Histamine elicits glutamate release from cultured

Journal of Pharmacological Sciences xxx (2018) 1e7

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Histamine elicits glutamate release from cultured astrocytes Ka rpa ti a, Takeo Yoshikawa a, *, Tadaho Nakamura a, b, Tomomitsu Iida a, Aniko Takuro Matsuzawa a, Haruna Kitano a, Ryuichi Harada a, Kazuhiko Yanai a a b

Department of Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan Division of Pharmacology, Tohoku Medical and Pharmaceutical University School of Medicine, 1-15-1 Fukumuro, Miyagino-ku, Sendai, 983-8536, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 January 2018 Received in revised form 30 April 2018 Accepted 8 May 2018 Available online xxx

Astrocytes play key roles in regulating brain homeostasis and neuronal activity. This is, in part, accomplished by the ability of neurotransmitters in the synaptic cleft to bind astrocyte membrane receptors, activating signalling cascades that regulate concentration of intracellular Ca2þ ([Ca2þ]i) and gliotransmitter release, including ATP and glutamate. Gliotransmitters contribute to dendrite formation and synaptic plasticity, and in some cases, exacerbate neurodegeneration. The neurotransmitter histamine participates in several physiological processes, such as the sleep-wake cycle and learning and memory. Previous studies have demonstrated the expression of histamine receptors on astrocytes, but until now, only a few studies have examined the effects of histamine on astrocyte intracellular signalling and gliotransmitter release. Here, we used the human astrocytoma cell line 1321N1 to study the role of histamine in astrocyte intracellular signalling and gliotransmitter release. We found that histamine activated astrocyte signalling through histamine H1 and H2 receptors, leading to distinct cellular responses. Activation of histamine H1 receptors caused concentration-dependent release of [Ca2þ]i from internal stores and concentrationdependent increase in glutamate release. Histamine H2 receptor activation increased cyclic adenosine monophosphate (cAMP) levels and phosphorylation of transcription factor cAMP response-element binding protein. Taken together, these data emphasize a role for histamine in neuron-glia communication. © 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Keywords: Astrocyte Calcium Gliotransmitter Glutamate Histamine

1. Introduction Astrocytes are the most abundant cell type in the central nervous system. They are involved in a wide range of physiological processes, including blood flow regulation,1 energy metabolism,2 ionic homoeostasis,3 and synaptic function.4,5 The ability of astrocytes to modulate synaptic function is, in part, mediated by their ability to bind locally released neurotransmitters, and respond with the release of gliotransmitters, such as adenosine triphosphate (ATP), glutamate, and D-serine.6,7 Gliotransmitter release appears to be disturbed under pathological conditions, and is associated with neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.8,9 Released neurotransmitters bind to various subclasses of G protein-coupled receptors (GPCRs) on astrocytes to activate intracellular signalling and regulate intracellular second messengers.10

* Corresponding author. Fax: þ81 22 717 8060. E-mail address: [email protected] (T. Yoshikawa). Peer review under responsibility of Japanese Pharmacological Society.

Signalling through Gq-coupled GPCRs can increase intracellular Ca2þ ([Ca2þ]i) concentration through the activation of phospholipase C (PLC); PLC cleaves the membrane phospholipid phosphatidylinositol 4,5-bis-phos-phate to yield 1,2-diacylglycerol (DAG) and inositol 1,4,5-triphosphate (InsP3), the latter of which can bind to InsP3 receptors leading to Ca2þ release from intracellular stores.11 In contrast, signalling through Gs-coupled GPCRs activates adenylyl cyclase, which increases the formation of cyclic AMP (cAMP). The importance of Gq-linked intracellular Ca2þ signalling for gliotransmitter release has been highlighted in several studies.8,9,12 Furthermore, a connection between cAMP treatment and enhanced glutamate release from astrocytes was reported.13 The neurotransmitter histamine plays a key role in the sleepwake cycle and in learning and memory.14 Alterations in brain histamine levels are closely connected with central nervous system dysfunction, and are thought to contribute to neurological disorders, including Alzheimer's disease and depression.15,16 Histamine binds to four distinct GPCRs (histamine H1eH4 receptors); of these, previous studies have reported the expression of H1eH3 receptors on astrocytes.17 In an early study, Inagaki et al. showed comparable

https://doi.org/10.1016/j.jphs.2018.05.002 1347-8613/© 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

rpa ti A, et al., Histamine elicits glutamate release from cultured astrocytes, Journal of Pharmacological Please cite this article in press as: Ka Sciences (2018), https://doi.org/10.1016/j.jphs.2018.05.002

