Histamine H1 Receptor Activation Stimulates ... - Springer Link

Journal of Neuro-Oncology 55: 81–89, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

Laboratory Investigation

Histamine H1 receptor activation stimulates mitogenesis in human astrocytoma U373 MG cells Adriana Hernández-Angeles1 , Luis-Enrique Soria-Jasso3 , Arturo Ortega2 and José-Antonio Arias-Montaño1 Departamento de Fisiolog´ıa, Biof´ısica y Neurociencias y, 2 Departamento de Genética y Biolog´ıa Molecular, Centro de Investigación y de Estudios Avanzados, 3 CICATA-IPN, Col. Irrigación, Mexico City, Mexico

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Key words: histamine, H1 receptor, astrocytoma cells, proliferation, glia, PKC Summary In human astrocytoma U373 MG cells that express histamine H1 receptors (180 ± 6 fmol/mg protein) but not H2 or H3 receptors, histamine stimulated mitogenesis as assessed by [3 H]-thymidine incorporation (173 ± 2% of basal; EC50 , 2.5 ± 0.4 µM). The effect of 100 µM histamine was fully blocked by the selective H1 antagonist mepyramine (1 µM) and was markedly reduced (93 ± 4% inhibition) by the phospholipase C inhibitor U73122 (10 µM). The activator of protein kinase C (PKC) phorbol 12-tetradecanoyl-13-acetate (TPA, 100 nM) stimulated [3 H]-thymidine incorporation (270±8% of basal), and this response was not additive with that to 100 µM histamine. The incorporation of [3 H]-thymidine induced by 100 µM histamine was partially reduced by the PKC inhibitor Ro 31-8220 (57 ± 7% inhibition at 300 nM) and by the compound PD 098,059 (30 µM, 62 ± 14% inhibition), an inhibitor of the mitogen-activated kinase (MAPK) kinases MEK1/MEK2. These results show that histamine H1 receptor activation stimulates the proliferation of human astrocytoma U373 MG cells. The action of histamine appears to be partially mediated by PKC stimulation and MAPK activation.

Introduction Histamine is a modulator in mammalian central nervous system, where regulates pre- and postsynaptically functions such as awareness, wakefulness, feeding and drinking behaviours, body temperature, analgesia and motor activity, among others [1,2]. Histaminergic neurones are located in the hypothalamus from where they send diffuse projections to almost all brain regions [3]. On the basis of their pharmacology and signal transduction mechanisms, histamine receptors have been subdivided into H1 , H2 and H3 subtypes, all members of the G-protein-coupled receptor family [2,4], although the molecular cloning of a novel histamine receptor (GPRv53) preferentially expressed in leukocytes was recently reported [5]. H1 receptors preferentially couple to the Gαq/11 family of G proteins and thus to Ca2+ mobilisation from intracellular stores, whereas H2 receptors stimulate cyclic AMP (cAMP) formation through the activation of GαS proteins [2,4]. H3 receptors regulate

the synthesis and release of histamine, as well as the release of other important neurotransmitters such as acetylcholine, noradrenaline, dopamine, serotonine [2] and γ -aminobutyric acid (GABA) [6]. H3 receptors appear to couple to both GαO and Gαi proteins, since H3 receptor activation produces inhibition of N-type voltage-sensitive Ca2+ channels and reduces forskolin-stimulated cAMP accumulation [2,7]. Gliomas, in particular those of astrocytic origin, are the most common of primary brain tumours [8]. Apart from being located on neurones, histamine H1 receptors are expressed by astrocytes [9–12]. The cell line U373 MG, derived from a human astrocytoma, expresses H1 receptors whose binding properties are similar to those of receptors present in mammalian brain [13]. Hence, this cell line appears to be a useful model on which to study astrocytic functions modulated by H1 receptor activation under physiological and pathological conditions. As stated above, H1 receptors are among those receptors coupled to Gαq/11 proteins which, when stimulated

