Olfactory coding in the antennal lobe of the bumble bee Bombus terrestris.

Sociality is classified as one of the major transitions in evolution, with the largest number of eusocial species found in the insect order Hymenoptera, including the Apini (honey bees) and the Bombini (bumble bees). Bumble bees and honey bees not only differ in their social organization and foraging strategies, but comparative analyses of their genomes demonstrated that bumble bees have a slightly less diverse family of olfactory receptors than honey bees, suggesting that their olfactory abilities have adapted to different social and/or ecological conditions. However, unfortunately, no precise comparison of olfactory coding has been performed so far between honey bees and bumble bees, and little is known about the rules underlying olfactory coding in the bumble bee brain. In this study, we used in vivo calcium imaging to study olfactory coding of a panel of floral odorants in the antennal lobe of the bumble bee Bombus terrestris. Our results show that odorants induce reproducible neuronal activity in the bumble bee antennal lobe. Each odorant evokes a different glomerular activity pattern revealing this molecule's chemical structure, i.e. its carbon chain length and functional group. In addition, pairwise similarity among odor representations are conserved in bumble bees and honey bees. This study thus suggests that bumble bees, like honey bees, are equipped to respond to odorants according to their chemical features.

Using FluoZin-3 and fura-2 to monitor acute accumulation of free intracellular Cd2+ in a pancreatic beta cell line.

The understanding of cellular Cd2+ accumulation and toxicity is hampered by a lack of fluorescent indicators selective for intracellular free Cd2+ ([Cd2+]i). In this study, we used depolarized MIN6 mouse pancreatic beta cells as a model for evaluating [Cd2+]i detection with commercially available fluorescent probes, most of which have been traditionally used to visualize [Ca2+]i and [Zn2+]i. We trialed a panel of 12 probes including fura-2, FluoZin-3, Leadmium Green, Rhod-5N, indo-1, Fluo-5N, and others. We found that the [Zn2+]i probe FluoZin-3 and the traditional [Ca2+]i probe fura-2 responded most consistently and robustly to [Cd2+]i accumulation mediated by voltage-gated calcium channels. While selective detection of [Cd2+]i by fura-2 required the omission of Ca2+ from extracellular buffers, FluoZin-3 responded to [Cd2+]i similarly in the presence or absence of extracellular Ca2+. Furthermore, we showed that FluoZin-3 and fura-2 can be used together for simultaneous monitoring of [Ca2+]i and [Cd2+]i in the same cells. None of the other fluorophores tested were effective [Cd2+]i detectors in this model.

Dual Detection System for Simultaneous Measurement of Intracellular Fluorescent Markers and Cellular Secretion.

Glucose-stimulated insulin secretion from pancreatic ß-cells within islets of Langerhans plays a critical role in maintaining glucose homeostasis. Although this process is essential for maintaining euglycemia, the underlying intracellular mechanisms that control it are still unclear. To allow simultaneous correlation between intracellular signal transduction events and extracellular secretion, an analytical system was developed that integrates fluorescence imaging of intracellular probes with high-speed automated insulin immunoassays. As a demonstration of the system, intracellular [Ca2+] ([Ca2+]i) was measured by imaging Fura-2 fluorescence simultaneously with insulin secretion from islets exposed to elevated glucose levels. Both [Ca2+]i and insulin were oscillatory during application of 10 mM glucose with temporal and quantitative profiles similar to what has been observed elsewhere. In previous work, sinusoidal glucose levels have been used to test the entrainment of islets while monitoring either [Ca2+]i or insulin levels; using this newly developed system, we show unambiguously that oscillations of both [Ca2+]i and insulin release are entrained to oscillatory glucose levels and that the temporal correlation of these are maintained throughout the experiment. It is expected that the developed analytical system can be expanded to investigate a number of other intracellular messengers in islets or other stimulus-secretion pathways in different cells.

NAD derived second messengers: Role in spontaneous diastolic Ca(2+) transients in murine cardiac myocytes.

