**3. Adenosine receptors and pain**

#### **3.1 Adenosine receptors**

628 Pharmacology

Györgi, 1929). Later, in 1970 Burnstock presented evidence that ATP acted as a neurotransmitter in nonadrenergic noncholinergic (NANC) nerves supplying the gut, and finally, in 1972, the purinergic neurotransmission hypothesis was proposed (Burnstock, 1972). With these discoveries, the number of publications involving ATP and its metabolites

Fig. 1. Total of publications with keywords ATP, Adenosine (Ado), Adenosine Receptors

Afterwards, it was established that the ATP acted as a cotransmitter with classical transmitters in both the peripheral nervous system and in the central and that purines are also powerful extracellular messengers to non-neuronal cells (Burnstock & Knight, 2004). Burnstock, in 1978, provided the basis for the distinction of two classes of purinergic receptors; adenosine-sensitive P1 and ATP-sensitive P2 receptor classes. In 1985, Burnstock and Kennedy proposed a basis for distinguishing two types of P2 purinoceptors, namely, P2X and P2Y. Afterwards, in 1994 Abbracchio and Burnstock through studies of transduction mechanisms and cloning of both P2X and P2Y receptors put forward a new nomenclature system, naming them, P2X ionotropic ligand-gated ion channel receptors and P2Y metabotropic G protein-coupled receptors, respectively. Currently, seven subtypes of P2X receptors (P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, P2X7) and eight subtypes of P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14) are clearly established. P2X and P2Y receptor activation by ATP stimulates cellular excitability, augments the release of excitatory amino acids, and consequently initiates pain responses (Burnstock, 2007; Burnstock & Williams, 2000). In the context of pain neurotransmission, preclinical studies show us that activation of P1 receptors by adenosine decreases pain, inflammation, and cellular excitability (McGaraughty & Jarvis, 2006). During the 80's and 90's research evaluating

(Ado Rec) and Inosine (Ino) from 1930 until 2010. Source: Pubmed

purinergic system in pain rocketed (Figure 2).

grew quickly and continues to do so (Figure 1).

Adenosine is the natural ligand of P1 receptors, also called adenosine receptors. All these receptors are G-protein coupled and are divided according to pharmacological, biochemical and molecular properties into four subtypes: A1, A2A, A2B and A3. Each receptor has a distinct distribution and due to its special features, has distinct roles as well (Burnstock et al., 2011; Fredholm et al., 2011; Ralevic & Burnstock, 1998; Ribeiro et al., 2002; Sawynok & Liu, 2003). Adenosine receptors were cloned and characterized in several mammal species (Burnstock, 2008).

#### **3.2 Distribution of adenosine receptors**

Adenosine receptors are present in several species and in distinct tissues. However, their distribution is quite irregular and different among species and mainly among tissues (Fredholm et al., 2011). A1 receptor (A1R) is a ubiquitous receptor. In the central nervous system, it is distributed in the cerebellum, cerebral cortex, hippocampus, thalamus, spinal cord (substantia gelatinosa), brain stem, olfactory bulb and other central sites. Peripherally A1R distribution is less wide than centrally, but there is a considerable density of A1R in sensory afferent fibers, mainly on C-fibers which are responsible for receiving and conducting the painful stimuli (Dixon et al., 1996; Sawynok, 2009). A2A receptor (A2AR)has an even distribution between central and peripheral nervous system, but mainly in central structures as nucleus accumbens, putamen, caudate and in immune tissues, vascular smooth muscle, endothelium, platelets and sensory neurons (Dixon et al., 1996; Fredholm, 1995; Ralevic & Burnstock, 1998; Sawynok, 2009). A2B receptor (A2BR) is also a ubiquitous receptor and it has been found either in many central or peripheral tissues. However, A2BR density is

The Involvement of Purinergic System in Pain:

by cAMP production enhancement (Table 1).

Sawynok and Liu, 2003; Jacobson and Guao, 2006.

**3.3.3 A2B receptor signaling** 

2006; Peakman and Hill, 1994).

