Hypothalamic TRPM8 and TRPA1 ion channel genes in the regulation of temperature homeostasis at water balance changes.

The role of the hypothalamic cold-sensitive ion channels - transient receptor potential melastatin 8 (TRPM8) and transient receptor potential ankyrin 1 (TRPA1) in homeostatic systems of thermoregulation and water-salt balance - is not clear. The interaction of homeostatic systems of thermoregulation and water-salt balance without additional temperature load did not receive due attention, too. On the models of water-balance disturbance, we tried to elucidate some aspect of these problems. Body temperature (Tbody), O2 consumption, CO2 excretion, electrical muscle activity (EMA), temperature of tail skin (Ttail), plasma osmolality, as well as gene expression of hypothalamic TRPM8 and TRPA1 have been registered in rats of 3 groups: control; water-deprived (3 days under dry-eating); and hyperhydrated (6 days without dry food, drinking liquid 4 % sucrose). No relationship was observed between plasma osmolality and gene expression of Trpm8 and Trpa1. In water-deprived rats, the constriction of skin vessels, increased fat metabolism by 10 % and increased EMA by 48 % allowed the animals to maintain Tbody unchanged. The hyperhydrated rats did not develop sufficient mechanisms, and their Tbody decreased by 0.8 °C. The development of reactions was correlated with the expression of genes of thermosensitive ion channels in the anterior hypothalamus. Ttail had a direct correlation with the expression of the Trpm8 gene, whereas EMA directly correlated with the expression of the Trpa1 gene in water-deprived group. The obtained data attract attention from the point of view of management and correction of physiological functions by modulating the ion channel gene expression.

Expression of Trpa1 and Trpv1 Genes in the Hypothalamus and Blood Pressure in Normotensive and Hypertensive Rats. Effect of Losartan and Captopril.

Analysis of the role of genomic regulation of systolic BP (SBP) in normal and hypertensive rats showed the presence of an inverse relationship between the level of Trpa1 gene expression in the anterior hypothalamus and SBP. Losartan, an antagonist of angiotensin II type 1 receptors, shifts it to the region of lower SBP and greater expression of the Trpa1 gene, which can attest to interaction of the TRPA1 ion channel in the anterior hypothalamus with angiotensin II type 1 receptors. No association was found between the expression of the Trpv1 gene in the hypothalamus and SBP. We have previously shown that activation of the peripheral ion channel TRPA1 in the skin also contributes to SBP decrease in hypertensive animals. Hence, activation of the TRPA1 ion channel both in the brain and at the periphery has similar effects on SBP and leads to its decrease.

Thermoregulatory Response to Cold at Various Levels of Activation of Peripheral TRPA1 Ion Channel.

The effect of TRPA1-ion channel on thermoregulatory responses depending on the level of its activity was studied in Wistar rats. To activate the TRPA1 ion channel localized in the skin, its agonist allyl isothiocyanate (AITC) was used in different concentrations (0.04, 0.4, 1, and 2.5%). Low concentration of AITC (0.04%) enhanced and high concentrations (1 and 2.5%), on the contrary, inhibited cold-defense responses (decreased their magnitude and led to their later initiation due to an increase in temperature thresholds). With an increase in TRPA1 activation, the increase in temperature thresholds (afferent link) was ahead of the decrease in the magnitude of responses (efferent link), which can attest to different sensitivity of these processes to TRPA1 activation.

Effects of activation of skin ion channels TRPM8, TRPV1, and TRPA1 on the immune response. Comparison with effects of cold and heat exposure.

