The influence of Prostaglandin E2 on bradykinin induced depolarization, generation and conduction of action potentials in the isolated vagus nerve

Dr Charles Murimi Mugera MSc Pharmacology 
The influence of PGE on bradykinin-induced depolarisation, generation and 
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conduction of action potentials in the isolated vagus nerve of the rat. 
Chapter 1.Introduction 
1.1. Inflammation and hyperalgesia 
Inflammation and tissue injury cause an enhanced response to noxious stimuli and a 
decrease of the pain threshold (Planells-Cases et al., 2005). Inflammatory mediators, 
released from the injured tissues are well known to produce hyperalgesia (Moriyama et 
al., 2005). Intradermal injection of some inflammatory mediators directly produces pain 
while others do not produce pain but potentiate the response to pain. We hypothesize that 
different inflammatory mediator’s act synergistically to produce the phenomenon of 
hyperalgesia. In our study we look at two of the most important inflammatory autacoids, 
bradykinin and PGE, and how their intracellular signalling pathways interact to induce 
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hyperalgesia. 
1.2. Bradykinin and hyperalgesia 
One of the most potent mediators of inflammation is the nonapeptide bradykinin (BK) 
which is generated in plasma and tissues following injury (Couture et al., 2001). 
Intradermal injection of bradykinin produces pain in animal models (Inoki et al., 1979) 
and in man (Meyer et al., 1992) . G-protein activation is an essential component of the 
bradykinin response, modulated by protein kinase C and lipoxygenase metabolites of 
arachidonic acid (McGuirk & Dolphin, 1992). 
BK exerts its biological effects through the activation of the bradykinin B2 receptor 
which belongs to the rhodopsin family of G protein-coupled receptors (Flavahan & 
Vanhoutte, 1990; Voyno-Yasenetskaya et al., 1989a; Voyno-Yasenetskaya et al., 1989b) 
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Dr Charles Murimi Mugera MSc Pharmacology 
The B2 receptor is ubiquitous and constitutively expressed. B1 receptor is a closely 
related receptor. It is activated by des –Arg (10)-kallidin or des-Arg (9)-BK, metabolites 
of kallidin and BK respectively. This receptor is expressed at a very low level in healthy 
tissues but is induced secondary to tissue injury and inflammation by various 
proinflammatory cytokines such as interleukin-1 beta and tumour necrosis factor alpha. 
Both B2 and B1 are coupled to the G alpha I and G alpha q G-protein subunits. 
Bradykinin activates phospholipase C epsilon and cyclooxygenase-1 through the 
bradykinin B receptor (Tang et al., 2004). 
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1.3. Bradykinin and TRPV1 
The TRPV 1 channel is thought to be pivotal in the BK induced hyperalgesia. The 
bradykinin B receptor sensitises vanilloid receptor 1 activity by facilitating both PKC 
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activity directly and PKA activity via PGE activity. Therefore, this suggests that 
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endogenous inflammatory autacoids produced by several enzymes may be capable of 
producing a synergistic response involving the TRPV1 channel. 
Bradykinin is thought to influence the TRPV1 channel in a number of ways. Firstly, G 
alpha q stimulates phospholipase C gamma causing hydrolysis of phosphatidylinositol-4, 
5-bisphosphate (PIP) and release of inositol trisphosphate (IP) and diacylglycerol 
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(DAG) (Venkatachalam et al., 2003). This hydrolysis of PIP to IP and DAG has been 
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 implicated in therelease of TRPV 1 from PIP-mediated inhibition (Chuang et al., 2001). 
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Diminution of plasma membrane phosphatidylinositol-4, 5-bisphosphate (PIP2) levels 
through antibody sequestration or PLC-mediated hydrolysis mimics the potentiating 
effects of bradykinin suggesting that PIP plays a role in inhibiting the TRPV1 channel 
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 (Chuang et al., 2001). The C-terminal domain of TRPV1 is thought to be the site required
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Dr Charles Murimi Mugera MSc Pharmacology 
for PIP-mediated inhibition of channel gating. Mutations thatweaken PIP-TRPV1 
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interaction increase spontaneous activity of the channel and therefore reduce thresholds 
for chemicalor thermal stimuli (Prescott & Julius, 2003). 
Secondly, DAG activates PKC epsilon. Activation of intracellular PKC activity results in 
increased TRPV1 sensitivity to capsaicin, low pH and heat (Di, V et al., 2002). Studies of 
cultured DRG cells have implicated PKC alpha as the isoforms most important for PKC 
effects on TRPV1 in vivo (Olah et al., 2002). PKC activation potentiates capsaicin-
induced activation of TRPV1 in a mechanism that involves direct phosphorylation of the 
TRPV1 protein on Serine residues 502 and 800 (Numazaki et al., 2003). 
