**1. Introduction**

It is widely accepted now that ATP, except well-known role as an intracellular source of energy, can regulate many important cell functions acting via specific extracellular receptors, namely P2 receptors [1]. P2 receptors are divided into two families, P2X and P2Y receptors, P2X receptors being a ligand-gated ion channel, while P2Y receptors are G protein-coupled. Seven subtypes of P2X and eight subtypes of P2Y receptors are well identified and put into current classification of receptors [2, 3].

P2X and P2Y receptors are widely distributed in animal and human tissues including smooth and skeletal muscles. In smooth muscles, stimulation of P2X receptors causes contractile responses, while stimulation of P2Y receptors usually leads to relaxant effects [4]. In contrast, in adult skeletal muscles, it has been established that, while stimulation of P2 receptors does not cause either contraction or relaxation, it significantly inhibits transmitter release at the neuromuscular junction [5, 6].

Although most experiments on P2 receptors were carried out on normal temperature conditions, we have shown in our publications that in several animal smooth and skeletal muscles, the responses mediated by both P2X and P2Y receptors are

**10**

*Adenosine Triphosphate in Health and Disease*

[1] Mózsik G, Szabo IL. Membranebound ATP-dependent Energy Systems and the Gastrointestinal Mucosal Damage and Prevention. Rijeka: InTech Open Acces/Open Minds Publishers; 2016. pp. 1-356. DOI: 105772/60095.

[2] Mózsik G, Szabo IL, Czimmer J. Membrane-bound ATP-dependent

gastrointestinal functions and their regulations in the gastrointestinal mucosa. Current Pharmaceutical

International Conferences on Ulcer Research (ICUR). Hungary: University

[4] Mózsik G, Szabó S, Takeuchi J. History of the Gastrointestinal Section of the International Union of Pharmacology (IUPHAR GI Section). Hungary: University Press of Pécs; 2006.

energy system, as extra- and intracellular key signals for

[3] Mózsik G. History of the

Press of Pécs; 2006. pp. 1-228

Design. 2017;**23**:1-31

pp. 1-164

ISBN: 978-953-51-2251-7

**References**

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 laboratories.

## **2. Guinea pig smooth muscle tissues**

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

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

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

**13**

conditions.

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

ecto-ATPase activity is temperature-dependent, with the optimum temperature of 37°C for warm-blood temperature animals [18]. However, this cannot explain results with the enzymatically stable P2X receptor agonist α,β-meATP, the effects of which are not affected by ecto-ATPases. Moreover, in taenia caeci when we used ATP, which is readily degraded by ecto-ATPases, we did not find any temperature dependency in agonist activity. Thus, it seems that supersensitivity of P2 receptors at a low temperature is a feature of receptor itself and is not dependent on ecto-

It was believed initially that PPADS was a selective P2X receptor antagonist [19, 20] although later antagonism of recombinant P2Y receptors by PPADS was reported [21]. In our earlier study, we established that in the guinea pig taenia caeci, substantial antagonism against P2Y receptor-mediated relaxation was obtained only at a concentration of 100 μM of PPADS [22]. Similarly, in these experiments we did not find any antagonism at P2Y receptors of PPADS at concentrations up to 30 μM on taenia caeci. Thus, it supports the view that at least in the pharmacological organ

bath experiments, PPADS shows relatively good selectivity to P2X receptors. It was an interesting finding that in taenia caeci responses to electrical field stimulation were clearly temperature-dependent, while the relaxation caused by exogenous ATP was statistically identical at different temperature conditions. Since it has been clearly shown that ATP is a transmitter which is released during electrical field stimulation of guinea pig taenia caeci to act on P2Y receptors, it seems that in this tissue only prejunctional mechanisms of transduction are sensitive to the

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

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

shifts of the temperature while postjunctional processes are not.

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

ATPase activity.

**3. Frog skeletal muscle**

#### *Temperature-Dependent Effects of ATP on Smooth and Skeletal Muscles DOI: http://dx.doi.org/10.5772/intechopen.80794*

ecto-ATPase activity is temperature-dependent, with the optimum temperature of 37°C for warm-blood temperature animals [18]. However, this cannot explain results with the enzymatically stable P2X receptor agonist α,β-meATP, the effects of which are not affected by ecto-ATPases. Moreover, in taenia caeci when we used ATP, which is readily degraded by ecto-ATPases, we did not find any temperature dependency in agonist activity. Thus, it seems that supersensitivity of P2 receptors at a low temperature is a feature of receptor itself and is not dependent on ecto-ATPase activity.

It was believed initially that PPADS was a selective P2X receptor antagonist [19, 20] although later antagonism of recombinant P2Y receptors by PPADS was reported [21]. In our earlier study, we established that in the guinea pig taenia caeci, substantial antagonism against P2Y receptor-mediated relaxation was obtained only at a concentration of 100 μM of PPADS [22]. Similarly, in these experiments we did not find any antagonism at P2Y receptors of PPADS at concentrations up to 30 μM on taenia caeci. Thus, it supports the view that at least in the pharmacological organ bath experiments, PPADS shows relatively good selectivity to P2X receptors.

It was an interesting finding that in taenia caeci responses to electrical field stimulation were clearly temperature-dependent, while the relaxation caused by exogenous ATP was statistically identical at different temperature conditions. Since it has been clearly shown that ATP is a transmitter which is released during electrical field stimulation of guinea pig taenia caeci to act on P2Y receptors, it seems that in this tissue only prejunctional mechanisms of transduction are sensitive to the shifts of the temperature while postjunctional processes are not.
