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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2001 Aug;133(8):1378–1386. doi: 10.1038/sj.bjp.0704200

Activation of the p38 and p42/p44 mitogen-activated protein kinase families by the histamine H1 receptor in DDT1MF-2 cells

Alex J Robinson 1, John M Dickenson 1,*
PMCID: PMC1621152  PMID: 11498525

Abstract

  1. The mitogen-activated protein kinases (MAPKs) consist of the p42/p44 MAPKs and the stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38 MAPK. In this study we have examined the effect of histamine H1 receptor activation on MAPK pathway activation in the smooth muscle cell line DDT1MF-2.

  2. Histamine stimulated time and concentration-dependent increases in p42/p44 MAPK activation in DDT1MF-2 cells. Responses to histamine were inhibited by the histamine H1 receptor antagonist mepyramine (KD 3.5 nM) and following pre-treatment with pertussis toxin (PTX; 57% inhibition).

  3. Histamine-induced increases in p42/p44 MAPK activation were blocked by inhibitors of MAPK kinase 1 (PD 98059), tyrosine kinase (genistein and tyrphostin A47), phosphatidylinositol 3-kinase (wortmannin and LY 294002) and protein kinase C (Ro 31-8220; 10 μM; 41% inhibition). Inhibitors of Src tyrosine kinase (PP2) and the epidermal growth factor tyrosine kinase (AG1478) were without effect. Removal of extracellular Ca2+, chelation of intracellular Ca2+ with BAPTA and inhibition of focal adhesion assembly (cytochalasin D) had no significant effect on histamine-induced p42/p44 MAPK activation.

  4. Histamine stimulated time and concentration-dependent increases in p38 MAPK activation in DDT1MF-2 cells but had no effect on JNK activation. Histamine-induced p38 MAPK activation was inhibited by pertussis toxin (74% inhibition) and the p38 MAPK inhibitor SB 203580 (95% inhibition).

  5. In summary, we have shown the histamine H1 receptor activates p42/p44 MAPK and p38 MAPK signalling pathways in DDT1MF-2 smooth muscle cells. Interestingly, signalling to both pathways appears to involve histamine H1 receptor coupling to Gi/Go-proteins.

Keywords: Histamine H1 receptor, mitogen-activated protein kinases, DDT1MF-2 cells

Introduction

Histamine H1 receptors belong to the G protein-coupled receptor (GPCR) superfamily and are linked to the activation of phospholipase C (PLC)-β isoforms via pertussis toxin-insensitive Gq/11-proteins (for review see Hill et al., 1997). Stimulation of PLC triggers the hydrolysis of the plasma membrane phospholipid, phosphatidylinositol 4,5-biphosphate producing two second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 stimulates the release of Ca2+ from intracellular stores, whereas DAG activates specific isoforms of protein kinase C (PKC). Furthermore, histamine H1 receptor-induced increases in intracellular Ca2+ are known to modulate the activity of Ca2+/calmodulin-dependent enzymes such as nitric oxide synthetase, phospholipase A2 (PLA2) and adenylyl cyclase (for review see Leurs et al., 1995). In addition to the expected coupling to phospholipase C, recent studies have shown that Gq-PCRs are also capable of activating other signalling pathways including members of the mitogen-activated protein kinase (MAPK) family and protein kinase B (Sugden & Clerk, 1997; Murga et al., 1998).

MAPKs are a group of serine/threonine protein kinases comprising three main subfamilies: the p42/p44 extracellular regulated kinases (ERKs), also known as p42/p44 MAPKs; the c-Jun N-terminal kinases (JNKs) which are also known as stress-activated protein kinases (SAPKs) and the p38 MAPKs (Denhardt, 1996; Widmann et al., 1999). The p42/p44 MAPKs are primarily associated with the regulation of cell proliferation and differentiation, whereas the JNKs and p38 MAPKs are involved in apoptosis, inflammation and responses to environmental stress (Paul et al., 1997; Ono & Han, 2000). The p42/p44 MAPK pathway is associated with activation by receptor tyrosine kinases (e.g., growth factor receptors), whereas p38 MAPK and JNK are activated by stimuli such as UV irradiation, osmotic stress and inflammatory cytokines (Malarkey et al., 1995; Paul et al., 1997). Recent studies indicate that a wide range of GPCRs, including members of the Gq-coupled family, are also involved in the regulation of p42/p44 MAPK, p38 MAPK and JNK pathways (for reviews see Sugden & Clerk, 1997; van Biesen et al., 1996). However, to our knowledge, there have been no reports describing the activation of MAPK signalling pathways by the histamine H1 receptor.

