Polyacrylic-Coated Solid Nanoparticles Increase the Aquaporin Permeability to Hydrogen Peroxide.

Aquaporins (AQPs) allow the diffusion of hydrogen peroxide (H2O2) and act as ROS scavenging systems, which are important for controlling the redox state of cells. Recently, cerium oxide nanoparticles were found to increase the water and H2O2 permeability by modulating AQPs. To further analyze the action of nanoparticles (NPs) on AQP, we examined the effect of the NPs presenting different core compositions (CeO2, Gd2O3, Fe3O4, and TiO2), hydrodynamic sizes, and surface functionalization. The NPs produced an increase in H2O and H2O2 permeability as a general trend. The hydrodynamic sizes of the NPs in the range of 22-100 nm did not produce any significant effect. The chemical nature of the NPs' core did not modify the effect and its intensity. On the other hand, the NPs' functionalized surface plays a major role in influencing both water and H2O2 permeability. The results suggest that NPs can play a significant role in controlling oxidative stress in cells and might represent an innovative approach in the treatment of a number of pathologies associated with an increased oxidative status.

Cerium Oxide Nanoparticles Regulate Oxidative Stress in HeLa Cells by Increasing the Aquaporin-Mediated Hydrogen Peroxide Permeability.

Some aquaporins (AQPs) allow the diffusion of hydrogen peroxide (H2O2), the most abundant ROS, through the cell membranes. Therefore, the possibility of regulating the AQP-mediated permeability to H2O2, and thus ROS scavenging, appears particularly important for controlling the redox state of cells in physiological and pathophysiological conditions. Several compounds have been screened and characterized for this purpose. This study aimed to analyze the effect of cerium oxide nanoparticles (CNPs) presenting antioxidant activity on AQP functioning. HeLa cells express AQP3, 6, 8, and 11, able to facilitate H2O2. AQP3, 6, and 8 are expressed in the plasma membrane and intracellularly, while AQP11 resides only in intracellular structures. CNPs but not cerium ions treatment significantly increased the water and H2O2 permeability by interacting with AQP3, 6, and especially with AQP8. CNPs increased considerably the AQP-mediated water diffusion in cells with oxidative stress. Functional experiments with silenced HeLa cells revealed that CNPs increased the H2O2 diffusion mainly by modulating the AQP8 permeability but also the AQP3 and AQP6, even if to a lesser extent. Current findings suggest that CNPs represent a promising pharmaceutical agent that might potentially be used in numerous pathologies involving oxidative stress as tumors and neurodegenerative diseases.

Nicotinic acid adenine dinucleotide phosphate activates two-pore channel TPC1 to mediate lysosomal Ca2+ release in endothelial colony-forming cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca2+ -releasing messenger that increases the intracellular Ca2+ concentration by mobilizing the lysosomal Ca2+ store through two-pore channels 1 (TPC1) and 2 (TPC2). NAADP-induced lysosomal Ca2+ release regulates multiple endothelial functions, including nitric oxide release and proliferation. A sizeable acidic Ca2+ pool endowed with TPC1 is also present in human endothelial colony-forming cells (ECFCs), which represent the only known truly endothelial precursors. Herein, we sought to explore the role of the lysosomal Ca2+ store and TPC1 in circulating ECFCs by harnessing Ca2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe ß-naphthylamide, and nigericin, which dissipates the proton gradient which drives Ca2+ sequestration by acidic organelles, caused endogenous Ca2+ release in the presence of a replete inositol-1,4,5-trisphosphate (InsP3 )-sensitive endoplasmic reticulum (ER) Ca2+ pool. Likewise, the amount of ER releasable Ca2+ was reduced by disrupting lysosomal Ca2+ content. Liposomal delivery of NAADP induced a transient Ca2+ signal that was abolished by disrupting the lysosomal Ca2+ store and by pharmacological and genetic blockade of TPC1. Pharmacological manipulation revealed that NAADP-induced Ca2+ release also required ER-embedded InsP3 receptors. Finally, NAADP-induced lysosomal Ca2+ release was found to trigger vascular endothelial growth factor-induced intracellular Ca2+ oscillations and proliferation, while it did not contribute to adenosine-5'-trisphosphate-induced Ca2+ signaling. These findings demonstrated that NAADP-induced TPC1-mediated Ca2+ release can selectively be recruited to induce the Ca2+ response to specific cues in circulating ECFCs.

