Fura-2 Ratio Research Articles

Abstract 67: Fractalkine is Increased in Cardiomyocyte-Specific EP4 Knockout Hearts and Decreases Cardiomyocyte Contractility

Introduction: Our laboratory has reported that approximately half of aged male mice with cardiomyocyte-selective knockout of the prostaglandin E2 EP4 receptor sub-type (EP4 KO) exhibit reduced cardiac function. Gene array on left ventricles (LV) of such mice showed elevated expression of fractalkine, a novel chemokine that is implicated in human heart failure, when compared with age-matched controls. Hypothesis: We therefore hypothesized that fractalkine protein is increased in EP4 KO hearts and contributes to depressed contractility. Methods: To test this hypothesis, echocardiography was performed on 28 week old male EP4 KO and wild type controls (WT), and fractalkine was measured in LV by ELISA. Additionally, the effect of fractalkine on single myocyte contractility and intracellular calcium was determined using the IonOptix myocyte contractility system. Myocytes (AVM) from male 16-20 week old C57Bl/6 mice were loaded with 1 μM Fura-2 AM and the response to 5 ng/ml fractalkine measured under basal conditions and after stimulation with 0.1 μM isoproterenol (Iso) with pacing at 3 Hz. Results: LV fractalkine was greater in EP4 KO than WT controls (0.34 vs 0.23 ng/mg protein, n=4, p <0.05). Under basal conditions, treatment of AVM for 10 min with 5 ng/ml fractalkine decreased both the speed of contraction and relaxation (from - 164.1 ± 22.8 to -100.4 ± 18.9 μm/sec, p < 0.05; and from 83.3 ± 16.8 to 38.2 ± 11.7 μm/sec respectively, p = 0.065, n = 22-33 cells). After stimulation with Iso, fractalkine also decreased both the speed of contraction and relaxation, from -568.8 ± 30.6 to -467.5 ± 40.5 μm/sec and from 407.0 ± 28.4 to 304.7 ± 37.5 μm/sec respectively, p < 0.05. In addition, fractalkine decreased the percent change in contraction after Iso stimulation from 10.3 ± 0.5 to 8.4 ± 0.8, p = 0.05. Surprisingly, fractalkine increased the percent change in the Fura-2 ratio after Iso stimulation from 115.8 ± 6.6 to 135.8 ± 6.2, p < 0.05 despite reducing contractility. Conclusion: Our results suggest that fractalkine directly depresses myocyte contractility by mechanisms that are downstream of changes in intracellular calcium. Furthermore, these effects may contribute to the impaired contractility observed in EP4 KO mouse hearts.

N-Methyl-d-aspartate Receptor Mechanosensitivity Is Governed by C Terminus of NR2B Subunit

N-methyl-D-aspartate receptors (NMDARs), critical mediators of both physiologic and pathologic neurological signaling, have previously been shown to be sensitive to mechanical stretch through the loss of its native Mg(2+) block. However, the regulation of this mechanosensitivity has yet to be further explored. Furthermore, as it has become apparent that NMDAR-mediated signaling is dependent on specific NMDAR subtypes, as governed by the identity of the NR2 subunit, a crucial unanswered question is the role of subunit composition in observed NMDAR mechanosensitivity. Here, we used a recombinant system to assess the mechanosensitivity of specific subtypes and demonstrate that the mechanosensitive property is uniquely governed by the NR2B subunit. NR1/NR2B NMDARs displayed significant stretch sensitivity, whereas NR1/NR2A NMDARs did not respond to stretch. Furthermore, NR2B mechanosensitivity was regulated by PKC activity, because PKC inhibition reduced stretch responses in transfected HEK 293 cells and primary cortical neurons. Finally, using NR2B point mutations, we identified a PKC phosphorylation site, Ser-1323 on NR2B, as a unique critical regulator of stretch sensitivity. These data suggest that the selective mechanosensitivity of NR2B can significantly impact neuronal response to traumatic brain injury and illustrate that the mechanical tone of the neuron can be dynamically regulated by PKC activity.

