Arachidonic Acid Evokes an Increase in Intracellular Ca2+ Concentration and Nitric Oxide Production in Endothelial Cells from Human Brain Microcirculation

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Article Arachidonic Acid Evokes an Increase in Intracellular Ca2+ Concentration and Nitric Oxide Production in Endothelial Cells from Human Brain Microcirculation

1 2,3, 2, 2 Roberto Berra-Romani , Pawan Faris † , Sharon Negri †, Laura Botta , Tullio Genova 4,5 and Francesco Moccia 2,* 1 Biomedicine School, Benemerita Universidad Autonoma de Puebla, 72000 Puebla, Mexico 2 Laboratory of General Physiology, Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, 27100 Pavia, Italy 3 Research Center, Salahaddin University, Erbil 44001, Iraq 4 Department of Life Sciences and Systems Biology, University of Torino, 10123 Torino, Italy 5 Department of Surgical Sciences, University of Torino, 10126 Torino, Italy * Correspondence: [email protected]; Tel.: +30-0382-987-613 These authors contributed equally to this manuscript. † Received: 21 June 2019; Accepted: 8 July 2019; Published: 9 July 2019

Abstract: It has long been known that the conditionally essential polyunsaturated arachidonic acid (AA) regulates cerebral blood flow (CBF) through its metabolites prostaglandin E2 and epoxyeicosatrienoic acid, which act on vascular smooth muscle cells and pericytes to vasorelax cerebral microvessels. However, AA may also elicit endothelial nitric oxide (NO) release through an increase 2+ 2+ 2+ in intracellular Ca concentration ([Ca ]i). Herein, we adopted Ca and NO imaging, combined with immunoblotting, to assess whether AA induces intracellular Ca2+ signals and NO release in the human brain microvascular endothelial cell line hCMEC/D3. AA caused a dose-dependent 2+ increase in [Ca ]i that was mimicked by the not-metabolizable analogue, eicosatetraynoic acid. The Ca2+ response to AA was patterned by endoplasmic reticulum Ca2+ release through type 3 inositol-1,4,5-trisphosphate receptors, lysosomal Ca2+ mobilization through two-pore channels 1 and 2 (TPC1-2), and extracellular Ca2+ influx through transient receptor potential vanilloid 4 (TRPV4). In addition, AA-evoked Ca2+ signals resulted in robust NO release, but this signal was considerably delayed as compared to the accompanying Ca2+ wave and was essentially mediated by TPC1-2 and TRPV4. Overall, these data provide the first evidence that AA elicits Ca2+-dependent NO release from a human cerebrovascular endothelial cell line, but they seemingly rule out the possibility that this NO signal could acutely modulate neurovascular coupling.

Keywords: arachidonic acid; brain microvascular endothelial cells; neurovascular coupling; cerebral blood ﬂow; Ca2+ signalling; nitric oxide; inositol-1,4,5-trisphosphate receptors; two-pore channels 1-2; transient receptor potential vanilloid 4

1. Introduction The 20-carbon omega-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) is incorporated in the plasma membrane esteriﬁed to phospholipids at the second alcohol of glycerol in the sn-2 position and exerts multiple homeostatic, structural, and signalling functions [1]. AA is released from the membrane pool by the activation of three distinct phospholipases, A2, C, and D, upon cell stimulation to exert either an autocrine and/or a paracrine eﬀect on adjoining cells [1,2]. For instance, synaptically released glutamate activates N-methyl-d-aspartate receptors (NMDARs), thereby raising intracellular Ca2+ levels within the dendritic tree and recruiting the Ca2+-dependent enzyme phospholipase A2

Cells 2019, 8, 689; doi:10.3390/cells8070689 www.mdpi.com/journal/cells Cells 2019, 8, 689 2 of 18

(PLA2). Neuronal PLA2, in turn, mobilizes AA from membrane phospholipids; hereafter, AA is converted by cyclooxygenase-2 (COX-2) into prostaglandin E2 (PGE2) to relax cerebral microvessels [3]. Likewise, neuronal activity causes an increase in astrocytic Ca2+ concentration, which also recruits PLA2 to stimulate the synthesis of a number of AA-derived metabolites that serve as vasodilatory mediators. These include PGE2 and epoxyeicosatrienoic acids (EETs) that are produced by COX-1 and cytochrome P450 epoxygenase [2,4], respectively. Alternately, astrocyte-derived AA is released onto neighbouring vascular smooth muscle cells (VSMCs) or pericytes where it can be metabolized into the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) by cytochrome P450 4A2 [2,4–6]. AA is thus regarded as a key regulator of neurovascular coupling (NVC), the mechanism whereby neuronal activity results in the elevation of local cerebral blood flow (CBF), which is indispensable to maintain proper supply of oxygen and nutrients to active brain regions [2–4,7]. For instance, the hemodynamic response to whisker stimulation in the somatosensory cortex was dramatically impaired by inhibiting the production of PGE2 and EETs by, respectively, pyramidal neurons and astrocytes [8]. Conversely, only EETs were responsible for the elevation in CBF evoked by stimulation of basal forebrain cholinergic neurons [9]. On the other hand, 20-HETE has been shown to induce capillary vasoconstriction in both the molecular [10] and granular [5] layer of the cerebellum, depending on the presence of nitric oxide (NO), that suppresses cytochrome P450 4A2 activity [2]. A recent series of studies demonstrated that cerebral endothelial cells, which line the lumen of brain microvessels and are in close apposition to both astrocyte end-feet and neuronal projections within the neurovascular unit, may also translate neuronal activity into NO-dependent vasodilation [11–13]. For instance, the neurotransmitter acetylcholine triggers a biphasic increase in intracellular Ca2+ 2+ 2+ concentration ([Ca ]i) which leads to the recruitment of Ca /calmodulin (CaM)-sensitive endothelial NO synthase (eNOS) in hCMEC/D3 cells [14], which are a widely utilized human brain microvascular endothelial cell line [15]. Likewise, acetylcholine and glutamate were found to induce repetitive 2+ oscillations in [Ca ]i and NO production in mouse brain microvascular endothelial cells [14,16]. The Ca2+ response to extracellular neurotransmitters in cerebrovascular endothelial cells is mainly mediated by inositol-1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) and two-pore channels 1 and 2 (TPC1-2), which mediate Ca2+ release from endoplasmic reticulum (ER) and lysosomes, respectively, and by store-operated Ca2+ entry (SOCE), a widespread Ca2+-permeable route activated following depletion of the ER Ca2+ reservoir [14,16,17]. Intriguingly, it has long been known that AA directly induces Ca2+-dependent NO release in vascular endothelial cells [18–21] and endothelial colony-forming cells (ECFCs) [22]. The Ca2+ response to AA is shaped by multiple components of the endothelial 2+ 2+ Ca toolkit, including InsP3Rs and TPC1, which mediate endogenous Ca release, respectively, and transient receptor potential vanilloid 4 (TRPV4), which mediates extracellular Ca2+ influx [18–21,23]. Conversely, AA is seemingly unable to activate SOCE [24–26]. A recent investigation suggested that AA may be converted by cytochrome P450 2U1 epoxygenase into 20-HETE also in hCMEC/D3 cells and rat brain microvessels [27]. Furthermore, a preliminary report showed that AA may trigger intracellular Ca2+ signals in primary human brain microvascular endothelial cells, but the underlying signalling pathway was not clearly elucidated and their ability to promote NO was not evaluated [23]. Herein, we investigated for the first time whether and how exogenous AA induces intracellular 2+ 2+ Ca signalling and NO release in hCMEC/D3 cells. AA elicited a biphasic increase in [Ca ]i which was shaped by endogenous Ca2+ release through type 3 inositol-1,4,5-trisphosphate receptor isoform 2+ (InsP3R3) and two-pore channels 1 and 2 (TPC1-2) and extracellular Ca influx through TRPV4. We further demonstrated that AA induced robust Ca2+-dependent NO release. However, AA-induced NO production was remarkably delayed as compared to intracellular Ca2+ signalling. In addition, AA-induced NO release was mainly dependent upon TPC1-2 and TRPV4 activation rather than InsP3R3. Overall, these data provide the evidence that AA is able to induce intracellular Ca2+ signalling and NO release in human brain microvascular endothelial cells, although this NO release is seemingly too slow to account for the vasorelaxing effects of AA observed in vivo. Cells 2019, 8, 689 3 of 18

2. Materials and Methods

2.1. Cell Culture Human cerebral microvascular endothelial cells (hCMEC/D3) were obtained from Institut National de la Santé et de la Recherche Médicale (INSERM, Paris, France) and cultured as extensively illustrated elsewhere [17]. Only hCMEC/D3 cells between passage 25 and 35 were employed in the present investigation. The cells were seeded at a concentration of 27,000 cells/cm2 and grown in tissue culture flasks coated with 0.1 mg/mL rat tail collagen type 1, in a medium having the following composition: EBM-2 medium (Lonza, Basel, Switzerland) supplemented with 5% foetal bovine serum, 1 ng/mL basic fibroblast growth factor, 1% penicillin–streptomycin, 5 µg/mL ascorbic acid, 1.4 µM hydrocortisone, 1/100 chemically defined lipid concentrate (Life Technologies, Milan, Italy), and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The cells were cultured at 37 ◦C, 5% CO2 saturated humidity.