Group 1 metabotropic glutamate receptors trigger glutamate-induced intracellular Ca2+ signals and nitric oxide release in human brain microvascular endothelial cells.

Neurovascular coupling (NVC) is the mechanism whereby an increase in neuronal activity causes an increase in local cerebral blood flow (CBF) to ensure local supply of oxygen and nutrients to the activated areas. The excitatory neurotransmitter glutamate gates post-synaptic N-methyl-D-aspartate receptors to mediate extracellular Ca2+ entry and stimulate neuronal nitric oxide (NO) synthase to release NO, thereby triggering NVC. Recent work suggested that endothelial Ca2+ signals could underpin NVC by recruiting the endothelial NO synthase. For instance, acetylcholine induced intracellular Ca2+ signals followed by NO release by activating muscarinic 5 receptors in hCMEC/D3 cells, a widely employed model of human brain microvascular endothelial cells. Herein, we sought to assess whether also glutamate elicits metabotropic Ca2+ signals and NO release in hCMEC/D3 cells. Glutamate induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) that was blocked by α-methyl-4-carboxyphenylglycine and phenocopied by trans-1-amino-1,3-cyclopentanedicarboxylic acid, which, respectively, block and activate group 1 metabotropic glutamate receptors (mGluRs). Accordingly, hCMEC/D3 expressed both mGluR1 and mGluR5 and the Ca2+ response to glutamate was inhibited by their pharmacological blockade with, respectively, CPCCOEt and MTEP hydrochloride. The Ca2+ response to glutamate was initiated by endogenous Ca2+ release from the endoplasmic reticulum and endolysosomal Ca2+ store through inositol-1,4,5-trisphosphate receptors and two-pore channels, respectively, and sustained by store-operated Ca2+ entry. In addition, glutamate induced robust NO release that was suppressed by pharmacological blockade of the accompanying increase in [Ca2+]i. These data demonstrate for the first time that glutamate may induce metabotropic Ca2+ signals in human brain microvascular endothelial cells. The Ca2+ response to glutamate is likely to support NVC during neuronal activity, thereby reinforcing the emerging role of brain microvascular endothelial cells in the regulation of CBF.

Sigma-1 Receptor Agonists Acting on Aquaporin-Mediated H2O2 Permeability: New Tools for Counteracting Oxidative Stress.

Sigma1 Receptor (S1R) is involved in oxidative stress, since its activation is triggered by oxidative or endoplasmic reticulum stress. Since specific aquaporins (AQP), called peroxiporins, play a relevant role in controlling H2O2 permeability and ensure reactive oxygen species wasted during oxidative stress, we studied the effect of S1R modulators on AQP-dependent water and hydrogen peroxide permeability in the presence and in the absence of oxidative stress. Applying stopped-flow light scattering and fluorescent probe methods, water and hydrogen peroxide permeability in HeLa cells have been studied. Results evidenced that S1R agonists can restore water permeability in heat-stressed cells and the co-administration with a S1R antagonist totally counteracted the ability to restore the water permeability. Moreover, compounds were able to counteract the oxidative stress of HeLa cells specifically knocked down for S1R. Taken together these results support the hypothesis that the antioxidant mechanism is mediated by both S1R and AQP-mediated H2O2 permeability. The finding that small molecules can act on both S1R and AQP-mediated H2O2 permeability opens a new direction toward the identification of innovative drugs able to regulate cell survival during oxidative stress in pathologic conditions, such as cancer and degenerative diseases.

Histamine induces intracellular Ca2+ oscillations and nitric oxide release in endothelial cells from brain microvascular circulation.

