Viktoriya Solodushko

Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections

Calcium (Ca2+) release through inositol 1,4,5-trisphosphate receptors (IP3Rs) regulates the function of virtually every mammalian cell. Unlike ryanodine receptors, which generate local Ca2+ events (“sparks”) that transmit signals to the juxtaposed cell membrane, a similar functional architecture has not been reported for IP3Rs. Here, we have identified spatially fixed, local Ca2+ release events (“pulsars”) in vascular endothelial membrane domains that project through the internal elastic lamina to adjacent smooth muscle membranes. Ca2+ pulsars are mediated by IP3Rs in the endothelial endoplasmic reticulum of these membrane projections. Elevation of IP3 by the endothelium-dependent vasodilator, acetylcholine, increased the frequency of Ca2+ pulsars, whereas blunting IP3 production, blocking IP3Rs, or depleting endoplasmic reticulum Ca2+ inhibited these events. The elementary properties of Ca2+ pulsars were distinct from ryanodine-receptor-mediated Ca2+ sparks in smooth muscle and from IP3-mediated Ca2+ puffs in Xenopus oocytes. The intermediate conductance, Ca2+-sensitive potassium (KCa3.1) channel also colocalized to the endothelial projections, and blockage of this channel caused an 8-mV depolarization. Inhibition of Ca2+ pulsars also depolarized to a similar extent, and blocking KCa3.1 channels was without effect in the absence of pulsars. Our results support a mechanism of IP3 signaling in which Ca2+ release is spatially restricted to transmit intercellular signals.

Taurine renders the cell resistant to ischemia-induced injury in cultured neonatal rat cardiomyocytes.

Taurine is found in very high concentration in the mammalian heart. Because chronic myocardial taurine loss produces myocardial injury, the effects of taurine supplementation on ischemia-induced necrosis and apoptosis were examined using a cardiomyocyte model of simulated ischemia. Neonatal rat heart cells were cultured for 24–72 h in a sealed flask, a condition that leads to simulated ischemia characterized by a decrease in the pH and oxygen content of the medium and a catabolite accumulation. The consequences of altered medium taurine on cellular apoptosis and necrosis were then evaluated. Exposure of cardiomyocytes to medium containing high extracellular concentrations of taurine (20 m M) significantly elevated intracellular taurine levels, reduced p53 content, and enhanced cellular Bcl-2 content. In the absence of taurine treatment, simulated ischemia led to cellular release of creatine phosphokinase (CPK), morphologic degeneration, and beating cessation by 24–72 h. Based on DNA ladder analysis and the Hoechst 33258 staining pattern, a significant number of cells placed in sealed flasks underwent apoptosis. CPK was lost from some of the cells during simulated ischemia. In contrast to the untreated ischemic cells, the cells that were incubated in medium supplemented with taurine exhibited significantly less ischemia-induced necrosis and apoptosis. The data suggest that taurine renders the cell resistant to ischemia-induced necrosis and apoptosis. The beneficial effects of taurine may be related to the elevation in cellular Bcl-2 content.

Protection from Palmitate-Induced Mitochondrial DNA Damage Prevents from Mitochondrial Oxidative Stress, Mitochondrial Dysfunction, Apoptosis, and Impaired Insulin Signaling in Rat L6 Skeletal Muscle Cells

Saturated free fatty acids have been implicated in the increase of oxidative stress, mitochondrial dysfunction, apoptosis, and insulin resistance seen in type 2 diabetes. The purpose of this study was to determine whether palmitate-induced mitochondrial DNA (mtDNA) damage contributed to increased oxidative stress, mitochondrial dysfunction, apoptosis, impaired insulin signaling, and reduced glucose uptake in skeletal muscle cells. Adenoviral vectors were used to deliver the DNA repair enzyme human 8-oxoguanine DNA glycosylase/(apurinic/apyrimidinic) lyase (hOGG1) to mitochondria in L6 myotubes. After palmitate exposure, we evaluated mtDNA damage, mitochondrial function, production of mitochondrial reactive oxygen species, apoptosis, insulin signaling pathways, and glucose uptake. Protection of mtDNA from palmitate-induced damage by overexpression of hOGG1 targeted to mitochondria significantly diminished palmitate-induced mitochondrial superoxide production, restored the decline in ATP levels, reduced activation of c-Jun N-terminal kinase (JNK) kinase, prevented cells from entering apoptosis, increased insulin-stimulated phosphorylation of serine-threonine kinase (Akt) (Ser473) and tyrosine phosphorylation of insulin receptor substrate-1, and thereby enhanced glucose transporter 4 translocation to plasma membrane, and restored insulin signaling. Addition of a specific inhibitor of JNK mimicked the effect of mitochondrial overexpression of hOGG1 and partially restored insulin sensitivity, thus confirming the involvement of mtDNA damage and subsequent increase of oxidative stress and JNK activation in insulin signaling in L6 myotubes. Our results are the first to report that mtDNA damage is the proximal cause in palmitate-induced mitochondrial dysfunction and impaired insulin signaling and provide strong evidence that targeting DNA repair enzymes into mitochondria in skeletal muscles could be a potential therapeutic treatment for insulin resistance.

