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

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 illustrated elsewhere (Zuccolo, Kheder, et al., 2019). Only hCMEC/D3 cells between passage 25 and 35 were employed in the present investigation. As described in, 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 the following medium: EBM-2 medium (Lonza, Basel, Switzerland) supplemented with 5% fetal bovine serum, 1% penicillin–streptomycin, 1.4 μM hydrocortisone, 5 μg/ml ascorbic acid, 1/100 chemically defined lipid concentrate (Life Technologies, Milan, Italy), 10 mM HEPES and 1 ng/ml basic fibroblast growth factor. The cells were cultured at 37°C, 5% CO₂ saturated humidity.

2.2 Solutions

Physiological salt solution (PSS) had the following composition (in mM): 150 NaCl, 6 KCl, 1.5 CaCl₂, 1 MgCl₂, 10 Glucose, 10 Hepes. In Ca²⁺-free solution (0Ca²⁺), Ca²⁺ was replaced with 2 mM NaCl, and 0.5 mM EGTA was added. Solutions were titrated to pH 7.4 with NaOH. The osmolality of PSS as measured with an osmometer (Wescor 5500, Logan, UT) was 339.5 mOsm/l.

2.3 [Ca²⁺]_i and NO measurements

Ca²⁺ imaging was carried out as described elsewhere (Zuccolo, Laforenza, et al., 2019). 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, 5% CO₂ saturated humidity. After de-esterification in PSS for 15 min, 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 alternately excited at 340 and 380 nM by using a filter wheel (Lambda 10, Sutter Instrument, Novato, CA). 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), each corresponding to a well defined single cell. (Ca²⁺)_i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when alternately exciting at 340 and 380 nm (Ratio [F₃₄₀/F₃₈₀]). An elevation in (Ca²⁺)_i induces an increase in the ratio (Zuccolo, Laforenza, et al., 2019). Ratio measurements were performed and plotted on-line every 3 s. The experiments were performed at room temperature (22°C).

NO was measured as also illustrated in (Zuccolo, Laforenza, et al., 2019). Briefly, hCMEC/D3 cells were loaded with the NO-sensitive fluorophore, 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) diacetate (10 µM) in PSS. The cells were maintained in DAF-FM for 1 hr, 37°C and 5% CO₂ saturated humidity. Subsequently, the dye was de-esterified in PSS for 1 hr. DAF-FM fluorescence was measured by using the same equipment described for Ca²⁺ recordings but with a different filter set, that is excitation at 480 nm and emission (termed “NO_i”) at 535 nm. Histamine-induced variations in DAF-FM fluorescence were recorded at room temperature (22°C) and plotted on-line every 5 s.

2.4 Immunoblotting

hCMEC/D3 cells were homogenized with a Teflon glass Potter-Elvehjem homogenizer in a solution containing: 250 mM Sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.6, 0.1 mg/ml phenylmethylsulfonyl fluoride, 100 mM β-mercaptoethanol, protease and phosphatase inhibitor cocktails (P8340 and P5726, P0044, Sigma-Aldrich Inc., Milan, Italy). The homogenate was heated at 80°C in Laemmli buffer (Laemmli, 1970), 30 µg proteins subjected to 12.5% SDS-polyacrylamide gel electrophoresis and transferred to the Hybond-P polyvinylidene fluoride membrane (GE Healthcare, Milan, Italy). After 1 hr blocking with Tris buffered saline containing 5% non fat dry milk (blocking solution) and 0.1% Tween (blocking solution), the membranes were incubated overnight with affinity purified anti-H₁R (sc-20633; Santa Cruz Biotechnology, Dallas, TX, Inc.) diluted 1:200 in the blocking solution. The membranes were washed and incubated for 1 hr with peroxidase-conjugated goat anti-rabbit immunoglobulin (AP132P; Merck Millipore, Milan, Italy) diluted 1:100.000. The bands were detected with ECL™ Select western blot analysis detection system (GE Healthcare, Milan, Italy). Blots were stripped with the method of Yeung and Stanley (2009) and reprobed with anti β-2-microglobulin (B2M) antibody (ab75853; Abcam, Cambridge, UK) as housekeeping. The antibody was diluted 1:10000 in blocking solution.

