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.

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.

[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.

[Effect of long-term cold adaptation on the mRNA expression of serotonin 5-HT1A and 5-HT2A receptors].

The mRNA expression of serotonin receptors 5-HT1A and 5-HT2A was investigated by the quantitative method RT-PCR in rats adapted to cold (5 weeks at +4 -(+6) degrees C) and in control (5 weeks at +20-22 degrees C). Four brain regions were examined: frontal cortex, hypothalamus, hippocampus, and midbrain. The influence of cold adaptation on the mRNA expression of 5-HT15 receptor was not found to be absent. The mRNA expression of 5-HT2A receptor changed under long-term cold exposure. These changes in different brain regions were various: in hypothalamus, there was an increase of the 5-HT2A receptor mRNA expression; in the cortex, a decrease; in the hippocampus and midbrain, significant changes of the mRNA expression were absent. The findings appear bo te adaptive and, according to their localization in the central nervous system, regulatory. They also suggest involvement of brain serotoninergic system in mechanism of adaptive reorganization of temperature regulation.

[Myocardial blood flow in different regions of the heart in patients with ischemic heart disease before and after revascularization]. / Miokardial'nyi krovotok na raznykh uchastkakh serdtsa u bol'nykh ishemicheskoi bolezn'iu serdtsa do i posle revaskuliarizatsii.

Laser doppler flowmetry was used for the intraoperative study of myocardial blood flow before and after revascularization in 116 patients with 2-3 vessel coronary artery disease and class II-IV angina. In patients without myocardial infarction, with microfocal myocardial infarction, or operated early after myocardial infarction revascularization caused no significant increase of myocardial blood flow. In patients with macrofocal infarction surgery was associated with significant increase of myocardial blood flow. Efficacy of revascularization could be assessed by lowering of flow gradients between various regions of the myocardium.

The brain tryptophan hydroxylase activity in the sleep-like states in frog.

The activity of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase, was determined in the brain stem in active awake frogs, and frogs in three sleep-like states: with plastic muscle tone (SLS-1), with rigid muscle tone (SLS-2), and with relaxed muscle tone (SLS-3). Significant decrease in the enzyme activity has been found in frogs in SLS-1 and SLS-2 compared to awake animals. The development in frogs a cataleptic-like immobility after treating the animals with rhythmic lighting was accompanied with a decrease in the brain tryptophan hydroxylase activity. These results provide strong evidence for the involvement of the brain serotonin in frogs in the control of evolutionary ancient sleep-like states, probably by the regulation of muscle tone.

Involvement of brain tryptophan hydroxylase in the mechanism of hibernation.

Marked changes were revealed in the activity of the key enzyme in serotonin biosynthesis, tryptophan hydroxylase (TPH), during entry into hibernation, hibernation, and arousal in ground squirrels (Citellus erythrogenys). An increase in TPH activity was found in the midbrain, hippocampus, and striatum during the prehibernation period in euthermic ground squirrels. A further increase in TPH activity was observed during the entry into hibernation. Significant elevation was found not only in potential TPH activity measured at the incubation temperature of 37 degrees C but also at incubation temperature of 7 degrees C, approximating the body temperature in hibernation. Vmax in the midbrain of hibernating animals was about 50% higher than in active ones without significant changes in Km. Thus, brain TPH maintains functionality during torpidity and is activated before the entry into hibernation. The results support the idea that brain serotonin is crucially involved in the transition to and the maintenance of the hibernation state.

[A sensitive fluorimetric method of determining tryptophan hydroxylase activity of structures in the brain]. / Chuvstvitel'nyi fliuorimetricheskii metod opredeleniia aktivnosti triptofangidroksilazy v strukturakh mozga.

An assay of tryptophane hydroxylase activity in brain was based on fluorometric measurement of serotonin after condensation with o-phthalic aldehyde in 10 N HCl. The method proved to be more sensitive and simple as compared with the previously reported technique and enabled to measure the enzyme activity in brain samples as little as 10 mg. The method is recommended for laboratory routine use.