The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arteries via endothelial histamine 1 receptors (H1 Rs) which result in the downstream production of nitric oxide (NO), the most powerful vasodilator transmitter in the brain. Although endothelial Ca 2+ signals drive histamine-induced NO release throughout the peripheral circulation, the mechanism by which histamine evokes NO production in human cerebrovascular endothelial cells is still unknown. Herein, we exploited the human cerebral microvascular endothelial cell line, hCMEC/D3, to assess the role of intracellular Ca 2+ signaling in histamine-induced NO release. To achieve this goal, hCMEC/D3 cells were loaded with the Ca 2+ - and NO-sensitive dyes, Fura-2/AM and DAF-FM/AM, respectively. Histamine elicited repetitive oscillations in intracellular Ca 2+ concentration in hCMEC/D3 cells throughout a concentration range spanning from 1 pM up to 300 µM. The oscillatory Ca 2+ response was suppressed by the inhibition of H 1 Rs with pyrilamine, whereas H 1 R was abundantly expressed at the protein level. We further found that histamine-induced intracellular Ca 2+ oscillations were initiated by endogenous Ca 2+ mobilization through inositol-1,4,5-trisphosphate- and nicotinic acid dinucleotide phosphate-sensitive channels and maintained over time by store-operated Ca 2+ entry. In addition, histamine evoked robust NO release that was prevented by interfering with the accompanying intracellular Ca 2+ oscillations, thereby confirming that the endothelial NO synthase is recruited by Ca 2+ spikes also in hCMEC/D3 cells. These data provide the first evidence that histamine evokes NO production from human cerebrovascular endothelial cells through intracellular Ca 2+ oscillations, thereby shedding novel light on the mechanisms by which this neuromodulator controls cerebral blood flow.

Setup and Validation of a Reliable Docking Protocol for the Development of Neuroprotective Agents by Targeting the Sigma-1 Receptor (S1R).

Sigma-1 receptor (S1R) is a promising molecular target for the development of novel effective therapies against neurodegenerative diseases. To speed up the discovery of new S1R modulators, herein we report the development of a reliable in silico protocol suitable to predict the affinity of small molecules against S1R. The docking method was validated by comparing the computational calculated Ki values of a test set of new aryl-aminoalkyl-ketone with experimental determined binding affinity. The druggability profile of the new compounds, with particular reference to the ability to cross the blood-brain barrier (BBB) was further predicted in silico. Moreover, the selectivity over Sigma-2 receptor (S2R) and N-methyl-D-aspartate (NMDA) receptor, another protein involved in neurodegeneration, was evaluated. 1-([1,1'-biphenyl]-4-yl)-4-(piperidin-1-yl)butan-1-one (12) performed as the best compound and was further investigated for acetylcholinesterase (AchE) inhibitor activity and determination of antioxidant activity mediated by aquaporins (AQPs). With a good affinity against both S1R and NMDA receptor, good selectivity over S2R and favorable BBB penetration potential together with its AChE inhibitory activity and its ability to exert antioxidant effects through modulation of AQPs, 12 represents a viable candidate for further development as a neuroprotective agent.

Muscarinic M5 receptors trigger acetylcholine-induced Ca2+ signals and nitric oxide release in human brain microvascular endothelial cells.

