Increase In Intracellular Ca2 Research Articles

Sperm gamma-aminobutyric acid type A receptor delta subunit (GABRD) and its interaction with purinergic P2X2 receptors in progesterone-induced acrosome reaction and male fertility.

The mechanism underlying the non-genomic action of progesterone in sperm functions and related Ca2+ mobilisation remains elusive. Herein we report the expression of gamma-aminobutyric acid type A receptor delta subunit (GABRD) in human and rodent sperm and its involvement in mediating the progesterone-induced acrosome reaction. GABRD was localised in the sperm head/neck region. A δ(392-422)-specific inhibitory peptide against GABRD blocked the progesterone-induced acrosome reaction and the associated increase in intracellular Ca2+. Similarly, an inhibitory effect against both progesterone-induced Ca2+ influx and the acrosome reaction was observed with a P2X2 receptor antagonist. The lack of synergism between the GABRD and P2X2 inhibitors suggests that these two receptors are playing a role in the same pathway. Furthermore, a co-immunoprecipitation experiment demonstrated that GABRD could undergo protein-protein interactions with the Ca2+-conducting P2X2 receptor. This interaction between the receptors could be reduced following progesterone (10μM) inducement. Significantly reduced GABRD expression was observed in spermatozoa from infertile patients with reduced acrosome reaction capacity, suggesting that normal expression of GABRD is critical for the sperm acrosome reaction and thus male fertility. The results of the present study indicate that GABRD represents a novel progesterone receptor or modulator in spermatozoa that is responsible for the progesterone-induced Ca2+ influx required for the acrosome reaction through its interaction with the P2X2 receptor.

Azadirachtin induced apoptosis in the prothoracic gland in Bombyx mori and a pronounced Ca2+ release effect in Sf9 cells.

Azadirachtin is a bio-rational insecticide used as an antifeedant and growth disruption agent against many insect species. However, recent studies have shown that there is a potential risk of this compound harming some beneficial insects. In such cases its application does not normally lead to death, but it may result in altered developmental regulation. Therefore, it is essential to obtain toxicological data to understand the mechanism of such sub-lethal effects, especially where they relate to important beneficial insects. Here, we found that azadirachtin could regulate growth and cocooning in silkworms, which may be associated with induced apoptotic effect on the prothoracic gland. However, azadirachtin treatment could not induce apoptosis in the prothoracic gland in vitro, in contrast to the effect of 20-hydroxyecdysone in vitro, which suggesting that the apoptosis might not be direct effect of azadirachtin. Then we examined the activity of Ca2+-Mg2+-ATPase and found that azadirachtin could trigger a significant increase in intracellular Ca2+ release in the Sf9 cell line, which suggested that the calcium signaling pathway might be involved in the process of apoptosis in prothoracic gland and growth regulation in vivo silkworms. Although more evidence is needed to fully understand the mechanism of azadirachtin in perturbing the growth of silkworms, this study provides some toxicological information and highlights the potential risks of azadirachtin in relation to silkworms.

Drebrin Regulation of Calcium Signaling in Immune Cells.

Store-operated Ca2+ channels are plasma membrane channels that are activated by depletion of intracellular Ca2+ stores, resulting in an increase in intracellular Ca2+; however, little is known about their regulation. Our work has shown that the immunosuppressant compound BTP2, which blocks Ca2+ influx into cells, interacts with the actin-reorganizing protein, drebrin. Here we review the role of drebrin in the regulation of calcium signaling, with a focus on immune cells.

Effect of Docosahexaenoic Acid on Voltage-Independent Ca2+ Entry Pathways in Cultured Vascular Smooth Muscle Cells Stimulated with 5-Hydroxytryptamine.

