Ca2+ waves require sequential activation of inositol trisphosphate receptors and ryanodine receptors in pancreatic acini

BACKGROUND & AIMS: The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) and the ryanodine receptor (RyR) are the principal Ca2+-release channels in cells and are believed to serve distinct roles in cytosolic Ca2+ (Ca(i)2+) signaling. This study investigated whether these receptors instead can release Ca2+ in a coordinated fashion. METHODS: Apical and basolateral Ca(i)2+ signals were monitored in rat pancreatic acinar cells by time-lapse confocal microscopy. Caged forms of second messengers were microinjected into individual cells and then photoreleased in a controlled fashion by either UV or 2-photon flash photolysis. RESULTS: InsP3 increased Ca(i)2+ primarily in the apical region of pancreatic acinar cells, whereas the RyR agonist cyclic adenosine diphosphate ribose (cADPR) increased Ca(i)2+ primarily in the basolateral region. Apical-to-basal Ca(i)2+ waves were induced by acetylcholine and initiation of these waves was blocked by the InsP3R inhibitor heparin, whereas propagation into the basolateral region was inhibited by the cADPR inhibitor 8-amino-cADPR. To examine integration of apical and basolateral Ca(i)2+ signals, Ca2+ was selectively released either apically or basolaterally using 2-photon flash photolysis. Ca(i)2+ increases were transient and localized in unstimulated cells. More complex Ca(i)2+ signaling patterns, including polarized Ca(i)2+ waves, were observed when Ca2+ was photoreleased in cells stimulated with subthreshold concentrations of acetylcholine. CONCLUSIONS: Polarized Ca(i)2+ waves are induced in acinar cells by serial activation of apical InsP3Rs and then basolateral RyRs, and subcellular release of Ca2+ coordinates the actions of these 2 types of Ca2+ channels. This subcellular integration of Ca2+-release channels shows a new level of complexity in the formation of Ca(i)2+ waves.     

Ablation of sarcolipin enhances sarcoplasmic reticulum calcium transport and atrial contractility

Sarcolipin is a novel regulator of cardiac sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and is expressed abundantly in atria. In this study we investigated the physiological significance of sarcolipin in the heart by generating a mouse model deficient for sarcolipin. The sarcolipin-null mice do not show any developmental abnormalities or any cardiac pathology. The absence of sarcolipin does not modify the expression level of other Ca2+ handling proteins, in particular phospholamban, and its phosphorylation status. Calcium uptake studies revealed that, in the atria, ablation of sarcolipin resulted in an increase in the affinity of the SERCA pump for Ca2+ and the maximum velocity of Ca2+ uptake rates. An important finding is that ablation of sarcolipin resulted in an increase in atrial Ca2+ transient amplitudes, and this resulted in enhanced atrial contractility. Furthermore, atria from sarcolipin-null mice showed a blunted response to isoproterenol stimulation, implicating sarcolipin as a mediator of beta-adrenergic responses in atria. Our study documented that sarcolipin is a key regulator of SERCA2a in atria. Importantly, our data demonstrate the existence of distinct modulators for the SERCA pump in the atria and ventricles.     

Targeted expression of the inositol 1,4,5-triphosphate receptor (IP3R) ligand-binding domain releases Ca2+ via endogenous IP3R channels

Virtually all functions of a cell are influenced by cytoplasmic [Ca(2+)] increases. Inositol 1,4,5-trisphosphate receptor (IP(3)R) channels, located in the endoplasmic reticulum (ER), release Ca(2+) in response to binding of the second messenger, IP(3).IP(3)Rs thus are part of the information chain interpreting external signals and transforming them into cytoplasmic Ca(2+) transients. IP(3)Rs function as tetramers, each unit comprising an N-terminal ligand-binding domain (LBD) and a C-terminal channel domain linked by a long regulatory region. It is not yet understood how the binding of IP(3) to the LBD regulates the gating properties of the channel. Here, we use the expression of IP(3) binding protein domains tethered to the surface of the endoplasmic reticulum (ER) to show that the all-helical domain of the IP(3)R LBD is capable of depleting the ER Ca(2+) pools by opening the endogenous IP(3)Rs, even without IP(3) binding. This effect requires the domain to be within 50 A of the ER membrane and is impaired by the presence of the N-terminal inhibitory segment on the LBD. These findings raise the possibility that the helical domain of the LBD functions as an effector module possibly interacting with the channel domain, thereby being part of the gating mechanisms by which the IP(3)-induced conformational change within the LBD regulates Ca(2+) release.     