Endothelin-1–Induced Proliferation Is Reduced and Ca2+Signaling Is Enhanced in Endothelin B–Deficient Optic Nerve Head Astrocytes

To characterize the influence of endothelin-1 (ET-1) on optic nerve head astrocyte (ONHA) proliferation and Ca²⁺ signaling in ONHAs lacking functional endothelin B (ETB) receptors. ONHAs were isolated from adult wild type (WT) and transgenic spotting lethal (TSL) rats, lacking functional ETB receptors. ONHA specificity was confirmed by positive glial fibrillary acidic protein (GFAP), negative A2B5 (a marker for type II astrocytes located outside the optic nerve head) and myelin basic protein (MBP) labeling. The mitogenic effects of 10⁻⁷ or 10⁻⁹ M ET-1, or vehicle were investigated for 48 or 72 hours in WT and TSL ONHAs. Intracellular calcium levels ([Ca²⁺](i)) were assessed in ONHAs loaded with fura-2 calcium indicator dye. ET-1-induced proliferation of TSL ONHAs was blunted at 48 hours (by 37% at 10⁻⁷ M and by 33% at 10⁻⁹ M) and 72 hours (by 117% at 10⁻⁷ M and by 100% at 10⁻⁹ M) compared with WT cells. ET-1-induced ONHA fura-2 ratio increases were significantly greater in TSL ONHAs (by 20% at 10⁻⁷ M and by 48% at 10⁻⁹ M) compared with WT ONHAs. ET-1-induced fura-2 ratio increases were blocked after pretreatment with BQ-610 (ETA antagonist) in WT and TSL ONHAs, but not by BQ-788 (ETB antagonist) in WT ONHAs. ET-1-induced ONHA proliferation is reduced in cells lacking functional ETB receptors, ET-1-induced [Ca²⁺](i) increases are enhanced in the absence of functional ETB receptors, and ETA, but not ETB, is required for ET-1-induced [Ca²⁺](i) elevation.

Alterations in intracellular free Ca 2+ concentrations in intact single muscle fibres from ALS mice

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease characterized by skeletal muscle atrophy, weakness and paralysis leading to death. The purpose of this study was to characterize the defects in excitation-contraction coupling in intact single fibres in a model of ALS. Single fibres were obtained from the flexor digitorum brevis muscle of control (CON) or G93A superoxide dismutase 1 Transgenic (Tg) mice at 9, 13 or 17–20 wks of age. Intracellular Ca2+ was assessed by the Fura-2 ratio at a range of stimulation frequencies (10–150Hz). Surprisingly there were no differences in peak tetanic Fura-2 ratio at any of the frequencies measured except at 10Hz in 17–20 wk old mice (0.585±0.02 vs. 0.670±0.02 in CON vs. Tg). There were however, changes in resting Fura-2 ratios at 13 wks (0.351±0.008 vs. 0.390±0.009) and 17–20 wks (0.374±0.001 vs. 0.415±0.001) in CON vs. Tg, respectively. These data indicate that single fibres from ALS mice retain the ability to respond to electrical activation even when animals are highly symptomatic (17–20 wks). However, there are elevations in resting intracellular Ca2+ at a pre-symptomatic age (13 wks) which may contribute to the atrophy process.

Effect of the SK/IK channel modulator 4,5‐dichloro‐1,3‐diethyl‐1,3‐dihydro‐benzoimidazol‐2‐one (NS4591) on contractile force in rat, pig and human detrusor smooth muscle

• To investigate the importance of small (SK)- and intermediate (IK)-conductance Ca2(+) -activated K(+) channels on bladder function, by studying the effects of 4,5-dichloro-1,3-diethyl-1,3-dihydro-benzoimidazol-2-one (NS4591), a new modulator of SK/IK channels, on contractions induced by electrical field stimulation (EFS) and carbachol in rat, pig and human detrusor. • Detrusor biopsies were obtained from rats, pigs and male patients undergoing cystectomy because of bladder cancer. • Force was recorded using myographs. • Intracellular free Ca(2+) was measured in myocytes using microfluorimetry. • In rat bladder rings subjected to EFS, cumulative addition of NS4591 (0.1-30 µM) decreased force by 82 ± 2.9% (n = 6).This effect was reduced by 64 ± 5.2% in the presence of 0.3 µM apamin, a specific inhibitor of SK channels. Apamin increased the force evoked by EFS significantly: force was increased by 14.2 ± 3.4% (n = 5) and 10.1 ± 2.6% (n = 7) in pig and human detrusor strips, respectively (P = 0.04 and P = 0.02). • The cumulative addition of NS4591 (0.3-30 µM) significantly reduced the amplitude of carbachol-induced rhythmic oscillations by 62.0 ± 12.0% (n = 12) and the minimum force between oscillations by 30 ± 5% (n = 9) in pig detrusor strips (P < 0.005). In the presence of 10 µM NS4591, carbachol (1 µM) induced rhythmic contractions with an amplitude and normalized mean power frequency (nmeanPF) of 8.4 ± 5.1% and 0.11 ± 0.06 mN root mean square (rms) Hz (n = 12), respectively, vs. 21 ± 3.4% and 0.17 ± 0.04 mN rms Hz in control strips (n = 13). Apamin induced 6- and 11-fold increases in amplitude and nmeanPF vs. 1.3- and 2-fold increases in control strips. • In human detrusor strips (n = 15), the cumulative addition of NS4591 (1-30 µM) significantly reduced the amplitude by 69 ± 11%, the nmeanPF by 78 ± 6% and the minimum force between carbachol-induced oscillations by 59 ± 5% (P < 0.008). The addition of apamin (0.3 µM) before application of 1 µM carbachol abolished the effects of NS4591 on amplitude and partially abolished its effect on nmeanPF by 41 ± 7%, vs. a 78 ± 6% reduction in the absence of apamin (n = 8). • In spontaneously active detrusor preparations, NS4591 reduced or abolished contractions. • Furthermore, NS4591 (10 µM) decreased the carbachol-induced increase in the fura-2 ratio by 43 ± 3% compared with control (n = 12) (P < 0.03). • The SK/IK channel modulator NS4591 inhibits EFS- and carbachol-induced contractions in rat, pig and human detrusor muscle. • NS4591 may have therapeutic potential for treatment of detrusor overactivity.