rpa ti et al. / Journal of Pharmacological Sciences xxx (2018) 1e7 A. Ka

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H1 receptor binding capacities in primary astrocyte cultures and brain tissue, where besides astrocytes neurons also express H1 receptors.18 Additionally, histamine stimulation increases [Ca2þ]i in astrocytes.19e21 Taken together, previous findings indicate a significant role for histamine in astrocyte signalling. Yet, only a few studies to date have examined the importance of histamine in astrocyte signalling, and the effect of histamine on gliotransmitter release is unknown. In the present study, we examined the role of histamine in astrocyte intracellular signalling and gliotransmitter release in the human astrocytoma cell line 1321N1. 2. Methods 2.1. Cell culture The 1321N1 human astrocytoma cell line was kindly donated by the late Professor Norimichi Nakahata, Tohoku University. Cells were cultured under a humidified atmosphere of 5% CO2 at 37 C in DMEM (Life Technologies, Carlsbad, CA, USA) supplemented with , France), 100 IU/ml 10% foetal bovine serum (Biowest, Nuaille benzylpenicillin potassium (Wako Pure Chemical Industries, Ltd., Osaka), 100 mg/ml streptomycin sulphate (Wako Pure Chemical Industries, Ltd.), and 2 mM GlutaMax-I (Life Technologies).

fluorescence was recorded for 20 s before the addition of histamine or a histamine receptor agonist. Fluorescence intensity was measured using a Flexstation® 3 microplate reader and analysed using Softmax Pro 5 software (Molecular Devices). Independent of the recording method, antagonist studies were performed as follows: after incubation with Fluo-4-AM, cells were incubated with a histamine receptor antagonist (H1 receptor: levocetirizine [Tokyo Chemical Industry, Tokyo], H2 receptor: ranitidine [SigmaeAldrich], H3 receptor: JNJ10181457, or H4 receptor: JNJ7777120 [both from Tocris]), an InsP3 receptor antagonist (aminoethoxydiphenyl borate [2-APB]), or a PLC inhibitor (U73122) (both from Cayman Chemicals, Ann Arbor, MI, USA) for 10 min, and subsequently, fluorescence was measured. The inhibitory drugs were present in pre-treatment solutions as well as in the experimental solutions. In some experiments, the sarcoendoplasmic reticulum Ca2þ transport ATPase (SERCA) inhibitor thapsigargin (Wako Pure Chemical Industries, Ltd.) was added for 10 min after the first histamine stimulation but prior to the second histamine stimulation. 2.4. Glutamate release assay

Total RNA isolation and reverse transcription of 1321N1 RNA were performed as described previously.22 Briefly, 1321N1, normal human astrocyte (Lonza, Basel, Switzerland), and whole human brain (Takara Bio Inc., Shiga) cDNAs were amplified using the KOD SYBR qPCR) real time-PCR master mix (Toyobo, Osaka), and run on 3-step-PCR for 40 cycles (98 C for 10 s, 60 C for 10 s, then 68 C for 30 s). Specific primers for the genes of H1eH4 receptors (HRH1e4) and b-actin (ACTB) were used for amplification (Table 1).

1321N1 cells cultured in 24-well plates were washed and incubated with HBSS for 30 min at 37 C 5% CO2. Subsequently, cells were washed again and incubated with HBSS vehicle or HBSS containing a histamine receptor antagonist (H1 receptor: levocetirizine and fexofenadine [Tokyo Chemical Industry] or H2 receptor: ranitidine and famotidine [Tokyo Chemical Industry]) and PLC-inhibitor U73122 or its inactive analogue U73343 (Abcam, Cambridge, UK) for 10 min, before the medium was exchanged with medium containing histamine or histamine receptor agonists (H1 receptor: 2-PEA; H2 receptor: dimaprit), or containing histamine together with inhibitory drugs. The supernatant was collected after 5 min and subjected to high-pressure liquid chromatography (HPLC).

