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Figueroa L.,Rush University | Shkryl V.M.,Aa Bogomoletz Institute Of Physiology | Blatter L.A.,Rush University | Rios E.,Rush University
PLoS ONE | Year: 2013

We extend the sensitivity of quantitative concentration imaging to an approximately 1000-fold range of concentrations by a method that uses two fluorescent dyes with the same fluorophore, having different affinity for the monitored species. While the formulation and illustration refer to a monitor of calcium concentration, the method is applicable to any species that binds to multiple indicators with the same spectral properties. The use of a common fluorophore has the virtue of leaving vast regions of the electromagnetic spectrum available for other applications. We provide the exact analytic expression relating measured fluorescence to [Ca2+] at equilibrium and an approximate analytic expression that does not require the equilibrium assumption. The sensitivity of the method is calculated numerically for two useful dye pairs. As illustrative application of the enhanced measurement, we use fluo-4 and fluo-4FF to image the calcium wave produced by a cardiac myocyte in response to a small artificial calcium spark. © 2013 Figueroa et al. Source


Shkryl V.M.,Rush University | Shkryl V.M.,Aa Bogomoletz Institute Of Physiology | Blatter L.A.,Rush University | Rios E.,Rush University
Journal of General Physiology | Year: 2012

Parameters (amplitude, width, kinetics) of Ca 2+ sparks imaged confocally are affected by errors when the spark source is not in focus. To identify sparks that were in focus, we used fast scanning (LSM 5 LIVE; Carl Zeiss) combined with fast piezoelectric focusing to acquire x-y images in three planes at 1-μm separation (x-y-z-t mode). In 3,000 x.y scans in each of 34 membrane-permeabilized cat atrial cardiomyocytes, 6,906 sparks were detected. 767 sparks were in focus. They had greater amplitude, but their spatial width and rise time were similar compared with all sparks recorded. Their distribution of amplitudes had a mode at ΔF/F 0 = 0.7. The Ca 2+ release current underlying in-focus sparks was 11 pA, requiring 20 to 30 open channels, a number at the high end of earlier estimates. Spark frequency was greater than in earlier imaging studies of permeabilized ventricular cells, suggesting a greater susceptibility to excitation, which could have functional relevance for atrial cells. Ca 2+ release flux peaked earlier than the time of peak fluorescence and then decayed, consistent with significant sarcoplasmic reticulum (SR) depletion. The evolution of fluorescence and release flux were strikingly similar for in-focus sparks of different rise time (T). Spark termination involves both depletion of Ca 2+ in the SR and channel closure, which may be synchronized by depletion. The observation of similar flux in sparks of different T requires either that channel closure and other termination processes be independent of the determinants of flux (including [Ca 2+] SR) or that different channel clusters respond to [Ca 2+] SR with different sensitivity. © 2012 Shkryl et al. Source


Soloviev A.I.,Institute of Pharmacology and Toxicology | Bondarenko A.I.,Aa Bogomoletz Institute Of Physiology | Kizub I.V.,Institute of Pharmacology and Toxicology
Vascular Pharmacology | Year: 2012

The goal of this study was to clarify the mechanisms of hypoxic pulmonary vasoconstriction (HPV) reversal following selective glycolysis blockade and to assess possible contribution of endothelial electrogenesis to this phenomenon as a trigger mechanism.We compared smooth muscle (SM) contractility and endothelial cell (EC) membrane potential (MP) during acute hypoxia before and after glycolysis blockade. MPs were recorded from the endothelium of guinea pig pulmonary artery (GPPA) and thoracic aorta (GPTA) using the patch-clamp technique. Acute hypoxia caused hyperpolarization in GPTA EC, while EC from GPPA were depolarized. Also, acute hypoxia elicited constriction in isolated GPPA and dilatation in GPTA. Selective glycolysis inhibition always reversed both electrical and contractile responses in GPPA to hypoxia, but in GPTA this only occurred in 30% of experiments. It is likely that an unknown glycolysis-driven mechanism in EC mediates vascular tone regulation under hypoxia and underlies the paradoxical difference in the response of pulmonary and systemic arterial SM to hypoxia. Our data suggest that HPV development in GPPA might, at least partially, be driven by EC depolarization spreading to the underlying SM cells. © 2012 Elsevier Inc.. Source


Maslov L.N.,Research Institute for Cardiology | Mrochek A.G.,National Academy of Sciences of Belarus | Khaliulin I.G.,University of Bristol | Krylatov A.V.,Research Institute for Cardiology | And 5 more authors.
Vestnik Rossiiskoi Akademii Meditsinskikh Nauk | Year: 2014

In Russia inhospital lethality after acute myocardial infarction is 16,5-16,7%. The part of patients perishes even after recanalisation of infarctrelated coronary artery as a result of reperfusion cardiac injury. Experimental data indicate that adenosine receptor agonists and opioids can prevent reperfusion damages of heart that is mimic postconditioning phenomena. Data of clinical observation show that adenosine during intravenous infusion or intracoronary administration during thrombolysis or percutaneous coronary intervention exert infarct reducing effect and eliminate manifestation of «no-reflow» phenomenon. Clinical data indicate that morphine is able to prevent cardiac reperfusion injury in human. Thus, analysis of published data testifies that adenosine and opioid receptor agonists can be prototype for development of drugs for prophylaxis of reperfusion heart injury. Source


Sukhanova K.Y.,Aa Bogomoletz Institute Of Physiology | Thugorka O.M.,Aa Bogomoletz Institute Of Physiology | Bouryi V.A.,Aa Bogomoletz Institute Of Physiology | Harhun M.I.,St Georges, University of London | And 2 more authors.
Pharmacological Reports | Year: 2014

Background ATP is one of the principal sympathetic neurotransmitters which contracts vascular smooth muscle cells (SMCs) via activation of ionotropic P2X receptors (P2XRs). We have recently demonstrated that contraction of the guinea pig small mesenteric arteries evoked by stimulation of P2XRs is sensitive to inhibitors of IP3 receptors (IP3Rs). Here we analyzed contribution of IP3Rs and ryanodine receptors (RyRs) to [Ca 2+]i transients induced by P2XR agonist αβ-meATP (10 μM) in single SMCs from these vessels. Methods The effects of inhibition of L-type Ca2+ channels (VGCCs), RyRs and IP3Rs (5 μM nicardipine, 100 μM tetracaine and 30 μM 2-APB, respectively) on αβ-meATP-induced [Ca2+]i transients were analyzed using fast x-y confocal Ca2+ imaging. Results The effect of IP3R inhibition on the [Ca2+] i transient was significantly stronger (67 ± 7%) than that of RyR inhibition (40 ± 5%) and was attenuated by block of VGCCs. The latter indicates that activation of VGCCs is linked to IP3R-mediated Ca2+ release. Immunostaining of RyRs and IP3Rs revealed that RyRs are located mainly in deeper sarcoplasmic reticulum (SR) while sub-plasma membrane (PM) SR elements are enriched with type 1 IP3Rs. This structural peculiarity makes IP3Rs more accessible to Ca 2+ entering the cell via VGCCs. Thus, IP3Rs may serve as an "intermediate amplifier" between voltage-gated Ca2+ entry and RyR-mediated Ca2+ release. Conclusions P2X receptor activation in mesenteric artery SMCs recruits IP3Rs-mediated Ca 2+ release from sub-PM SR, which is facilitated by activation of VGCCs. Sensitivity of IP3R-mediated release to VGCC antagonists in vascular SMCs makes this mechanism of special therapeutic significance. © 2014 Institute of Pharmacology, Polish Academy of Sciences. All rights reserved. Source

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