**2. PAR-2 activation in the gastrointestinal tract**

PAR-2 is activated through proteolytic cleavage by specific serine proteases, such as trypsin and mast cell (MC)-tryptase [4] and lysosomal macrophagic cysteine protease cathepsin-S [5, 6]. PAR-2 is generally expressed in the basolateral and apical side of epithelial cells [20], fibroblasts, MCs, smooth muscle cells, endothelial cells of the GI tract [21], enteric sensory neurons, terminals of mesenteric afferent nerves, and immune cells [17]. The higher number of mast cells and mast cell tryptase in biopsied colonic tissues enhanced the PAR-2 activity to regulate CGRP, SP, and VIP expressions resulting in symptoms associated with IBD [22]. Recently, Hassler et al. [23] suggested that PAR2-expressed sensory neurons are a key target for mechanical and spontaneous pain triggered by the release of endogenous proteases from the many immune cells. In-vitro study exhibits the up-regulation of PAR-2 expression in cultured endothelial cells of human umbilical vein treated with TNF-α, IL-1α, and bacterial lipopolysaccharide in a dose-dependent manner [24]. Therefore, it is important to note that PAR-2 activation on intestinal immunocytes induces acute enteritis [9, 25] while its neuronal expression incites neurogenic inflammation [26, 27].

#### **2.1 Role of PAR-2 in inflammation**

PAR-2 seems essential in the interplay between nerves, immunocytes, MCs, and epithelial cells within the luminal wall during GI diseases [17]. Histopathologically, PAR-2-agonists (SLIGRL) induced acute colitis has been observed with erythema,

*Intervention of PAR-2 Mediated CGRP in Animal Model of Visceral Hyperalgesia DOI: http://dx.doi.org/10.5772/intechopen.106859*

granulocyte infiltrations and thickened colonic wall [25, 28], the colonic tissue sampled from the PAR-2 knockout mice that are infused intracolonically with 2,4,6-trinitrobenzene sulfonic acid (TNBS) showed lower myeloperoxidase activities, microscopic- and macroscopic-damage scores [29]. Mediators such as intracellularand vascular cell adhesion-molecule-1 were decreased while cyclooxygenase-1 was increased in the PAR-2 knockout mice, which clearly confirms the pro-inflammatory role of PAR-2. Notably, PAR-2, inactive during colitis, has been expressed for inducing VH after resolution of colitis [30]. Furthermore, PAR-2 has also been overexpressed in biopsies obtained from ulcerative colitis (UC) and CD patients, which strongly suggests its intricate role in IBDs [31–33].

#### **2.2 Effects of PAR-2 on gastrointestinal functions**

PAR-2 modulates GI functions, such as motility, ionic exchange, paracellular permeability, sensory functions, and inflammation [34]. The excitatory, as well as inhibitory actions of PAR-2-agonists on isolated smooth muscles, have been devised earlier [35, 36]. In-vitro, PAR-2 activation shows a region-specific role because it enhances the contractibility of gastric smooth muscles and reduces the contractility of circular and longitudinal colonic smooth muscles in mice [35, 37]. However, the intraperitoneal administration of PAR-2-agonists accelerated GI transit in mice [38]. Moreover, Mall et al. (2002) reported that PAR-2 activation on the enterocytes triggers intestinal water secretion through a direct cellular mechanism, while Kong et al. [20] described the same by a prostaglandin E2-dependent mechanism. Additionally, activated PAR-2 stimulates mucus secretion by a nerve-mediated mechanism [39]. It weakens the intestinal barrier, resulting in an increased passage of fluids or even microorganisms across the gut mucosa. The intracolonic administration of PAR-2 agonist in mice increases colonic permeability and results in a general inflammatory response [25, 34].

### **3. CGRP-receptors and their distribution**

CGRP-receptor is a heterotrimeric complex, composed of calcitonin receptor-like receptor (CLR), receptor activity-modifying protein-1, and a small intracellular protein component and receptor component protein. CLR, a classical G-protein linked receptor, couples through adenylyl cyclase [40]. CGRP is expressed throughout the peripheral and central nervous systems (CNS). Of the two forms, α-CGRP is mainly expressed in the CNS, especially in striatum, amygdalae, hypothalamus, colliculi, brainstem, cerebellum, and trigeminal complex [41–43], while β-CGRP is primarily expressed in the enteric neurons and vascular smooth muscle cells [44, 45]. Interestingly, α-CGRP is also found to be expressed in primary spinal afferent C- and Aδ-fibers [46].