82 by agonist-occupied receptors, activate a membranebound, PIP2 -specific phospholipase C (PLC) that breaks down phosphatidylinositol 4,5-biphosphate (PIP2 ) into inositol 1,4,5-triphosphate (Ins(1,4,5)P3 ) and diacylglicerol (DAG). Ins(1,4,5)P3 mobilises Ca2+ ions from intracellular stores while DAG activates protein kinase C, (PKC) [14]. Growth factors are involved in the regulation of normal cell division and in the generation of tumours, and some, such as endothelin-1 and angiotensin II, act on cell-surface, Gprotein-coupled receptors [15]. These receptors appear to regulate cellular growth by activating PLC and hence Ins(1,4,5)P3 and DAG production, leading to PKC activation [15–18]. We report herein that in the U373 MG cell line, derived from a human astrocytoma, H1 receptor activation results in increased cell proliferation, as assessed by [3 H]-thymidine incorporation, an effect that appears to be partially related to PKC stimulation and to the activation of mitogen-activated protein kinases (MAPKs). Experimental procedures Cell culture Human astrocytoma U373 MG cells were a kind gift from Dr. J.M. Young (Department of Pharmacology, University of Cambridge, UK). Cells were grown in Dulbecco’s modified Eagle Medium (DMEM)/nutrient mixture F-12 (1 : 1 v : v) supplemented with 10% bovine foetal serum, penicillin (50 UI/ml) and streptomycin (0.1 mg/ml). Cells were grown as monolayers in either 150 cm2 flasks or 24-well plates in a humidified atmosphere (5% CO2 ) at 37◦ C.

[3 H]-Inositol phosphate ([3 H]-IPs) accumulation Measurements of [3 H]-IPs accumulation in [3 H]inositol-labelled cell monolayers were carried out as described in detail elsewhere [13]. [3 H]-Thymidine incorporation Cells were plated in 24-well plates at a density of 3 × 105 cells/well and were grown for 24 h in normal medium before changing to serum-free medium for 48 h. Incubation in serum-free medium resulted in a marked decrease in cell growth since cell counting after 72 h yielded 117 ± 5% of plating values whereas the number of cells grown in sister wells in serumcontaining medium was 221 ± 26% of plating values. After 48 h in serum-free medium, fresh medium (serum-free) containing drugs under test was added for 1 h (except for time-course experiments), the medium was then replaced by drug- and serum-free medium and incubations were continued for a further 23 h with [3 H]-thymidine (1 µCi/well) being present for the last 4 h. For the determination of [3 H]-thymidine incorporation into the trichloroacetic acid (TCA)-insoluble fraction, cells were washed twice with Krebs–Henseleit buffer (composition in mM: NaCl 116, KCl 4.7, MgSO4 1, KH2 PO4 1.2, NaHCO3 25, CaCl2 2, d-glucose 11; pH 7.4 after saturation with O2 /CO2 , 95 : 5%), before adding 1 ml ice-cold 5% TCA (w : v). After 20 min on ice the TCA-insoluble material was washed twice with ethanol before adding 0.5 ml 0.1 M NaOH (2% Na2 CO3 ). Plates were allowed to stand overnight at room temperature, samples were then neutralised with 0.1 M HCl, scintillation liquid was added and the tritium content was determined by liquid scintillation counting.

Radioligand binding Data analysis Cells were lysed in 10 mM Tris–HCl buffer (pH 7.4) containing 1 mM EGTA, by using a Polytron (set 7, 10 s). Membranes were obtaining by two cycles of centrifugation (30 min, 20,000 × g) and resuspension in 50 mM Tris–HCl solution (pH 7.4). Radioligand binding was assayed in 50 mM Tris– HCl, 5 mM MgCl2 essentially as described elsewhere [13], in the presence of 0.1–10 nM [3 H]-mepyramine (H1 receptors), 20 nM [3 H]-tiotidine (H2 receptors) or 4 nM N -α-[methyl-3 H]-histamine (H3 receptors). Nonspecific binding was defined by 1 µM mepyramine, 10 µM tiotidine and 1 µM thioperamide, respectively.