We previously reported that docosahexaenoic acid (DHA) inhibits an increase in intracellular Ca2+ concentration ([Ca2+]i) in cultured rat vascular smooth muscle cells (VSMCs) through a mechanism involving mainly voltage-dependent Ca2+ channels; however, the effect of DHA on voltage-independent pathways, such as store-operated and receptor-operated Ca2+ entry, and Ca2+ entry through Na+/Ca2+ exchanger (NCX), has not been clarified. In the present study, we investigated the effect of DHA treatment on the expression of transient receptor potential canonical (TRPC) channels, capacitative Ca2+ entry, and Ca2+ entry through NCX in rat cultured VSMCs stimulated with 5-hydroxytryptamine (5-HT). RT-PCR analysis detected TRPC1, TRPC4, and TRPC6 mRNA in cultured VSMCs. DHA treatment for 2 d slightly but significantly decreased TRPC1, but not TRPC4 and TRPC6, mRNA expression. Sarpogrelate, a selective serotonin 5-HT2A receptor inhibitor, completely inhibited the 5-HT-induced increase in [Ca2+]i in cultured VSMCs. Ca2+ influx by adding extracellular Ca2+ (1.3 mM) to the Ca2+-free condition in the presence of 5-HT was partially but significantly inhibited by sarpogrelate. DHA treatment for 2 d had no effect on Ca2+ influx when extracellular Ca2+ was added to the Ca2+-free condition in the presence of either 5-HT alone or 5-HT with sarpogrelate. KB-R7943, a selective inhibitor of reverse mode NCX, significantly suppressed the 5-HT-induced increase of [Ca2+]i. Furthermore, DHA treatment for 2 d significantly decreased NCX1 mRNA expression. These results suggest that DHA seems to have little effect on capacitative Ca2+ entry. Through decreasing NCX1 expression, DHA may suppress the 5-HT-induced increase in [Ca2+]i.

ATP2B1 gene Silencing Increases Insulin Sensitivity through Facilitating Akt Activation via the Ca2+/calmodulin Signaling Pathway and Ca2+-associated eNOS Activation in Endothelial Cells.

Endothelial cell insulin resistance may be partially responsible for the higher risk of atherosclerosis and cardiovascular disease in populations with insulin resistance and type 2 diabetes mellitus (T2DM). A genome-wide association study revealed a significant association between the ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) gene and T2DM in two community-based cohorts from the Korea Association Resource Project. However, little is known about the implication of the ATP2B1 gene on T2DM. In the present study, we investigated the role of the ATP2B1 gene in endothelial cell insulin sensitivity. ATP2B1 gene silencing resulted in enhanced intracellular calcium concentrations and increased insulin-induced Akt activation compared to that in the negative siRNA-transfected HUVECs (Human Umbilical Vein Endothelial Cells). The elevated insulin sensitivity mediated by ATP2B1 gene silencing was Ca2+/calmodulin-dependent, as verified by administration of the calcium chelator BAPTA-AM or the calmodulin-specific antagonist W7. Moreover, higher levels of phosphorylation of eNOS (Ser1177) were observed in ATP2B1-silenced HUVECs. In addition to BAPTA-AM and W7, L-NAME, an eNOS antagonist, abolished insulin-induced Akt phosphorylation at Ser473 in both si-Neg and si-ATP2B1-transfected endothelial cells. These results indicate that the enhanced insulin sensitivity in ATP2B1-silenced endothelial cells is alternatively dependent on an increase in intracellular Ca2+ and the subsequent activation of the Ca2+/calmodulin/eNOS/Akt signaling pathway. In summary, ATP2B1 gene silencing increased insulin sensitivity in endothelial cells by directly modulating the Ca2+/calmodulin signaling pathway and via the Ca2+/calmodulin/eNOS/Akt signaling pathway alternatively.

5,6-δ-DHTL, a stable metabolite of arachidonic acid, is a potential EDHF that mediates microvascular dilation