Basal forebrain neurons control cerebral blood flow (CBF) by releasing acetylcholine (Ach), which binds to endothelial muscarinic receptors to induce nitric (NO) release and vasodilation in intraparenchymal arterioles. Nevertheless, the mechanism whereby Ach stimulates human brain microvascular endothelial cells to produce NO is still unknown. Herein, we sought to assess whether Ach stimulates NO production in a Ca2+ -dependent manner in hCMEC/D3 cells, a widespread model of human brain microvascular endothelial cells. Ach induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+ ]i ) that was prevented by the genetic blockade of M5 muscarinic receptors (M5-mAchRs), which was the only mAchR isoform coupled to phospholipase Cß (PLCß) present in hCMEC/D3 cells. A comprehensive real-time polymerase chain reaction analysis revealed the expression of the transcripts encoding for type 3 inositol-1,4,5-trisphosphate receptors (InsP3 R3), two-pore channels 1 and 2 (TPC1-2), Stim2, Orai1-3. Pharmacological manipulation showed that the Ca2+ response to Ach was mediated by InsP3 R3, TPC1-2, and store-operated Ca2+ entry (SOCE). Ach-induced NO release, in turn, was inhibited in cells deficient of M5-mAchRs. Likewise, Ach failed to increase NO levels in the presence of l-NAME, a selective NOS inhibitor, or BAPTA, a membrane-permeant intracellular Ca2+ buffer. Moreover, the pharmacological blockade of the Ca2+ response to Ach also inhibited the accompanying NO production. These data demonstrate for the first time that synaptically released Ach may trigger NO release in human brain microvascular endothelial cells by stimulating a Ca2+ signal via M5-mAchRs.

Propolis Induces AQP3 Expression: A Possible Way of Action in Wound Healing.

Propolis is the generic name of a complex of resinous compound collected by honeybees and it has been utilized for many years in folk medicine. As other products generated by honeybees (such as royal jelly, pollen, honey), propolis has great therapeutic properties, but very little scientific information is available. Therefore, this study was aimed at exploring the potential wound healing properties of propolis. To that end, we utilized an in vitro scratch wound healing model consisting of human immortalized keratinocytes. Our scratch wound data clearly demonstrated that propolis induced a pronounced increase in the wound repair abilities of keratinocytes. A cell migration assay showed that propolis stimulated keratinocytes to close the wound. We revealed the role of H2O2 as the main mediator of propolis regenerative properties. We showed that this extracellularly released H2O2 could pass across the plasma membrane through a specific aquaporin (i.e., AQP3) modulating intracellular responses. The data offer a biological characterization of propolis positive effects suggesting that propolis could also be utilized in wound treatment within clinical settings.

Regulation of Aquaporin Functional Properties Mediated by the Antioxidant Effects of Natural Compounds.

Some aquaporins (AQPs) have been recently demonstrated to facilitate the diffusion of hydrogen peroxide (H2O2) from the producing cells to the extracellular fluid, and their reactive oxygen species scavenging properties have been defined. Nevertheless, the identification of different AQPs acting as peroxiporins, their functional role in eustress and distress, and the identification of antioxidant compounds able to regulate AQP gating, remain unsolved. This study aims to investigate, in HeLa cells: (1) the expression of different AQPs; (2) the evaluation of naringenin, quercetin, (R)-aloesaponol III 8-methyl ether, marrubiin, and curcumin antioxidant profiles, via α,α-diphenyl-ß-picrylhydrazyl assay; (3) the effect of the compounds on the water permeability in the presence and in the absence of oxidative stress; and (4) the effect of pre- and post-treatment with the compounds on the H2O2 content in heat-stressed cells. Results showed that HeLa cells expressed AQP1, 3, 8, and 11 proteins. The oxidative stress reduced the water transport, and both pre- and post-treatment with the natural compounds recovering the water permeability, with the exception of curcumin. Moreover, the pre- and post-treatment with all the compounds reduced the H2O2 content of heat-stressed cells. This study confirms that oxidative stress reduced water AQP-mediated permeability, reversed by some chemical antioxidant compounds. Moreover, curcumin was shown to regulate AQP gating. This suggests a novel mechanism to regulate cell signaling and survival during stress, and to manipulate key signaling pathways in cancer and degenerative diseases.

Aquaporin-Mediated Water and Hydrogen Peroxide Transport Is Involved in Normal Human Spermatozoa Functioning.

