Release Of Stores Research Articles

Targeting Zinc(II) Signalling to Prevent Cancer.

Zinc is an important element that is gaining momentum as a potential target for cancer therapy. In recent years zinc has been accepted as a second messenger that is now recognized to be able to activate many signalling pathways within a few minutes of an extracellular stimulus by release of zinc(II) from intracellular stores. One of the major effects of this store release of zinc is to inhibit a multitude of tyrosine phosphatases which will prevent the inactivation of tyrosine kinases and hence, encourage further activation of tyrosine kinasedependent signalling pathways. Most of these signalling pathways are not only known to be involved in driving aberrant cancer growth, they are usually the main driving force. All this data together now positions zinc and zinc signalling as potentially important new targets to prevent aggressive cancer growth.

Temporal reprogramming of calcium signalling via crosstalk of gonadotrophin receptors that associate as functionally asymmetric heteromers

Signal crosstalk between distinct G protein-coupled receptors (GPCRs) is one mechanism that underlies pleiotropic signalling. Such crosstalk is also pertinent for GPCRs activated by gonadotrophic hormones; follicle-stimulating hormone (FSH) and luteinising hormone (LH), with specific relevance to female reproduction. Here, we demonstrate that gonadotrophin receptor crosstalk alters LH-induced Gαq/11-calcium profiles. LH-induced calcium signals in both heterologous and primary human granulosa cells were prolonged by FSHR coexpression via influx of extracellular calcium in a receptor specific manner. LHR/FSHR crosstalk involves Gαq/11 activation as a Gαq/11 inhibitor abolished calcium responses. Interestingly, the enhanced LH-mediated calcium signalling induced by FSHR co-expression was dependent on intracellular calcium store release and involved Gβγ. Biophysical analysis of receptor and Gαq interactions indicated that ligand-dependent association between LHR and Gαq was rearranged in the presence of FSHR, enabling FSHR to closely associate with Gαq following LHR activation. This suggests that crosstalk may occur via close associations as heteromers. Super-resolution imaging revealed that LHR and FSHR formed constitutive heteromers at the plasma membrane. Intriguingly, the ratio of LHR:FSHR in heterotetramers was specifically altered following LH treatment. We propose that functionally significant FSHR/LHR crosstalk reprograms LH-mediated calcium signalling at the interface of receptor-G protein via formation of asymmetric complexes.

Improving the Cooling Efficiency of Heat Sinks through the Use of Different Types of Phase Change Materials

As the temperature of electronic devices increases, their failure rate increases. That is why electrical devices should be cooled. One of the promising cooling techniques is using Phase Change Materials (PCMs). A new passive temperature management technique, that involves the direct placement of PCMs on the chip, has been explored and developed. PCMs are potential temperature regulators that can store thermal energy and release it during melting and freezing respectively. PCM-based heat sinks can efficiently store the heat dissipated from the electronic components to delay the peak temperature of the electronic devices as much as possible and then release the stored energy during the off period. This paper compares the temperature distribution on a heat sink with and without PCM with different magnitudes of heat flux. Also, two different PCMs with different densities, namely salt-hydrate and wax, have been investigated in cooling electronic devices.

Seismogenic structures of the 2006 ML4.0 Dangan Island earthquake offshore Hong Kong