2.3. Intracellular Ca2þ measurement

2.5. HPLC measurement

1321N1 cells, cultured on glass-bottom dishes, were incubated with 5 mM Fluo-4-AM dye (Dojindo, Kumamoto) for 30 min, followed by incubation with Hank's balances salt solution (HBSS) for 10 min. The buffer was replaced with fresh HBSS, and fluorescence intensity was detected using an inverted microscope (Olympus, Tokyo). Histamine (SigmaeAldrich, St. Louis, MO, USA) or a histamine receptor agonist (H1 receptor: 2-pyridylethylamine [2-PEA], H2 receptor: dimaprit, H3 receptor: immethridine [all purchased from Tocris, Bristol, UK], or H4 receptor: 4-methylhistamine [Santa Cruz Biotechnology, Dallas, TX, USA]) was added after 30 s. Images were analysed using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA). For fluorescent assay, cells were seeded into 96-well clearbottom microplates and prepared as described above. Baseline

Glutamate was measured using an HPLC system, as described previously.23 In this study, samples were analysed using an FA-3ODS separation column, HTEC-500 electrochemical detection system, and M-504G auto-sampling injector (Eicom, Kyoto).

2.2. PCR

Table 1 Primer sequences for human histamine receptor genes. Gene HRH1 HRH2 HRH3 HRH4 ACTB

Primer Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense

5 50 50 50 50 50 50 50 50 50

1321N1 cells cultured in 6-well plates were washed and treated with 0.1 mM histamine, with or without 0.2 mM ranitidine, for 5 min. The reaction was terminated, and the cells were lysed in radioimmunoprecipitation assay buffer containing 1 mM sodium orthovanadate (New England Biolabs, Ipswich, MA, USA) and cOmplete™ Protease Inhibitor Cocktail® (Roche, Basel, Switzerland). Total protein concentrations were measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). 2.7. Western blot

Sequence 0

2.6. cAMP response-element binding protein phosphorylation assay

Product size (bp) 0

-TACTGAAACCAGCCAGGGAGT -3 -ACTGAAAATGGACGCTGTGC -30 -TGGTTTCCCTACTTCACCGC -30 -AAGCCATGGTCTGTCTGTGG -30 -TCACCCGAGCGGTCTCATAC -30 -TCACCCACCCCATACCTGTG -30 -TTTGTGGGTGTGATCTCCATTCC -30 -TCCACAGATGTTGGAAGAGACAG -30 -CGCCCTATAAAACCCAGCGG -30 -AACATGATCTGGGTCATCTTCTCG -30

402 348 430 489 489

Ten mg of extracted protein was separated on a 12% sodium dodecyl sulphate-polyacrylamide gel, and transferred onto a polyvinylidene fluoride membrane. The membrane was blocked in 5% non-fat dry milk solution for 1 h, and incubated with primary IgG rabbit anti-cAMP response-element binding protein (CREB) (1:5000), IgG rabbit anti-phospho-CREB (1:5000), or IgG rabbit anti-b-actin (1:1000) antibody (all purchased from Cell Signalling Technology, Danvers, MA, USA) overnight at 4 C. After washing, the membrane was incubated with peroxidase-linked anti-rabbit IgG antibody (Jackson ImmunoResearch Inc., West Grove, PA, USA; 1:2000), and



the blots were developed using electrochemiluminescence reagent (BioRad, Hercules, CA, USA) and visualized using an Ez-Capture MG luminescent image analyser (Atto, Tokyo). 2.8. cAMP measurement The cAMP levels were measured using the Cyclic AMP ELISA Kit (Cayman Chemicals). Briefly, cells were cultured overnight in 6-well plates before incubation with HBSS for 30 min. Cells were washed and treated with 0.5 mM 3-isobutyl-1-methylxanthine (Nacalai Tesque, Kyoto) for 10 min followed by treatment with histamine or histamine and ranitidine for 5 min. The reaction was stopped with 0.1 M HCl and immediate cell lysis. After centrifugation at 1000 g for 5 min, the cAMP levels in the supernatants were determined using the Cyclic AMP ELISA Kit. Total protein concentrations were measured using a Pierce™ BCA Protein Assay Kit. 2.9. Statistical analysis All statistical analyses were performed using Prism 5 software (GraphPad, La Jolla, CA, USA). Significant differences were tested using one-way analysis of variance (ANOVA) with Tukey's post-hoc test, two-way ANOVA with Bonferroni post-hoc test, or Student's t tests; p < 0.05 was the threshold for statistical significance. Results are expressed as the mean ± standard error. 3. Results 3.1. 1321N1 cells express histamine receptor genes We detected the expression of HRH1 and HRH2, but neither HRH3 nor HRH4, in 1321N1 cells and normal human astrocytes (Fig. 1). In comparison, PCR using cDNA obtained from the human whole brain showed the expression of HRH3. Additionally obtained data using rat astrocyte cDNA from different brain regions (the hippocampus, cortex, cerebellum, and diencephalon) confirmed our observation. In general, the genes for rat H1 (Hrh1) and H2 receptors (Hrh2) were expressed in all brain areas, but the gene expression levels were region-dependent (Supplementary Fig. 1). We detected the highest expression level of Hrh1 in the cortex, while the lowest levels were detected in the hippocampus and cerebellum; Hrh2 and Hrh3 were highly expressed in hippocampal astrocytes. 3.2. Histamine increases [Ca2þ]i and cAMP concentration in cultured astrocytes The 1321N1 cells showed significant increases in intracellular calcium ([Ca2þ]i) and intracellular cAMP concentration in response to histamine treatment. Within seconds, [Ca2þ]i increased in a