Our previous studies have shown that the histamine H1 receptor stimulates inositol phospholipid hydrolysis, release of Ca2+ from intracellular Ca2+ stores and Ca2+ influx in the hamster vas deferens derived smooth muscle cell line, DDT1MF-2 (White et al., 1993; Dickenson & Hill, 1991; 1992). In the present study we have investigated whether the histamine H1 receptor regulates p42/p44 MAPK, p38 MAPK and JNK signalling pathways in DDT1MF-2 cells. Preliminary data from this study has been presented to the British Pharmacological Society (Robinson & Dickenson, 2000).

Methods

Cell culture

The hamster vas deferens smooth muscle cell line (DDT1MF-2) was obtained from the European Collection of Animal Cell Cultures (Porton Down, Salisbury, U.K.). DDT1MF-2 cells were cultured in 75 cm2 flasks in Dulbecco's modified Eagles medium (DMEM) supplemented with 2 mM L-glutamine and 10% (v v−1) foetal calf serum. Cells were maintained at 37°C in a humidified 5% CO2 atmosphere until confluency and subcultured (1 : 10 split ratio) using trypsin (0.05% w v−1)/EDTA (0.02% w v−1). Cells for determination of MAPK activation were grown in 6-well cluster dishes.

Western blot analysis

DDT1MF-2 cells were grown in 6-well plate cluster dishes and when 80–90% confluent placed in DMEM medium containing 0.1% bovine serum albumin for 16 h. Serum-starved cells were then washed once with Hanks/HEPES buffer, pH 7.4, and incubated at 37°C for 30 min in 500 μl well−1 of the same medium. Where appropriate kinase inhibitors were added during this incubation period. Agonists were subsequently added in 500 μl of medium and the incubation continued for 5 min (unless otherwise stated) at 37°C. Stimulation's were terminated by aspiration of the medium and the addition of 300 μl of ice-cold lysis buffer (mM): NaCl 150, Tris.HCl 50, EDTA 5, 1% (v v−1) IGEPAL CA-630, 0.5% (w v−1) sodium deoxycholate, 0.1% (w v−1) SDS, Na3VO4 1, NaF 1, benzamidine 1, phenylmethylsulphonylfluoride 0.1, 10 μg/ml aprotinin and 5 μg/ml leupeptin. Cells were then incubated on ice for 5 min, after which the cell lysates were removed and placed into Eppendorf microcentrifuge tubes and vortexed. Insoluble material was removed by centrifugation and 250 μl of the cell lysate removed and stored at −20°C until required. Protein determinations were made using the method of Lowry et al. (1951) using bovine serum albumin as the standard and samples stored at −20°C until required.

Protein samples (20 μg) were separated by Sodium Dodecyl Sulphate/Polyacrylamide Gel Electrophoresis (SDS/PAGE; 10% acrylamide gel) using a Bio-Rad Mini-Protean II system (1 h at 200 V). Proteins were transferred to nitrocellulose membranes using a Bio-Rad Trans-Blot system (1 h at 100 V in 25 mM Tris, 192 mM glycine and 20% MeOH). Following transfer, the membranes were washed with phosphate buffered saline (PBS) and blocked for 1 h at room temperature with 5% (w v−1) skimmed milk powder in PBS. Blots were then incubated overnight at 4°C with primary antibodies in 5% (w v−1) skimmed milk powder dissolved in PBS-Tween 20 (0.5% by vol). Primary antibodies were removed and the blot extensively washed with PBS/Tween 20. Blots were then incubated for 2 h at room temperature with the secondary antibody (goat anti-rabbit/mouse IgG coupled to horseradish peroxidase) at 1 : 1000 dilution in 5% (w v−1) skimmed milk powder dissolved in PBS/Tween 20. Following removal of the secondary antibody, blots were extensively washed as above and developed using the Enhanced Chemiluminescence detection system (Amersham) and quantified using the programme QuantiScan (BioSoft). The uniform transfer of proteins to the nitrocellulose membrane was routinely monitored by transiently staining the membranes with Ponceau S stain (Sigma Chemical Co.) prior to application of the primary antibody.