The chronic effects of neonatal alloxan-induced diabetes mellitus on ventricular myocyte shortening and cytosolic Ca2+

Diabetes mellitus is a serious global health problem, and cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. The chronic effects of neonatal alloxan- (ALX) induced diabetes mellitus on ventricular myocyte contraction and intracellular Ca(2+) transport have been investigated. Ventricular myocyte shortening was measured with a video edge detection system and intracellular Ca(2+) was measured in fura-2 loaded cells by fluorescence photometry. Diabetes was induced in 5-day old male Wistar rats by a single intraperitoneal injection of ALX (200 mg/kg body weight). Experiments were performed 12 months after ALX treatment. Fasting blood glucose was elevated and blood glucose at 60, 120 and 180 min after a glucose challenge (2 g/kg body weight, intraperitoneal) was elevated in diabetic rats compared to age-matched controls. Amplitude of shortening was significantly (P < 0.05) reduced in electrically stimulated myocytes from diabetic hearts (5.70 ± 0.24%) compared to controls (6.48 ± 0.28%). Amplitude of electrically evoked Ca(2+) transients was also significantly (P < 0.05) reduced in myocytes from diabetic hearts (0.11 ± 0.01 fura-2 ratio units) compared to controls (0.15 ± 0.01 fura-2 ratio units). Fractional sarcoplasmic reticulum Ca(2+) release was not significantly (P > 0.05) altered in myocytes from diabetic heart (0.70 ± 0.03 fura-2 ratio units) compared to controls (0.72 ± 0.03 fura-2 ratio units). Amplitude of caffeine-stimulated Ca(2+) transients was significantly (P < 0.05) reduced in myocytes from diabetic hearts (0.43 ± 0.02 fura-2 ratio units) compared to controls (0.51 ± 0.03 fura-2 ratio units). Area under the caffeine-evoked Ca(2+) transient was significantly (P < 0.05) reduced in myocytes from diabetic heart (0.77 ± 0.06 Vsec) compared to controls (1.14 ± 0.12 Vsec). Intracellular Ca(2+) refilling rate during electrical stimulation following application of caffeine was significantly (P < 0.05) slower in myocytes from diabetic heart (0.013 ± 0.001 V/sec) compared to controls (0.031 ± 0.007 V/sec). Depressed shortening may be partly attributed to depressed sarcoplasmic reticulum Ca(2+) transport in myocytes from neonatal ALX-induced diabetic rat heart.