The northern margin of the South China Sea, as a typical extensional continental margin, has relatively strong intraplate seismicity. Compared with the active zones of Nanao Island, Yangjiang, and Heyuan, seismicity in the Pearl River Estuary is relatively low. However, a ML4.0 earthquake in 2006 occurred near Dangan Island (DI) offshore Hong Kong, and this site was adjacent to the source of the historical M5.8 earthquake in 1874. To reveal the seismogenic mechanism of intraplate earthquakes in DI, we systematically analyzed the structural characteristics in the source area of the 2006 DI earthquake using integrated 24-channel seismic profiles, onshore–offshore wide-angle seismic tomography, and natural earthquake parameters. We ascertained the locations of NW- and NE-trending faults in the DI sea and found that the NE-trending DI fault mainly dipped southeast at a high angle and cut through the crust with an obvious low-velocity anomaly. The NW-trending fault dipped southwest with a similar high angle. The 2006 DI earthquake was adjacent to the intersection of the NE- and NW-trending faults, which suggested that the intersection of the two faults with different strikes could provide a favorable condition for the generation and triggering of intraplate earthquakes. Crustal velocity model showed that the high-velocity anomaly was imaged in the west of DI, but a distinct entity with low-velocity anomaly in the upper crust and high-velocity anomaly in the lower crust was found in the south of DI. Both the 1874 and 2006 DI earthquakes occurred along the edge of the distinct entity. Two vertical cross-sections nearly perpendicular to the strikes of the intersecting faults revealed good spatial correlations between the 2006 DI earthquake and the low to high speed transition in the distinct entity. This result indicated that the transitional zone might be a weakly structural body that can store strain energy and release it as a brittle failure, resulting in an earthquake-prone area.

Study on the coupling heating system of floor radiation and sunspace based on energy storage technology

With the gradual increase of building energy consumption in recent years, the application of phase change energy storage materials in buildings has become the focus of researches. Phase change material (PCM) embedded in the envelope can increase the building inertia to keep the temperature stable. It can also store energy and release energy when needed. In this paper, the system of PCM radiant floor combined with PCM wall attached with sunspace was established. The thermal characteristics and the energy consumption of the building are studied through analyzing the data from the experimental investigation including the temperature and the heat flux. Two different buildings were built between the PCM room and the conventional room with the same dimension and building. The main test conditions are divided into passive and active control stages by comparing the air temperatures, envelope temperatures, phase transition temperatures, heat fluxes and power consumptions between the two rooms. It is concluded that the average room temperature of the experimental room is 7.15°C higher than that of the conventional room in the passive energy storage stage. While the average energy-saving rate of PCM room was 54.27% compared with the conventional room in the active control stage.

Linking muscarinic receptor activation to UPS-mediated object memory destabilization: Implications for long-term memory modification and storage

Consolidated memories can become destabilized during reactivation, resulting in a transient state of instability, a process that has been hypothesized to underlie long-term memory updating. Consistent with this notion, relatively remote memories, which are resistant to standard destabilization procedures, are reliably destabilized when novel information (i.e., the opportunity for memory updating) is present during reactivation. We have also shown that cholinergic muscarinic receptor (mAChR) activation can similarly destabilize consolidated object memories. Synaptic protein degradation via the ubiquitin proteasome system (UPS) has previously been linked to destabilization of fear and object-location memories. Given the role of calcium in regulating proteasome activity, we hypothesized that activation of cholinergic receptors, specifically M1 mAChRs, stimulates the UPS via inositol triphosphate receptor (IP3R)-mediated release of intracellular calcium stores to facilitate object memory destabilization. We present converging evidence for this hypothesis, which we tested using a modified spontaneous object recognition task for rats and microinfusions into perirhinal cortex (PRh), a brain region strongly implicated in object memory. We extend our previous findings by demonstrating that M1 mAChRs are necessary for novelty-induced object memory destabilization. We also show that proteasome inhibition or IP3R antagonism in PRh prevents object memory destabilization induced by novelty or M1 mAChR stimulation. These results establish an intracellular pathway linking M1 receptors, IP3Rs, and UPS activity to object memory destabilization and suggest a previously unacknowledged role for cholinergic signaling in long-term memory modification and storage.

Epithelial Chloride Transport by CFTR Requires TMEM16A

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is the secretory chloride/bicarbonate channel in airways and intestine that is activated through ATP binding and phosphorylation by protein kinase A, but fails to operate in cystic fibrosis (CF). TMEM16A (also known as anoctamin 1, ANO1) is thought to function as the Ca2+ activated secretory chloride channel independent of CFTR. Here we report that tissue specific knockout of the TMEM16A gene in mouse intestine and airways not only eliminates Ca2+-activated Cl− currents, but unexpectedly also abrogates CFTR-mediated Cl− secretion and completely abolishes cAMP-activated whole cell currents. The data demonstrate fundamentally new roles of TMEM16A in differentiated epithelial cells: TMEM16A provides a mechanism for enhanced ER Ca2+ store release, possibly engaging Store Operated cAMP Signaling (SOcAMPS) and activating Ca2+ regulated adenylyl cyclases. TMEM16A is shown to be essential for proper activation and membrane expression of CFTR. This intimate regulatory relationship is the cause for the functional overlap of CFTR and Ca2+-dependent chloride transport.

