To clarify whether these abnormal Ca2+ transients can be attributed to abnormal action potential generation or abnormal Ca2+ handling/excitation-contraction (EC) coupling, we developed a procedure to determine Ca2+ and action potential signals at the cellular level in isolated heart tissues.Abnormal Ca2+ transients. For the failed and alternating Ca2+ transient generation, there were two types of causes, i.e., failed or abnormal action potential generation and abnormal EC coupling. In cells showing delayed initiation of Ca2+ transients with field stimulation, action potential onset was delayed and the rate of rise was slower than in healthy cells. Similar delayed onset was also observed in the presence of heptanol, an inhibitor of gap junction channels but having a non-specific channel blocking effect. A Na+ channel blocker, on the other hand, reduced the rate of rise of the action potentials but did not result in desynchronization of the action potentials. The delayed onset of action potentials can be explained primarily by impaired gap junctions and partly by Na+ channel inactivation.After loading ventricular papillary muscle with rhod-2 and di-4-ANEPPS, mono-wavelength fluorescence images from rhod-2 and ratiometric images of two wavelengths of emission from di-4-ANEPPS were sequentially obtained. To mimic the ventricular tachycardia, the ventricular muscles were field-stimulated in non-flowing Krebs solution which elicited abnormal Ca2+ signals and that our methods are useful for investigating the physiology and pathophysiology of heart muscle.Our results indicate that there are multiple patterns for the causes of abnormal Ca This Ca2+ influx, in turn, triggers the opening of ryanodine receptors in the sarcoplasmic reticulum (SR) and results in a transient elevation of cytosolic Ca2+ sufficient for muscular contraction. Concomitantly with this excitation-contraction (EC) coupling process, the action potentials in those cells evoke further action potentials in adjacent cells by current flux through gap junction channels. A serial propagation of this process occurs rapidly with a rate of ∼1 mm/ms at 37°C in ventricular muscle. Therefore, when Ca2+ signals in heart tissues are observed with a light microscope, the onset of action potential induced Ca2+ transients in individual cells within the field view appear synchronized.Action potentials generated in cardiac cells initiate Ca2+ transient alternans and desynchronized Ca2+ transients have been reported 2+ transient alternans is a phenomenon in which an alternating Ca2+ transient occurs from beat-to-beat. Two major mechanisms that can account for these alternans have been proposed 2+ transient accompanies the broader action potentials. Secondly, the Ca2+ transient alternans can be also induced even when action potential waveforms are uniform. In this case, the amount of Ca2+ release from the SR alternates from beat-to-beat, i.e., the process of EC coupling is not constant 2+ released from SR may inactivate VDCC to reduce Ca2+ current. Identifying how these two mechanisms interact to produce cellular alternans has remained difficult since Ca2+ and membrane potentials are bidirectionally coupled Under a variety of pathophysiological conditions, including heart failure, ischemia-reperfusion, and ventricular tachycardia, the occurrence of Ca2+ signals in cardiac muscles paced at ∼2 Hz in non-flowing Krebs solution 2+ transients and occasional missing Ca2+ transients in addition to Ca2+ transient alternans. The desynchronized onsets of Ca2+ transients, with lags of several tens of ms, were detected in some cells in a small area, 0.3 mm×0.3 mm of field of view of microscope. These observations can be explained by delayed onset of the action potentials that cause the Ca2+ transients, although another possible explanation would be that EC coupling was desynchronized whereas onset of action potential was synchronized.We have been studying abnormal Ca2 consumption but O2 supply is compromised by cardiac dysfunction 2+ dynamics, are not well known. It is important to determine the mechanisms of abnormal Ca2+ and action potential signals under such pathophysiological conditions.Our preparation resembles the condition of sustained ventricular tachycardia (sustained VT) where cardiac cells are hyperactive in terms of O2+ responses can be attributed to abnormal action potentials or other factors such as failure of EC coupling. We monitored membrane potential and Ca2+ signals using the ratiometric fluorescent membrane potential indicator di-4-ANEPPS and the Ca2+ indicator rhod-2 in the same preparation. Our results indicate that there are multiple patterns for the causes of abnormal Ca2+ signals and that determination of both signals are necessary for investigation of mechanisms of cardiac arrhythmia. Causes of abnormal signals will be discussed based on effects of inhibitors of gap junctions and Na+ channels and of reduced current influx into isolated single cells.Our main aim in this study was to investigate whether the abnormal Ca−1). The heart was quickly removed from the chest and perfused via the aorta with a high-K+ Krebs solution. Papillary muscle bundles  were excised from the left and right ventricles. For membrane potential imaging, papillary muscles were incubated in a normal Krebs solution containing 10 µM di-4-ANEPPS at room temperature for 40 min. For Ca2+ and membrane potential imaging in the same muscles, they were incubated with 5 µM rhod-2 AM in normal Krebs solution for 2 hr and 10 µM di-4-ANEPPS was added to the solution for the last 40 min. After washing out the dyes, the preparations were kept in Krebs solution and used within three hours.All experiments were carried out in accordance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences, and approved by the Committee for Animal Experimentation of Juntendo University and the Saga University Animal Care and Use Committee. Cardiac muscle preparations were obtained following the procedure described previously −1 . Experiments were carried out at 25°C.For imaging experiments, a muscle bundle was connected with silk thread to hooks in a chamber , stretched to its slack length, and gently pushed toward the bottom with a Plexiglas plate so that the lower surface of the bundle was 5–10 µm above the bottom. This gap allowed the cells at the lower surface access to the bathing solution. The muscle was superfused with normal Krebs solution at a rate of 2 ml min2+ signals, ventricular muscles were stimulated at 2 Hz for 10∼20 min in non-flowing Krebs solution as described previously After mounting, the muscles were conditioned by stimulating with a pair of platinum wire electrodes  with current pulses of suprathreshold voltage (2 ms duration) at 0.5 Hz for >10 min. In control recording, muscles were stimulated at 0.5 or 2 Hz in flowing Krebs solution. To induce abnormal CaWhen local stimulation was used, a pair of stimulation electrodes  were placed at one end of the muscle and signals at a region 2∼3 mm from the electrodes were monitored.2+ measurements, rhod-2 was excited by 568 nm and emission light was passed through the BP620/80 filter and imaged with the same camera at the one side  equipped with an Argon Krypton Ion Laser (488 and 568 nm excitation)  and a 20one side . The z-a2+ signals from rhod-2 were visually detected and captured as a sequence of images, and then a sequence of di-4-ANEPPS signals were obtained from the same region. Ca2+ signals were again monitored after the di-4-ANEPPS sequence to confirm the abnormal Ca2+ signals were still observed. The interval between two imaging sequences was 30 s∼1 min.Data acquisition was performed using AquaCosmos software . In typical experiments, each image (0.16 mm×0.32 mm in size) was taken with 64×128 pixels every 8.7 ms (exposure time was 8 ms), or with 256×512 pixels every 17 ms (exposure time was 16 ms) and 500∼1000 images were captured during one measurement. Because di-4-ANEPPS fluorescence intensity changes produced by an action potential were small, 10% or less of total fluorescence, it was difficult to detect abnormal action potential signals by direct visual observation of fluorescence. Consequently, in this study, abnormal Ca2+ signals because the fluorescence change in di-4-ANEPPS due to an action potential was very small, less than 1% of the rhod-2 signal change, during a Ca2+ transient.The cross-talk signal of each dye was determined. Contamination of the rhod-2 signal by the di-4 ANEPPS signals (Ex488/Em525 & Ex488/Em620) was negligible (less than 1%). Conversely, fluorescence from di-4-ANEPPS significantly increased background fluorescence of the rhod-2 signal (568Ex/620Em) depending on the loading level of di-4-ANEPPS. However, changes in membrane potential had no effect on the profile of active Ca2+ and membrane potential signals were obtained from fluorescence and ratio-signal changes from ROIs (Region of interest) which are contours of individual cells and from a whole field of view. Because ventricular cells have abundant transverse-tubule (TT) membranes, most of the membrane potential signals were assumed to derive from TT. As we did not use any reagent to inhibit muscle contractions, healthy muscles sometimes generated considerable movement during measurements. In general, Ca2+ signals were more affected than action potential signals by movement artifacts because of the mono-wavelength determination. Even with the movement, however, the onset of action potentials and Ca2+ transients could be correctly captured because they preceded muscle contractions.The time course of CaSingle ventricular cells of the guinea pig were isolated by a standard enzymatic method 3, 1 NaH2PO4, 2 CaCl2, 1 MgCl2 and 10 glucose and was saturated with 95% O2-5% CO2. High-K+ Krebs solution used for muscle preparation contained 25 mM KCl instead of 5 mM. Normal Tyrode solution used for whole cell clamp experiments contained (mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.33 NaH2PO4, 11 glucose, and 5 HEPES-NaOH (pH 7.4). Pipette solution contained (mM): 110 K-aspartate, 30 KCl, 10 NaCl, 5 Mg-ATP, 0.1 Tris-GTP, and 20 HEPES-KOH (pH 7.2). Di-4-ANEPPS and rhod-2 AM were obtained from Invitrogen/Molecular Probes . Pilsicainide (SUN 1165), a Class Ic pure sodium channel blocker Normal Krebs solution contained (mM): 120 NaCl, 5 KCl, 25 NaHCOData were analyzed using Student's t-test and reported as mean±SEM. P values below 0.05 were considered to be significant.2+ transients and action potential signals were sequentially determined in a muscle. Under control condition, cells in the field of view showed global Ca2+ transients in response to every field stimuli although a later part of the signal was contaminated with a movement artifact  much shorter than image acquisition interval (8.7 ms). This is reasonable because the rate of rise of action potential in isolated single cells was 100∼200 mV/ms.2+ signals in cardiac muscles could be induced by pacing at ∼2 Hz in non-flowing Krebs solution 2+ signals induced by the protocol, which was obtained in an experiments similar to 2+ transients in a control muscle. Cells in the muscle showed normal synchronized Ca2+ transients in response to the stimulation. When the muscle was stimulated at 2 Hz for 20 minutes in non-flowing Krebs solution, cells showed delayed, failed and alternating amplitudes of Ca2+ transients upon electrical