**3.3.4 A3 receptor signaling** 

**3.3.2 A2A receptor signaling** 

cell protection (Boison et al., 2010; Shulte & Fredholm, 2003).

Adenosine Receptors and Inosine as Pharmacological Tools in Future Treatments 631

(PKC), phospholipase D (PLD), phospholipase A2 (PLA2) and others. Furthermore, adenosine or adenosine agonists can activate K+ channels (Jacobson & Gao, 2006; Megson et al., 1995; Ralevic & Burnstock, 1998). A1R activation can also activate PI3K and MAPK pathways, more specifically ERK1/2 and MEK inducing gene expression changes and glial

A2AR is coupled to Gs (the most part) and Golf protein (mainly in striatum). The main intracellular event after activation of these proteins is adenylate cyclase activation followed

Table 1. Adenosine receptors signaling. Adapted from Ralevic and Burnstock, 1998;

The increasing of cAMP stimulates cAMP-dependent kinase (PKA). Thus, PKA becomes able to activate several pathways through PKC, calcium channels, potassium channels, cAMP responsive element-binding (CREB), MAPK, PLC activation (Burnstock, 2007; Cunha et al., 2008; Fredholm et al., 2003, 2007; Jacobson & Gao, 2006; Ralevic & Burnstock, 1998).

Through Gs and Gq activation A2B receptor induces adenylate cyclase and PLC. In humans, A2BR can increase intracellular calcium by IP3 activation. Moreover, the arachidonic acid pathway is also involved in A2BR signaling (Feoktistov & Biaggioni, 2011; Jacobson & Gao,

A3R like A1R is coupled to Gi/0 and also to Gq/11 protein. Its main signaling transduction is the inhibition of adenylate cyclase and stimulation of PLC, IP3, DAG, PKC and PLD. Also, like other adenosine receptors, A3R activates MAPK pathway, mainly ERK1/2 (Abbracchio

et al., 1995; Armstrong & Ganote, 1994; Palmer et al., 1995; Shneyvays et al., 2004).

very low and it has been found in great density only in bowel and bladder. A3 receptors (A3R)are widely distributed in several mammals, however, few studies have indicated specific roles for this receptor (Dixon et al., 1996; Ralevic & Burnstock, 1998; Salvatore et al., 1993).

#### **3.3 General adenosine receptor signaling**

All adenosine receptors are coupled to G-protein. Nevertheless there are many kinds of Gprotein and each one may activate a distinct pathway. Thus, the four adenosine receptors can stimulate or inhibit several pathways and consequently exert many physiological actions (Jacobson & Gao, 2006; Ralevic & Burnstock, 1998). We show below the main signaling characteristics of each adenosine receptor.

#### **3.3.1 A1 receptor signaling**

A1R is coupled to Gi/0 protein family which is pertussis toxin-sensitive. Most of the biological effects induced by A1R activation are due to inhibition of cAMP second messenger (Burnstock, 2007; Jacobson & Gao, 2006; Ralevic & Burnstock, 1998; Sawynok, 1998) (Figure 3 and Table 1).

Fig. 3. Adenosine A1 receptors and its main pathways. A1R, adenosine A1 receptor; AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate; DAG, diacylglicerol; ERK1/2, extracellular signal-regulated kinases 1 and 2; IP3, inositol triphosphate; K+, potassium channels; MAPK, mitogen-activated protein kinase; MEK, MAPK and ERK kinase; PKA, protein kinase; PLC, phospholipase C.

Beta and gamma subunities of A1R, when activated stimulate phospholipase C (PLC). Activation of PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) leading to increased levels Ca+2. Moreover, the enhancement of intracellular calcium can induce some enzymes such as protein kinase C (PKC), phospholipase D (PLD), phospholipase A2 (PLA2) and others. Furthermore, adenosine or adenosine agonists can activate K+ channels (Jacobson & Gao, 2006; Megson et al., 1995; Ralevic & Burnstock, 1998). A1R activation can also activate PI3K and MAPK pathways, more specifically ERK1/2 and MEK inducing gene expression changes and glial cell protection (Boison et al., 2010; Shulte & Fredholm, 2003).