Different aquaporins (AQPs) are expressed in human sperm cells and with a different localization. Their function has been related to cell volume control in response to the osmotic changes encountered passing from the epididymal fluid to the cervical mucus or involved in the end stage of cytoplasm removal during sperm maturation. Recently, AQPs have also shown hydrogen peroxide (H2O2) permeability properties. Here, we investigate the expression, localization and functioning of AQPs in human sperm cells with particular attention to their role as peroxiporins in reactive oxygen species (ROS) scavenging in both normospermic and sub-fertile human subjects. Western blotting and immunocytochemistry were used to confirm and clarify the AQPs expression and localization. Water and H2O2 permeability was tested by stopped flow light scattering method and by the CM-H2DCFDA (5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate, acetyl ester) H2O2 fluorescence probe, respectively. AQP3, -7, -8, and -11 proteins were found in human sperm cells and localized in the head (AQP7), in the middle piece (AQP8) and in the tail (AQP3 and -11) in both the plasma membrane and in intracellular structures. Sperm cells showed water and H2O2 permeability which was reversibly inhibited by H2O2, heat stress and the AQP inhibitor HgCl2. Reduced functionality was observed in patients with compromised basal semen parameters. Present findings suggest that AQPs are involved in both volume regulation and ROS elimination. The relationship between sperm number and motility and AQP functioning was also demonstrated.

Sigma receptor and aquaporin modulators: chiral resolution, configurational assignment, and preliminary biological profile of RC752 enantiomers.

The key role of chiral small molecules in drug discovery programs has been deeply investigated throughout last decades. In this context, our previous studies highlighted the influence of the absolute configuration of different stereocenters on the pharmacokinetic, pharmacodynamic and functional properties of promising Sigma receptor (SR) modulators. Thus, starting from the racemic SR ligand RC752, we report herein the isolation of the enantiomers via enantioselective separation with both HPLC and SFC. After optimization of the eco-sustainable chiral SFC method, both enantiomers were obtained in sufficient amount (tens of mg) and purity (ee up to 95%) to allow their characterization and initial biological investigation. Both enantiomers a) displayed a high affinity for the S1R subtype (Ki = 15.0 ± 1.7 and 6.0 ± 1.2 nM for the (S)- and (R)-enantiomer, respectively), but only negligible affinity toward the S2R (> 350 nM), and b) were rapidly metabolized when incubated with mouse and human hepatic microsomes. Furthermore, the activity on AQP-mediated water permeability indicated a different functional profile for the enantiomers in terms of modulatory effect on the peroxiporins gating.

Allyl Isothiocianate Induces Ca2+ Signals and Nitric Oxide Release by Inducing Reactive Oxygen Species Production in the Human Cerebrovascular Endothelial Cell Line hCMEC/D3.

Nitric oxide (NO) represents a crucial mediator to regulate cerebral blood flow (CBF) in the human brain both under basal conditions and in response to somatosensory stimulation. An increase in intracellular Ca2+ concentrations ([Ca2+]i) stimulates the endothelial NO synthase to produce NO in human cerebrovascular endothelial cells. Therefore, targeting the endothelial ion channel machinery could represent a promising strategy to rescue endothelial NO signalling in traumatic brain injury and neurodegenerative disorders. Allyl isothiocyanate (AITC), a major active constituent of cruciferous vegetables, was found to increase CBF in non-human preclinical models, but it is still unknown whether it stimulates NO release in human brain capillary endothelial cells. In the present investigation, we showed that AITC evoked a Ca2+-dependent NO release in the human cerebrovascular endothelial cell line, hCMEC/D3. The Ca2+ response to AITC was shaped by both intra- and extracellular Ca2+ sources, although it was insensitive to the pharmacological blockade of transient receptor potential ankyrin 1, which is regarded to be among the main molecular targets of AITC. In accord, AITC failed to induce transmembrane currents or to elicit membrane hyperpolarization, although NS309, a selective opener of the small- and intermediate-conductance Ca2+-activated K+ channels, induced a significant membrane hyperpolarization. The AITC-evoked Ca2+ signal was triggered by the production of cytosolic, but not mitochondrial, reactive oxygen species (ROS), and was supported by store-operated Ca2+ entry (SOCE). Conversely, the Ca2+ response to AITC did not require Ca2+ mobilization from the endoplasmic reticulum, lysosomes or mitochondria. However, pharmacological manipulation revealed that AITC-dependent ROS generation inhibited plasma membrane Ca2+-ATPase (PMCA) activity, thereby attenuating Ca2+ removal across the plasma membrane and resulting in a sustained increase in [Ca2+]i. In accord, the AITC-evoked NO release was driven by ROS generation and required ROS-dependent inhibition of PMCA activity. These data suggest that AITC could be exploited to restore NO signalling and restore CBF in brain disorders that feature neurovascular dysfunction.