Fig. 1. Expression of histamine receptor genes. Representative results of PCR using 1321N1 and normal human astrocyte cDNA were compared to those of human whole brain cDNA. HRH1-HRH4: genes for histamine H1eH4 receptors, ACTB: gene for b-actin.

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concentration-dependent manner (EC50 ¼ 800.7 nM) (Fig. 2A). Pharmacological assays revealed that increases in [Ca2þ]i were H1 receptor-dependent (Fig. 2B); treatment with 100 nM levocetirizine, a selective H1 receptor antagonist, completely abolished [Ca2þ]i responses (IC50 ¼ 35.89 nM; Fig. 2C), whereas the H2 receptor antagonist ranitidine (0.2 mM) increased [Ca2þ]i, and H3 and H4 receptor antagonists (JNJ10181457 and JNJ7777120, respectively) had no effects. These findings were confirmed with agonist experiments (Fig. 2D); the H1 receptor partial agonist 2-PEA (1 mM) elicited a 40% increase in [Ca2þ]i compared to histamine, whereas H2eH4 receptor agonists (dimaprit, immethridine, and 4-methylhistamine) had no effects. To test whether H1 receptor signalling in astrocytes was Gq-coupled as previously reported in other cell types,24 we treated cells with the PLC inhibitor U73122 (5 mM), InsP3 receptor antagonist 2-APB (300 mM), or SERCA inhibitor thapsigargin (500 nM), and then measured changes in [Ca2þ]i in response to histamine. The inhibition of Gq-coupled signalling completely prevented histamine-induced increases in [Ca2þ]i (Fig. 2E). These results suggested that the activation of Gq-coupled H1 receptor signalling is sufficient to increase [Ca2þ]i. We also measured cAMP levels in histamine-treated 1321N1 cells, and showed that cAMP levels increased in response to histamine treatment (Fig. 2F). In contrast, increases in cAMP levels were significantly attenuated by pre-treatment with the H2 receptor antagonist ranitidine. Taken together, these data suggested that H2 receptor activation leads to cAMP accumulation in 1321N1 cells.

3.3. Histamine H1 receptor signalling increases glutamate release Since previous studies have established that increased [Ca2þ]i induces glutamate release from astrocytes, we examined whether histamine can enhance the release of glutamate. Extracellular glutamate level was significantly increased in response to histamine (Fig. 3A). We further investigated the link between H1 receptor signalling and glutamate release. Levocetirizine, at a concentration of 100 nM, prevented the effect of histamine on glutamate release (Fig. 3B). The H1 receptor antagonist fexofenadine also inhibited histamine-induced glutamate release (Fig. 3C), while the H1 receptor agonist 2-PEA failed to significantly increase glutamate release from 1321N1 cells (Fig. 3D). To further confirm the role of H1 receptor signalling, we treated 1321N1 cells with the PLC inhibitor U73122 (5 mM), and subsequently measured glutamate release in response to histamine. In the presence of U73122, histamine did not enhance glutamate release, although U73122 itself enhanced glutamate release in the absence of histamine (Fig. 3E). The inactive analogue U73343 did not alter histamine-dependent glutamate release (Supplementary Fig. 2). A previous study showed that increased cAMP concentrations could promote astrocytic glutamate release.13 Thus, we examined the effect of adenylyl cyclase activator forskolin on glutamate release. Forskolin (100 mM) significantly enhanced glutamate release, which was further increased during histamine co-treatment (Fig. 3F). However, the H2 receptor agonist dimaprit did not have any impact on glutamate release (Fig. 3G). Moreover, the selective H2 receptor antagonists ranitidine (0.2 mM) and famotidine (10 mM) had no effects on histamine-induced glutamate release (Fig. 3H and I). These data suggest that H1 receptor activation stimulates PLC and elicits the release of glutamate from 1321N1 cells, while the H2 receptor is not involved in this process. We similarly confirmed that histamine elevates glutamate release, using primary cortical rat astrocytes (Supplementary Fig. 3); H1 receptor signalling was associated with Gq activation and Ca2þ signalling in primary rat cortical astrocytes.