2.2. Solutions The composition of the physiological salt solution (PSS) used to load the cells with the Ca2+- and NO-sensitive dyes and to bath the cells during recordings had the following composition: 150 mM NaCl, 6 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES. In order to obtain an extracellular solution devoid of Ca2+ (0Ca2+), Ca2+ was substituted with 2 mM NaCl and 0.5 mM EGTA was added. Solutions were titrated to pH 7.4 with NaOH. The osmolality of PSS was measured with an osmometer (Wescor 5500, Wescor, Logan, UT, USA) and resulted to be 300–310 mOsm/L.

2+ 2.3. [Ca ]i and NO Imaging Ca2+ imaging was carried out as described elsewhere [17]. Briefly, hCMEC/D3 cells were loaded with 4 µM Fura-2 acetoxymethyl ester (Fura-2/AM; 1 mM stock in dimethyl sulfoxide) in PSS. The cells were maintained in the presence of Fura-2 for 30 min at 37 ◦C and 5% CO2 saturated humidity. After de-esterification in PSS, the coverslip (8 mm) was mounted on the bottom of a Petri dish and the cells observed by an upright epifluorescence Axiolab microscope (Carl Zeiss, Oberkochen, Germany) equipped with a Zeiss 40 Achroplan objective (water immersion, 2.0 mm working distance, × 0.9 numerical aperture). The cells were excited alternately at 340 and 380 nm by using a filter wheel (Lambda 10, Sutter Instrument, Novato, CA, USA). The emitted fluorescence was detected at 510 nm by using an Extended-ISIS CCD camera (Photonic Science, Millham, UK). Custom-made software, working in the LINUX environment, was used to drive the CCD camera and the filter wheel, and to measure and plot on-line the fluorescence from 15 to 25 rectangular “regions of interest” (ROI), 2+ each corresponding to well defined single cells. [Ca ]i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm 2+ (Ratio (F340/F380)). An increase in [Ca ]i causes an increase in the ratio [17]. Ratio measurements were performed and plotted on-line every 3 s. The experiments were performed at room temperature (22 ◦C), as discussed in [28]. NO was measured as described in [17]. Briefly, hCMEC/D3 cells were loaded with the membrane permeable NO-sensitive dye 4-amino-5-methylamino-2’,7’-difluorofluorescein (DAF-FM) diacetate (10 µM) in PSS. The cells were maintained in the presence of DAF-FM for 1 h at 37 ◦C and 5% CO2 saturated humidity. Subsequently, the dye was de-esterified in PSS for a further hour. DAF-FM fluorescence was measured by using the same equipment described for Ca2+ recordings but with a different filter set, i.e., excitation at 480 nm and emission at 535 nm wavelength (emission intensity was shortly termed “NOi”). The changes in DAF-FM fluorescence induced by AA were recorded and plotted on-line every 5 s. Again, off-line analysis was performed by using custom-made macros developed by Microsoft Office Excel software. The experiments were performed at room temperature (22 ◦C). Cells 2019, 8, 689 4 of 18

2.4. Western Blotting Semi-confluent adherent cells were carefully lysate by using ice-cold RIPA buffer (150 mM NaCl, 25 mM Tris-HCl pH 7.6, 1% sodium deoxycholate, 1% NP-40, and 0.1% SDS) adding protease inhibitor cocktail (Sigma-Aldrich, Milan, Italy). In order to determine the protein concentration, a bicinchoninic acid kit (Thermo Fisher Scientific, Waltham, MA, USA) was used following the manufacturer’s instructions. Antibodies against TRPV4 (ab94868; Abcam, Cambridge, UK) and antibodies anti–β-actin (Sigma-Aldrich) were used as indicated by the manufacturer. Chemiluminescence assays were conducted by using western blotting plus ECL (Perkin Elmer, Milano, Italy).

2.5. Statistical Analysis All the data were obtained from hCMEC/D3 cells from no fewer than three independent experiments. The peak amplitude of AA-evoked Ca2+ signals was measured as the diﬀerence between the ratio at the Ca2+ peak and the mean ratio of 30 sec baseline before the peak. The dose– response relationship was ﬁtted by using the following equation:

100 Y = (1) + EC50 1 [AA]

2+ where Y is the amplitude of the Ca response, [AA] is the AA concentration, and EC50 is the half-maximal effective concentration, i.e., the [AA] that elicited 50% of the maximal response. The peak amplitude of AA-induced NO production was evaluated as the difference between the maximal increase in DAF-FM fluorescence and the average of 1 min baseline before the peak. Pooled data are given as average standard error (SE) and statistical significance (p < 0.05) was evaluated by ± two-tailed Student’s t test for unpaired observations or one-way ANOVA analysis followed by the post-hoc Dunnett’s test as appropriate. Data relative to both Ca2+ and NO signals are summarized as mean SE, whereas the number of cells analysed for each condition is indicated in the corresponding ± bar histograms.

2.6. Chemicals Fura-2/AM and DAF-FM were purchased from Molecular Probes (Molecular Probes Europe BV, Leiden, The Netherlands). GSK1016790A (GSK) was obtained from Tocris (Bristol, UK). All the chemicals were of analytical grade and obtained from Sigma Chemical Co. (St. Louis, MO, USA).

3. Results

2+ 3.1. AA Triggers an Increase in [Ca ]i in hCMEC/D3 Cells In order to assess whether AA induces intracellular Ca2+ signals, hCMEC/D3 cells were loaded with the Ca2+-sensitive ﬂuorophore, Fura-2, as described in paragraph 2.3. Neuronal activity leads to remarkable increase in synaptic AA concentration, which can rise up to 50–200 µM[29–31]. Accordingly, 2+ AA induced an increase in [Ca ]i in hCMEC/D3 cells that started at 1 µM (Figure1A), reached the maximal response at 300 µM, and whose dose–response relationship could be ﬁtted by a sigmoidal 2+ curve with an EC50 of 8.4 µM (Figure1B). A careful inspection of the Ca traces revealed that 3 µM 2+ AA elicited a slow elevation in [Ca ]i that remained stable during the continuous presence of the agonist in the bath (Figure1A). Conversely, when applied at 30 µM and 300 µM, AA induced a rapid 2+ 2+ Ca transient that slowly decayed to a sustained plateau of elevated [Ca ]i (Figure1A), as recently shown in ECFCs [22]. As synaptic AA concentration may achieve the mid-to-high micromolar range [29–31], we used 30 µM AA throughout the remainder of the study. The Ca2+ response to AA (30 µM) was reversible as, 2+ 2+ when AA was removed from the bath at the peak of the Ca rise, the [Ca ]i rapidly returned to the baseline, whereas the subsequent addition of AA raised intracellular Ca2+ levels again (Figure1C). Cells 2019, 8, 689 5 of 18

AA-induced endothelial Ca2+ signals may be triggered by many of its derivatives, such as 5,6- epoxyeicosatrienoic (5,6-EET) and 8,9-EET and leukotriene C4, which are synthesized, respectively, by cytochromeCells 2019, 8 P450, x epoxygenase and glutathione S-transferase 2 [32–36]. Therefore, we evaluated5 of the20 Ca2+ response to the non-metabolized AA analogue, eicosatetraynoic acid (ETYA), as described in sigmoidal curve with an EC50 of 8.4 μM (Figure 1B). A careful inspection of the Ca2+ traces revealed other types of endothelial cells [22,37]. As shown in Figure2, ETYA (30 µM) induced an increase in that 3 μM AA elicited a slow elevation in [Ca2+]i that remained stable during the continuous presence [Ca2+] whose kinetics and amplitude were similar to those of the Ca2+ signal induced by AA (30 µM) of thei agonist in the bath (Figure 1A). Conversely, when applied at 30 μM and 300 μM, AA induced in the same batches of hCMEC/D3 cells (Figure2A,B). Taken together, these ﬁndings demonstrate that a rapid Ca2+ transient that slowly decayed to a sustained plateau of elevated [Ca2+]i (Figure 1A), as 2+ AArecently has no need shown to in be ECFCs metabolized [22]. to induce intracellular Ca signals in human brain microvascular endothelial cells. A B 1.4 0.6 AA 30 100 300

