| α2-Adrenoceptor(α2-AR), one of the classic adrenergic receptors, belongs to class A G protein-coupled receptors(GPCR). Epinephrine and noradrenaline(NE) are endogenous ligands of α2-AR. α2-AR is widespread both in the central nervous system and in the peripheral tissues and mediates lots of physiological functions, like analgesia, sedation, hypotension and lowered body temperature. α2-AR is an important target for drug development. Dexmedetomidine, an α2-adrenoceptor agonist, which can induce a state resembling natural sleep is being used in clinical anesthesia and ICU. The distribution of the three subtypes: α2A-AR、α2B-AR and α2C-AR is significantly different. It was not clear about the physiological roles of the α2-adrenoceptor subtypes until now. Which subtype mediated sedation after DMED administration? α2-AR activated by DMED can couple with Gαi and mediate a series of changes of signaling pathways. How do these changes relate to sedation? Accordingly, we carryed out research as follows.Purpose: To illuminate the subtype(s) and postreceptor signaling pathway(s) of α2-AR mediating sedation.Content: 1. In vitro, we constructed cell models for screening drugs of specific α2-AR subtype and then evaluated the pharmacodynamics of α2-AR agonists and antagonists. The subtype(s) of α2-AR mediated sedation were studied in mice. 2. The signaling pathway(s) involved in α2-AR sedation were studied in mice.Methods: 1. pc DNA3.1/Hygro-HA-α2A-AR and pc DNA3.1/Hygro-HA-α2C-AR recombinant plasmids were constructed and transfected into CHO-PKAcat-EGFP cells. A single clone expressing α2A/2C-AR was selected by cultivating in the presence of 200 mg/L hygromycin B followed by PKA redistribution assay. Z’ factors of CHO-PKAcat-α2A-AR and CHO-PKAcat-α2C-AR cell line were calculated to verify the reliability of the models. 2. The transcriptional expression of two subtypes was determined by quantitative Real-time PCR(q RT-PCR). Radioligand receptor binding assay was used to determine the densities of expressed receptor subtypes. Time-resolved fluorescence resonance energy transfer(TR-FRET) immunoassay was used to identify the function of inhibiting c AMP accumulation of α2A-AR and α2C-AR. 3. The α2-AR agonists(DMED, medetomidine, clonidine, NE, guanfacine, xylazine and moxonidine) and antagonists(atipamezole, BRL44408, JP-1302 and ARC239) were evaluated by PKA redistribution assay in the CHO-PKAcat-α2A-AR and CHO-PKAcat-α2C-AR cell models. 4. The sedation of DMED was evaluated in the loss of righting reflex(LORR) and locomotor activity models of mice. Mice were pretreated with different antagonists(i.c.v) to investigate the receptor subtypes mediated sedation of DMED. 5. The effects of different α2-AR agonists(DMED, clonidine, guanfacine, xylazine) on the locomotor activity of mice were evaluated. The effects of these α2-AR agonists on the blood pressure and heart rate of anesthetized rat were tested to exclude the influence of hypotension on locomotor activity. The mice were pretreated by microinjection in lateral ventricles with c AMP analogue- dibutyryl-c AMP(dbc AMP) and G protein gated inwardly rectifying K+ channels inhibitor-Tertiapin-Q trifluoroacetate salt(T-Q) to investigate the blockade on the sedation in the response to DMED.Results: 1. pc DNA3.1/Hygro-HA-α2A-AR and pc DNA3.1/Hygro-HA-α2C-AR recombinant plasmids were constructed successfully and verified by restriction enzyme digestion and gene sequencing. The cell line No.7 of CHO-PKAcat-α2A-AR and No.60 of CHO-PKAcat-α2C-AR exhibited stably response in PKA redistribution assay. Z’ factors of two cell lines were both between 0.5~1, which proved the reliability of CHO-PKAcat-α2A/2C-AR cell models. 