2.5 Statistical analysis

The amplitude of histamine-induced NO production was evaluated as the difference between the maximal increase in DAF fluorescence and an average of 1 min baseline before the peak. Pooled data are given as average ± standard error (SE) and statistical significance (p < .05) was evaluated by two-tailed Student's t test for unpaired observations or one-way analysis of variance analysis followed by the post-hoc Dunnett's test as appropriate. Data relative to both Ca²⁺ and NO signals are presented as mean ± SE, whereas the number of cells analyzed 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). All the chemicals were of analytical grade and obtained from Sigma Chemical Co. (St. Louis, MO).

3.1 Histamine induces intracellular Ca²⁺ oscillations in hCMEC/D3 cells

To assess whether histamine increases the [Ca²⁺]_i, hCMEC/D3 cells were loaded with the Ca²⁺-sensitive fluorescent indicator, Fura-2/AM, as described in Materials and methods. Unlike the mouse brain endothelial cell line bEND5 (Zuccolo et al., 2017; Zuccolo, Kheder, et al., 2019), hCMEC/D3 did not undergo any unsolicited elevation in [Ca²⁺]_i when no agonist was present in the perfusate (data not presented). Early work demonstrated that histamine could trigger repetitive Ca²⁺ spiking in human vascular endothelial cells at concentrations spanning from the low nanomolar up to the middle micromolar range (Q. Hu, Deshpande, Irani, & Ziegelstein, 1999; Jacob et al., 1988; Morgan & Jacob, 1996; Oike, Droogmans, & Nilius, 1994). Likewise, histamine evoked intracellular Ca²⁺ oscillations in hCMEC/D3 cells in a dose-dependent manner. Histamine did not elicit any discernible increase in [Ca²⁺]_i at very low concentrations, such as 3fM (not shown). The Ca²⁺ response to histamine appeared at 1 pM (Figure 1 and Figure 2a), when the majority of hCMEC/D3 cells produced a single Ca²⁺ transient in response to agonist stimulation (Figure 1 and Figure 2a). However, a low (≈10%) percentage of hCMEC/D3 cells challenged with 1 pM histamine displayed a short train of consecutive Ca²⁺ transients (1–4 transients, Ca²⁺ traces not shown). The dose-response relationship showed that the fraction of oscillating cells augmented with a minimum effective dose of 1 pM histamine, a maximal response at 1 nM, and an EC₅₀ of 4.26 pM (Figure 1 and Figure 2a). The dose-response relationship of the percentage of oscillating cells was fitted by a sigmoidal curve, showing that the percentage of oscillating cells did not further increase at histamine concentrations higher than 1 μM (Figure 2a). Nearly all (>92%) the cells displayed intracellular Ca²⁺ oscillations at histamine concentrations ranging from 1 nM to 300 μM (Figure 1 and Figure 2a). The dose-response relationship of the amplitude of the 1st Ca²⁺ spike and of the number of Ca²⁺ oscillations over 45 min also followed a sigmoidal curve. The magnitude of the 1st Ca²⁺ transient reached a maximal response at 1 μM and showed an EC₅₀ of 54.07 nM (Figure 2b), whereas the number of Ca²⁺ oscillations over 45 min also attained a maximal response at 1 μM and displayed an EC₅₀ of 91.83 nM (Figure 2c). The amplitudes of consecutive Ca²⁺ spikes and the interspike interval (ISI) of the oscillatory responses to 1 nM, 1 and 30 μM histamine were further evaluated. As shown in Figure 2d, the amplitude of the Ca²⁺ spikes remarkably decreased after the initial Ca²⁺ response (spike number 1), but it remained relatively constant during the Ca²⁺ train induced by each dose of histamine. Likewise, the ISI did not undergo significant variations during 45 min recording at 1 and 30 μM, while the interspike period at 1 nM was significantly larger as compared to higher doses, although the frequency of Ca²⁺ spikes accelerated after the eight spikes (Figure 2e). This statistical analysis, therefore, suggests that 1 μM histamine represents the most suitable dose to explore the mechanisms that underlie the Ca²⁺-dependent production of NO by histamine in hCMEC/D3 cells. As expected, the oscillatory Ca²⁺ response to 1 μM histamine ceased upon agonist removal from the bath, but rapidly resumed on its reapplication (Figure 2f), thereby confirming that histamine was necessary for the onset of the repetitive spikes.

