Characterization of Substance P processing in mouse spinal cord S9 fractions using high-resolution Quadrupole-Orbitrap mass spectrometry.

Tachykinins are a family of pronociceptive neuropeptides with a specific role in pain and inflammation. Several mechanisms regulate endogenous tachykinins and Substance P (SP) levels, including the differential expression of protachykinin mRNA and the controlled secretion of tachykinins from neurons. Proteolysis is suspected to regulate extracellular SP concentrations but few studies were conducted on the metabolism of proneuropeptides and neuropeptides. Here, we provide evidence that proteolysis controls SP levels in the spinal cord leading to the formation of active C-terminal fragments. Using high-resolution mass spectrometry, specific tachykinins fragments were characterized and quantified. The metabolic stability of ß-Tachykinin58-71 and SP were very short resulting in half-life of 5.7 and 3.5min respectively. Several C-terminal fragments were identified, including SP3-11, SP5-11 and SP8-11, which conserve affinity for the Neurokinin 1 receptor. Interestingly, the metabolic stability of C-terminal fragments was significantly superior. Two specific Prolyl endopeptidase inhibitors were used and showed a significant reduction in the rate of formation of SP3-11 and SP5-11 providing strong evidence that Prolyl endopeptidase is involved into N-terminal processing of SP in the spinal cord.

Study on the distribution sites and the molecular mechanism of analgesia after intracerebroventricular injection of rat/mouse hemokinin-1 in mice.

Hemokinin-1 is a peptide encoded by Pptc, which belongs to the family of mammalian tachykinins. Our previous results showed that rat/mouse hemokinin-1 (r/m HK-1) produced striking analgesia after intracerebroventricular (i.c.v.) injection in mice, and the analgesia could be blocked by the NK1 receptor antagonist and the opioid receptor antagonist, respectively. However, the precise distribution sites and the molecular mechanism involved in the analgesic effect after i.c.v. administration of r/m HK-1 are needed to be further investigated deeply. Using the fluorescence labeling method, our present results directly showed that r/m HK-1 peptides were mainly distributed at the ventricular walls and several juxta-ventricular structures for the first time. Our results showed that the mRNA expressions of NK1 receptor, PPT-A, PPT-C, KOR, PDYN, DOR and PENK were not changed markedly, as well as the protein expression of NK1 receptor was hardly changed. However, both the transcripts and proteins of MOR and POMC were up-regulated significantly, indicating that the analgesic effect induced by i.c.v. administration of r/m HK-1 is related to the activation of NK1 receptor first, then it is related to the release of endogenous proopiomelanocortin, as well as the increased expression level of µ opioid receptor. These results should facilitate further the analysis of the analgesia of r/m HK-1 in the central nerval system in acute pain and may open novel pharmacological interventions.

Hemokinin-1 has Substance P-like function in U-251 MG astrocytoma cells: a pharmacological and functional study.

Substance P (SP) triggers responses in astrocytoma cells, which are considered important for proliferation and neuroimmunomodulatory activity. In this study, we compared the effects of SP with those of the novel tachykinin Hemokinin-1 (HK-1) in the human astrocytoma cell line U-251 MG. We show that U-251 MG cells express high levels of Neurokinin-1 (NK-1) receptors. The binding affinities of 125I-SP and 125I-mHK-1 to these receptors were in a similar, subnanomolar range. HK-1 and SP stimulated Ca2+ mobilization and induced increased cytokine mRNA expression. A specific NK-1 receptor antagonist blocked the observed effects. We conclude that there are no qualitative differences in SP and HK-1-evoked responses, suggesting that both peptides act through NK-1 receptors in U-251 MG cells. Moreover, we show TAC4 mRNA expression in gliomas, indicating a possible involvement of HK-1 in glioma biology.

Two NK-3 receptor subtypes: demonstration by biological and binding assays.