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Taken together, these findings indicate that histamine can activate astrocyte signalling and induce the release of glutamate from a human astrocytoma cell line and cultured rat cortical astrocytes. 3.4. Histamine H2 receptor activation increases CREB phosphorylation in 1321N1 cells Finally, we determined whether histamine could activate the transcription factor CREB, downstream of the H2 receptor second messenger, cAMP. Treatment with histamine for 5 min significantly enhanced CREB phosphorylation compared to control (no treatment). In contrast, the phospho-CREB/CREB ratio was unchanged when cells were pre-treated with the H2 receptor antagonist ranitidine prior to histamine stimulation (Fig. 4A and B). 4. Discussion

Fig. 2. Increased intracellular concentrations of the second messengers Ca2þ and cAMP in response to histamine treatment. Changes in intracellular calcium ([Ca2þ]i) concentration (AeE) and cyclic adenosine monophosphate (cAMP) (F) levels. A. Changes in [Ca2þ]i were measured after stimulation with the indicated concentrations of histamine. Cells were loaded with Fluo-4-AM to detect changes in [Ca2þ]i; n ¼ 6. B. Cells were incubated with the indicated H1eH4 receptor antagonists before and during stimulation with 10 mM histamine. H1 receptor: 100 nM levocetirizine; H2 receptor: 0.2 mM ranitidine; H3 receptor: 1 mM JNJ10181457; and H4 receptor: 1 mM JNJ 7777120; n ¼ 6. Statistical significance was tested using one-way analysis of variance (ANOVA) with Tukey's post-hoc test (*p < 0.05); n.d. ¼ not detected. C. Changes in [Ca2þ]i in the presence of 10 mM histamine and increasing concentrations of levocetirizine; n ¼ 6. D. Cells were treated with either 10 mM histamine or H1eH4 receptor agonists. H1 receptor: 1 mM 2-pyridylethylamine (2-PEA); H2 receptor: 1 mM dimaprit; H3 receptor: 1 mM immethridine; and H4 receptor: 1 mM 4-methylhistamine; n ¼ 6. Statistical significance was calculated using one-way ANOVA with Tukey's post-hoc test (**p < 0.01); n.d. ¼ not detected. E. Cells were pre-treated with a phospholipase C inhibitor (5 mM U73122), an inositol 1,4,5-triphosphate receptor antagonist (300 mM aminoethoxydiphenyl borate [2-APB]), or a sarcoendoplasmic reticulum Ca2þ transport ATPase inhibitor (500 nM thapsigargin), and subsequently, the cells were stimulated with 100 mM histamine in the presence of the previously mentioned compounds to measure Ca2þ responses; n ¼ 8. F. Cells were treated with various concentrations of histamine, with or without the H2 receptor antagonist ranitidine, before they were lysed and the levels of cyclic adenosine monophosphate (cAMP) were determined; n ¼ 6. Statistical significance was determined using one-way ANOVA with Tukey's post-hoc test and t-test (*p < 0.05, **p < 0.01, and ***p < 0.001).