3.2 Histamine-induced intracellular Ca²⁺ oscillations are triggered by H₁Rs

H₁Rs have long been known to trigger the endothelial Ca²⁺ response to histamine due to their tight coupling to PLCβ and InsP₃-dependent ER Ca²⁺ mobilization (Q. Hu et al., 1999; Kishi et al., 1996; Lin et al., 2000; Oike et al., 1994; Sheng & Braun, 2007; Toda et al., 1991). Likewise, histamine-induced intracellular Ca²⁺ oscillations in hCMEC/D3 cells were blocked by pyrilamine (10 μM, 10 min; Figure 3a,c), a selective H₁R antagonist (Adderley, Zhang, & Breslin, 2015). This treatment abrogated the oscillatory Ca²⁺ response to histamine (1 μM) in all the cells (Figure 3b). Likewise, immunoblots displayed a major band of about 56 kDa for H₁Rs (Figure 3d), as previously shown by Adderley et al. (2015). Therefore, these findings confirm that histamine-induced repetitive Ca²⁺ spikes are initiated by H₁Rs also in hCMEC/D3 cells.

3.3 Histamine-induced intracellular Ca²⁺ oscillations require extracellular Ca²⁺ entry through the SOCE pathway

Extracellular stimulation with physiological autacoids, such as acetylcholine, triggers endogenous Ca²⁺ release followed by extracellular Ca²⁺ entry in hCMEC/D3 cells (Zuccolo, Laforenza et al. 2019). Therefore, to disentangle the contribution of intra- and extracellular Ca²⁺ sources to histamine-induced intracellular Ca²⁺ oscillations (Figure 4a), we stimulated the cells upon removal of extracellular Ca²⁺ (0Ca²⁺). The Ca²⁺ response to histamine (1 μM) always arose under 0Ca²⁺ conditions, but rapidly run down despite the continuous presence of agonist (Figure 4b). Intracellular Ca²⁺ oscillations, however, could be reinitiated by exposure to 1.5 mM Ca²⁺ in the presence of histamine in 142 out of 144 cells (Figure 4b). The amplitude of the first histamine-induced Ca²⁺ transient was not reduced by removal of extracellular Ca²⁺ (Figure 4c). This behavior is different as compared to acetylcholine, which did not evoke sizeable Ca²⁺ signals in the majority of hCMEC/D3 cells bathed under 0Ca²⁺ conditions (Zuccolo, Laforenza, et al., 2019). Likewise, removal of extracellular Ca²⁺ during ongoing oscillations reversibly suppressed histamine-induced intracellular Ca²⁺ spikes in 65 of 84 cells (Figure 4d). SOCE represents the major Ca²⁺ entry pathway underlying agonizts-evoked extracellular Ca²⁺ entry in cerebrovascular endothelial cells (G. C. Brailoiu et al., 2016; E. Brailoiu, Shipsky, Yan, Abood, & Brailoiu, 2017; Kito et al., 2015; Moccia et al., 2015; Zuccolo et al., 2017; Zuccolo, Kheder, et al., 2019; Zuccolo, Laforenza, et al., 2019) and is mediated by the interaction between STIM2, the ER Ca²⁺ sensor, and Orai1, the pore-forming subunit on the plasma membrane, in hCMEC/D3 cells (Zuccolo, Laforenza, et al., 2019). Accordingly, when extracellular Ca²⁺ was restituted to the bath in the absence of the agonist, the subsequent increase in [Ca²⁺]_i did not adopt an oscillatory waveform but appeared as the Ca²⁺ bump typical of SOCE activation (Lodola et al., 2017; Sanchez-Hernandez et al., 2010). Therefore, we first assessed whether intracellular Ca²⁺ oscillations restart upon restoration of extracellular Ca²⁺ levels in the presence of Pyr6 (10 μM, 10 min), a selective Orai1 inhibitor (Moccia et al., 2016). This treatment, however, prevented the restart of Ca²⁺ oscillations following return to the Ca²⁺-containing perfusate in 80 of 80 cells (Figure 4e). Then, we found that the addition of Pyr6 (10 μM) during the ongoing spiking response irreversibly blocked histamine-induced intracellular Ca²⁺ oscillations (Figure 4f). These findings, therefore, demonstrate that histamine-induced repetitive Ca²⁺ transients are initiated by endogenous Ca²⁺ mobilization, whereas they are sustained by extracellular Ca²⁺ entry through the SOCE pathway.