ObjectiveProminent among the endothelium-derived hyperpolarizing factors (EDHFs) are the Cytochrome P450 (CYP) epoxygenase-derived arachidonic acid metabolites—the epoxyeicosatrienoic acids (EETs), that are known as vasodilators in the microcirculation. Among the EET isomers, 5,6-EET undergoes rapid lactonization in aqueous solution to the more stable 5,6-δ DHTL (5,6-dihydroxytrienoic lactone) isomer. It is unclear whether this metabolic transformation maintains its vasodilator potential and what is the mechanism of action. Thus, the aim of this study was to investigate the capacity of the lactone isomer, 5,6- δ DHTL, to induce dilation of arterioles and explore the endothelial Ca2+ response mechanism. Approach and resultsIn isolated human microvessels 5,6- δ DHTL induced a dose dependent vasodilation, that was inhibited by mechanical denudation of the endothelial layer. This 5,6- δ DHTL -dependent dilation was partially reduced in the presence of L-NAME (NOS inhibitor) or the NO-scavenger, cPTIO (by 19.7%, which was not statistically significantly). In human endothelial cells, 5,6- δ DHTL induced an increase in intracellular Ca2+([Ca2+]i) in a dose dependent manner. This increase in [Ca2+]i was similar to that induced by the 5,6-EET isomer, and significantly higher than observed by administering the hydrolytic dihydroxy isomer, 5,6-DHET. Further experiments aimed to investigate the mechanism of action revealed, that the 5,6-δ DHTL-mediated ([Ca2+]i elevation was reduced by IP3 and ryanodine antagonists, but not by antagonists to the TRPV4 membrane channel. Similar to their effect on the dilation response in the arteries, NO inhibitors reduced the 5,6-δ DHTL-mediated ([Ca2+]i elevation by 20%. Subsequent 5,6-δ DHTL -dependent K+ ion efflux from endothelial cells, was abolished by the inhibition of small and intermediate conductance KCa. ConclusionsThe present study shows that 5,6-δ DHTL is a potential EDHF, that dilates microvessels through a mechanism that involves endothelial dependent Ca2+ entry, requiring endothelial hyperpolarization. These results suggest the existence of additional lactone-containing metabolites that can be derived from the PUFA metabolism and which may function as novel EDHFs.

Epac activation inhibits IL-6-induced cardiac myocyte dysfunction

Pro-inflammatory cytokines are released in septic shock and impair cardiac function via the Jak-STAT pathway. It is well known that sympathetic and thus catecholamine signaling is activated thereafter to compensate for cardiac dysfunction. The mechanism of such compensation by catecholamine signaling has been traditionally understood to be cyclic AMP-dependent protein kinase (PKA)-mediated enforcement of cardiac contractility. We hypothesized that the exchange protein activated by cAMP (Epac), a newly identified target of cAMP signaling that functions independently of PKA, also plays a key role in this mechanism. In cultured cardiac myocytes, activation of Epac attenuated the inhibitory effect of interleukin-6 on the increase of intracellular Ca2+ concentration and contractility in response to isoproterenol, most likely through inhibition of the Jak-STAT pathway via SOCS3, with subsequent changes in inducible nitric oxide synthase expression. These findings suggest a new role of catecholamine signaling in compensating for cardiac dysfunction in heart failure. Epac and its downstream pathway may be a novel target for treating cardiac dysfunction in endotoxemia.

Optochemokine Tandem for Light-Control of Intracellular Ca2.

An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca2+-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca2+ by tandem endosomes into the cytosol via CatCh was visualized using the Ca2+-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca2+ in response to light.

Protease induced plasticity: matrix metalloproteinase-1 promotes neurostructural changes through activation of protease activated receptor 1.

Matrix metalloproteinases (MMPs) are a family of secreted endopeptidases expressed by neurons and glia. Regulated MMP activity contributes to physiological synaptic plasticity, while dysregulated activity can stimulate injury. Disentangling the role individual MMPs play in synaptic plasticity is difficult due to overlapping structure and function as well as cell-type specific expression. Here, we develop a novel system to investigate the selective overexpression of a single MMP driven by GFAP expressing cells in vivo. We show that MMP-1 induces cellular and behavioral phenotypes consistent with enhanced signaling through the G-protein coupled protease activated receptor 1 (PAR1). Application of exogenous MMP-1, in vitro, stimulates PAR1 dependent increases in intracellular Ca2+ concentration and dendritic arborization. Overexpression of MMP-1, in vivo, increases dendritic complexity and induces biochemical and behavioral endpoints consistent with increased GPCR signaling. These data are exciting because we demonstrate that an astrocyte-derived protease can influence neuronal plasticity through an extracellular matrix independent mechanism.