Strong ß-adrenergic stimulation induced spontaneous diastolic Ca(2+) transients (SCTs) in electrically paced murine cardiac myocytes [28]. To obtain further insights into the underlying mechanism, we developed a method for a simultaneous analysis, in which the free luminal Ca(2+) concentration in the sarcoplasmic reticulum (SR) ([Ca(2+)]SR) and the free cytosolic Ca(2+) concentration ([Ca(2+)]i) were measured in parallel in the same cell. Each spontaneous diastolic Ca(2+) transient was exactly mirrored by a decrease of [Ca(2+)]SR. Since antagonism of the Ca(2+) mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) was shown to block SCTs in single cardiac myocytes [28], we analyzed the effect of the novel ADP-ribosyl cyclase inhibitor SAN4825 on both cytosolic and intra-luminal Ca(2+) transients upon strong ß-adrenergic stimulation. A strong antagonist effect of SAN4825 on SCTs at low micromolar concentrations was observed. Our results suggest that the underlying mechanism of spontaneous diastolic Ca(2+) transients observed upon strong ß-adrenergic stimulation is sensitization of type 2 ryanodine receptor by the Ca(2+) releasing activity of the products of ADP-ribosyl cyclase activity.

Direct measurements of SR free Ca reveal the mechanism underlying the transient effects of RyR potentiation under physiological conditions.

AIMS: Most of the calcium that activates contraction is released from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR). It is controversial whether activators of the RyR produce a maintained increase in the amplitude of the systolic Ca transient. We therefore aimed to examine the effects of activation of the RyR in large animals under conditions designed to be as physiological as possible while simultaneously measuring SR and cytoplasmic Ca. METHODS AND RESULTS: Experiments were performed on ventricular myocytes from canine and ovine hearts. Cytoplasmic Ca was measured with fluo-3 and SR Ca with mag-fura-2. Application of caffeine resulted in a brief increase in the amplitude of the systolic Ca transient accompanied by an increase of action potential duration. These effects disappeared with a rate constant of ∼3 s(-1). Similar effects were seen in cells taken from sheep in which heart failure had been induced by rapid pacing. The decrease of Ca transient amplitude was accompanied by a decrease of SR Ca content. During this phase, the maximum (end-diastolic) SR Ca content fell while the minimum systolic increased. CONCLUSIONS: This study shows that, under conditions designed to be as physiological as possible, potentiation of RyR opening has no maintained effect on the systolic Ca transient. This result makes it unlikely that potentiation of the RyR has a maintained role in positive inotropy.

Sequential double fluorescent detections of total proteins and phosphoproteins in SDS-PAGE.

A fluorescent staining technique, using selective chelation with fluorophore and metal ion to the phosphate groups of phosphoproteins in SDS-PAGE is described. As a fluorescent dye and a metal ion, Fura 2 pentapotassium salt and Al(3+) were employed, respectively. The staining method, Fura 2 stain, has sensitivities of 16-32 ng of α-casein and ß-casein, 62 ng of ovalbumin, phosvitin, and κ-casein using an ultraviolet transilluminator. Furthermore, Fura 2 stain is able to carry out continuative double detection of total proteins and phosphoproteins on the same gel within 3.5 h. Consequently, selective phosphoprotein and total protein detections could be obtained without other poststaining. Considering the low cost, simplicity, and speed, Fura 2 staining may provide great practicalities in routine phosphoproteomics research.

Optimization of fluorescence assay of cellular manganese status for high throughput screening.

The advent of high throughput screening (HTS) technology permits identification of compounds that influence various cellular phenotypes. However, screening for small molecule chemical modifiers of neurotoxicants has been limited by the scalability of existing phenotyping assays. Furthermore, the adaptation of existing cellular assays to HTS format requires substantial modification of experimental parameters and analysis methodology to meet the necessary statistical requirements. Here we describe the successful optimization of the Cellular Fura-2 Manganese Extraction Assay (CFMEA) for HTS. By optimizing cellular density, manganese (Mn) exposure conditions, and extraction parameters, the sensitivity and dynamic range of the fura-2 Mn response was enhanced to permit detection of positive and negative modulators of cellular manganese status. Finally, we quantify and report strategies to control sources of intra- and interplate variability by batch level and plate-geometric level analysis. Our goal is to enable HTS with the CFMEA to identify novel modulators of Mn transport.

Phosphoinositide-3-kinase/akt - dependent signaling is required for maintenance of [Ca(2+)](i), I(Ca), and Ca(2+) transients in HL-1 cardiomyocytes.