3.4 InsP₃ and NAADP trigger histamine-induced endogenous Ca²⁺ release

We next assessed whether histamine-induced endogenous Ca²⁺ release was supported by InsP₃Rs following PLCβ recruitment by H₁Rs (Figure 5a), as demonstrated in endothelial cells from a variety of vascular beds (Q. Hu et al., 1999; Kishi et al., 1996; Lin et al., 2000; Oike et al., 1994; Sheng & Braun, 2007; Toda et al., 1991). As illustrated in Figure 5b, histamine-evoked intracellular Ca²⁺ mobilization was prevented by U73122 (10 µM; 30 min), a widespread employed PLC inhibitor (Dragoni et al., 2013; Zuccolo, Laforenza, et al., 2019), and 2-aminoethoxydiphenyl borate (2-APB; 50 µM, 30 min), which only blocks InsP₃Rs in the absence of extracellular Ca²⁺ (0Ca²⁺) (Zuccolo, Laforenza, et al., 2019). In addition, the acute addition of 2-APB (50 μM) during an established response to histamine (1 μM) caused the immediate interruption of the spiking activity in 68 out of 70 cells (Figure 5c). It is, however, worth mentioning that under such conditions (i.e., Ca²⁺ present in the bath), 2-APB could also target SOCE (Moccia et al., 2014; Moccia et al., 2016). Histamine-induced intracellular Ca²⁺ release was also suppressed by emptying the ER Ca²⁺ reservoir with cyclopiazonic acid (CPA; 10 µM), which specifically interferes with sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) activity (Berra-Romani et al., 2008). As reported elsewhere (Zuccolo,Laforenza, et al., 2019), CPA induced a transient elevation in [Ca²⁺]_i due to passive ER Ca²⁺ efflux followed by Ca²⁺ extrusion across the plasma membrane through the Na⁺/Ca²⁺ exchanger and the Plasma Membrane Ca²⁺-ATPase. The subsequent addition of histamine (10 µM) failed to increase the [Ca²⁺]_i in hCMEC/D3 cells (Figure 5d). Moreover, SERCA blockade during ongoing Ca²⁺ oscillations caused a large elevation in [Ca²⁺]_i followed by abrogation of histamine-evoked intracellular Ca²⁺ transients in 78 out of 79 cells (Figure 5e). Notably, after the Ca²⁺ peak caused by CPA-evoked ER Ca²⁺ release, the [Ca²⁺]_i declined to a sustained plateau level which was sustained by SOCE (Zuccolo et al., 2017; Zuccolo, Kheder, et al., 2019). Figure 5f reports the statistical analysis of these experiments. Collectively, these findings indicate that histamine-induced intracellular Ca²⁺ oscillations require repetitive cycles of InsP₃-dependent Ca²⁺ mobilization from the ER followed by Ca²⁺ reuptake and that SOCE is required to ensure proper refilling of endogenous stores to sustain the ongoing Ca²⁺ transients.

