Pi incorporation into troponin I (TnI) and myosin light chain 2 (MLC2), which was inhibited by preincubation with the protein kinase C antagonist calphostin C. In addition, the ability of these agonists to stimulate phosphorylation of TnI or MLC2 did not require extracellular Ca2+ or intact intracellular Ca2+ stores. The effects of AA and ET together on phosphorylation of TnI or MLC2 were not additive.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of Ca2+ cycling in cardiac myocytes by arachidonic acid

It is believed that inotropic agents exert their effects in cardiac muscle via a modulation of Ca2+ cycling; however, the involvement of phospholipase activation and the biochemical pathways participating in inotropic responsiveness remain unclear. The aim of the current study was to determine whether arachidonic acid and/or eicosanoids participate in inotropic responses by modulating Ca2+ cycling in cardiac myocytes. Experiments were performed with populations of freshly isolated, fura-2-loaded adult rat ventricular myocytes. Arachidonic acid stimulated a transient increase in cytosolic free Ca2+, which was still present after addition of EGTA but was significantly reduced by pretreatment with caffeine. Addition of arachidonic acid to either electrically stimulated or quiescent myocytes enhanced the amplitude of the ATP-induced Ca2+ transient. This effect was still observed in the presence of inhibitors of cyclooxygenase, lipoxygenase, and epoxygenase pathways but was significantly diminished after pretreatment with inhibitors of protein kinase C. In contrast, arachidonic acid attenuated the amplitude of electrically induced Ca2+ transients. This effect was mimicked by eicosatetraynoic acid and by the K+ channel agonist pinacidil. The inhibitory effect of eicosatetraynoic acid and arachidonic acid was reversed by addition of fatty acid-free bovine serum albumin. Together, these results suggest that arachidonic acid may play a physiological role in cardiac muscle excitation-contraction coupling as a modulator of sarcolemmal ion channels and/or Ca2+ release from the sarcoplasmic reticulum.

Modulation of intracellular Ca2+ via α1B-adrenoreceptor signaling molecules, Gαh (transglutaminase II) and phospholipase C-δ1

Abstract We characterized the α1B-adrenoreceptor (α1B-AR)-mediated intracellular Ca2+ signaling involving Gαh (transglutaminase II, TGII) and phospholipase C (PLC)-δ1 using DDT1-MF2 cell. Expression of wild-type TGII and a TGII mutant lacking transglutaminase activity resulted in significant increases in a rapid peak and a sustained level of intracellular Ca2+ concentration ([Ca2+]i) in response to activation of the α1B-AR. Expression of a TGII mutant lacking the interaction with the receptor or PLC-δ1 substantially reduced both the peak and sustained levels of [Ca2+]i. Expression of TGII mutants lacking the interaction with PLC-δ1 resulted in a reduced capacitative Ca2+ entry. Reduced expression of PLC-δ1 displayed a transient elevation of [Ca2+]i and a reduction in capacitative Ca2+ entry. Expression of the C2-domain of PLC-δ1, which contains the TGII interaction site, resulted in reduction of the α1B-AR-evoked peak increase in [Ca2+]i, while the sustained elevation in [Ca2+]i and capacitative Ca2+ entry remained unchanged. These findings demonstrate that stimulation of PLC-δ1 via coupling of the α1B-AR with TGII evokes both Ca2+ release and capacitative Ca2+ entry and that capacitative Ca2+ entry is mediated by the interaction of TGII with PLC-δ1.

Propofol attenuates capacitative calcium entry in pulmonary artery smooth muscle cells.

BackgroundDepletion of intracellular Ca2+ stores results in capacitative Ca2+ entry (CCE) in pulmonary artery smooth muscle cells (PASMCs). The authors aimed to investigate the effects of propofol on CCE and to assess the extent to which protein kinase C (PKC) and tyrosine kinases mediate propofol-induced changes in CCE. MethodsPulmonary artery smooth muscle cells were cultured from explants of canine intrapulmonary artery. Fura 2–loaded PASMCs were placed in a dish (37°C) on an inverted fluorescence microscope. Intracellular Ca2+ concentration was measured using fura 2 in PASMCs using a dual-wavelength spectrofluorometer. Thapsigargin (1 &mgr;m), a sarcoplasmic reticulum Ca2+–adenosine triphosphatase inhibitor, was used to deplete intracellular Ca2+ stores after removing extracellular Ca2+. CCE was activated when extracellular Ca2+ (2.2 mm) was restored. ResultsThapsigargin caused a transient increase in intracellular Ca2+ concentration (182 ± 11%). Restoring extracellular calcium (to induce CCE) resulted in a peak (246 ± 12% of baseline) and a sustained (187 ± 7% of baseline) increase in intracellular Ca2+ concentration. Propofol (1, 10, 100 &mgr;m) attenuated CCE in a dose-dependent manner (peak: 85 ± 3, 70 ± 4, 62 ± 4%; sustained: 94 ± 5, 80 ± 5, 72 ± 5% of control, respectively). Tyrosine kinase inhibition (tyrphostin 23) attenuated CCE (peak: 67 ± 4%; sustained: 74 ± 5% of control), but the propofol-induced decrease in CCE was still apparent after tyrosine kinases inhibition. PKC activation (phorbol 12-myristate 13-acetate) attenuated CCE (peak: 48 ± 1%; sustained: 53 ± 3% of control), whereas PKC inhibition (bisindolylmaleimide) potentiated CCE (peak: 132 ± 11%; sustained: 120 ± 4% of control). Moreover, PKC inhibition abolished the propofol-induced attenuation of CCE. ConclusionTyrosine kinases activate and PKC inhibits CCE in PASMCs. Propofol attenuates CCE primarily via a PKC-dependent pathway. CCE should be considered a possible cellular target for anesthetic agents that alter vascular smooth muscle tone.