A method for estimating intracellular ion concentration using optical nanosensors and ratiometric imaging

Optical nanoparticle (NP)-based sensors have been widely implemented as tools for detection of targeted ions and biomolecules. The NP sensing platform offer a modular design that can incorporate different sensing components for greater target specificity and the ability to tune the dynamic range, as well as encapsulation of multiple dyes to generate a ratiometric signal with varying spectra. Despite these advantages, demonstrating quantitative ion imaging for intracellular measurement still possess a major challenge. Here, we describe fundamentals that enable intracellular validation of this approach using ion-selective nanosensors for investigating calcium (Ca2+) as a model ion. While conventional indicators can improve individual aspects of indicator performance such as Kd, wavelength, and ratiometric measurements, the use of NP sensors can achieve combined benefits of addressing these issues simultaneously. The nanosensor incorporates highly calcium-selective ionophores and two fluorescence indicators that act as signal transducers to facilitate quantitative ratiometric imaging. For intracellular Ca2+ application, the sensors are fine-tuned to physiological sensing range, and live-cell imaging and quantification are demonstrated in HeLa cells loaded with nanosensors and their responsiveness to carbachol-evoked store release (~400 nM). The current nanosensor design thus provides a promising sensing platform for real-time detection and optical determination of intracellular ions.

Rodent Models of Non-classical Progesterone Action Regulating Ovulation.

It is becoming clear that steroid hormones act not only by binding to nuclear receptors that associate with specific response elements in the nucleus but also by binding to receptors on the cell membrane. In this newly discovered manner, steroid hormones can initiate intracellular signaling cascades which elicit rapid effects such as release of internal calcium stores and activation of kinases. We have learned much about the translocation and signaling of steroid hormone receptors from investigations into estrogen receptor α, which can be trafficked to, and signal from, the cell membrane. It is now clear that progesterone (P4) can also elicit effects that cannot be exclusively explained by transcriptional changes. Similar to E2 and its receptors, P4 can initiate signaling at the cell membrane, both through progesterone receptor and via a host of newly discovered membrane receptors (e.g., membrane progesterone receptors, progesterone receptor membrane components). This review discusses the parallels between neurotransmitter-like E2 action and the more recently investigated non-classical P4 signaling, in the context of reproductive behaviors in the rodent.

231 Super resolution imaging unveiling the dyadic ultrastructure in atrial and ventricular cardiomyocytes

Calcium-induced calcium release drives contraction in cardiomyocytes. Located on the sarcoplasmic reticulum, ryanodine receptors (RyR) are responsible for the release of intracellular calcium stores. It has recently become apparent that...

Phosphatidylinositol 4,5-bisphosphate (PIP2 ) modulates afterhyperpolarizations in oxytocin neurons of the supraoptic nucleus.