Data analysis

Agonist pEC50 values (−log EC50; concentration of drug producing 50% of the maximal response) were obtained by computer assisted curve fitting by use of the computer programme Prism (GraphPAD, CA, U.S.A.). Statistical significance was determined by Student's unpaired t-test or ANOVA followed by Dunnett's multiple comparison tests. All statistical analysis was performed using GraphPAD Software and P<0.05 was considered statistically significant. All data are presented as mean±s.e.mean. The n in the text refers to the number of separate experiments. Antagonist dissociation constants (KD) were estimated by a modification of the method of Lazareno & Roberts (1987). A concentration-response curve to an agonist was generated and a concentration (C) of the agonist under study chosen which gave a response greater than 50% of the maximal response. The concentration of antagonist (IC50) required to reduce the response of this concentration (C) of CPA by 50% was then determined. The agonist concentration-response curve was fitted to a logistic equation as described above and a concentration of the agonist identified (C1) which yielded a response equivalent to 50% of that produced by concentration C (in the absence of antagonist). The apparent KD was then determined from the following relationship: C/C1=IC50/KD+1.

Materials

Aprotinin, bovine serum albumin, Dulbecco's modified Eagles medium, foetal calf serum, histamine, leupeptin, mepyramine, and pertussis toxin were obtained from Sigma Chemical Co. (Poole, Dorset, U.K.). AG 1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline), BAPTA/AM, cytochalasin D, daidzein, epidermal growth factor, genistein, LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), PD 98059 (2′-amino-3′-methoxyflavone), PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), Ro 31-8220 (3-{1-[3-(2-isothioureido) propyl]indol-3-yl}-4-(1-methylindol-3-yl)-3-pyrrolin-2,5-dione), SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole) and tyrphostin A47 (α-cyano-(3,4-dihydroxy)thiocinnamide) were from Calbiochem (Nottingham, U.K.). Tiotidine and thioperamide were obtained from Tocris (Semat Technical (U.K.) Ltd). Phospho-specific p42/p44 MAPK and p38 MAPK antibodies were purchased from Sigma Chemical Co. Phospho-specific JNK antibody was from Promega. All other chemicals were of analytical grade.

Results

The regulation of MAPKs (p42/p44 MAPK, JNK and p38 MAPK) by the histamine H1 receptor has been investigated in the hamster vas deferens derived smooth muscle cell line, DDT1MF-2. The activities of MAPKs are controlled by dual phosphorylation within the amino acid sequence Thr-X-Tyr (Widmann et al., 1999). Increases in MAPK activation were determined by Western blot analysis using phospho-specific p42/p44 MAPK (Thr202/Tyr204), JNK (Thr183/Tyr185) and p38 MAPK (Thr180/Tyr182) antibodies.

Histamine H1 receptor-mediated activation of p42/p44 MAPK

Stimulation of DDT1MF-2 cells with histamine produced a rapid and transient increase in p42 and p44 MAPK phosphorylation with dominant activation of the p42 MAPK isoform (Figure 1a). The response to histamine was concentration-dependent (p[EC50]=6.1±0.3; n=4; Figure 1b) and time-dependent with peak activation occurring at 5 min. p42/p44 MAPK responses to histamine (100 μM) were antagonized by the histamine H1 receptor antagonist, mepyramine, yielding an apparent KD value of 3.1±0.8 nM (n=3; Figure 1c). Furthermore, pre-treatment with tiotidine (1 μM) and thioperamide (1 μM), histamine H2 and H3 receptor antagonists respectively, had no effect on histamine (100 μM) induced p42/p44 MAPK activation (Figure 1d). These data indicate that increases in p42/p44 MAPK activation following stimulation of DDT1MF-2 cells with histamine are mediated through the histamine H1 receptor. Our previous studies have shown that histamine H1 receptor activation in DDT1MF-2 cells stimulates PTX-insensitive increases in inositol phosphate accumulation and calcium mobilization (White et al., 1993; Dickenson & Hill, 1993). These observations reflect the proposed coupling of histamine H1 receptors to phospholipase C activation via Gq-proteins (Hill et al., 1997). However, in this study, pre-treatment with PTX (100 ng ml−1 for 16 h) significantly reduced histamine-induced p42/p44 MAPK activation (57±9% inhibition; n=7; P<0.05; Figure 2a) suggesting the involvement of Gi/Go proteins. In addition PTX pre-treatment inhibited basal p42/p44 MAPK phosphorylation (60±6% inhibition; n=7). Histamine (100 μM) induced p42/p44 MAPK activation was also inhibited by pre-treatment (30 min) of cells with the selective MAPK kinase inhibitor, PD 98059 (Dudley et al., 1995; 50 μM; 89±5% inhibition; n=3; P<0.05; Figure 2b).