CaBP1, a neuronal Ca2+ sensor protein,inhibits inositol trisphosphate receptors by clamping intersubunit interactions

Calcium-binding protein 1 (CaBP1) is a neuron-specific member of the calmodulin superfamily that regulates several Ca²⁺ channels, including inositol 1,4,5-trisphosphate receptors (InsP₃Rs). CaBP1 alone does not affect InsP₃R activity, but it inhibits InsP₃-evoked Ca²⁺ release by slowing the rate of InsP₃R opening. The inhibition is enhanced by Ca²⁺ binding to both the InsP₃R and CaBP1. CaBP1 binds via its C lobe to the cytosolic N-terminal region (NT; residues 1–604) of InsP₃R1. NMR paramagnetic relaxation enhancement analysis demonstrates that a cluster of hydrophobic residues (V101, L104, and V162) within the C lobe of CaBP1 that are exposed after Ca²⁺ binding interact with a complementary cluster of hydrophobic residues (L302, I364, and L393) in the β-domain of the InsP₃-binding core. These residues are essential for CaBP1 binding to the NT and for inhibition of InsP₃R activity by CaBP1. Docking analyses and paramagnetic relaxation enhancement structural restraints suggest that CaBP1 forms an extended tetrameric turret attached by the tetrameric NT to the cytosolic vestibule of the InsP₃R pore. InsP₃ activates InsP₃Rs by initiating conformational changes that lead to disruption of an intersubunit interaction between a “hot-spot” loop in the suppressor domain (residues 1–223) and the InsP₃-binding core β-domain. Targeted cross-linking of residues that contribute to this interface show that InsP₃ attenuates cross-linking, whereas CaBP1 promotes it. We conclude that CaBP1 inhibits InsP₃R activity by restricting the intersubunit movements that initiate gating.     

Structural basis for the high Ca2+ affinity of the ubiquitous SERCA2b Ca2+ pump

Sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA) Ca²⁺ transporters pump cytosolic Ca²⁺ into the endoplasmic reticulum, maintaining a Ca²⁺ gradient that controls vital cell functions ranging from proliferation to death. To meet the physiological demand of the cell, SERCA activity is regulated by adjusting the affinity for Ca²⁺ ions. Of all SERCA isoforms, the housekeeping SERCA2b isoform displays the highest Ca²⁺ affinity because of a unique C-terminal extension (2b-tail). Here, an extensive structure–function analysis of SERCA2b mutants and SERCA1a2b chimera revealed how the 2b-tail controls Ca²⁺ affinity. Its transmembrane (TM) segment (TM11) and luminal extension functionally cooperate and interact with TM7/TM10 and luminal loops of SERCA2b, respectively. This stabilizes the Ca²⁺-bound E1 conformation and alters Ca²⁺-transport kinetics, which provides the rationale for the higher apparent Ca²⁺ affinity. Based on our NMR structure of TM11 and guided by mutagenesis results, a structural model was developed for SERCA2b that supports the proposed 2b-tail mechanism and is reminiscent of the interaction between the α- and β-subunits of Na⁺,K⁺-ATPase. The 2b-tail interaction site may represent a novel target to increase the Ca²⁺ affinity of malfunctioning SERCA2a in the failing heart to improve contractility.     

Overexpression of diacylglycerol kinase zeta inhibits endothelin-1-induced decreases in Ca2+ transients and cell shortening in mouse ventricular myocytes

