**3. Frog skeletal muscle**

*Adenosine Triphosphate in Health and Disease*

**2. Guinea pig smooth muscle tissues**

eliminated at a temperature of 27°C [12].

and the maximum effect was registered at around 20°C [13].

laboratories.

significantly affected by changing the temperature conditions. In this chapter, we will review our earlier and recent studies as well as those findings done in other

Our first publication on temperature dependency of the P2 receptor-mediated processes was about guinea pig smooth muscle tissue [7]. We registered responses of isolated guinea pig urinary bladder and vas deferens (P2X receptors) and taenia caeci (P2Y receptors) at the three temperature conditions of 30, 37, and 42°C. We found that the contractile responses of both urinary bladder and vas deferens to a P2X receptor agonist α,β-methylene ATP (α,β-meATP) and to electrical field stimulation in the presence of atropine and phentolamine were markedly more prominent at a temperature of 30°C than at 37 or 42°C. Similarly, relaxation of carbachol-precontracted taenia caeci caused by electrical field stimulation temperature dependently increased with decrease of temperature, while relaxation of this tissue by exogenous ATP was not affected by the temperature. A P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) at all three temperature conditions concentration-dependently antagonized contractile responses to α,β-meATP and electrical field stimulation in both urinary bladder and vas deferens. PPADS, even at the highest concentration tested, had no effect on the relaxant responses of the taenia caeci either to electrical field stimulation or ATP, and its action was not affected by the change of temperature. It was concluded that the effectiveness of P2 receptor-mediated responses in guinea pig urinary bladder,

vas deferens, and taenia caeci increases by the decrease of temperature.

Temperature dependency for some receptor-mediated responses has been tested earlier on several animal and human tissues. Using guinea pig ileum and trachea and rat vas deferens and atria preparations, hypothermia-induced supersensitivity to adenosine has been established for responses mediated via adenosine A1, but not adenosine A2, receptors [8]. It has been shown that in the rabbit central ear artery, but not femoral artery, cooling to 24°C reduces contraction, increases the relaxation caused by histamine [9], and enhances the relaxation caused by cholinoceptor stimulation [10]. Later in the study from the same laboratory, it was shown that in rabbit central ear artery at 30°C α1-adrenoceptor-mediated response is reduced and the P2 receptor-mediated component becomes more prominent [11]. On the other hand, it was found that the release of ATP from rabbit pulmonary artery induced by methoxamine, an α1-adrenoceptor agonist, being observed at 37°C, was completely

The increase of bladder contractility at low temperature might be due to activation of cold receptors in the bladder, the presence of which has been shown both in animal and human urinary bladder [13, 14]. However, it is unlikely that cold receptors are involved in the effects which we registered in the present study since the threshold temperature to stimulate these receptors was found to be less than 30°C,

It is generally accepted that in the presence of adreno- and cholinoceptor blockers, the contractions of guinea pig vas deferens and urinary bladder are mediated by P2X receptors, while in guinea pig taenia caeci, low-frequency electrical field stimulation evokes the relaxation via P2Y receptors [15–17]. We have found that both P2X and P2Y receptor-mediated responses elicited by electrical field stimulation are increased at low temperature. It could be suggested that this effect occurs due to the decrease of activity of the transmitter-metabolizing enzymes, namely, ecto-ATPase and ectonucleotidases during cooling, since it is generally accepted that

**12**

Next, we decided to test the P2 receptor-mediated effects in tissues of coldblooded animals. For that the contractile responses of isolated *Rana ridibunda* frog sartorius muscle contractions evoked by electrical field stimulation (EFS) were studied at three temperature conditions of 17, 22, and 27°C [23]. ATP concentration dependently inhibited the electrical field stimulation-evoked contractions of sartorius muscle at all three temperatures; this effect was significantly more prominent at a temperature of 17°C than at the other two temperatures. Adenosine also caused inhibition of electrical field stimulation-evoked contractions which was statistically identical at all three temperature conditions tested. A P2 receptor antagonist, PPADS, reduced the inhibitory effect of ATP at all three temperatures but did not affect inhibitory action of adenosine. In contrast, 8-(p-sulfophenyl)theophylline (8-SPT), a nonselective P1 receptor antagonist, abolished inhibitory effects of adenosine at all three temperature conditions but did not antagonize inhibition caused by ATP. In electrophysiological experiments, ATP and adenosine temperature dependently reduced end-plate currents recorded in sartorius neuromuscular junction by voltage clamp technique. The inhibitory effects of both agonists were enhanced with the decrease of temperature. 8-SPT abolished the inhibitory effect of adenosine but not ATP on end-plate currents. Suramin, a nonselective P2 receptor antagonist, inhibited the action of ATP but not adenosine, while PPADS had no influence on the effects of either ATP or adenosine. It was concluded from this study that the effectiveness of P2 receptor-mediated inhibition of frog skeletal muscle contraction in contrast to that of adenosine is dependent on the temperature conditions.

Thus, we had demonstrated that presynaptic P2 receptor-mediated inhibition of the frog skeletal muscle contractions produced by nerve stimulation has a clear temperature-dependent feature—lowering the temperature leads to the increase of 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] or processes of energy production and transfer [32, 33].

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 end-plate current reduction of the muscle contraction.

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 receptors cannot be ruled out at present.

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

**15**

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

could be a reflection of their past role in the early stage of evolution.

[7] and amphibian tissues [23], is a fundamental feature of these receptors which

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

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

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 tempera-

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

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

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

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

skeletal muscles, namely rat soleus muscle [38].

extent, inhibitory presynaptic effect [38].

ture differences of the environment [39].

by several degrees Celsius [44–46].

observed in practice [49–51].

**4. Rodent skeletal muscles**

[7] and amphibian tissues [23], is a fundamental feature of these receptors which could be a reflection of their past role in the early stage of evolution.
