Histamine 1 receptor-Gβγ-cAMP/PKA-CFTR pathway mediates the histamine-induced resetting of the suprachiasmatic circadian clock

Abstract

Background: Recent evidence indicates that histamine, acting on histamine 1 receptor (H1R), resets the circadian clock in the mouse suprachiasmatic nucleus (SCN) by increasing intracellular Ca(2+) concentration ([Ca(2+)]i) through the activation of CaV1.3 L-type Ca(2+) channels and Ca(2+)-induced Ca(2+) release from ryanodine receptor-mediated internal stores.

Results: In the current study, we explored the underlying mechanisms with various techniques including Ca(2+)- and Cl(-)-imaging and extracellular single-unit recording. Our hypothesis was that histamine causes Cl(-) efflux through cystic fibrosis transmembrane conductance regulator (CFTR) to elicit membrane depolarization needed for the activation of CaV1.3 Ca(2+) channels in SCN neurons. We found that histamine elicited Cl(-) efflux and increased [Ca(2+)]i in dissociated mouse SCN cells. Both of these events were suppressed by bumetanide [Na(+)-K(+)-2Cl(-) cotransporter isotype 1 (NKCC1) blocker], CFTRinh-172 (CFTR inhibitor), gallein (Gβγ protein inhibitor) and H89 [protein kinase A (PKA) inhibitor]. By itself, H1R activation with 2-pyridylethylamine increased the level of cAMP in the SCN and this regulation was prevented by gallein. Finally, histamine-evoked phase shifts of the circadian neural activity rhythm in the mouse SCN slice were blocked by bumetanide, CFTRinh-172, gallein or H89 and were not observed in NKCC1 or CFTR KO mice.

Conclusions: Taken together, these results indicate that histamine recruits the H1R-Gβγ-cAMP/PKA pathway in the SCN neurons to activate CaV1.3 channels through CFTR-mediated Cl(-) efflux and ultimately to phase-shift the circadian clock. This pathway and NKCC1 may well be potential targets for agents designed to treat problems resulting from the disturbance of the circadian system.

Keywords: CFTR; Calcium; Chloride; Circadian rhythm; Histamine; NKCC1; Suprachiasmatic nucleus.

Fig. 1

Histamine induces the efflux of NKCC1-accumulated Cl⁻, leading to intracellular Ca²⁺ rise in SCN neurons. (a, left panel) Traces from a Ca²⁺-imaging experiment which show the effect of bumetanide on histamine-elicited increase in [Ca²⁺]_i in an SCN neuron. (a, right panel) Graphs summarizing the effects of bumetanide on the histamine-elicited Ca²⁺ responses in 105 SCN neurons from 3 mice. The bar charts indicate the mean (± SEM) peak Ca²⁺ responses. b Traces from Cl⁻-imaging experiments show the effects of H1R agonist on [Cl⁻]_i in SCN cells. Up- and downward deflections of the trace denote decrease and increase in [Cl⁻]_i, respectively. c Traces and summary graphs showing the effects of bumetanide on H1R agonist-induced decrease (n = 81) and increase (n = 80) in [Cl⁻]_i in SCN cells. The bar charts indicate the mean (± SEM) peak Cl⁻ responses. The symbols connected by lines in (a) and (c) denote data from the same cells. **: p < 0.001, paired t-test

Fig. 2

H1R activation increases the cAMP level in the SCN through the G_βγ protein. Graph showing the effects on the content of cAMP in the mouse SCN tissue, of H1R (2-pyridylethylamine, 100 μM, n = 5 mice) and H2R (amthamine, 20 μM, n = 5 mice) agonists alone or in combination with the G_βγ inhibitor gallein (100 μM, n = 6 mice). Control: n = 5 mice. One-way ANOVA followed by Student-Newman-Keuls pairwise comparison test (*: p < 0.05)