2. The m RNA of α2A/2C-AR in the CHO-PKAcat-α2A/2C-AR cell lines remained stable after several generations by RT-q PCR analysis. The Bmax and Kd were(4.92±5.27) pmol/mg and(12.67±11.16) n M respectively of CHO-PKAcat-α2A-AR by [3H]RX821002 binding assay. The c AMP accumulation caused by forskolin(10 μM) was effectively inhibited by α2-AR agonist DMED(10-6~ 10-4 M) in CHO-PKAcat-α2A-AR. The c AMP content in CHO-PKAcat-EGFP cells and CHO-PKAcat-α2C-AR cells was increasing after stimulated by forskolin(10 μM). DMED(10-8~ 10-4 M) dose-dependently inhibited c AMP accumulation in CHO-PKAcat-α2C-AR cells. It showed that the receptors on the models functionally well. 3. In the CHO-PKAcat-α2A/2C-AR cells, the α2-AR agonists(DMED, medetomidine,clonidine, NE, guanfacine, xylazine and moxonidine) showed no significant selectivity on the α2A/2C-AR subtypes by PKA redistribution assay. DMED represented more effectively than other agonists on two subtypes. Antagonist atipamezole showed high efficiency on α2A/2C-AR with no selectivity. BRL44408 can selectively blocked α2A-AR(IC50=242.30±26.00 n M). JP-1302(IC50=2813.00±521.64 n M) can blocked α2C-AR effectively. α2C-AR can also be blocked by 10-5 M ARC239. 4. DMED(0.1~0.22 mg/kg, i.v) dose-dependently induced LORR in mice with ED50 = 0.12 mg/kg,ED100 = 0.22 mg/kg. There was no significant difference of the induction time, but the immobilization time had the prolonged tendency with the increasing of the dose. Locomotor activity of mice could be inhibited dose-dependently by DMED(0.000391~0.1 mg/kg) with significant difference at doses of 0.00625~0.1 mg/kg compared with control. Atipamezole(0.05~1 μg each, i.c.v) could dose-dependently diminish LORR in the response to DMED(0.25 mg/kg, i.v), so did the α2A-AR selective antagonist BRL44408(2.5~20 μg each, i.c.v). There was no significant difference of the induction time, but the immobilization time was decreased respectively. However, neithor JP-1302(88 μg) nor ARC239(48 μg) could reverse LORR induced by DMED(0.25 mg/kg,i.v). Antagonist atipamezole(0.2, 0.8, 3.2 μg each, i.c.v) had no effect on locomotor activity of mice administrated alone. Atipamezole(0.8, 3.2 μg each, i.c.v) could effectively antagonize the inhibition of DMED(0.0125 mg/kg, i.v) on the locomotor activity. Antagonist BRL44408(2.5, 5 and 10 μg each, i.c.v) administrated alone could dose-dependently inhibit locomotor activity of mice with no significant difference. 5 μg BRL44408 had significantly antagonism to DMED(0.0125 mg/kg, i.v) induced inhibition of locomotor activity. But 10 μg BRL44408 showed no effect on DMED induced inhibition of locomotor activity. JP-1302(5.5, 22, 88 μg each, i.c.v)administrated alone had tendency to inhibit locomotor activity of mice, but could not antagonize DMED(0.0125 mg/kg, i.v). ARC239(3, 12, 48 μg each, i.c.v)administrated alone could dose-dependently inhibite locomotor activity of mice, especially at dose of 48 μg. However, ARC239 showed no antagonism to DMED(0.0125 mg/kg, i.v) induced inhibition of locomotor activity. 5. The sedation of clonidine, guanfacine and xylazine was weaker than DMED. The rank order of inhibition of locomotor activity was DMED > clonidine > guanfacine ≈ xylazine. Inhibition of locomotor activity of these drugs belonged to sedation of central inhibition and was irrelevant to hypotension. Dbc AMP(20~40 μg each, i.c.v)can dose-dependently inhibited LORR in the response to DMED(0.20 mg/kg, i.v). LORR induced by DMED could be totally diminish by 40 μg dbc AMP. T-Q(250, 300 pmol each, i.c.v) could attenuate LORR induced by DMED. There was no significant difference of the induction time, but the immobilization time was decreased compared DMED alone. The locomotor activity of mice was slightly inhibited by dbc AMP(1.25, 5, 20 μg each, i.c.v). There were no effect of dbc AMP on the DMED inhibition of locomotor activity. T-Q(40, 100, 250 pmol each, i.c.v) administrated alone had no effect on locomotor activity, but T-Q could not antagonize DMED inhibition of locomotor activity as well. The effects of dbc AMP and T-Q on the DMED inhibition of locomotor activity were not similar with that in model of LORR. The mechanism need further study.Conclusion: 1. CHO-PKAcat-α2A/2C-AR cell lines were successfully constructed. 2. Antagonist BRL44408 could selectively block α2A-AR. Both JP-1302 and ARC239 could block α2C-AR effectively. 3. α2A-AR played a key role in sedation of α2-AR. 4. Inhibition of AC-c AMP-PKA pathway and activition of G protein gated inwardly rectifying K+ channels might be involved in the mechanism of sedation of α2-AR agonists. |
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