All data are expressed as means±SEM. Concentrationresponse curves were fitted by non-linear regression to a logistic (Hill) equation using the program Prism (GraphPad Software, San Diego, CA). Materials [3 H]-Thymidine, myo[3 H]-inositol and fura 2-AM were purchased from Sigma Chemical Co. (St. Louis, MO). [3 H]-tiotidine and N -α-[methyl-3 H]-histamine were from New England Nuclear (Boston, MA).

Mepyramine (pyrilamine maleate), histamine dihydrochloride, thioperamide maleate, TPA (phorbol 12-tetradecanoyl-13-acetate), Ro 31-8220 (bisindoylmaleimidine IX), U73122 (1-[6-[(17b)-3 methoxyestra-1,3,5(10)-trien-17-yl]hexyl]-1H-pyrrole2,5-dione) and PD 098,059 (2-(2-amino-3methoxyphenyl)-4H-1-benzopyran-4-one) were from Research Biochemicals Inc. (Natick, MA). TPA, Ro 31-8220, PD 098,059 and U73122 were dissolved in ethanol. All other drugs were dissolved in water.

[3H]-Thymidine incorporation ( % basal )

83 180 160 140 120 100 -7

Radioligand binding to cell membranes In line with previous results [13], significant specific [3 H]-mepyramine binding to membranes of U373 MG cells was detected with best-fit values of 0.61±0.10 nM for the dissociation constant (Kd ) and 180 ± 6 fmol/mg protein for maximum binding (combined data from three experiments). In contrast, specific binding for either 20 nM [3 H]-tiotidine or 4 nM N -α-[methyl-3 H]histamine was not detected. [3 H]-Thymidine incorporation Effect of histamine Since three batches of [3 H]-thymidine with different specific activity were used through this study, the incorporation of the labelled compound was expressed as a percentage of controls. Time-course experiments showed that [3 H]-thymidine incorporation stimulated by histamine (100 µM) was statistically different from controls after 30-min incubations, and increased steadily up to 4 h reaching a plateau that remained for 2 h before decreasing markedly afterwards (data not shown). In subsequent experiments incubation with histamine was thus set at 1 h. Histamine-induced [3 H]-thymidine incorporation was concentration-dependent with EC50 2.5 ± 0.4 µM, maximum effect 173 ± 2% of basal and Hill coefficient (nH ) 1.1±0.2 (Figure 1). In agreement with an H1 receptor-mediated effect, the action of histamine was fully blocked by the selective H1 antagonist mepyramine (Figure 2). The concentration of the antagonist (1 µM) was that calculated to block >97% of the effect of the agonist, assuming that the EC50 value for histamine-induced [3 H]-thymidine incorporation approximates the Kd of the agonist.

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Figure 1. Concentration-response curve for histamine-induced [3 H]-thymidine incorporation. Cells were maintained in serumfree medium for 48 h, histamine was added for 1 h and incubations were continued for a further 23 h in serum-free medium with [3 H]-thymidine being present for the last 4 h. The incorporation of [3 H]-thymidine is expressed as a percentage of basal. Values are means ± SEM of the combined values from three experiments with three replicates for each concentration. The line drawn is the best-fit to a logistic (Hill) equation. Best estimates are given in the text.


Results

-6 -5 -4 Log [ Histamine (M) ]

175

a

150 125 100 75 50 25 0

Basal 100 µM 1 µM Hist mepy

Hist + mepy

Figure 2. Inhibition by mepyramine of histamine-induced [3 H]-thymidine incorporation. Histamine (100 µM; Hist) was present for 1 h. When required, mepyramine (1 µM; mepy) was added 15 min before histamine. [3 H]-Thymidine incorporation is expressed as a percentage of basal incorporation. Values are means ± SEM of the combined values from three experiments with six replicates for each condition. a Significantly different (P < 0.05) from basal; ANOVA and post hoc Dunnett test.