The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca(2+)](i), Ca(2+) transients and membrane Ca(2+) current, I(Ca), in cultured murine HL-1 cardiomyocytes. LY294002 (1-20 µM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca(2+)](i), Ca(2+) transients and I(Ca). We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2-8 nM); ß (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca(2+)](i) regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca(2+)](i), and inhibited Ca(2+) transients. Triciribine (1-20 µM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca(2+)](i), and Ca(2+) transients and I(Ca). We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca(2+)](i) in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca(2+)](i) required for excitation-contraction coupling in cardiomyoctyes.

Real-time detection of acetylcholine release from the human endocrine pancreas.

Neurons, sensory cells and endocrine cells secrete neurotransmitters and hormones to communicate with other cells and to coordinate organ and system function. Validation that a substance is used as an extracellular signaling molecule by a given cell requires a direct demonstration of its secretion. In this protocol we describe the use of biosensor cells to detect neurotransmitter release from endocrine cells in real-time. Chinese hamster ovary cells expressing the muscarinic acetylcholine (ACh) receptor M3 were used as ACh biosensors to record ACh release from human pancreatic islets. We show how ACh biosensors loaded with the Ca(2+) indicator Fura-2 and pressed against isolated human pancreatic islets allow the detection of ACh release. The biosensor approach is simple; the Ca(2+) signal generated in the biosensor cell reflects the presence (release) of a neurotransmitter. The technique is versatile because biosensor cells expressing a variety of receptors can be used in many applications. The protocol takes ∼3 h.

Arrestins 2 and 3 differentially regulate ETA and P2Y2 receptor-mediated cell signaling and migration in arterial smooth muscle.

Overstimulation of endothelin type A (ET(A)) and nucleotide (P2Y) Gα(q)-coupled receptors in vascular smooth muscle causes vasoconstriction, hypertension, and, eventually, hypertrophy and vascular occlusion. G protein-coupled receptor kinases (GRKs) and arrestin proteins are sequentially recruited by agonist-occupied Gα(q)-coupled receptors to terminate phospholipase C signaling, preventing prolonged/inappropriate contractile signaling. However, these proteins also play roles in the regulation of several mitogen-activated protein kinase (MAPK) signaling cascades known to be essential for vascular remodeling. Here we investigated whether different arrestin isoforms regulate endothelin and nucleotide receptor MAPK signaling in rat aortic smooth muscle cells (ASMCs). When intracellular Ca(2+) levels were assessed in isolated ASMCs loaded with Ca(2+)-sensitive dyes, P2Y(2) and ET(A) receptor desensitization was attenuated by selective small-interfering (si)RNA-mediated depletion of G protein-coupled receptor kinase 2 (GRK2). Using similar siRNA techniques, knockdown of arrestin2 prevented P2Y(2) receptor desensitization and enhanced and prolonged p38 and ERK MAPK signals, while arrestin3 depletion was ineffective. Conversely, arrestin3 knockdown prevented ET(A) receptor desensitization and attenuated ET1-stimulated p38 and ERK signals, while arrestin2 depletion had no effect. Using Transwell assays to assess agonist-stimulated ASMC migration, we found that UTP-stimulated migration was markedly attenuated following arrestin2 depletion, while ET1-stimulated migration was attenuated following knockdown of either arrestin. These data highlight a differential arrestin-dependent regulation of ET(A) and P2Y(2) receptor-stimulated MAPK signaling. GRK2 and arrestin expression are essential for agonist-stimulated ASMC migration, which, as a key process in vascular remodeling, highlights the potential roles of GRK2 and arrestin proteins in the progression of vascular disease.

Multi-target novel neuroprotective compound ITH33/IQM9.21 inhibits calcium entry, calcium signals and exocytosis.

Compound ITH33/IQM9.21 (ITH/IQM) belongs to a new family of l-glutamic acid derivatives with antioxidant and neuroprotective properties on in vitro and in vivo models of stroke. Because neuronal damage after brain ischemia is tightly linked to excess Ca2+ entry and neuronal Ca2+ overload, we have investigated whether compound ITH/IQM antagonises the elevations of the cytosolic Ca2+ concentrations ([Ca2+]c) and the ensuing exocytotic responses triggered by depolarisation of bovine chromaffin cells. In fluo-4-loaded cell populations, ITH/IQM reduced the K+-evoked [Ca2+]c transients with an IC50 of 5.31 µM. At 10 µM, the compound decreased the amplitude and area of the Ca2+ transient elicited by challenging single fura-2-loaded cells with high K+, by 40% and 80%, respectively. This concentration also caused a blockade of K+-induced catecholamine release at the single-cell level (78%) and cell populations (55%). These effects are likely due to blockade of the whole-cell inward Ca2+ currents (IC50=6.52 µM). At 10 µM, ITH/IQM also inhibited the Ca2+-dependent outward K+ current, leaving untouched the voltage-dependent component of IK. The inward Na+ current was unaffected. Inhibition of depolarisation-elicited Ca2+ entry, [Ca2+]c elevation and exocytosis could contribute to the neuroprotective effects of ITH/IQM in vulnerable neurons undergoing depolarisation during brain ischemia.