380) 1.2 0.3 µM 0.4 (269)

/F (73) 3 µM (125) 340 1.0 30 µM Fura-2 Ratio) 0.2 3 EC = 8.4 µM 300 µM Δ 50 1 0.8 0.3 (75) 10 Ratio(F 0.0 (83) (83) (50) Peak ( Peak 0.6 0 200 400 600 800 -6.5 -5.5 -4.5 -3.5 Time (sec) log [AA] C 1.4 AA AA

380) 1.2 /F

340 1.0

0.8 Ratio (F

0.6 0 300 600 900 1200 1500 1800 Time (sec)

2+ 2+ Figure 1. Arachidonic acid (AA) induces an increase in intracellular Ca concentration ([Ca ]i) in 2+ 2+ hCMECFigure/D3 1. cells. Arachidonic (A) Intracellular acid (AA) Ca induces2+ signals an increase evoked byin intracellular increasing doses Ca concentration of AA in the hCMEC([Ca ]i) /inD3 2+ cellhCMEC/D3 line. In this cells. and (A the) Intracellular following figures,Ca signals agonists evoked and by drugs increasing were doses administered of AA in the as indicatedhCMEC/D3 by cell line. In this and the following figures, agonists and drugs were administered as indicated by the the horizontal bars above the traces. (B) Dose–response relationship of the amplitude of AA-evoked horizontal bars above the traces. (B) Dose–response relationship of the amplitude of AA-evoked Ca2+ Ca2+ signals in hCMEC/D3 cells. The continuous line was obtained from a fit to a sigmoidal signals in hCMEC/D3 cells. The continuous line was obtained from a fit to a sigmoidal2+ concentration-response curve by using Equation (1). (C) 30 µM AA evoked a rapid increase in [Ca ]i concentration-response curve by using Equation (1). (C) 30 μM AA evoked a rapid increase in [Ca2+]i that decayed to the baseline upon agonist removal from the perfusate. When AA was re-added at that decayed to the baseline upon agonist removal2 +from the perfusate. When AA was re-added at the the same dose, it induced a second elevation in [Ca ]i that achieved the same peak amplitude as the same dose, it induced a second elevation in [Ca2+]i that achieved the same peak amplitude as the first first one. one. 3.2. AA-Induced Intracellular Ca2+ Signals Required Endogenous Ca2+ Release and Extracellular Ca2+ Influx As synaptic AA concentration may achieve the mid-to-high micromolar range [29–31], we used 2+ 2+ 30As μM mentioned AA throughout earlier, the AA-induced remainder endothelialof the study. Ca The signalsCa2+ response impinge to onAA endogenous (30 μM) was Ca reversiblerelease 2+ 2+ andas, extracellular when AA was Ca removedinflux [18from–21 the]. In bath order at the to addresspeak of thisthe Ca issue,2+ rise, we the evaluated [Ca2+]i rapidly the Ca returnedresponse to to AAthe inbaseline, hCMEC whereas/D3 cells the in thesubsequent absence a ofddition extracellular of AA raised Ca2+ (0Caintracellular2+), after Ca which2+ levels extracellular again (Figure Ca2 + was1C). restituted AA-induced while endothelial maintaining Ca the2+ signals agonist may in be the triggered perfusate. by Undermany of 0Ca its2 +derivatives,conditions, such 30 µasM 5,6- AA 2+ 2+ inducedepoxyeicosatrienoic a transient elevation (5,6-EET) in and [Ca 8,9-EET]i, which and was leukotriene due to Ca C4, whichmobilization are synthesized, from the respectively, intracellular stores,by cytochrome whereas reintroduction P450 epoxygenase of external and glutathione Ca2+ caused S-transferase a second intracellular 2 [32–36]. Therefore, Ca2+ wave we (Figure evaluated3A), whichthe wasCa2+ sustainedresponse to by the extracellular non-metabolized Ca2+ influx AA analogue [22]. , eicosatetraynoic acid (ETYA), as described inThe other same types results of endothelial were obtained cells [22,37]. when As AA shown concentration in Figure 2, was ETYA raised (30 μ upM) to induced 50 µM an (Figure increase3B). 2+ 2+ As expectedin [Ca ]i whose [22], endogenous kinetics and Ca amplitude2+ release were and similar extracellular to those Ca of2+ theinflux Ca were signal significantly induced by( pAA< 0.05)(30 higherμM) at thein the highest same AA batches concentration of hCMEC/D3 (Figure3 C).cells Overall, (Figure these 2A,B). findings Taken demonstrate together, thatthese intracellular findings demonstrate that AA has no need to be metabolized to induce intracellular Ca2+ signals in human Ca2+ release and extracellular Ca2+ influx mediate AA-evoked Ca2+ signals in hCMEC/D3 cells. brain microvascular endothelial cells.

3.2. AA-Induced Intracellular Ca2+ Signals Required Endogenous Ca2+ Release and Extracellular Ca2+ Influx

Cells 2019, 8, x 6 of 20

As mentioned earlier, AA-induced endothelial Ca2+ signals impinge on endogenous Ca2+ release and extracellular Ca2+ influx [18–21]. In order to address this issue, we evaluated the Ca2+ response to AA in hCMEC/D3 cells in the absence of extracellular Ca2+ (0Ca2+), after which extracellular Ca2+ was restituted while maintaining the agonist in the perfusate. Under 0Ca2+ conditions, 30 μM AA induced a transient elevation in [Ca2+]i, which was due to Ca2+ mobilization from the intracellular stores,Cells whereas2019, 8, 689 reintroduction of external Ca2+ caused a second intracellular Ca2+ wave (Figure 3A),6 of 18 which was sustained by extracellular Ca2+ influx [22]. A B 1.6 AA 30 μM / ETYA 30 μM 0.60

1.4

380) 0.45

/F 1.2 340 0.30

1.0 Ratio) Fura-2 73 Δ 52 0.15

Ratio (F 0.8

0.6 ( Peak 0.00 0 200 400 600 800 1000 1200 1400 Time (sec) AA ETYA

Figure 2. Arachidonic acid (AA) metabolism is not necessary for arachidonic acid-induced elevation in 2+ [Ca ]i in hCMEC/D3 cells. (A) 30 µM eicosatetraynoic acid (ETYA), a non-metabolizable analogue of AA, elicits an intracellular Ca2+ signal which overlaps with the Ca2+ response to 30 µM AA in CellshCMEC 2019, 8, /xD3 cells. (B) Mean SE of the amplitude of ETYA- and AA-induced elevations in [Ca2+] . 7 of 20 Figure 2. Arachidonic acid (AA)± metabolism is not necessary for arachidonic acid-induced elevation i in [Ca2+]i in hCMEC/D3 cells. (A) 30 μM eicosatetraynoic acid (ETYA), a non-metabolizable analogue of AA, elicits an intracellular Ca2+ signal which overlaps with the Ca2+ response to 30 μM AA in hCMEC/D3A cells. (B) Mean ± SE of the amplitude of ETYA-B and AA-induced elevations in [Ca2+]i. 1.4 AA 50 µM AA 30 µM 1.6 2+ +Ca2+ 2+ The same results were+Ca obtained when AA+Ca concentration2+ was1.4 raised up to 50 μM (Figure+Ca 3B). As 380) 2+ 380) 0Ca2+ /F 1.2 2+0Ca 2+ expected [22], endogenous Ca release and extracellular Ca/F 1.2 influx were significantly (p < 0.05) 340 higher at the highest AA concentration (Figure 3C). Overall,340 these findings demonstrate that 1.0 intracellular Ca2+ release1.0 and extracellular Ca2+ influx mediate AA-evoked Ca2+ signals in hCMEC/D3 Ratio (F cells. Ratio(F 0.8 0.8 0.6 0 500 1000 1500 0 500 1000 1500 2000 Time (sec) Time (sec) C 1.0 Ca2+ release 0.8 * Ca2+ entry * 2+ 0.6 Ca release * Ca2+ entry

