Introduction

Physiological systems of the body are equipped with compensatory feedback mechanisms to prevent excessive use of cellular energy and maintain metabolism, which is necessary to avoid threats, for example, in the case of a "fight or flight" response to stress¹. Catecholamines are the triggering factor in stress response. Catecholamines (epinephrine, norepinephrine) are released from the adrenal medulla and neuronal synapses and enter the bloodstream1. High concentrations of catecholamines in the blood can contribute to arrhythmias. Catecholamines act through adrenergic receptors. Adrenergic receptors mediate various physiological and pharmacological actions of endogenous catecholamines and their synthetic analogs. Heart nerve terminals can also contribute to an increase in the local level of catecholamines and participate both in response to stress and in the basal regulation of the heart and cardiogenesis². Aberrant feedback control of the sympathetic response can lead to a whole range of pathologies¹.

Adrenergic receptors are G-protein coupled receptors. As a result of the study of pharmacological and molecular properties, adrenergic receptors have been subdivided into three types, α1-AR, α2-AR, and β-AR. These types, in turn, are represented by three (or more) receptor subtypes. α2-adrenergic receptors are distributed throughout the central nervous system and peripheral tissues. The noradrenergic system and α2-adrenergic receptors are involved in the regulation of various physiological functions such as excitation, attention, cardiovascular diseases, and activation of the peripheral sympathetic and parasympathetic systems³.

Cellular reactions accompanied by exposure to catecholamines are associated with cellular signalling pathways related to various heterogeneous complexes of Gα/Gβγ proteins. Currently, 20 Gα, 6 Gβ, 12 Gγ proteins are known, which provide almost 1500 combinatorial variants of signal transduction; they are additionally multiplied by various effector isoforms⁴. It is also known that effectors can be regulated either by Gα or Gβγ subunits or by their dual or synergistic effects⁵.

Adrenergic receptors use two different signalling cascades to amplify their regulatory signals. β-adrenergic receptors act through Gαs, and α2-adrenergic receptors through Gαi proteins. Activation of α2 and β-adrenergic receptors counteract each other by decreasing and increasing the activity of adenylate cyclase, providing homeostatic regulation of intracellular cyclic adenosine monophosphate (cAMP) and its downstream cascades⁶.

In contrast to the presynaptic α1 and β-ARs, which control neuronal activity and neurotransmitter release, α2-ARs play an overwhelming role. Activation of presynaptic α2-AR, leads to dissociation of the Gi protein and to the reduction of cAMP levels, the opening of internally rectifying K+ channels, and inhibition of voltage-driven Ca2+ channels that directly affect the exocytosis mechanism7. Thus, α2-ARs have been recognized as short-loop feedback inhibitors for the release of sympathetic and adrenal catecholamines, and in general, they have an inhibitory effect on sympathoadrenergic regulation⁸. Specific α2-AR agonists are mainly involved in antinociceptive, sedative, central hypotensive, hypothermic, and behavioral effects⁹. One such specific α2-AR agonist is clonidine, which is used for hypertension. The α2-adrenergic receptor agonist, clonidine, is widely used in both anesthesia and intensive care. However, clonidine can cause severe hemodynamic side effects such as hypotension and bradycardia¹⁰.

MATERIAL AND METHODS

The experiment involved eighteen male and female 20-week-old outbred rats, which were kept in cages in a specially designated room - vivarium. All animals were kept under the same conditions in which the light and temperature were constantly controlled. The animals received proper care: they were fed and watered daily, the cages were cleaned once a week, and washed once a month.

For the experiment, rats were anesthetized with urethane injected intraperitoneally (solution 25%, 800 mg/kg). Urethane does not affect the activity of the rat heart. The effect of anesthesia was determined according to such indicators as blood breathing and the cessation of vibrissae oscillations. The anesthetized rat was placed on the operating table with its limbs fixed, and electrodes were placed to record an electrocardiogram (ECG), the operating field was cut out, which was disinfected with an alcohol solution. Operations were performed under microscope guidance. The selective agonist of α2-adrenergic receptors ST-91 (Tocris) was used for the experiment, at an effective dose of the agonist is 0.01 mg/kg. The ST-91 preparation was injected into the right femoral vein; an insulin syringe was used for accurate dosing. The signal from the electrograph was sent to the S1-83 oscilloscope and then to the personal computer. The ECG data were subjected to computer-assisted mathematical analysis. As a result of the analysis, we obtained the values of the parameters of the variation pulseogram. In our work, we analyzed the average cardiointerval (Xav, ms), mode (Mo, ms), the amplitude of the mode (AMo, %), variation amplitude (∆X, ms), rms deviation (δ, ms), as well as conventional units (conventional units), the stress index, the vegetative balance index, the vegetative rhythm index, the indicator of the adequacy of the regulatory processes (regulatory system stress index, vegetative balance index, vegetative rhythm index, regulation adequacy) were determined. These indicators allow us to determine the important mechanisms of regulation of the heart.

Data storage, graphical inference, and statistical analysis were performed using Statistica data analysis software. A value of p<0.05 was considered significant.

