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The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) in the target cells, which activates the Ca 2+ /Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca 2+ ] i and NO production. The current study assessed whether and how glutamate drives Ca 2+dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca 2+ ] i , which was maximally activated at 200 μM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca 2+ oscillations were triggered by rhythmic endogenous Ca 2+ mobilization and maintained over time by extracellular Ca 2+ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca 2+ release was mediated by InsP 3 -sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) J Cell Physiol. 2019;234:3538-3554. wileyonlinelibrary.com/journal/jcp 3538 | gated two-pore channel 1. Constitutive store-operated Ca 2+ entry mediated Ca 2+ entry during ongoing Ca 2+ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca 2+ signals. Of note, glutamate induced Ca 2+ -dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca 2+ oscillations and NO release have the potential to impact on neurovascular coupling in the brain. K E Y W O R D S Ca 2+ oscillations, endothelial cells, glutamate, neurovascular coupling (NVC), nitric oxide

Store-operated Ca2+ entry (SOCE) provides a major Ca2+ entry route in cancer cells. SOCE is mediated by the assembly of Stim and Orai proteins at endoplasmic reticulum (ER)-plasma membrane junctions upon depletion of the ER Ca2+ store. Additionally, Stim and Orai proteins underpin constitutive Ca2+ entry in a growing number of cancer cell types due to the partial depletion of their ER Ca2+ reservoir. Herein, we investigated for the first time the structure and function of SOCE in primary cultures of colorectal carcinoma (CRC) established from primary tumor (pCRC) and metastatic lesions (mCRC) of human subjects. Stim1-2 and Orai1-3 transcripts were equally expressed in pCRC and mCRC cells, although Stim1 and Orai3 proteins were up-regulated in mCRC cells. The Mn2+-quenching technique revealed that constitutive Ca2+ entry was significantly enhanced in pCRC cells and was inhibited by the pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3. The larger resting Ca2+ influx in pCRC was associated to their lower ER Ca2+ content as compared to mCRC cells. Pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3 prevented ER-dependent Ca2+ release, thereby suggesting that constitutive SOCE maintains ER Ca2+ levels. Nevertheless, pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3 did not affect CRC cell proliferation and migration. These data provide the first evidence that Stim and Orai proteins mediate constitutive Ca2+ entry and replenish ER with Ca2+ in primary cultures of CRC cells. However, SOCE is not a promising target to design alternative therapies for CRC.

Stromal cell-derived factor-1α (SDF-1α) drives endothelial colony-forming cell (ECFC) homing and incorporation within neovessels, thereby restoring tissue perfusion in ischemic tissues and favoring tumor vascularization and metastasis. SDF-1α stimulates ECFC migration by activating the G-protein-coupled receptor, CXCR4, and then engaging the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. Sporadic evidence showed that SDF-1α may also act through an increase in intracellular Ca concentration ([Ca]) in bone marrow-derived hematopoietic progenitor cells and fully differentiated endothelial cells. Of note, recent evidence demonstrated that intracellular Ca signals play a key role in controlling the proangiogenic activity of ECFCs. The present investigation was, therefore, undertaken to assess whether and how SDF-1α induces ECFC motility by triggering intracellular Ca signals. We found that SDF-1α caused a dose-dependent increase in [Ca] that was inhibited by ADM3100, a selective CXCR4 antagonist. Pharmacological manipulation revealed that the Ca response to [Ca] was shaped by an initial intracellular Ca release through inositol-1,4,5-trisphosphate receptors (InsPRs), followed by a sustained phase of extracellular Ca entry through store-operated Ca channels. InsP-dependent Ca release and store-operated Ca entry (SOCE) were both necessary for SDF-1α-induced extracellular signal-regulated kinases 1/2 (ERK 1/2) and AKT phosphorylation. Finally, SDF-1α employed intracellular Ca signals, ERK 1/2, and PI3K/AKT to promote ECFC migration in vitro and neovessel formation in vivo. These data, therefore, provide the first evidence that SDF-1α induces ECFC migration through the Ca-dependent activation of the ERK 1/2 and PI3K/AKT pathways.

Inhalation of vapors from a hot tea of Eucalyptus camaldulensis Dehnh. leaves is considered by Iraqi–Kurdistan people an effective spasmolytic and antipyretic remedy for the treatment of respiratory diseases. The constituents of volatile fractions isolated by hydrodistillation from dried leaves of the plant collected in Kurdistan were determined by GC-FID and GC-MS analyses. More than 90% components were identified. The most abundant constituents were 1,8-cineole, p-cymene, α-pinene, terpinen-4-ol, aromadendrene, and α-terpineol. The different volatile fractions induced relaxation on rat isolated aortic and tracheal rings in concentration-dependent manner. These effects appeared to be due to a complex interaction between various terpenoid components rather than being only due to the main oil constituent, 1,8-cineole. The KCa channel and the NO pathway were not significantly involved in the relaxation mechanism, while Ca2+ channels played a major role in the spasmolytic effects.

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ABSTRACT:The monoterpene, α-terpinyle acetate (TA) is a constituent of essential oils present in aromatic plants. Since the role of ion channels and endothelial hyperpolarizing factors in TA induced relaxation in rat's aorta is unknown, the current study aimed to study the mechanism underlying the vasodilatory effect of TA in isolated aortic rings. Terpinyle acetate induced a potent vasodilation in rat aortic rings with a percentage of relaxation of 63.79 %. The results of the role of K + channel subtypes in vasorelaxation revealed that both Kv and KATP played a major role since GLIB produced a maximum percent of inhibition in the relaxation produced by TA to 8.91 %; this was followed by 4-AP in which the percent of inhibition reduced to 14.95. On the other hand, Kir played no role in the TA induced vasorelaxation since BaCl2 did not produce any inhibition in aortic relaxation. Furthermore, also L-type Ca 2+ channel played no role in TA induced relaxation since the L-type Ca 2+ channel inhibitor Nifedipine did not reduce the percent of relaxation. Endothelium also played a considerable role in the induced vasorelaxation since, in denuded aorta, the percent of relaxation was reduced to 36%. Preincubation of the aortic ring with methylene blue, a soluble cGMP inhibitor also significantly reduced the TA induced relaxation to 16.39%. In contrast, preincubation with cyclooxygenase inhibitor Indomethacin did not produce any inhibitory effect on AT induced vasorelaxation. It can be concluded from these novel results that AT induced vasorelaxation involve the activation of KV, KATP channels and at least partly dependent on endothelium via the activation NOcGMP signal transduction pathway.