Fura-2 Ratio) Fura-2 0.4 Δ 51 51 81 0.2 81 Peak ( Peak 0.0 30 μM 50 μM

Figure 3. Arachidonic acid induces endogenous Ca2+ release and extracellular Ca2+ inﬂux in hCMEC/D3 cells.Figure (A )3. 30 ArachidonicµM AA evoked acid endogenous induces endogenous Ca2+ release Ca under2+ release absence and of extracellular extracellular Ca22++ (0Cainflux2+ )in hCMEC/D3 cells. (A) 30 μM AA evoked endogenous2+ Ca2+2 +release under absence of extracellular Ca2+ conditions followed by a second increase in [Ca ]i upon Ca restitution to the bath in the continuous presence(0Ca2+) conditions of the agonist. followed (B) 50 byµ aM second AA evoked increase endogenous in [Ca2+]i upon Ca2+ Carelease2+ restitution under 0Ca to 2the+ conditions bath in the 2+ 2+ 2+ 2+ followedcontinuous by a secondpresence increase of the in agonist. [Ca ]i upon(B) 50 Ca μM restitutionAA evoked to theendogenous bath in the Ca continuous release presenceunder 0Ca of theconditions agonist. (followedC) Mean bySE a second of the amplitude increase in of [Ca endogenous2+]i upon Ca Ca2+2 restitution+ release and to the extracellular bath in the Ca continuous2+ inﬂux ± inducedpresence by of each the concentrationagonist. (C) Mean of AA. ± SE The of asteriskthe amplitude indicates of pendogenous< 0.05. Ca2+ release and extracellular Ca2+ influx induced by each concentration of AA. The asterisk indicates p < 0.05. 2+ 3.3. InsP3R3 and TPC1-2 Mediate AA-Induced Endogenous Ca Release

3.3. InsP3R3 and TPC1-2 Mediate AA-Induced Endogenous Ca2+ Release 2+ Recent studies demonstrated that InsP3Rs and TPC1-2 mediate AA-induced endogenous Ca

releaseRecent in human studies ECFCs demonstrated [22] and rat pancreatic that InsP3βRscells and [38 TPC1-2]. Of note, mediate a thorough AA-induced characterization endogenous of their Ca2+ 2+ Careleasetoolkit in human disclosed ECFCs that hCMEC[22] and/D3 rat cells pancreatic express β only cells one [38]. of theOf note, three a InsP thorough3R isoforms, characterization i.e. InsP3R3, of andtheir TPC1-2, Ca2+ toolkit while disclosed they lack InsPthat 3hCMEC/D3R1-2 and RyR1-3 cells express [17]. We only ﬁrst one focused of the on three the contributionInsP3R isoforms, of ER i.e. InsP3R3, and TPC1-2, while they lack InsP3R1-2 and RyR1-3 [17]. We first focused on the contribution of ER Ca2+ release through InsP3R3 to AA-induced intracellular Ca2+ release. As shown in Figure 4A, the endogenous Ca2+ response to AA (30 μM) was abrogated by 2-aminoethoxydiphenyl borate (2-APB) (50 μM, 20 min) (Figure 4B), which inhibits InsP3Rs under 0Ca2+ conditions [17,39], and by depletion of the ER Ca2+ content with cyclopiazonic acid (CPA) (10 μM) (Figure 4C). As previously described [17], CPA caused a transient elevation in [Ca2+]i by unmasking the passive ER Ca2+ leak followed by Ca2+ extrusion across the plasma membrane under 0Ca2+ conditions (Figure 4C). The following addition of AA (30 μM) under these conditions failed to increase the [Ca2+]i, thereby confirming the role of ER Ca2+ in AA-evoked intracellular Ca2+ response. Next, we evaluated the contribution of lysosomal Ca2+ release through TPC1-2. The Ca2+ response to AA (30 μM) was prevented upon disruption of the lysosomal Ca2+ pool with glycyl-L-phenylalanine 2-naphthylamide (GPN) (200 μM) administered under 0Ca2+ conditions (Figure 4D). In addition, AA-evoked intracellular Ca2+ mobilization was suppressed by NED-19 (100 μM, 30 min) (Figure 4E), which selectively blocks TPC1-2 [17,40]. The statistical analysis of these results is shown in Figure 4F. In aggregate, these data provide the evidence that AA-evoked intracellular Ca2+ mobilization in hCMEC/D3 cells is mediated by InsP3R3 and TPC1-2.

Cells 2019, 8, 689 7 of 18

2+ 2+ Ca release through InsP3R3 to AA-induced intracellular Ca release. As shown in Figure4A, the endogenous Ca2+ response to AA (30 µM) was abrogated by 2-aminoethoxydiphenyl borate (2-APB) 2+ (50 µM, 20 min) (Figure4B), which inhibits InsP 3Rs under 0Ca conditions [17,39], and by depletion of the ER Ca2+ content with cyclopiazonic acid (CPA) (10 µM) (Figure4C). As previously described [ 17], 2+ 2+ 2+ CPA caused a transient elevation in [Ca ]i by unmasking the passive ER Ca leak followed by Ca extrusion across the plasma membrane under 0Ca2+ conditions (Figure4C). The following addition of 2+ AA (30 µM) under these conditions failed to increase the [Ca ]i, thereby conﬁrming the role of ER Ca2+ in AA-evoked intracellular Ca2+ response. Next, we evaluated the contribution of lysosomal Ca2+ release through TPC1-2. The Ca2+ response to AA (30 µM) was prevented upon disruption of the lysosomal Ca2+ pool with glycyl-L-phenylalanine 2-naphthylamide (GPN) (200 µM) administered under 0Ca2+ conditions (Figure4D). In addition, AA-evoked intracellular Ca2+ mobilization was suppressed by NED-19 (100 µM, 30 min) (Figure4E), which selectively blocks TPC1-2 [17,40]. The statistical analysis of these results is shown in Figure4F. In aggregate, these data provide the evidence that AA-evoked intracellular Ca2+ mobilization in Cells 2019, 8, x 8 of 20 hCMEC/D3 cells is mediated by InsP3R3 and TPC1-2. A B C 1.6 1.4 μ 1.4 2-APB 50 M 2+ 0Ca2+ 0Ca 1.4 0Ca2+ 380) 380) 1.2 380) 1.2 CPA 10 μM /F /F μ /F 1.2 AA 30 M AA 30 μM 340 340

340 μ 1.0 1.0 AA 30 M 1.0 0.8 0.8

Ratio(F 0.8 Ratio(F Ratio (F

0.6 0.6 0.6 0 500 1000 1500 0 200 400 600 800 1000 0 500 1000 1500 2000 2500 3000 Time (sec) Time (sec) Time (sec)

D E F 1.6 1.4 0Ca2+ NED-19 100 μM 0.25 *

1.4 0Ca2+ 0.20 380) GPN 200 μM 380) 1.2 380) /F /F 1.2 AA 30 μM /F 0.15 340 340 AA 30 μM 1.0 340 1.0 0.10 81 77 65 62 95 0.8

Ratio (F 0.8 Ratio (F

Ratio (F 0.05

0.6 0.6 0.00 + 0 500 1000 1500 2000 2500 0 200 400 600 800 1000 2 ) Time (sec) Time (sec) PB -A CPA GPN D-19 2 E N Ctrl (0Ca

Figure 4. Type 3 inositol-1,4,5-trisphosphate receptor 3 InsP3R3 and two-pore channels 1 and 2 (TPC1-2) 2+ mediateFigure endogenous4. Type 3 inositol-1,4,5-trisphosphate Ca release in response to arachidonicreceptor 3 InsP acid3 inR3 hCMEC and two-pore/D3 cells. channels (A) Endogenous 1 and 2 Ca(TPC1-2)2+ mobilization mediate endogenous induced by AACa2+ (30releaseµM) in under response 0Ca2 to+ conditions.arachidonic (acidB) 2-aminoethoxydiphenyl in hCMEC/D3 cells. (A) borateEndogenous (2-APB; Ca 50 2+µ M,mobilization 30 min) prevents induced AA-evoked by AA endogenous (30 μM) Caunder2+ release. 0Ca2+ In conditions. this and in the(B) following2-aminoethoxydiphenyl panels, AA has alwaysborate been(2-APB; applied 50 atμM, 30 µ30M. min) (C) Cyclopiazonic prevents AA-evoked acid (CPA; endogenous 10 µM) inhibited Ca2+ Sarco-Endoplasmicrelease. In this and Reticulumin the following Ca2+-ATPase panels, AA (SERCA) has always activity, been thereby applied eliciting at 30 μ aM. transient (C) Cyclopiazonic elevation 2+ 2+ inacid [Ca (CPA;]i followed 10 μM) byinhibited Ca removal Sarco-Endoplasmic from the cytosol. Reticulum The following Ca2+-ATPase addition (SERCA) of AA activity, did not thereby result 2+ ineliciting an increase a transient in [Ca elevation]i.(D) Glycyl-L-phenylalaninein [Ca2+]i followed by Ca 2-naphthylamide2+ removal from the (GPN; cytosol. 200 µ TheM) elicitedfollowing a 2+ 2+ transientaddition elevationof AA did in not [Ca result]i due in an to increase the disruption in [Ca2+ of]i. ( theD) Glycyl-L-phenylalani lysosomal Ca poolne and 2-naphthylamide the following 2+ liberation(GPN; 200 of μ lysosomalM) elicited Caa transientcontent elevation into the in cytosol [Ca2+]i [due41,42 to]. the The disruption following of addition the lysosomal of AA Ca did2+ pool not 2+ 2+ resultand the in anfollowing increase liberation in [Ca ]ofi.( lysosomalE) NED-19) Ca (1002+ contentµM, 30 into min) the blocked cytosol AA-evoked[41,42]. The following intracellular addition Ca 2+ release.of AA (didF) Meannot resultSE ofin thean amplitudeincrease in of [Ca the2+ intracellular]i. (E) NED-19) Ca (100response μM, 30 to min) AA under blocked the designatedAA-evoked ± treatments.intracellular The Ca asterisk2+ release. indicates (F) Meanp < ±0.05. SE of the amplitude of the intracellular Ca2+ response to AA under the designated treatments. The asterisk indicates p < 0.05.

