Papers by Umberto Laforenza
Journal of Biological Chemistry, 2005
Phospholipase C signaling stimulates Ca 2؉ entry across the plasma membrane through multiple mech... more Phospholipase C signaling stimulates Ca 2؉ entry across the plasma membrane through multiple mechanisms. Ca 2؉ store depletion stimulates store-operated Ca 2؉-selective channels, or alternatively, other phospholipase C-dependent events activate Ca 2؉-permeable non-selective cation channels. Transient receptor potential 7 (TRPC7) is a non-selective cation channel that can be activated by both mechanisms when ectopically expressed, but the regulation of native TRPC7 channels is not known. We knocked out TRPC7 in DT40 B-cells, which expresses both forms of Ca 2؉ entry. No difference in the store-operated current I crac was detected between TRPC7 ؊/؊ and wild-type cells. Wild-type cells demonstrated nonstore-operated cation entry and currents in response to activation of the B-cell receptor or protease-activated receptor 2, intracellular dialysis with GTP␥S, or application of the synthetic diacylglycerol oleyl-acetyl-glycerol. These responses were absent in TRPC7 ؊/؊ cells but could be restored by transfection with human TRPC7. In conclusion, in B-lymphocytes, TRPC7 appeared to participate in the formation of ion channels that could be activated by phospholipase Clinked receptors. This represents the first demonstration of a physiological function for endogenous TRPC7 channels.
Stem Cells and Development, Dec 1, 2010
Endothelial progenitor cells (EPCs) may be recruited from the bone marrow to sites of tissue rege... more Endothelial progenitor cells (EPCs) may be recruited from the bone marrow to sites of tissue regeneration to sustain neovascularization and reendothelialization after acute vascular injury. This feature makes them particularly suitable for cell-based therapy. In mature endothelium, store-operated Ca 2+ entry (SOCE) is activated following emptying of inositol-1,4,5-trisphosphate-sensitive stores, which controls a wide number of functions, including proliferation, nitric oxide synthesis, and vascular permeability. The present work aimed at investigating SOCE expression in EPCs harvested from both peripheral blood (PB-EPCs) and umbilical cord blood (UCB-EPCs) by employing both Ca 2+ imaging and molecular biology techniques. SOCE was induced upon either pharmacological (ie, cyclopiazonic acid) or physiological (ie, ATP) depletion of the intracellular Ca 2+ pool. Further, store-dependent Ca 2+ entry was inhibited by the SOCE inhibitor, AU1 c BTP-2. Real-time reverse transcriptionpolymerase chain reaction and western blot analyses showed that both PB-EPCs and UCB-EPCs express all the molecular candidates to mediate SOCE in differentiated cells, including TRPC1, TRPC4, Orai1, and Stim1. Moreover, pharmacological maneuvers demonstrated that, as well as in differentiated endothelial cells, the signal transduction pathway leading to depletion of the intracellular Ca 2+ pool impinged on the phospholipase C=inositol-1,4,5-trisphosphate pathway. Finally, blockage of SOCE with AU1 c BTP-2 impaired PB-EPC proliferation. These findings provide the first evidence that EPCs express SOCE, which might thus be regarded as a novel target to enhance the regenerative outcome of cell-based therapy.
Cells, Jun 27, 2023
Isothiocianate Induces Ca 2+ Signals and Nitric Oxide Release by Inducing Reactive Oxygen Species... more Isothiocianate Induces Ca 2+ Signals and Nitric Oxide Release by Inducing Reactive Oxygen Species Production in the Human Cerebrovascular Endothelial Cell Line hCMEC/D3.
Biochimie, Sep 1, 2021
Aquaporins (AQPs) are transmembrane water channels and some of them are permeable in addition to ... more 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.
Methods in Enzymology, 1997
ABSTRACT
Cell Calcium, Nov 1, 2021
The excitatory neurotransmitter glutamate gates post-synaptic N-methyl-d-aspartate (NMDA) recepto... more 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.
Journal of Cellular Physiology, Jul 16, 2019
The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arter... more The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arteries via endothelial histamine 1 receptors (H1Rs) 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 1Rs with pyrilamine, whereas H 1R 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.