U373 MG cells express NK1 receptors coupled to PLC activation and whose activation stimulates DNA synthesis and cell proliferation [19]. In a series of experiments in which histamine (100 µM) stimulated [3 H]-thymidine incorporation to 166 ± 7% of basal, the selective NK1 agonist Sar9 -O11 -met-substance P

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Effect of PLC inhibition on histamine-induced [3 H]-IPs accumulation and [3 H]-thymidine incorporation In [3 H]-inositol-labelled cells and in the presence of 20 mM LiCl, basal [3 H]-IPs formation after 30-min incubations was 380 ± 20 dpm/well (n = 4 experiments). The addition of histamine resulted in a concentration-dependent increase in [3 H]-IPs accumulation (maximum effect 495 ± 12% of basal, EC50 2.8 ± 0.4 µM, nH 1.3 ± 0.2). The effect of 100 µM histamine (483 ± 26% of basal) was reversed (98 ± 1% inhibition) by the H1 antagonist mepyramine (1 µM). Figure 3A shows that pre-incubation with the PLC inhibitor U73122 resulted in a concentrationdependent inhibition of the [3 H]-IPs accumulation induced by either histamine or the NK1 agonist Sar9 -O11 -met-SP (IC50 estimates 2.5 ± 0.2 µM and 1.9 ± 0.1 µM, respectively). The incubation with a single concentration (10 µM) of U73122 that resulted in 92 ± 1% inhibition of histamine-stimulated [3 H]-IPs reduced by 93 ± 4% the incorporation of [3 H]thymidine induced by 100 µM histamine (Figure 3B). Effect of PKC activation and inhibition on [3 H]-thymidine incorporation The drug phorbol TPA, known to activate directly PKC, was also able to stimulate [3 H]-thymidine incorporation (Figure 4). The simultaneous addition of TPA (100 nM) and histamine (100 µM) increased [3 H]-thymidine incorporation to levels (359 ± 21% of basal, 98 ± 9% of TPA alone) statistically different (P < 0.05, Student’s t test) of those expected for additive actions (expected value, 461 ± 30% of basal). In order to assess further the involvement of PKC, the effect of histamine was tested in the presence of the inhibitor Ro 31-8220 (Figure 5). Whereas no significant effect was observed on basal [3 H]-thymidine incorporation, 0.3 µM Ro 31-8220 inhibited by 61±6% the action of 100 µM histamine. Increasing the concentration of the PKC inhibitor up to 1 µM resulted in no further inhibition (57 ± 7% reduction).

A [3H]-IPs ( % control )

100 75 50 25

Histamine Sar9-O11-met-SP

0 -7.0

B [3H]-Thymidine incorporation ( % basa l )

(Sar9 -O11 -met-SP; 100 nM) increased [3 H]-thymidine incorporation to 183 ± 16% of basal. The simultaneous addition of Sar9 -O11 -met-SP and histamine-increased [3 H]-thymidine incorporation to levels (194 ± 14% of basal) statistically different (P < 0.05, Student’s t test) from those expected for additive actions (expected value, 249 ± 17% of basal).

200

-6.5 -6.0 -5.5 Log [ U73122 (M) ]

-5.0

a

150 100 50 0

Basal 10 µM 100 µM Hist + U73122 Hist U73122

Figure 3. Inhibition by U73122 of agonist-stimulated [3 H]-IPs accumulation and histamine-induced [3 H]-thymidine incorporation. (A) Effect of U73122 on [3 H]-IPs accumulation. [3 H]-IPs formation stimulated by histamine (100 µM) or Sar9 -O11 -metsubstance P (Sar9 -O11 -met-SP, 100 nM) was determined in the presence of the indicated concentrations of the PLC inhibitor U73122. Values are means±SEM of the combined data from three experiments with three replicates for each condition. (B) Effect of U73122 on histamine-induced [3 H]-thymidine incorporation. Histamine (100 µM) was present for 1 h. When required, U73122 (10 µM) was added 15 min before histamine. [3 H]-thymidine incorporation is expressed as a percentage of basal incorporation. Values are means ± SEM of the combined values from three experiments with six replicates for each condition. a Significantly different (P < 0.05) from basal; ANOVA and post hoc Dunnett test.