Recent studies revealed that NAADP-induced lysosomal Ca²⁺ release through TPC1–2 supports agonist-induced intracellular Ca²⁺ signals in multiple types of endothelial cells (Brailoiu et al., 2010; Gambara et al., 2008; Zuccolo, Kheder, et al., 2019), including the hCMEC/D3 cell line (Zuccolo, Laforenza, et al., 2019). In agreement with these observations, histamine-evoked intracellular Ca²⁺ transients were suppressed by NED-19 (100 µM, 30 min), a selective TPC1–2 blocker (Di Nezza et al., 2017; Pitt, Reilly-O'Donnell, & Sitsapesan, 2016) (Figure 6a, b and f). Moreover, the acute addition of NED-19 (100 μM) abrogated the established spiking response to histamine (1 μM) in 60 of 71 cells (Figure 6c). Subsequently, we depleted the lysosomal Ca²⁺ store with glycyl-l-phenylalanine 2-naphthylamide (GPN), which is selectively hydrolyzed by cathepsin C to induce the osmotic lysis of acidic vesicles and liberate their Ca²⁺ content (Moccia, Billington, & Santella, 2006; Ronco et al., 2015). As also shown in (Zuccolo, Laforenza et al., 2019), GPN (200 μM) elicited a transient elevation in [Ca²⁺]_i due to lysosomal Ca²⁺ disruption (Figure 6d). The following addition of histamine (1 μM) failed to induce intracellular Ca²⁺ oscillations (Figure 6d,f) thereby confirming that NAADP sustains the spiking response by inducing EL Ca²⁺ release through TPC1–2. Consistently, the acute addition of GPN (200 μM) during an established oscillatory Ca²⁺ signal caused a transient elevation in [Ca²⁺]_i followed by the interruption of Ca²⁺ activity (Figure 6e).

3.5 Histamine-evoked intracellular Ca²⁺ oscillations drive NO production in hCMEC/D3 cells

With the aim to evaluate whether histamine-induced intracellular Ca²⁺ oscillations stimulate NO production, hCMEC/D3 cells were led with the NO-sensitive fluorescent indicator, DAF-FM, as illustrated in Materials and Methods. Histamine (1 µM) evoked a rapid elevation in DAF-FM fluorescence that was prevented by N(ω)-nitro-l-arginine methyl ester (L-NAME) (100 µM, 1 hr; Figure 7a), an established NOS inhibitor, or BAPTA (30 µM, 2 hr; Figure 7a), an intracellular Ca²⁺ buffer (Zuccolo, Laforenza, et al., 2019). In addition, histamine-induced NO release was abrogated by pyrilamine (10 μM, 10 min), which inhibits H₁Rs (Figure 7b). Moreover, histamine-induced NO release occurred also under 0Ca²⁺ conditions, although DAF-FM fluorescence underwent a significantly (p < .05) lower increase compared to that recorded when Ca²⁺ was present in the perfusate (Figure 7c,e). Accordingly, the subsequent addition of Ca²⁺ to the recording chamber caused a further NO signal (Figure 7c), which confirms that both intra- and extracellular Ca²⁺ sources support histamine-evoked NO production in hCMEC/D3 cells. In agreement with this finding, blockage of SOCE with Pyr6 (10 μM; 10 min) attenuated, but not did fully suppress, histamine-induced NO production (Figure 7d,e). Figure 7e reports the statistical analysis of these experiments. These data, therefore, demonstrate that histamine impinges on intracellular Ca²⁺ signals to stimulate NO release via H₁Rs also in endothelial cells from human brain microcirculation.

As expected based upon the Fura-2/AM recordings in 0Ca²⁺, histamine (1 μM) failed to induce NO synthesis in the presence of U73122 (10 µM, 30 min), 2-APB (50 µM, 30 min), and CPA (10 μM) (Figure 8a,b). Moreover, histamine-evoked NO release was abrogated by emptying the lysosomal Ca²⁺ store with GPN (200 µM, 30 min) and by blocking TPC1–2 with NED-19 (100 µM, 30 min; Figure 8c). Figure 8d reports the statistical analysis of these experiments. It is, therefore, possible to conclude that histamine-induced NO release in endothelial cells from human brain microcirculation is supported by intracellular Ca²⁺ oscillations supported by NAADP, InsP_3, and SOCE.

