Resibufogenin and cinobufagin activate central neurons through an ouabain-like action

Abstract

Cinobufagin and resibufogenin are two major effective bufadienolides of Chan su (toad venom), which is a Chinese medicine obtained from the skin venom gland of toads and is used as a cardiotonic and central nervous system (CNS) respiratory agent, an analgesic and anesthetic, and as a remedy for ulcers. Many clinical cases showed that Chan su has severe side-effects on the CNS, causing shortness of breath, breathlessness, seizure, coma and cardiac arrhythmia. We used whole-cell recordings from brain slices to determine the effects of bufadienolides on excitability of a principal neuron in main olfactory bulb (MOB), mitral cells (MCs), and the cellular mechanism underlying the excitation. At higher concentrations, cinobufagin and resibufogenin induced irreversible over-excitation of MCs indicating a toxic effect. At lower concentrations, they concentration-dependently increased spontaneous firing rate, depolarized the membrane potential of MCs, and elicited inward currents. The excitatory effects were due to a direct action on MCs rather than an indirect phasic action. Bufadienolides and ouabain had similar effects on firing of MCs which suggested that bufadienolides activated neuron through a ouabain-like effect, most likely by inhibiting Na+/K+-ATPase. The direct action of bufadienolide on brain Na+ channels was tested by recordings from stably Nav1.2-transfected cells. Bufadienolides failed to make significant changes of the main properties of Nav1.2 channels in current amplitude, current-voltage (I-V) relationships, activation and inactivation. Our results suggest that inhibition of Na+/K+-ATPase may be involved in both the pharmacological and toxic effects of bufadienolide-evoked CNS excitation.

Figure 1. Chemical structures of cinobufagin and resibufogenin.

Figure 2. Over-excitation of MC evoked by cinobufagin.

Arrow indicates the time point of adding 10 µM cinobufagin. The recording trace at the (1) and (2) time point in the upper trace are shown at an extended time scale in the middle trace and lower trace.

Figure 3. Bufadienolides and ouabain enhanced the firing activity of MCs in a concentration-dependent manner.

A Original recording from a representative MC illustrated the increase in firing rate and depolarization following application of cinobufagin at 1 µM and 5 µM, respectively. B1 Concentration-response curves for spiking of MCs evoked by various concentrations of bufadienolides and ouabain. The firing rates in the presence of bufadienolides and ouabain were normalized with respect to the control condition and were averaged (each point was the mean ± SEM of 3 to 7 cells). Error bars have been left out for clarity. The lines are fits for each set of data to the Hill equation: y  =  Axⁿ/(K _d ⁿ + xⁿ), where y is the increase of spiking rate, A is the maximal increase, K _d is the apparent dissociation constant for bufadienolides, and n is the Hill coefficient. K _d and A were estimated using a Marquardt nonlinear least squares routine. B2 Relationship between concentrations of bufadienolides or ouabain and evoked depolarization of membrane potential. Error bars were left out for clarity.

Figure 4. Cinobufagin activated MCs in the presence of synaptic blockers.

Original recording from representative MCs illustrated the enhancement of firing rate and depolarization following application of cinobufagin. The two traces are from different MCs.

Figure 5. Ouabain had an excitatory effect on MCs.

Original recording from representative MCs illustrated the increase in firing rate and depolarization following application of ouabain. The two traces are from different MCs.

Figure 6. Both bufadienolides and oubain elicited inward currents in MCs.

Original recordings illustrated cinobufagin-, resibufogenin- and ouabain-evoked inward currents of MCs. Data are from different cells.

Figure 7. Cinobufagin had no effects on Na_v1.2 channels.

A, B Recording traces of activation of Na_v1.2 currents in control (A) and in 100 µM cinobufagin (B). The currents were elicited by stepping to various depolarized potentials (ranging from −80 to +100 mV in 10-mV increments) for 9 msec, and then returning to the holding potential of −100 mV. C The current-voltage (I–V) relationships in control and in the presence of various concentrations of cinobufagin. Peak currents at each depolarized potential were measured. The data are from a representative cell. D Cinobufagin did not change Na_v1.2 channel activation and inactivation. Activation: From the peak Na⁺ currents as obtained in A and B, the Na⁺ conductance values (G) were calculated (n = 3–5 for each point), normalized to the maximum in control and plotted as a function of membrane potentials (V). Inactivation: The voltage dependence of steady-state inactivation (h _∞) was examined by applying 500-msec prepulse potentials from −100 mV to 0 mV in 10-mV increment from a holding potential of −100 mV before stepping to the test potential (0 mV) for 35 msec (n = 5 for each point). The peak current (I) for each cell was normalized with respect to the first value measured at test potential (0 mV). Conductance–voltage relationships or inactivation–voltage relationships from individual cells were fitted with a Boltzmann function, y  =  1/{1 + exp[(V – V _.5)/k]} or y  = 1- 1/{1 + exp[(V – V _.5)/k]}, where V is membrane potential, V _.5 is the half-activation (V _a) or half inactivation (V _h) voltage, and k is a slope factor.