Altered Calcium Signaling in an Experimental Model of Glaucoma

To investigate calcium signaling in a rat experimental model of glaucoma. A method for labeling ganglion cell layer (GCL) neurons with the calcium indicator Fura-2 in flat-mounted retinas of adult rats was established. Pharmacologically evoked responses in laser-induced glaucomatous and control retinas were imaged 2 weeks after the initial laser treatment. The optic nerves of the same eyes were evaluated for neurodegenerative changes. After laser treatment, intraocular pressure (IOP) was elevated 1.5- to 4.9-fold (24.70 ± 15.57 mm Hg) compared with control eyes (8.71 ± 1.53 mm Hg), and the area of neurodegenerative axons in optic nerve sections of laser-treated eyes was increased by 1.2- to 13.3-fold. The basal intracellular Ca(2+) level, as revealed by the Fura-2 ratio, was elevated in GCL neurons of laser-treated eyes compared with controls. This might suggest a mild degree of damage at the level of the soma in the GCL neurons of eyes with elevated IOP. Although glaucomatous GCL neurons remained functional as assessed pharmacologically, analysis of imaging data revealed that responses evoked by a brief application of ATP were slightly reduced rather than increased in the cells of laser-treated eyes compared with controls. No significant relationships were found between IOP/optic nerve damage and functional characteristics (basal intracellular Ca(2+) level or response to carbachol/elevated K(+)/ATP) within cells of laser-treated eyes. Ca(2+) imaging is a useful tool to map altered physiological characteristics of individual GCL neurons in the glaucomatous eye.

Distinguishing receptor‐ versus store‐operated calcium entry in arteriolar endothelium

The rise in [Ca+2]i for native endothelial cells responding to acetylcholine (ACh) consists of an initial rapid peak (internal release) succeeded by a sustained plateau (influx). Whereas the plateau reflects receptor‐activated Ca+2 entry (RACE) plus store‐operated Ca+2 entry (SOCE), respective contributions are not well defined in the microcirculation. The goal of this study was to distinguish the contributions of RACE and SOCE in arteriolar endothelium. Abdominal muscle feed arterioles were dissected from anesthetized C57BL/6 mice (n=6). Intact endothelial tubes were isolated enzymatically with gentle trituration, loaded with Fura‐2 dye and superfused (physiological saline, PSS; 2.5 ml/min; pH 7.4, 32 °C). With zero [Ca+2] in PSS, cells were emptied of Ca+2 using ACh (1 μM) or cyclopiazonic acid (CPA; 5 μM). Total Ca+2 influx (RACE + SOCE) was recorded by adding back 2 mM Ca+2 PSS with ACh; SOCE was distinguished by adding back Ca+2 with CPA. Peak Fura‐2 ratios were greater with ACh (2.73±0.18) vs. CPA (1.92±0.04, p&lt;0.05; n=3). Integrated responses (arbitrary ratio units) were greater when restoring Ca+2 with ACh (240±27) than with CPA (89±9, p&lt;0.05; n=3). By subtraction, SOCE accounted for 37% while RACE accounted for 63% of total Ca+2 influx. We suggest that ACh‐stimulated Ca+2 influx in mouse arteriolar endothelium occurs predominantly via receptor‐activated signaling events. (NIH HL41026 &amp; T32‐AR048523)

Regulation of Cerebromicrovascular Permeability by Lysophosphatidic Acid

Lysophosphatidic acid (LPA) increases permeability of cerebral endothelium in culture, but it has been suggested that histamine release is required in vivo. Cerebral venular permeability was measured by using the single-vessel micro-occlusion technique, and fura-2 ratios were used to track changes in endothelial [Ca(2+)]. Topical acute LPA application dose-dependently increased permeability (log EC(50)-9.4; similar to the K(d) of the LPA1 receptor). The calcium response to LPA was similar to histamine, but the permeability response was unaffected by H(2)-histamine receptor antagonism, and was blocked by Ki16425, a LPA1 receptor antagonist. The permeability response was blocked by nitric oxide synthase and free radical scavenging, which were carried out together, but not separately. Intravascular LPA bolus injection increased permeability. Whole serum albumin, or plasma albumin co-applied with LPA, increased permeability, but less potently than LPA itself (log EC(50) 5.1 and 6.1, respectively). Tachyphylaxis of the LPA1 receptor was demonstrated by LPA application for 10 minutes, which resulted in suppression of the response to subsequent applications for the following 15 minutes. Lysophosphatidic acid increases cerebrovascular permeability by acting directly on the endothelium and utilizes both nitric oxide and free radical signaling pathways.