Recruitment of Dynamic Endothelial Ca2+ Signals by the TRPA1 Channel Activator AITC in Rat Cerebral Arteries

Stimulation of endothelial TRP channels, specifically TRPA1, promotes vasodilation of cerebral arteries through activation of Ca2+‐dependent effectors along the myoendothelial interface. However, presumed TRPA1‐triggered endothelial Ca2+ signals have not been described. We investigated whether TRPA1 activation induces specific spatial and temporal changes in Ca2+ signals along the intima that correlates with incremental vasodilation.

Positive Feedback Regulation of Agonist-Stimulated Endothelial Ca2+ Dynamics by KCa3.1 Channels in Mouse Mesenteric Arteries

Objective— Intermediate and small conductance KCa channels IK1 (KCa3.1) and SK3 (KCa2.3) are primary targets of endothelial Ca2+ signals in the arterial vasculature, and their ablation results in increased arterial tone and hypertension. Activation of IK1 channels by local Ca2+ transients from internal stores or plasma membrane channels promotes arterial hyperpolarization and vasodilation. Here, we assess arteries from genetically altered IK1 knockout mice (IK1−/−) to determine whether IK1 channels exert a positive feedback influence on endothelial Ca2+ dynamics. Approach and Results— Using confocal imaging and custom data analysis software, we found that although the occurrence of basal endothelial Ca2+ dynamics was not different between IK1−/− and wild-type mice (P>0.05), the frequency of acetylcholine-stimulated (2 &mgr;mol/L) Ca2+ dynamics was greatly decreased in IK1−/− endothelium (515±153 versus 1860±319 events; P<0.01). In IK1−/−/SK3T/T mice, ancillary suppression (+Dox) or overexpression (−Dox) of SK3 channels had little additional effect on the occurrence of events under basal or acetylcholine-stimulated conditions. However, SK3 overexpression did restore the decreased event amplitudes. Removal of extracellular Ca2+ reduced acetylcholine-induced Ca2+ dynamics to the same level in wild-type and IK1−/− arteries. Blockade of IK1 and SK3 with the combination of charybdotoxin (0.1 &mgr;mol/L) and apamin (0.5 &mgr;mol/L) or transient receptor potential vanilloid 4 channels with HC-067047 (1 &mgr;mol/L) reduced acetylcholine Ca2+ dynamics in wild-type arteries to the level of IK1−/−/SK3T/T+Dox arteries. These drug effects were not additive. Conclusions— IK1, and to some extent SK3, channels exert a substantial positive feedback influence on endothelial Ca2+ dynamics.

Taurine-deficient cardiomyopathy: role of phospholipids, calcium and osmotic stress.

The concentration of taurine in excitable tissues is extremely high, usually in the mM range17. Although this amino acid is synthesized in the liver, the major source of taurine for the maintenance of the large intracellular taurine pools is the diet. In some species, such as infant monkeys, cats and the fox, taurine is an essential nutrient. Without an adequate dietary source of taurine, these animals develo numerous defects, including growth retarda-

Possible cause of taurine-deficient cardiomyopathy: potentiation of angiotensin II action.

Taurine, an amino acid that exhibits anti-angiotensin II and osmoregulatory activity, is found in very high concentration in the heart. When the intracellular content of taurine is dramatically reduced, the heart develops contractile defects and undergoes an eccentric form of hypertrophy. The development of myocyte hypertrophy has been largely attributed to angiotensin II, whose growth properties are antagonized by taurine. Overt heart failure is usually associated with myocyte death, including death due to angiotensin II–induced apoptosis. However, the effect of taurine deficiency on angiotensin II–induced apoptosis has not been examined. To investigate this effect, taurine-deficient cells, produced by incubating rat neonatal cardiomyocytes with medium containing the taurine transport inhibitor, &bgr;-alanine, were exposed to angiotensin II. The peptide increased terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining and caspase 9 activation more in the taurine-deficient than the normal cell. Angiotensin II also promoted the translocation of protein kinase C (PKC)ε and PKC&dgr;, the expression of Bax, and the activation of c-Jun N-terminal kinase (JNK), effects that were greater in the taurine-deficient cell. However, the data ruled out a role for extracellular signal-related kinase (ERK), Bad, and p38 mitogen-activated protein kinase in the &bgr;-alanine-angiotensin II interaction. Because PKC and JNK affect the expression and phosphorylation state of certain Bcl-2 family members, they appear to contribute to the potentiation of angiotensin II–induced apoptosis by taurine deficiency.

