Expression of histamine receptor genes Hrh3 and Hrh4 in rat brain endothelial cells

Abstract

Background and purpose: Brain vascular endothelial cells express histamine H1 and H2 receptors, which regulate brain capillary permeability. We investigated whether H3 and H4 receptors are also expressed in these cells and may thus play a role in permeability regulation.

Experimental approach: An immortalized rat brain endothelial cell line RBE4 was used to assess the presence of H3 and H4 receptors. Reverse transcription-PCR (RT-PCR) and sequencing were used to identify the receptor mRNAs. The receptors were stimulated with histamine and immepip, and specific inverse agonists/antagonists ciproxifan and JNJ 7777120 were used to block H3 and H4 receptors, respectively.

Key results: RT-PCR of mRNA extracted from cultured immortalized RBE4 cells revealed two rat H4 receptor gene (Hrh4) transcripts, one full-length (coding sequence 1173 bp), and one with a 164 bp deletion. Also, two rat H3 receptor gene (Hrh3) isoform mRNAs were expressed in RBE4 cells, and sequencing showed they were the full-length H3 receptor and the 144 bp deletion form. Both histamine and immepip (H3 and H4 receptor agonists) activated the Erk1/2 MAPK pathway in the RBE4 cells and in vivo in brain blood vessels by activating H4 receptors, as the H4 receptor-specific inverse agonists/antagonist JNJ 7777120, but not ciproxifan, H3 receptor antagonist, dose-dependently blocked this effect in RBE4 cells.

Conclusions and implications: Both Hrh3 and Hrh4 receptors are expressed in rat brain endothelial cells, and activation of the histamine H4 receptor activates the Erk1/2 cascade. H3 and H4 receptors in endothelial cells are potentially important for regulation of blood-brain barrier permeability, including trafficking of immunocompetent cells.

Keywords: Erk1/Erk2; GPCR; MAPK; blood-brain barrier; histamine.

Figure 1

RT-PCR amplification of two variants of the histamine H₄ receptor messages in RBE4 cells, as well as in a rat spleen sample. Culturing RBE4 cells in regular medium supplemented with 10% calf serum revealed the same two H₄ receptor transcripts that are seen in cells after 12 h of starvation (no calf serum supplement). White arrows indicate the transcripts with 1073 bp and 1009 bp CDS, respectively. The contr/RT lane indicates the negative control sample with mRNA omitted from the amplification reaction.

Figure 2

A schematic drawing of the tree exon rat histamine H₄ receptor gene and the partial sequence of the two isolated H₄ isoforms. (A) The E1, E2 and E3 exons are indicated and the deleted region in the shorter isoform is marked in green. (B) A part of the cloned sequences are aligned and identical accept for the 164 bp deletion in the shorter cDNA (gap marked in green). The potential new stop codon for the frame shifted shorter message is marked in red.

Figure 3

RT-PCR amplification of the histamine H₃ receptor messages in RBE4 cells. The presence of the H_3A (full-length transcript) and H_3C (144 bp deletion) isoforms are verified by cloning and sequencing.

Figure 4

Time dependence of 10 nM histamine (HA)-induced MAPK (Erk1/2, p44/p42) phosphorylation in RBE4 cells. Sample n ≥ 3 per time point, *P < 0.05.

Figure 5

Histamine (HA)- and immepip-induced MAPK (Erk1/2, p44/p42) phosphorylation in RBE4 cells. (A) Immunostaining detecting phosphorylated MAPK in cultured and differentially treated RBE4 cells. (B) Representative quantification of MAPK phosphorylation in HA and histamine H₄ receptor antagonist (JNJ 7777120) treated RBE4 cells. (C) Representative quantification of MAPK phosphorylation in immepip and JNJ 7777120-treated RBE4 cells. The quantifications were done on Western blot films (see inserts), and results are indicated as the ratio between phosphorylated and non-phosphorylated MAPK. Untreated cells show a weak but consistent level of phosphorylated MAPK (base line), the degree of background phosphorylation was moderately affected by JNJ 7777120. Histamine (10 nM) as well as immepip (10 μM) increased the level of phosphorylation, and this activation of MAPK was inhibited by JNJ 7777120. In (B) and (C) n = 2, SD indicated.

Figure 6

Immepip-induced Erk1/2 phosporylation in RBE4 cells is not affected by the histamine H₃ receptor antagonist ciproxifan.

Figure 7

MAPK (Erk1/2, p44/p42) phosphorylation in rat brain endothelial cells in vivo. (A) Modest phospho-MAPK immunoreactivity in the brainstem of untreated control rats. (B) MAPK phosphorylation in brain capillaries in dorsomedial tegmental area after perfusion with 10 μM histamine (HA). (C) Preperfusion with 10 μM JNJ 4444120, a histamine H₄ receptor antagonist, down-regulated locally the histamine-induced MAPK phosphorylation.