Figure 1.

Figure 1

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Histamine H1 receptor stimulation of p42/p44 MAPK in DDT1MF-2 cells. Cell lysates (20 μg) were analysed for p42/p44 MAPK activation by Western blotting using a phospho-specific p42/p44 MAPK antibody. Representative Western blots for each experiment are shown in the upper panels. (a) Time-course profile for histamine-induced p42/p44 MAPK activation in DDT1MF-2 cells treated with vehicle (time zero) or histamine (100 μM) for the indicated periods of time. (b) Concentration-response curve for histamine in DDT1MF-2 cells treated with vehicle (control; C) or the indicated concentrations of histamine for 5 min. (c) Inhibition of histamine-induced p42/p44 MAPK activation by the selective histamine H1 receptor antagonist mepyramine. Cells were pre-incubated for 30 min with the indicated concentrations of mepyramine before stimulating with 100 μM histamine for 5 min. (d) Effect of pre-incubation (30 min) with tiotidine (1 μM; H2 receptor antagonist) and thioperamide (1 μM; H3 receptor antagonist) on histamine (100 μM)-induced p42/p44 MAPK activation. Combined results represent the mean±s.e.mean from three (a), four (b), three (c), and three (d) independent experiments.

Figure 2.

Figure 2

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Effect of pertussis toxin and the MAPK kinase inhibitor PD 98059 on histamine H1 receptor-induced p42/p44 MAPK activation in DDT1MF-2 cells. DDT1MF-2 cells were pre-treated with (a) pertussis toxin (PTX; 16 h 100 ng ml−1) and (b) the MAPK kinase inhibitor PD 98059 (30 min; 50 μM) prior to stimulating with histamine (100 μM) for 5 min. Similar results were obtained in at least four independent experiments.

Tyrosine kinases belonging to the Src family are involved in the regulation of p42/p44 MAPKs by both Gq and Gi/Go-protein-coupled receptors (Lopez-Ilasaca, 1998). In this study we explored the involvement of tyrosine kinases in histamine H1 receptor-induced p42/p44 MAPK activation using the broad range tyrosine kinase inhibitors genistein and tyrphostin A47 and the Src-family tyrosine kinase inhibitor, PP2 (Hanke et al., 1996). Responses to histamine (100 μM) were significantly reduced by genistein (100 μM; 87±8% inhibition; n=6; P<0.05; Figure 3a) and tyrphostin A47 (100 μM; 95±9% inhibition; n=6; P<0.05; Figure 3b). Daidzein (100 μM), the inactive analogue of genistein, had no significant effect on histamine-induced p42/p44 MAPK activation (95±8% of control response; n=4; P<0.05; Figure 3a). Similarly, the Src-family tyrosine kinase inhibitor PP2 also had no significant effect on histamine-induced p42/p44 MAPK activation (95±8% of control response; n=4; P<0.05; Figure 3a). Recent studies have also demonstrated that certain GPCRs activate the p42/p44 MAPK pathway via transactivation (ligand-independent) of the epidermal growth factor receptor tyrosine kinase (Zwick et al., 1999). In this study, pre-incubation of cells with AG1478 (1 μM; 30 min), an inhibitor specific for the epidermal growth factor receptor tyrosine kinase had no significant effect on histamine (100 μM)-mediated p42/p44 MAPK activation. In contrast, epidermal growth factor (10 nM)-induced p42/p44 MAPK activation was completely inhibited (10±8% of control response; n=4; Figure 3c) by AG1478 (1 μM). These data suggest that epidermal growth factor receptor transactivation is not involved in histamine H1 receptor-mediated p42/p44 MAPK activation in DDT1MF-2 cells.

Figure 3.

Figure 3

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Effect of tyrosine kinase inhibitors on histamine H1 receptor stimulation of p42/p44 MAPK in DDT1MF-2 cells. Representative immunoblots showing the effects of pre-treating DDT1MF-2 cells for 30 min with (a) the broad range tyrosine kinase inhibitor genistein (100 μM) and daidzein (100 μM), the inactive analogue of genistein, (b) the broad range tyrosine kinase inhibitor tyrphostin A47 (100 μM) and the Src tyrosine kinase inhibitor PP2 (10 μM) and (c) AG1478 (1 μM) the EGF receptor tyrosine kinase inhibitor. Cells were stimulated with histamine (100 μM) or EGF (10 nM) for 5 min. Similar results were obtained in at least three independent experiments.