3.4. TRPV4 is Expressed and Mediates AA-Evoked Extracellular Ca2+ Influx

TRPV4 is virtually the only Ca2+-permeable pathway responsible for AA-induced extracellular Ca2+ influx in endothelial cells [22,37,43,44]. Therefore, we first assessed whether a functional TRPV4 protein was expressed in hCMEC/D3 cells. We carried out a Western Blot analysis by exploiting an affinity-purified antibody, which detected a major band at 91 kDa (Figure 5A), the expected molecular weight for TRPV4 [45]. Thereafter, we exposed hCMEC/D3 cells to GSK (20 nM), a specific TRPV4 agonist [45,46]. GSK (20 nM) caused a robust increase in [Ca2+]i in the presence, but not in the absence, of extracellular Ca2+ (Figure 5B), which is consistent with the notion that TRPV4 mediates extracellular Ca2+ influx. Notably, GSK-induced Ca2+ influx was suppressed by RN-1734 (20 μM, 20 min), a selective TRPV4 blocker [45,47], in the majority of the cells, whereas it was remarkably inhibited in the remaining (Figure 5C,D). These preliminary experiments provide the evidence that TRPV4 is expressed and mediates extracellular Ca2+ influx in hCMEC/D3 cells.

Cells 2019, 8, 689 8 of 18

3.4. TRPV4 is Expressed and Mediates AA-Evoked Extracellular Ca2+ Influx TRPV4 is virtually the only Ca2+-permeable pathway responsible for AA-induced extracellular Ca2+ influx in endothelial cells [22,37,43,44]. Therefore, we first assessed whether a functional TRPV4 protein was expressed in hCMEC/D3 cells. We carried out a Western Blot analysis by exploiting an affinity-purified antibody, which detected a major band at 91 kDa (Figure5A), the expected molecular weight for TRPV4 [45]. Thereafter, we exposed hCMEC/D3 cells to GSK (20 nM), a specific TRPV4 2+ agonist [45,46]. GSK (20 nM) caused a robust increase in [Ca ]i in the presence, but not in the absence, of extracellular Ca2+ (Figure5B), which is consistent with the notion that TRPV4 mediates extracellular Ca2+ influx. Notably, GSK-induced Ca2+ influx was suppressed by RN-1734 (20 µM, 20 min), a selective TRPV4 blocker [45,47], in the majority of the cells, whereas it was remarkably inhibited in the remaining (Figure5C,D). These preliminary experiments provide the evidence that TRPV4 is expressed and mediatesCells 2019, 8 extracellular, x Ca2+ influx in hCMEC/D3 cells. 9 of 20

Figure 5.5. TransientTransient Receptor Receptor Potential Potential Vanilloid Vanilloid 4 (TRPV4) protein is expressedexpressed andand mediatesmediates 2+ extracellularextracellular Ca Ca2+ influxinflux in inhCMEC/D3 hCMEC/D3 cells. cells. (A) Left, (A) representative Left, representative Western Western blots illustrating blots illustrating TRPV4 TRPV4and β-actin and β -actinexpression expression in HEK-293 in HEK-293 cells cells (HEK) (HEK) and and hCMEC/D3 hCMEC/D3 cells. cells. Left, Left, quantification quantification ofof TRPV4TRPV4//β-actin protein levels in hCMEChCMEC/D3/D3 cells normalizednormalized on HEK cellscells fromfrom threethree independentindependent experiments. (B) GSK1016790A (GSK; 20 nM) increased the [Ca2+] in the presence, but not in the experiments.(B) GSK1016790A (GSK; 20 nM) increased the [Ca2+]i iin the presence, but not in the 2+ 2+ µ absenceabsence (0Ca(0Ca2+),), of of extracellular extracellular Ca 2+. .((CC) )RN-1734 RN-1734 (20 (20 μM,M, 20 20 min) min) blocked blocked GSK-evoked GSK-evoked extracellular 2+ 2+ Ca 2+ influx. GSK was administered at 20 nM. (D) Mean ± SE of the amplitude of GSK-induced Ca 2+ Ca influx. GSK was administered at 20 nM. (D) Mean ± SE of the amplitude of GSK-induced Ca influxinflux inin thethe absenceabsence andand inin thethe presencepresence ofof RN-1734RN-1734 (RN).(RN). TheThe totaltotal numbernumber ofof cellscells analysedanalysed inin thethe p presencepresence ofof RNRN isis indicatedindicated inin thethe brackets.brackets. TheThe asteriskasterisk indicatesindicates p << 0.05. Next, we explored the role of TRPV4 in AA-evoked intracellular Ca2+ signals. In agreement Next, we explored the role of TRPV4 in AA-evoked intracellular Ca2+ signals. In agreement with with previous reports in endothelial cells [22,37,43,44], the pharmacological blockade of TRPV4 with previous reports in endothelial cells [22,37,43,44], the pharmacological blockade of TRPV4 with RN-1734 (20 µM, 20 min) decreased the magnitude and curtailed the duration of AA-evoked Ca2+ RN-1734 (20 μM, 20 min) decreased the magnitude and curtailed the duration of AA-evoked Ca2+ response (Figure6A,B). To further confirm this observation, we assessed whether AA could increase response2+ (Figure 6A,B). To further confirm2+ this observation, we assessed whether AA could increase the [Ca ]i after exhaustion of the ER Ca pool and full SOCE activation with CPA (10 µM) in the the [Ca2+]i after exhaustion of the ER Ca2+ pool and full SOCE activation with CPA (10 μM) in the presence of extracellular Ca2+. Accordingly, SOCE is elicited by pharmacological depletion of the presence of extracellular Ca2+. Accordingly, SOCE is elicited by pharmacological depletion of the ER ER Ca2+ reservoir with CPA and can no longer be activated if ER cisternae are not replenished with Ca2+ reservoir with CPA and can no longer be activated if ER cisternae are not replenished with Ca2+ because of CPA-dependent SERCA inhibition [17,48]. Therefore, under these conditions, any extracellular stimulation will be able to increase the [Ca2+]i only by mainly acting on store-independent Ca2+-permeable channels, such as TRPV4, located on the plasma membrane [22,24]. Accordingly, previous experiments revealed that AA did not evoke any Ca2+ signal under 0Ca2+ conditions and following treatment with CPA (Figure 4C). Indeed, when AA (30 μM) was added upon SOCE activation with CPA (10 μM) for 30 min (early response to CPA not shown) in the presence of extracellular Ca2+, it elicited a sustained elevation in [Ca2+]i that was suppressed by RN-1734 (20 μM, 20 min) (Figure 6C,D). Therefore, these findings clearly show that TRPV4 mediates AA-induced Ca2+ influx in hCMEC/D3 cells.

Cells 2019, 8, 689 9 of 18

Ca2+ because of CPA-dependent SERCA inhibition [17,48]. Therefore, under these conditions, any 2+ extracellular stimulation will be able to increase the [Ca ]i only by mainly acting on store-independent Ca2+-permeable channels, such as TRPV4, located on the plasma membrane [22,24]. Accordingly, previous experiments revealed that AA did not evoke any Ca2+ signal under 0Ca2+ conditions and following treatment with CPA (Figure4C). Indeed, when AA (30 µM) was added upon SOCE activation with CPA (10 µM) for 30 min (early response to CPA not shown) in the presence of extracellular 2+ 2+ Ca , it elicited a sustained elevation in [Ca ]i that was suppressed by RN-1734 (20 µM, 20 min) (Figure6C,D). Therefore, these ﬁndings clearly show that TRPV4 mediates AA-induced Ca 2+ inﬂux in Cells 2019, 8, x 10 of 20 hCMEC/D3 cells.