Cancers, Apr 15, 2019
Nicotinic acid adenine dinucleotide phosphate (NAADP) gates two-pore channels 1 and 2 (TPC1 and T... more Nicotinic acid adenine dinucleotide phosphate (NAADP) gates two-pore channels 1 and 2 (TPC1 and TPC2) to elicit endo-lysosomal (EL) Ca 2+ release. NAADP-induced EL Ca 2+ signals may be amplified by the endoplasmic reticulum (ER) through the Ca 2+-induced Ca 2+ release mechanism (CICR). Herein, we aimed at assessing for the first time the role of EL Ca 2+ signaling in primary cultures of human metastatic colorectal carcinoma (mCRC) by exploiting Ca 2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe β-naphthylamide (GPN), and nigericin, which dissipates the ∆pH which drives Ca 2+ refilling of acidic organelles, caused massive Ca 2+ release in the presence of a functional inositol-1,4,5-trisphosphate (InsP 3)-sensitive ER Ca 2+ store. Liposomal delivery of NAADP induced a transient Ca 2+ release that was reduced by GPN and NED-19, a selective TPC antagonist. Pharmacological and genetic manipulations revealed that the Ca 2+ response to NAADP was triggered by TPC1, the most expressed TPC isoform in mCRC cells, and required ER-embedded InsP 3 receptors. Finally, NED-19 and genetic silencing of TPC1 reduced fetal calf serum-induced Ca 2+ signals, proliferation, and extracellular signal-regulated kinase and Akt phoshorylation in mCRC cells. These data demonstrate that NAADP-gated TPC1 could be regarded as a novel target for alternative therapies to treat mCRC.
Proceedings of the Society for Experimental Biology and Medicine, Sep 1, 2000
In the intestinal lumen thiamine is in free form and very low concentrations. Absorption takes pl... more In the intestinal lumen thiamine is in free form and very low concentrations. Absorption takes place primarily in the proximal part of the small intestine by means of a dual mechanism, which is saturable at low (physiological) concentrations and diffusive at higher. Thiamine undergoes intracellular phosphorylation mainly to thiamine pyrophosphate, while at the serosal side only free thiamine is present. Thiamine uptake is enhanced by thiamine deficiency, and reduced by thyroid hormone and diabetes. The entry of thiamine into the enterocyte, as evaluated in brush border membrane vesicles of rat small intestine in the absence of H+ gradient, is Na+- and biotransformation-independent, completely inhibited by thiamine analogs and reduced by ethanol administration and aging. The transport involves a saturable mechanism at low concentrations of vitamin and simple diffusion at higher. Outwardly oriented H+ gradients enhance thiamine transport, whose saturable component is a Na+-independent electroneutral uphill process utilizing energy supplied by the H+ gradient, and involving a thiamine/ H+ 1:1 stoichiometric exchange. The exit of thiamine from the enterocyte, as evaluated in basolateral membrane vesicles, is Na+-dependent, directly coupled to ATP hydrolysis by Na+-K+-ATPase, and inhibited by thiamine analogs. Transport of thiamine by renal brush border membrane vesicles is similar to the intestinal as far as both H+ gradient influence and specificity are concerned. In the erythrocyte thiamine transport is a Na+-independent, electroneutral process yet with two components: saturable, prevailing at low thiamine concentrations, and diffusive at higher. The saturable (specific) component is missing in patients of the rare disease known as thiamine-responsive megaloblastic anaemia (TRMA), producing a general disturbance of thiamine transport up to thiamine deficiency. The TRMA gene is located in chromosome 1q23.3. Recently, the thiamine transporter has been cloned: it is a protein of 497 amino acid residues with high homology with the reduced-folate transporter.
Vascular Pharmacology, Apr 1, 2018
Cell death discovery, Jul 1, 2023
Colorectal carcinoma (CRC) represents the fourth most common cancer worldwide and is the third mo... more 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. Prooxidant 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 Ca 2+ 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 Ca 2+ signals can either engage an antiapoptotic pro-survival signaling pathway or to promote mitochondrial Ca 2+ 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 (H 2 O 2)-induced Ca 2+ 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. TRPA1mediated Ca 2+ entry in response to H 2 O 2 and 4-HNE results in mitochondrial Ca 2+ 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.
Cells, Mar 19, 2022
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Molecules, Apr 19, 2019
Propolis is the generic name of a complex of resinous compound collected by honeybees and it has ... more 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 H 2 O 2 as the main mediator of propolis regenerative properties. We showed that this extracellularly released H 2 O 2 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.
Alcohol and Alcoholism, Nov 1, 1988
ABSTRACT
Cells, Jun 10, 2022
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Pharmaceuticals
Neuropathic pain (NP) is a chronic condition resulting from damaged pain-signaling pathways. It i... more 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 func...
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Papers by Umberto Laforenza