Dexmedetomidine Modulates Histamine-induced Ca(2+) Signaling and Pro-inflammatory Cytokine Expression

Abstract

Dexmedetomidine is a sedative and analgesic agent that exerts its effects by selectively agonizing α2 adrenoceptor. Histamine is a pathophysiological amine that activates G protein-coupled receptors, to induce Ca(2+) release and subsequent mediate or progress inflammation. Dexmedetomidine has been reported to exert inhibitory effect on inflammation both in vitro and in vivo studies. However, it is unclear that dexmedetomidine modulates histamine-induced signaling and pro-inflammatory cytokine expression. This study was carried out to assess how dexmedetomidine modulates histamine-induced Ca(2+) signaling and regulates the expression of pro-inflammatory cytokine genes encoding interleukin (IL)-6 and -8. To elucidate the regulatory role of dexmedetomidine on histamine signaling, HeLa cells and human salivary gland cells which are endogenously expressed histamine 1 receptor were used. Dexmedetomidine itself did not trigger Ca(2+) peak or increase in the presence or absence of external Ca(2+). When cells were stimulated with histamine after pretreatment with various concentrations of dexmedetomidine, we observed inhibited histamine-induced [Ca(2+)]i signal in both cell types. Histamine stimulated IL-6 mRNA expression not IL-8 mRNA within 2 hrs, however this effect was attenuated by dexmedetomidine. Collectively, these findings suggest that dexmedetomidine modulates histamine-induced Ca(2+) signaling and IL-6 expression and will be useful for understanding the antagonistic properties of dexmedetomidine on histamine-induced signaling beyond its sedative effect.

Keywords: Ca2+ signaling; Dexmedetomidine; Histamine; Interleukin; Pro-inflammatory cytokines.

Fig. 1. Dexmedetomidine did not elicit [Ca²⁺]_i signals in HeLa cells. Changes in [Ca²⁺]_i induced by 10 ng/mL dexmedetomidine in (A) 1 mM Ca²⁺ medium and (B) Ca²⁺ free medium (0 Ca²⁺). The upper bars indicate the extracellular solutions applied to the cells.

Fig. 2. Histamine-induced [Ca²⁺]_i signals were inhibited by dexmedetomidine. (A) Expression levels of histamine receptors mRNA (H1R to H4R) in HeLa cells. (B) Changes in [Ca²⁺]_i induced by 100 nM histamine. (C) Changes in [Ca²⁺]_i after pretreatment with 10 ng/mL dexmedetomidine (Dex) and subsequent treatment with 100 nM histamine. (D) Changes in [Ca²⁺]_i after pretreatment with 10 µM histamine (E) Changes in [Ca²⁺]_i after pretreatment with 100 ng/mL dexmedetomidine and subsequent treatment with 10 µM histamine. The upper bars indicate the extracellular solutions applied to the cells and traces were selected randomly. Three separate experiments were performed and each point represents a mean±SEM. (-): no RNA, M: DNA ladder (bp).

Fig. 3. The effect of dexmedetomidine on CPA-induced [Ca²⁺]_i release and Ca²⁺ entry. The CPA-induced [Ca²⁺]_i increase in the presence of 2 mM extracellular Ca²⁺ in the absence (A) or presence (B) of 100 ng/mL dexmedetomidine. (C) Analysis of CPA-induced Ca²⁺ release and influx as determined using R_340/380 fluorescence ratios. The upper bars indicate the extracellular solutions applied to the cells. Three separate experiments were performed and each point represents a mean±SEM.

Fig. 4. Effects of dexmedetomidine on histamine-induced IL-6 and IL-8 mRNA expression. (A) After pre-treatment with 100 ng/mL dexmedetomidine (Dex) for 30 min, HeLa cells were stimulated with PBS and 100 nM histamine (His) for 90 min and then total RNA was extracted and amplified with primers specific for IL-6, IL-8, and GAPDH. Data are from one of experimental replicates. IL-6 (B) and IL-8 mRNA (C) expressions were quantified after normalizing to GAPDH levels as a loading control (n=6 and n=3, respectively). Results are the means±SEMs of independent experiments. M: DNA ladder (bp), PBS: phosphate buffered saline. ^*p<0.01 was considered statistically significant.

Fig. 5. Histamine-induced [Ca²⁺]_i signals were inhibited by dexmedetomidine in HSG cells. (A) Expression levels of histamine receptor mRNA (H1R to H4R) in HSG cells. (B) Changes in [Ca²⁺]_i induced by 30 µM histamine. (C) Changes in [Ca²⁺]_i after pretreatment with 100 ng/mL dexmedetomidine (Dex) and subsequent treatment with 30 µM histamine (His). (D) Evoked [Ca²⁺]_i (Δ Ca²⁺) calculated by the peak value of 30 µM histamine stimulation in the presence or absence of 100 ng/mL dexmedetomidine. The upper bars indicate the extracellular solutions applied to the cells and traces were represented with average value. (E, F) After pre-treatment with 100 ng/mL dexmedetomidine for 30 min, HSG cells were stimulated with PBS or with 30 µM histamine for 90 min and then total RNAs were extracted and amplified with primers specific for IL-6, IL-8, and GAPDH. Data are from one of three experimental replicates. IL-6 and IL-8 mRNA expressions were quantified after normalizing to GAPDH levels as a loading control. Results are the mean±SEMs of three independent experiments. (-): no RNA, M: DNA ladder (bp), ^*p<0.01 was considered statistically significant.