**4. Rodent skeletal muscles**

*Adenosine Triphosphate in Health and Disease*

P2 receptor-mediated inhibition. The depressant effect of exogenous ATP on neuromuscular transmission was demonstrated for the first time more than 40 years ago [24], although for a long time, it was believed that inhibitory action of ATP is indirect and depends on degradation to adenosine [25, 26]. In mammalian tissues, the existence of presynaptic P2 receptors at neuromuscular junction was suggested by immunohistochemical analysis [27], and electrophysiologically, it was established that ATP but not adenosine inhibited nonquantal release of acetylcholine [5]. At frog neuromuscular junction, it was shown that ATP inhibited transmitter release via presynaptic P2 receptors [6], and it was proposed that ATP produces its effect via P2Y2-like receptors coupled to multiple intracellular cascades [28].

Temperature dependency of skeletal muscle contractility is a known phenomenon. It has been shown that this phenomenon has an endothermic nature, and raising the temperature increases the force and the strain of the myosin heads attached in the isometric contraction [29]. The decrease of contractile force at lower temperature could be due to the attenuation of metabolic enzyme activities [30, 31]

In contrast to adenosine, we have found that the effect of ATP on neuromuscular

transmission was temperature-dependent in functional experiments. Lowering the temperature caused the increase of ATP-induced inhibition of electrical field stimulation-evoked contractions, and this effect was highly sensitive to P2 receptor antagonist PPADS and not sensitive to 8-SPT, a P1 antagonist. These differences between two purines are thought to be coupled at their action mechanism. Both ATP and adenosine reduce quantal release of acetylcholine [6], thereby decreasing amplitude of postsynaptic end-plate currents. However, the temperature-mediated effect of ATP is more prominent and can achieve corresponding to amplitude of

To find the nature of receptors involved, we used PPADS, a P2 receptor antagonist with a preferential effect on P2X receptors in functional whole tissue experiments [19, 20], and found that ATP-evoked inhibition of muscle contraction was highly sensitive to this antagonist, while in electrophysiological study, it failed to affect responses to ATP. However, another nonselective P2 receptor antagonist suramin [15] significantly reduced ATP-induced inhibition. Although both PPADS and suramin are considered as nonselective P2 receptor antagonists, it has been shown that suramin, compared to PPADS, has a more broad P2 receptor antagonist activity, affecting most of P2X and P2Y receptor subtypes [34]. For instance, it has been shown that recombinant P2Y2 receptors are sensitive to antagonistic effect of suramin but not of PPADS [35]. In addition, in organ bath pharmacological experiments, PPADS tends to antagonize mostly P2X receptor subtypes [16, 19, 20], blocking P2Y receptor-mediated processes only at higher concentrations [22]. Neither PPADS nor suramin affects inhibition caused by adenosine. These results support the view that ATP inhibited the electrical field stimulation-evoked contractions of frog skeletal muscle by acting on presynaptic P2 receptors. It is most likely that these receptors belong to P2Y family, but involving some subtypes of P2X

It has been proposed that purine nucleotides and nucleosides were among the first neurotransmitters in the evolution and development of the living cells [36, 37]. Thus, it is possible that, in phylogenetically older animals, in which organism is functioning in low-temperature conditions, the transmitter role of purine nucleosides and nucleotides in cell-to-cell communications is as important as well-known intracellular metabolic actions of purines (production of energy, involvement in synthesis of nucleic acids). Thus, we suggest that supersensitivity of P2 receptormediated responses at lower temperature, which we have demonstrated in mammal

or processes of energy production and transfer [32, 33].

end-plate current reduction of the muscle contraction.

receptors cannot be ruled out at present.

**14**

When we found that, similar to that in guinea pig smooth muscle tissues, P2 receptor-mediating processes in amphibian skeletal muscles are markedly more pronounced in low-temperature condition, we did the next study using rodent skeletal muscles, namely rat soleus muscle [38].