Targeting Stim and Orai Proteins as an Alternative Approach in Anticancer Therapy.

An increase in intracellular Ca2+ concentration plays a key role in the establishment of many cancer hallmarks, including aberrant proliferation, migration, invasion, resistance to apoptosis and angiogenesis. The dysregulation of Ca2+ entry is one of the most subtle mechanisms by which cancer cells overwhelm their normal counterparts and gain the adaptive advantages that result in tumour growth, vascularisation and dissemination throughout the organism. Both constitutive and agonist-induced Ca2+ influx may be mediated by store-dependent as well as store-independent Ca2+ entry routes. A growing body of evidences have shown that different isoforms of Stromal Interaction Molecules (Stim1) and Orai proteins, i.e. Stim1, Stim2, Orai1 and Orai3, underlie both pathways in cancer cells. The alteration in either the expression or the activity of Stim and Orai proteins has been linked to the onset and maintenance of tumour phenotype in many solid malignancies, including prostate, breast, kidney, esophageal, skin, brain, colorectal, lung and liver cancers. Herein, we survey the existing data in support of Stim and Orai involvement in tumourigenesis and provide the rationale to target them in cancer patients. Besides, we summarize the most recent advances in the identification of novel pharmacological tools that could be successfully used in clinical therapy.

N-Acetyl-L-cysteine inhibits sulfur mustard-induced and TRPA1-dependent calcium influx.

Transient receptor potential family channels (TRPs) have been identified as relevant targets in many pharmacological as well as toxicological studies. TRP channels are ubiquitously expressed in different tissues and act among others as sensors for different external stimuli, such as mechanical stress or noxious impacts. Recent studies suggest that one member of this family, the transient receptor potential ankyrin 1 cation channel (TRPA1), is involved in pain, itch, and various diseases, suggesting TRPA1 as a potential therapeutic target. As a nociceptor, TRPA1 is mainly activated by noxious or electrophilic compounds, including alkylating substances. Previous studies already revealed an impact of 2-chloroethyl-ethyl sulfide on the ion channel TRPA1. In this study, we demonstrate that sulfur mustard (bis-(2-chloroethyl) sulfide, SM) activates the human TRPA1 (hTRPA1) in a dose-dependent manner measured by the increase in intracellular Ca2+ concentration ([Ca2+]i). Besides that, SM-induced toxicity was attenuated by antioxidants. However, very little is known about the underlying mechanisms. Here, we demonstrate that N-acetyl-L-cysteine (NAC) prevents SM-induced hTRPA1-activation. HEK293-A1-E cells, overexpressing hTRPA1, show a distinct increase in [Ca2+]i immediately after SM exposure, whereas this increase is reduced in cells pretreated with NAC in a dose-dependent manner. Interestingly, glutathione, although being highly related to NAC, did not show an effect on hTRPA1 channel activity. Taken together, our results provide evidence that SM-dependent activation of hTRPA1 can be diminished by NAC treatment, suggesting a direct interaction of NAC and the hTRPA1 cation channel. Our previous studies already showed a correlation of hTRPA1-activation with cell damage after exposure to alkylating agents. Therefore, NAC might be a feasible approach mitigating hTRPA1-related dysregulations after exposure to SM.

Specific combinations of ion channel inhibitors reduce excessive Ca2+ influx as a consequence of oxidative stress and increase neuronal and glial cell viability in vitro