Fig. 3

G_βγ-cAMP/PKA signaling pathway plays a crucial role in H1R agonist-elicited Ca²⁺ rise and Cl⁻ fluxes. a Summary graphs showing the effects of the G_βγ blocker gallein on H1R agonist-elicited increase in [Ca²⁺]_i. The bar charts indicate the mean (± SEM) peak Ca²⁺ responses. **: p < 0.001 [n = 47 neurons from 3 mice, t(46) = 10.409, paired t-test]. b Summary graphs showing the effects of gallein on H1R agonist-induced Cl⁻ efflux [left panel, n = 21 cells from 4 mice, t(20) = 6.783, p < 0.001, paired t-test] and influx [right panel, n = 50 cells from 4 mice, t(49) = −14.644, p < 0.001, paired t-test]. The bar charts indicate the mean (± SEM) peak Cl⁻ responses. **: p < 0.001. c Summary graphs showing the effects of the PKA inhibitor H89 on H1R agonist-elicited increase in [Ca²⁺]_i. The bar charts indicate the mean (± SEM) peak Ca²⁺ responses. **: p < 0.001 [n = 90 neurons from 3 mice, t(89) = 7.272, paired t-test]. d Summary graphs showing the effects of H89 on H1R agonist-induced Cl⁻ efflux [left panel, n = 277 cells from 8 mice, t(276) = 19.895, paired t-test] and influx [right panel, n = 90 cells from 8 mice, t(89) = −11.458, paired t-test]. The bar charts indicate the mean (± SEM) peak Cl⁻ responses. **: p < 0.001. The symbols connected by lines in (a-d) denote data from the same cells

Fig. 4

The CFTR inhibitor CFTR_inh-172 suppresses histamine-elicited Cl⁻ efflux and Ca²⁺ rise in SCN cells. a Traces from Cl⁻-imaging experiments that show the inhibitory effects of CFTR_inh-172 on 2-pyridylethylamine (H1R agonist)-induced Cl⁻ efflux in an SCN cell. b Summary graph showing the effect of CFTR_inh-172 on H1R agonist-induced Cl⁻ efflux (n = 41 cells from 4 mice; left panel) and influx (n = 38 cells from 4 mice; right panel) in SCN cells. c Summary graph showing the effect of CFTR_inh-172 on histamine-elicited increase in [Ca²⁺]_i in SCN neurons (n = 31 neurons from 3 mice). The bar charts in (b) and (c), respectively, indicate the mean (± SEM) Cl⁻ and Ca²⁺ responses elicited by H1R agonist or histamine in the absence or presence of CFTR_inh-172. The symbols connected by lines in (b) and (c) denote data from the same cells. **: p < 0.001

Fig. 5

Effect of blockade/KO of G_βγ, PKA, CFTR or NKCC1 on histamine-induced resetting of circadian clock. a-j Plots against ZT of the firing rate of SCN neurons recorded in different experimental conditions. Each plot shows the representative result of 6 repeated experiments. The projected light and dark phases of the animal room are indicated with open and filled horizontal bars, respectively. The dashed vertical line in each plot indicates the average time of peak neural activity for control slices. The filled square denotes the time of slice preparation, while the arrow indicates the time of drug application. k Graph summarizing the effects of various experimental treatments on the time of peak of circadian firing activity rhythm. Student-Newman-Keuls comparison tests were performed after Kruskal-Wallis one-way ANOVA on Ranks (p < 0.001). The results of pair-wise comparisons of the value of each experimental group with those of control and histamine groups are denoted with asterisk and spade, respectively. *, ♠: p < 0.05

Fig. 6

Proposed signaling pathway for histamine-induced resetting of the circadian pacemaker in the SCN. ① A subset of SCN neurons are loaded with Cl⁻ by the action of the Cl⁻ importer NKCC1. ② H1R stimulation by histamine at early night results in the production of activated G_βγ protein in these cells. ③ The activated G_βγ protein stimulates adenylate cyclase (AC) to produce cAMP from ATP. ④ PKA activated by cAMP opens the Cl⁻ channel CFTR. ⑤ The efflux of Cl⁻ through CFTR down the electrochemical gradient results in membrane depolarization. ⑥ In response to this membrane depolarization, the Ca_V1.3 L-type voltage-gated Ca²⁺ channel (VGCC) is activated. ⑦ The resulting Ca²⁺ influx through Ca_V1.3 VGCC induces Ca²⁺ release from the endoplasmic reticulum (ER) through RyR. ⑧ The consequent increase in [Ca²⁺]_i leads to phase delay of the circadian clock