Role of Ca2+, PKC and focal adhesion kinases in histamine H1 receptor-induced p42/p44 MAPK activation

PLC activation generates IP3 and DAG, which mobilize intracellular Ca2+ and activate certain PKC isoforms, respectively (Berridge, 1993). Indeed, histamine H1 receptor activation in DDT1MF-2 cells stimulates intracellular Ca2+ release and Ca2+ influx (Dickenson & Hill, 1991; 1992). In this study we have examined the role of Ca2+ and PKC in the regulation of p42/p44 MAPK by the histamine H1 receptor. The role Ca2+ influx was explored by measuring p42/p44 MAPK responses in the absence of extracellular Ca2+ (using nominally Ca2+-free Hanks/HEPES buffer containing 0.1 mM EGTA). This procedure prevents histamine H1 receptor-mediated Ca2+ influx in DDT1MF-2 cells (Dickenson & Hill, 1992). As shown in Figure 4a, removal of extracellular Ca2+ had no significant effect on histamine (100 μM; 81±9% of control response; n=8; P>0.05) induced p42/p44 MAPK activation. The potential role of Ca2+ derived from intracellular stores was investigated using the Ca2+ chelator BAPTA (cells pre-incubated for 30 min with 50 μM BAPTA/AM) in the absence of extracellular Ca2+. Loading cells with BAPTA in the absence of extracellular Ca2+ did not inhibit histamine (90±12% of control; n=4) induced p42/p44 MAPK activation (Figure 4b). These observations demonstrate that p42/p44 MAPK activation by histamine H1 receptors is independent of Ca2+ elevation in DDT1MF-2 cells.

Figure 4.

Figure 4

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Role of Ca2+, PKC and focal adhesion kinase in histamine H1 receptor stimulation of p42/p44 MAPK in DDT1MF-2 cells. Measurements of p42/p44 MAPK activation (5 min stimulation with 100 μM histamine) were made as follows: (a) in the presence (1.3 mM CaCl2) and absence of extracellular Ca2+ (nominally Ca2+-free Hanks/HEPES buffer containing 0.1 mM EGTA) to prevent Ca2+ influx; (b) using BAPTA (cells pre-incubated for 30 min with 50 μM BAPTA/AM) in the absence of extracellular Ca2+ (nominally Ca2+-free Hanks/HEPES buffer containing 0.1 mM EGTA) to chelate Ca2+-released from intracellular stores; (c) in cells pre-incubated with the PKC inhibitor Ro 31-8220 (10 μM) and (d) in cells pre-incubated for 1 h with cytochalasin D (1 μM) to disrupt focal adhesion-based signalling. Similar results were obtained in at least four independent experiments.

The role of PKC in the regulation of p42/p44 MAPK by the histamine H1 receptor was explored using the PKC inhibitor Ro 31–8220 (Davis et al., 1989). The PKC activator, phorbol 12-myristate 13-acetate (100 nM) induced p42/p44 MAPK activation in DDT1MF-2 cells and as expected Ro 31–8220 (10 μM) inhibited this response (85±5% inhibition; n=5). Responses to histamine (100 μM) were partially sensitive to PKC inhibition (41±7% inhibition; n=6) indicating the possible involvement of a PKC-dependent pathway (Figure 4c).

Recent reports have shown that depolymerization of the actin cytoskeleton using cytochalasin D blocks GPCR-mediated p42/p44 MAPK activation suggesting the involvement of focal adhesion based signalling (Luttrell et al., 1997; 1999; Della Rocca et al., 1999a). However, as shown in Figure 4d, pre-treatment of DDT1MF-2 cells with cytochalasin D (1 μM for 1 h) had no significant effect on histamine (100 μM; 95±7% of control response; n=4) induced p42/p44 MAPK activation.

Effect of PI-3K inhibition on histamine H1 receptor-induced p42/p44 MAPK activation

Phosphatidylinositol 3-kinase (PI-3K) has been implicated in Gi-PCR-induced regulation of p42/p44 MAPK (Sugden & Clerk, 1997; van Biesen et al., 1996). In this study we have examined the role of PI-3K in the regulation of p42/p44 MAPK by the histamine H1 receptor in DDT1MF-2 cells. As shown in Figure 5 responses to histamine (100 μM) were inhibited following pre-treatment (30 min) with wortmannin (p[IC50]=7.6±0.06; n=4) and LY 294002 (p[IC50]=4.8±0.1; n=4). In these experiments, wortmannin (30 nM) and LY 294002 (30 μM) inhibited histamine (100 μM) p42/p44 MAPK responses by 64±10% (n=4) and 85±8% (n=4) respectively. These observations clearly demonstrate that a PI-3K-dependent pathway is involved in histamine H1 receptor-mediated p42/p44 MAPK activation in DDT1MF-2 cells.