Endothelin-1 (ET-1) is released in various cardiovascular disorders including congestive heart failure, and may modulate significantly the disease process by its potent action on vascular and cardiac muscle cell function and gene regulation. In adult mouse ventricular cardiomyocytes loaded with indo-1, ET-1 induced a sustained negative inotropic effect (NIE) in association with decreases in Ca²⁺ transients. The ET-1-induced effects on Ca²⁺ transients and cell shortening were abolished in diacylglycerol (DAG) kinase ζ-overexpressing mouse ventricular myocytes. A nonselective protein kinase C (PKC) inhibitor, GF109203X, inhibited the ET-1-induced decreases in Ca²⁺ transients and cell shortening in concentration-dependent manners, whereas a selective Ca²⁺-dependent PKC inhibitor, Gö6976, did not affect the ET-1-induced effects. A phospholipase Cβ inhibitor, U73122, and an inhibitor of phospholipase D, C₂-ceramide, partially, but significantly, attenuated the ET-1-induced effects. Derivatives of the respective inhibitors with no specific effects, U73343 and dihydro-C₂-ceramide, did not affect the ET-1-induced effects. Taken together, these results indicate that activation of a Ca²⁺-independent PKC isozyme by 1,2-DAG, which is generated by phospholipase Cβ and phospholipase D activation and inactivated by phosphorylation via DAG kinase, is responsible for the ET-1-induced decreases in Ca²⁺ transients and cell shortening in mouse ventricular cardiomyocytes.     

Loss of inositol 1,4,5-trisphosphate receptors from bile duct epithelia is a common event in cholestasis

BACKGROUND AND AIMS: Cholestasis is one of the principal manifestations of liver disease and often results from disorders involving bile duct epithelia rather than hepatocytes. A range of disorders affects biliary epithelia, and no unifying pathophysiologic event in these cells has been identified as the cause of cholestasis. Here we examined the role of the inositol 1,4,5-trisphosphate receptor (InsP3R)/Ca(2+) release channel in Ca(2+) signaling and ductular secretion in animal models of cholestasis and in patients with cholestatic disorders. METHODS: The expression and distribution of the InsP3R and related proteins were examined in rat cholangiocytes before and after bile duct ligation or treatment with endotoxin. Ca(2+) signaling was examined in isolated bile ducts from these animals, whereas ductular bicarbonate secretion was examined in isolated perfused livers. Confocal immunofluorescence was used to examine cholangiocyte InsP3R expression in human liver biopsy specimens. RESULTS: Expression of the InsP3R was selectively lost from biliary epithelia after bile duct ligation or endotoxin treatment. As a result, Ca(2+) signaling and Ca(2+)-mediated bicarbonate secretion were lost as well, although other components of the Ca(2+) signaling pathway and adenosine 3',5'-cyclic monophosphate (cAMP)-mediated bicarbonate secretion both were preserved. Examination of human liver biopsy specimens showed that InsP3Rs also were lost from bile duct epithelia in a range of human cholestatic disorders, although InsP3R expression was intact in noncholestatic liver disease. CONCLUSIONS: InsP3-mediated Ca(2+) signaling in bile duct epithelia appears to be important for normal bile secretion in the liver, and loss of InsP3Rs may be a final common pathway for cholestasis.     

Interplay of Mg2+, ADP,and ATP in the cytosol and mitochondria: Unravelling the role of Mg2+ in cell respiration