stimuli along with a prolonged decay of Ca2+ transients and an elevated diastolic Ca2+ inferred from rhod-2 fluorescence (Panel B).We have previously shown that abnormal Ca2+ transients. In 2+ transients with every second stimuli. Determination of di-4-ANEPPS signal revealed that action potentials also occurred every second stimulus. This indicates that the refractory period of the myocytes was prolonged which prevented the action potentials and Ca2+ transients from following every stimulation.In experiments shown in 2+ transients still occurred once every two stimuli  with a big Ca2+ release  was ∼20% shorter than that with a lesser Ca2+ release . This phenomenon represents a kind of action potential-, Ca2+- and mechanical alternans 2+ and mechanical alternans with mild action potential alternans. In this muscle, cells in the field of view responded with synchronized Ca2+ transients. When the stimulation interval was further prolonged, notable alternating Ca2+ transients with large and small amplitudes appeared on every stimulation  and action potentials (b) under control condition. After stimulation in non-flowing solution, cell 2 showed delayed smaller Ca2+ transients  was significantly longer than that from individual cells (23.8±1.0 ms). This indicates that the rising phase of the action potentials was desynchronized from cell to cell in the field of view.Both in 2+ transients between two aligned cells could be reproduced in the presence of heptanol 2+ transients could be correlated with the action potentials, action potential signals were monitored in ventricular muscles loaded with di-4-ANEPPS. Under control conditions, the onsets of action potentials were simultaneous in three aligned cells  similarly. The prolonged latency probably reflects suppressed conduction velocity, indicating suppressed Na+ channel activity. In the presence of 40 µM pilsicainide, latency was ∼16 ms after stimulation, and the average t10–90  was significantly slower than control (<8 ms) but similar to that in cells showing desynchronized Ca2+ transients . In spite of the delayed onset of action potentials, action potentials in all cells were synchronized within a field of view of 0.3 mm×0.15 mm  and in the whole field of view .Another possibility for the desynchronized onset of action potentials is the inactivation of Na×0.15 mm  as revea10–90 in individual cells was significantly reduced in muscles after the pacing protocol, in the presence of heptanol and pilsicainide. However, significant differences in the t10–90 between individual cells and whole field of view were detected in muscles with desynchronized onset of Ca2+ transients and with heptanol, but not in the presence of pilsicainide. These results indicate that the inhibition of Na+ channels alone could not cause desynchronized onset of action potentials.max) on action potential latency. The dV/dtmax values were independent of latency period and remained relatively constant in individual cells although their absolute values were variable among cells. It is demonstrated that a delay of 100 ms or more in action potential generation can occur in association with decreased current influx into myocytes with little change in dV/dtmax.The results shown above indicate that the desynchronized onset of action potentials can be attributed to the suppression of gap junction channels which causes a decrease in depolarizing current influx from neighboring cells. To evaluate the possibility that reduced depolarizing current influx can mimic a slow onset of action potential generation, isolated single cardiac myocytes were injected with a very small amount of current. As shown in 2+ transients in pathophysiological cardiac muscle, we carried out serial measurements of Ca2+ and membrane potential signals in the same cardiac muscle preparations. In general, there were two reasons for abnormal Ca2+ transients, i.e., abnormal action potential generation and abnormal EC coupling. Determination of both signals at the cellular level is important for the investigation of the physiology and pathology of heart muscles.To clarify the cause of abnormal Ca2+ and membrane potential signals simultaneously from large numbers of pixels in the same heart tissue have been made by several groups 2+ indicator and potentiometric dye that have been used are, indo-1 and di-4-ANEPPS 2+ and membrane potential signals at the cellular level in rat hearts using RH237 and fluo-4 2+ signals. Compared to mono-wavelength determination, ratiometric signals of two wavelengths of emission from di-4-ANEPPS has higher signal-to-noise ratio without using a smoothing filter as noise from the light source tends to be cancelled 2+ and action potential signals could not be measured simultaneously with this procedure, we were able to get insight into relation between Ca2+ and membrane potentials because, in most cases, muscle conditions were relatively stable for several minutes, which was longer than the period of determination of both signals.Attempts to capture Ca+ channels which reduces the rate of rise of the action potentials. One probable mechanism is suggested by the response of the cells to elevated [Ca2+]i2+]i was considerably higher than in normal muscles and the decay time of Ca2+ transients was significantly prolonged2+ extrusion by Na+-Ca2+ exchanger would be enhanced and would lead to depolarization of the resting membrane potential, which, in turn, would result in inactivation of Na+ channels. In addition to this, activation of KATP channels also may be involved in the reduced excitability because APD tended to be shortened after the pacing protocol The alternating suppression of the action potential generation indicates that cardiac cells are less responsive to triggering stimulation. The decreased responsiveness can be explained by partial inactivation of Na2+ transients were observed once every two stimuli. This is an extreme case of Ca2+ alternans, where a large and small amplitude of Ca2+ transients take turn from beat-to-beat. The mechanisms of the Ca2+ alternans is explained by depletion of diastolic SR Ca2+ content after the big Ca2+ release and/or fluctuation of Ca2+ releasing activity from RyR2 which is regulated by luminal SR Ca2+2+ during action potential generation even when Ca2+ transients and contractions were missed every two stimuli.The instances shown in 2+ transients; (1) individual cells take turns with independent, out-of-phase type Ca2+ transients and (2) cells in a cluster show synchronous alternating Ca2+ transients. The latter case would contribute more to mechanical alternans. The reason for the synchronized alternating Ca2+ transients is unclear, but a probable explanation is that once a failure of action potential generation occurs, it may reset the Ca2+ release mode in all the cells to the big release with high luminal Ca2+. Further study is needed to examine this hypothesis.There were two patterns for the alternating missing of Ca2+ transients Although we sometimes observed two-aligned cells taking turn showing alternating out-of-phase Ca2+ transients were detected, a similar delay was also detected in action potential signals in the same cells, indicating that the delayed Ca2+ transient onset is attributed to slow onset of action potential. Because the duration of the field stimulation pulse was 2 ms, action potentials occurring with several tens of ms delay could not have been induced directly by the stimulation. Instead, they must have resulted from propagated action potentials from adjacent cells. This phenomenon can be expected when responsiveness of some cells to field stimulation is reduced and the rate of current flow from adjacent cells is limited. Actually, similar desynchronized and delayed onsets could be mimicked by the gap junction inhibitor heptanol. Moreover, our previous data indicated that, in muscles showing desynchronized Ca2+ transients, non-phosphorylated Cx43 was significantly elevated in peripheral and intracellular regions, suggesting suppression of gap junction channel activity Where delayed onset of Ca+ channels, although blocking Na+ channels alone could not reproduce desynchronized action potentials within a field of view of microscope. It is reasonable to assume that both inhibition of gap channel activity and inactivation of Na+ channels occur in these muscles.The decreased responsiveness is also reasonable because, in muscles showing delayed initiation of action potentials, those signals were, short-lasting, noisy and the rate of rise was slower than in the control. Those cells seem to be functionally compromised with inactivated Na2+ responses and action potentials at the cellular level in tissue would provide valuable information for investigation of arrhythmias.In normal heart muscle, cell-to-cell propagation of action potentials in two-dimensional images is too fast to capture with a confocal microscope. However, under some pathophysiological conditions, the propagation is slowed sufficiently to be detected by optical imaging. The region of reduced conductivity can induce conduction block, action potential dispersion and/or T-wave alternans which lead to malignant arrhythmias. Further development of optical technologies for determination of CaFigure S12+ signals before (A) and after (B) pacing protocol in non-flowing Krebs solution. Left panel: Surface images of a cardiac muscle. Right panel: Time-based scan images. The muscle was stimulated at 2 Hz (arrows). Inset is a expanded time-based scan image showing delayed onset of Ca2+ transient.Ca(0.80 MB TIF)Click here for additional data file.Figure S22+ transients and membrane potential signals. A. Response to 1.43 Hz stimulation after 2+ and action potentials obtained from a different muscle. In this case, alternating large and small Ca2+ transients are attributed to immature action potentials with every second stimulus.Examples of alternating Ca(2.31 MB TIF)Click here for additional data file.Movie S12+ response at 1.67 Hz stimulation after the pacing protocol in non-flowing Krebs solution. This movie corresponds to the upper panel in Ca(9.08 MB MOV)Click here for additional data file.Movie S2Ratiometric action potential signals at 1.67 Hz stimulation after the pacing protocol in non-flowing Krebs solution. This movie corresponds to the lower panel in (8.82 MB MOV)Click here for additional data file.


We attempted combined use of angioscopy and intravascularultrasonography (IVUS) to localize the coronary ostia anddetermine the aortic segment to be replaced in patients withannuloaortic ectasia, because these preoperative informations areimportant for selection of an appropriate technique forreconstructing the coronary artery, to prevent complications, andalso to postoperative follow-up. Two cases with annuloaorticectasia underwent angioscopy and IVUS both pre- andpost-operatively. Structure of aortic cusps, position of coronaryostia, the extent of ectasia with very thin wall were clearlyobserved by IVUS. Angioscopy showed milky white luminal surface ofthe ectasic segment. After Cabrol’s operation, the sutured portionof native aorta and graft was clearly identified by IVUS and muralthrombus and naked surface of graft were observed by angioscopy.Complications were observed in none. The results indicatefeasibility of combined use of angioscopy and IVUS fordetermination of surgical approach and follow-up in patients withAAE.


