Involvement of the H1 Histamine Receptor, p38 MAP Kinase, Myosin Light Chains Kinase, and Rho/ROCK in Histamine-Induced Endothelial Barrier Dysfunction

Abstract

Objective: The mechanisms by which histamine increases microvascular permeability remain poorly understood. We tested the hypothesis that H1 receptor activation disrupts the endothelial barrier and investigated potential downstream signals.

Methods: We used confluent EC monolayers, assessing TER as an index of barrier function. HUVEC, HCMEC, and HDMEC were compared. Receptor expression was investigated using Western blotting, IF confocal microscopy and RT-PCR. Receptor function and downstream signaling pathways were tested using pharmacologic antagonists and inhibitors, respectively.

Results: We identified H1-H4 receptors on all three EC types. H1 antagonists did not affect basal TER but prevented the histamine-induced decrease in TER. Blockade of H2 or H3 attenuated the histamine response only in HDMEC, while inhibition of H4 attenuated the response only in HUVEC. Combined inhibition of both PKC and PI3K caused exaggerated histamine-induced barrier dysfunction in HDMEC, whereas inhibition of p38 MAP kinase attenuated the histamine response in all three EC types. Inhibition of RhoA, ROCK, or MLCK also prevented the histamine-induced decrease in TER in HDMEC.

Conclusion: The data suggest that multiple signaling pathways contribute to histamine-induced endothelial barrier dysfunction via the H1 receptor.

Keywords: barrier dysfunction; endothelial cells; histamine; permeability.

Fig. 1. Comparison of Histamine induced changes in TER

Representative tracing of TER for HUVEC (A), HCMEC (B) and HDMEC (C). (D) Maximum change in resistance of HUVEC, HCMEC and HDMEC upon treatment with 10 μM histamine (n=4 for each group). *P<0.01 compared to respective control. (E) Comparison of the recovery time in HUVEC, HCMEC and HDMEC (n=4 for each group).

Fig. 2. Western Analysis identifying the Histamine receptors on EC

(A) Blots are shown for the H1R (A) H2R (B) H3R (C) and H4R (D). All blots shown are representative of three experiments. Rat brain endothelial cells were used as a positive control.

Fig. 3. Identification the Histamine receptor on EC using immunocytochemistry

HUVEC, HCMEC, and HDMEC were immunolabeled to visualize localization of the H1R (A), H2R (B), H3R (C) and H4R (D), all labeled in green. Nuclei are labeled in blue. Images are projections of confocal z-stacks, and are representative of three separate experiments for each EC type. Images of the negative controls for labeling are provided in the supplemental data (Suppl. Fig. 1).

Fig. 4. Blockade of H1 and Histamine-Induced Changes in TER

(A) Mean maximal changes in TER of HUVEC pretreated with 10 μM mepyramine (top, n=12 for all groups) or 100 nM cetirizine (bottom, n=8 for all groups) for 30 minutes prior to stimulation with 10 μM histamine for 10 minutes (B) Mean maximal changes in TER of HCMEC pretreated with 10 μM mepyramine (top, n=14 for all groups) or 100 nM cetirizine (bottom, n=7 for all groups) for 30 minutes prior to stimulation with 10 μM histamine for 10 minutes. (C) Mean maximal changes in TER of HDMEC pretreated with 10 μM mepyramine (top, n=8) or 100 nM cetirizine (bottom, n=4) for 30 minutes prior to stimulation with 10 μM histamine for 10 minutes. *P<0.01 versus control group. †P<0.01 versus all other groups.

Fig. 5. Blockade of H2 and Histamine-Induced Changes in TER

Mean changes in TER of HUVEC (A), HCMEC (B), and HDMEC (C) pretreated with 10 μM cimetidine for 30 minutes prior to stimulation with 10 μM histamine for 10 minutes. †P<0.01 versus all other groups. *P<0.05 versus control. For all HUVEC groups, n=16; for all HCMEC groups, n=12; for all HDMEC groups, n=10.

Fig. 6. Blockade of H3 and Histamine-Induced Changes in TER

(A) Mean changes in TER of HUVEC (A), HCMEC (B), and HDMEC (C) pretreated with 100 nM ciproxifan or vehicle for 30 minutes prior to stimulation with 10 μM histamine for 10 minutes. *P<0.001 versus control. †P<0.01 versus all other groups. For all HUVEC and HCMEC groups, n=8; for all HDMEC groups, n=12.

Fig. 7. Blockade of H4 and Histamine-Induced Changes in TER

(A) Mean changes in TER of HUVEC (A), HCMEC (B), and HDMEC (C) pretreated with 10 μM JNJ or vehicle for 30 minutes prior to stimulation with 10 μM histamine for 10 minutes. Values are means ± SE. *P<0.001 versus control. †P<0.001 versus all other groups. For all HUVEC groups, n=8; for all HCMEC groups, n=4; for all HDMEC groups, n=16.

Fig. 8. Effect of inhibitors of PKC and PI3K on TER of HDMEC treated with histamine

HDMEC were pretreated with 100 nM GFX and/or 100 nM PI 828 in the presence of 10 μM histamine for 30 minutes (n=28 each group). Values are means ± SE. *P<0.001 versus control. †P<0.05 versus all other groups.

Fig. 9. Blockade of p38 MAP kinase and Histamine-Induced Changes in TER

Mean changes in TER of HUVEC (A), HCMEC (B), and HDMEC (C) pretreated with 10 μM SB203580 or vehicle followed by 10 μM histamine for 30 minutes *P<0.01 versus control. †P0.05 compared to all other groups. For all HUVEC groups, n=8; for all HCMEC groups, n=24; for all HDMEC groups, n=8.

Fig. 10. Inhibition of MLCK, RhoA and ROCK and Histamine-Induced Changes in TER

HDMEC were pretreated with either (A) 1 μM ML-7 (n=16), (B) 10 μM Y16 (n=8), (C) 100 nM H1152 (n=8) or vehicle followed by histamine (10 μM) for 30 minutes. **P<0.001 compared to control. †P<0.01 compared to all other groups.