4.1 Histamine-induced intracellular Ca²⁺ oscillations via H₁Rs in hCMEC/D3 cells

Intracellular Ca²⁺ oscillations control a plethora of endothelial functions (McCarron, Lee, & Wilson, 2017), including angiogenesis (Noren et al., 2016), vasculogenesis (Dragoni et al., 2013), gene expression (Zhu et al., 2011), vascular tone (Francis et al., 2016), and NO release (Zuccolo et al., 2017; Zuccolo, Kheder, et al., 2019). Likewise, histamine-induced repetitive Ca²⁺ spikes were reported in multiple types of endothelial cells, such as human umbilical vein endothelial cells (HUVEC; Jacob et al., 1988; Morgan & Jacob, 1996; Oike et al., 1994), HUVEC-derived EA.hy926 cells (Hadri, Pavoine, Lipskaia, Yacoubi, & Lompre, 2006) and human aortic endothelial cells (Zhu et al., 2008). The present investigation demonstrated that histamine evoked intracellular Ca²⁺ oscillations also in the human cerebrovascular endothelial cell line hCMEC/D3. Previous observations on human cranial arteries (Jansen-Olesen et al., 1997) demonstrated H₁R expression at the protein level, a finding that was confirmed in the present investigation. Furthermore, pyrilamine, a selective H₁R antagonist, prevented histamine-induced intracellular Ca²⁺ oscillations, which is fully consistent with the tight coupling between this receptor and PLCβ (H. Haas & Panula, 2003). Likewise, pharmacological blockade of H₁Rs inhibited histamine-induced oscillatory and biphasic Ca²⁺ signals in, respectively, HUVEC (Ikeda et al., 1999), EA.hy926 cells (Hadri et al., 2006) and bovine corneal endothelial cells (Crawford et al., 1992). The dose-response relationship disclosed that the oscillatory Ca²⁺ signal arose at 1 pM histamine, whereas the percentage of oscillating cells attained a peak at 1 nM. However, the amplitude of the 1st spike and the number of Ca²⁺ oscillations over 45 min achieved their peak at 1 µM. The percentage of oscillating cells, the magnitude of the 1st Ca²⁺ spike and the frequency of Ca²⁺ transients did not significantly increase by raising histamine concentration above 1 µM. These observations concur with existing data showing that low micromolar doses of histamine are already able to induce repetitive Ca²⁺ spikes in vascular endothelial cells (Q. Hu et al., 1999; Jacob et al., 1988; Morgan & Jacob, 1996; Oike et al., 1994). A recent investigation measured histamine levels in the cerebrospinal fluid and in the parenchyma of human brain reporting a nanomolar concentration which is well above the threshold required to induce the spiking Ca²⁺ response documented here (Croyal et al., 2011). Nevertheless, histamine levels may vary depending on the brain area, reaching a concentration as high as ≈1.4 μg/g in the hypothalamus (Lipinski, Schaumberg, & Baldessarini, 1973). Furthermore, histamine levels in the brain dramatically fluctuate according to the circadian rhythm as the firing of TMS neurons significantly increases during waking (John, Wu, Boehmer, & Siegel, 2004). Finally, varicosities of histaminergic fibers are closely apposed to cerebral blood vessels, including arteries, arterioles, and capillaries (W. W. Hu & Chen, 2012; Wada, Inagaki, Yamatodani, & Watanabe, 1991). It is, therefore, likely that local histamine concentration undergoes a significant increase following the activation of TMN neurons, as demonstrated for glutamatergic synapses (Lee et al., 2007). These observations prompted us to use 1 μM histamine to stimulate hCMEC/D3 cells, as reported in several types of vascular endothelial cells (Q. Hu et al., 1999; Jacob et al., 1988; Oike et al., 1994).