Transient receptor potential ankyrin 1 (TRPA1) mediates reactive oxygen species-induced Ca2+ entry, mitochondrial dysfunction, and caspase-3/7 activation in primary cultures of metastatic colorectal carcinoma cells.

Colorectal carcinoma (CRC) represents the fourth most common cancer worldwide and is the third most common cause of malignancy-associated mortality. Distant metastases to the liver and lungs are the main drivers of CRC-dependent death. Pro-oxidant therapies, which halt disease progression by exacerbating oxidative stress, represent an antitumour strategy that is currently exploited by chemotherapy and ionizing radiation. A more selective strategy to therapeutically exploit reactive oxygen species (ROS) signaling would consist in targeting a redox sensor that is up-regulated in metastatic cells and is tightly coupled to the stimulation of cancer cell death programs. The non-selective cation channel, Transient Receptor Potential Ankyrin 1 (TRPA1), serves as a sensor of the cellular redox state, being activated to promote extracellular Ca2+ entry by an increase in oxidative stress. Recent work demonstrated that TRPA1 channel protein is up-regulated in several cancer types and that TRPA1-mediated Ca2+ signals can either engage an antiapoptotic pro-survival signaling pathway or to promote mitochondrial Ca2+ dysfunction and apoptosis. Herein, we sought to assess for the first time the outcome of TRPA1 activation by ROS on primary cultures of metastatic colorectal carcinoma (mCRC cells). We found that TRPA1 channel protein is up-regulated and mediates enhanced hydrogen peroxide (H2O2)-induced Ca2+ entry in mCRC cells as compared to non-neoplastic control cells. The lipid peroxidation product 4-hydroxynonenal (4-HNE) is the main ROS responsible for TRPA1 activation upon mCRC cell exposure to oxidative stress. TRPA1-mediated Ca2+ entry in response to H2O2 and 4-HNE results in mitochondrial Ca2+ overload, followed by mitochondrial depolarization and caspase-3/7 activation. Therefore, targeting TRPA1 could represent an alternative strategy to eradicate metastatic CRC by enhancing its sensitivity to oxidative stress.

Discovery of RC-752, a Novel Sigma-1 Receptor Antagonist with Antinociceptive Activity: A Promising Tool for Fighting Neuropathic Pain.

Neuropathic pain (NP) is a chronic condition resulting from damaged pain-signaling pathways. It is a debilitating disorder that affects up to 10% of the world's population. Although opioid analgesics are effective in reducing pain, they present severe risks; so, there is a pressing need for non-opioid pain-relieving drugs. One potential alternative is represented by sigma-1 receptor (S1R) antagonists due to their promising analgesic effects. Here, we report the synthesis and biological evaluation of a series of S1R antagonists based on a 2-aryl-4-aminobutanol scaffold. After assessing affinity toward the S1R and selectivity over the sigma-2 receptor (S2R), we evaluated the agonist/antagonist profile of the compounds by investigating their effects on nerve growth factor-induced neurite outgrowth and aquaporin-mediated water permeability in the presence and absence of oxidative stress. (R/S)-RC-752 emerged as the most interesting compound for S1R affinity (Ki S1R = 6.2 ± 0.9) and functional antagonist activity. Furthermore, it showed no cytotoxic effect in two normal human cell lines or in an in vivo zebrafish model and was stable after incubation in mouse plasma. (R/S)-RC-752 was then evaluated in two animal models of NP: the formalin test and the spinal nerve ligation model. The results clearly demonstrated that compound (R/S)-RC-752 effectively alleviated pain in both animal models, thus providing the proof of concept of its efficacy as an antinociceptive agent.