Figure 5.

Figure 5

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Role of PI-3K in histamine H1 receptor stimulation of p42/p44 MAPK in DDT1MF-2 cells. DDT1MF-2 cells were pre-incubated (30 min) with the indicated concentrations of the PI-3K inhibitors (a) wortmannin and (b) LY 294002 before stimulating (5 min) with vehicle (control; C) or 100 μM histamine. Representative immunoblots for wortmannin (a) and LY 294002 (b) are shown in the upper panels. Combined results obtained from four independent experiments (mean±s.e.mean) are shown in the lower panels. Data are presented as the percentage of the control response to 100 μM histamine (100%) in the absence of PI-3K inhibitor. *Significantly different (P<0.05) from the control response to 100 μM histamine.

Effect of histamine H1 receptor activation on JNK and p38 MAPK

The p38 MAPK and JNK signalling pathways are activated by stimuli such as UV irradiation, osmotic stress and inflammatory cytokines (Paul et al., 1997). In our initial experiments we determined whether osmotic stress activates p38 MAPK and JNK in DDT1MF-2 cells. Sorbitol (0.5 M) treatment activated p38 MAPK (Figure 6a) and the 46 kDa and 54 kDa isoforms of JNK (Figure 6b) in DDT1MF-2 cells, although with different time-course profiles. Stimulation of DDT1MF-2 cells with histamine produced concentration-dependent (p[EC50]=5.8±0.4; n=5; Figure 6d) and time-dependent (peak activation occurring at 5 min; Figure 6c) increases in p38 MAPK activation. In contrast, histamine did not stimulate JNK phosphorylation in DDT1MF-2 cells (data not shown). The histamine H1 receptor antagonist mepyramine (1 μM) blocked histamine (100 μM)-induced p38 MAPK activation (95±9% inhibition; n=4; Figure 7a), whereas tiotidine (1 μM) and thioperamide (1 μM), histamine H2 and H3 receptor antagonists respectively, had no effect. These data indicate that histamine stimulated increases in p38 MAPK activation are mediated through the histamine H1 receptor in DDT1MF-2 cells. The p38 MAPK inhibitor SB 203580 (20 μM) blocked 100 μM histamine-induced p38 MAPK activation (95±7% inhibition; n=4; Figure 7b). Finally, pre-treatment with PTX (100 ng ml−1) significantly reduced histamine-induced p38 MAPK activation (74±11% inhibition; n=6; P<0.05; Figure 7c).

Figure 6.

Figure 6

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Histamine H1 receptor stimulation of p38 MAPK in DDT1MF-2 cells. DDT1MF-2 cells were treated with 0.5 M sorbitol for the indicated periods of time. Cell lysates were resolved by SDS–PAGE and transferred to nitrocellulose before being probed with antisera specific for (a) phospho p38 MAPK and (b) phospho JNK. Stimulation of DDT1MF-2 cells with histamine produced (c) time-dependent (using 100 μM histamine) and (d) concentration-dependent increases in p38 MAPK activation (5 min stimulation). Control cells (C) were treated with vehicle. Combined results obtained from four (c) and five (d) independent experiments (mean±s.e.mean) are shown in the lower panels.

Figure 7.

Figure 7

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Effect of the histamine receptor antagonists, pertussis toxin and SB 203580 on histamine-induced p38 MAPK activation. DDT1MF-2 cells were pre-incubated with (a) mepyramine (1 μM), tiotidine (1 μM; H2 receptor antagonist) and thioperamide (1 μM; H3 receptor antagonist) for 30 min, (b) the p38 MAPK inhibitor SB 203580 (20 μM) for 30 min or (c) pertussis toxin (100 ng ml−1 for 16 h). Cells were stimulated with 100 μM histamine for 5 min. Similar results were obtained in at least four independent experiments.

Discussion

Histamine H1 receptors trigger the activation of phospholipase C (PLC)-β isoforms via pertussis toxin-insensitive Gq/11-proteins in a variety of cell types (for review see Hill et al., 1997). In this study we have investigated whether the histamine H1 receptor stimulates MAPK signalling (p42/p44 MAPK, JNK and p38 MAPK activation) in the hamster vas deferens smooth muscle cell line, DDT1MF-2.