We registered carbachol- and electric field stimulation-induced contractions of rat soleus muscle in norm and in the presence of ATP under different temperature conditions—37 and 14°C. We found that with decreasing temperature, both the force and the time of contractions are increased. ATP inhibited the amplitude of contraction caused by indirect stimulation by an electric field; in this case, the combined pre- and postsynaptic modulation effect of this purine was observed. To separate these effects, we investigated the effect of ATP on carbachol-induced contraction. In this mode, ATP increased the contraction of the "slow" muscle. With a decrease in temperature, both pre- and postsynaptic effects of ATP are enhanced, but not equivalent. The increasing potentiating effect of ATP with the use of postsynaptic P2 receptors overlaps and masks an increased, but to a lesser extent, inhibitory presynaptic effect [38].

Maintaining the body temperature in certain range provides for warm-blooded animals the ability to move and perform motor activity in a wide range of temperature differences of the environment [39].

It is known that in the absence of significant fluctuations in the temperature of the internal organs of mammals, the peripheral parts of their body can experience significant changes in temperature, for example, up to 15°C decrease in humans [40, 41]. Thus, the peripheral skeletal muscles of warm-blooded animals retain the ability to contractile activity even with a significant decrease in their temperature.

According to modern ideas, the strength, speed of contraction, and relaxation of skeletal muscles of warm-blooded animals as a rule increase with increasing temperature [42, 43]. This was observed, for example, during physical exercises when the temperature of skeletal muscles on the periphery of the human body increased by several degrees Celsius [44–46].

At the same time, the above studies did not attach special importance to the types of skeletal muscle examined. It is known that several types of phase skeletal muscle fibers are distinguished from which "slow" and "fast" are distinguished in all classifications [47, 48]. It is understandable that these muscles differing in their very function—maintaining pose ("slow" muscles) and performing subtle movements ("fast")—are made to react differently to temperature changes which is observed in practice [49–51].

Molecular non-quantum secretion of the mediator still has not given a fundamental importance due to the lack of a generalized action. Indeed, despite its large value, non-quantum secretion only depolarizes the end-plate region by ~5 mV which can be determined by hyperpolarization in the presence of postsynaptic receptor blockers—the "H effect" [52–54]. However, non-quantum secretion is extremely important and is crucial for the functioning of the synapse. It should be noted that the evaluation of non-quantum secretion of the myoneural synapse in the coldblooded is difficult because of the small value of the registered H effect [54, 55].

The temperature dependence of the magnitude of non-quantum secretion in neuromuscular preparations of rodents is complex. It was found that in the range from 10 to 35°C, the size of non-quantum secretion has two relative peaks at 20 and 35°C and two minima at 25 and 10°C (in the latter case, the H effect is not expressed at all) [54–56].

The frequency of spontaneous single-quantum responses in synapses of warmblooded animals increases as the temperature increases [56–58] without changing the amplitude of these responses [59]. Thus, two processes of acetylcholine release, namely quantum and non-quantum, have a different temperature dependence, which indicates the presence of independent mechanisms [55, 58].

The analysis of the published data leads to an unequivocal conclusion about the rise of the synaptic delay of postsynaptic responses with decreasing temperature on rodent preparations [60, 61]—the same as in cold-blooded.

It is known that in the neuromuscular synaptic cleft, there is an acetylcholinesterase which rapidly cleaves the neurotransmitter acetylcholine [62]. It was shown that when the temperature of the rat diaphragm preparation was reduced from 37 to 17°C, the activity of acetylcholinesterase decreased by 34% [63]. A similar pattern was observed in experiments with the preparation of the frog sartorius muscle which led to a suggestion that a decrease in the activity of acetylcholinesterase is responsible for an increase of the time course of the end-plate current at hypothermia [64].

To study the state of postsynaptic cholinergic receptors, cholinomimetics (primarily the slowly decaying cholinomimetic agent—carbachol) and cholinolytics are used. In experiments with slow muscle preparations such as rat m. soleus, the amplitude of the miniature potentials of the terminal plate did not change after application of carbachol at a temperature range from 18 to 38°C. On the other hand, at temperatures 37–38°C, there was a 40% decrease in the incidence of spontaneous postsynaptic responses in the presence of this cholinomimetic (in which combination indicates presynaptic nature of the effect) [65].