Aquaporin-6 May Increase the Resistance to Oxidative Stress of Malignant Pleural Mesothelioma Cells.

Malignant pleural mesothelioma (MPM) is an aggressive cancer of the pleural surface and is associated with previous asbestos exposure. The chemotherapy drug is one of the main treatments, but the median survival ranges from 8 to 14 months from diagnosis. The redox homeostasis of tumor cells should be carefully considered since elevated levels of ROS favor cancer cell progression (proliferation and migration), while a further elevation leads to ferroptosis. This study aims to analyze the functioning/role of aquaporins (AQPs) as a hydrogen peroxide (H2O2) channel in epithelial and biphasic MPM cell lines, as well as their possible involvement in chemotherapy drug resistance. Results show that AQP-3, -5, -6, -9, and -11 were expressed at mRNA and protein levels. AQP-6 was localized in the plasma membrane and intracellular structures. Compared to normal mesothelial cells, the water permeability of mesothelioma cells is not reduced by exogenous oxidative stress, but it is considerably increased by heat stress, making these cells resistant to ferroptosis. Functional experiments performed in mesothelioma cells silenced for aquaporin-6 revealed that it is responsible, at least in part, for the increase in H2O2 efflux caused by heat stress. Moreover, mesothelioma cells knocked down for AQP-6 showed a reduced proliferation compared to mock cells. Current findings suggest the major role of AQP-6 in providing mesothelioma cells with the ability to resist oxidative stress that underlies their resistance to chemotherapy drugs.

Histamine activates an intracellular Ca2+ signal in normal human lung fibroblast WI-38 cells.

Histamine is an inflammatory mediator that can be released from mast cells to induce airway remodeling and cause persistent airflow limitation in asthma. In addition to stimulating airway smooth muscle cell constriction and hyperplasia, histamine promotes pulmonary remodeling by inducing fibroblast proliferation, contraction, and migration. It has long been known that histamine receptor 1 (H1R) mediates the effects of histamine on human pulmonary fibroblasts through an increase in intracellular Ca2+ concentration ([Ca2+]i), but the underlying signaling mechanisms are still unknown. Herein, we exploited single-cell Ca2+ imaging to assess the signal transduction pathways whereby histamine generates intracellular Ca2+ signals in the human fetal lung fibroblast cell line, WI-38. WI-38 fibroblasts were loaded with the Ca2+-sensitive fluorophore, FURA-2/AM, and challenged with histamine in the absence and presence of specific pharmacological inhibitors to dissect the Ca2+ release/entry pathways responsible for the onset of the Ca2+ response. Histamine elicited complex intracellular Ca2+ signatures in WI-38 fibroblasts throughout a concentration range spanning between 1 µM and 1 mM. In accord, the Ca2+ response to histamine adopted four main temporal patterns, which were, respectively, termed peak, peak-oscillations, peak-plateau-oscillations, and peak-plateau. Histamine-evoked intracellular Ca2+ signals were abolished by pyrilamine, which selectively blocks H1R, and significantly reduced by ranitidine, which selectively inhibits H2R. Conversely, the pharmacological blockade of H3R and H4R did not affect the complex increase in [Ca2+]i evoked by histamine in WI-38 fibroblasts. In agreement with these findings, histamine-induced intracellular Ca2+ signals were initiated by intracellular Ca2+ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate (InsP3) receptors (InsP3R) and sustained by store-operated Ca2+ channels (SOCs). Conversely, L-type voltage-operated Ca2+ channels did not support histamine-induced extracellular Ca2+ entry. A preliminary transcriptomic analysis confirmed that WI-38 human lung fibroblasts express all the three InsP3R isoforms as well as STIM2 and Orai3, which represent the molecular components of SOCs. The pharmacological blockade of InsP3 and SOC, therefore, could represent an alternative strategy to prevent the pernicious effects of histamine on lung fibroblasts in asthmatic patients.