In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg²⁺ concentrations must be considered as well. Here we developed in vivo/in vitro techniques using ³¹P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg²⁺ concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg²⁺ in the mitochondrial matrix, where [Mg²⁺] is tenfold higher. In contrast, owing to a much higher affinity for Mg²⁺, ATP is mostly complexed by Mg²⁺ in both compartments. Mg²⁺ starvation used to alter cytosolic and mitochondrial [Mg²⁺] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg²⁺ concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.In heterotrophic and well-oxygenated plant cells, ATP is regenerated from ADP principally by glycolysis and mitochondrial oxidative phosphorylation. Surprisingly, although ATP synthesis mechanisms have been deciphered for decades, whether cell respiration is controlled by [ATP]/[ADP] or [ATP]/[ADP][Pi] ratios (1, 2), by the adenylate energy charge ([ATP + 0.5 ADP]/[ATP + ADP + AMP]) (3, 4), and/or by the concentration of ATP or ADP in the cytosol (5, 6) remains a matter of debate. To our knowledge, the determining factor for controlling cell respiration in response to the energy demand has not yet been unambiguously characterized.MgATP is the substrate of numerous phosphorylating enzymes and the principal energy source of the cell. Indeed, any increase in metabolic activity increases the rate of MgATP use and, consequently, the rate of ADP and magnesium release, and vice versa. In normoxia, the MgATP concentration should be essentially balanced by the ADP phosphorylation catalyzed by mitochondrial ATP synthase, thereby adjusting oxidative phosphorylation to cell ATP needs. The ADP/ATP carrier (AAC) of the inner mitochondrial membrane, which exchanges free nucleotides, and adenylate kinase (EC 2.7.4.3), which interconverts MgADP and free ADP with MgATP and free AMP in the presence of Mg²⁺ (7), participate in this regulation (reviewed in ref. 8). Clearly, to better understand the interplay of free and Mg-complexed ADP and ATP in the regulation of cell respiration it is necessary to know their concentrations, as well as the concentration of Mg²⁺ in the cytosol and mitochondrial matrix.Nucleotides can be measured using ³¹P-NMR spectroscopy both in vitro, from cell extracts, and in vivo, in perfused material. After 1 h of data accumulation time, detection thresholds are approximately 20 nmol in vitro and 50 nmol in vivo (9). Various techniques for measuring intracellular [Mg²⁺] and free/Mg-complexed nucleotides have been proposed (10–12), but none allows measurement in different intracellular compartments. In vivo ³¹P-NMR spectroscopy offers this possibility, because the chemical shift (δ) of the γ- and β-phosphorus resonances of ATP and the β-phosphorus resonance of ADP depend on pH and [Mg²⁺] (13). We adapted this noninvasive technique to the simultaneous in vivo measurement of cytosolic and mitochondrial Mg²⁺ and free/Mg-complexed nucleotides concentrations in culture cells.We used homogenous cells cultivated on liquid nutrient media (NM) so as to narrow resonance peaks on in vivo NMR spectra, thus improving the signal-to-noise ratios and the accuracy of chemical shift measurements and limiting peak overlaps. In addition, the heterotrophic sycamore (Acer pseudoplatanus L.) cells of cambial origin used in this study contain no large chloroplasts, but only small plastids (14, 15) with low amounts of nucleotides (16), thus permitting more precise measurement of the cytosolic and mitochondrial nucleotide pools.To modify nucleotide concentrations without using inhibitors that may interfere with mitochondrial functioning, we varied the cell culture media: standard, adenine-supplied, Pi-starved, and Mg-starved. In this paper, we refer to cytoplasm as the cell compartment exterior to the vacuole and cytosol as the cell compartment exterior to the vacuole and the organelles bounded by a double membrane (mitochondria and plastids).The aim of the present study was to determine the role of ADP, ATP, and Mg²⁺ concentrations in the in vivo control of mitochondrial respiration. We show that the balance between cytosolic and mitochondrial free ADP, depending on the concentration of Mg²⁺ in the cytosol and matrix, mediates this regulation.     

Beta-cell hubs maintain Ca2+ oscillations in human and mouse islet simulations

Islet β-cells are responsible for secreting all circulating insulin in response to rising plasma glucose concentrations. These cells are a phenotypically diverse population that express great functional heterogeneity. In mice, certain β-cells (termed ‘hubs’) have been shown to be crucial for dictating the islet response to high glucose, with inhibition of these hub cells abolishing the coordinated Ca²⁺ oscillations necessary for driving insulin secretion. These β-cell hubs were found to be highly metabolic and susceptible to pro-inflammatory and glucolipotoxic insults. In this study, we explored the importance of hub cells in human by constructing mathematical models of Ca²⁺ activity in human islets. Our simulations revealed that hubs dictate the coordinated Ca²⁺ response in both mouse and human islets; silencing a small proportion of hubs abolished whole-islet Ca²⁺ activity. We also observed that if hubs are assumed to be preferentially gap junction coupled, then the simulations better adhere to the available experimental data. Our simulations of 16 size-matched mouse and human islet architectures revealed that there are species differences in the role of hubs; Ca²⁺ activity in human islets was more vulnerable to hub inhibition than mouse islets. These simulation results not only substantiate the existence of β-cell hubs, but also suggest that hubs may be favorably coupled in the electrical and metabolic network of the islet, and that targeted destruction of these cells would greatly impair human islet function.     