The multifunctional signal molecule calmodulin kinase II (CaMKII) has been associated with cardiac arrhythmogenesis under conditions where its activity is chronically elevated. Recent studies report that its activity is also acutely elevated during acidosis. We test a hypothesis implicating CaMKII in the arrhythmogenesis accompanying metabolic acidification.2+release events during metabolic acidification were investigated using confocal microscope imaging of Fluo-4-loaded ventricular myocytes.We obtained monophasic action potential recordings from Langendorff-perfused whole heart preparations and single cell action potentials (AP) using whole-cell patch-clamped ventricular myocytes. Spontaneous sarcoplasmic reticular (SR) Cam), reversibly suppressed this spontaneous arrhythmogenesis during intrinsic rhythm and regular 8 Hz pacing. However, it failed to suppress arrhythmia evoked by programmed electrical stimulation. These findings paralleled a CaMKII-independent reduction in the transmural repolarization gradients during acidosis, which previously has been associated with the re-entrant substrate under other conditions. Similar acidification produced spontaneous AP firing and membrane potential oscillations in patch-clamped isolated ventricular myocytes when pipette solutions permitted cytosolic Ca2+ to increase following acidification. However, these were abolished by both KN-93 and use of pipette solutions that held cytosolic Ca2+ constant during acidosis. Acidosis also induced spontaneous Ca2+ waves in isolated intact Fluo-4-loaded myocytes studied using confocal microscopy that were abolished by KN-93.In Langendorff-perfused murine hearts, introduction of lactic acid into the Krebs-Henseleit perfusate resulted in abnormal electrical activity and ventricular tachycardia. The CaMKII inhibitor, KN-93 (2 μ2+ waves in spontaneous arrhythmic events during metabolic acidification.These findings together implicate CaMKII-dependent SR Ca Previous experiments in isolated whole hearts  have con2+-dependent signal molecule calmodulin kinase II (CaMKII) -evoked Ca2+ transients   under cohythmias . Taken tad libitum and kept under a constant temperature environment with 12 : 12 h light : dark cycles. Wild-type male or female mice (age 3–7 months) mice were killed by cervical dislocation  Act 1986). Hearts were rapidly excised, submerged under ice-cold Krebs-Henseleit solution and the aorta cannulated and fixed to a size 21-gauge cannula using a micro-aneurysm clip .All animal handling followed UK animal welfare regulations. Mice were fed −1. The flow rate was kept constant using a peristaltic pump . The heated perfusate (37 °C) was passed through a 2 μm filtering membrane . The control Krebs-Henseleit solution contained (in mm): 119 NaCl, 25 NaHCO3, 4.0 KCl, 1.2 KH2PO4, 1.0 MgCl2, 1.8 CaCl2, 10 glucose, and 2.0 sodium pyruvate. In the solution designed to cause metabolic acidification, 20 mm NaHCO3 was exchanged with 20 mm sodium l-lactate. During all experiments, solutions were continuously bubbled using 95%/5% O2/CO2 resulting in pH ∼7.4 for control solutions and ∼6.7 for solutions causing metabolic acidification.Cannulated hearts were retrogradely perfused in a Langendorff perfusion set-up with a flow rate of 2–3 mL minplus interface and spike software . Electrical activity was recorded from hearts during three types of stimulation: (1) intrinsic rhythm, (2) regular 8 Hz pacing and (3) programmed electrical stimulation (PES)  electrodes . Recordings were bandpass filtered (0.005–1 kHz) and sampled at 5 kHz using a Neurolog amplifier , a 1401 −1 (37 °C) in a Langendorff set-up similar to that used for MAP recordings. All solutions used in the isolation procedure and for storing isolated myocytes during experiments were made from the same basic solution containing (in mm): 120 NaCl, 5.4 KCl, 5 MgSO4, 10 glucose, 5.4 sodium pyruvate, 20 taurine and 10 HEPES. During the first 2 min of the isolation procedure, hearts were perfused with a nominally Ca2+-free solution (pH 6.95), made from the basic solution but also containing 5 mm NTA  intended to sequester extracellular Ca2+. Subsequently, hearts were perfused for 10 min with a digestion solution modified from the basic solution by additionally containing 250 μm CaCl2, collagenase type II (13 000 units in 50 mL) and hyaluronidase (22 000 units in 50 mL), pH 7.4. The ventricles were then isolated and placed in a modified basic solution that additionally contained 250 μm CaCl2, 2 mg mL−1 BSA and pH 7.4 to stop enzymatic digestion. Ventricles were then gently cut and triturated using disposable Pasteur pipettes to separate the myocytes. After 15–20 min rest the myocytes were transferred to the storing solution. This was essentially the basic solution but additionally contained 1 mg mL−1 BSA and 1 mm CaCl2.Cannulated hearts were retrogradely perfused at 2–3 mL min−1, gravity driven) and visualization of myocytes was achieved using an inverted microscope . All recordings were performed at 37 °C. All recordings were performed without applying holding current and APs were triggered by applying small constant current pulses through the recording electrode. Recordings were low-pass filtered at 10 kHz and sampled at 20 kHz. The external control solution contained (in mm): 145 NaCl, 5 KCl, 10 HEPES, 10 glucose, 1 MgCl2, 1 CaCl2; pH was adjusted to 7.4 with NaOH. In solutions designed to produce metabolic acidification, 20 mm NaCl was replaced by 20 mm sodium l-lactate, and pH was adjusted to 6.7 with NaOH.Single cell APs were recorded using whole-cell patch-clamping in the current clamp mode . Pipettes (2–4 MΩ) were pulled from filamented borosilicate glass  using a vertical pipette puller . Myocytes were placed in a perfusion chamber  that allowed a complete solution change within ∼2 min (flow rate ∼2 mL min2+ during acidification was prevented by using 5 mm BAPTA as the Ca2+ chelating agent (BAPTA solution)  that during acidosis the change in free Ca2+ could replicate that occurring in acidified, intact myocytes. In both solutions the free Ca2+ was 50 nm at pH 7.2. When acidified, the BAPTA solution could be assumed to maintain the free Ca2+ constant  . In contpH range . Accordiconstant  while in2+ remained unchanged during acidosis, the concentration of BAPTA that was required in the EGTA-BAPTA solution could be calculated as: Assuming that the total Ca6 m−1 is the apparent affinity constant of BAPTA at 36 °C and an ionic strength of 194 mm  for 30 min followed by a 20 min wash. Cells were placed in a custom-designed experimental chamber placed on an inverted Leica confocal microscope . The chamber was perfused at 2–3 mL min−1 and the bathing solution could be completely exchanged within ∼2 min. Cells were stimulated by field stimulation at 0.5 Hz using constant voltage pulses . All recordings were done in frames (64 × 64) with a frame speed of ∼16 Hz. External solutions were similar to patch-clamp experiments. All experiments were performed at room temperature (∼24 °C). For quantification, the fluorescence in a cell-free region of the image  was subtracted from the fluorescence of resting cells in control conditions . This gave the F0 value. Amplitudes of fluorescence signals during AP-evoked Ca2+ transients and during Ca2+ waves, Fpeak,test, were related to this F0 value after subtracting the resting fluorescence of the cell under the conditions when the transients or waves were recorded, Frest,test. For confocal microscope imaging of intact myocytes, cells were pre-loaded with 10 μm cell-impermeant Fluo-4 was added to the pipette solution and the fluorescence was recorded using a Cairns photometric Optoscan system .For imaging of patch-clamped cells, 100 μAll chemicals were of analytical grade. Fluo-4-AM, cell-impermeant Fluo-4 and pluronic DMSO were from Molecular Probes . Collagenase type II was from Worthington . KN-93 was from Tocris ; all other chemicals were from Sigma-Aldrich .t-test was used to test whether two data groups, which were assumed to be normally distributed, had identical means. All t-tests were preceded by F-tests to test for equal variance between two data groups concerned. Average data are presented as means ± SEM. P < 0.05 was considered the limit of significance.Fisher’s exact test was used to assess whether association between two categorical data groups was non-random. Student’s P < 0.01 and < 0.05, testing findings obtaining in the presence vs. the absence of KN-93 for spontaneous APs and spontaneous VT during metabolic acidosis respectively: Fisher’s exact test). During intrinsic rhythm, metabolic acidification was similarly associated with spontaneous VT in 53% of the hearts when KN-93 was absent  but CaMKII inhibition did not affect the frequency of the intrinsic rhythm under control or acidic conditions. Thus, under control conditions of normal extracellular pH, the intrinsic rhythm was 228 ± 64 beats min−1 and 197 ± 41 beats min−1 in the absence and presence of KN-93 respectively (P < 0.69). Under acidified conditions the intrinsic rhythm was 127 ± 35 and 197 ± 56 beats min−1 in the absence and presence of KN-93 respectively (P < 0.33). Taken together these findings suggest that CaMKII inhibition reduced spontaneous arrhythmogenicity caused by metabolic acidification whether during regular 8 Hz pacing or during intrinsic activity.Arrhythmogenicity produced by metabolic acidification was initially assessed by recording epicardial MAPs from intact, Langendorff-perfused, hearts. All hearts were both initially examined under control conditions of normal extracellular pH and then during subsequent metabolic acidification. s absent . HoweverComplementary experiments on three hearts exposed to 8 Hz regular pacing showed that the anti-arrhythmic effects of KN-93 during metabolic acidosis were reversible. In these experiments KN-93 was initially introduced under conditions of normal pH  and duriData of the kind illustrated in 90). Such experiments demonstrated that metabolic acidification significantly prolonged epicardial APD90 regardless of whether KN-93 was absent  or during metabolic acidification (P = 0.95 and 0.66 respectively).The experiments that followed tested for the possible relationships between such substrate, the arrhythmogenecity brought about by acidosis and possible contrasting effects of KN-93 upon these. They investigated the effects of metabolic acidification and KN-93 on left ventricular epicardial and endocardial MAPs in intact hearts during regular 8 Hz pacing. 90 = endocardial APD90 − epicardial APD90) but demonstrate that these were independent of CaMKII inhibition .These findings associate metabolic acidification with significantly reduced left ventricular transmural gradients of repolarization . The episodes of VT during metabolic acidification that were observed either using regular pacing or PES, generally had a monomorphic appearance with activation cycles of 76 ± 8 ms and 71 ± 7 ms in the absence and presence of KN-93 respectively. This indicates that KN-93 did not markedly affect the arrhythmic substrate during acidosis.The above measurements of left ventricular ΔAPDogenesis , PES couogenesis  consisteogenesis . Howeverogenesis . Thus, dtriggering mechanisms without affecting the arrhythmic substrate that arises during metabolic acidification.Taken together the whole heart experiments suggest that CaMKII inhibition acts to reduce AP 2+ handling both under conditions of normal extracellular pH and during metabolic acidification in the absence and presence of KN-93.The cellular mechanisms behind the arrhythmogenic trigger during acidosis and the role of CaMKII in this triggering were next explored by confocal microscope imaging to compare SR Ca2+ transients evoked by 0.5 Hz stimulation of typical ventricular myocytes in the absence . absence  or prese absence  of KN-93of KN-93 . This deof KN-93 , which w2+ waves . Remarka2+ (2+ had increased by 38 ± 18% in the absence of KN-93 and by 22 ± 11% in the presence of KN-93 (P = 0.62).Secondly, as reported in other studies, acidification was associated with a CaMKII-independent increase in the free cytosolic Ca2+  dropped from 530 ± 43 V s−1 before acidification to 426 ± 36 V s−1 after 5 min acidification . However, the APD90 remained unchanged . In the experiments with EGTA-BAPTA solution the presence of KN-93 did not affect either dv/dt or APD90 under control conditions or during acidosis. Thus, dv/dt dropped from 503 ± 48 to 476 ± 24 V s−1 after 5 min acidification in the absence of KN-93  and from 492 ± 71 to 440 ± 85 V s−1 in the presence of KN-93 , also suggesting an absence of significant effects of KN-93 by itself on dv/dt either before or after acidification. With EGTA-BAPTA solution APD90 was 51 ± 6 ms under control conditions and 40 ± 7 ms after 5 min of metabolic acidification in the absence of KN-93  and in the presence of KN-93 APD90 was 65 ± 12 ms under control conditions and 43 ± 9 ms after 5 min of metabolic acidification .In cells investigated with the BAPTA solution the maximal rate of rise in the APs  the enhanced arrhythmic triggering, (2) the re-entrant substrate and finally (3) the role of CaMKII in the arrhythmogenecity associated with metabolic acidification.Metabolic acidification is a key cellular alteration in a number of clinically important situations including the cardiac ischaemia that occurs with acute coronary artery occlusion. Such situations are associated with increased arrhythmogenicity (whole heart level separated arrhythmic tendency during metabolic acidification that could be attributed to triggered activity from arrhythmic tendency arising from re-entrant substrate as detected using PES stimulation. During intrinsic rhythm a significant proportion of acidified hearts developed spontaneous VT. With regular 8 Hz pacing, all acidified hearts fired spontaneous APs and a significant proportion showed VT. These findings associated metabolic acidification with the appearance of spontaneous APs that could act as triggers for VT. To study the role of SR Ca2+ handling in this arrhythmic triggering during metabolic acidification confocal imaging of intact myocytes was performed during initial conditions of normal extracellular pH and subsequently during metabolic acidification. These experiments directly demonstrated that acidosis led to the appearance of large Ca2+ waves, known to cause DADs via an increased Na+-Ca2+ exchange current (2+ waves were compatible with a markedly increased SR Ca2+ content during acidification. The latter would agree with other studies focusing on the effect of acidification on SR Ca2+ loading (2+ waves and DADs. Such a notion is further supported by the patch-clamp experiments, which could study spontaneous electrical activity in single isolated ventricular myocytes in isolation from re-entrant substrate. Results were compared between cells patch-clamped using pipettes that contained either BAPTA solution or EGTA-BAPTA solution. In the first case, metabolic acidification altered neither cytosolic free [Ca2+], reflected in the fluorescence of a loaded Fluo-4, nor the normal pattern of AP firing, in so doing additionally excluding significant arrhythmogenic actions of the lactate ion itself in the present experiments. In the second case using the EGTA-BAPTA solution, metabolic acidification increased intracellular Ca2+ similar to the rise in baseline Ca2+ in the confocal imaging during acidosis, in so doing confirming successful cell acidification, and produced DADs and spontaneous APs. The above findings together suggest in vivo situations in which acidosis elevates free cytosolic Ca2+ that in turn produces altered SR Ca2+ handling and triggered arrhythmic phenomena.First, the experiments at the  loading . It is t90 arising from preferential prolongation of epicardial MAPs. This is in common with the re-entrant arrhythmic substrate reported in murine models of LQT3 and hypokalaemia (90. This is further supported by the reduced dv/dt of the single cell APs during acidosis, which indicates that acidification caused a reduction in Na+ current during the AP that may add to closure of gap junctions in generating a reduced AP conduction velocity.Secondly, in addition, metabolic acidification also resulted in re-entrant substrate that might result in persistent arrhythmogenesis following such spontaneous APs. Thus, epicardial and endocardial MAP recordings suggested an association between metabolic acidification and reduced ΔAPD2+ during acidosis greatly reduced DADs and completely abolished spontaneous APs during acidosis. In confocal imaging experiments, KN-93 caused a larger decline in the electrically evoked Ca2+ transients and abolished the spontaneous SR Ca2+ waves during the metabolic acidification despite acidosis still causing a marked rise in baseline Ca2+ in the presence of KN-93. However, it did not prevent the arrhythmias and abnormal electrical activity during PES. Nor did it affect epicardial or endocardial APD90, or ΔAPD90 whether during control conditions or during metabolic acidification. Together these findings indicate that whilst CaMKII did not affect the substrate for arrhythmia that arose with metabolic acidification, it enhanced AP triggering during metabolic acidosis by the generation of spontaneous SR Ca2+ waves.Thirdly, acidification has also been associated with elevated CaMKII activity (2+ waves are generally believed to occur when the free luminal SR Ca2+ reaches a threshold concentration for RyR2A opening (2+ uptake or reduced thresholds for RyR2A opening. The increased phosphorylation of the CaMKII-specific site on phospholamban during acidosis (2+ content usually observed during acidosis (2+ that leads to RyR2A opening. However, a recent study suggested that CaMKII phosphorylation of RyR2A actually reduces its opening probability (2+ waves observed in this study in metabolically acidified myocytes could arise from SR Ca2+ overloading due to CaMKII-mediated phospholamban phosphorylation and possibly from CaMKII-mediated RYR2a phosphorylation causing increased opening probability of RyR2a.Spontaneous SR CaIt is well established that the activity of CaMKII becomes elevated during structural heart disease (We report no conflict of interest.


Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca2+ release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP3Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca2+]i. The initial abrupt sub-PM [Ca2+]i upstroke was all but abolished by block of VGCCs (by 5 μM nicardipine), depletion of intracellular Ca2+ stores (with 10 μM cyclopiazonic acid) or inhibition of IP3Rs (by 2 μM xestospongin C or 30 μM 2-APB), but was not affected by block of RyRs (by 50–100 μM tetracaine or 100 μM ryanodine). Inhibition of either IP3Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation–contraction coupling in this phasic visceral smooth muscle occurs by Ca2+ entry through VGCCs which evokes an initial IP3R-mediated Ca2+ release activated via a CICR mechanism.Membrane depolarization triggers Ca From here Ca2+ can be released either by a process of Ca2+-induced Ca2+ release (CICR) through ryanodine receptors (RyRs) upon Ca2+ entry during an AP 3) generated by a Gq/11-link from an activated stimulant receptor to phospholipase C. The entry of Ca2+ through VGCCs is believed to load SR calcium stores which, upon reaching a critical level of loading 2+ which in the majority of smooth muscles generate transient high local concentrations of sub-plasmalemmal (sub-PM) Ca2+ that open Ca2+-dependent potassium (BK) channels, present in abundance in the cell membrane of smooth muscles. Bursts of openings of these BK channels are seen as spontaneous transient outward currents (STOCs) in voltage-clamped SMCs 2+ indicator, the transient localized sub-PM increase in Ca2+ concentration ([Ca2+]i) is seen as a flash of emitted light  are stimulated to contract either by depolarization of the cell membrane , often in the form of an action potential (AP), or by activation of a variety of receptors  usually coupled to G-proteins, or by a combination of these mechanisms  “spark” . The tra3 receptors (IP3Rs) in the generation of smooth muscle myocyte tension is an area of active investigation. As smooth muscles do not have a sarcomeric organization of their contractile proteins and calcium stores, the sites of Ca2+ release and re-storage are necessarily very different from those of striated muscles. In vas deferens and bladder SMCs an AP generates Ca2+ release from sub-PM ‘hot spots’ which then generate a cell-wide Ca2+ wave presumably by CICR. This is followed by SMC contraction 3Rs seems to be to facilitate CICR within and between RyRs domains 3R-evoked Ca2+ release did not activate RyRs but RyR blockers inhibited IP3R-mediated Ca2+ signals 2+ waves could still be evoked by strong activation of adrenoceptors leading to myocyte and vessel contraction; thus CICR release apparently can occur among IP3Rs alone although normally, at low levels of adrenoceptor activation RyRs were necessary to trigger IP3R dependent Ca2+ waves 3R Ca2+ release is important as in visceral smooth muscles. The present work examines the relative roles of RyRs, IP3Rs and VGCCs in intracellular Ca2+ mobilization in longitudinal intestinal smooth muscle myocytes in response to strong activation of muscarinic receptors. A preliminary account of some of this work has previously been published in an abstract form The respective role of SR RyRs and IP2Experiments were performed on preparations of the longitudinal muscle layer of the guinea-pig ileum: (1) freshly isolated SMCs or (2) freshly dissected strips of smooth muscles (see below). Adult male guinea-pigs (300–500 g) were killed by decapitation after cervical dislocation as approved under Schedule 1 of the UK Animals (Scientific Procedures) Act 1986.2.12+-free physiological saline solution . The pieces of the tissue were transferred into the same solution supplemented with (mg/ml): protease (Type X) 0.5, collagenase (Type 1A) 1.5, soybean trypsin inhibitor 1 and bovine serum albumin 1, and incubated for 20 min at 37 °C. The pieces of the tissue were then rinsed for 10 min in an enzyme-free Ca2+-free solution and triturated with a wide bore glass pipette. Small aliquots of the cell suspension were transferred to the experimental chambers and diluted with PSS composed of (mM): NaCl 120, KCl 6, CaCl2 2.5, MgCl2 1.2, glucose 12, HEPES 10; pH adjusted to 7.4 with NaOH. Experiments on isolated cells were conducted at 22–24 °C within 8 h of cell isolation.The longitudinal muscle layer of the ileum was dissected and cut into small pieces, which were placed in Ca2.2Smooth muscle strips (∼10 mm in length) were dissected from longitudinal layer of guinea-pig ileum, transferred to a homemade organ bath (3 ml volume) and attached to an isometric force transducer at a resting tension load of 5 mN and bathed in the PSS at 37 °C. The output of the force transducer was connected to a custom-made amplifier. The signals were digitized using a Digidata 1320 AD/DA converter  hosted by a PC running Axoscope 8.0 software (Molecular Devices Co.).2.32+]i) in isolated SMCs were imaged using the high-affinity (kd(Ca) = 345 nM) fluorescent Ca2+ indicator fluo-4, which was loaded by 20-min incubation of the SMCs with 5 μM fluo-4 acetoxymethyl ester (fluo-4 AM) followed by a 40-min wash in PSS to allow time for de-esterification. To minimize SMC contraction, 40 μM of wortmannin was added to the bathing solution 10 min before imaging commenced.Changes in intracellular concentration of ionized calcium  which was either superfused through the experimental bath or applied as a brief (≤600 ms) pulse through a glass micropipette (located within 100–200 μm of the cell) attached to the outlet of pressure ejector Picospritzer III . Similar application of the control solution (without agonist) had no effect on [CaF/F0). The temporal profiles of CCh-induced [Ca2+]i mobilization are illustrated by the plots showing (1) the time course of the normalized fluo-4 fluorescence intensity (F/F0) averaged within multiple sub-PM regions (outlined in the corresponding images) where F/F0 changes were initiated and rose above 2.5 or (2) the time course of F/F0 averaged within the entire confocal optical slice.In the figures the intensity of fluo-4 fluorescence was normalized to the average fluorescence intensity in the images captured before CCh application and colour coded as indicated by the bars (2.4kd(Ca) = 42 μM) fluorescent Ca2+ indicator fluo-3FF, which was loaded by 90-min incubation of the SMCs with 5 μM fluo-3FF AM followed by 60-min wash in PSS.Distribution of intracellular calcium stores within isolated SMCs was visualized using the low-affinity  for 16 h at 4 °C. Following a 10-min rinse (four times) in PSS supplemented with 3% (w/v) BSA, primary antibody-specific binding was visualized by incubating SMCs for 2 h at room temperature with Alexa Fluor 488 conjugated to chicken anti-rabbit IgG  and Alexa Fluor 633 conjugated to goat anti-mouse IgG . In controls, primary antibodies were omitted from the experimental media during the first (16 h) incubation.Isolated SMCs were fixed by 5–10-min incubation with 4% (w/v) paraformaldehyde. Nonspecific binding was blocked by incubating the SMCs with 3% (w/v) bovine serum albumin (BSA) and 0.3% (w/v) Triton X-100 (a cell permeabilizing agent) for 1 h at room temperature. Primary and secondary antibodies were diluted in PSS supplemented with 3% (w/v) BSA, 0.3% (w/v) Triton X-100, 20 U/ml penicillin and 20 μg/ml streptomycin. To visualize the distribution of IP2.6x–y confocal images of fluo-4 fluorescence were acquired at 19–42 Hz using a Zeiss plan-Apochromat 40 × 1.3 N.A. oil-immersion objective. Fluo-4 and fluo-3FF fluorescence was excited by the 488 nm line of a 200 mW argon ion laser  and was captured at wavelengths above 505 nm. The pinhole was set to provide a confocal optical slice below 1.2 μm.Experimental chambers were placed on the stage of an Axiovert 100 M inverted microscope attached to a LSM 510 laser-scanning unit . The Em = 519 nm) and Alexa Fluor 633 (Em = 647 nm). Dual excitation, using the multitrack mode of an LSM 510, was performed by the 488 nm line of a 200 mW argon ion laser and the 633 nm line of a 15 mW helium/neon ion laser, respectively. The emitted fluorescence signal was filtered using 505–550 nm bandpass filter (for the green IP3R label) and 650 nm longpass filter (for the red RyR label). The adequacy of the imaging protocol applied to the double-labelled SMCs was confirmed by control experiments on the single-labelled cells.To avoid any bleed-through in immunofluorescence experiments, SMCs were double labelled using fluorophores with extremely well separated emission spectra: Alexa Fluor 488 : KCl 120, KH2PO4 5, MgSO4 5, Na2ATP 5, Li2GTP, Hepes 10; pH was adjusted to 7.4 with KOH. Recording of the cell membrane potential was synchronized with confocal imaging using a TTL synchronizing pulse generated by the confocal scanner at the beginning of the time series protocol.The cell membrane potential was monitored using perforated-patch (200 μg/ml amphotericin B) tight seal recording in the current-clamp mode of an Axopatch 200A (Molecular Devices Co.) patch-clamp amplifier. This allowed low resistance access to the cell while minimally interfering with i at 100 nM with 4.6 mM CaCl2/ 10 mM BAPTA buffer added to the pipette solution Muscarinic cationic current : NaCl 135, CsCl 6, CaCl2 2.5, MgCl2 1.2, glucose 12, Hepes 10 (pH 7.4 adjusted with NaOH) and the composition of the pipette solution was (in mM): CsCl 126, MgSO4 5, Na2ATP 5, Li2GTP 1 (pH adjusted to7.4 with CsOH and [Ca2+]i was clamped at 20 nM with 2.6 mM CaCl2/10 mM EGTA buffer).Voltage-gated Ca2.8N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid (HEPES), 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA), ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), carbamylcholine chloride (carbachol), 1,3,7-trimethylxanthine (caffeine), dimethyl sulfoxide (DMSO), paraformaldehyde and Triton X-100 were obtained from Sigma Chemical Co., Poole, Dorset, UK. Fluo-4 acetoxymethyl ester, Alexa Fluor 488 conjugated chicken anti-rabbit IgG (H + L), Alexa Fluor 633 conjugated goat anti-mouse IgG (H + L) and pluronic F-127 were obtained from Invitrogen Ltd., Paisley, UK. Fluo-3FF acetoxymethyl ester was from TefLabs, Austin, Texas, USA. All other chemicals were from BDH Laboratory Supplies , Pool, UK.Protease (Type X), collagenase (type 1A), soybean trypsin inhibitor (Type II-S), bovine serum albumin, adenosine 5′ triphosphate , guanosine 5’ triphosphate , creatine, 2.9n) analysed. Comparative analysis of the data groups was performed using Student's t-test for paired or unpaired samples, as appropriate, with the threshold for statistical significance set at the 0.05 level.Image processing was carried out using an Indy workstation  with custom routines written in IDL . The final figures were produced using MicroCal Origin  and CorelDraw 7.0 . Where appropriate, data are expressed as mean values ± S.E.M. for the number of cells (33.12+ concentration ([Ca2+]i) following muscarinic receptor activation were related to the changes in the cell membrane potential (Vm). SMCs freshly isolated from the guinea-pig ileum were preloaded with the high affinity Ca2+ indicator fluo-4 and bathed in PSS  was combined with recording of Vm in perforated-patch configuration to minimally