Role for PKCß in enhanced endothelin-1-induced pulmonary vasoconstrictor reactivity following intermittent hypoxia.

Intermittent hypoxia (IH) resulting from sleep apnea causes both systemic and pulmonary hypertension. Enhanced endothelin-1 (ET-1)-induced vasoconstrictor reactivity is thought to play a central role in the systemic hypertensive response to IH. However, whether IH similarly increases pulmonary vasoreactivity and the signaling mechanisms involved are unknown. The objective of the present study was to test the hypothesis that IH augments ET-1-induced pulmonary vasoconstrictor reactivity through a PKCß-dependent signaling pathway. Responses to ET-1 were assessed in endothelium-disrupted, pressurized pulmonary arteries (∼150 µm inner diameter) from eucapnic-IH [(E-IH) 3 min cycles, 5% O(2)-5% CO(2)/air flush, 7 h/day; 4 wk] and sham (air-cycled) rats. Arteries were loaded with fura-2 AM to monitor vascular smooth muscle (VSM) intracellular Ca(2+) concentration ([Ca(2+)](i)). E-IH increased vasoconstrictor reactivity without altering Ca(2+) responses, suggestive of myofilament Ca(2+) sensitization. Consistent with our hypothesis, inhibitors of both PKCα/ß (myr-PKC) and PKCß (LY-333-531) selectively decreased vasoconstriction to ET-1 in arteries from E-IH rats and normalized responses between groups, whereas Rho kinase (fasudil) and PKCδ (rottlerin) inhibition were without effect. Although E-IH did not alter arterial PKCα/ß mRNA or protein expression, E-IH increased basal PKCßI/II membrane localization and caused ET-1-induced translocation of these isoforms away from the membrane fraction. We conclude that E-IH augments pulmonary vasoconstrictor reactivity to ET-1 through a novel PKCß-dependent mechanism that is independent of altered PKC expression. These findings provide new insights into signaling mechanisms that contribute to vasoconstriction in the hypertensive pulmonary circulation.

Store-operated calcium entry and non-capacitative calcium entry have distinct roles in thrombin-induced calcium signalling in human platelets.

Phosphatidylserine (PS)-exposing platelets accelerate coagulation at sites of vascular injury. PS exposure requires sustained Ca2+ signalling. Two distinct Ca2+ entry pathways amplify and sustain platelet Ca2+ signalling, but their relative importance in human platelets is not known. Here we examined the relative roles of store-operated Ca2+ entry (SOCE) and non-capacitative Ca2+ entry (NCCE) in thrombin-induced Ca2+ signalling and PS exposure by using two Ca2+ channel blockers. BTP-2 showed marked selectivity for SOCE over NCCE. LOE-908 specifically blocked NCCE under our conditions. Using these agents we found that SOCE is important at low thrombin concentrations whereas NCCE became increasingly important as thrombin concentration was increased. PS exposure was reduced by LOE-908, and only activated at thrombin concentrations that also activate NCCE. In contrast, BTP-2 had no effect on PS exposure. We suggest that SOCE amplifies and sustains Ca2+ signalling in response to low concentrations of thrombin whereas both NCCE and SOCE are important contributors to Ca2+ signalling at higher thrombin concentrations. However, despite being involved in Ca2+ signalling at high thrombin concentrations, SOCE is not important for thrombin-induced PS exposure in human platelets. This suggests that the route of Ca2+ entry is an important regulator of thrombin-induced PS exposure in platelets.

Vagus nerve stimulation inhibits the increase in Ca2+ transient and left ventricular force caused by sympathetic nerve stimulation but has no direct effects alone--epicardial Ca2+ fluorescence studies using fura-2 AM in the isolated innervated beating rabbit heart.