There are several studies on the temperature dependence of the contractile apparatus of "slow" skeletal muscles with conflicting data on the muscles of the same animals [66–70]. Thus, according to some sources, the temperature dependence of the "slow" muscle fibers of the rat is much more pronounced than the "fast" ones [42, 71–79], while others found that the temperature sensitivity of the myosin of the "slow" muscle fibers of the rat does not differ from the "fast ones" [80, 81].

In experiments on demembranized muscle fibers, where the temperature effects of electromechanical coupling are not relevant and only the modulation of the mechanical function itself plays a role, it was clarified that the dependence of the reduction force of "slow" and "fast" fibers on temperature is similar [80]. With an increase of temperature from 10 to 35°C, the force of contraction of "slow" fibers increased threefold and the "fast" ones by three and a half. The situation is different with the rate of contraction. Thus, the rate of reduction of "fast" fibers increased with increasing temperature from 10 to 35°C, while for "slow" fibers this parameter changed insignificantly [80].

We found that as the temperature is lowered, the force of contractions of the slow skeletal muscle of the rat increases [38], while the fast one decreases. With carbachol-induced contraction (when only receptors of the postsynaptic membrane are stimulated), as the temperature decreases, the amplitude of contractions of the slow muscle of the rat also increases [38].

It is known that a decrease in Ca2+ concentration, which provides exocytosis of the neurotransmitter quanta [82–84], reduces the strength of the contraction of the skeletal muscle over time—in contrast to the rapid effect on cardiomyocytes [85]. This action is temperature-dependent; in rat fast muscle fibers, the contraction force increased with increasing concentration of calcium ions; the lower the temperature, the more is the effect. A rat slow muscle did not produce similar effect [86].

**17**

**Author details**

**Acknowledgements**

**Conflict of interest**

provided the original work is properly cited.

Ayrat U. Ziganshin\* and Sergey N. Grishin Kazan State Medical University, Kazan, Russia

grant nos. 16-04-00101 and 18-44-160009.

\*Address all correspondence to: auziganshin@gmail.com

The authors declare the absence of conflict of interest.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Temperature-Dependent Effects of ATP on Smooth and Skeletal Muscles*

That is, the reason for the phenomenon being discussed is synaptic.

of changes in muscle responses with decreasing temperature.

effects of certain drugs might be altered by this nonphysiologic state.

Despite a large number of studies, the direct mechanisms of temperature effects on the functioning of the muscle remain unsolved [87, 88]. Moreover, if the muscle is stimulated directly, then the temperature change no longer has such an effect.

It was believed that changes in motor units at low temperature are due to deple-

In conclusion, we believe that studying the effects of hypothermic conditions has not only theoretical significance but also potentially important clinical implications since hypothermia is widely used in clinical practice for cerebral protection during surgical interventions or resuscitation of critically ill patients [92–96]. This underlines the importance of studying the reaction of other organs and tissues to hypothermia and especially the effect that low temperatures have on receptor-based interactions. Such studies add important information regarding the activity of P2 receptors under hypothermic conditions in mammalian muscles. Although these results cannot be directly transferred to human muscle tissues, they provide important insight into how activation of human P2 receptors might behave under hypothermia and predict how

This study was partly supported by the Russian Foundation for Basic Research

tion of the activity of metabolic enzyme systems [30, 31] or energy synthesis and transfer processes [89, 90]. However, it was clear that the contribution of the temperature sensitivity of the muscle biochemical processes cannot justify such dramatic changes in the nature of the contraction of the whole muscular organ with a change in temperature [91]. We suggest that the temperature-sensitive tonic effects of endogenous ATP during the contraction can underlie the phenomenology

*DOI: http://dx.doi.org/10.5772/intechopen.80794*

**5. Conclusion**