perturb endogenous [Ca2+]i (n = 4). Muscarinic receptors were stimulated by brief applications of 10 μM carbachol (CCh) through a glass micropipette positioned within 100–200 μm of the cell surface. This revealed that the discharge of each action potential (AP) following muscarinic receptor activation was associated with a [Ca2+]i transient which was initiated by an abrupt increase of [Ca2+]i at multiple sub-plasmalemmal (sub-PM) regions. The dynamics of [Ca2+]i changes in these sub-PM regions of initiation was therefore analysed. In the example shown in 2+]i  these signals reflect Ca2+ release from non-uniformly distributed sub-PM Ca2+ stores. Because of their location and robust onset kinetics, we refer to these events hereafter as the “sub-PM [Ca2+]i upstroke (SPCU)”.The dynamics of the change in intracellular Ca2+]i mobilization in non-patched SMCs are illustrated by 2+]i changes, while red traces show the dynamics of [Ca2+]i changes averaged at multiple sub-PM regions  where CCh-induced [Ca2+]i transients were initiated. The galleries show sequential confocal images of fluo-4 fluorescence acquired during the periods marked by a grey background in the plots, respectively. In all cases CCh-induced [Ca2+]i transients were initiated by a SPCU . Muscarinic receptor activation evoked a SPCU independently of the extent of myocyte contraction (n = 147), suggesting that this phenomenon is not a result of the increase in the local density of Ca2+-release units, which could be caused by change in SMC geometry. In the vast majority of cases (97%) two phases can be clearly distinguished in the Ca2+ responses to CCh: (1) an initial high-amplitude [Ca2+]i transient and (2) a delayed increase of global [Ca2+]i characterized by a smaller amplitude and a tendency to oscillation.Spatio-temporal patterns of CCh-induced i mobilization. In this and all subsequent experiments we analysed the dynamics of [Ca2+]i changes at multiple sub-PM regions of initiation of CCh-induced [Ca2+]i mobilization. In control, the response to CCh was initiated by a SPCU (2+]i transient was followed by a lower amplitude sustained phase with two [Ca2+]i oscillations (2+ channels with 5 μM nicardipine (30-s incubation) eliminated the SPCU (2+]i transient (2+]i transient (green trace) was reduced by 60%, time-to-peak was increased from 0.8 to 1.3 s and the Ca2+ wave propagated more slowly than in control (2+ entry and (2) Ca2+ entry through VGCCs is not the only source of Ca2+ upon muscarinic [Ca2+]i mobilization.Muscarinic cationic channels in gastro-intestinal smooth muscles have very low, if any, permeability for Ca2+ release from intracellular stores to CCh-induced [Ca2+]i mobilization, the effect of Ca2+ store depletion was tested. In control, CCh induced a rapidly propagating [Ca2+]i wave , resulted in complete depletion of the intracellular Ca2+ stores in this cell type, as we have previously demonstrated using flash release of “caged” IP3 preloaded into the cells through the patch pipette 2+ stores with CPA treatment eliminated the SPCU, reduced the peak amplitude of the initial CCh-induced [Ca2+]i transient by 80%, increased time-to-peak from 0.7 to 1.4 s and virtually completely abolished the delayed phase of the response  completely abolished [Ca2+]i mobilization in response to CCh  abolished the SPCU, inhibited the initial [Ca2+]i triggered by CCh in the presence of tetracaine and xestospongin C was caused by Ca2+ entry through VGCCs.Subsequent block of VGCCs with 5 μM nicardipine (1-min incubation) completely abolished the residual response to CCh , may affect some other mechanisms of intracellular Ca2+ homeostasis. Since the key event in muscarinic [Ca2+]i mobilization seems to be the Ca2+ entry through VGCCs, we tested the effect of 2 μM xestospongin C and 30 μM 2-APB on the voltage-gated Ca2+ current (ICa) evoked by a 500-ms voltage steps from −80 to +0 mV under whole-cell voltage clamp conditions (+/Na+-containing solutions (see Section ICa. The peak amplitude of ICa (ICa evoked by the first voltage step (Itest/Ifirst) and plotted over time (n = 7) and following application of 2 μM xestospongin C (n = 4) or 30 μM 2-APB (n = 5). This revealed that after allowing for ICa rundown, the reduction of the current amplitude by a further 74% during 10 min  can account for the inhibitory effect of xestospongin C on VGCCs. In contrast, 2-APB during the same period augmented ICa (taking into account the ICa rundown) on average by 35% . Thus, the inhibitory effect of 2 μM xestospongin C on CCh-induced [Ca2+]i mobilization may partially  of the control mIcat (cat (n = 6) of the control mIcat (cat by 61.5% (significant difference: p = 0.00019).Taking into account that activation of VGCCs in response to muscarinic receptor activation is caused by depolarization evoked by mIon mIcat A were tetion cat Aa constiol mIcat Ac. Incubcat  as the level of the cell membrane depolarization caused by this current. Indeed, any ionic current cannot change the cell membrane potential (Vm) beyond the level of the reversal potential of the current. In the case when the input resistance of the cell is relatively high (such as at resting condition) even a small amplitude current can produce a substantial shift in Vm. We therefore tested the effect of 30 μM 2-APB on CCh-induced membrane depolarization (+/Na+-containing solution (see Section Vm from −33 ± 2 mV to −4 ± 1 mV (n = 5) in control (Ba) and from −29 ± 4 mV to −5 ± 1 mV (n = 5) in 30 μM 2-APB (Bb), (n = 5). The values of the magnitude (ΔVm) of membrane depolarization (Bc) caused by CCh in control (29 ± 2 mV) and in 2-APB (24 ± 4 mV), as well as extent of the CCh-induced depolarization in control and in 2-APB were not significantly different . This validates 2-APB as pharmacological tool for study of the role of IP3Rs in CCh-induced [Ca2+]i mobilization.However, in terms of CCh-induced i transient by 79% and increased time-to-peak from 0.5 to 4.5 s  averaged at multiple sub-PM regions of initiation of the CCh-induced [Ca2+]i transient was then plotted over time. The response to the succeeding CCh application (Test) was related to the response to the first CCh application (Control). The test response was obtained either in the control external solution  or following a 10-min incubation of the SMC with a drug or combination of several drugs. When the effect of VGCC block on muscarinic [Ca2+]i mobilization was tested, nicardipine was applied 30 s–1 min before the pulse of CCh to minimize possible effect of VGCC block on the SR Ca2+ load. Two parameters were examined and are summarized in the bar diagram plots: (1) relative change in peak amplitude (ΔF/F0)Test/(ΔF/F0)Control (tpeak)Test/(tpeak)Control (n = 29) of the control response (F/F0)Test/(ΔF/F0)Control = 84 ± 10%, (tpeak)Test/(tpeak)Control = 116 ± 15%, n = 7) or with 50–100 μM tetracaine ((ΔF/F0)Test/(ΔF/F0)Control = 85 ± 6%, (tpeak)Test/(tpeak)Control = 108 ± 5%, n = 14) had no significant effect on either amplitude  or kinetics  of the initial [Ca2+]i transient; (iii) depletion of intracellular Ca2+ stores with 10 μM CPA ((ΔF/F0)Test/(ΔF/F0)Control = 11.5 ± 6%, (tpeak)Test/(tpeak)Control = 284 ± 18%, n = 6) reduced the peak amplitude by 87.5% (p = 1.3 × 10−7) and increased time-to-peak by 178% (p = 1.7 × 10−15); (iv) block of VGCCs with 5 μM nicardipine ((ΔF/F0)Test/(ΔF/F0)Control = 9 ± 1%, (tpeak)Test/(tpeak)Control = 334 ± 27%, n = 18) reduced the peak amplitude by 90% (p = 5.7 × 10−18) and increased time-to-peak by 227% (p = 5.2 × 10−14); (v) simultaneous block of VGCCs and RyRs with 5 μM nicardipine/50–100 μM tetracaine ((ΔF/F0)Test/(ΔF/F0)Control = 9 ± 3%, (tpeak)Test/(tpeak)Control = 360 ± 60%, n = 6) reduced the peak amplitude by 90% (p = 2.2 × 10−9) and increased time-to-peak by 253% (p = 8.7 × 10−11); (vi) block of IP3Rs with 2 μM xestospongin C ((ΔF/F0)Test/(ΔF/F0)Control = 22 ± 6%, (tpeak)Test/(tpeak)Control = 261 ± 26%, n = 15) reduced the peak amplitude by 76% (p = 1.2 × 10−11) and increased time-to-peak by 159% (p = 3.3 × 10−10); block of IP3Rs with 30 μM 2-APB ((ΔF/F0)Test/(ΔF/F0)Control = 23 ± 3%, (tpeak)Test/(tpeak)Control = 283 ± 22%, n = 16) reduced the peak amplitude by 75% (p = 2.0 × 10−13) and increased time-to-peak by 177% (p = 3.6 × 10−13); (vii) simultaneous block of RyRs and IP3Rs with 50–100 μM tetracaine / 2 μM xestospongin C ((ΔF/F0)Test/(ΔF/F0)Control = 10 ± 4%, (tpeak)Test/(tpeak)Control = 287 ± 16%, n = 6) reduced the peak amplitude by 89% (p = 3.4 × 10−9) and increased time-to-peak by 181% (p = 2.1 × 10−16); (viii) simultaneous block of RyRs and IP3Rs with 50–100 μM tetracaine / 30 μM 2-APB ((ΔF/F0)Test/(ΔF/F0)Control = 9 ± 2%, (tpeak)Test/(tpeak)Control = 291 ± 46%, n = 6) reduced the peak amplitude by 90% (p = 2.1 × 10−8) and increased time-to-peak by 185% (p = 2.1 × 10−10); (ix) block of VGCCs (with 5 μM nicardipine) following intracellular Ca2+ store depletion (with 10 μM CPA), or simultaneous block of VGCCs and IP3Rs (with either 5 μM nicardipine / 2 μM xestospongin C or 5 μM nicardipine / 30 μM 2-APB) completely abolished [Ca2+]i mobilization in response to CCh. Thus, significant inhibition of the CCh-induced [Ca2+]i transient was associated with significant reduction in its rate of rise, which is indicative of the elimination of the SPCU. Altogether these results strongly suggest that following muscarinic stimulation, SPCU results from Ca2+ release mediated via IP3Rs, which are activated synergistically by (1) IP3 mobilized via the M3- Gq/11-PLCβ pathway, and (2) Ca2+ entering the cell through VGCCs activated via the M2- Go- mIcat-membrane depolarization pathway.The effects of the Ca)Control B and (2))Control C. This r)Control Aa constiresponse B, while response C; (ii) b3.43Rs in these SR elements.The above hypothesis suggests: (1) a sub-PM location of the SR elements in ileal SMCs and (2) the expression of IP2+ stores visualized with the low-affinity (kd(Ca) = 42 μM) Ca2+ indicator fluo-3FF (n = 47). This spatial organization of intracellular Ca2+ stores is generally similar to that we have previously demonstrated in the rabbit portal vein myocytes using DiOC6 and BODIPY TR-X ryanodine Intracellular Cafluo-3FF A consist3Rs (with antibody specific for type 1 IP3R) and RyRs  by double labelling (n = 18) of the same SMC  and only then spreads to the cell periphery , the global [Ca2+]i mobilization within the SMC is substantially attenuated  of the control response; (ii) inhibition of IP3Rs with 30 μM 2-APB ((Fo)Test/(Fo)Control = 26.5 ± 5%, n = 4) reduced the peak isometric force by 73% (p = 0.0006); (iii) inhibition of RyRs with 100 μM tetracaine ((Fo)Test/(Fo)Control = 29 ± 10%, n = 3) reduced the peak isometric force by 70% (p = 0.001). This demonstrates that both IP3R- and RyR-mediated Ca2+ release are required for full scale contractile response to 2 μM CCh. However, in contrast to tetracaine, 2-APB also abolished spontaneous oscillations of isometric tension observed in control  in the cell membrane, which may lead to membrane hyperpolarization and, as a result, to a decrease of Ca2+ entry through VGCCs 2+]i mobilization and perhaps the contractile response. This, however, was not observed. Indeed, even though the peak of the CCh-induced [Ca2+]i