The role of calcium in apoptosis induced by 7β‐hydroxycholesterol and cholesterol‐5β,6β‐epoxide

Oxysterols, such as 7beta-hydroxy-cholesterol (7beta-OH) and cholesterol-5beta,6beta-epoxide (beta-epoxide), may have a central role in promoting atherogenesis. This is thought to be predominantly due to their ability to induce apoptosis in cells of the vascular wall and in monocytes/macrophages. Although there has been extensive research regarding the mechanisms through which oxysterols induce apoptosis, much remains to be clarified. Given that experimental evidence has long associated alterations of calcium (Ca(2+)) homeostasis to apoptotic cell death, the aim of the present study was to determine the influence of intracellular Ca(2+) changes on apoptosis induced by 7beta-OH and beta-epoxide. Ca(2+) responses in differentiated U937 cells were assessed by epifluorescence video microscopy, using the ratiometric dye fura-2. Over 15-min exposure of differentiated U937 cells to 30 muM of 7beta-OH induced a slow but significant rise in fura-2 ratio. The Ca(2+) channel blocker nifedipine and the chelating agent EGTA blocked the increase in cytoplasmic Ca(2+). Moreover, dihydropyridine (DHP) binding sites identified with BODIPY-FLX-DHP were blocked following pretreatment with nifedipine, indicating that the influx of Ca(2+) occurred through L-type channels. However, following long-term incubation with 7beta-OH, elevated levels of cytoplasmic Ca(2+) were not maintained and nifedipine did not provide protection against apoptotic cell death. Our results indicate that the increase in Ca(2+) may be an initial trigger of 7beta-OH-induced apoptosis, but following chronic exposure to the oxysterol, the influence of Ca(2+) on apoptotic cell death appears to be less significant. In contrast, Ca(2+) did not appear to be involved in beta-epoxide-induced apoptosis.

4-Hydroxy-2-nonenal Induces Calcium Overload via the Generation of Reactive Oxygen Species in Isolated Rat Cardiac Myocytes

Background It has been reported that that the amount of 4-hydroxy-2-nonenal (HNE), which is a major lipid peroxidation product and a cytotoxic aldehyde, is increased in the human failing myocardium. This study was designed to determine whether HNE has a pro-oxidant effect in cardiac myocytes and whether HNE causes Ca 2+ overload. Methods and Results Exposure to HNE for 10 minutes in the presence of ferric nitrilotriacetate induced the production of hydroxyl radical (·OH) in the rat myocardium as assessed by electron spin resonance spectroscopy, and HNE induced the generation of reactive oxygen species (ROS) in a dose-dependent manner as assessed by 2′, 7′-dichlorofluorescein diacetate fluorescence. HNE increased intracellular Ca 2+ concentration ([Ca 2+] i) as assessed by fura-2 ratio in a dose- and time-dependent manner. After 20 minutes of HNE (400 μmol/L) exposure, hypercontracture was induced in 67% of the cells. Catalase, an antioxidative enzyme that can decompose hydrogen peroxide (H 2O 2), significantly attenuated the increase in [Ca 2+] i and completely inhibited hypercontracture. Carvedilol, a β-blocker with potent antioxidant activity, also significantly attenuated the increase in [Ca 2+] i and completely inhibited hypercontracture, but propranolol had no effect on either [Ca 2+] i increase or hypercontracture. Conclusions HNE induces the formation of ROS, especially H 2O 2 and ·OH, in cardiomyocytes and subsequently ROS cause intracellular Ca 2+ overload. HNE formation may play an important role as a mediator of oxidative stress in heart failure.

Activation by Ca2+/calmodulin of an exogenous myosin light chain kinase in mouse arteries

Activation of myosin light chain kinase (MLCK) and other kinases was studied in the arteries of transgenic mice that express an optical fluorescence resonance energy transfer (FRET) MLCK activity biosensor. Binding of Ca(2+)/calmodulin (Ca(2+)/CaM) induces an increase in MLCK activity and a change in FRET. After exposure to high external [K(+)], intracellular [Ca(2+)] (fura-2 ratio or fluo-4 fluorescence) and MLCK activity both increased rapidly to an initial peak and then declined, rapidly at first and then very slowly. After an initial peak ('phasic') force was constant or increased slowly (termed 'tonic' force). Inhibition of rho-kinase (Y-27632) decreased tonic force more than phasic, but had little effect on [Ca(2+)] and MLCK activation. Inhibition of PKCalpha and PKCbeta with Gö6976 had no effect. KN-93, an inhibitor of CaMK II, markedly reduced force, MLCK FRET and [Ca(2+)]. Applied during tonic force, forskolin caused a rapid decrease in MLCK FRET ratio and force, but no change in Ca(2+), suggesting a cAMP mediated decrease in affinity of MLCK for Ca(2+)/CaM. However, receptor (beta-adrenergic) activated increases in cAMP during KCl were ineffective in causing relaxation, changes in [Ca(2+)], or MLCK FRET. At the same tonic force, MLCK FRET ratio activated by alpha(1)-adrenoceptors was approximately 60% of that activated by KCl. In conclusion, MLCK activity of arterial smooth muscle during KCl-induced contraction is determined primarily by Ca(2+)/CaM. Rho-kinase is activated, by unknown mechanisms, and increases 'Ca(2+) sensitivity' significantly. Forskolin mediated increases in cAMP, but not receptor mediated increases in cAMP cause a rapid decrease in the affinity of MLCK for Ca(2+)/CaM.