Results and Discussion

To study the role of α2-adrenergic receptors in the regulation of cardiovascular activity of the heart, we injected the selective α2-AR agonist ST-91 into the right femoral vein of rats at a dose of 0.01 mg/kg of animal weight. This was the minimum dose of the agonist that led to changes in the mean cardiointerval (Xav).

In vivo stimulation of α2-AR with ST-91 causes a short-term negative chronotropic. In effect, α2-AR activation with ST-91 increases the mean cardio interval (Xav) from 209.88 ± 14.9 ms to 327.13 ± 31.59 ms (p≤0.01) after the agonist administration. By the first minute of observation, Хm is 263.5 ± 14.2 ms (p≤0.05). During the 3rd minute of recording, Xav is 229 ± 12.2 ms, which corresponds to an increase of 10.14 ± 2.4% (p≤0.01) from the initial value. By the 5th minute of registration, the value of Хm does not change. During the 7th and 10th minutes after the administration of the agonist, Xav is 243.88 ± 13.6 ms and 238.6 ± 17.2 ms, respectively. And by the 20th minute of recording, the average cardio interval indicator reaches its initial value and is 208.5 ± 17 ms. Further, during the experiment, no significant changes in Xav were observed (Figure 1).

In the experiment, the α.-AR agonist ST-91 affects the heart rate and the parameters of autonomic balance. The initial mode was 208.63 ± 14.2 ms. after the start of α.-AR stimulation, this value increased to 308.75 ± 27.8 ms (p≤0.01), and 1 minute of observations was 254 ± 14.4 ms (p≤0.05). Such indicators as the variation range (∆ X) and the standard deviation (δ) also tended to increase. ∆Х increased from 18.63 ± 3.2 ms to 127.63 ± 25.6 ms (p≤0.01), and δ from 21.75 ± 7.44 ms to 1504.38 ± 498.7 ms (p≤ 0.05). Further, the indices and modes and ∆Х gradually returned to the initial value. The mode amplitude immediately after the introduction of ST-91 decreased from 14.24 ± 1.92% to 8.25 ± 0.5% (p≤0.05), followed by a gradual recovery of the indicator to the initial value. Other indicators of heart rate variability decreased during the experiment. Vegetative balance index decreased from 1094±318.3 conventional units to 114.38±42.3 conventional units (р≤0.05), regulatory system stress index decreased from 2925.7±94.5 conventional units to 212 ± 76 conventional units (p≤0.05), vegetative rhythm index decreased from 344.63 ± 71.6 conventional units to 44.7±14.4 conventional units (p≤0.01), regulation adequacy decreased from 73.5±13.8 conventional units up to 28.8±4.3 conventional units (p≤0.05). After a decrease in these indicators, the values were restored to the initial level.

In the present work (Figure 1) as reported in our earlier works¹⁰, activation of α.-AR with clonidine hydrochloride at a dose of 0.01 mg/kg (Tocris), showed a long-lasting increase in the average cardiointerval¹⁰ which persisted throughout the experiment.

Thus, activation of α.-adrenergic receptors in the whole organism (in vivo) with ST-91 and clonidine hydrochloride at doses of 0.01 mg/kg of animal weight leads to a decrease in the cardiac activity of sexually mature rats. At the same time, ST-91 has a short-term decrease in heart rate, and clonidine hydrochloride has a longer-lasting effect on heart contractions.

Figure 1
The effect of activation of alpha2-adrenoceptors on average cardio intervals (Xavg). ***р≤0.001, **р≤0.01, *р≤0.05.

In conclusion, when we compared the effects of two α₂-adrenergic agonists, ST-91 and clonidine, on cardiac activity in rats, we demonstrated that stimulation of α₂-adrenergic receptors with an ST-91 agonist in 20-week-old rats resulted in a short-term decrease in heart rate. At the same time, the analysis of indicators of heart rate variability indicates an increase in the tone of the parasympathetic channel of regulation of the heart. Our previous experiments showed that clonidine reduces heart rate and this bradycardic effect persists throughout the experiment¹⁰. To date, three different subtypes of α₂-AR have been identified, designated as α₂A/D, α2B, and α₂C¹¹. They differ in their pharmacological properties and tissue distribution¹¹. To understand the biological significance of each subtype of α₂-AR, genetically engineered mice helped to a large extent^12,13. All three subtypes of α₂-AR are approximately 75% identical in their transmembrane segments, but they perform different physiological functions. α2A/D-AR play an important role in the regulation of sympathetic tone and pain perception and also mediates sedative effects in α2 -agonists. α₂B-ARs are responsible for vasoconstriction in some vascular beds. α2C-AR and α₂A/D-AR regulate the release of catecholamines as presynaptic autoreceptors^14-16.

We hypothesize that the differences in the effects of ST-91 and clonidine are associated with a significant affinity of ST-91 for one of the three subtypes of α₂-AR, which is responsible for short-term cardiac activity at the beginning of the experiment. This assumption requires further research.

Acknowledgments

Acknowledgments

This study was prepared in accordance with the Russian state program of competitive growth of Kazan Federal University and supported by the RFBR and Government of the Republic of № 18-44-160022.

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