[Sar9] Substance P

The autoradiographic distribution of neurokinin (NK)-1 receptors was visualized in the rat brain using the highly selective ligand, [3H]-[Sar9,Met(O2)11]-substance P. This ligand apparently binds to a single class of high affinity (Kd = 1.4 +/- 0.5 nM), low capacity (Bmax = 160 +/- 3.0 fmol/mg protein) sites in rat brain membrane preparations. The ligand selectivity profile reveals that substance P (SP) and unlabeled [Sar9,Met(O2)11]-SP are potent competitors of [3H]-[Sar9,Met(O2)11]-SP binding while NK-2 and NK-3 analogues are virtually inactive demonstrating the selectivity of this radioligand for the NK-1 receptor class. Autoradiographic data show that [3H]-[Sar9,Met(O2)11]-SP binding sites are broadly but discretely distributed in rat brain, the highest densities of sites being located in the external plexiform layer of the olfactory bulb, striatum, olfactory tubercule, amygdala-hippocampal area, endopiriform and entorhinal cortices, superior colliculus, locus coeruleus and substantia gelatinosa of the spinal cord. This distribution is similar, but not identical, to that previously reported for NK-1 sites using less selective ligands such as [125I]Bolton-Hunter SP. For example, some difference in labelling patterns are observed in the hippocampal formation. This could be explained by the existence of NK-1 receptor subtypes, only one of them being recognized by [3H]-[Sar9,Met(O2)11]-SP or by the greater selectivity of this radioligand for NK-1 over NK-2 and NK-3 receptor classes.

Peripheral inflammation produces pain hypersensitivity by sensitizing nociceptors. Potentiation of P2X3 receptor activity in nociceptors may play an important role in this peripheral sensitization. However, we do not fully understand how P2X3 activity is elevated in inflammation. Thus, we investigated whether P2X3 activity in trigeminal nociceptive neurons is regulated by the neurokinin-1 (NK-1) receptor that is activated by an inflammatory mediator, substance P. Single-cell RT-PCR and immunohistochemistry revealed that NK-1 in nociceptive neurons was mainly co-expressed with P2X3. Ca(2+) imaging and whole-cell patch-clamp recordings indicated that both substance P and Sar-substance P, a selective NK-1 agonist, significantly potentiated α,β-meATP-induced currents and [Ca(2+)](i) responses in nociceptive neurons. These potentiating effects were completely blocked by GR82334, a specific NK-1 antagonist. Our results demonstrate that substance P sensitizes P2X3 receptor through the activation of NK-1, thus warranting these receptors as possible targets for pain therapy in the orofacial region.Abbreviations:α,β-methylene adenosine 5'-triphosphate (ATP), α,β-meATP; neurokinin-1, NK-1; single-cell reverse-transcription polymerase chain-reaction, single-cell RT-PCR; [Sar(9),Met(O(2))(11)]-substance P, Sar-substance P.

Lumbar intrathecal injections of substance P-saporin (SP-sap) destroy dorsal horn neurons that express the neurokinin-1 receptor (NK-1R) resulting in decreased responses to a range of noxious stimuli and decreased hyperalgesia and allodynia. Forebrain injections of SP-sap produce considerable non-specific damage raising some concern about use of this toxin in vivo. The more stable and selective substance P congener, [Sar9,Met(O2)11]substance P coupled to saporin (SSP-sap) produces much more selective forebrain lesions at significantly lower doses. The present study sought to determine the anatomic and nocifensive behavioral effects of lumbar intrathecal injections of the more precisely targeted SSP-sap. On the basis of loss of lamina I NK-1R staining, lumbar intrathecal SSP-sap was seven times more potent than SP-sap and produced no loss of NK-1R expressing neurons in deeper laminae (III-VI or X). Transient decreases in hotplate responding occurred at 44 degrees C and 47 degrees C but not 52 degrees C during the first 3 weeks after SSP-sap injection with return to baseline by 4 weeks. Operant escape responses were reduced at 0.3 degrees C, 44 degrees C and 47 degrees C for at least 4 months. In the formalin test, SSP-sap also was about seven times more potent than SP-sap in reducing phase two behavior in both female Long Evans and male Sprague-Dawley rats. Both SSP-sap and SP-sap reduced formalin-induced FOS expression in deep and superficial laminae of the L4 dorsal horn in parallel with the reduction in phase 2 behavior. In summary, SSP-sap is highly effective in destroying lamina I NK-1R expressing neurons, without loss of deep NK-1R neurons. The behavioral effects of SSP-sap are similar to SP-sap suggesting that the antinociceptive effects of both toxins are indeed due to selective loss of NK-1R neurons in lamina I. SSP-sap is an attractive agent for possible treatment of chronic pain.