4.2 InsP₃, NAADP, and SOCE mediate histamine-evoked intracellular Ca²⁺ oscillations

A number of observations support the view that InsP₃- and NAADP-driven periodical Ca²⁺ mobilization initiates histamine-induced repetitive Ca²⁺ spikes, whereas SOCE maintains the oscillatory signal over time. First, histamine triggered 1–2 Ca²⁺ transients in a Ca²⁺-free solution, whereas Ca²⁺ transients immediately resumed when extracellular Ca²⁺ was restituted to the bath. This observation demonstrates that endogenous Ca²⁺ release initiates the oscillatory Ca²⁺ response, whereas extracellular Ca²⁺ entry supports the Ca²⁺ transients over time, as previously demonstrated for acetylcholine-induced Ca²⁺ transients in the hCMEC/D3 cell line (Zuccolo, Laforenza, et al., 2019) and glutamate-evoked intracellular Ca²⁺ oscillations in mouse cerebrovascular endothelial cells (Zuccolo, Kheder, et al., 2019). Second, histamine-induced extracellular Ca²⁺ entry was inhibited upon pharmacological blockade of SOCE with Pyr6. Likewise, SOCE inhibition resulted in complete abrogation of the histamine-induced intracellular Ca²⁺ oscillations. This observation concurs with the notion that hCMEC/D3 cells lack transient receptor potential (TRP) canonical (TRPC) channels (Zuccolo, Laforenza, et al., 2019), which contribute to mediate Ca²⁺ influx following PLC recruitment in vascular endothelial cells (Moccia, Berra-Romani, & Tanzi, 2012). It should be pointed out that neither removal of extracellular Ca²⁺ nor acute addition of Pyr6 resulted in the immediate inhibition of ongoing oscillations, which persisted for at least one spike. As discussed in (Berra-Romani et al., 2012; Berridge, 2007), this finding indicates that, rather than triggering repetitive Ca²⁺ release from the oscillating stores, SOCE is required to recharge the endogenous stores during prolonged stimulation. We have previously shown that SOCE is constitutively activated in hCMEC/D3 cells and may be further recruited by physiological depletion of the ER Ca²⁺ pool with acetylcholine (Zuccolo, Laforenza, et al., 2019). These findings, therefore, indicate that constitutive SOCE can be also engaged by histamine. Third, the spiking Ca²⁺ response elicited by histamine was initiated by H₁Rs, which are coupled by G_q/11 to PLCβ and InsP₃-dependent ER Ca²⁺ mobilization (H. Haas & Panula, 2003). Fourth, histamine-induced intracellular Ca²⁺ oscillations were suppressed by inhibiting PLCβ with U73122, by blocking InsP₃R3, the only InsP₃R isoform present in the hCMEC/D3 cell line (Zuccolo, Laforenza, et al., 2019), with 2-APB and by emptying the ER Ca²⁺ pool with CPA. Fifth, histamine-induced intracellular Ca²⁺ oscillations were suppressed by blocking NAADP-gated TPC1–2 with the selective inhibitor, NED-19 (Pitt et al., 2016), and by emptying the lysosomal Ca²⁺ pool with GPN. NAADP has also been shown to gate type 1 RyRs (Hohenegger, Suko, Gscheidlinger, Drobny, & Zidar, 2002; Wolf et al., 2015). However, RyRs are absent and caffeine did not trigger an increase in [Ca²⁺]_i in hCMEC/D3 cells. It is, therefore, possible to conclude that TPC1–2 are targeted by NAADP to shape histamine-induced intracellular Ca²⁺ oscillations. Notably, earlier work demonstrated that H₁Rs are also coupled to NAADP production and lysosomal Ca²⁺ release in HUVEC and EA.hy926 (Esposito et al., 2011). The ongoing intracellular Ca²⁺ spikes were also abrogated by preventing SERCA-mediated Ca²⁺ sequestration into the ER with CPA and by impeding Ca²⁺ reuptake into acidic vesicles with GPN. As discussed (Dai et al., 2007; Dai, Kuo, Leo, van Breemen, & Lee, 2006), these findings suggest that once delivered into the cytosol, Ca²⁺ must be sequestered back into endogenous organelles to give rise to the next cycle of Ca²⁺ mobilization. These findings are in strong agreement with the pioneering proposal by Galione and coworkers according to which the finely tuned interplay between NAADP and InsP₃ is the most suitable mechanism to initiate recurrent sequences of Ca²⁺ fluxes via the two-pool (i.e., lysosomal and ER Ca²⁺ stores) model (Churchill & Galione, 2001). Likewise, NAADP and InsP₃ were recently found to mediate repetitive Ca²⁺ oscillations also in mouse cerebrovascular endothelial cells challenged with acetylcholine and glutamate (Zuccolo et al., 2017; Zuccolo, Kheder et al., 2019). The exact mechanism by which these two Ca²⁺-releasing second messengers interact to shape intracellular Ca²⁺ signals is yet to be fully elucidated. It has been originally suggested that NAADP-mediated local Ca²⁺ release is amplified by the recruitment of juxtaposed InsP₃Rs and/or RyRs into a regenerative cytosolic Ca²⁺ wave (Morgan, 2016; Morgan, Platt, Lloyd-Evans, & Galione, 2011). Recent studies, however, demonstrated that InsP₃-dependent ER Ca²⁺ release may, in turn, be necessary to refill the EL Ca²⁺ pool (Atakpa, Thillaiappan, Mataragka, Prole, & Taylor, 2018; Ronco et al., 2015). Therefore, one could envisage that InsP₃-induced Ca²⁺ release serves to load the lysosomal Ca²⁺ store, thereby sustaining Ca²⁺ release through TPC1–2. Moreover, an increase in intraluminal Ca²⁺ could, in turn, activate TPC2 and, sometimes, also TPC1 (Pitt et al., 2016). Alternately, InsP₃-induced ER Ca²⁺ release could induce the Ca²⁺-dependent production of NAADP (Morgan et al., 2013), the physiological agonist of TPC1–2.