Combinations of Ca2+ channel inhibitors have been proposed as an effective means to prevent excess Ca2+ flux and death of neurons and glia following neurotrauma in vivo. However, it is not yet known if beneficial outcomes such as improved viability have been due to direct effects on intracellular Ca2+ concentrations. Here, the effects of combinations of Lomerizine (Lom), 2,3-dioxo-7-(1H-imidazol-1-yl)6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl]acetic acid monohydrate (YM872), 3,5-dimethyl-1-adamantanamine (memantine (Mem)) and/or adenosine 5′-triphosphate periodate oxidized sodium salt (oxATP) to block voltage-gated Ca2+ channels, Ca2+ permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, NMDA receptors and purinergic P2X7 receptors (P2X7R) respectively, on Ca2+ concentration and viability of rat primary mixed cortical (MC) cultures exposed to hydrogen peroxide (H2O2) insult, were assessed. The contribution of ryanodine-sensitive intracellular stores to intracellular Ca2+ concentration was also assessed. Live cell calcium imaging revealed that a 30min H2O2 insult induced a slow increase in intracellular Ca2+, in part from intracellular sources, associated with loss of cell viability by 6h. Most combinations of inhibitors that included oxATP significantly decreased Ca2+ influx and increased cell viability when administered simultaneously with H2O2. However, reductions in intracellular Ca2+ concentration were not always linked to improved cell viability. Examination of the density of specific cell subpopulations demonstrated that most combinations of inhibitors that included oxATP preserved NG2+ non-oligodendroglial cells, but preservation of astrocytes and neurons required additional inhibitors. Olig2+ oligodendroglia and ED-1+ activated microglia/macrophages were not preserved by any of the inhibitor combinations. These data indicate that following H2O2 insult, limiting intracellular Ca2+ entry via P2X7R is generally associated with increased cell viability. Protection of NG2+ non-oligodendroglial cells by Ca2+ channel inhibitor combinations may contribute to observed beneficial outcomes in vivo.

Development of Ca2+-release mechanisms during oocyte maturation of the starfish Asterina pectinifera.

An important step for successful fertilization and further development is the increase in intracellular Ca2+ in the activated oocyte. It has been known that starfish oocytes become increasingly sensitive to inositol 1,4,5-trisphosphate (IP3) during meiotic maturation to exhibit highly efficient IP3-induced Ca2+ release (IICR) by the time of germinal vesicle breakdown (GVBD). However, we noted that the peak level of intracellular Ca2+ increase after insemination is already high in the maturing oocytes before GVBD. Using maturing oocytes before GVBD, we investigated Ca2+ release mechanisms other than IICR. We report here that Ca2+-release mechanisms dependent on nicotinic acid adenine dinucleotide phosphate (NAADP) and nicotinamide adenine dinucleotide (NADP), the precursor of NAADP, became functional prior to the development of IICR mechanisms. As with IP3, but unlike NAADP, the Ca2+ stores responsive to NADP are sensitized during the meiotic maturation induced by 1-methyladenine (1-MA). This suggests that the process may represent a physiological response to the maturation hormone. NADP-dependent Ca2+ release in immature oocytes, however, did not induce oocyte maturation by itself, but was enhanced by the conditions mimicking the increases of intracellular Ca2+ and pH that take place in the maturing oocytes of starfish.

Extracellular ATP Induces Calcium Signaling in Odontoblasts

Odontoblasts form dentin at the outermost surface of tooth pulp. An increasing level of evidence in recent years, along with their locational advantage, implicates odontoblasts as a secondary role as sensory or immune cells. Extracellular adenosine triphosphate (ATP) is a well-characterized signaling molecule in the neuronal and immune systems, and its potential involvement in interodontoblast communications was recently demonstrated. In an effort to elaborate the ATP-mediated signaling pathway in odontoblasts, the current study performed single-cell reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescent detection to investigate the expression of ATP receptors related to calcium signal in odontoblasts from incisal teeth of 8- to 10-wk-old rats, and demonstrated an in vitro response to ATP application via calcium imaging experiments. While whole tissue RT-PCR analysis detected P2Y2, P2Y4, and all 7 subtypes (P2X1 to P2X7) in tooth pulp, single-cell RT-PCR analysis of acutely isolated rat odontoblasts revealed P2Y2, P2Y4, P2X2, P2X4, P2X6, and P2X7 expression in only a subset (23% to 47%) of cells tested, with no evidence for P2X1, P2X3, and P2X5 expression. An increase of intracellular Ca2+ concentration in response to 100μM ATP, which was repeated after pretreatment of thapsigargin or under the Ca2+-free condition, suggested function of both ionotropic and metabotropic ATP receptors in odontoblasts. The enhancement of ATP-induced calcium response by ivermectin and inhibition by 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) confirmed a functional P2X4 subtype in odontoblasts. Positive calcium response to 2′,3′-O-(benzoyl-4-benzoyl)-ATP (BzATP) and negative response to α,β-methylene ATP suggested P2X2, P2X4, and P2X7 as functional subunits in rat odontoblasts. Single-cell RT-PCR analysis of the cells with confirmed calcium response and immunofluorescent detection further corroborated the expression of P2X4 and P2X7 in odontoblasts. Overall, this study demonstrated heterogeneous expression of calcium-related ATP receptor subtypes in subsets of individual odontoblasts, suggesting extracellular ATP as a potential signal mediator for odontoblastic functions.