transient was associated with some minor (about 8 mV) transient repolarization of the cell membrane Control = 167 ± 11%, n = 8) by 70% Test/(tpeak)Control) in control  and following paxilline treatment  were not significantly different (p = 0.92).On the other hand, a SPCU should activate large conductance Camembrane C, inhibi42+]i is a primary signal for contraction in all types of muscles, the nature of the mechanisms linking excitation to [Ca2+]i mobilization, as well as the spatial organization and molecular composition of intracellular Ca2+ release units are important determinants of the contractile response. However, the mechanisms coupling excitation to [Ca2+]i mobilization in skeletal, cardiac and smooth muscles are different. While in skeletal muscles membrane depolarization triggers Ca2+ release from the SR via direct interaction between the voltage sensors in the T-tubules  and ryanodine receptors (RyRs) expressed in the terminal sacs of the SR (reviewed in Refs. 2+ entry through VGCCs triggers RyR-mediated Ca2+ release via a Ca2+-induced Ca2+ release (CICR) mechanism (reviewed in Refs. As an increase of i, may be further augmented by RyR-mediated Ca2+ release activated via CICR 3Rs to intracellular [Ca2+]i mobilization and the role of these receptors in the genesis of localized Ca2+-release events (sparks or puffs), propagating Ca2+ waves and [Ca2+]i oscillations varies in different types of SMCs, and often depends on the strengths and mechanism of SMC stimulation. In some phasic SMCs, e.g. urinary bladder and vas deferens, Ca2+ entry through VGCCs is tightly coupled to RyR-mediated Ca2+ release. Electrical stimulations of these SMCs (under current- or voltage-clamp conditions) triggered an abrupt RyR-mediated Ca2+-release at multiple sub-PM regions  induced by stimulation of SMCs with neurotransmitters and hormones 3R-mediated Ca2+ release Cytosolic Ca2+]i upstroke (SPCU) produced by IP3R-mediated Ca2+ release from sub-PM SR elements. These events were closely associated with action potentials  of single ileal SMCs triggers an abrupt sub-PM i transient was spatially uniform, which is characteristic of Ca2+ entry through VGCCs 2+ stores depleted) completely abolished [Ca2+]i mobilization in response to CCh  Ca2+ mobilized upon the initial [Ca2+]i transient activates RyRs to release Ca2+ and (3) the sustained rise of [Ca2+]i and/or [Ca2+]i oscillations are the result of interplay between IP3Rs and RyRs.In contrast with Cain C 3Rs . It shou [Ca2+]i C. On thes by 90% B. Altoge3Rs to SPCU may affect voltage-gated Ca2+ entry induced by muscarinic receptor activation, namely: (1) xestospongin C was shown to inhibit barium current through VGCCs in guinea-pig ileal myocytes but had no effect on mIcatcat in SMCs from murine stomach ICa by 74% , the global [Ca2+]i mobilization within the SMC is substantially attenuated  3Rs with 2-APB (which eliminates SPCU) nor direct inhibition of BK channels with 0.1 μM paxilline accelerated the contractile response to CCh (2+ entry though VGCCs facilitated by inhibition of BK channels (via the resulting greater membrane depolarization) does not alter [Ca2+]i, but instead it increases the SR Ca2+ load, thus causing an increase of CCh-induced isometric force. Thus, it seems likely that overall physiological effect of SPCU is to engage a rapid full-scale [Ca2+]i mobilization, rather than to control contractile activity of intestinal smooth muscles via modulation of the BK channel activity.Another important target for sub-PM Cae to CCh . Paxillie to CCh B and C be to CCh B. A like3R-mediated Ca2+ release from sub-PM SR elements facilitated by Ca2+ influx through VGCCs and serves to augment intracellular Ca2+ mobilization via CICE (acting on muscarinic cationic channels) and via CICR (acting on RyRs) mechanisms, and represents the key element in the chain of the signalling events in cholinergic contraction.In conclusion, SPCU is caused by an abrupt IP


Lysophosphatidic acid (LPA), an unrelated agonist, evoked Ca2+ release without inducing robust Ca2+ entry. Both LPA and UTP induced the redistribution of STIM1 into puncta which localized to regions near or at the plasma membrane, as well as within the cytoplasm. Plasma membrane-associated STIM1 remained high for up to 10 min after UTP stimulation, whereas it had returned almost to baseline by that time point in LPA-stimulated cells. This correlated with faster reloading of the endoplasmic reticulum Ca2+ stores in LPA treated cells. Thus by differentially regulating store-refilling after agonist-mediated depletion, LPA and UTP may exert distinct effects on the duration of STIM1 localization at the plasma membrane, and thus, on the magnitude and duration of ACE.The mechanisms of agonist-induced calcium entry (ACE) following depletion of intracellular calcium stores have not been fully established. We report here that calcium-independent phospholipase A (iPLA This is usually followed by Ca2+ influx across the plasma membrane (PM), or agonist-induced Ca2+ entry (ACE) (2+-ATPase (SERCA) with thapsigargin (TG) evokes store-operated Ca2+ entry (SOCE)  . The latry (ACE) . Similary (SOCE) .2) which generates lysophospholipids that activate SOCE at the PM by an uncharacterized process. In their studies, SOCE was impaired by knockdown of iPLA2 using RNA interference or by pharmacological inhibition of iPLA2 with bromoenol lactone (BEL), a specific iPLA2 inhibitor  in the N-terminal domain  (2+ release-activated Ca2+ current (ICRAC) in Jurkat T cells  . Althoug the PM) , and pre T cells , the ext T cells . Indeed  T cells . Notablychannels .2 and STIM1 in ACE in HaCaT keratinocytes. We have found that inhibition of iPLA2 with BEL impaired UTP and ATP-induced Ca2+ entry. We have also observed that stimulation with physiological agonists triggered restructuring of STIM1 into puncta that were assembled at or near the PM. The duration of STIM1 localization to the PM appeared to be agonist dependent, with UTP promoting sustained targeting of STIM1 to the PM whereas lysophosphatidic acid (LPA) induced only transient association of STIM1 with the PM. Together, our findings suggest that divergent signaling pathways differentially regulate ACE by controlling the duration of STIM1 localization to the PM.In this study, we have examined the role of iPLAFluo-4-AM was obtained from Invitrogen , bromoenol lactone (BEL) from Sigma . All other reagents, including MCDB153 medium were from Sigma unless indicated otherwise. The dsRed-ER vector was from Clontech .6 cells were resuspended in 100 µl of nucleofection solution with 5 µg of YFP-STIM1 plasmids, transferred to a cuvette and nucleofected on program U20. The cells were then resuspended in 500 µl of complete medium and seeded in Willco glass-bottomed microwell dishes . In some experiments, transfections were performed with Lipofectamine Plus ) according to the manufacturer's protocol.HaCaT keratinocytes, a kind gift from Dr. NE Fusenig , were grown in DMEM supplemented with 10% fetal calf serum (FCS) and antibiotics. The YFP-STIM1 expression construct was a generous gift from Tobias Meyer . Cells were nucleofected  according to the manufacturer's instructions. Briefly, 102+ unless indicated otherwise. To minimize compartmentalization of the dye, 200 µM of the anion transport inhibitor sulphinpyrazone in dimethylsulphoxide (DMSO) was included in the medium during loading and de-esterification (Di 2 (prepared in PBS) at a final concentration of 500 µM. For Ca2+-free assays, cells were loaded as above using nominally Ca2+-free Krebs-Henseleit buffer.HaCaT keratinocytes were seeded in Willco glass-bottomed microwell dishes  the day prior to experimentation. Cells were loaded with 3 µM of Fluo-4 acetoxymethyl (AM) ester for 45 min in supplemented MCBD153 medium  with 70 ation Di . After l2+]i were monitored at 4-sec intervals with a Leica TCS SP2 confocal laser scanning microscope equipped with an argon laser . A heated stage was used to maintain the cells at 37°C during image acquisition, and images were captured using a 63× Plan Apo objective (NA1.32). Fluo-4 was excited with the 488-nm line of the laser, collecting emitted fluorescence through a 500–550 nm window of the detector. Quantification was performed with Leica confocal software, and changes in [Ca2+]i expressed as the ratio of the initial fluorescence to the temporal fluorescence (Ft/F0).Changes in i oscillations of decreasing amplitude in the absence of external Ca2+ (2+ ([Ca2+]o) in the medium, we observed that stimulation with UTP evoked a rapid increase in [Ca2+]i that remained elevated for the duration of recording (2+ entry. To confirm that the sustained [Ca2+]i elevation was due to Ca2+ entry (and not for instance inhibition of Ca2+ pumps), paired assays were performed in which cells were stimulated with UTP alone or UTP and the Ca2+ chelator EGTA. Simultaneous addition of UTP and EGTA resulted in a gradual return of the [Ca2+]i to basal levels (2+]o abolishes the sustained [Ca2+]i plateau. Similar results were obtained with ATP (data not shown).Extracellular nucleotides enhance proliferation of keratinocytes and other cells . Stimulanal Ca2+ . With onecording , suggestl levels , indicat2+]i was due to Ca2+ entry across the PM, we performed fluorescence quenching assays in which Mn2+ was added to the medium after stimulation. Addition of Mn2+ led to a return of the [Ca2+]i signal baseline levels  PLA2 , an enzyme involved in DAG turnover (2 and not PAP-1.Inhibition of iPLADa) PLA2  has beenDa) PLA2 . Howeverted SOCE . Therefowith ATP . In additurnover . Treatmeturnover ), did no2+ release in many cells and modulates keratinocyte growth and migration  was similar to that obtained with UTP and ATP  . In addi2+ entry compared to LPA. This suggested that even though the respective UTP and LPA signaling pathways were equipotent for Ca2+ release on HaCaT keratinocytes  to the ER, and endocytic recycling of transferrin receptors (2 activity as incubation of cells expressing YFP-STIM1 with BEL for 30 min did not have any discernable effect on the translocation of YFP-STIM1 (data not shown). This does not, however, imply that BEL treatment has no effect on STIM1-mediated Ca2+ entry. For instance, BEL  could potentially inhibit the fundamental mechanism, as yet unknown, by which STIM1 activates Orai1. Further investigations will be required to establish whether there is any crosstalk between iPLA2 and STIM1-dependent Ca2+ entry.Several studies have shown that PLAeceptors , 2001. T2+ stores seem to be reloaded more rapidly after LPA stimulation compared to UTP stimulation? Significant amounts of the Ca2+ released from intracellular reservoirs are extruded from cells by the plasma membrane Ca2+-ATPase (PMCA). Inhibit of PMCA . Both LPaT cells . AlternaaT cells . Determi2+ influx. We can consider these moieties as functional elements of a complex Ca2+ influx network (CaIN) that provides the cell with robustness with respect to Ca2+ entry. In this model, we envisage several routes to robustness, each designed to promote Ca2+ entry following PLC activation, regardless of variables in other parts of the CaIN. Thus, as illustrated in Figure 2 activation, and (c) evoke substantial Ca2+ entry through cell surface IP3Rs channels (2+ entry through TRP channels (see The current understanding of ACE implicates a variety of cell surface receptors, phospholipases, ion channels, small molecules, and regulatory proteins in the activation, first of store depletion, and then of Cachannels  as well nels see , altogetnels see  on the C2 activity is required for ACE, and that UTP-induced Ca2+ entry in HaCaT keratinocytes is associated with remodeling of STIM1 into puncta. The duration of STIM1 puncta localization to the PM appears to be agonist-dependent, with UTP but not LPA promoting sustained re-organization of STIM1. Further investigations will be required to clarify the basis for differential spatiotemporal dynamics of STIM1 in response to stimulation with Ca2+ mobilizing agonists.In conclusion, we have shown in this study that iPLA