Afterhyperpolarizations (AHPs) generated by repetitive action potentials in supraoptic magnocellular neurons regulate repetitive firing and spike frequency adaptation but relatively little is known about PIP2 's control of these AHPs. We examined how changes in PIP2 levels affected AHPs, somatic [Ca2+ ]i , and whole cell Ca2+ currents. Manipulations of PIP2 levels affected both medium and slow AHP currents in oxytocin (OT) neurons of the supraoptic nucleus. Manipulations of PIP2 levels did not modulate AHPs by influencing Ca2+ release from IP3 -triggered Ca2+ stores, suggesting more direct modulation of channels by PIP2 . PIP2 depletion reduced spike-evoked Ca2+ entry and voltage-gated Ca2+ currents. PIP2 appears to influence AHPs in OT neurons by reducing Ca2+ influx during spiking. Oxytocin (OT)- and vasopressin (VP)-secreting magnocellular neurons of the supraoptic nucleus (SON) display calcium-dependent afterhyperpolarizations (AHPs) following a train of action potentials that are critical to shaping the firing patterns of these cells. Previous work demonstrated that the lipid phosphatidylinositol 4,5-bisphosphate (PIP2 ) enabled the slow AHP component (sAHP) in cortical pyramidal neurons. We investigated whether this phenomenon occurred in OT and VP neurons of the SON. Using whole cell recordings in coronal hypothalamic slices from adult female rats, we demonstrated that inhibition of PIP2 synthesis with wortmannin robustly blocked both the medium and slow AHP currents (ImAHP and IsAHP ) of OT, but not VP neurons with high affinity. We further tested this by introducing a water-soluble PIP2 analogue (diC8 -PIP2 ) into neurons, which in OT neurons not only prevented wortmannin's inhibitory effect, but slowed rundown of the ImAHP and IsAHP . Inhibition of phospholipase C (PLC) with U73122 did not inhibit either ImAHP or IsAHP in OT neurons, consistent with wortmannin's effects not being due to reducing diacylglycerol (DAG) or IP3 availability, i.e. PIP2 modulation of AHPs is not likely to involve downstream Ca2+ release from inositol 1,4,5-trisphosphate (IP3 )-triggered Ca2+ -store release, or channel modulation via DAG and protein kinase C (PKC). We found that wortmannin reduced [Ca2+ ]i increase induced by spike trains in OT neurons, but had no effect on AHPs evoked by uncaging intracellular Ca2+ . Finally, wortmannin selectively reduced whole cell Ca2+ currents in OT neurons while leaving VP neurons unaffected. The results indicate that PIP2 modulates both the ImAHP and IsAHP in OT neurons, most likely by controlling Ca2+ entry through voltage-gated Ca2+ channels opened during spike trains.

Effects of latent heat storage and controls on stability and performance of a solar assisted heat pump system for domestic hot water production

Solar assisted heat pump systems have been widely applied in domestic hot water productions due to their sustainability and stability in operations. However, their performance efficiency requires further improvement using advanced technologies such as energy storage with phase change materials (PCM) and optimal system controls. Accordingly, a test rig of a new indirect solar assisted heat pump (IDX-SAHP) system has been designed, built and instrumented. The IDX-SAHP system consists of three operational loops: solar thermal, solar assisted heat pump and load profile. A PCM heat exchanger tank was purposely designed and installed in the system solar thermal loop to store solar energy, when applicable, and release heat when required by the heat pump. In addition, an air cooling heat exchanger was also installed in the solar thermal loop to absorb heat from the ambient air for the heat pump. A detailed control strategy has been designed and implemented for the system for efficient operation in different modes and maintain constant load supply water temperature during an operational day. In different weather conditions of sunny and overcast, comprehensive measurements were carried out on the test rig and system structures with and without the PCM tank. Test results showed that the system could efficiently meet the daily domestic hot water demand and the PCM heat exchanger integration exerted a significant effect on system stability and performance efficiency. Quantitatively, the average COP of the IDX-SHAP system with a PCM tank could increase 6.1% and 14.0% on sunny and cloudy days respectively compared to systems without PCM tank integration.

Flight-Test Experiments on Cavity Flow in an SUU-41 Pod

The conceptual development, design, ground test, and flight test of a novel weapons-cavity acoustics store separation pod led to several positive outcomes to aid in the understanding of cavity–store interaction. The weapons-cavity acoustics store separation pod was a modified SUU-41 pod (suspended utility unit) instrumented to collect forces and accelerations on a scaled model missile. In addition, time-resolved pressure and qualitative flowfield imaging were captured for the scaled cavity. This research included seven test missions on the F-16 Fighting Falcon. The flight-test data indicate that there is a clear correlation between loads and missile acceleration. The normal force, pitch moment, and acceleration data acquired for difference missile model configurations within the cavity yielded considerable insight into possible complications relating to store release. Pressure transducer data indicated the frequency of Rossiter tones and corresponded closely to results anticipated from prior flights involving the SUU-41 pod. Correlation between pressure transducer and loads/acceleration acting on the missile model was found to occur most commonly around Rossiter mode 2, indicating mode matching.

