Modulating Effect of the Cardiotonic Steroid Marinobufagenin on Slow Sodium Channels

The objective of this study was to determine the potential role of the cardiotonic steroid marinobufagenin in the mechanisms of nociceptive signal modulation. It is known that the first phase of the formation of a painful sensation is related to activation of tissue peripheral receptors that are capable of encoding information not only about painful stimulation, but also about signals of other modalities. Nociceptive signals emerge in free nerve endings innervated by A and C afferents of nociceptive neurons of dorsal ganglia. Slow sodium channels (Na_ν1.8) insensitive to tetrodotoxin, which play an exclusive role in encoding nociceptive signals, have been identified in membranes of nociceptive neurons. Therefore, search for the specific blocker of these channels is important not only for clarification of the mechanisms of sensory information encoding, but also for medical practice.

Structure of marinobufagenin – CAS 470-42-8

It is known that the target for endogenous marinobufagenin is Na⁺, K⁺ ATPase. In addition to its main pump function, this enzyme can also function as a signal transducer. Therefore, the target for the agent studied may be Na⁺, K⁺ ATPase (transducermediated effect), as well as the amino acid sequence of the Na_ν1.8 channel (activation of a “modulated receptor”).

In the present study, it has been proved that marinobufagenin, acting at a low, “endogenous” concentration, is capable of reducing the voltage sensitivity of the activation gate structure of Na_ν1.8 channels. We will explore the molecular mechanism of this effect in our following studies.

The effect of the cardiotonic steroid marinobufagenin from the class of bufadienolides on Na_ν1.8 sodium channels was studied using the patch clamp method. We have modified this method in order to measure the effective charge value of the activation gate structure of slow sodium channels. Experiments were carried out on cultured sensory neurons isolated from the areas L₅–S₁ of the spinal cord ganglia of newborn Wistar rats with the use of standard solutions. Cultivation of isolated neurons for 2 h in the standard culture media with the use of СО₂ temperaturecontrolled chamber allows obtaining intact cells.

The results were processed statistically using Student’s t test. The differences were considered significant at р ≤ 0.05.

The responses of Na_ν1.8 slow sodium channels were measured in the control and after application of marinobufagenin to the culture medium at a concentration of 10 nmol/L. The experimental agent reduced the amplitude of the currents. This was caused mainly by the socalled “rundown effect” slightly limiting the applicability of the patch clamp method. The voltage sensitivity of the activation gate structure of slow sodium channels was estimated by the modified Almers method, giving satisfactory measurement of the effective charge value (Z_eff) of the activation gate structure of slow sodium channels. This is due to a rather slow development of Na_ν1.8 channel conductivity inactivation. The tangent of the slope angle of the regression line determines the limiting logarithmic sensitivity to voltage variation of the channel gate structure.

A significant (p < 0.05) decrease in the effective charge value (Z_eff = 4.7 ± 0.4, n = 15) after application of the steroid in comparison to the control (Z_eff =6.8 ± 0.4, n = 15) has been found in studying the effect of marinobufagenin on the membrane of nociceptive neuron.

Pharmacological agents capable of reducing the nociceptors excitability due to a decrease in the voltage sensitivity of the activation gate structure of Na_ν1.8 slow sodium channels could play the role of analgesics. Our data suggest that marinobufagenin could play the role of the modulator of slow sodium channel activity. The discovered effect of the reduction of Na_ν1.8 channel voltage sensitivity by marinobufagenin may be caused by one of the two mechanisms. The first of them, transducermediated one, is possibly triggered by the effect of the experimental agent on its wellknown molecular target, Na⁺, K⁺ ATPase linked to slow sodium channels. The second mechanism may be activated by the direct interaction of marinobufagenin with Na_ν1.8 channels. Further experiments will allow choosing one of these alternative hypotheses. However, it is evident that any of the two possible mechanisms of action of the given bufadienolide would lead to a decrease in nociceptor excitability. The physiological significance of this effect cannot be overestimated, because it is known that the endogenous cardiotonic steroid ouabain is a strong analgesic. Another specific modulator of nociceptive neuron membrane excitability, comenic acid, which affects Na_ν1.8 channels by selectively binding to the specific receptor, also could successfully play the role of a safe analgesic capable of replacing opiates. All these data suggest that the study of the physiological role of endogenous marinobufagenin deserves close attention.

Reference：

N. Shelykh, V. B. Plakhova, S. A. Podzorova, A. Ya. Bagrov, and B. V. Krylov. Doklady Biological Sciences, 2014, Vol. 458, pp. 278–280

 

Related  products：