Ethanol while being introduced into the organism mainly through ingestion or skin application affects many functional systems. The basis for the influence of ethanol on the body functioning is its direct effect on the cell metabolic processes. Ethyl alcohol affects the cell membrane by separating fatty acid chains of phospholipids and changing membrane fluidity; it also alters the permeability of volt- and ligand-gated ion channels and directly alters the electrical potentials of neurolemma.

Ambient temperature is one of the major factors affecting the living organism. The start thermal afferent information under the external cooling primarily is given by the peripheral skin thermo-afferents. In recent years there have been numerous studies concerning the cellular and molecular mechanisms of cold sensitivity. It is believed that thermosensitive TRP channels are the basis for temperature sensation1. Perception of cold in the physiologically relevant temperature range is performed with participation of the cold-sensitive ion channel TRPM8, which is activated by cooling in the range of 28-8ºC. Menthol is one of known agonists of the TRPM8 ion channel. The expression of the gene of the TRPM8 ion channel has been proved on the endings of sensory neurons, spinal ganglia and brain structures234. Free endings of sensory neurons are the peripheral thermoreceptors (thermo-afferents). Thus the perception of cold and the afferent signal depend on the activity of cold-sensitive ion channels, which increase its permeability in response to the decrease in temperature15.

Afferent signal determines the character and sequence of various physiological responses initiation to some effect. Temperature thresholds for cold-defense responses, aimed at maintaining temperature homeostasis, characterize thermal afferent signal at external cold exposure. The study of the thermal afferent signal role that is formed under the effect of pharmacological modulation of the skin thermoreceptor activity is of particular interest. We have previously shown that pharmacological activation of cold-sensitive TRPM8 ion channels leads to the shift in temperature thresholds of responses to cooling6. What are the effects of ethanol on the temperature thresholds as well as the structure of thermoregulatory response of the whole organism to cold exposure is not currently known. Ethanol, being able quickly to penetrate the lipid-rich membrane, and causing the conformation of the protein molecules, can alter the function of ion channels. Currently, there are data that ethanol interacts with the molecules such as TRP ion channels789. So, it was shown in vitro on HEK293T cells that ethanol inhibits cold-sensitive TRPM8, but activates the warm-sensitive TRPV1 ion channel7. The possible interaction of ethanol and the TRPM8 ion channel in the whole living organism is not known.

In the present research we try to find the answer to following questions1. How the application of ethanol to skin (the area of thermosensitive afferents concentration) affects temperature thresholds and thermoregulatory blood vessels and metabolic response to rapid and slow cooling?2. What is the interrelation of the ethanol effect and effect of the TRPM8 ion channel activation in the formation of cold-defense metabolic responses?

In thermoneutral conditions application to the skin of 50% ethanol or 1% solution of menthol in 50% ethanol had no effect on the registered parameters (Table 1).

There is a definite sequence of thermoregulatory responses characteristic to rapid and slow deep cooling6. At rapid cooling (Figure 1, left panel), to begin with, when only skin temperature decreases, the first phase of metabolic response characterized by rising respiratory coefficient develops, i.e. increased utilization of carbohydrates. Then, with greater cooling of the skin, but also without change in core temperature, a constrictor response of the skin blood vessels has been initiated. The second phase of metabolic response characterized by a decrease in the respiratory coefficient (increase of lipid metabolism) enfolds when not only skin, but also deep body temperature falls, this is associated with an increase in thermogenic muscle activity (shivering). At slow cooling (Figure 2, left panel), the first phase of metabolic response associated with the dynamic activity of skin thermoreceptors is absent. Cold-defense response includes the constriction of skin blood vessels and metabolic response, the character of which is similar to the second phase of the metabolic response at rapid cooling.

The obtained results prove that thermoregulatory response to cooling are largely modified by the preliminary application of the ethanol to skin. It can be assumed that the effect of ethanol in our experiments was caused mainly through the peripheral skin afferents. Previously we have shown that the modulation of the peripheral thermal inputs by various biologically active substances results in changes of the thermal thresholds and other parameters of thermoregulatory responses131415.

As it was mentioned above, thermoregulatory responses to rapid and slow cooling are characterized by different components. In current experiments, the application of ethanol and solution of menthol in ethanol differently affects thermoregulatory metabolic response depending on the cooling rate.