The data presented has shown for the first time that the histamine H1 receptor stimulates p42/p44 MAPK activation. Recent studies suggest that Gq/G11-PCRs preferentially employ PKC and/or Ca2+ signals to activate p42/p44 MAPK in a Raf-dependent but Ras-independent manner (Sugden & Clerk, 1997). Hence, in this study we examined the role of PKC and Ca2+ in the regulation of p42/p44 MAPK by the histamine H1 receptor in DDT1MF-2 cells. The data presented indicate that histamine H1 receptor-induced p42/p44 MAPK responses in DDT1MF-2 cells are independent of Ca2+ released from intracellular stores. However, the removal of extracellular Ca2+ did produce a small but non-significant reduction (c. 20%) in histamine-induced p42/44 MAPK activation suggesting a role for Ca2+ influx. In addition, p42/p44 MAPK responses to histamine were partially blocked by the selective PKC inhibitor Ro 31-8220 (c. 40% inhibition) indicating the involvement of a PKC-dependent pathway. Previous studies have shown that histamine H1 receptor-mediated inositol phosphate accumulation in DDT1MF-2 cells is reduced in the absence of extracellular Ca2+ (White et al., 1993). Hence the small but non-significant reduction in p42/p44 MAPK activation in the absence of extracellular Ca2+ may be a consequence of reduced inositol phospholipid breakdown ultimately leading to a decrease in PKC activation (via DAG generation). This seems plausible given the involvement of PKC in the overall p42/p44 MAPK response to histamine in DDT1MF-2 cells.

It is notable that the response to histamine was sensitive to PTX pre-treatment (c. 60% inhibition) indicating the involvement of Gi/Go proteins in coupling the histamine H1 receptor to p42/p44 MAPK. In contrast, our previous studies have shown that the histamine H1 receptor stimulates PTX-insensitive increases in inositol phosphate accumulation and calcium mobilization in DDT1MF-2 cells (Dickenson & Hill, 1993; White et al., 1993). Interestingly, several other studies have reported the involvement of Gi/Go proteins in histamine H1 receptor-mediated cell signalling. For example, the guinea-pig histamine H1 receptor in transfected Chinese hamster ovary cells stimulates arachidonic acid (AA) release, which is partially sensitive to PTX (Leurs et al., 1994). Similarly, histamine H1 receptor activation in rabbit platelets triggers AA release, which is completely blocked by PTX (Murayama et al., 1990). These observations suggest that the histamine H1 receptor may activate different signalling pathways through coupling to various subtypes of G-protein, i.e. Gq/G11-mediated PLC activation and Gi/Go-mediated PLA2 and p42/p44 MAPK. In view of the prominent role of PI-3K in Gi/Go-PCR-mediated p42/p44 MAPK activation we investigated the role of PI-3K in histamine H1 receptor-induced p42/p44 MAPK activation. The PI-3K inhibitors, wortmannin and LY 294002, significantly reduced histamine-induced p42/p44 MAPK activation suggesting a role for PI-3K activation. In summary, the data presented suggest that Gi/Go-protein coupling and PI-3K activation are involved in histamine H1 receptor-mediated p42/p44 MAPK activation in DDT1MF-2 cells.

The requirement for non-receptor tyrosine kinases (Src family and the calcium-regulated focal adhesion kinase, Pyk2) in GPCR-mediated p42/p44 MAPK activation has been well-documented (Sugden & Clerk, 1997). In this study we explored the role of tyrosine kinases in histamine H1 receptor-induced p42/p44 MAPK activation in DDT1MF-2 cells. Responses to histamine were sensitive to the broad range tyrosine kinase inhibitors genistein and tyrphostin A47 but not to the specific Src tyrosine kinase inhibitor PP2. These data suggest that tyrosine kinases other than Src contribute to histamine H1 receptor-mediated p42/p44 MAPK activation in DDT1MF-2 cells. Furthermore, since histamine H1 receptor-induced p42/p44 MAPK responses were independent of Ca2+ influx and intracellular Ca2+ release it would appear that the Ca2+-activated tyrosine kinase PyK2 is not involved. Finally, the non-calcium dependent focal adhesion kinase p125FAK has been implicated in GPCR-mediated p42/p44 MAPK activation (Luttrell et al., 1997; Della Rocca et al., 1999a). However, this kinase appears not be involved in histamine H1 receptor-mediated p42/p44 MAPK in DDT1MF-2 cells since depolymerization of the actin cytoskeleton with cytochalasin D had no effect.