Calumenin Knock-down (kd) Enhances Ca2+ Cycling Ability In Hl-1 Cells

Calumenin is a multiple EF-hand Ca2+-binding protein localized in the sarcoplasmic reticulum (SR) lumen. Evidence of the interaction between calumenin and SERCA2 in rat cardiac SR was shown recently (Mol. Cells, 26:265-269, 2008). To elucidate the possible role of calumenin in cardiac excitation-contraction (E-C) coupling, calumenin was knocked down by transfection of mouse cardiac cell line (HL-1 cells) with calumenin specific siRNA oligonucleotides. After 72 hrs of transfection, calumenin protein level was reduced by 75% without any obvious changes in the expression levels of other E-C coupling proteins such as RyR2, SERCA2, NCX, CSQ and PLB. A field stimulation (1Hz) of KD cells (n = 58) led to significantly increased Ca2+ transient peak height (1.02 ± 0.02 vs. 0.82 ± 0.03 fura-2 ratio at 340 and 380 nm, p < 0.05), decreased time to peak (0.093 ± 0.001 vs. 0.107 ± 0.003 s, p < 0.05) and time to 50% baseline (0.172 ± 0.005 vs. 0.235 ± 0.006 s, p < 0.05) as compared to control cells (n = 44). On the other hand, the SR Ca2+ load remained unchanged in KD cells. Pull-down experiments with GST fusion proteins showed that calumenin interacts with both RyR2 and SERCA2 in a Ca2+ dependent manner. Taken together, the present results suggest that calumenin is related to SR Ca2+ homeostasis. Currently, the molecular interactions between calumenin and SERCA2 or RyR2 are being examined by using various deletion mutants.

UV Ratiometric Imaging Of Isolated Ventricular Cardiomyocytes Using An LED Based Illuminator

Ratiometric fluorescence microscopy methods allow researchers to obtain calibrated images of dynamic changes in the physical properties of cells and tissues independent of dye concentration.The wavelength changes required for ratiometric imaging are routinely achieved using a short-arc source in combination either with a diffraction grating or interference filters mounted in a filter wheel. Filter wheels typically switch positions in around 50ms, scanning monochromators can achieve wavelength changes within a ms. These approaches generally limit ratio imaging rates to a little over 10Hz, and have the inherent drawbacks of short lamp life and high thermal emissions. An LED based system has much higher stability than a short-arc source, and with sub-microsecond wavelength switching times allows the very highest speeds to be obtained. Here we made use of the popular Ca2+ probe Fura-2 to record images of intracellular [Ca2+] in isolated ventricular cardiomyocytes at frame rates in excess of 100Hz using a simple and inexpensive LED based system.Transient spatial gradients of Ca2+ can exist in single ventricular cardiomyocytes during spontaneous release of Ca2+ from the sarcoplasmic reticulum. This can result intracellular Ca2+ waves traveling at 100-200microns/s along the length of the cell. Rapid imaging is required to resolve the time course and pattern of intracellular Ca2+ release.Conventionally Fura-2 ratios are calculated by monitoring the fluorescence signal elicited from excitation at 340 and 380nm, however short wavelength LEDs (340nm) are not available currently. Alternatively, reliable ratiometric measurements can be made by exciting Fura-2 at its isobestic point (360nm) and 380nm. We have followed fast spatial changes in Ca2+ by switching wavelength in the microsecond time domain using commercial LEDs emitting at peak wavelengths of 365nm and 385nm.

Myofilament sensitivity to Ca2+ in ventricular myocytes from the Goto–Kakizaki diabetic rat