Fast interaction between AMPA and NMDA receptors by intracellular calcium

Suppression of NMDA receptor (NMDAR)-mediated currents by intracellular Ca2+ has been described as a negative feedback loop in NMDAR modulation. In the time scale of tenths of milliseconds the depth of the suppression does not depend on the Ca2+ source. It may be caused by Ca2+ influx through voltage-gated calcium channels, NMDAR channels or release from intracellular stores. However, NMDARs are often co-expressed in synapses with Ca2+-permeable AMPA receptors (AMPARs). Due to significant differences in activation kinetics between these two types of glutamate receptors (GluRs), Ca2+ entry through AMPARs precedes full activation of NMDARs, and therefore, might have an impact on the amplitude of NMDAR-mediated currents. The study of Ca2+-mediated crosstalk between AMPAR and NMDAR in native synapses is challenging due to high NMDAR Ca2+ permeability. Therefore, recombinant Ca2+-permeable AMPAR and Ca2+-impermeable NMDAR mutant channels were co-expressed in HEK 293 cells to examine their interaction. An AMPAR-mediated increase in intracellular Ca2+ concentration ([Ca2+]i) reversibly reduced the size of NMDAR-mediated whole-cell currents. The time course of the NMDAR channel inactivation and recovery from inactivation followed the time course of the [Ca2+]i transient. When brief (1ms) pulses of glutamate were applied to outside-out patches, the degree of NMDAR inactivation increased with the increase in charge carried by the currents through co-activated AMPARs. However, AMPAR-mediated NMDAR inactivation was abolished in the presence of intracellular fast Ca2+ buffer BAPTA or in Ca2+-free extracellular solution. We conclude that Ca2+ entering through AMPARs inactivates co-localized NMDARs in the time range of excitatory postsynaptic currents.

Olfactory signaling components and olfactory receptors are expressed in tubule cells of the human kidney

Cells of the renal tubule system are in direct contact with compounds dissolved in the urine, such as short chain fatty acids (SCFA). Murine OR78, a member of the olfactory receptor (OR) family, is involved in SCFA-related regulation of renal blood pressure in mice. It is still unclear whether OR signaling has an impact on human renal physiology. In our study, we showed that OR51E1 and OR11H7, both of which can be activated by the SCFA isovaleric acid, are expressed in the HK-2 human proximal tubule cell line. We observed a transient increase in intracellular Ca2+ when isovaleric acid and 4-methylvaleric acid were added to HK-2 cells. The isovaleric acid-induced response was dependent on extracellular Ca2+ and adenylyl cyclase (AC) activation. Furthermore, we demonstrated that the canonical olfactory signaling components Gαolf and ACIII are co-localized with OR51E1. The number of cells responding to isovaleric acid correlated with the presence of primary cilia on HK-2 cells. OR51E1 protein expression was confirmed in the tubule system of human kidney tissue. Our study is the first to show the expression of ORs and olfactory signaling components in human kidney cells. Additionally, we discuss ORs as potential modulators of the renal physiology.

Transient exposure to calcium ionophore enables in vitro fertilization in sterile mouse models.

Mammalian sperm acquire fertilizing capacity in the female tract in a process called capacitation. At the molecular level, capacitation requires protein kinase A activation, changes in membrane potential and an increase in intracellular calcium. Inhibition of these pathways results in loss of fertilizing ability in vivo and in vitro. We demonstrated that transient incubation of mouse sperm with Ca2+ ionophore accelerated capacitation and rescued fertilizing capacity in sperm with inactivated PKA function. We now show that a pulse of Ca2+ ionophore induces fertilizing capacity in sperm from infertile CatSper1 (Ca2+ channel), Adcy10 (soluble adenylyl cyclase) and Slo3 (K+ channel) KO mice. In contrast, sperm from infertile mice lacking the Ca2+ efflux pump PMACA4 were not rescued. These results indicate that a transient increase in intracellular Ca2+ can overcome genetic infertility in mice and suggest this approach may prove adaptable to rescue sperm function in certain cases of human male infertility.