AB2.0 AA 30 μM 0.7 * Ctrl 380) 1.7

/F RN-1734 20 μM 0.5

340 1.4 139

Fura-2 Ratio) 0.3 1.1 Δ Ratio (F 0.1 51

0.8 Peak( 0 200 400 600 800 1000 1200 1400 trl Time (sec) C RN C D 1.9 CPA 10 μM 0.7 AA 30 μM *

380) 1.6

/F Ctrl 0.5 340 1.3 RN-1734

20 μM Fura-2 Ratio) 0.3 1.0 Δ 64 49 Ratio (F 0.1

0.7 Peak( 0 500 1000 1500 Time (sec) N Ctrl R 2+ Figure 6. TRPV4 mediates arachidonic acid-induced extracellular Ca inﬂux in hCMEC/D3cells. (A) Intracellular Ca2+ response to AA (30 µM) recorded in the presence and in the absence of RN-1734 2+ (20FigureµM, 6. 20 TRPV4 min). (mediatesB) Mean arachidonicSE of the amplitude acid-induced of AA-evoked extracellular Ca Ca2+ inﬂux influx in in the hCMEC/D3 absence and cells. in the(A) 2+ ± presenceIntracellular of RN-1734 Ca response (RN). to The AA asterisk (30 μM) indicates recordedp

Cells 2019, 8, 689 10 of 18

(Figure7B). These data, therefore, indicate that AA-evoked Ca 2+ signalling results in NO release also Cells 2019, 8, x 11 of 20 in hCMEC/D3 cells. AB 0.50 AA 30 μM 0.08 *

0.06 ) 0.45 Ctrl 535 BAPTA DAF-FM)

(F 0.04 i L-NAME Δ

NO 0.40 0.02 214 19 NR Peak ( Peak (143) (127) 0.35 0.00 0 200 400 600 800 1000 1200 1400 rl E Time (sec) Ct AM -N C D BAPTA L 0.60 AA 30 μM / ETYA 30 μM 0.15 0.55

) 0.10

535 0.50 DAF-FM) (F i 0.45 Δ 100 130

NO 0.05 0.40 Peak ( Peak

0.35 0.00 0 400 800 1200 1600 A Time (sec) AA ETY Figure 7. Arachidonic acid induces NO release in a Ca2+-dependent manner in hCMEC/D3 cells. (FigureA) AA 7. (20 ArachidonicµM) evoked acid a robustinduces increase NO release in the in fluorescencea Ca2+-dependent in hCMEC manner/D3 in cellshCMEC/D3 loaded cells. with the(A) NO-selectiveAA (20 μM) fluorophore,evoked a robust 4-amino-5-methylamino-2’,7’-difluorofluorescein increase in the fluorescence in hCMEC/D3(DAF-FM). cells loaded AA-elicited with the G NONO-selective release was fluorophore, abrogated by4-amin L-N o-5-methylamino-2’,7’-difluorofl-Nitro-L-arginine methyl ester (L-NAME;uorescein (DAF-FM). 100 µM, 1 h) AA-elicited or BAPTA (30 µM, 2 h). (B) Mean SE of the amplitudeG of AA-elicited NO release under the designated treatments. NO release was abrogated± by L-N -Nitro-L-arginine methyl ester (L-NAME; 100 μM, 1 h) or BAPTA For(30 L-NAMEμM, 2 h). and (B) BAPTA,Mean ± the SE number of the ofamplitude total cells of analysed AA-elicited is indicated NO release in the under brackets. the Thedesignated asterisk indicatestreatments.p < For0.05. L-NAME (C) AA and (30 BAPTA,µM) and the ETYA number (30 µ ofM) total induced cells ananalysed increase is indicated in DAF-FM in fluorescencethe brackets. with overlapping kinetics and amplitude. (D) Mean SE of the amplitude of AA- and ETYA-evoked The asterisk indicates p < 0.05. (C) AA (30 μM) and ±ETYA (30 μM) induced an increase in DAF-FM NOfluorescence release in with hCMEC overlapping/D3 cells. kinetics NR means and no amplitude. response. (D) Mean ± SE of the amplitude of AA- and ETYA-evoked NO release in hCMEC/D3 cells. NR means no response. In order to dissect the contribution of extra- and intracellular Ca2+ sources to NO production, hCMEC D3 cells were challenged with AA (30 µM) under 0Ca2+ conditions. Removal of external Ca2+ In order/ to dissect the contribution of extra- and intracellular Ca2+ sources to NO production, p 2+ significantlyhCMEC/D3 cells ( < 0.05)were reducedchallenged AA-elicited with AA NO (30 release,μM) under whereas 0Ca2+ restoration conditions. of Removal extracellular of external Ca levels Ca2+ causedsignificantly a further (p < elevation 0.05) reduced in DAF-FM AA-elicited fluorescence NO release, (Figure 8whereasB). Accordingly, restoration also theof extracellular pharmacological Ca2+ µ blockagelevels caused of TRPV4 a further with RN-1734elevation (20 in DAF-FMM, 30 min) fluorescence remarkably (Figure impaired 8B). AA-elicited Accordingly, NO synthesisalso the (Figurepharmacological8C). We adopted blockage the same of pharmacologicalTRPV4 with RN-1734 strategy as(20 that μM, described 30 min) in Sectionremarkably 3.3 to dissectimpaired the 2+ contributionAA-elicited NO of the synthesis ER and lysosomal(Figure 8C). Ca Westores adopted to AA-induced the same NO pharmacological release. Surprisingly, strategy inhibition as that µ 2+ ofdescribed InsP3R3 in with Section 2-APB 3.3 (50 to M,dissect 20 min) the (Figure contribution8D) and of depletion the ER ofand the lysosomal ER Ca storeCa2+ withstores CPA to (10 µM, 30 min) (Figure8E) did not a ffect the NO signal. Conversely, preventing lysosomal Ca2+ release AA-induced NO release. Surprisingly, inhibition of InsP3R3 with 2-APB (50 μM, 20 min) (Figure 8D) µ µ throughand depletion TPC1-2 of withthe ER GPN Ca2+ (200 store withM, 30 CPA min) (10 (Figure μM, 308F) min) and (Figure NED-19 8E) (100 did notM, affect 30 min) the (Figure NO signal.8G) abolishedConversely, AA-induced preventing NO lysosomal release. Altogether,Ca2+ release these through findings TPC1-2 strongly with suggestGPN (200 that μ TPC1-2M, 30 min) and (Figure TRPV4 2+ are8F) mainlyand NED-19 responsible (100 μM, for 30 the min) Ca (Figure-dependent 8G) abolished recruitment AA-induced of eNOS induced NO release. by AA Altogether, in hCMEC these/D3 cells.findings However, strongly the suggest slow kinetics that TPC1-2 of NO and release TRPV4 are notare compatible mainly responsible with a direct for the involvement Ca2+-dependent of AA inrecruitment NVC. of eNOS induced by AA in hCMEC/D3 cells. However, the slow kinetics of NO release are not compatible with a direct involvement of AA in NVC.

Cells 2019, 8, 689 11 of 18 Cells 2019, 8, x 12 of 20

A B AA 30 μM 0.55 AA 30 μM 0.6 Ca2+ Ca2+ 2+ 0.50 0Ca ) ) 535 535 0.5 0.45 (F (F i i NO 0.40 NO 0.4

0.35 0 500 1000 1500 2000 2500 0 400 800 1200 1600 Time (sec) Time (sec) CD AA 30 μM 0.55 AA 30 μM 0.55 2-APB 50 μM μ 0.50 RN-1734 20 M 0.50 ) ) 535 535 0.45 (F 0.45 (F i i NO NO 0.40 0.40

0.35 0 500 1000 1500 0 500 1000 1500 Time (sec) Time (sec)

E AA 30 μM F 0.55 CPA 10 μM 0.55 AA 30 μM GPN 200 μM 0.50 0.50 ) ) 535 535 0.45 0.45 (F (F i i NO NO 0.40 0.40

0.35 0 400 800 1200 1600 2000 2400 2800 0 200 400 600 800 1000 Time (sec) Time (sec) G H 0.55 μ * AA 30 M 0.15 * NED-19 100 μM 0.50 * ) * 0.10 79