The chronic effects of type 2 diabetes mellitus on myofilament sensitivity to Ca(2+) in ventricular myocytes from the Goto-Kakizaki (GK) rat have been investigated. Experiments were performed in ventricular myocytes isolated from 17-month GK rats and age-matched Wistar controls. Myocytes were loaded with fura-2 (an indicator for intracellular Ca(2+) concentration) and the fura-2 ratio (340/380 nm), and shortening were measured simultaneously in electrically stimulated myocytes. Myofilament sensitivity to Ca(2+) was assessed from phase-plane diagrams of fura-2 versus cell length by measuring the gradient of the fura-2-cell length trajectory during late relaxation of the twitch contraction. Non-fasting and fasting blood glucose were elevated in GK rats compared to controls. Fasting blood glucose was 151.5 +/- 15.3 mg/dl (n = 8) in GK rats compared to 72.1 +/- 3.6 mg/dl (n = 9) in controls. At 120 min after intraperitoneal injection of glucose (2 g/kg body weight), blood glucose was 570.8 +/- 36.8 mg/dl in GK rats compared to 148 +/- 8.6 mg/dl in controls. Amplitude of shortening was significantly increased in myocytes from GK rats (6.56 +/- 0.54%, n = 31) compared to controls (5.05 +/- 0.43%, n = 36), and the amplitude of the Ca(2+) transient was decreased in myocytes from GK rats (0.23 +/- 0.02 RU, n = 31) compared to controls (0.30 +/- 0.02 RU, n = 36). The fura-2-cell length trajectory during the late stages of relaxation of the twitch contraction was steeper in myocytes from GK rats (89.2 +/- 16.6 microm/RU, n = 27) compared to controls (31.9 +/- 5.9 microm/RU, n = 35). Increased amplitude of shortening, accompanied by a decrease in amplitude of the Ca(2+) transient, might be explained by an increased myofilament sensitivity to Ca(2+).

Anionic Selectivity Sequence of the Cl−–H+ Symporter in the Synaptosomal Preparation from Rat Brain Cortex

The Na(+)/H(+) exchanger has been the only unequivocally demonstrated H(+)-transport mechanism in the synaptosomal preparation. We had previously suggested that a Cl(-)-H(+) symporter (in its acidifying mode) is involved in cytosolic pH regulation in the synaptosomal preparation. Supporting this suggestion, we now show that: (1) when synaptosomes are transferred from PSS to either gluconate or sulfate solutions, the Fura-2 ratio remains stable instead of increasing as it does in 50 mM K solution. This indicates that these anions do not promote a plasma membrane depolarization. (2) Based in the recovery rate from the cytosolic alkalinization, the anionic selectivity of the Cl(-)-H(+) symporter is NO(3)(-) > Br(-) > Cl(-) >> I(-) = isethionate = sulfate = methanesulfonate = gluconate. (3) PCMB 10 muM inhibits the gluconate-dependent alkalinization by 30 +/- 6%. (4) Neither Niflumic acid, 9AC, Bumetanide nor CCCP inhibits the recovery from the cytosolic alkalinization.

Misinterpretation of the effect of amlodipine on cytosolic calcium concentration with fura-2 fluorospectrometry

Due to its high sensitivity, reproducibility and reliability, fura-2 is one of the most preferred tools to quantify Ca2+ levels. Recently, using fura-2/AM, some calcium channel blockers (CCBs) have been reported to have inhibitory effects on intracellular Ca2+ signaling even in nonexcitable cells that do not possess voltage-dependent Ca2+ channels (VDCC). Some CCBs are known to be photosensitive. Since photosensitive chemicals often have autofluorescent property, it is likely that CCBs with autofluorescence interfere with the fluorescent dye probes for Ca2+, which may cause misinterpretation of the results. The aim of this study was to clarify the characteristics of various CCBs on Ca2+ fluorescent probes and to identify proper fluorescent probes for each CCB to allow reliable Ca2+ measurement using endothelial cells (ECs), which possess no VDCC. Thapsigargin (TG, Ca2+-ATPase inhibitor on endoplasmic reticulum) increased fura-2 F340/F380 ratio from 0.75+/-0.06 to 4.28+/-0.32, suggesting the increase in cytosolic Ca2+ concentrations in ECs. Verapamil, nifedipine and nicardipine did not affect TG-induced increase in fura-2 F340/F380 ratio; however, amlodipine dose dependently inhibited the rise of fura-2 ratio by 24% at 100 nM, by 55% at 1 microM and by 82% at 10 microM, respectively, in ECs. In ex vivo experiments without cells, due to the autofluorescence of amlodipine excited by ultraviolet (UV light), amlodipine itself boosted the intensity of F380 much more than it did that of F340, which results in a decrease in F340/F380 ratio of fura-2 fluorescence. Rhod-2/AM, a fluorescent probe of which the excitation wavelength is out of the excitation spectrum of amlodipine, showed that amlodipine did not affect TG-induced intracellular Ca2+ increase in ECs. When the effect of amlodipine on intracellular Ca2+ concentration is assessed, fura-2 fluorospectrometry should not be used due to fluorescent interaction between amlodipine and fura-2, and using rhod-2 is strongly recommended.

