Bilirubin modulates acetylcholine receptors in rat superior cervical ganglionic neurons in a bidirectional manner

Abstract

Autonomic dysfunction as a partial contributing factor to cardiovascular instability in jaundiced patients is often associated with increased serum bilirubin levels. Whether increased serum bilirubin levels could directly inhibit sympathetic ganglion transmission by blocking neuronal nicotinic acetylcholine receptors (nAChRs) remains to be elucidated. Conventional patch-clamp recordings were used to study the effect of bilirubin on nAChRs currents from enzymatically dissociated rat superior cervical ganglia (SCG) neurons. The results showed that low concnetrations (0.5 and 2 μM) of bilirubin enhanced the peak ACh-evoked currents, while high concentrations (3 to 5.5 µM) of bilirubin suppressed the currents with an IC50 of 4 ± 0.5 μM. In addition, bilirubin decreased the extent of desensitization of nAChRs in a concentration-dependent manner. This inhibitory effect of bilirubin on nAChRs channel currents was non-competitive and voltage independent. Bilirubin partly improved the inhibitory effect of forskolin on ACh-induced currents without affecting the action of H-89. These data suggest that the dual effects of enhancement and suppression of bilirubin on nAChR function may be ascribed to the action mechanism of positive allosteric modulation and direct blockade. Thus, suppression of sympathetic ganglionic transmission through postganglionic nAChRs inhibition may partially contribute to the adverse cardiovascular effects in jaundiced patients.

Figure 1. Dose–response curve of rat SCG nAChR currents.

(A), representative nAChR current traces evoked by 10, 20, 50, 100, 200 and 400 μM ACh. (B), dose–response curve of nAChR currents fitted by nonlinear regression with logistic equation using Origin 8.0 software. Each data point represents mean ± SEMs (n = 9).

Figure 2. Specificity of nAChRs-mediated currents in SCG neurons.

Representative nAChR current traces evoked by ACh alone (control), coapplied with 100 µM hexamethonium bromide, and ACh alone (recovery), respectively. 100 µM hexamethonium bromide, a special nAChR antagonist, completely blocked the currents induced by 200 µM acetylcholine.

Figure 3. Bilirubin enhances and inhibits nAChR currents in rat SCG neurons in a dose-dependent manner.

(A) Representative traces of nAChR currents enhanced and inhibited by different concentrations of bilirubin (0.5, 2, 3, 4 and 5 μM). nAChR currents were activated by 100 μM ACh, and the duration of fast perfusion ACh by gravity was about 8 s. (B) The dose-response relationship for bilirubin on ACh-induced peak currents. The response rates of ACh-induced currents by bilirubin were normalized to the control response induced by 100 μM ACh. (C) The dose-response relationship for bilirubin on desensitization of nAChRs. Desensitization of nAChRs by bilirubin was normalized to the control response induced by 100 μM ACh. Each data point is expressed as means ± SEMs (n = 5).

Figure 4. The dose-response curve for bilirubin.

The inhibitory rate of nAChR currents by bilirubin in SCG neurons was normalized to the control response induced by 100 μM ACh. Each data point is expressed as means ± SEMs (n = 5).

Figure 5. Voltage-independent suppression of nAChRs in rat SCG neurons.

(A) Current-voltage relationships of nAChRs induced by 100 μM ACh were derived from the ramp stimulation protocol (membrane potentials increased from −70 mV to +30 mV at a rate of 333 mV/s) in the presence () or absence () of 4.5 μM bilirubin. (B) There was no voltage dependency regarding the effect of 4.5 μM bilirubin on peak nAChRs currents. Each response was normalized to that without bilirubin at each membrane potential from −70 mV to −20 mV. All data points are expressed as means ± SEMs (n = 5).

Figure 6. Curves represent the dose-responses of nAChR currents activated by ACh in the presence () or absence () of 4 μM bilirubin.

All nAChR currents evoked by ACh in SCG neurons were normalized to that induced by 100 μM ACh alone (referred to as Relative IACh). Administration of 4 μM bilirubin shifted the dose-responses of nAChR currents to the right, and markedly increased the potency and maximum efficacy of ACh (400 and 800 µM)-induced response. All data points are expressed as means ± SEMs (n = 3).

Figure 7. Enhanced inhibition of nAChRs by bilirubin pre-application.

(A), representative nAChR current traces evoked by ACh alone (control), coapplied with bilirubin, bilirubin pre-application, and ACh alone (recovery), respectively. (B), graph showing the percentage of current inhibition when 100 μM ACh was coapplied with 4.5 μM bilirubin with or without 4.5 μM bilirubin preapplication. All data points are expressed as mean ± SEMs (n = 6).

Figure 8. Bilirubin is involved in the cAMP and PKA pathway in suppressing nAChR currents.

The inhibitory effect of bilirubin on nAChR currents persisted in the presence of 10 μM forskolin. (A) Representative traces of nAChR currents inhibited by 4 μM bilirubin persisted in the presence of 10 μM forskolin. (B) The dose-response relationship for bilirubin on ACh-induced peak currents persisted in the presence of 10 μM forskolin. The response rates of ACh-induced currents by bilirubin were normalized to the control response induced by 100 μM ACh. (C) The dose-response relationship for bilirubin on desensitization of nAChR persisted in the presence of 10 μM forskolin. Desensitization of nAChRs by bilirubin was normalized to the control response induced by 100 μM ACh. Each data point is expressed as means ± SEMs (n = 5).

Figure 9. The inhibitory effect of bilirubin on nAChR currents persisted in the presence of 1 μM H-89.

(A) Representative traces of nAChR currents inhibited by 4 μM bilirubin persisted in the presence of 1 μM H-89. (B) The dose-response relationship for bilirubin on ACh-induced peak currents persisted in the presence of 1 μM H-89. The response rates of ACh-induced currents by bilirubin were normalized to the control response induced by 100 μM ACh. (C) The dose-response relationship for bilirubin on desensitization of nAChR persisted in the presence of 1 μM H-89. Desensitization of nAChRs by bilirubin was normalized to the control response induced by 100 μM ACh. Each data point is expressed as means ± SEMs (n = 5).