The effects of pharmacological stimulation of skin ion channels TRPA1, TRPM8, TRPV1 on the immune response are presented. These effects are compared with the effects of different types of temperature exposures - skin cooling, deep cooling, and deep heating. This analysis allows us to clear the differences in the influence on the immune response of thermosensitive ion channels localized in the skin; (2) whether the changes in the immune response under temperature exposures are due to these thermosensitive ion channels. Experiments were performed on Wistar rats. For stimulation of TRPM8 ion channel, an application to the skin of 1% menthol was used, for TRPA1 - 0.04% allylisotiocianate, and for TRPV1 - capsaicin in a concentration of 0.001.The antigen binding in the spleen was two-times stimulated by activation of the cold-sensitive ion channel TRPM8 and much weaker by activation of warm-sensitive TRPV1 (by 15%), and another cold-sensitive ion channel TRPA1 (by 40%). Only the stimulation of TRPA1 significantly (by 140%) increased antibody formation in the spleen, while TRPM8 had practically no effect on this process, and activation of TRPV1 significantly (by 60%) inhibited antibody formation. Stimulation of the TRPM8 ion channel significantly (by 60%) reduced the level of IgG in the blood, which is believed to control of infectious diseases.The obtained results show that pharmacological activation of the skin TRPA1, TRPM8, TRPV1 ion channels can differently affect the immune system. At the epicenter of changes there were the antigen binding and antibody formation in the spleen, as well as the level of IgG in the blood. Exactly stimulation of the TRPM8 ion channel determines the changes in the immune response when only the skin is cooling, while at deep body heating, the changes in the immune response are mostly determined by the activation of the skin TRPV1 ion channel.

Skin TRPA1 ion channel participates in thermoregulatory response to cold. Comparison with the effect of TRPM8.

There was no clear evidence of the TRPA1 ion channel involvement in the formation of thermoregulatory responses. The present results convincingly show that the skin TRPA1 ion channel activation has significant influence on the formation of thermoregulatory responses of the body to cooling; it is especially strongly manifested for the metabolic component. At the TRPA1 activation by its agonist AITC (0.04%), an enhancement in thermoregulatory responses is observed: the temperature thresholds for the first phase and the second one of the metabolic response decrease, the values of all components of the metabolic response considerably increase: the increment of oxygen consumption in the first phase increases from 1.8 ± 0.24 in the control to 2.9 ± 0.35 ml/min*kg under AITC, P = 0.04; the increment of oxygen consumption in the second phase increases from 6.2 ± 2.06 to 17.4 ± 1.20 ml/min*kg, P = 0.002, as well as shivering rises from 7.8 ± 1.79 to 15.4 ± 1.87 mV, P = 0.011. In consideration of our previous results on the influence of TRPM8 ion channel activation on thermoregulatory responses (Kozyreva et al., J. Therm.Biol., 2010) it is obvious that the TRPM8 and TRPA1 ion channels have a pronounced, but unequal effects on the values of different phases of the metabolic response to cold. The TRPM8 activation manifests itself in an increase of value only the urgent first phase, this phase is associated with carbohydrate metabolism. As the recent results have shown the influence of the TRPA1 activation is realized predominantly in the clearly marked increase in the second phase of the metabolic response associated with lipid metabolism, as well as in evident shivering gain. The ability to predominantly control different parameters of thermoregulatory responses to cold may indicate the importance of both the TRPM8 and the TRPA1 ion channels in the processes of maintaining temperature homeostasis. The obtained data testify to the joint sequential operation of these thermosensitive ion channels.

Effect of Activation of Peripheral Ion Channel TRPM8 on Gene Expression of Thermosensitive TRP Ion Channels in the Hypothalamus. Comparison with the Effect of Cooling.

Experiments on rats showed that activation of the peripheral ion channel TRPM8 with menthol and rapid cooling (decrease in core temperature by 3°C) led to 1.5-fold activation of the expression of TRPV3 ion channel gene in the posterior hypothalamus, but had no effect on the expression of this gene in the anterior hypothalamus. Neither stimulation of peripheral TRPМ8, nor acute cooling affected the expression of genes for other thermosensitive ion channels (TRPV1, TRPV2, TRPV4, TRPA1, and TRPМ8) in the hypothalamus. Enhanced expression of Trpv3 gene can indicate increased sensitivity of hypothalamic neurons in the range of TRPV3 ion channel functioning (31-39oC). The relationship between the changes in Trpv3 gene expression and the shift of thermoregulatory reaction thresholds is discussed. Our findings attest to the presence of a functional relationship between TRP ion channels of the peripheral nervous system and TRP channels in the central structures of the brain.