Our study demonstrates that histamine interacts with membranous histamine receptors on astrocytes to activate intracellular signalling cascades and elicit the release of glutamate in a H1 receptor- and concentration-dependent fashion. Histamine has been implicated in astrocyte signalling since the discovery of histamine receptors on astrocytes more than three decades ago.25 Yet, only a few studies have evaluated the effects of histamine on astrocyte signalling. Here, we demonstrated the expression of HRH1 and HRH2 in the 1321N1 cell line and normal human astrocytes, confirming that the cell line maintained its characteristic gene expression pattern. The H1 receptors were shown to possess a similar binding capacity for histamine in pure astrocyte cultures and the brain, where both astrocytes and neurons express H1 receptors, suggesting astrocytes could be one of the main targets of the histaminergic system in the brain.18 Indeed, 1321N1 and normal human astrocytes showed an abundant expression of HRH1, further underlining the importance of H1 receptors in astrocytes. Comparable to our own result, previous studies have shown the expression of Hrh1 and Hrh2 in rat primary astrocytes.18,25 Although a more recent study by Mele and Juric26 suggested the expression of Hrh3 in rat cortical astrocytes, our data did not confirm their results. However, we observed expression of Hrh3 in rat hippocampal and cerebellar astrocytes, suggesting that the expression of Hrh3 on astrocytes is restricted to certain parts of the rodent brain and is rodent-specific. The H1 and H2 receptors are coupled to the Gq- and Gs-proteins, respectively.24 We confirmed that astrocytes respond to histamine via Gq-coupled Ca2þ signalling related to H1 receptor activation.20 The Ca2þ signalling was concentration-dependent and demonstrated a high sensitivity to histamine in 1321N1 cells. The ability of histamine to modulate astrocytic [Ca2þ]i is of great importance, as previous studies have outlined the roles of Ca2þ signalling under normal as well as pathological conditions.8,9,27 Furthermore, we showed that histamine induced Gs-coupled H2 receptor signalling and increased cAMP levels in a concentration-dependent manner. In astrocytes, the neurotransmitter glutamate is known to promote [Ca2þ]i through GPCR activation, thereby eliciting the release of a wide range of gliotransmitters.4 We provide evidence, for the first time, that histamine can induce gliotransmitter release. Glutamate was released in a concentration-dependent manner, and relied on H1 receptor activation. The H1 receptor antagonists completely attenuated the histamine-induced glutamate release. As no potent H1 receptor agonists are currently available, we treated 1321N1 cells with a partial agonist, 2-PEA, but observed only a slight increase in the glutamate released from 1321N1 cells. This may be because 2-PEA acts as a partial agonist, even at high concentrations, and moreover, it possesses low affinity for the



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Fig. 3. Histamine induced glutamate release. A. Glutamate concentrations were measured after treatment with different concentrations of histamine (pH 7.4) for 5 min; n ¼ 6. Statistical significance was calculated using one-way analysis of variance (ANOVA) with Tukey's post-hoc test (*p < 0.05 and ***p < 0.001). B. Glutamate concentrations were measured after treatment with histamine and the indicated concentrations of the H1 receptor antagonist, levocetirizine; n ¼ 12. Statistical significance was calculated using one-way ANOVA with Tukey's post-hoc test: (*p < 0.05, **p < 0.01, and ***p < 0.001). C. Glutamate concentrations were measured after treatment with histamine in the presence or absence of the H1 receptor antagonist fexofenadine (1 mM); n ¼ 12. Two-way ANOVA and Bonferroni post-test was used for statistical analysis within the 0 mM and 1 mM fexofenadine groups, and t-test was used for statistical analysis between the different histamine concentrations (*p < 0.05 and **p < 0.01). D. Cells were treated with histamine or the H1 receptor agonist 2-pyridylethylamine (2-PEA) (1 mM) prior to the measurement of glutamate concentrations; n ¼ 12. Statistical significance was calculated using one-way ANOVA with Tukey's post-hoc test (*p < 0.05). E. Glutamate concentrations were measured after treatment with histamine in the presence of 5 mM U73122; n ¼ 12. Two-way ANOVA and Bonferroni post-test were used for statistical analysis within the 0 mM and 5 mM U73122 groups, and t-test was used for statistical analysis between the control groups (*p < 0.05). F. Glutamate concentrations were measured in the presence of the adenylyl cyclase activator forskolin (100 mM) and histamine; n ¼ 12. Two-way ANOVA and Bonferroni post-test were used for statistical analysis within the 0 mM and 100 mM forskolin groups, and t-test was used for statistical analysis between the control groups and the different histamine concentrations (**p < 0.01 and ***p < 0.001). G. Cells were treated with histamine or the H2 receptor agonist dimaprit (10 mM) prior to the measurement of glutamate concentrations; n ¼ 16. Statistical significance was calculated using one-way ANOVA with Tukey's post-hoc test (***p < 0.001). H. Glutamate concentrations were measured after treatment with histamine in the presence of the H2 receptor antagonist ranitidine (0.2 mM); n ¼ 12. Two-way ANOVA and Bonferroni post-test were used for statistical analysis within the 0 mM and 0.2 mM ranitidine groups, and t-test was used for statistical analysis between the different histamine concentrations (**p < 0.01). I. Glutamate concentrations were measured after treatment with histamine in the presence of the H2 receptor antagonist famotidine (10 mM); n ¼ 12. Two-way ANOVA and Bonferroni post-test were used for statistical analysis between the 0 mM and 10 mM famotidine groups, and t-test was used for statistical analysis between the different histamine concentrations (**p < 0.01 and ***p < 0.001).

