Effects Of Bilirubin On Nicotinic Acetylcholine Receptor Channels Currents In Rat Superior Cervical Ganglionic Cells And Its Mechanisms

Obejective:. Sympathetic nerve activity of patients with jaundice was decreased andthe decline of autonomic nerve function was correlated with blood bilirubin concentration.Acetylcholine (ACh), released by preganglionic firbers of SCG, modulates release ofneurotransmitters (catecholamine) from post-ganglionic firbers via neuronal nicotinicacetylcholine receptors (nAChRs) of postsynaptic membrane to regulate the function ofcardiovascular system. nAChRs on the SCG are vital for processing and transmission ofsynaptic information. In order to illustrate the declined function of autonomic nervoussystem in patients with jaundice, we study the effects of bilirubin on the nicotinicacetylcholine receptors(nAChRs) channels currents in rat SCG neurons and relatedmechanism.Method: Superior cervical ganglions were acutely dissociated from5-12-day-old SDrats and digested enzymatically in well-oxygenated standard solution containingcollagenase I (0.5~1.0mg/ml), protease E (1.0~1.5mg/ml) and DNase I (0.05~0.1mg/ml)for25-40min at31.5℃. Then the neurons were mechanically dissociated with pipettes andsingle neuron was adhered to the bottom of the dish within30min for whole-cellpatch-clamp recording. All electrical measurements were carried out in whole-cellpatch-clamp recording mode at room temperature (22-25℃). The patch pipettes werepulled from the glass capillaries on a four-stage puller. The patch pipette was positioned ona neuron using a miromanipulator. When sealing resistance beyond1G, whole-cellsealing was succeeded by the methods of sucking cell membrane and compensatingresistance. The currents were recorded with patch-clamp amplifier at a holding potential-60mV, filtered at1kHz. The drugs, including ACh and bilirubin, were administered witha rapid application system by self-made Y-tube on the surface of cells.1μM atropine wasadded in the standard solution to prevent possible activation of endogenous muscarinic receptors. The results were expressed as mean±standard deviation (SD). Statisticalanalysis was performed using paired Student’s t test by the Patch Master and Origin8.0.P<0.05was considered statistically significant.Results: At a holding potential (Vh)-60mV, acetylcholine evoked an inward currentof rapid activation and then desensitization decay, which were nAChRs channel currents.Bilirubin (0.5μM) enhanced the100μM ACh-evoked peak current by1.13(P<0.05) times,and reduced the rate of desensitization by37.9%(P<0.05). However, bilirubin at highconcentration (5μM) inhibited the100μM ACh-evoked peak currents by72.6%(P<0.05),and decreased the rate of desensitization by96.5%(P<0.05). Under the intervention of4μM bilirubin, the peak currents evoked by100、200、400and800μM ACh were635.3±172.7,1669.6±177.0,4370.8±206.2and6916.9±73.6(pA) respectively. Forskolin(a protein kinase A activator) caused a100μM ACh-evoked relative peak currents (IACh)decreased from0.44±0.11to0.30±0.96(p<0.05), whereas its relative rate ofdesensitization increased from0.05±0.07to0.30±0.29(p<0.05); Furthermore the100μMACh-evoked relative peak currents (IACh) increased from0.38±0.05to0.43±0.08(p>0.05)after administration of H-89(a protein kinase A inhibitor), and its relative rate ofdesensitization increased from0.004±0.002to0.014±0.003(p>0.05).Conclusion: Bilirubin can affect IAChof SCG by the manner of reversible recovery.Bilirubin at low concentration significantly enhance IACh, and this action may be ascribedto the decrease of its rate of desensitization. However, high concentration bilirubinsignificantly suppresses IAChand this action may be due to the competitive inhibition ofbilirubin. Bilirubin decreases the desensitization rate of IAchon concentration dependentmanner. Involvement of the cyclic AMP(cAMP) and PKA pathway during suppression ofIAChby bilirubin.