Sarcoplasmic Reticulum Function in Murine Ventricular Myocytes Overexpressing SR CaATPase

To examine the effects of the overexpression of sarcoplasmic reticulum (SR) CaATPase on function of the SR and Ca²⁺homeostasis, we measured [Ca²⁺]_itransients (fluo-3), and L-type Ca²⁺currents (I_Ca,L), Na/Ca exchanger currents (I_Na/Ca), and SR Ca²⁺content with voltage clamp in ventricular myocytes isolated from wild type (WT) mice and transgenic (SRTG) mice. The amplitude of [Ca²⁺]_itransients was insignificantly increased in SRTG myocytes, while the diastolic [Ca²⁺]_itended to be lower. The initial and terminal declines of [Ca²⁺]_itransients were significantly accelerated in SRTG myocytes, implying a functional upregulation of the SR CaATPase. We examined the functional contribution of only the SR CaATPase to the initial and the terminal phase of the decline of [Ca²⁺]_i, by abruptly inhibiting Na/Ca exchange with a rapid switcher device. The rate of [Ca²⁺] decline mediated by the SR CaATPase was increased by 40% in SRTG compared with WT myocytes. The function of the L-type Ca²⁺channel was unchanged in SRTG myocytes, while INa/Ca density was slightly (10%) decreased. Measured SR Ca²⁺content was significantly increased by 29% in SRTG myocytes. Thus, overexpression of SR CaATPase markedly accelerates the decline of [Ca²⁺]_itransients, and induces an increase in SR Ca²⁺content, with some downregulation of the Na/Ca exchanger.     

Lipid rafts establish calcium waves in hepatocytes

BACKGROUND & AIMS: Polarity is critical for hepatocyte function. Ca(2+) waves are polarized in hepatocytes because the inositol 1,4,5-trisphosphate receptor (InsP3R) is concentrated in the pericanalicular region, but the basis for this localization is unknown. We examined whether pericanalicular localization of the InsP3R and its action to trigger Ca(2+) waves depends on lipid rafts. METHODS: Experiments were performed using isolated rat hepatocyte couplets and pancreatic acini, plus SkHep1 cells as nonpolarized controls. The cholesterol depleting agent methyl-beta-cyclodextrin (mbetaCD) was used to disrupt lipid rafts. InsP3R isoforms were examined by immunoblot and immunofluorescence. Ca(2+) waves were examined by confocal microscopy. RESULTS: Type II InsP3Rs initially were localized to only some endoplasmic reticulum fractions in hepatocytes, but redistributed into all fractions in mbetaCD-treated cells. This InsP3R isoform was concentrated in the pericanalicular region, but redistributed throughout the cell after mbetaCD treatment. Vasopressin-induced Ca(2+) signals began as apical-to-basal Ca(2+) waves, and mbetaCD slowed the wave speed and prolonged the rise time. MbetaCD had a similar effect on Ca(2+) waves in acinar cells but did not affect Ca(2+) signals in SkHep1 cells, suggesting that cholesterol depletion has similar effects among polarized epithelia, but this is not a nonspecific effect of mbetaCD. CONCLUSIONS: Lipid rafts are responsible for the pericanalicular accumulation of InsP3R in hepatocytes, and for the polarized Ca(2+) waves that result. Signaling microdomains exist not only in the plasma membrane, but also in the nearby endoplasmic reticulum, which in turn, helps establish and maintain structural and functional polarity.     

Oscillations in cytoplasmic free calcium concentration in human pancreatic islets from subjects with normal and impaired glucose tolerance