The majority of spinal afferents innervated into the GI tract express CGRP and SP [47]. CGRP has been reported to be expressed markedly higher in the lumbosacral DRG and spinal cord dorsal horn (SCDH) during visceral inflammation [11, 48]. Zhang et al. [49] confirmed the absence of secondary hyperalgesia in the mice missing α-CGRP expression in the CNS. The SP and CGRP released from afferent terminals lead to neurogenic inflammation at the peripheral sites, resulting in MCs degranulation, plasma extravasation, and arteriolar vasodilation [50]. CGRP causes vasodilatation via its receptors on the smooth muscle cells at peripheral synapses. However, at

central synapses, it acts postsynaptically on the second-order neurons to transmit pain via the brainstem and midbrain to higher cortical pain regions [51].

#### **3.1 CGRP modulates mast cell functions**

CGRP is secreted from non-myelinated C-fibers and thinly myelinated Aδ-fibers originating from DRG neurons [52]. Sun et al. [53] showed peak CGRP levels in the colonic tissues, spinal cord, and hypothalamus of rats with IBS, and its correlation with VH. Our earlier studies also demonstrated the remarkably higher CGRP expression in DRG and spinal cord that was correlated with VH in the TNBSinduced ileitis rats and goats, respectively [13, 15]. Therefore, CGRP and CGRPreceptors are found to be involved in the transmission and modulation of pain in the periphery and CNS [54, 55].

MCs that reside near the nerve fibers are true candidates for modulating neural activity and nociception [56]. The mediators such as SP, CGRP, vasoactive intestinal protein (VIP), dopamine, and arachidonic acid are able to influence MCs activation. The aforementioned mediators act on nociceptors, send signals to the CNS, and cause the simultaneous central release of SP and CGRP [57], which further activate MCs, and create a bidirectional positive feedback-loop for resultant neurogenic inflammation [58].

#### **3.2 CGRP-release mediated by PAR-2**

Activated PAR-2 sensitizes Transient Receptor Potential Vanilloid subtype-1 receptors (TRPV-1) and triggers the release of sensory CGRP and SP [59]. CGRP and SP released from intestinal afferent terminals cause vascular dilatation, plasma extravasation, granulocyte infiltrations, and neurogenic inflammation [8, 9, 60]. An earlier study [8] reported that PAR-2-agonists-induced edema was entirely mediated by the release of SP and CGRP from sensory neurons and further activation of neurokinin-1 (NK-1)- and CGRP-receptors on endothelial cells. In DRG, PAR-2 co-expresses with TRPV-1, TRPV-4, TRPA-1 (Transient Receptor Potential Cation Channel, Subfamily-A, Member-1), SP and CGRP [8, 61, 62]. It is also reported that 63% of sensory neurons express PAR-2 and up to 40% of them express both SP and CGRP [8]. Activated PAR-2 transmits C-fiber afferent input to the SCDH for the release of excitatory amino acids and neuropeptides from the central terminals [63].

#### **3.3 Role of CGRP in sensitization**

Afferent fibers innervating the gut vessels have cell bodies in the DRG. These fibers are peptidergic, containing both CGRP and SP, and have collaterals in enteric ganglia, mucosa, muscularis externa, and sympathetic prevertebral ganglia [64]. SP, CGRP, VIP, and somatostatin act as mediators of neurogenic inflammation in IBDs [65–67]. After stimulation, TRPV-1 depolarizes sensory neurons either directly or indirectly to initiate the release of these neuropeptides from the afferent terminals [68]. TRPV-1-positive nerve fibers co-express with SP, NK-1, and CGRP in mucosa, submucosal layer, deep muscular plexus, circular muscle, myenteric plexus, and longitudinal muscle layer in the rectum and colon of mice [69]. CGRP which is expressed largely in splanchnic afferents and CGRP-immunoreactivities from the GI tract disappears with capsaicin treatment [70]. Interestingly, about 50% of CGRP-immunoreactive extrinsic afferent neurons express SP- or NK-1-immunoreactivities [71] and their expressions fluctuate during

#### *Intervention of PAR-2 Mediated CGRP in Animal Model of Visceral Hyperalgesia DOI: http://dx.doi.org/10.5772/intechopen.106859*

colitis [72]. The earlier decrease of the above neuropeptides may be due to their depletion from the peripheral nerve terminals or the damaged nerves at the initial inflammatory stage. CGRP and SP increase during inflammation or afferent nerve stimulation. TNBS-induced colitis/ileitis and or colorectal distension (CRD) results in higher expression of neural activation markers (such as c-Fos, pERK) as well as releases of SP and CGRP in the SCDH that are commonly linked with pain signaling [15, 73, 74].

Plourde et al. [75] confirmed the role of CGRP in pain modulation because intravenously administered CGRP-1-receptor-antagonists (h-CGRP8-37) reversed the sensitization provoked by infusion of intracolonic acetic acid. SP and CGRP may either increase the peripheral sensory gain of extrinsic afferents within the gut or contribute to primary afferent transmission within the CNS [16, 76]. Despite irritation, immune challenge and inflammation cause the release of CGRP and SP from extrinsic afferents and intrinsic neurons within the gut [45, 77], the precise site at which CGRP-receptor and NK-1 mediate visceral pain is not known.