Effect of PD 098,059 on histamine-induced [3 H]-thymidine incorporation Figure 6 shows the effect of the MEK1/2 inhibitor PD 098,059 on histamine-stimulated [3 H]-thymidine incorporation. A single concentration (30 µM) of PD 098,059 shown to inhibit circa 85% of the [3 H]-thymidine incorporation stimulated by plateletderived growth factor (PDGF) in 3T3 mouse fibroblasts [20], had no significant effect on basal [3 H]-thymidine

a

400

b

300 a 200 100 0

Basal 100 µM 100 nM Hist + Hist TPA TPA


Figure 4. Effect of TPA on [3 H]-thymidine incorporation. TPA (100 nM), histamine (100 µM; Hist) or TPA + histamine were present for 1 h. [3 H]-Thymidine incorporation is expressed as a percentage of basal incorporation. Values are means ± SEM of the combined data from three experiments with six replicates for each condition. a Significantly different (P < 0.05) from basal; b Not significantly different from TPA alone; ANOVA and post hoc Student–Newman–Keuls test. a

175 150

a,b

125 100 75 50 25 0 Basal

100 µM 0.3 µM Ro318220 Hist Ro318220 + Hist

Figure 5. Effect of the PKC inhibitor Ro 31-8220 on histamineinduced [3 H]-thymidine incorporation. The effect of histamine (100 µM) was evaluated in the absence or presence of 0.3 µM Ro 31-8220 added 15 min before histamine. [3 H]-Thymidine incorporation is expressed as a percentage of basal incorporation. Values are means ± SEM of the combined data from three experiments with six replicates for each condition. a Significantly different (P < 0.05) from basal; b Significantly different (P < 0.05) from histamine alone; ANOVA and post hoc Student–Newman– Keuls test.

incorporation, but inhibited by 62 ± 14% the action of 100 µM histamine. Discussion Histamine is closely associated with mast cells in almost all tissues with a long established role



85 a

175

a,b

150 125 100 75 50 25 0 Basal

100 µM 30 µM PD098,059 Hist PD098,059 + Hist

Figure 6. Effect of the MEK1/MEK2 inhibitor PD 098,059 on histamine-induced [3 H]-thymidine incorporation. The effect of histamine (100 µM) was evaluated in the absence or presence of 30 µM PD 098,059 added 15 min before histamine. [3 H]-Thymidine incorporation is expressed as a percentage of basal incorporation. Values are means ± SEM of the combined data from three experiments with six replicates for each condition. a Significantly different (P < 0.05) from basal; b Significantly different (P < 0.05) from histamine alone; ANOVA and post hoc Student–Newman–Keuls test.

as mediator of inflammation. Other functions in which histamine is involved include smooth muscle contraction, regulation of endothelial cell function, gastric acid secretion, hormone release and heart muscle contraction [4]. In addition, histamine is widely distributed within the mammalian central nervous system where its function as neuromodulator is strongly supported by experimental evidence [1,2]. The activity of the enzyme histidine decarboxylase, which catalyses the formation of histamine from histidine, has been shown to be higher in tissues undergoing rapid growth or repair [21,22] suggesting also a role for histamine in cell proliferation. Astrocytes are involved in a variety of mammalian central nervous system functions such as terminating the action of neurotransmitters, buffering extracellular K+ and providing energetic substrate for neuronal metabolism [23,24]. In addition, damage to brain tissue induces reactive gliosis characterised by both cell hyperplasia and hypertrophy as well as by the presence of large numbers of reactive astrocytes, derived from pre-existing astrocytes as recent data indicate [25,26]. Accordingly, astrocyte proliferation has been demonstrated in adult rats and mice [27]. Gliomas are the most common primary tumours of the human central nervous system and more than 70% of malignant gliomas correspond to astrocytomas and their most malignant form, gliobastoma multiforme