Summary Plasma insulin levels in healthy subjects oscillate and non-insulin-dependent diabetic patients display an irregular pattern of such oscillations. Since an increase in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) in the pancreatic beta cell is the major stimulus for insulin release, this study was undertaken to investigate the dynamics of electrical activity, [Ca²⁺]_i-changes and insulin release, in stimulated islets from subjects of varying glucose tolerance. In four patients it was possible to investigate more than one of these three parameters. Stimulation of pancreatic islets with glucose and tolbutamide sometimes resulted in the appearance of oscillations in [Ca²⁺]_i, lasting 2–3 min. Such oscillations were observed even in some islets from patients with impaired glucose tolerance. In one islet from a diabetic patient there was no response to glucose, whereas that islet displayed [Ca²⁺]_i-oscillations in response to tolbutamide, suggesting that sulphonylurea treatment can mimic the complex pattern of glucose-induced [Ca²⁺]_i-oscillations. We also, for the first time, made patch-clamp recordings of membrane currents in beta-cells in situ in the islet. Stimulation with glucose and tolbutamide resulted in depolarization and appearance of action potentials. The islet preparations responded to stimulation with a number of different secretagogues with release of insulin. The present study shows that human islets can respond to stimulation with glucose and sulphonylurea with oscillations in [Ca²⁺]_i, which is the signal probably underlying the oscillations in plasma insulin levels observed in healthy subjects. Interestingly, even subjects with impaired glucose tolerance had islets that responded with oscillations in [Ca²⁺]_i upon glucose stimulation, although it is not known to what extent the response of these islets was representative of most islets in these patients.Abbreviations [Ca²⁺]_i Cytoplasmic free Ca²⁺ - NIDDM non-insulin-dependent diabetes mellitus - DMSO dimethylsulphoxide - PC pancreatic cancer     

Relationship between Ca2+-affinity and shielding of bulk water in the Ca2+-pump from molecular dynamics simulations

The sarcoplasmic reticulum Ca(2+)-ATPase transports two Ca(2+) per ATP hydrolyzed from the cytoplasm to the lumen against a large concentration gradient. During transport, the pump alters the affinity and accessibility for Ca(2+) by rearrangements of transmembrane helices. In this study, all-atom molecular dynamics simulations were performed for wild-type Ca(2+)-ATPase in the Ca(2+)-bound form and the Gln mutants of Glu771 and Glu908. Both of them contribute only one carboxyl oxygen to site I Ca(2+), but only Glu771Gln completely looses the Ca(2+)-binding ability. The simulations show that: (i) For Glu771Gln, but not Glu908Gln, coordination of Ca(2+) was critically disrupted. (ii) Coordination broke at site II first, although Glu771 and Glu908 only contribute to site I. (iii) A water molecule bound to site I Ca(2+) and hydrogen bonded to Glu771 in wild-type, drastically changed the coordination of Ca(2+) in the mutant. (iv) Water molecules flooded the binding sites from the lumenal side. (v) The side chain conformation of Ile775, located at the head of a hydrophobic cluster near the lumenal surface, appears critical for keeping out bulk water. Thus the simulations highlight the importance of the water molecule bound to site I Ca(2+) and point to a strong relationship between Ca(2+)-coordination and shielding of bulk water, providing insights into the mechanism of gating of ion pathways in cation pumps.     

High glucose oxidizes SERCA cysteine-674 and prevents inhibition by nitric oxide of smooth muscle cell migration

Nitric oxide (NO) causes S-glutathiolation of the reactive cysteine-674 in the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA), thus increasing SERCA activity, and inhibiting Ca²⁺ influx and migration of vascular smooth muscle cells (VSMC). Because increased VSMC migration contributes to accelerated neointimal growth and atherosclerosis in diabetes, the effect of culture of VSMC in high glucose (HG) was determined. Rat aortic VSMC were exposed to normal (5.5 mmol/L) or high (25 mmol/L) glucose for 3 days, and serum-induced cell migration during 6 h into a wounded cell monolayer was measured 5 min after adding the NO donor S-nitroso-N-acetylpenicillamine (SNAP) or 24 h after interleukin-1β (IL-1β) to express inducible nitric oxide synthase (iNOS). In normal glucose, SNAP or IL-1β significantly inhibited migration in cells infected with adenovirus to express GFP or SERCA wild type (WT), but not with a C674S SERCA mutant. After HG, NO failed to inhibit migration, nor did it decrease calcium-dependent association of calmodulin with calcineurin, indicating that NO failed to decrease intracellular calcium levels via SERCA. In contrast, overexpression of SERCA WT, but not the SERCA C674S mutant, preserved the ability for NO to inhibit migration despite exposing the cells to HG. The antioxidant, Tempol, or overexpression of superoxide dismutase also prevented the effects of HG. Further studies showed that both biotinylated-iodoacetamide and NO-induced biotinylated glutathione labeling of SERCA C674 were decreased by HG, and a sequence-specific sulfonic acid antibody detected oxidation of the C674 SERCA thiol. These results indicate that failure of NO to inhibit migration in VSMC exposed to HG is due to oxidation of the SERCA reactive cysteine-674.     

