Effect of peptidases on the ability of exogenous and endogenous neurokinins to produce neurokinin 1 receptor internalization in the rat spinal cord

Abstract

1 The ability of peptidases to restrict neurokinin 1 receptor (NK1R) activation by exogenously applied or endogenously released neurokinins was investigated by measuring NK1R internalization in rat spinal cord slices. 2 Concentration -response curves for substance P and neurokinin A were obtained in the presence and absence of 10 mM thiorphan, an inhibitor of neutral endopeptidase (EC 3.4.24.11), plus 10 mM captopril, an inhibitor of dipeptidyl carboxypeptidase (EC 3.4.15.1). These inhibitors significantly decreased the EC 50 of substance P to produce NK1R internalization from 32 to 9 nM, and the EC 50 of neurokinin A from 170 to 60 nM. 3 Substance P was significantly more potent than neurokinin A, both with and without these peptidase inhibitors. 4 In the presence of peptidase inhibitors, neurokinin B was 10 times less potent than neurokinin A and 64 times less potent than substance P (EC 50 ¼ 573 nM). 5 Several aminopeptidase inhibitors (actinonin, amastatin, bacitracin, bestatin and puromycin) failed to further increase the effect of thiorphan plus captopril on the NK1R internalization produced by 10 nM substance P. 6 Electrical stimulation of the dorsal root produced NK1R internalization by releasing endogenous neurokinins. Thiorphan plus captopril increased NK1R internalization produced by 1 Hz stimulation, but not by 30 Hz stimulation. 7 Therefore, NEN and DCP restrict NK1R activation by endogenous neurokinins when they are gradually released by low-frequency firing of primary afferents, but become saturated or inhibited when primary afferents fire at a high frequency.

μ-Opioid receptor inhibition of substance P release from primary afferents disappears in neuropathic pain but not inflammatory pain

“…NK1R internalization was evoked by electrically stimulating the dorsal root ipsilateral to the surgery with 3000 pulses of 20 V and 0.4 ms delivered at 100 Hz (Adelson et al, 2009). The slices were superfused with the peptidase inhibitors captopril and thiorphan (10 μM) to reduce substance P degradation (Marvizon et al, 2003b). Root stimulation produced slightly less NK1R internalization in slices from CCI rats (70.7 ± 3.59 %, n = 14 slices) than in slices from the sham rats (85.8 ± 4.13 %, n = 10 slices, Fig.…”

“…NK1R internalization may also detect the release of neurokinin A, but not the release of neurokinin B. These tachykinins were 5–7 times and 64 times less potent than substance P to induce NK1R internalization, respectively (Marvizon et al, 2003b). Since substance P and neurokinin A are co-released from primary afferents (Trafton et al, 2001), their detection is functionally equivalent.…”

NMDA receptors in primary afferents require phosphorylation by Src family kinases to induce substance P release in the rat spinal cord

“…Slices were incubated with drugs using established procedures (Marvizon et al, 1997; Marvizon et al, 1999; Lao et al, 2003; Marvizon et al, 2003a; Marvizon et al, 2003b). The slices were placed on a nylon net glued to a plastic ring inserted halfway down a plastic tube containing 5 ml aCSF.…”

Inhibition by Spinal μ- and δ-Opioid Agonists of Afferent-Evoked Substance P Release

“…Accordingly, NK1R internalization indicates SP release evoked by noxious stimuli (Abbadie et al, 1997;Allen et al, 1997;Liu et al, 1997;Honore et al, 1999;Trafton et al, 1999Trafton et al, , 2001 or by primary afferent stimulation (Allen et al, 1999;Marvizon et al, 1999bMarvizon et al, , 2003a. Trafton et al (1999), using this elegant methodology, reported that percutaneous intrathecal delivery of mor-phine, at doses said to be analgesic, produced only a modest (Յ20%) block of NK1R internalization evoked by noxious stimuli.…”