As mentioned above, histamine may induce vasorelaxation in human cerebral, meningeal and temporal arteries, thereby increasing CBF (Jansen-Olesen et al., 1997; Lassen et al., 2003). The hemodynamic response to histamine is triggered by endothelial H₁Rs, which promote the release of NO, the most powerful vasorelaxing mediator in the brain (Guerra et al., 2018; Lourenco, Ledo, Barbosa, & Laranjinha, 2017; Mapelli et al., 2017). Oscillations in [Ca²⁺]_i stimulate eNOS to synthesize NO in multiple types of vascular endothelial cells (Kasai, Yamazawa, Sakurai, Taketani, & Iino, 1997; Sandow, Senadheera, Grayson, Welsh, & Murphy, 2012), including mouse cerebrovascular endothelial cells (Zuccolo et al., 2017; Zuccolo, Kheder, et al., 2019). Histamine-evoked NO production in hCMEC/D3 cells was suppressed by inhibiting H₁Rs with pyrilamine and by interfering with the activity of the main components of the endothelial Ca²⁺ machinery, that is InsP₃R3, TPC1–2, and SOCE. Unlike acetylcholine (Zuccolo, Laforenza, et al., 2019), histamine was able to induce NO release under 0Ca²⁺ conditions, whereas restitution of extracellular Ca²⁺ evoked the second bump in intracellular NO release. Therefore, both endogenous Ca²⁺ mobilization and SOCE are required to activate eNOS in hCMEC/D3 cells exposed to histamine. These observations suggest that the pool of endogenous Ca²⁺-releasing channels, that is InsP₃R3 and/or TPC1–2, engaged by histamine is also coupled to plasmalemmal eNOS and is, therefore, distinct from that recruited by acetylcholine. Consistently, emerging evidence has been provided in favor of the heterogeneity of ER microenvironment and that, for instance, InsP₃Rs can be coupled to distinct effectors and/or plasmalemmal proteins (Berridge, 2002; Taylor & Machaca, 2018). Moreover, functionally distinct eNOS pools have been described in the vascular wall (Biwer et al., 2016). In addition, endothelial Ca²⁺ signals could engage vasorelaxing pathways other than eNOS, including prostacyclin and endothelium-dependent hyperpolarization (Behringer, 2017; Guerra et al., 2018). For instance, a recent investigation demonstrated that cultured human brain microvascular endothelial cells release considerable amounts of prostacyclin (Yoshida, Kurita, Eto, Kumagai, & Kaji, 2017), although its role in histamine-induced vasodilation in human brain circulation remains to be elucidated. Furthermore, H₁Rs selectively contributed to the decrease in transendothelial resistance elicited by histamine in monolayers of human cardiac endothelial cells (Adderley et al., 2015). Conversely, H₂R, H₃R, and H₄R contribute to regulating the endothelial permeability in HUVEC (Adderley et al., 2015) and rat brain microvascular endothelial cells (Karlstedt et al., 2013). The increase in endothelial permeability is mediated by the Ca²⁺-sensitive effector, myosin light chain kinase, thereby suggesting that the intracellular Ca²⁺ oscillations described in the present investigation also control the blood–brain barrier permeability. Future work might be devoted to manipulating the intracellular Ca²⁺ machinery to assess the role of each signaling pathway, that is InsP₃R3, TPC1–2, and SOCE, in the regulation of endothelial permeability by histamine.