Participation of Purinergic P2X Receptors in the Thermoregulatory Response to Cooling.

We studied the role of purinergic P2X receptors in the body response to cooling. In experiments on rats, P2X receptor antagonist PPADS was administered in different modes, which resulted in changes of different characteristics of the thermoregulatory response to cold. Iontophoresis of P2X antagonist into the skin decreased the thermal thresholds of all thermoregulatory responses to cooling, which can attest to a modulating effect of P2X receptors on peripheral thermosensitive afferents. Intraperitoneal administration of P2X antagonist suppressed thermoregulatory activity of skeletal muscles (shivering) developing during cooling without changing the thresholds of thermoregulatory responses. The findings suggest that ATP and P2X receptors play an important role in the formation of the response to cooling.

Calcium ions facilitate body heat emission response to warming.

Involvement of various areas of the body surface in heat emission response to warming is characterized by a certain succession. The first response preceding the deep body temperature rise is dilation of ear skin vessels. Then, an increase in deep body temperature is counterbalanced by vascular reaction in the tail region, which plays the leading role in up-regulation of heat emission. Calcium ions accelerate the vascular response to warming in both regions, although they produce no effect on the maximum level of heat emission. Our findings confirm the involvement of Ca(2+)-dependent mechanisms in activation of the processes aimed at stabilization of body temperature in warm-blooded animals. The role of heat-sensitive TRPV1 ion channels determining modality of the temperature signal and direction of effector reactions is discussed.

[Respiratory system response to cooling in subjects with single nucleotide polymorphism rs11562975 in gene of thermosensitive TRPM8 ion channel].

On the basis of genomic studies in subjects belonged to Russian ethnic group it was identified individuals with heterozygous genotype, containing the C-allele in single nucleotide polymorphism rs11562975, located in 6 exon of the gene encoding the temperature-sensitive ion channel TRPM8. Subjects with heterozygous genotype GC were characterized by not only increased sensation to cold but also hypometabolic response to local skin cooling and non-temperature activation of TRPM8 ion channel by menthol--decrease in total metabolism, pulmonary ventilation and coefficient of oxygen extraction. The subjects with homozygous genotype GG had a lower cold sensation and adequate response to local skin cooling in respect to thermoregulation--decrease in respiratory heat loss and increase in the lipid metabolism.

Effects of pharmacological activation of TRPM8 ion channels on the thermoregulatory responses during warming.

Preliminary non-thermal activation of cold-sensitive TRPM8 ion channel facilitates initiation of the heat-defense responses in homoiothermal animals by decreasing temperature threshold of the vasodilatory response. TRPM8 activation leads to earlier heat-initiated increase in oxygen consumption, but reduces its magnitude. Warming inhibits the lipolytic effect of menthol activation of TRPM8 observed under thermoneutral conditions. Thus, modulation of the skin temperature afferent signal by ion channel agonist TRPM8 changes not only cold-defense, but also heat-defense responses of the body.

[Effect of alpha2-adrenergic receptor antagonist yohimbine on thermoregulatory responses to cooling].

Ionoforetic yohimbine application to skin has a modulating effect on the thermoregulatory parameters. In thermoneutral conditions, yohimbine increases heat production and heat dissipation. At subsequent cooling, yohimbine facilitates the initiation of vascular and metabolic response by reducing the temperature threshold of both nonshivering and shivering thermogenesis. Under the influence of yohimbine the maximum value of both vascular and metabolic response increases. In strengthening the metabolic response the skeletal muscles shivering significantly contributes. In contrast to yohimbine, the norepinephrine thermogenic effect occurs by strengthening nonshivering thermogenesis. Features of the yohimbine influence on thermoregulatory parameters due to the dual localization and function of the alpha2-adrenergic receptors.

