The development of novel analgesics would be facilitated if the mechanisms
underlying nociception and inflammatory pain were fully understood. Adenosine
5'triphosphate (ATP) and adenosine can cause pain in humans when applied to a
blister base, but the algogenic mechanism of action is still unclear. Cells contain
millimolar concentrations of ATP, which is released into the extracellular space
when the cells are damaged, and is subsequently metabolised to adenosine.
Consequently, levels of the purines are increased in damaged, inflamed or ischemic
tissues and this makes them ideal candidates to signal the presence of tissue injury. It
is thought that ATP and adenosine might be involved in the initiation of pain by
directly or indirectly activating distinct subtypes of P2 or P 1 receptors respectively.
In this thesis, behavioural, electrophysiological, and immunohistochemical
techniques were used to test the hypothesis that ATP and adenosine are involved in
the initiation of pain by directly and /or indirectly activating nociceptors innervating
the cornea and the knee joint in vivo.
ATP and ATP analogues were administered to the normal cat cornea and the
normal rat knee joint under pentobarbitone anaesthesia and their effects on the
discharge of nociceptors innervating these tissues were recorded. The effects of
inflammation caused by photorefractive keratectomy of the cornea or Freund's
adjuvant induced monoarthritis of the knee joint on the sensitivity to the purines was
also determined. In behavioural studies, ATP analogues were instilled into the eyes
of conscious rats and any changes in behaviour indicative of pain were assessed. To
establish whether the P2X3 receptor subtype for ATP was expressed in the cell
bodies of mouse corneal and rat knee joint neurones in the trigeminal and dorsal root
ganglia respectively, these cells were retrogradely labelled using fluorogold and
subsequently examined for co- localisation of fluorogold fluorescence with P2X3
immunoreactivity. Adenosine and adenosine analogues were also administered to the
normal and arthritic rat knee joint and, in behavioural studies, the effect of adenosine
agonists, adenosine antagonists and increasing the levels of endogenous adenosine on
the pain and inflammation associated with experimental arthritis were determined.
Immunoreactivity to P2X3 receptors was found in cell bodies of mouse
corneal nociceptors, but none of the ATP analogues tested excited cat corneal
nociceptors or caused pain when instilled into the eyes of conscious rats. The P2X3
subtype was also expressed in knee joint neurones in the dorsal root ganglia. ATP,
the stable P2X1 and P2X3 selective agonist, aß- methylene ATP and the P2 agonists,
ATPyS and benzoylbenzoyl ATP (BzATP), caused a rapid- onset, short- lasting
increase in action potential discharge from nociceptors innervating the rat knee joint.
These responses were antagonised by the P2 antagonist PPADS. ATP and ATPyS
also caused a delayed- onset, long- lasting increase in firing which was probably
mediated by adenosine Al receptors since adenosine, and the Al selective agonists
GR79236 and CPA evoked a similar response. These slow -onset responses were
antagonised by the Al selective antagonist DPCPX. Paradoxically, systemic
injections of DPCPX were not analgesic in behavioural studies, while the adenosine
uptake inhibitor, dipyridamole, which increases the extracellular levels of
endogenous adenosine, was. GR79236 had no effect on the pain of arthritis but did
possess anti -inflammatory properties. The ability of ATP to indirectly activate rat
knee joint nociceptors via P2X7 receptors expressed on inflammatory cells was
assessed by injecting high concentrations of BzATP, ATPyS and ATP intraiv
articularly to the knee joint and monitoring their effects on spontaneous and
bradykinin- evoked neural discharge. BzATP did not cause any increase the basal
action potential discharge rate nor did it sensitise the nociceptors to bradykinin. The
data from the other agonists was complicated by their metabolism to adenosine but,
like BzATP, no evidence was found for a sensitising effect.
This data supports the hypothesis that ATP and its metabolite, adenosine can
directly excite nociceptors innervating the rat knee joint via P2X and Al receptor
subtype(s), respectively. However, it does not support a role for P2X mediated
initiation of pain from the cat or rat cornea nor does it indicate that ATP could cause
pain via an indirect action on inflammatory cells. These findings have implications
for the development of novel therapies for the treatment of pain.