535 133

0.45 DAF-FM) (F i Δ 81 56 82

NO 0.05 150 (121)(133) 104 0.40 (181) (220) Peak ( Peak

0.35 0.00 0 400 800 1200 1600 a Time (sec) RN PB Ctrl 0C A CPA GPN 2- NED-19

Figure 8. TPC1-2 and TRPV4 support arachidonic acid-induced NO production in hCMEC/D3 cells. (AFigure) AA (308. TPC1-2µM) evoked and TRPV4 robust support NO release arachidonic under controlacid-induced conditions NO production in hCMEC in/D3 hCMEC/D3 cells. (B) AAcells. (30(Aµ) M)AA induced (30 μM) NO evoked release robust under NO 0Ca release2+ conditions, under control although conditions this signal in hCMEC/D3 was lower ascells. compared (B) AA to(30 2+ controlμM) induced conditions. NO However, release under readdition 0Ca of conditions, Ca2+ to the although perfusate this caused sign aal second was lower increase as compared in DAF-FM to 2+ fluorescence.control conditions. (C) RN-1734 However, (20 µ readditionM, 20 min) of reduced Ca to AA-evokedthe perfusate NO caused release. a second AA was increase administered in DAF-FM at 30fluorescence.µM. (D) 2-APB (C) (50RN-1734µM, 30 (20 min) μM, did 20 not min) aff ectreduced AA-induced AA-evoked NO production. NO release. AA AA was was administered administered at at 3030µ M.μM. (E ()D CPA) 2-APB (10 µ (50M, 30μM, min) 30 min) did not did impair not affect AA-evoked AA-induced NO release. NO production. AA was administered AA was administered at 30 µM. (Fat) GPN30 μM. (200 (Eµ) M,CPA 30 (10 min) μM, remarkably 30 min) did reduced not impair AA-induced AA-evoked NO NO production. release. AA AA was was administered administered at at30 30 μµM.M. (GF)) GPN NED-19 (200 (100 μM,µM, 30 30 min) min) remarkably impaired AA-evoked reduced AA NO-induced release. NO AA wasproduction. administered AA was at 30administeredµM. (H) Mean at 30SE μM. of the(G) amplitude NED-19 (100 of AA-evoked μM, 30 min) NO impaired release underAA-evoked the designated NO release. treatments. AA was ± ± Theadministered asterisk indicates at 30 pμM.< 0.05. (H) TheMean total numberSE of the of analysedamplitude cells of isAA-evoked indicatedin NO the release brackets. under the designated treatments. The asterisk indicates p < 0.05. The total number of analysed cells is indicated 4. Discussionin the brackets. Herein, we demonstrate for the first time that the conditionally essential PUFA AA is able to elicit NO4. Discussion release from a human brain microvascular endothelial cell line in a Ca2+-dependent manner. The Ca2+ responseHerein, towe AA demonstrate involves endogenous for the first Ca time2+ release that the from conditionally the ER and lysosomal essential CaPUFA2+ pools AA through,is able to 2+ respectively,elicit NO release InsP3R3 from and TPC1-2a human and brain extracellular microvascular Ca influx endothelial through TRPV4.cell line However, in a Ca only2+-dependent TPC1-2 channelsmanner. and The TRPV4 Ca2+ response are seemingly to AA coupledinvolves to endogenous eNOS activation Ca2+ release and NO from release. the AAER and is the lysosomal precursor Ca of2+ multiplepools through, vasoactive respectively, mediators shapingInsP3R3 the and hemodynamic TPC1-2 and response extracellular to neuronal Ca2+ influx activity. through However, TRPV4. the slowHowever, kinetics only of AA-evoked TPC1-2 channels NO release and makeTRPV4 it unlikelyare seemingly that this coupled signal directlyto eNOS contributes activation to and NVC. NO release. AA is the precursor of multiple vasoactive mediators shaping the hemodynamic response to

Cells 2019, 8, 689 12 of 18

4.1. Intracellular Ca2+ Signals Induced by AA in hCMEC/D3 Cells It has long been known that synaptic activity leads to a signiﬁcant increase in the local AA concentration, which can raise up to 50–200 µM following the Ca2+-dependent recruitment of PLA2 in both postsynaptic neurons and perisynaptic astrocytes [29–31]. AA, in turn, regulates multiple synaptic processes, including neurotransmitter release [50], long-term potentiation [29,30], and local increase in CBF [2,7]. Intriguingly, a recent investigation demonstrated that 50 µM AA could elicit an increase in 2+ 2+ [Ca ]i in primary human brain endothelial cells [23]. As endothelial Ca signals are emerging as an unsuspected regulator of NVC [11,12,14,17,51] and AA may directly trigger Ca2+-dependent NO release in other endothelial cell types [18–22], we sought to assess whether it could elicit intracellular Ca2+ signals and NO release also in human cerebrovascular endothelial cells [15].

4.2. The Dose–Response Relationship of AA-Evoked Intracellular Ca2+ Signals in hCMEC/D3 Cells

2+ We found that AA induced a dose-dependent increase in [Ca ]i within the physiological 2+ concentration range that it reaches during synaptic activity. The EC50 of AA-evoked Ca signal was indeed 8.4 µM, whereas the peak Ca2+ response was attained at 300 µM. A similar analysis revealed that breast tumor-derived endothelial cells (b-TECs) displayed a higher sensitivity as compared to hCMEC/D3 cells, as the Ca2+ response to AA arose at 0.5 µM and achieved its peak at 10 µM[37]. However, AA-evoked Ca2+ signals in ECFCs appeared at concentrations higher than 2 µM and attained 2+ the maximal amplitude at 100 µM[22,45]. Likewise, a sizeable elevation in [Ca ]i was detected in human umbilical vein endothelial cells (HUVECs) challenged with 10–40 µM AA [36]. The endothelial Ca2+ response to AA can, however, be induced by many of its metabolites. For instance, 5,6-EET and 8,9-EET mediate AA-evoked Ca2+ response in mouse aortic endothelial cells [32,33], whereas leukotriene C4 is responsible for AA-induced Ca2+ inﬂux in HUVECs [36]. Nevertheless, ETYA, 2+ a non-metabolizable analogue of AA, evoked an increase in in [Ca ]i in hCMEC/D3 cells whose magnitude and kinetics were similar to those induced by AA, as also observed in bovine aortic endothelial cells [21], b-TECs [37] and ECFCs [22]. It is, therefore, possible to conclude that AA directly induces intracellular Ca2+ signalling in hCMEC/D3 cells.

2+ 4.3. The Complex Mechanism of AA-Evoked Intracellular Ca Signals in hCMEC/D3 Cells: InsP3R3, TPC1-2, and TRPV4

2+ A wide repertoire of mechanisms may be exploited by AA to increase the [Ca ]i in endothelial 2+ 2+ cells. For instance, the Ca response to AA is mediated by intracellular Ca release through InsP3Rs and TPCs and by extracellular Ca2+ influx through TRPV4 in ECFCs [22], whereas TRPV4 mediates AA-evoked Ca2+ signals in b-TECs [43], mouse aortic [32,33] and pulmonary artery [35] endothelial cells. A preliminary report showed that AA induced both endogenous Ca2+ release and extracellular Ca2+ influx in primary human brain microvascular endothelial cells. This investigation suggested 2+ 2+ the involvement of ER Ca release through InsP3Rs, but some Ca mobilization still occurred in the presence of 2-APB. Furthermore, the pathway responsible for extracellular Ca2+ influx was not evaluated [23].

4.3.1. InsP3R3 and TPC1-2 Herein, manipulation of extracellular Ca2+ levels revealed that the Ca2+ response to AA in hCMEC/D3 cells required both intra- and extracellular Ca2+ sources. AA-evoked Ca2+ release was 2+ strongly impaired by preventing InsP3R3-dependent ER Ca mobilization with 2-APB and CPA. In agreement with these observations, it has beed demonstrated that AA directly activates InsP3Rs in several cellular types [52–54]. Besides the ER Ca2+ pool, AA is capable of mobilizing lysosomal Ca2+ through the recently identiﬁed TPC1-2, as demonstrated in rat pancreatic β-cells [38] and ECFCs [22]. Accordingly, AA-evoked intracellular Ca2+ release was impaired by disrupting the lysosomal Ca2+ reservoir with GPN and by blocking TPC1-2 with NED-19. It is not clear how AA induces lysosomal Cells 2019, 8, 689 13 of 18

Ca2+ release in hCMEC/D3 cells. It has recently been demonstrated that a remarkable Ca2+-mediated cross-talk is established between the ER and lysosomal Ca2+ pools [55–57]. For instance, the ER Ca2+ mobilized by InsP3 may be directly transferred into the acidic vesicles at ER-lysosome contact sites, thereby increasing the lysosomal Ca2+ content [58]. An increase in intraluminal Ca2+ could in turn activate TPC2 and, sometimes, also TPC1 [40]. An alternative hypothesis, which does not rule out the 2+ 2+ previous one, implies that InsP3-dependent Ca release stimulates the Ca -dependent production of nicotinic acid adenine dinucleotide phosphate (NAADP), the physiological agonist of TPCs [40,59]. 2+ 2+ Finally, TPC1-2 could trigger InsP3-dependent Ca release from the ER through the Ca -induced 2+ Ca release process [60,61]. Whatever the interaction mode, it is clear that both InsP3R3 and TPC1-2 contribute to AA-evoked intracellular Ca2+ release in hCMEC/D3 cells.