However, the lack of effective and selective blockers has hindered the study of these channels, mostly due to the lack of good assay system. Here, we have developed a reliable drug screening method for better blockers of CaCCs, using the endogeneous CaCCs in Xenopus laevis oocytes and two-electrode voltage-clamp (TEVC) technique.Ca2+ ionophore, which was followed by a treatment of thapsigargin which depletes Ca2+ stores to eliminate any contribution of Ca2+ release. TEVC was performed with micropipette containing chelerythrine to prevent PKC dependent run-up or run-down. Under these conditions, Ca2+-activated Cl- currents induced by bath application of Ca2+ to oocytes showed stable peak amplitude when repetitively activated, allowing us to test several concentrations of a test compound from one oocyte. Inhibitory activities of commercially available blockers and synthesized anthranilic acid derivatives were tested using this method. As a result, newly synthesized N-(4-trifluoromethylphenyl)anthranilic acid with trifluoromethyl group (-CF3) at para position on the benzene ring showed the lowest IC50.Oocytes were prepared with a treatment of CaN-(4-trifluoromethylphenyl)anthranilic acid as an improved CaCC blocker.Our results provide an optimal drug screening strategy suitable for high throughput screening, and propose  CaCCs serve a number of important physiological roles in a variety of cell types. These functions include vascular tone regulation, cardiac excitability, smooth muscle contraction, fast block of polyspermy in certain eggs [+ channel current [2+ concentration ([Ca2+]i) in several cell types, which could elicit other cellular responses o, the amplitude of 2nd response was smaller compared to the initial response  piperazine-N'-[ethanesulfonic acid]) was obtained from J.T Baker .Chemical compounds including the following reagents were purchased from Sigma-Aldrich Co. ; Collagenase type 1A, ionomycin-Ca2+ induced Cl- current applied before blocker application. Normalized and average data were fitted to the SigmaPlot's Logistic, 3 Parameter curve to determine dose-response relationship and IC50. All data are expressed as mean ± standard error of mean and statistical analysis was performed using a 2-tailed t-test.Currents were digitally recorded with AxoScope software  and data analysis was done with SigmaPlot 10.0 . All the current responses during a blocker were normalized to the average of a CaThe authors declare that they have no competing interests.SJO carried out electrophysiological recordings, data analysis and manuscript preparation. JHP, JKL and EJR designed and synthesized chemical compounds. SKH participated in electrophysiological recordings and synthesis of chemical compounds. CJL conceived the idea, coordinated the study, carried our data interpretation and drafted the manuscript. All authors have read and approved the manuscript.Methods for synthesis of anthranilic acid derivatives.Click here for file


However, the mechanisms of agonist-induced calcium entry (ACE) in keratinocytes are not fully understood.In many cells, depletion of intracellular calcium (Ca2) impairs ACE in normal human epidermal keratinocytes.This study was designed to determine if pharmacological inhibition of calcium-independent phospholipase A  or uridine triphosphate (UTP)] or with lysophosphatidic acid (LPA), a bioactive lipid that regulates keratinocyte proliferation and differentiation.Confocal laser scanning microscopy was used to monitor the dynamics of Ca2+ release in primary human keratinocytes. This was not followed by robust Ca2+ influx when the experiments were performed in low Ca2+ (70 μmol L−1) medium. Upon elevation of extracellular Ca2+ to 1·2 mmol L−1, however, a biphasic response consisting of an initial Ca2+ peak followed by an elevated plateau was observed. The plateau phase was inhibited when cells were treated with bromoenol lactone, a specific pharmacological inhibitor of iPLA2. These findings indicate that iPLA2 activity is required for ACE in keratinocytes. LPA also evoked Ca2+ release in keratinocytes but failed to induce sustained Ca2+ entry even when extracellular Ca2+ was elevated to 1·2 mmol L−1.Both ATP and UTP induced Ca2 in regulating ACE in primary human keratinocytes.Our results demonstrate for the first time an important role for iPLA Stimulation of G protein-coupled receptors (GPCRs), tyrosine kinase receptors and nonreceptor tyrosine kinases activate phospholipase C (PLC) which in turn hydrolyses phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol 1,4,5-trisphosphate (IP3).3 to its receptors (IP3R) on the ER triggers the release of Ca2+ from the ER lumen leading to store depletion.2+ across the PM, a phenomenon known as store-operated calcium entry (SOCE), which is the dominant form of Ca2+ entry in nonexcitable cells.Calcium is a ubiquitous second messenger that regulates numerous cellular processes such as gene transcription, cell proliferation, exocytosis and contraction.sphate IP.3 to its2) by displacement of inhibitory calmodulin (CaM) from iPLA2.2 activity appears to be required for SOCE.One model of SOCE involves a diffusible messenger or ‘calcium influx factor’ (CIF) that is released from the ER upon store depletion.2+-sensing protein localized predominantly to the ER contains a low-affinity Ca2+-binding EF hand which resides in the ER lumen when the stores are full. Depletion of the stores by IP3-mediated Ca2+ release, or by inhibition of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump with thapsigargin (TG) causes Ca2+ to dissociate from STIM1 inducing the re-organization of STIM1 into discrete puncta.15A second model for SOCE has emerged recently, involving STIM1 and Orai1. STIM1, a Ca2+ entry in keratinocytes. A requirement for PLCγ for SOCE has been demonstrated,3R. However, the putative role of iPLA2 in Ca2+ entry in keratinocytes has not been examined. In the present study therefore, we have investigated the role of iPLA2 in agonist-induced Ca2+ entry (ACE) in normal human epidermal keratinocytes (NHEKs). We have performed our studies using physiological agonists such as adenosine triphosphate (ATP) and uridine triphosphate (UTP), which have been reported to promote NHEK proliferation in vitrosn-glycerol-3-phosphate), which can promote proliferation or differentiation depending on cell density.2.Little is known about the mechanisms of Ca2 antibody from Santa Cruz . The iPLA2 antibody recognizes iPLA2β but not iPLA2γ according to the manufacturer. All other reagents were obtained from Sigma unless stated otherwise.Fluo-4-AM was purchased from Molecular Probes , bromoenol lactone (BEL) from Sigma  and the iPLANHEK were prepared from redundant foreskin with the approval of the Newcastle and North Tyneside local ethical committee. The cells were cultured in supplemented MCDB 153 culture medium as previously described.−1 of Fluo-4 acetoxymethyl (AM) ester (Molecular Probes) for 45 min at 37 °C. Dye loading and all subsequent steps were performed with MCDB153 medium (Sigma) containing 70 μmol L−1 Ca2+ unless indicated otherwise. To minimize uptake of the dye into organelles, 200 μmol L−1 of the anion transport inhibitor sulphinpyrazone was dissolved in dimethyl sulphoxide (DMSO) and included in the medium during loading and de-esterification. After loading, the cells were washed in Ca2+ and Mg2+ free phosphate-buffered saline (PBS) and incubated in MCDB 153 medium with 70 μmol L−1 Ca2+ for 1 h at 37 °C to allow de-esterification of the intracellular dye. Where indicated, Ca2+ in the medium was raised to 1·2 mmol L−1 at the start of the de-esterification phase. The iPLA2 inhibitor BEL (10 or 20 μmol L−1) or vehicle (0·1% DMSO) was added for the last 30 min of de-esterification.Subconfluent monolayers of cells, seeded in Willco glass-bottomed microwell dishes  1 day before experiments, were loaded with 3 μmol L2+]i were detected with a Leica TCS SP2 confocal laser scanning microscope equipped with an argon laser . Fluorescence excitation was performed with the 488 nm line of the laser. Fluorescence emission was collected through a 500–550 nm window of the detector. Images were captured with a 63X Plan Apo objective (NA1.32) at 4-s intervals as 12-bit frames of 512 × 512 pixels. The perimeter of each cell was outlined to define the region of interest whose mean fluorescence intensity in each frame was determined by Leica confocal software. The changes in [Ca2+]i were expressed as the ratio of the temporal fluorescence to the initial fluorescence (Ft/F0).The cells were maintained at 37 °C during image acquisition with a heated stage. Changes in i elevation in primary keratinocytes,2+]i (indicative of Ca2+entry) was not clear. When preconfluent monolayers of NHEKs were stimulated with UTP, a single [Ca2+]i transient was observed in the majority of responsive cells , a second transient of smaller amplitude was observed at later time points  level needs to be in the millimolar range before the electrochemical gradient is sufficiently high to promote robust ACE in keratinocytes following stimulation with exogenous nucleotides.In these experiments, which were performed in medium containing 70 μmol L.2+entry . Taken t2 impaired TG-induced entry in mouse smooth muscle cells.2 activity was required for ACE in NHEKs. For these studies, we used the suicide substrate, BEL, to inhibit iPLA2 activity. BEL is a specific inhibitor of iPLA2, with a 1000-fold selectivity for iPLA2 over cytosolic PLA2.