Differential effects of anoctamins on intracellular calcium signals.

The Ca2+-activated Cl- channel TMEM16A [anoctamin (ANO)1] is homologous to yeast Ist2 and has been shown to tether the cortical endoplasmic reticulum (ER) to the plasma membrane. We therefore examined whether ANO1 and other members of the ANO family affect intracellular Ca2+ ([Ca2+]i) signals. It is shown that expression of ANO1 augments Ca2+ store release upon stimulation of GPCRs, whereas knockdown of ANO1, or lack of Ano1 expression in Ano1-/- animals, as shown in an earlier report, inhibits Ca2+ release. ANO6, and -10 show similar effects, whereas expression of ANO4, -8, and -9 attenuate filling of the ER store. The impact of ANO1 and -4 were examined in more detail. ANO1 colocalized and interacted with IP3R, whereas ANO4 colocalized with SERCA Ca2+ pumps and interacted with ORAI-1 channels, respectively. ANO1 Cl currents were rapidly activated exclusively through Ca2+ store release, and remained untouched by influx of extracellular Ca2+ In contrast expression of ANO4 caused a delayed activation of membrane-localized ANO6 channels, solely through store-operated Ca2+ entry via ORAI. Ca2+ signals were inhibited by knocking down expression of endogenous ANO1, -5, -6, and -10 and were also reduced in epithelial cells from Ano10-/- mice. The data suggest that ANOs affect compartmentalized [Ca2+]i signals, which may explain some of the cellular defects related to ANO mutations.-Cabrita, I., Benedetto, R., Fonseca, A., Wanitchakool, P., Sirianant, L., Skryabin, B. V., Schenk, L. K., Pavenstädt, H., Schreiber, R., Kunzelmann, K. Differential effects of anoctamins on intracellular calcium signals.

An experimental system for release simulation of internal stores in a supersonic wind tunnel

Aerodynamic parameters obtained from separation experiments of internal stores in a wind tunnel are significant in aircraft designs. Accurate wind tunnel tests can help to improve the release stability of the stores and in-flight safety of the aircrafts in supersonic environments. A simulative system for free drop experiments of internal stores based on a practical project is provided in this paper. The system contains a store release mechanism, a control system and an attitude measurement system. The release mechanism adopts a six-bar linkage driven by a cylinder, which ensures the release stability. The structure and initial aerodynamic parameters of the stores are also designed and adjusted. A high speed vision measurement system for high speed rolling targets is utilized to measure the pose parameters of the internal store models and an optimizing method for the coordinates of markers is presented based on a priori model. The experimental results show excellent repeatability of the system, and indicate that the position measurement precision is less than 0.13mm, and the attitude measurement precision for pitch and yaw angles is less than 0.126°, satisfying the requirements of practical wind tunnel tests. A separation experiment for the internal stores is also conducted in the FL-3 wind tunnel of China Aerodynamics Research Institute.

Aristoyunnolin H attenuates extracellular matrix secretion in cardiac fibroblasts by inhibiting calcium influx.

Aristoyunnolin H is a novel aristophyllene sesquiterpenoid isolated from the traditional Chinese medicine Aristolochia yunnanensis Franch. The present research was designed to explore the anti-fibrotic effects of aristoyunnolin H in adult rat cardiac fibroblasts (CFs) stimulated with angiotensin II (Ang II). Western blot analysis data showed that aristoyunnolin H reduced the upregulation of fibronectin (FN), connective tissue growth factor and collagen I(Col I) production induced by Ang II in CFs. By studying the dynamic intracellular changes of Ca2+, we further found that while aristoyunnolin H relieved the calcium influx, it has no effect on intracellular calcium store release. Meanwhile, aristoyunnolin H also inhibited the Ang II-stimulated phosphorylation of Ca2+/calmodulin-dependent protein kinase II. In conclusion, aristoyunnolin H may attenuate extracellular matrix secretion in vitro by inhibiting Ang II-induced calcium signaling.

The effect of imiquimod on taste bud calcium transients and transmitter secretion.