Transactivation of receptor tyrosine kinases such as the receptor for epidermal growth factor receptor has also been implicated in GPCR-mediated activation of p42/p44 MAPK (Zwick et al., 1999). However, in this study the epidermal growth factor receptor tyrosine kinase specific tyrphostin AG1478 had no significant effect on histamine-induced p42/p44 MAPK activation.

Overall the results presented in this study suggest that histamine H1 receptor-induced p42/p44 MAPK activation in DDT1MF-2 cells proceeds via two different pathways; one dependent on Gq and the other involving Gi/Go protein coupling. The Gq protein dependent pathway presumably involves the activation of Raf via PKC (Ras-independent) as reported for other GqPCRs (Sugden & Clerk, 1997). The second route appears to involve histamine H1 receptor coupling to Gi/Go proteins. Previous studies have shown that the activation of p42/p44 MAPK by Gi/Go-PCRs involves G protein-derived βγ subunits, genistein-sensitive c-Src-related protein tyrosine kinase(s) and PI-3K activation in a Ras-dependent manner (Koch et al., 1994; Hawes et al., 1995; 1996; Garnovskaya et al., 1996; Luttrell et al., 1996; Igishi & Gutkind, 1998). Our findings indicate that the Gi/Go-protein component of the histamine H1 receptor response signals through a similar pathway. However, further studies are required in order to firmly establish the role of G-protein βγ subunits, Raf and Ras in histamine H1 receptor-mediated p42/p44 MAPK activation in DDT1MF-2 cells.

Recent studies have indicated a role of clathrin-mediated endocytosis in GPCR-induced p42/p44 MAPK activation (Lefkowitz, 1998). Blocking receptor endocytosis prevents p42/p44 MAPK signalling elicited by a range of receptors including the β2 adrenergic, 5-HT1A receptor, m1 muscarinic and δ-opioid receptors (Daaka et al., 1998; Della Rocca et al., 1999b; Vogler et al., 1999; Ignatova et al., 1999). Studies are currently in progress to determine whether internalization of the histamine H1 receptor is important for histamine induced p42/p44 MAPK activation in DDT1MF-2 cells.

In this study we have also explored the regulation of p38 MAPK and JNK signalling pathways by the histamine H1 receptor. The p38 MAPK and JNK signalling pathways are typically activated by stimuli such as UV irradiation, osmotic stress and inflammatory cytokines, although an increasing number of GPCRs have been shown to activate these pathways (Paul et al., 1997). The data presented in this study indicate that the histamine H1 receptor can activate p38 MAPK signalling in DDT1MF-2 cells. Signalling pathways linking GPCRs to p42/p44 MAPK activation have been extensively studied and characterized, in contrast the mechanisms by which GPCRs stimulate p38 MAPK are largely unknown. Recent studies have shown that m2 muscarinic (Gi-coupled) and β-adrenergic (Gs-coupled) receptor activation of p38 MAPK is mediated via Gβγ, whereas activation by the Gq-coupled m1 muscarinic receptor involves Gαq/11 and Gβγ (Yamauchi et al., 1997). Subsequent studies have shown that PKC and Src family kinases are involved in Gαq/11 stimulated p38 MAPK activation (Nagao et al., 1998). In this study we have shown the activation of p38 MAPK by histamine was sensitive to PTX pre-treatment indicating the involvement of Gi/Go proteins in coupling the histamine H1 receptor to p38 MAPK in DDT1MF-2 cells. However, further studies are required in order to identify the molecular mechanisms mediating p38 MAPK activation by the histamine H1 receptor.

In summary, we have shown that the histamine H1 receptor activates p38 MAPK and p42/p44 MAPK in the smooth muscle cell line DDT1MF-2. Given that p38 MAPK and p42/44 MAPK regulate many cellular processes including inflammation, cell differentiation, cell growth and death (Paul et al., 1997; Ono & Han, 2000) future experiments will investigate the effects of histamine H1 receptor activation on cell proliferation and apoptosis.

Acknowledgments

This work was funded by The Nottingham Trent University.

Abbreviations

DAG

diacylglycerol

GPCR

G protein-coupled receptor

IP3

inositol 1,4,5-trisphosphate

JNK

C-Jun N-terminal kinase

MAPK

mitogen activated protein kinase

PKC

protein kinase C

PLA2

phospholipase A2

PLC

phospholipase C

PTX

pertussis toxin

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