Receptor activated bladder and spinal ATP release in neurally intact and chronic spinal cord injured rats

Abstract

Neurally intact (NI) rats and chronic spinal cord injured (SCI) rats were studied to determine how activation of mechanosensory or cholinergic receptors in the bladder urothelium evokes ATP release from afferent terminals in the bladder as well as in the spinal cord. Spinal cord transection was performed at the T(9)-T(10) level 2-3 weeks prior to the experiment and a microdialysis fiber was inserted in the L(6)-S(1) lumbosacral spinal cord one day before the experiments. Mechanically evoked (i.e. 10 cm/W bladder pressure) ATP release into the bladder lumen was approximately 6.5-fold higher in SCI compared to NI rats (p<0.05). Intravesical carbachol (CCh) induced a significantly greater release of ATP in the bladder from SCI as compared to NI rats (3424.32+/-1255.57 pmol/ml versus 613.74+/-470.44 pmol/ml, respectively, p<0.05). However, ATP release in NI or SCI rats to intravesical CCh was not affected by the muscarinic antagonist atropine (Atr). Spinal release of ATP to bladder stimulation with 10 cm/W pressure was five-fold higher in SCI compared to NI rats (p<0.05). CCh also induced a significantly greater release of spinal ATP in SCI rats compared to controls (4.3+/-0.9 pmol versus 0.90+/-0.15 pmol, p<0.05). Surprisingly, the percent inhibitory effect of Atr on CCh-induced ATP release was less pronounced in SCI as compared to NI rats (49% versus 89%, respectively). SCI induces a dramatic increase in intravesical pressure and cholinergic receptor evoked bladder and spinal ATP release. Muscarinic receptors do not mediate intravesical CCh-induced ATP release into the bladder lumen in NI or SCI rats. In NI rats sensory muscarinic receptors are the predominant mechanism by which CCh induces ATP release from primary afferents within the lumbosacral spinal cord. Following SCI, however, nicotinic or purinergic receptor mechanisms become active, as evidenced by the fact that Atr was only partially effective in inhibiting CCh-induced spinal ATP release.

Figure 1

Schematic representation showing the protocol used for the measurement of ATP released into the bladder lumen and spinal cord.

Figure 2

Macroscopic view of the rat spinal cord showing the microdialysis probe placed between the L₆-S₁ spinal cord segments.

Figure 3

Release of ATP into the bladder lumen in neurally intact (NI) (n=5) and spinal cord injured (SCI) (n=6) rats after raising the intravesical pressure to 10cm/w. Bladder ATP concentration was significantly higher in SCI compared to NI rats (p<0.05, unpaired t-test).

Figure 4

Bladder lumen ATP release to chemical stimulation with carbachol. NI=neurally intact rat; SCI=spinal cord injured rat; C=carbachol; A=atropine. Carbachol stimulation evoked a greater increase in bladder ATP concentration in SCI (n=6) compared to NI (n=5) rats (p<0.05, unpaired t-test). Atropine, however, did not alter bladder ATP concentration to carbachol stimulation in either group tested.

Figure 5

ATP release measured by a microdialysis probe implanted in the L6-S1 spinal cord of rats during 10cm/w pressure in neurally intact (NI) and spinal cord injured (SCI) rats. Note the greater than 5-fold increase in ATP levels with pressure stimulation in SCI rats (n=6) compared to NI rats (n=5) (p<0.01, unpaired t-test).

Figure 6

Bar graph depicting L6-S1 spinal cord ATP release collected with a microdialysis probe during intravesical chemical stimulation with carbachol or carbachol + atropine in NI (n=5) and SCI (n=6) rats. NI=neurally intact rat; SCI=spinal cord injured rat; C=carbachol; A=atropine. Carbachol evoked a significantly higher spinal ATP release in SCI versus NI rats (p<0.01, unpaired t-test). In addition, atropine significantly inhibited carbachol evoked spinal ATP release in both groups (p<0.05, paired t-test). * denotes significant change compared to C-NI group. # denotes significant change compared to C-SCI group.