Arachidonic acid-evoked Ca2 + signals promote nitric oxide release and proliferation in human endothelial colony forming cells

Arachidonic acid (AA) stimulates endothelial cell (EC) proliferation through an increase in intracellular Ca2+ concentration ([Ca2+]i), that, in turn, promotes nitric oxide (NO) release. AA-evoked Ca2+ signals are mainly mediated by Transient Receptor Potential Vanilloid 4 (TRPV4) channels. Circulating endothelial colony forming cells (ECFCs) represent the only established precursors of ECs. In the present study, we, therefore, sought to elucidate whether AA promotes human ECFC (hECFC) proliferation through an increase in [Ca2+]i and the following activation of the endothelial NO synthase (eNOS). AA induced a dose-dependent [Ca2+]i raise that was mimicked by its non-metabolizable analogue eicosatetraynoic acid. AA-evoked Ca2+ signals required both intracellular Ca2+ release and external Ca2+ inflow. AA-induced Ca2+ release was mediated by inositol-1,4,5-trisphosphate receptors from the endoplasmic reticulum and by two pore channel 1 from the acidic stores of the endolysosomal system. AA-evoked Ca2+ entry was, in turn, mediated by TRPV4, while it did not involve store-operated Ca2+ entry. Moreover, AA caused an increase in NO levels which was blocked by preventing the concomitant increase in [Ca2+]i and by inhibiting eNOS activity with NG-nitro-l-arginine methyl ester (l-NAME). Finally, AA per se did not stimulate hECFC growth, but potentiated growth factors-induced hECFC proliferation in a Ca2+- and NO-dependent manner. Therefore, AA-evoked Ca2+ signals emerge as an additional target to prevent cancer vascularisation, which may be sustained by ECFC recruitment.

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.

Inhibitory action of linoleamide and oleamide toward sarco/endoplasmic reticulum Ca2+-ATPase

BackgroundSERCA maintains intracellular Ca2+ homeostasis by sequestering cytosolic Ca2+ into SR/ER stores. Two primary fatty acid amides (PFAAs), oleamide (18:19-cis) and linoleamide (18:29,12-cis), induce an increase in intracellular Ca2+ levels, which might be caused by their inhibition of SERCA. MethodsThree major SERCA isoforms, rSERCA1a, hSERCA2b, and hSERCA3a, were individually overexpressed in COS-1 cells, and the inhibitory action of PFAAs on Ca2+-ATPase activity of SERCA was examined. ResultsThe Ca2+-ATPase activity of each SERCA was inhibited in a concentration-dependent manner strongly by linoleamide (IC50 15–53μM) and partially by oleamide (IC50 8.3–34μM). Inhibition by other PFAAs, such as stearamide (18:0) and elaidamide (18:19-trans), was hardly or slightly observed. With increasing dose, linoleamide decreased the apparent affinity for Ca2+ and the apparent maximum velocity of Ca2+-ATPase activity of all SERCAs tested. Oleamide also lowered these values for hSERCA3a. Meanwhile, oleamide uniquely reduced the apparent Ca2+ affinity of rSERCA1a and hSERCA2b: the reduction was considerably attenuated above certain concentrations of oleamide. The dissociation constants for SERCA interaction varied from 6 to 45μM in linoleamide and from 1.6 to 55μM in oleamide depending on the isoform. ConclusionsLinoleamide and oleamide inhibit SERCA activity in the micromolar concentration range, and in a different manner. Both amides mainly suppress SERCA activity by lowering the Ca2+ affinity of the enzyme. General significanceOur findings imply a novel role of these PFAAs as modulators of intracellular Ca2+ homeostasis via regulation of SERCA activity.