Thirdly, BK also causes release of prostaglandins and this may be the key mechanism for 
BK-mediated hyperalgesia (Vasko et al., 1994). BK acts on G alpha i to inhibit adenylate 
cyclase and stimulate the mitogen-activated protein MAP kinase pathways. This is 
associated with increased expression of cytosolic phospholipase A . 
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BK increases PGE in a three-step process .The process involves the production of 
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prostaglandins from arachidonic acid (AA) by the combined actions of phospholipase A 
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(PLA), cyclooxygenase (COX) and specific terminal prostaglandin synthases. PLA 
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mobilises AA from membrane phospholipids . AA is then converted to PGH by COX. 
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PGH is converted to PGE by prostaglandin E synthase (PGES). 
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 1.4. Prostaglandins and Hyperalgesia 
Prostanoids, the cyclooxygenase metabolites of arachidonic acid, are synthesised and 
released upon cellstimulation and act on cells in the vicinity of their synthesisto exert 
their actions. Prostanoids include PGD, PGE, PGF, PGI,and thromboxne A. 
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Dr Charles Murimi Mugera MSc Pharmacology 
Prostanoids, in particular prostaglandin I (PGI) and prostaglandin E (PGE), are 
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generated in response to tissue injury or inflammation (Bombardieri et al., 1981). 
Prostanoids, unlike bradykinin, do not produce pain but potentiate the response to pain. 
PGE has a potentiating effect on the sensitivity of vagal C fiber activity in response to 
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chemical or mechanical stimulation (Lee & Morton, 1995; Ho et al., 2000). Analgesic 
effects of non-steroidal anti-inflammatory drugs (NSAIDs) are attributed predominantly 
to inhibition of prostaglandin synthesis. Therefore, this implies that there is huge scope 
for development of far more potent analgesics by furthering our understanding of the 
interaction between the mediators that produce pain, such as bradykinin, and the 
mediators that potentiate the pain such as PGE. 
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 Receptors mediating the actions of prostanoidshave been identified and cloned from 
human tissues (Pierce et al., 1995; Ushikubi et al., 1995) . They are G protein-coupled
rhodopsin-type receptor with seven transmembrane domains. There are eight types of 
prostanoid receptors TP, IP, EP, EP, EP, EP FP, and DP. TP and IP are coupled to the 
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G alpha s and G alpha q respectively. EP1 acts by an unknown mechanism to cause an 
2+
increase in Ca, EP2 and EP4 are coupled to G alpha s and cause increased activity of 
adenylate cyclase. EP 3 is predominantly coupled to G alpha s or G alpha I (Narumiya et 
al., 1999). The different types of prostanoids receptors and their second messenger 
systems have been summarised on Table 1.1. It has been suggested that prostanoids 
contribute to the development of pain mainly through EP1 or IP receptors (Minami et al., 
2001; Stock et al., 2001; Moriyama et al., 2005). 
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Dr Charles Murimi Mugera MSc Pharmacology 
Prostaglandins produce excitatory effects on sensory neurones by several different 
known mechanisms. Firstly, by reducing the activation threshold of a tetrodotoxin-
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resistant voltage-gated Na current (TTX-R I). TTX-R I appears to be the current 
NaNa
 primarily responsible for the action potentialgeneration in the cell body and terminals of 
nociceptive afferent sensory neurons. Its significance, however, on the nerve a trunk of 
afferent sensory neurons is not clear. Therefore , PGE reduces the activation threshold 
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+
of this tetrodotoxin-insensitive voltage-gated Na channels (England et al., 1996; Gold et 
al., 1996a; Gold et al., 1996b; Gold et al., 1998) thus activating them at more negative 
potentials.(Sangameswaran et al., 1996; Akopian et al., 1996). Evidence supporting a 
role for TTX- R I currents in the sensitisation of primary afferent neurons and 
Na
inflammatory hyperalgesia is derived from studies on the distribution, biophysical 
properties of the channel currents and the effect of anti nociceptive agents. Studies of the 
distribution of TTX-R I among primary afferent neurons and other tissues of the body 
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suggest that these currents are expressed only in a subpopulation of primary afferent 
neurons that are likely to be involved in nociception. In situ hybridisation , in neonatal 
and adult dorsal root and trigeminal ganglia, shows that the channel is expressed only by 
small-diameter sensory neurons (Akopian et al., 1996) . In addition studies of the 
electrophysiological and pharmacological properties of the channels show these channels 
to be ideally suited to mediate the repetitive discharge associated with prolonged 
membrane depolarisations. Finally, there is evidence that modulation of these currents, 
with inflammatory agents and anti-nociceptive agents, is an underlying mechanism of 
primary afferent neuron sensitisation. (Chabal et al., 1992; Gold et al., 1996; Gold, 
1999). TTX-R I is modulated by both PKA and PKC, however, PKC activity is 
Na
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