86 [8,28,29]. For this reason alone, astrocytoma-derived cells continue to be a major focus of neurobiological and neuro-oncological research. In particular, much effort is being directed at understanding the cellular mechanisms involved in the regulation of proliferation of astrocytic cancer cells. The cell line U373 MG, derived from a human astrocytoma, expresses histamine H1 receptors whose affinity for [3 H]-mepyramine (Kd 0.61 ± 0.10 nM) is similar to that reported for mammalian brain [4], and whose activation results in phosphoinositide hydrolysis and Ca2+ mobilisation from intracellular stores (data presented herein and ref. [30]). Furthermore, U373 MG cells are immunoreactive to glial fibrilar acidic protein [31] and take up the neurotransmitter [3 H]-GABA in a Na+ and Cl− -dependent manner (E. Sánchez-Lemus and J.A. Arias-Montaño, unpublished observations). This astrocyte-derived cell line appears thus to be a useful model on which to study the effect of histamine on glial functions related to physiological and pathological conditions, and in this work we show that histamine H1 receptor activation results in stimulation of the proliferation of U373 MG cells, as assessed by the incorporation of [3 H]-thymidine. Growth factors are involved in the regulation of normal cell division and in the generation of tumours, and some agonists at G-protein-coupled receptors can act as growth factors by stimulating PKC through the production of the second messengers Ins(1,4,5)P3 and DAG. PKC appears to phosphorylate and activate the protein Raf-1 which in turn stimulates MEK1/MEK2 (MAPK) kinases. The phosphorylation by MEK1/MEK2 is required for activation of MAPKs, a subfamily of extracellularly-responsive or extracellular signalregulated kinases (ERKs), the final result being the phosphorylation and activation of transcription factors and gene activation [15,18,32–34]. U373 MG cells express the PKC isoforms α, β, γ , δ and ε [35]. Isoforms α, β and γ belong to the family of conventional PKCs, activated by phosphatidylserine (PS) in a Ca2+ -dependent manner. These PKCs bind DAG, which increases their specificity for PS and shifts the affinity for Ca2+ into the physiological range. Isoforms δ and ε (novel PKCs) are Ca2+ -insensitive, but still activated by DAG or phorbol esters in the presence of PS [36]. Some pieces of evidence suggest that the effect of histamine on [3 H]-thymidine incorporation in U373 MG cells could be due, at least in part, to one or more PKC isoforms being activated by DAG. Firstly, H1 receptor activation stimulates PIP2 hydrolysis and thus Ins(1,4,5)P3

and DAG production. The latter would then activate PKC either by itself or in conjunction with Ca2+ ions released by Ins(1,4,5)P3 , and our results show that histamine-stimulated [3 H]-thymidine incorporation is almost abolished by PLC inhibition. In addition, histamine stimulates [3 H]-thymidine incorporation with an EC50 estimate (2.5 ± 0.4 µM) in good agreement with values obtained for histamine-stimulated [3 H]-IPs accumulation (2.8 ± 0.4 µM) and Ca2+ mobilisation (1.86 or 4.6 µM) [37,38]. Further, [3 H]-thymidine incorporation was also stimulated by the activation of NK1 receptors coupled to PIP2 hydrolysis, and the effect of the NK1 agonist Sar9 -O11 -met-SP was not additive with that of histamine. Secondly, stimulation of cell proliferation was also seen when U373 MG cells were incubated with TPA, known to activate directly PKC by acting on the DAG-binding site. The actions of histamine and TPA on [3 H]-thymidine incorporation were not additive, suggesting a common pathway for their stimulatory effects. Finally, the action of histamine was markedly (but not fully) inhibited by Ro 31-8220, a selective and potent PKC inhibitor [39]. In primary glial cells phorbol ester treatment stimulates DNA synthesis and proliferation, an effect prevented by the PKC inhibitor H7 [40], and in 132-1N1 human astrocytoma cells that express muscarinic receptors (M3 and M5 ) coupled to phosphoinositide hydrolysis, the stimulatory effect of the agonist carbachol on DNA synthesis is attenuated by PKC inhibition [41,42]. PKC-mediated Raf activation results in the phosphorylation and stimulation of MEK1/MEK2. It is well established that the sequential phosphorylation by MEK1/MEK2 of the tyrosine and threonine residues within a Tyr-X-Thr MAPK activation motif is required for MAPK stimulation [34], and in U373 MG cells histamine-induced mitogenesis might also involve MAPK activation since histaminestimulated [3 H]-thymidine incorporation was significantly blocked by PD 098,059, a drug shown to inhibit MEK1/2 activation and thus MAPK42,44 stimulation [20]. However, and as stated above, we found a significant fraction (∼40%) of histamine-induced [3 H]-thymidine incorporation to U373 MG cells to be insensitive to PKC blockade, in line with other studies that report that in DDT1 MF-2 smooth muscle cells histamine H1 receptor activation results in MAPK activation only partially blocked (41 ± 7% inhibition) by the PKC inhibitor Ro 31-8220 [43], and that in C6 glioma cells that express P2Y2 receptors coupled to PLC activation, UTP-induced [3 H]-thymidine incorporation was