Slingshot Isoform-Specific Regulation of Cofilin-Mediated Vascular Smooth Muscle Cell Migration and Neointima Formation

Objective—We hypothesized that cofilin activation by members of the slingshot (SSH) phosphatase family is a key mechanism regulating vascular smooth muscle cell (VSMC) migration and neoinitima formation following vascular injury. Methods and Results—Scratch wound and modified Boyden chamber assays were used to assess VSMC migration following downregulation of the expression of cofilin and each SSH phosphatase isoform (SSH1, SSH2, and SSH3) by small interfering RNA (siRNA), respectively. Cofilin siRNA greatly attenuated the ability of VSMC migration into the “wound,” and platelet-derived growth factor (PDGF)–induced migration was virtually eliminated versus a 3.5-fold increase in nontreated VSMCs, establishing a critical role for cofilin in VSMC migration. Cofilin activation (dephosphorylation) was increased in PDGF-stimulated VSMCs. Thus, we assessed the role of the SSH family of phosphatases on cofilin activation and VSMC migration. Treatment with either SSH1 or SSH2 siRNA attenuated cofilin activation, whereas SSH3 siRNA had no effect. Only SSH1 siRNA significantly reduced wound healing and PDGF-induced VSMC migration. Both SSH1 expression (4.7-fold) and cofilin expression (3.9-fold) were increased in balloon injured versus noninjured carotid arteries, and expression was prevalent in the neointima. Conclusion—These studies demonstrate that the regulation of VSMC migration by cofilin is SSH1 dependent and that this mechanism potentially contributes to neointima formation following vascular injury in vivo.

Effect of ischemia, calcium depletion and repletion, acidosis and hypoxia on cellular taurine content.

Summary. Occlusion of the left main coronary artery led to a time-dependent release of taurine from the heart. Upon reperfusion, there was a second phase of taurine release, which exceeded the amount of taurine that exited the heart during the 45 min ischemic insult. To obtain information on the mechanism underlying the release of taurine, three variables were examined, acidosis, hypoxia and calcium overload. It was found that large amounts of taurine also leave the cell during the calcium paradox, a condition induced by perfusing the heart with calcium containing buffer following a period of calcium free perfusion. However, little taurine effluxes the hearts exposed to buffer whose pH was lowered to 6.6. Isolated neonatal cardiomyocytes subjected to chemical hypoxia also lost large amounts of taurine. However, the amount of taurine leaving the cells appeared to be correlated with the intracellular sodium concentration, [Na+]i. The data suggest that taurine efflux is regulated by [Na+]i and cellular osmolality, but not by cellular pH.

Functional Tuning of Intrinsic Endothelial Ca2+ Dynamics in Swine Coronary Arteries

RATIONALE Recent data from mesenteric and cerebral beds have revealed spatially restricted Ca(2+) transients occurring along the vascular intima that control effector recruitment and vasodilation. Although Ca(2+) is pivotal for coronary artery endothelial function, spatial and temporal regulation of functional Ca(2+) signals in the coronary endothelium is poorly understood. OBJECTIVE We aimed to determine whether a discrete spatial and temporal profile of Ca(2+) dynamics underlies endothelium-dependent relaxation of swine coronary arteries. METHODS AND RESULTS Using confocal imaging, custom automated image analysis, and myography, we show that the swine coronary artery endothelium generates discrete basal Ca(2+) dynamics, including isolated transients and whole-cell propagating waves. These events are suppressed by depletion of internal stores or inhibition of inositol 1,4,5-trisphosphate receptors but not by inhibition of ryanodine receptors or removal of extracellular Ca(2+). In vessel rings, inhibition of specific Ca(2+)-dependent endothelial effectors, namely, small and intermediate conductance K(+) channels (K(Ca)3.1 and K(Ca)2.3) and endothelial nitric oxide synthase, produces additive tone, which is blunted by internal store depletion or inositol 1,4,5-trisphosphate receptor blockade. Stimulation of endothelial inositol 1,4,5-trisphosphate-dependent signaling with substance P causes idiosyncratic changes in dynamic Ca(2+) signal parameters (active sites, event frequency, amplitude, duration, and spatial spread). Overall, substance P-induced vasorelaxation corresponded poorly with whole-field endothelial Ca(2+) measurements but corresponded precisely with the concentration-dependent change in Ca(2+) dynamics (linearly translated composite of dynamic parameters). CONCLUSIONS Our findings show that endothelium-dependent control of swine coronary artery tone is determined by spatial and temporal titration of inherent endothelial Ca(2+) dynamics that are not represented by tissue-level averaged Ca(2+) changes.