H1 receptor (pKi ¼ 3.7)28,29. Further, we tested the role of PLC in H1 receptor-mediated glutamate release. Glutamate release was elevated in the presence of the PLC inhibitor U73122, but did not increase further when the cells were co-treated with histamine, indicating the importance of PLC activity for glutamate release. The increased glutamate release in the absence of histamine is most likely attributable to the nature of U73122, which facilitates influx of extracellular Ca2þ at inhibitory concentrations.30 The molecular mechanism of gliotransmitter release is not fully understood until now, and previous studies have emphasized that the elevation of [Ca2þ]i triggers vesicular exocytosis of glutamate. Parpura et al. reported that the essential role of Ca2þ release from internal stores in glutamate release.4 The H1 receptor antagonist assays showed that 10 nM levocetirizine neither inhibited Ca2þ signalling nor glutamate release, while at a higher concentration both cellular responses were inhibited, indicating the contribution of the intracellular Ca2þ release caused by H1 receptors to glutamate release. In contrast, H2 receptor antagonist ranitidine increased [Ca2þ]i, but not glutamate release, in the presence of 10 mM histamine, suggesting that ranitidine could induce [Ca2þ]i elevation

independently of internal Ca2þ stores through a yet unknown mechanism. We also revealed that histamine-induced glutamate release was increased after Ca2þ response reached a plateau. This result suggests that Ca2þ-independent signalling was also involved in histamine-induced glutamate release. Various reports have described the involvement of other intracellular signalling pathways in glutamate release (reviewed by Malarkey and Parpura31). Since our results indicate that PLC plays a crucial role in glutamate release, DAG, which is produced through PLC activation, independently of Ca2þ signalling, becomes a potential facilitator for H1 receptordependent glutamate release. This hypothesis was previously proven by Mungenast et al. showing DAG-induced gliotransmitter release from cultured astrocytes.10 However, in order to fully understand the involved mechanism, additional studies are required. Till this day, it remains unknown whether cAMP regulation by Gs and Gi-proteins contributes to gliotransmitter release. In a previous study it was argued that increased cAMP concentrations could promote astrocytic glutamate release.13 Our study showed that forskolin positively affected glutamate release, supporting the involvement of cAMP in glutamate release. Interestingly, forskolin further enhanced


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monoamine neurotransmitters in brain function in the context of astrocyte signalling. Further in vivo studies will reveal new possibilities in drug design for the treatment of different neurological disorders. Conflict of interest The authors declare no conflict of interest. Acknowledgements

Fig. 4. CREB is activated as a consequence of histamine-induced signalling. A. Cells were treated with 0.1 mM histamine, with or without 0.2 mM ranitidine, for 5 min before the reaction was stopped, and the cells were lysed and analysed for phospho-cAMP response-element binding protein (CREB)/CREB using western blotting. Forskolin was used as positive control. The control value was set to 1; n ¼ 4e6, t-test (*p < 0.05 and **p < 0.01). B. Western blot with samples analysed in A. Lanes (left to right): (1e3) control; (4e6) 0.1 mM forskolin; (7e9) 0.1 mM histamine, 5 min; (10e12) 0.1 mM histamine þ 0.2 mM ranitidine, 5 min.

glutamate release in the presence of histamine, indicating that the contribution of cAMP elevation to histamine-induced glutamate release was negligible. In fact, the level of cAMP after histamine or forskolin treatment was significantly different. Moreover, the H2 receptor agonist dimaprit as well as the two selective H2 receptor antagonists ranitidine and famotidine did not have any impact on glutamate release. Therefore, H1 receptor plays a predominant role in histamine-induced glutamate release, and the H1 and H2 receptor signals are not cross-linked. In contrast, it appears that histamine H2 receptor signalling increases cAMP to levels that were sufficient to initiate further downstream signalling to activate CREB. Moreover, CREB can be activated by different upstream signals, and responds quickly to different stimuli selectively, thereby contributing to a variety of functions, including learning and memory.32 Future investigations will provide a better understanding of the ways in which histamine-induced CREB activation affects brain function. Summarizing these data, histamine activates astrocyte signalling in the 1321N1 cell line through two distinct receptors, H1 and H2 receptors. We used primary rat astrocytes to validate our results from an astrocytic cell line, supporting our hypothesis that histamine-induced Ca2þ signalling and gliotransmitter release are crucial for brain function, and are therefore, largely conserved across mammals. Our findings emphasize the need for in vivo studies to further investigate the effects of histamine-dependent astrocyte signalling on brain function. Previous studies have shown that histamine binding capacity decreases in various neurological disorders, including Alzheimer's disease,33 especially in the cortex. Based on our in vitro results, it is likely that H1 receptors are the main regulators of histamine-dependent astrocyte signalling in vivo. Future studies should use novel conditional H1 receptor knock-out mouse models that lack H1 receptors specifically on astrocytes or neurons to better address this possibility. In conclusion, we demonstrate, for the first time, that histamine plays a key role in regulating astrocyte function and gliotransmitter release, and we now must re-consider the roles of