[Effect of transfer of fragment of chromosome 13, containing gene i16st on parameters of mouse temperature homeostasis].

Mechanisms of maintenance of temperature homeostasis in warm and under effect of cold were studied in mice of AKR strain and of its coherent strain AKR.CBA-D13Mit76 with the changed gene i16st encoding the gp 130 receptor, via which IL-6 performs its action. Under thermoneutral conditions and under action of cold, there were recorded temperature parameters, total oxygen consumption, carbon dioxide release, respiratory coefficient, and electrical muscle activity. Animals of the studied strains demonstrated different reactions to equal cold effect. At cooling, all mice of the AKR strain entered the state of hypothermia by decreasing metabolism. Mice of the AKR.CBA-D13Mit76 line showed 2 different types of reaction: 39 % of the animals of this strain reacted like mice of the AKR strain, but the majority (61 %) resisted actively to the cold action, which was manifested as a marked increase of metabolism. Taking into account the gene penetrance, this can indicate effect of the gene i16st on choice of the active ("regulated") or passive ("dependent") way of the organism reacting to temperature actions.

[Thermosensitive TRP channels gene expression in hypothalamus of normal rats and rats adapted to cold].

The gene expression of thermosensitive TRP (transient receptor potential) ion channels was studied by the method of quantitative reverse transcription-polymerase-chain-reaction (RT-PCR) in hypothalamus of control (normal) rats (5 weeks at the room temperature 20 +/- 22 degrees C) and rats adapted to cold (5 weeks at the room temperature 4 +/- 6 degrees C). For the first time in this brain region the expression of Trpv3 and Trpm8 genes was found, that was the evidence of the presence of TRPV3 and TRPM8 ion channels in the hypothalamus; it was made a comparison of the gene expression of the 6 thermosensitive ion channels (TRPV1, TRPV2, TRPV3, TRPV4, TRPA1, TRPM8) and found that there was rather high expression of the genes of thermosensitive ion channels activated at temperatures above 30 degrees C, while, the gene expression level of cold sensitive TRPM8 and TRPA1 proved to be much lower; it was found that long-term cold adaptation modified the gene expression of TRPV3 ion channel. The findings support the assumption that thermosensitive TRP ion channels of hypothalamus may be involved in the mechanisms of thermal adaptation at the level of gene expression.

[Relationship of single-nucleotide polymorphism rs11562975 in thermo-sensitive ion channel TRPM8 gene with human sensitivity to cold and menthol].

The examination of people belonging to the Russian ethnic group revealed that 20.3% of subjects had heterozygous genotype, containing the C-allele in single nucleotide polymorphism rs11562975, located in exon 7 of the gene encoding the temperature-sensitive ion channel TRPM8. Functional differences, associated with sensitivity to cold and menthol were identified between subjects with different genotypes of the polymorphism rs11562975 (GG and GC). Subjects with heterozygous genotype GC were characterized by increased sensitivity to cold and reduced sensitivity to menthol, agonist of the ion channel TRPM8, compared with subjects with homozygous genotype GG.

[Effect of TRPM8 ion channel activation on thermoregulatory response to cooling].

In rats, the effect of activation of the cold- and menthol-sensitive TRPM8 ion channel on different thermoregulatory parameters: total oxygen consumption, carbon dioxide release, respiration coefficient, constriction response of skin blood vessels, muscle activity, was studied. Activation of TRPM8 with menthol even in thermoneutral conditions produces an increase in oxygen consumption and a decrease in respiratory coefficient, which may suggest enhanced non-shivering thermogenesis and lipolysis. Rapid cooling against the background of TRPM8 activation is characterized by a decrease in the temperature thresholds of all thermoregulatory responses without associated changes in sequences of their initiation as well as in enhancement of metabolic component of emergency thermogenesis which leads to improved maintenance of core temperature in conditions when external cold acts on the organism. The obtained data on the effect of TRPM8 activation on metabolic parameters in thermoneutral conditions and under cooling suggest acontinuous involvement of this receptor in regulation of total metabolism and, possibly, in determination of the type of organism's metabolism as well as in determination of organism's response to external cooling.