Involvement of the sarcoplasmic reticulum calcium pump in myocardial contractile dysfunction: Comparison between chronic pressure-overload and stunning

Summary Acute as well as chronic forms of heart failure involve mechanical dysfunction during systole and/or diastole. The rapid Ca²⁺ release from and Ca²⁺ reuptake into the tubuli of the sarcoplasmic reticulum are processes that critically determine normal systolic and diastolic myocardial function, which explains why in the last fifteen years so much attention has been paid to understand the performance of the sarcoplasmic reticulum Ca²⁺ pump during myocardial contractile dysfunction. In this communication we have reviewed the literature data on sarcoplasmic reticulum Ca²⁺ pump function in the chronically pressure-overloaded hypertrophied and stunned (post-ischemic reversibly injured) myocardium in the light of some new data from our laboratory. Results on the pressure-overloaded hypertrophied myocardium provide evidence that impaired relaxation is most likely due to a low capacity of the sarcoplasmic reticulum to pump Ca²⁺, a consequence of a lower density of Ca²⁺-pumping sites within the sarcotubular membranes. Contractile dysfunction in stunned myocardium is accompanied by an upregulation of the sarcoplasmic reticulum Ca²⁺ ATPase gene resulting in a slight increase of the Ca²⁺ pumping activity. The latter increase is likely an adaptive response of the reversibly injured myocardium which may contribute to the slow recovery of contractile function.     

Theoretical investigation of action potential duration dependence on extracellular Ca in human cardiomyocytes

Reduction in [Ca²⁺]_o prolongs the AP in ventricular cardiomyocytes and the QT_c interval in patients. Although this phenomenon is relevant to arrhythmogenesis in the clinical setting, its mechanisms are counterintuitive and incompletely understood. To evaluate in silico the mechanisms of APD modulation by [Ca²⁺]_o in human cardiomyocytes. We implemented the Ten Tusscher-Noble-Noble-Panfilov model of the human ventricular myocyte and modified the formulations of the rapidly and slowly activating delayed rectifier K⁺ currents (I_Kr and I_Ks) and L-type Ca²⁺ current (I_CaL) to incorporate their known sensitivity to intra- or extracellular Ca²⁺. Simulations were run with the original and modified models at variable [Ca²⁺]_o in the clinically relevant 1 to 3 mM range. The original model responds with APD shortening to decrease in [Ca²⁺]_o, i.e. opposite to the experimental observations. Incorporation of Ca²⁺ dependency of K⁺ currents cannot reproduce the inverse relation between APD and [Ca²⁺]_o. Only when I_CaL inactivation process was modified, by enhancing its dependency on Ca²⁺, simulations predict APD prolongation at lower [Ca²⁺]_o. Although Ca²⁺-dependent I_CaL inactivation is the primary mechanism, secondary changes in electrogenic Ca²⁺ transport (by Na⁺/Ca²⁺ exchanger and plasmalemmal Ca²⁺-ATPase) contribute to the reversal of APD dependency on [Ca²⁺]_o. This theoretical investigation points to Ca²⁺-dependent inactivation of I_CaL as a mechanism primarily responsible for the dependency of APD on [Ca²⁺]_o. The modifications implemented here make the model more suitable to analyze repolarization mechanisms when Ca²⁺ levels are altered.     

Polyol pathway impairs the function of SERCA and RyR in ischemic-reperfused rat hearts by increasing oxidative modifications of these proteins