Imiquimod is an immunomodulator approved for the treatment of basal cell carcinoma and has adverse side effects, including taste disturbances. Paracrine transmission, representing cell-cell communication within taste buds, has the potential to shape the final signals that taste buds transmit to the brain. Here, we tested the underlying assumption that imiquimod modifies taste transmitter secretion in taste buds of mice. Taste buds were isolated from C57BL/6J mice. The effects of imiquimod on transmitter release in taste buds were measured using calcium imaging with cellular biosensors, and examining the net effect of imiquimod on taste-evoked ATP secretion from mouse taste buds. Up to 72% of presynaptic (Type III) taste cells responded to 100μM imiquimod with an increase in intracellular Ca2+ concentrations. These Ca2+ responses were inhibited by thapsigargin, an inhibitor of the sarco/endoplasmic reticulum Ca2+ -ATPase, and by U73122, a PLC inhibitor, suggesting that the Ca2+ mobilization elicited by imiquimod was dependent on release from internal Ca2+ stores. Moreover, combining studies of Ca2+ imaging with cellular biosensors showed that imiquimod evoked secretion of 5-HT, which then provided negative feedback onto receptor (Type II) cells to reduce taste-evoked ATP secretion. Our results provide evidence that there is a subset of taste cells equipped with a range of intracellular mechanisms that respond to imiquimod. The findings are also consistent with a role of imiquimod as an immune response modifier, which shapes peripheral taste responses via 5-HT signalling.

Intracellular calcium increases in vascular smooth muscle cells with progression of chronic kidney disease in a rat model.

Vascular smooth muscle cells (VSMCs) exhibit phenotypic plasticity, promoting vascular calcification and increasing cardiovascular risk. Changes in VSMC intracellular calcium ([Ca 2+ ] i ) are a major determinant of plasticity, but little is known about changes in [Ca 2+ ] i in chronic kidney disease (CKD). We have previously demonstrated such plasticity in aortas from our rat model of CKD and therefore sought to examine changes in [Ca 2+ ] i during CKD progression. We examined freshly isolated VSMCs from aortas of normal rats, Cy/+ rats (CKD) with early and advanced CKD, and advanced CKD rats treated without and with 3% calcium gluconate (CKD + Ca 2+ ) to lower parathyroid hormone (PTH) levels. [Ca 2+ ] i was measured with fura-2. Cy/+ rats developed progressive CKD, as assessed by plasma levels of blood urea nitrogen, calcium, phosphorus, parathyroid hormone and fibroblast growth factor 23. VSMCs isolated from rats with CKD demonstrated biphasic alterations in resting [Ca 2+ ] i : VSMCs from rats with early CKD exhibited reduced resting [Ca 2+ ] i , while VSMCs from rats with advanced CKD exhibited elevated resting [Ca 2+ ] i . Caffeine-induced sarcoplasmic reticulum (SR) Ca 2+ store release was modestly increased in early CKD and was more drastically increased in advanced CKD. The advanced CKD elevation in SR Ca 2+ store release was associated with a significant increase in the activity of the sarco-endoplasmic reticulum Ca 2+ ATPase (SERCA); however, SERCA2a protein expression was decreased in advanced CKD. Following SR Ca 2+ store release, recovery of [Ca 2+ ] i in the presence of caffeine and extracellular Ca 2+ was attenuated in VSMCs from rats with advanced CKD. This impairment, together with reductions in expression of the Na + /Ca 2+ exchanger, suggest a reduction in Ca 2+ extrusion capability. Finally, store-operated Ca 2+ entry (SOCE) was assessed following SR Ca 2+ store depletion. Ca 2+ entry during recovery from caffeine-induced SR Ca 2+ store release was elevated in advanced CKD, suggesting a role for exacerbated SOCE with progressing CKD. With progressive CKD in the Cy/+ rat there is increased resting [Ca 2+ ] i in VSMCs due, in part, to increased SOCE and impaired calcium extrusion from the cell. Such changes may predispose VSMCs to phenotypic changes that are a prerequisite to calcification.