−1 [Ca2+]o led to a sustained [Ca2+]i plateau in control cells exposed to vehicle (DMSO), consistent with the results shown in 2+]i phase was impaired in cells treated with BEL  evoked a single [Ca2+]i transient that reached a peak about 20 s thereafter  [Ca2+]o was exerting a nonspecific effect enhancing effect on ACE in keratinocytes. In addition, the duration of the transient was not significantly altered by the inclusion of 10 mmol L−1 EGTA in the medium, indicating that sustained Ca2+ entry did not occur (data not shown). Thus LPA does not appear to induce significant Ca2+ influx in keratinocytes. This observation is consistent with the findings of others on T cells and fibroblasts.2+ release peaked at about 1 μmol L−1  domain that binds lipids2 enhance the localization of TRP proteins to the cell surface of keratinocytes, and that inhibition of iPLA2 impairs this process. However, we cannot exclude a role for arachidonic acid generated by iPLA2 activity, given that it has also been implicated in ACE2+ entry in keratinocytes.How does iPLA2+]i plateau in primary keratinocytes under low (70 μmol L−1) [Ca2+]o conditions, the [Ca2+]i transient induced by LPA under these conditions was significantly longer than that induced by UTP. The duration of a [Ca2+]i transient depends on the balance of [Ca2+]i extrusion from the cytosol by [Ca2+]i pumps  and [Ca2+]i re-entry into the ER through SERCA pumps. Both PMCA and SERCA activity may be impaired in NHEK stimulated with LPA, given that LPA has been shown to induce H2O2 production in HaCaT keratinocytes2O2 can inhibit PMCAAlthough none of the agonists used in our experiments induced a sustained i peak generated by the application of exogenous nucleotides was followed by an elevated plateau. Importantly, addition of Mn2+ to the medium after UTP stimulation led to quenching of the Fluo-4 signal (data not shown), indicating that under these conditions UTP activated Ca2+ entry. In contrast to extracellular nucleotides, LPA stimulation did not produce an elevated Ca2+ plateau in keratinocytes even in the presence of millimolar levels of [Ca2+]o (2+ entry in these cells.2+ from internal stores, LPA does not promote robust Ca2+ entry in several distinct cell types. The contrast between UTP and LPA induced [Ca2+]i mobilization may be due to differential coupling of LPA receptor activation to STIM1 or Orai1/CRACM1, two newly discovered mediators of SOCE.39When extracellular Ca [Ca2+]o . The rea2+ release in cultured human keratinocytes, and when external Ca2+ is in the millimolar range, Ca2+ release follows sustained ACE. Stimulation with LPA also evoked Ca2+ release, but without inducing robust Ca2+ influx. Pharmacological inhibition of iPLA2 impaired ACE, highlighting the importance of iPLA2 in ACE in keratinocytes.In conclusion, the work presented here demonstrates that extracellular nucleotides trigger Ca


The peroxynitrite production by LA was found to depend on mitochondrial Ca2+ efflux. Downregulation of hsp90β1, which has been previously shown to be essential for polyunsaturated fatty acid-induced mitochondrial Ca2+ efflux, significantly diminished LA-responsive mitochondrial Ca2+ efflux and the coupled peroxynitrite generation, implicating a critical role of hsp90β1 in the LA responses. Our results further demonstrated that mitochondrial complexes I and III were directly involved in the LA-induced peroxynitrite generation. Using the well established type 2 diabetic animal model db/db mice, we observed a dramatically enhanced LA responsive mitochondrial Ca2+ efflux and protein nitrotyrosylation in the kidney. Our study thus demonstrates a cause-effect relationship between LA and peroxynitrite or protein nitrotyrosylation and provides a novel mechanism for lipid-induced nephropathy in diabetes.It is well known that excessive non-esterified fatty acids in diabetes contribute to the pathogenesis of renal complications although the mechanism remains elusive. Enhanced oxidative stress has been hypothesized as a unified factor contributing to diabetic complications and increased protein nitrotyrosylation has been reported in the kidneys of diabetic patients. In the current manuscript we described that linoleic acid (LA) caused mitochondrial Ca In the current study, we demonstrated that linoleic acid -responsive mitochondrial Ca2+ efflux caused peroxynitrite generation in HM cells. Our results also showed increased nitrotyrosylation of proteins in LA-treated HM cells and in the kidney of db/db diabetic mice, which may in turn impair kidney function and contribute to the pathogenesis of diabetic renal complications.NEFA may influence mitochondrial function by altering gene expression 2+]i rise in HM cells and the amplitude of the LA responses was dose dependent (2+]i response of LA (30 µM) was associated with ROS generation in HM cells  preferentially reacts with peroxynitrite 2+]i rise, but significantly attenuated the ROS production , suggesting that the generated ROS were predominantly peroxynitrite. The data also indicated that the LA responsive peroxynitrite generation was either unrelated or occurred downstream of the [Ca2+]i rise.Because nitrosative injury as indexed by protein nitrotyrosine was dramatically increased in diabetic nephropathy 2+]i mobilization by measuring dihydroethidium , and diaminofluorescein-2 diacetate  fluorescence, respectively. As shown in 2+]i mobilization was associated with the generation of superoxide and NO. The specificity of DHE, DAF-2, and DCF was further demonstrated by using the exogenous NO donor . The NO donor had no effect on DHE fluorescence . As shown in 2+]i mobilization by activation of Ca2+ release from the ER and store-operated Ca2+ influx through inositol-1, 4, 5-trisphosphate dependent and independent mechanisms, respectively. However, as shown in 2+]i mobilization induced by both BK and TG was not associated with any detectable peroxynitrite production. These results indicated that the rise of [Ca2+]i from the endoplasmic reticulum (ER) Ca2+ release and store-operated Ca2+ influx did not lead to peroxynitrite generation.Bradykinin (BK) and thapsigargin (TG) are known to induce [Ca2+]i mobilization was observed in the absence of extracellular Ca2+, chelation of extracellular Ca2+ significantly diminished LA-induced [Ca2+]i signal ethane-N,N,N',N'-tetraacetic acid  in the absence of extracellular Ca2+, abolished LA-induced [Ca2+]i signal but had no significant effect on peroxynitrite generation , indicating that LA responsive peroxynitrite generation occurred independently of [Ca2+]i rise. Because BAPTA does not prevent Ca2+ release from the internal Ca2+ stores, including ER and mitochondria, these experiments did not exclude the role of Ca2+ release from the internal Ca2+ stores in peroxynitrite generation.Even though LA-induced i mobilization and had no significant effect on peroxynitrite generation  in the absence of extracellular Caneration . These r2+, pretreatment of the cells with carbonyl cyanide-p-trifluoromethoxyphenylhydrazone , a well-known mitochondrial uncoupler, completely inhibited LA-induced peroxynitrite production and also attenuated the [Ca2+]i response -17-demethoxygeldanamycin (17-DMAG), and hsp90β1 RNAi. As shown in 2+]m efflux  but also attenuated peroxynitrite generation  without significant effect on LA-induced [Ca2+]i mobilization . Blocking complex III with either antimycin A  or myxothiazol  also did not alter the [Ca2+]i responses  to LA but inhibited peroxynitrite generation . These results indicate that the mitochondrial complexes I and III are responsible for LA-induced peroxynitrite generation.It has been known that in the mitochondria ROS are produced predominantly by complexes I and III 2+ efflux . Western blot analysis indicated that nitrotyrosylation in a protein with a MW about 22 kDa was significantly enhanced in db/db mice . Analysis of the nitrotyrosine level in kidney sections with immunohistochemistry further confirmed that protein nitrotyrosylation was enhanced in db/db mice kidney  or L-NG-monomethyl arginine (0.5 mM) (data not shown), indicating that the LA responsive NO, a required substrate for peroxynitrite generation, was produced in an L-arginine independent manner. Mitochondrial complex III has been shown to possess nitrite reductase activity and is responsible for NO generation in the mitochondria by an L-arginine independent manner Peroxynitrite is a highly reactive oxidant and is formed by the reaction between superoxide and NO. It has been reported that the mitochondrial complexes I and III cause superoxide production 2+]m efflux and peroxynitrite generation were inhibited by 17-DMAG and, more specifically, hsp90β1 RNAi, indicating the hsp90β1 involvement  cells  were prepared and cultured as previously described 2+]i or together with DHE (1 µM), DAF-2 (0.5 nM), or DCF (1 µM) as needed at 37°C for 30 min in RPMI 1640 medium to measure [Ca2+]i and superoxide, NO, or peroxynitrite simultaneously. Loaded cells were harvested and resuspended in PBS1Ca buffer . [Ca2+]i mobilization and superoxide, NO or peroxynitrite generation were monitored in a fluorometer  using a cuvette with the temperature stabilized at room temperature. The ratio of fluorescence excited at 340 and 380 nm with emission of 510 nm was recorded and used to index the [Ca2+]i change as previously reported 6 cells/ml in these experiments. The interference of LA to DHE, DAF-2, and DCF fluorescence was calibrated by subtracting the signal resulted from palmitic acid, which does not cause [Ca2+]i mobilization in HM cells.HM cells grown in 100 mm dishes were labeled with fura-2 (1 µM) alone to measure [Ca2+]i mobilization was measured in the absence of extracellular Ca2+, the loaded cells were washed and measured in PBS0Ca buffer (containing 200 µM EGTA instead of 1 mM CaCl2 as in PBS1Ca).In experiments in which m and peroxynitrite generation were measured with fluorescence excited at 578 or 506 nm and emitted at 602 or 529 nm in a PTI QM-6 fluorometer, respectively. In these experiments, mitochondrial preparations equal to 40 µg/ml proteins were used.Mitochondria were prepared from HM cells or the kidney of 12–16 wk old db/+ or db/db mice as previously described 2+]m efflux and peroxynitrite generation were determined in the absence of Ca2+ refilling, the mitochondrial preparations were suspended in MB1 buffer and diluted with nine volumes of HKG-EGTA buffer (containing 200 µM EGTA instead of 1 mM CaCl2 as in HKG), then labeled with X-rhod-1 and DCF and measured as described above.In experiments in which LA-induced i mobilization and peroxynitrite generation in treated HM cells were assessed as described above.The effects of 17-DMAG and hsp90β1 RNAi on LA-induced [Ca3VO4 1 mM, NaF 1 mM, pH 7.4), and kept on ice for 60 min. The mixture was centrifuged at 12,000×g for 15 min at 4°C. Western blot analysis of the proteins in the supernatant was performed with mouse mAb to nitrotyrosine  and α-tubulin was used to control protein loading.HM cells in 100 mm dishes were treated with LA , lysed in ice-cold RIPA buffer  of kidney tissues were homogenized in 1 ml RIPA buffer with a glass-glass homogenizer manually for 30 strokes. The homogenates were centrifuged at 150×g for 15 min at 4°C and the supernatants were collected. The nitrotyrosine levels were determined by western blot analysis.2O2. The mouse mAb to nitrotyrosine was added for 1 h at room temperature. Tissue sections were treated with VECTASTAIN® ABC-Peroxidase Kit . The DAB Substrate Kit (Vector Laboratories) was used as the enzyme substrate. Sections were counterstained with CAT Hematoxylin . Procedures involving the use of mice were approved by the Institutional Animal Care and Use Committee, University of Texas Health Science Center at San Antonio (0707-001).The kidneys of 12–16 wk old db/+ and db/db mice were fixed in formalin, embedded in paraffin and sectioned (4 µm). Paraffin sections were deparaffinized with xylene and hydrated with a series of graded ethanol washes. Endogenous peroxidase was inactivated by incubation for 30 min in 0.3% HP<0.05.