H2S preconditioning-induced PKC activation regulates intracellular calcium handling in rat cardiomyocytes

The present study was aimed to investigate the regulatory effect of protein kinase C (PKC) on intracellular Ca(2+) handling in hydrogen sulfide (H(2)S)-preconditioned cardiomyocytes and its consequent effects on ischemia challenge. Immunoblot analysis was used to assess PKC isoform translocation in the rat cardiomyocytes 20 h after NaHS (an H(2)S donor, 10(-4) M) preconditioning (SP, 30 min). Intracellular Ca(2+) was measured with a spectrofluorometric method using fura-2 ratio as an indicator. Cell length was compared before and after ischemia-reperfusion insults to indicate the extent of hypercontracture. SP motivated translocation of PKCalpha, PKCepsilon, and PKCdelta to membrane fraction but only translocation of PKCepsilon and PKCdelta was abolished by an ATP-sensitive potassium channel blocker glibenclamide. It was also found that SP significantly accelerated the decay of both electrically and caffeine-induced intracellular [Ca(2+)] transients, which were reversed by a selective PKC inhibitor chelerythrine. These data suggest that SP facilitated Ca(2+) removal via both accelerating uptake of Ca(2+) into sarcoplasmic reticulum and enhancing Ca(2+) extrusion through Na(+)/Ca(2+) exchanger in a PKC-dependent manner. Furthermore, blockade of PKC also attenuated the protective effects of SP against Ca(2+) overload during ischemia and against myocyte hypercontracture at the onset of reperfusion. We demonstrate for the first time that SP activates PKCalpha, PKCepsilon, and PKCdelta in cardiomyocytes via different signaling mechanisms. Such PKC activation, in turn, protects the heart against ischemia-reperfusion insults at least partly by ameliorating intracellular Ca(2+) handling.

Dynamic Regulation, Desensitization, and Cross-talk in Discrete Subcellular Microdomains during β2-Adrenoceptor and Prostanoid Receptor cAMP Signaling

Dynamic and localized actions of cAMP are central to the generation of discrete cellular events in response to a range of G(s)-coupled receptor agonists. In the present study we have employed a cyclic nucleotide-gated channel sensor to report acute changes in cAMP in the restricted cellular microdomains adjacent to two different G(s)-coupled receptor pathways, beta(2)-adrenoceptors and prostanoid receptors that are expressed endogenously in HEK293 cells. We probed by either selective small interference RNA-mediated knockdown or dominant negative overexpression the contribution of key signaling components in the rapid attenuation of the local cAMP signaling and subsequent desensitization of each of these G-protein-coupled receptor signaling pathways immediately following receptor activation. Direct measurements of cAMP changes just beneath the plasma membrane of single HEK293 cells reveal novel insights into key regulatory roles provided by protein kinase A-RII, beta-arrestin2, cAMP phosphodiesterase-4D3, and cAMP phosphodiesterase-4D5. We provide new evidence for distinct modes of cAMP down-regulation in these two G(s)-linked pathways and show that these distinct G-protein-coupled receptor signaling systems are subject to unidirectional, heterologous desensitization that allows for limited cross-talk between distinct, dynamically regulated pools of cAMP.

Effects of (-)-stepholidine on NMDA receptors: comparison with haloperidol and clozapine

To examine whether (-)-stepholidine (SPD) has a direct effect on the N-methyl- D-aspartic acid receptors (NMDAR) containing the NMDA receptor subunits NR2A or NR2B and to compare its effect with those of haloperidol (Hal) and clozapine (Cloz). NMDAR was transiently expressed in human embryonic kidney 293 (HEK293) cells. Changes in intracellular calcium concentration ([Ca2+]i) induced by NMDAR activation were monitored with Fura-2 ratio imaging techniques. SPD had no significant effects on either subunit of NMDAR at a concentration of less than 100 micromol/L. Hal selectively inhibited NMDAR containing the NR2B subunit, whereas Cloz inhibited both subunits of NMDAR. Although both Hal and Cloz inhibited NR1a/NR2B receptor-mediated Ca2+ influx, their effects were different. Hal was more potent and had a faster peak effect than Cloz. Both Hal and Cloz inhibit NMDAR-mediated function, whereas SPD produced only a little inhibition at a high concentration. Based on our other studies, the modulation of SPD on NMDAR function may be via D1 receptor action underlying an indirect mechanism.