A number of studies have shown that the polyol pathway, consisting of aldose reductase (AR) and sorbitol dehydrogenase (SDH), contributes to ischemia-reperfusion (I/R)-induced myocardial infarction due to depletion of ATP. In this report we show that the polyol pathway in I/R heart also contributes to the impairment of sacro/endoplasmic reticulum Ca²⁺-ATPase (SERCA) and ryanodine receptor (RyR), two key players in Ca²⁺ signaling that regulate cardiac contraction. Rat hearts were isolated and retrogradely perfused with either Krebs' buffer containing 1 μM AR inhibitor, zopolrestat, or 200 nM SDH inhibitor, CP-170,711, and challenged by 30 min of regional ischemia and 45 min of reperfusion. We found that post-ischemic contractile function of the isolated perfused hearts was improved by pharmacological inhibition of the polyol pathway. I/R-induced contractile dysfunction is most likely due to impairment in Ca²⁺ signaling and the activities of SERCA and RyR. All these abnormalities were significantly ameliorated by treatment with ARI or SDI. We showed that the polyol pathway activities increase the level of peroxynitrite, which enhances the tyrosine nitration of SERCA and irreversibly modifies it to form SERCAC674-SO₃H. This leads to reduced level of S-glutathiolated SERCA, contributing to its inactivation. The polyol pathway activities also deplete the level of GSH, leading to decreased active RyR, the S-glutathiolated RyR. Thus, in I/R heart, inhibition of polyol pathway improved the function of SERCA and RyR by protecting them from irreversible oxidation.     

Taurocholate-stimulated leukotriene C4 biosynthesis and leukotriene C4-stimulated choleresis in isolated rat liver

Cysteinyl-containing leukotrienes seem to exert a cholestatic effect. However, leukotriene inhibitors were found to reduce bile salt efflux in isolated rat hepatocytes, suggesting a role for leukotrienes in bile flow formation. In the isolated rat liver, the effects of two different concentrations of leukotriene C₄ on bile flow and bile salt excretion are analyzed, as well as the possible effect of taurocholate on the hepatic production of cysteinyl-containing leukotrienes. Leukotriene C₄ (0.25 fmol) increased bile salt excretion (+22.2%; P < 0.05), whereas a much higher dose (0.25 × 10⁶ fmol) showed the known cholestatic effect, reducing bile salt excretion (−25.9%; P < 0.01). These dose-dependent biphasic effects were specific because they could be prevented by the simultaneous administration of cysteinyl-containing leukotriene antagonists. On the other hand, taurocholate administration induced a dose-dependent increase in biliary excretion of cysteinyl-containing leukotrienes. Furthermore, taurocholate increased messenger RNA levels of 5-lipoxygenase, a key enzyme in leukotriene biosynthesis. Taurocholate increase of hepatocyte intracellular calcium was not significant, suggesting that taurocholate effects are not mediated by stimulation of calcium metabolism. These results constitute evidence for the existence of a positive feedback mechanism by which bile salts stimulate the synthesis of leukotrienes that, in turn, stimulate bile salt excretion.     

Intracellular Ca- and PKC-dependent upregulation of T-type Ca channels in LPC-stimulated cardiomyocytes

Lysophosphatidylcholine (LPC) accumulation in intracellular and/or interstitial space in cardiomyocytes may underlie as a mechanism for tachycardia and various arrhythmias during cardiac ischemia, which is usually accompanied by elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The present study was therefore designed to investigate possible mechanisms responsible for [Ca²⁺]_i elevation by LPC focusing on T-type Ca²⁺ channel current (I_Ca.T). LPC as well as phorbol 12-myristate 13-acetate (PMA) significantly accelerated the beating rates of neonatal rat cardiomyocytes. Augmentation of I_Ca.T by LPC was dependent on the intracellular Ca²⁺ concentration: an increase of I_Ca.T was significantly larger in high [Ca²⁺]_i condition (pCa = 7) than those in low [Ca²⁺]_i condition (pCa = 11). In heterologous expression system by use of human cardiac Ca_V3.1 and Ca_V3.2 channels expressed in HEK293 cells, LPC augmented Ca_V3.2 channel current (I_Cav3.2) in a concentration-dependent manner but not Ca_V3.1 channel current (I_Cav3.1). Augmentation of I_Cav3.2 by LPC was highly [Ca²⁺]_i dependent: I_Cav3.2 was unchanged when pCa was 11 but was markedly increased when [Ca²⁺]_i was higher than 10⁻¹⁰ M (pCa ≤ 10) by LPC application (10-50 μM). A specific inhibitor of protein kinase Cα (Ro-32-0432) attenuated the increase of I_Cav3.2 by LPC. LPC stimulates I_Ca.T in a [Ca²⁺]_i-dependent manner via PKCα activation, which may play a role in triggering arrhythmias in pathophysiological conditions of the heart.