Mechanisms of modulation of thermoregulatory reactions during cooling in hypertensive rats by the sympathetic nervous system.

Susceptibility of thermoregulatory responses to cold to blockage of α(1)- and ß-adrenoreceptors differs in health and hypertension. α(1)-Adrenoceptor blockade reduces vessel reactivity during cooling and vessel reaction to cold becomes similar to that in intact normotensive rats. Changes in the structure of metabolic response to cold in favor of non-shivering thermogenesis typical of hypertensive animals becomes even more pronounced under conditions of α(1)-adrenoceptor blockade due to inhibition of cold shivering. Blockage of ß-adrenoceptors does not affect parameters of vascular response to cooling. In hypertensive rats, in contrast to normotensive animals, ß-adrenoceptor blockade during cooling increased temperature thresholds for total metabolic reaction and shivering. The maximum shivering intensity also increased, which partially compensated inhibition of non-shivering thermogenesis. In the whole organism, blockade of one type of adrenoceptors during cooling leads to intensification of compensatory mechanisms realized through adrenoceptors of the other type. In hypertensive rats, compensatory capacities of thermogenic processes controlled by α(1)- and ß-adrenoceptors are impaired in comparison with normotensive animals under conditions of inhibition of both shivering and non-shivering thermogenesis.

Alpha1- and beta-adrenoblockers effects on immunogenesis in rats under thermoneutral conditions and after cooling of various extent.

Experiments with rats showed that ionophoretic delivery of alpha1-adrenoblocker into the skin does not change effects of superficial and deep cooling and did not affect the amount of the antibody producing cells in spleen and a blood level of circulating antibodies under thermoneutral conditions. Meanwhile alpha1-adrenoblocker abolished inhibitory effect of fast deep cooling on antigen-binding properties of spleen cells and peritoneal extraction cells, and markedly stimulated it. beta-Adrenoblocker had no effect on immunogenesis under thermoneutral conditions and changes modulating effect of cooling on immune response. It abolished immunosuppressive effect of deep cooling and enhanced the stimulating effect of superficial cooling. It was true both for antigen-binding processes in spleen and peritoneal cavity and for antibody production in spleen. The results obtained indicate an involvement of alpha1--and beta-adrenoreceptors in immune response inhibition after deep cooling. Adrenoblockers effects on various immune competent cells are ambiguous and depend on temperature.

Modulating effects of calcium on immune response of homoiothermal animal under thermoneutral conditions and during deep cooling.

Preliminary ionophoretic administration of Ca(2+) ions into the skin prevents the inhibitory effects of deep cooling on some processes characterizing the immune response. Differently directed changes in some immune response parameters induced by exogenous calcium and deep cooling suggest that competitive interactions between calcium-dependent processes can serve as mechanisms of functional changes in various physiological systems during the formation of the systemic reaction of a homoiothermal organism to cold.

The role of alpha1- and beta-adrenoceptors in initiation and development of thermoregulatory reactions during fast and slow cooling.

Ionophoretic application of alpha1- and beta-adrenoceptor blockers into the skin produces no effect on the parameters of thermal homeostasis under thermoneutral conditions. alpha1-Adrenoblocker verapamil inhibits cold shivering during fast and slow cold exposure; it elevates the temperature threshold and moderates the vasoconstrictor response during rapid cooling. These changes are accompanied by a compensatory decrease in the threshold and stimulation of non-shivering thermogenesis. Application of non-selective beta-blocker propranolol had no effect on the temperature thresholds of the thermoregulatory reactions, but augmented the maximum amplitude of shivering during both cooling protocols, thereby compensating the decrease in non-shivering thermogenesis. In the whole organism, block of one type adrenoceptors during cold exposure is accompanied by activation of the compensatory mechanisms mediated by adrenoceptors of the other type.