4.3.2. TRPV4 As for extracellular Ca2+ influx, the available evidence hints at the involvement of TRPV4. First, a functional TRPV4 protein is expressed in these cells, as testified by Western Blot analysis and the Ca2+ response to GSK, a selective TRPV4 agonist [47]. In addition, GSK was found to induce exclusively extracellular Ca2+ influx and its action was inhibited by RN-1734, a specific TRPV4 blocker. Second, RN-1734 mimicked extracellular Ca2+ removal, by reducing the amplitude and curtailing the Ca2+ response to AA. Third, AA was still able to induce RN-1734-sensitive Ca2+ influx upon full activation of SOCE, which represents an alternative pathway for Ca2+ influx in hCMEC/D3 cells [17]. In addition, AA has long been known to inhibit [18,22,24,25], rather than inducing, SOCE. Finally, Orai3, which is expressed in hCMEC/D3 cells [17] and mediates AA-induced extracellular Ca2+ influx in HUVECs [36] and rat carotid artery vascular smooth muscle cells [62], is not directly gated by AA, but by leukotriene C4. Conversely, extracellular Ca2+ influx in hCMEC/D3 cells is not promoted by AA metabolites (see Paragraph 4.1). There is, therefore, strong evidence to support the conclusion that TRPV4 is responsible for AA-induced Ca2+ influx in hCMEC/D3 cells.

4.4. AA Induces NO Release in hCMEC/D3 Cells: The Rationale for the Present Investigation The physiological role of AA metabolites in NVC has recently been extensively reviewed [2,7]. It has been largely accepted that neuronal activity leads to PGE2 synthesis via COX-2 and COX-1 in neurons and astrocytes, respectively, to EET production via cytochrome P450 epoxygenase in glial cells, and 20-HETE synthesis in VSMCs and pericytes via cytochrome P450 4A2 [2,4–6]. Intriguingly, the vasomotor effect of AA metabolites is finely tuned by NO, which suppresses 20-HETE formation, thereby enabling PGE2-dependent vasodilation in the somatosensory cortex [9] and the molecular layer of the cerebellum [63]. It has, however, long been known that the pharmacological blockade of all the signalling pathways leading to an increase in CBF, i.e., NO, PGE2, EET, and K+ signalling, does not lead to a complete inhibition of the hemodynamic response [4]. While the molecular mechanisms driving CBF may vary along the cerebrovascular tree, the signalling mechanisms of NVC could be highly redundant in order to ensure proper blood supply to the working brain [64]. Quite unexpectedly [4], it has been recently demonstrated that the hemodynamic response may be initiated at capillary level by various mechanisms [5,10,63], including the endothelial cell ability to detect neuronal activity [13]. In addition, synaptic activity was found to induce NO-mediated vasorelaxation in penetrating arterioles and capillaries through the activation of endothelial NMDARs [11]. These in vivo findings were confirmed by the evidence, provided in vitro, that glutamate and acetylcholine were able to induce repetitive Ca2+ transients in mouse brain endothelial cells [14,16]. Likewise, acetylcholine-induced intracellular Ca2+ signals and NO release were reported in hCMEC/D3 cells [17].

4.4.1. AA Induces Delayed NO Release in hCMEC/D3 Cells As it has long been known that AA directly induces Ca2+-dependent NO release throughout peripheral circulation [18,65] and in ECFCs [22], which represent the only known truly endothelial precursors in flowing blood [66], we reasoned it was worth assessing whether this mechanism was at Cells 2019, 8, 689 14 of 18 work also in brain microvascular endothelial cells. However, NO release started with a significantly longer latency (~150 sec) as compared to the onset of the Ca2+ signal, which was immediate. The delayed kinetics of the increase in DAF-FM noted in response to AA are similar to observations in mouse brain microvascular endothelial cells challenged with glutamate [14]. As discussed elsewhere [5,14,67], DAF-FM does not truly detect NO, but it rather reacts with multiple nitrogen derivatives, including ONOO, NO2, and N2O3. We cannot, therefore, rule out the hypothesis that the NO signal arises before the increase in DAF-FM fluorescence is detectable by our imaging system. Yet, it seems unlikely that such a delayed NO release may induce the rapid increase in CBF that follows neuronal activity with a latency of ~600 msec [68]. So, it is conceivable that the major role of AA-induced NO release from human cerebrovascular endothelial cell is other than controlling NVC. Early studies demonstrated that AA-dependent NO release stimulated angiogenesis in different types of endothelial cells, including bovine aortic endothelial cells [18], b-TECs [19,43], and ECFCs [22]. Of note, AA promotes proliferation and migration also in rat brain microvascular endothelial cells [69,70]. Future investigation is required to assess whether AA-induced NO production also stimulates angiogenesis in hCMEC/D3 cells.

4.4.2. AA Induced NO Release via TPC1-2 and TRPV4 in hCMEC/D3 Cells The mechanism whereby AA induces NO release in hCMEC/D3 cells is, however, somehow different from that reported in other endothelial cell types. For instance, TRPV4 provides the source of Ca2+ necessary for AA-dependent recruitment of eNOS in b-TECs [19,43], while TRPV4 is supported by InsP3Rs and TPC1 in ECFCs [22]. Herein, we found that AA-evoked NO production in hCMEC/D3 cells was mainly driven by TPC1-2 and TRPV4 channels. Accordingly, the increase in DAF-FM fluorescence induced by AA was significantly reduced both by removal of extracellular Ca2+ and by pharmacological blockade of TRPV4 with RN-1734. Likewise, the impairment of lysosomal Ca2+ mobilization with GPN, which disrupts acidic vesicles, or NED-19, which blocks TPC1-2, suppressed AA-evoked NO release in hCMEC/D3 cells. These observations support the emerging role of lysosomal Ca2+ release in driving eNOS activation throughout vascular endothelium: TPC1 and/or TPC2 were indeed found to stimulate NO production in human and rat aortic endothelial cells [71], mouse [14] and human [17] brain microvascular endothelial cells, and ECFCs [22]. Conversely, preventing InsP3-dependent ER 2+ 2+ Ca release with 2-APB, which inhibits InsP3R3, or with CPA, which depletes the ER Ca store [17], did not significantly affect eNOS activation by AA. This result was somehow unexpected as InsP3R3 supports acetylcholine-induced NO production in hCMEC/D3 cells [17]. It is, therefore, conceivable that the pool of ER-embedded InsP3R3 is not coupled to eNOS.

5. Conclusions We provided the first evidence that AA, whose metabolites are known to modulate CBF, is per se able to induce Ca2+-dependent NO release in a human cerebrovascular endothelial cell line. The Ca2+ 2+ response to AA requires endogenous Ca release through ER-embedded InsP3R3 and lysosomal TPC1-2 and extracellular Ca2+ influx through TRPV4. However, only TPC1-2 and TRPV4 drive NO production, while InsP3R3 plays no sizeable role in this signal. This finding suggests that, besides being metabolized into the vasorelaxing mediators PGE2 and EETs, AA could induce NO release from human brain microvascular endothelial cells. It is, however, unlikely that AA-induced endothelial-dependent NO release is directly involved in the onset of the hemodynamic response to neuronal activity. These findings should, however, be confirmed in vivo by taking advantage of the availability of mouse models expressing endothelial genetic Ca2+ indicators [72]. However, a recent report confirmed that acetylcholine was able to induce endothelial NO release in vivo [73], as suggested by previous in vitro observations in hCMEC/D3 cells [17]. This same report provided the evidence that also synaptically released glutamate induced endothelial NO production [73], as previously observed in a mouse brain cerebrovascular endothelial cell line [14]. Cells 2019, 8, 689 15 of 18

Author Contributions: R.B.-R., P.F., S.N., L.B. and T.G. performed and analysed the experiments. F.M. writing— original draft preparation, supervision and funding acquisition. All the authors read and reviewed the final draft of the manuscript. Funding: This research was funded by: Italian Ministry of Education, University and Research (MIUR): Dipartimenti di Eccellenza Program (2018–2022)—Dept. of Biology and Biotechnology “L. Spallanzani”, University of Pavia (F.M. and L.B.), and by Fondo Ricerca Giovani from the University of Pavia (F.M. and L.B.). P.F. was supported by MAECI (Ministero degli Affari Esteri e della Cooperazione Internazionale). Conflicts of Interest: The authors declare no conflict of interest.

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