At rapid cooling, ethanol has no effect on the first phase of the metabolic response, which takes place only at rapid changes of the skin temperature, i.e. in the presence of the dynamic activity of the skin cold thermoreceptors. This suggests that ethanol does not influence the processes caused by the dynamic activity of the skin thermoreceptors. It should also be noted, that these responses are initiated when only skin temperature decreases before the fall of deep body temperature 6.

On the contrary, according the above mentioned data, ethanol has clear inhibitory effect on the component of the metabolic response to rapid cooling, which normally develops when the deep body temperature falls. Thus, under the influence of ethanol, the second phase of the metabolic response to rapid cooling and shivering thermogenesis which accompanies this phase does not develop and is completely absent. At slow cooling, ethanol similar rapid cooling supresses the metabolic response and shivering does not develop. Inhibition in metabolic response to cold exposure is accompanied by the acceleration of the deep body temperature fall. Thus, at cooling with different rates, ethanol inhibits the most powerful thermogenic component of the metabolic response to cold which develops when the deep body temperature decreases, and has no influence on the earlier started responses which are initiated without considerable decrease in deep body temperature, but only at the rapid decrease in skin temperature. Moreover, on the background of ethanol, instead of increasing thermogenic metabolism during cooling, a significant decrease in oxygen consumption is observed. A number of researchers have also noted the hypothermic effect of the intrinsic administration of ethanol in thermoneutral conditions16,17as well as its inhibitory effect mainly on thermogenesis and not on heat loss18.

Decrease of the main phase of the cold thermogenesis which we observed in our experiments, when applying ethanol to skin, may be due to inhibition of non-shivering thermogenesis in brown adipose tissue, and inhibition of muscle thermogenic activity (shivering). It is known that mitochondrial UCP1 in brown adipose tissue (BAT) is a key molecule for non-shivering thermogenesis. The data on the influence of ethanol on the level of noradrenaline and uncoupling in BAT give evidence for the possibility to change non-shivering thermogenesis by ethanol19. So it was shown, that at room temperature, ethanol did not significantly alter the level of noradrenaline or UCP1 mRNA in BAT, whereas at cold exposure (+4ºC) the noradrenaline level in rats drinking ethanol was significantly lower than in control animals20. Earlier, it was noted that drinking alcohol can delay the onset of shivering and reduce its duration, and this may also result in the decrease of thermogenesis21. The inhibitory effect of ethanol on the contractile activity of skeletal muscles during cooling can be mediated by α-adrenoreceptors, since the important role of these receptors in modulating effects of ethanol is known, as well as their role in muscle contraction22. Previously we also have shown that α-adrenoreceptor antagonist completely abolishes the development of shivering in the cold23.

Recent studies allow us to believe that ethanol can modulate and interrelate with thermosensitive TRP ion channels, which are responsible for the afferent thermal information and the initiation of thermoregulatory responses79. However, presented data as well as our previous results12 have shown that menthol, agonist of the cold-sensitive TRPM8 ion channel, when dissolved in ethanol or saline has the mostly pronounced stimulating effect on the first phase of metabolic response to rapid cooling and the vascular response. So, ethanol does not prevent the effect of menthol. At the same time, the activation of the cold-sensitive TRPM8 ion channel by menthol has no obvious influence on effects of ethanol – inhibition the most powerful thermogenic component of the metabolic response to cold. Consequently, there are no evidences on the inhibiting effect of ethanol on the activity of the TRPM8 ion channel. The available literary data7 on the effect of ethanol on the TRPM8 ion channel were obtained in vitro on the culture of the specific HEK293T cells and may not fully reproduce the processes of the whole living organism.

Thus, the present results prove that modulatory effects of ethanol and activation of the cold-sensitive TRPM8 ion channel are mainly directed at different metabolic components of thermoregulatory response to cold. Menthol, as agonist of the TRPM8 ion channel, predominantly stimulates the emergency first phase of metabolic response which appears only at rapid cooling in the presence of the dynamic activity of the skin cold receptors before the decrease of deep body temperature, while ethanol inhibits the most powerful phase of metabolic response to cold which appears at deep body cooling and is associated with the development of shivering thermogenesis in muscles and non-shivering thermogenesis in brown adipose tissue. It is possible that various effects of ethanol and menthol are due to their impact on different groups of the skin thermal afferents.