Effects of Fatty Acids on Intracellular [Ca2+], Mitochondrial Uncoupling and Apoptosis in Rat Pachytene Spermatocytes and Round Spermatids.

The aim of this work was to explore the ability of free arachidonic acid, palmitic acid and the unsaturated fatty acids oleic acid and docosahexaenoic acid to modify calcium homeostasis and mitochondrial function in rat pachytene spermatocytes and round spermatids. In contrast to palmitic acid, unsaturated fatty acids produced significant increases in intracellular calcium concentrations ([Ca2+]i) in both cell types. Increases were fatty acid specific, dose-dependent and different for each cell type. The arachidonic acid effects on [Ca2+]i were higher in spermatids than in spermatocytes and persisted when residual extracellular Ca2+ was chelated by EGTA, indicating that the increase in [Ca2+]i originated from release of intracellular calcium stores. At the concentrations required for these increases, unsaturated fatty acids produced no significant changes in the plasma membrane potential of or non-specific permeability in spermatogenic cells. For the case of arachidonic acid, the [Ca2+]i increases were not caused by its metabolic conversion to eicosanoids or anandamide; thus we attribute this effect to the fatty acid itself. As estimated with fluorescent probes, unsaturated fatty acids did not affect the intracellular pH but were able to induce a progressive decrease in the mitochondrial membrane potential. The association of this decrease with reduced reactive oxygen species (ROS) production strongly suggests that unsaturated fatty acids induced mitochondrial uncoupling. This effect was stronger in spermatids than in spermatocytes. As a late event, arachidonic acid induced caspase 3 activation in a dose-dependent manner both in the absence and presence of external Ca2+. The concurrent but differential effects of unsaturated fatty acids on [Ca2+]i and mitochondrial functions are additional manifestations of the metabolic changes that germ cells undergo during their differentiation.

Reactive oxygen species facilitate the EDH response in arterioles by potentiating intracellular endothelial Ca2+ release

There is abundant evidence that H2O2 can act as an endothelium-derived hyperpolarizing factor in the resistance vasculature. However, whilst scavenging H2O2 can abolish endothelial dependent hyperpolarization (EDH) and the associated vascular relaxation in some arteries, EDH-dependent vasorelaxation can often be mimicked only by using relatively high concentrations of H2O2. We have examined the role of H2O2 in EDH-dependent vasodilatation by simultaneously measuring vascular diameter and changes in endothelial cell (EC) [Ca2+]i during the application of H2O2 or carbachol, which triggers EDH. Carbachol (10µM) induced dilatation of phenylephrine-preconstricted rat cremaster arterioles was largely (73%) preserved in the presence of indomethacin (3µM) and l-NAME (300µM). This residual NO- and prostacyclin-independent dilatation was reduced by 89% upon addition of apamin (0.5µM) and TRAM-34 (10µM), and by 74% when an extracellular ROS scavenging mixture of SOD and catalase (S&C; 100Uml−1 each) was present. S&C also reduced the carbachol-induced EC [Ca2+]i increase by 74%. When applied in Ca2+-free external medium, carbachol caused a transient increase in EC [Ca2+]i. This was reduced by catalase, and was enhanced when 1µM H2O2 was present in the bath. H2O2 -induced dilatation, which occurred only at concentrations ≥100µM, was reduced by a blocking antibody to TRPM2, which had no effect on carbachol-induced responses. Similarly, iberotoxin and Rp-8bromo cGMP reduced the vasodilatation induced by H2O2, but not by carbachol. Inhibiting PLC, PLA2 or CYP450 2C9 each greatly reduced the carbachol-induced increase in EC [Ca2+]i and vasodilatation, but adding 10µM H2O2 during PLA2 or CYP450 2C9 inhibition completely restored both responses. The nature of the effective ROS species was investigated by using Fe2+ chelators to block the formation of ∙OH. A cell permeant chelator was able to inhibit EC Ca2+ store release, but cell impermeant chelators reduced both the vasodilatation and EC Ca2+ influx, implying that ∙OH is required for these responses. The results indicate that rather than mediating EDH by acting directly on smooth muscle, H2O2 promotes EDH by acting within EC to enhance Ca2+ release.