The existence of two neurokinin NK-3 receptor subtypes has been suggested on the basis of results obtained in binding assays. In the present study, we have confirmed the two NK-3 receptor subtypes by using data obtained in both biological and binding assays. Experiments have been performed in the rat portal vein and in the guinea-pig ileum treated with NK-1 and NK-2 selective antagonists, namely CP 96345 and SR 48968. Orders of potency of agonists on the rat portal vein are as follows: for neurokinins, NKB > NKA > SP; for tachykinins, KAS > ELE > PHY; and for selective agonist: [MePhe7]NKB >> senktide. On the guinea-pig ileum, the agonist rank orders of potency are: NKB > SP > NKA, ELE > KAS > PHY; and for selective agonist: [MePhe7]NKB = senktide. The apparent affinity of antagonists shows differences in both biological and binding assays. In fact, on the rat portal vein, SR 48968 is almost inactive (pA2 or IC50 approximately 4.8), while R-486 [Trp7, beta Ala8]NKA(4-10) shows a pA2 value of 7.45 and an IC50 of 5.6. An opposite pattern of activity is observed in the guinea-pig ileum, where SR 48968 shows a pA2 of 6.05 and an IC50 of 6.7, while R-486 has a pA2 of 6.1 and an IC50 of < 5.0. These results confirm the existence of two NK-3 sites differing pharmacologically. It is proposed to name NK-3A the receptor of the guinea-pig ileum and NK-3B the receptor of the rat portal vein.

Injection of tachykinins and selective neurokinin receptor ligands into the substantia nigra reticulata increases striatal dopamine and 5-hydroxytryptamine metabolism.

Bilateral injection of endogenous tachykinins (substance P (SP), neurokinin A (NKA)) and selective neurokinin receptor ligands (senktide, [Sar9,Met(O2)11]SP, [MePhe7]NKB) into the substantia nigra reticulata increased striatal dopamine and serotonin metabolism. The increase in dopamine metabolism in the dorsal striatum at a low dose of the substances may be a direct effect on dopamine neurons in the substantia nigra reticulata via NK1 and NK3 receptors. The lack of effect at intermediate doses may be due to inhibitory mechanisms or desensitization. The changes after high doses in the dorsal and ventral striatum may be due to actions on dopaminergic neurons in the substantia nigra as well as in the ventral tegmental area, since there was considerable diffusion from the site of injection. An apparent rapid degradation of injected SP or NKA indicates that N-terminal SP fragments may participate in the SP response. The increased serotonin metabolism that occurs only at a high dose may involve all three neurokinin receptors.

Occurrence, release, and effects of multiple tachykinins in cat colonic tissues and nerves.

Neurokinin A (NKA)-immunoreactivities and substance P (SP)-immunoreactivities were found in picomolar amounts in colonic tissues and almost an order of magnitude higher amounts in vagal, pelvic, splanchnic, and lumbar colonic nerves of the cat. Continuous electric stimulation of the pelvic nerve at 4 Hz or intermittent electric burst stimulation of the pelvic nerve at 40 Hz during 1 second with 10-second rest periods produced a marked release of NKA-like immunoreactivity (NKA-LI) and SP-LI from the colon to blood (P less than 0.001). Reflex activation of the pelvic nerve by mechanical stimulation of the anus or rectal distension produced a less pronounced release of NKA-LI and SP-LI from the colon to blood (P less than 0.01). There was a simultaneous colonic contraction and vasodilation during each nerve stimulation. Reverse-phase high performance liquid chromatography showed presence of NKA, NKA oxide, NKA (3-10)/NKA (4-10), and neuropeptide K (NPK) in colonic tissues and release of all these molecular forms except NPK on nerve stimulation. Substance P and SP oxide were present both in colonic tissue extracts and in released material. Close intraarterial infusions of NKA, neurokinin B, SP, NPK, eledoisin, and physalaemin at doses of 0.1-100 pmol/min induced dose-dependent contractions of the proximal and distal colon (P less than 0.001) and vasodilatation (P less than 0.001), NKA being the most potent. The effects of the tachykinins were reduced after tetrodotoxin (P less than 0.05) and atropine (P less than 0.05) but unchanged after treatment with hexamethonium. Our findings indicate that tachykinins are released from the pelvic nerve to induce a nonadrenergic noncholinergic contraction and vasodilatation of the colon in the cat.

Label distribution after injection of labelled tachykinins into the rat lateral cerebroventricle.

The present study investigated the label distribution in several brain regions, as well as in the peripheral circulation, following injection of labelled tachykinins [( 3H]-substance P, [3H]-eledoisin or [125I]-neurokinin A) into the lateral cerebroventricle of the rat. A widespread label distribution, extending as far as to the brainstem, was detected. Hypothalamus, striatum and hippocampus were the most labelled regions by the 3 labels; however the patterns of distribution of the 3 labelled tachykinins showed marked differences. Distribution in the brain was rapid, reaching a maximum usually within 2 min after injection and declining slightly afterwards. Large amounts of label (1/6-1/15 of the total amount injected) were detected in serum even at 2 min after injection and increased thereafter, reaching a maximum at 10-15 min.