We acknowledge the support of the Biomedical Research Core of Tohoku University Graduate School of Medicine and the Biomedical Research Unit of Tohoku University Hospital. This work was supported by a Grant-in-Aid for Scientific Research (A) (26253016) from the Japan Society for the Promotion of Science (JSPS), a Grant-in-Aid for Young Scientists (B) (16K18389) from JSPS, and a Grant-in-Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network) from the Ministry of Education, Science, Sports and Culture of Japan. We also acknowledge the support of the Tohoku University Division for Interdisciplinary Advanced Research and Education, and the Nishinomiya Basic Research Fund, Japan. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jphs.2018.05.002. References 1. Howarth C. The contribution of astrocytes to the regulation of cerebral blood flow. Front Neurosci. 2014;8(103). 2. Brown AM, Ransom BR. Astrocyte glycogen and brain energy metabolism. Glia. 2007;55(12):1263e1271. 3. Olsen ML, Khakh BS, Skatchkov SN, Zhou M, Lee CJ, Rouach N. New insights on astrocyte ion channels: critical for homeostasis and neuron-glia signaling. J Neurosci. 2015;35(41):13827e13835. 4. Parpura V, Basarsky TA, Liu F, Jeftinija K, Jeftinija S, Haydon PG. Glutamatemediated astrocyte-neuron signalling. Nature. 1994;369(6483):744e747. 5. Zhang JM, Wang HK, Ye CQ, et al. ATP released by astrocytes mediates glutamatergic activity-dependent heterosynaptic suppression. Neuron. 2003;40(5): 971e982. 6. Hamilton NB, Attwell D. Do astrocytes really exocytose neurotransmitters? Nat Rev Neurosci. 2010;11(4):227e238. 7. Volterra A, Meldolesi J. Astrocytes, from brain glue to communication elements: the revolution continues. Nat Rev Neurosci. 2005;6(8):626e640. 8. Agulhon C, Sun MY, Murphy T, Myers T, Lauderdale K, Fiacco TA. Calcium signaling and gliotransmission in normal vs. Reactive astrocytes. Front Pharmacol. 2012;3(139). 9. Harada K, Kamiya T, Tsuboi T. Gliotransmitter release from astrocytes: functional, developmental, and pathological implications in the brain. Front Neurosci. 2016;9(499). 10. Mungenast AE. Diacylglycerol signaling underlies astrocytic ATP release. Neural Plast. 2011;537659(10):13. 11. Tamamushi S, Nakamura T, Inoue T, et al. Type 2 inositol 1,4,5-trisphosphate receptor is predominantly involved in agonist-induced Ca(2þ) signaling in Bergmann glia. Neurosci Res. 2012;74(1):32e41. 12. Bazargani N, Attwell D. Astrocyte calcium signaling: the third wave. Nat Neurosci. 2016;19(2):182e189. 13. Shiga H, Murakami J, Nagao T, et al. Glutamate release from astrocytes is stimulated via the appearance of exocytosis during cyclic AMP-induced morphologic changes. J Neurosci Res. 2006;84(2):338e347. 14. Haas H, Panula P. The role of histamine and the tuberomamillary nucleus in the nervous system. Nat Rev Neurosci. 2003;4(2):121e130. 15. Fernandez-Novoa L, Cacabelos R. Histamine function in brain disorders. Behav Brain Res. 2001;124(2):213e233. 16. Nuutinen S, Panula P. Histamine in neurotransmission and brain diseases. Adv Exp Med Biol. 2011;709:95e107. 17. Juric DM, Krzan M, Lipnik-Stangelj M. Histamine and astrocyte function. Pharmacol Res. 2016;111:774e783. 18. Inagaki N, Fukui H, Taguchi Y, Wang NP, Yamatodani A, Wada H. Characterization of histamine H1-receptors on astrocytes in primary culture: [3H] mepyramine binding studies. Eur J Pharmacol. 1989;173(1):43e51.


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