87 significantly, but not fully blocked by PKC inhibitors [44]. In addition, in rat cortical astrocytes endotelin-1 stimulates phosphoinositide hydrolysis and Ca2+ mobilisation, as well as [3 H]-thymidine incorporation that is fully prevented by the PLC inhibitor U73122, but not by PKC inhibitors [45]. Taken together, these pieces of information suggest that additional Ca2+ -dependent, PKC-independent mechanisms are involved in the mitogenic signals activated by PLC-coupled receptors. It has been reported that ligand-independent (transactivation) of the epidermal-growth-factor receptor is a general phenomenon evoked by various Gq/11 -coupled receptors in different cellular settings, and a rise in [Ca2+ ]i appears to be sufficient to trigger growth factor-independent phosphorylation of receptor tyrosine kinases in some cell lines [18]. With this background in mind, we tested the effect of genistein, a general inhibitor of tyrosine kinases [46], on histamine-induced [3 H]-thymidine incorporation. However, a concentration of 100 µM of the inhibitor resulted in a marked increase in [3 H]-thymidine incorporation (280 ± 40% of basal), making the compound unsuitable for testing the involvement of tyrosine kinase transactivation in the proliferative action of histamine. We have no explanation for the stimulatory action of genistein on [3 H]-thymidine incorporation. An alternative mechanism for the PKC-insensitive component of histamine-induced mitogenesis would be the release by U373 MG cells of proliferative substances in response to H1 receptor activation. For instance, it has been shown that H1 receptor activation stimulates arachidonic acid release [47] and products of the arachidonic acid metabolism such as leukotriene B4 have been shown to stimulate the proliferation of astrocytoma cells [48]. Histamine also induces the release of nerve growth factor from primary cultured astrocytes [49]. In addition, reactive astrocytes and U373 MG cells release cytokines to the extracellular medium [50,51] and recombinant human interleukin-1β stimulates mitogenesis in U373 MG cells [52]. Clearly, further research will be required to understand the precise role and mechanisms of glial mitogenesis induced by histamine and other neuroactive substances. Finally, it must be mentioned that other studies have provided results similar to those presented here. Histamine has been shown to stimulate the growth of cultured human glioma tumour cells [53] as well as [3 H]-thymidine incorporation to the DNA of primary cerebellar and forebrain astrocytes [54,55], although the mechanisms involved were not studied.

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Address for offprints: José-Antonio Arias-Montaño, Departamento de Neurociencias, CINVESTAV Apdo. postal 14-740, 07000 México, D.F., México; Tel.: (+525) 7 477 000 Ext. 5717, 5718; Fax: (+525) 7 473 754; E-mail: [email protected]