Human sperm functioning is related to the aquaporin-mediated water and hydrogen peroxide transport regulation.

Aquaporins (AQPs) are transmembrane water channels and some of them are permeable in addition to water to other small solutes including hydrogen peroxide. The sperm cells of mammals and fishes express different AQPs, although there is no agreement in the literature on their localization. In humans, AQP3 and AQP11 are expressed mainly in the tail, AQP7 in the head and AQP8 in the midpiece. Thanks to the results of experiments with KO mice and to data obtained by comparing sub-fertile patients with normospermic subjects, the importance of AQPs for the normal functioning of sperms to ensure normal fertility emerged. AQP3, AQP7 and AQP11 appeared involved in the sperm volume regulation, a key role for fertility because osmoadaptation protect the sperm against a swelling and tail bending that could affect sperm motility. AQP8 seems to have a fundamental role in regulating the elimination of hydrogen peroxide, the most abundant reactive oxygen species (ROS), and therefore in the response to oxidative stress. In this review, the human AQPs expression, their localization and functions, as well as their relevance in normal fertility are discussed. To understand better the AQPs role in human sperm functionality, the results of studies obtained in other animal species were also considered.

NMDA receptors elicit flux-independent intracellular Ca2+ signals via metabotropic glutamate receptors and flux-dependent nitric oxide release in human brain microvascular endothelial cells.

The excitatory neurotransmitter glutamate gates post-synaptic N-methyl-d-aspartate (NMDA) receptors (NMDARs) to mediate extracellular Ca2+ entry and stimulate neuronal nitric oxide (NO) synthase to release NO and trigger neurovascular coupling (NVC). Neuronal and glial NMDARs may also operate in a flux-independent manner, although it is unclear whether their non-ionotropic mode of action is involved in NVC. Recently, endothelial NMDARs were found to trigger Ca2+-dependent NO production and induce NVC, but the underlying mode of signaling remains elusive. Herein, we report that GluN1 protein, as well as GluN2C and GluN3B transcripts and proteins, were expressed and that NMDA did not elicit inward currents, but induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) in the human brain microvascular endothelial cell line, hCMEC/D3. A multidisciplinary approach, including live cell imaging, whole-cell patch-clamp recordings, pharmacological manipulation and gene targeting, revealed that NMDARs increase the [Ca2+]i in a flux-independent manner in hCMEC/D3 cells. The Ca2+ response to NMDA was triggered by endogenous Ca2+ release from the endoplasmic reticulum and the lysosomal Ca2+ stores and sustained by store-operated Ca2+ entry. Unexpectedly, pharmacological and genetic blockade of mGluR1 and mGluR5 dramatically impaired NMDARs-mediated Ca2+ signals. These findings indicate that NMDARs may increase the endothelial [Ca2+]i in a flux-independent manner via group 1 mGluRs. However, imaging of DAF-FM fluorescence revealed that NMDARs may also induce Ca2+-dependent NO release by signaling in a flux-dependent manner. These findings, therefore, shed novel light on the mechanisms whereby brain microvascular endothelium decodes glutamatergic signaling and regulates NVC.

Manuka Honey Induces Apoptosis of Epithelial Cancer Cells through Aquaporin-3 and Calcium Signaling.

Honey is a natural product with a long use in traditional medicine and is well recognized to regulate different biological events. It is an important source of various biological or pharmacological molecules and, therefore, there is a strong interest to explore their properties. Evidence is growing that honey may have the potential to be an anticancer agent acting through several mechanisms. Here we observed for the first time in a cancer cell line a possible mechanism through which honey could induce an alteration in the intracellular reactive oxygen species and homeostatic balance of intracellular calcium concentration leading to cell death by apoptosis. This mechanism seems to be enhanced by manuka honey's ability to maintain high H2O2 permeability through aquaporin-3.