The effects of direct autonomic nerve stimulation on the heart may be quite different to those of perfusion with pharmacological neuromodulating agents. This study was designed to investigate the effect of autonomic nerve stimulation on intracellular calcium fluorescence using fura-2 AM in the isolated Langendorff-perfused rabbit heart preparation with intact dual autonomic innervation. The effects of autonomic nerve stimulation on cardiac force and calcium transients were more obvious during intrinsic sinus rhythm. High-frequency (15 Hz, n = 5) right vagus nerve stimulation (VS) decreased heart rate from 142.7 +/- 2.6 to 75.5 +/- 10.2 beats min(-1) and left ventricular pressure from 36.4 +/- 3.2 to 25.9 +/- 1.9 mmHg, whilst simultaneously decreasing the diastolic and systolic level of the calcium transient. Direct sympathetic nerve stimulation (7 Hz, n = 8) increased heart rate (from 144.7 +/- 10.5 to 213.2 +/- 4.9 beats min(-1)) and left ventricular pressure (from 37.5 +/- 3.6 to 43.7 +/- 2.8 mmHg), whilst simultaneously increasing the diastolic and systolic level of the calcium transient. During constant ventricular pacing, the high-frequency right vagus nerve stimulation did not have any direct effect on ventricular force or the calcium transient (n = 8), but was effective in reducing the effect of direct sympathetic nerve stimulation.

Hydroxylated PCB induces Ca2+ oscillations and alterations of membrane potential in cultured cortical cells.

Polychlorinated biphenyls (PCBs) are known as environmental pollutants that may cause adverse health effects. Although some congeners have been shown to affect brain development or function, the molecular mechanisms mediating their toxicity are not yet fully understood. Since signal transduction via intracellular Ca(2+) is crucial for neuronal development and plasticity, we investigated the effect of PCBs on Ca(2+) homeostasis and membrane potential in cultured mouse cortical cells. Acute exposure to hydroxylated PCB 106 [4(OH)-2',3,3',4',5'-pentachlorobiphenyl, OH-PCB 106, 0.1 microM] caused recurring Ca(2+) oscillations that were classified into three prototypes. Although extracellular Ca(2+) deprivation significantly reduced the oscillations, 54% of the cells still showed different patterns of oscillations or gradual increase in the intracellular Ca(2+) concentration, indicating possible involvement of multiple Ca(2+) channels in a cell-specific manner. Such a possibility was further confirmed by differential responses to several channel/receptor blockers, including nifedipine, ryanodine, xestospongine and tetrodotoxin. Although all chemicals had partial inhibition action in different subsets of neurons, nifedipine blocked the OH-PCB 106 action in the largest subpopulation of cells and with the greatest magnitude. Ryanodine also blocked the action with a similar magnitude, but in a smaller subpopulation of cells. Moreover, OH-PCB 106 induced depolarization of the plasma membrane in all the recorded cells. Taken together, our results indicate that OH-PCB 106 alters membrane potential as well as Ca(2+) dynamics in part by inducing extracellular influx and/or intracellular release of Ca(2+). These mechanisms may be responsible for their neurotoxicity.

Attenuation of maitotoxin-induced cytotoxicity in rat aortic smooth muscle cells by inhibitors of Na+/Ca2+ exchange, and calpain activation.

The highly potent marine toxin maitotoxin (MTX) evoked an increase in cytosolic Ca(2+) levels in fura-2 loaded rat aortic smooth muscle cells, which was dependent on extracellular Ca(2+). This increase was almost fully inhibited by KB-R7943, a potent selective inhibitor of the reverse mode of the Na(+)/Ca(2+) exchanger (NCX). Cell viability was assessed using ethidium bromide uptake and the alamarBlue cytotoxicity assay. In both assays MTX-induced toxicity was attenuated by KB-R7943, as well as by MDL 28170, a membrane permeable calpain inhibitor. Maitotoxin-evoked contractions of rat aortic strip preparations in vitro, which persist following washout of the toxin, were relaxed by subsequent addition of KB-R7943 or MDL 28170, either in the presence of, or following washout of MTX. These results suggest that MTX targets the Na(+)/Ca(2+) exchanger and causes it to operate in reverse mode (Na(+) efflux/Ca(2+) influx), thus leading to calpain activation, NCX cleavage, secondary Ca(2+) overload and cell death.