Experimental Conditions Are Important Determinants of Cardiac Inotropic Effects of Propofol

Background:The rationale for this study is that the depressant effect of propofol on cardiac function in vitro is highly variable but may be explained by differences in the temperature and stimulation frequency used for the study. Both temperature and stimulation frequency are known to modulate cellular mechanisms that regulate intracellular free Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity in cardiac muscle. The authors hypothesized that temperature and stimulation frequency play a major role in determining propofol-induced alterations in [Ca2+]i and contraction in individual, electrically stimulated cardiomyocytes and the function of isolated perfused hearts. Methods:Freshly isolated myocytes were obtained from adult rat hearts, loaded with fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+]i and myocyte shortening were simultaneously measured in individual cells at 28° or 37°C at various stimulation frequencies (0.3, 0.5, 1, 2, and 3 Hz) with and without propofol. Langendorff perfused hearts paced at 180 or 330 beats/min were used to assess the effects of propofol on overall cardiac function. Results:At 28°C (hypothermic) and, to a lesser extent, at 37°C (normothermic), increasing stimulation frequency increased peak shortening and [Ca2+]i. Times to peak shortening and rate of relengthening were more prolonged at 28°C compared with 37°C at low stimulation frequencies (0.3 Hz), whereas the same conditions for [Ca2+]i were not altered by temperature. At 0.3 Hz and 28°C, propofol caused a dose-dependent decrease in peak shortening and peak [Ca2+]i. These changes were greater at 28°C compared with 37°C and involved activation of protein kinase C. At a frequency of 2 Hz, there was a rightward shift in the dose-response relation for propofol on [Ca2+]i and shortening at both 37° and 28°C compared with that observed at 0.3 Hz. In Langendorff perfused hearts paced at 330 beats/min, clinically relevant concentrations of propofol decreased left ventricular developed pressure, with the effect being less at 28°C compared with 37°C. In contrast, only a supraclinical concentration of propofol decreased left ventricular developed pressure at 28°C at either stimulation frequency. Conclusion:These results demonstrate that temperature and stimulation frequency alter the inhibitory effect of propofol on cardiomyocyte [Ca2+]i and contraction. In isolated cardiomyocytes, the inhibitory effects of propofol are more pronounced during hypothermia and at higher stimulation frequencies and involve activation of protein kinase C. In Langendorff perfused hearts at constant heart rate, the inhibitory effects of propofol at clinically relevant concentrations are more pronounced during normothermic conditions.

Intravenous Anesthetics Attenuate Phenylephrine-induced Calcium Oscillations in Individual Pulmonary Artery Smooth Muscle Cells

Background: The authors investigated the effects of intravenous anesthetics on alpha‐adrenergic‐induced oscillations in intracellular free calcium concentration ([Ca2+]i) in individual pulmonary artery smooth muscle cells (PASMCs). Methods: PASMCs were cultured from explants of canine intrapulmonary artery. Fura‐2‐loaded PASMCs were continuously superfused with phenylephrine (10 micro Meter) at 37 [degree sign] Celsius on the stage of an inverted fluorescence microscope. Measurement of [Ca2+] sub i was via a dual wavelength spectrofluorometer. Intravenous anesthetics were added to the superfusate to assess their effects on the phenylephrine‐induced [Ca2+]i oscillations. Results: Resting [Ca2+]i was 103 +/‐ 6 nM. Phenylephrine stimulated [Ca2+]i oscillations, reaching a peak concentration of 632 +/‐ 20 nM and a frequency of 1.53 +/‐ 0.14 transients/min. The effects of phenylephrine were dose‐dependent. The effects of intravenous anesthetics on phenylephrine‐induced [Ca2+]i oscillations were dose‐dependent. Ketamine (100 micro Meter) reduced the amplitude (221 +/‐ 22 nM) but not the frequency (1.48 +/‐ 0.11/min) of the oscillations, whereas thiopental (100 micro Meter) decreased the amplitude (270 +/‐ 20 nM) and the frequency (1.04 +/‐ 0.10/min). Propofol (100 micro Meter) and the Intralipid[registered sign] vehicle inhibited the amplitude (274 +/‐ 11 nM) but not the frequency (1.39 +/‐ 0.11/min) of the oscillations. The effects of ketamine and thiopental, but not propofol, were evident at clinically relevant concentrations. Conclusion: Ketamine, thiopental, and propofol exerted differential effects to inhibit the amplitude or the frequency of phenylephrine‐induced [Ca2+]i oscillations in individual PASMCs. Thus, intravenous anesthetics may alter the pulmonary vascular response to alpha‐adrenoreceptor activation by directly inhibiting [Ca sup 2+]i signaling in PASMCs.

Erratum: Sphingosylphosphorylcholine is a ligand for ovarian cancer G-protein-coupled receptor1. (Nature Cell Biology (2000) vol. 2 (261-267))

Owing to the serious concerns about the validity of the data published in the paper, the authors would like to retract this paper. All authors, apart from Dr. Kui Zhu, have signed this retraction. Dr. Zhu did not return a signature.