Effects of equinatoxin II on isolated guinea pig taenia caeci muscle contractility and intracellular Ca2+.

Equinatoxin II (EqT II) is a approximately 20kDa cytotoxic and cytolytic protein isolated from the sea anemone Actinia equina. When injected intravenously to rats the toxin has been reported to produce a rapid cardiorespiratory arrest. In the present study, we show that EqT II increases the tension of spontaneous contractions and induces long-lasting contracture of guinea pig taenia caeci muscle. In taenia caeci, dissociated smooth muscle cells, microspectrofluorometric measurements, using the Ca(2+) indicator fura-2/AM, revealed that the toxin causes a marked increase in intracellular calcium, provided Ca(2+) is present in the external medium. The increase in intracellular Ca(2+) by EqT II was not blocked or diminished by the calcium channel blocker verapamil. Furthermore, pre-treatment of smooth muscle cells with Ca(2+)-ATPase inhibitor thapsigargin, or exposure of the cells to a high K(+) (75 mM) medium did not prevent EqT II-induced intracellular Ca(2+) increases. Replacement of external sodium by sucrose markedly modified the time course of Ca(2+) signals suggesting the involvement of the Na(+)/Ca(2+) exchanger in EqT II action. Our results strongly suggest that EqT II-induced increase in intracellular Ca(2+) and muscle tension are both dependent on the ability of EqT II to insert into the membrane and form pores allowing Ca(2+) influx into the cells. To our knowledge this is the first report showing that EqT II causes contraction and contracture of taenia caeci muscles and increases intracellular Ca(2+) in smooth muscle cells.

Role of reverse mode Na+/Ca2+ exchanger in the cardioprotection of metabolic inhibition preconditioning in rat ventricular myocytes.

This study determined the role of the reverse mode Na(+)/Ca(2+) exchanger (NCX) in cardioprotection of metabolic inhibition preconditioning in isolated ventricular myocyctes. Activity of the reverse mode NCX was assessed by changes of [Ca(2+)](i) upon withdrawal of extracellular Na(+). [Ca(2+)](i) was measured by spectrofluorometry, using Fura-2 as Ca(2+) indicator. The amplitude of contraction and exclusion of trypan blue by myocytes served as indices of contractile function and viability, respectively. Firstly, NCX activity significantly decreased during simulated reperfusion after severe metabolic inhibition (index ischaemia) in myocytes subjected to metabolic inhibition preconditioning. This inhibitory effect on NCX activity correlated with the enhancing effect of metabolic inhibition preconditioning on cell viability following ischaemic insult. Treatment myocytes with E4031, an activator of reverse mode NCX, during index ischaemia and reperfusion attenuated the enhancing effects of metabolic inhibition preconditioning on cell contraction and viability. Secondly, NCX activity was significantly higher at the end of metabolic inhibition preconditioning. More importantly, E4031 pretreatment mimicked the beneficial effects of metabolic inhibition preconditioning in myocytes and ischaemic preconditioning in the isolated perfused heart, respectively, and these effects were abolished by KB-R7943, an inhibitor of reverse mode NCX. The results indicate that increased reverse mode NCX activity during preconditioning triggered cardioprotection, and reduced reverse mode NCX activity during reperfusion after index ischaemia conferred cardioprotection.

A remarkable increase in the pHi sensitivity of voltage-dependent calcium channels occurs in human sperm incubated in capacitating conditions.

Human sperm are endowed with voltage-dependent calcium channels (VDCC) that produce increases in [Ca2+]i in response to depolarization with KCl. These channels are stimulated during "capacitation", a complex biochemical process, accompanied by a slight pHi alkalization, that sperm must accomplish to acquire the ability to fertilize the egg. The stimulation can be explained in part by the fact that in non-capacitated sperm, calcium influx through VDCC is stimulated by pHi alkalization in the range of pHi observed during capacitation. In this work, we explored the effect of pHi on VDCC in capacitated sperm loaded with fura ff. Strikingly, the pHi sensitivity of VDCC increased approximately 7-fold when sperm was capacitated, as compared with non-capacitated sperm. This finding suggests that the pHi sensitivity of VDCC can be modulated during capacitation so that a combined effect of pHi alkalization and biochemical regulation enhances calcium influx through these channels.