A role for tachykinins in the regulation of human sperm motility.

BACKGROUND: Tachykinins and tachykinin receptors are widely distributed in the male reproductive tract and appear to be involved in reproduction. However, the function and expression of tachykinins and their receptors in human spermatozoa remain poorly studied. We analysed the effects of tachykinins on sperm motility and characterized the population of tachykinin receptors in human spermatozoa. METHODS AND RESULTS: Motility analysis was performed following World Health Organization guidelines and we found that substance P (SP), human hemokinin-1 (hHK-1), neurokinin A (NKA) and neurokinin B (NKB) produced concentration-dependent increases in sperm progressive motility. The effects of tachykinins were antagonized by the NK(1) receptor-selective antagonist SR 140333, the NK(2) receptor-selective antagonist, SR 48968 and, to a lesser extent, also by the NK(3) receptor-selective antagonist SR 142801. Immunocytochemistry studies showed expression of the NK(1), NK(2) and NK(3) tachykinin receptor proteins in spermatozoa with different major sites of localization for each receptor. Western blot analysis confirmed the presence of tachykinin receptors in sperm cell homogenates. RT-PCR demonstrated expression of the genes that encode SP/NKA (TAC1), NKB (TAC3) and hHK-1 (TAC4) but not the genes TACR1, TACR2 and TACR3 encoding NK(1), NK(2) and NK(3) receptors, respectively. CONCLUSIONS: These results show for the first time that the NK(1), NK(2) and NK(3) tachykinin receptor proteins are present in human spermatozoa. Our findings suggest that tachykinins, probably acting through these three tachykinin receptors, play a role in the regulation of human sperm motility.

A 25 year adventure in the field of tachykinins.

Several aspects of our 25 year adventure in the field of tachykinins will be successively described. They concern: substance P (SP) synthesis and release in the basal ganglia, the identification and pharmacological characterization of central tachykinin NK(1), NK(2) and NK(3) binding sites and their topographical distribution, the description of some new biological tests for corresponding receptors, the identification of tachykinin NK(1) receptor subtypes or conformers sensitive to all endogenous tachykinins (substance P, neurokinin A (NKA), neurokinin B (NKB), neuropeptide gamma (NP gamma) and neuropeptide K (NPK)) and finally, the functional involvement of these receptors and their subtypes in tachykinin-induced regulations of dopamine and acetylcholine release in the striatum.

Tachykinins regulate the function of platelets.

Evidence has been mounting for peripheral functions for tachykinins, a family of neuropeptides including substance P (SP), neurokinin A, and neurokinin B, which are recognized for their roles in the central and peripheral nervous system. The recent discovery of 4 new members of this family, the endokinins (EKA, B, C, and D), which are distributed peripherally, adds support to the notion that tachykinins have physiologic/endocrine roles in the periphery. In the present study we report a fundamental new function for tachykinins in the regulation of platelet function. We show that SP stimulates platelet aggregation, and underlying this is the intracellular mobilization of calcium and degranulation. We demonstrate the presence of the tachykinin receptors NK1 and NK3 in platelets and present evidence for the involvement of NK1 in SP-mediated platelet aggregation. Platelets were found to contain SP-like immunoreactivity that is secreted upon activation implicating SP-like substances in the autocrine/paracrine regulation of these cells. Indeed, NK1-blocking antibodies inhibited aggregation in response to other agonists. Of particular note is the observation that EKA/B cross-react in the SP immunoassay and are also able to stimulate platelet activation. Together our data implicate tachykinins, specifically SP and EKA/B, in the regulation of platelet function.

The role of neurokinin B in pre-eclampsia.

Pre-eclampsia, a life-threatening disease unique to pregnancy, has been called a disease of theories. To date, there has been no widely accepted predictive test or therapeutic intervention to prevent or delay pre-eclampsia. The discovery of a new placental hormone, neurokinin B, may finally help to answer some of the past mysteries.

Effect of post-mortem delay on density of tachykinin receptors in rat peripheral tissues.

The effect of post-mortem delay on the affinity and density of tachykinin NK1 and NK2 receptors was examined in the rat submandibular gland and gastric fundus, respectively, using saturation binding studies with the radioligands [125I]Bolton-Hunter [Sar9, Met(O2)11]SP and [125I][Lys5, Tyr(I2)7, MeLeu9, Nle10]NKA(4-10). For NK1 receptors, no significant changes were seen in either Kd (control 375 +/- 35 pM, n = 5; 32 h post-mortem 390 +/- 59 pM, n = 5) or Bmax (control 96 +/- 16 fmol/mg protein, n = 5; 32 h post-mortem 62 +/- 10 fmol/mg protein, n = 5). For NK2 receptors, no alterations were seen up to 16 h post-mortem. However, significant (p < 0.001) changes were seen at 32 h post-mortem (n = 4), where values for Kd were increased (3.0 +/- 0.2 nM) and those for Bmax were reduced (42 +/- 5.9 fmol/mg protein), relative to control (Kd = 1.3 +/- 0.2 nM; Bmax = 208 +/- 30 fmol/mg protein, n = 5). These changes are probably related to observed histological deterioration. This study demonstrates the stability of tachykinin receptors in these peripheral tissues and indicates the suitability of post-mortem tissue as a valid control in future tachykinin receptor studies.

Substance P enhances cytokine-induced vascular cell adhesion molecule-1 (VCAM-1) expression on cultured rheumatoid fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of the synovial membrane of multiple joints. This inflammatory microenvironment allows fibroblast-like synoviocytes (FLS) to express or enhance several adhesion or costimulatory molecules. This phenotypic shift, under proinflammatory cytokines, seems to be related to functional consequences for antigen presentation to T cells. The sensory neuropeptide substance P (SP), present at high levels, is able to act on FLS proliferation and enzyme secretion. These data led us to investigate whether SP could also provoke a phenotypic change of FLS. Using flow cytometry and a three-step cellular ELISA method, we determined whether SP has an influence on the expression of MHC class II, intercellular adhesion molecule-1 (ICAM-1), VCAM-1, LFA-3, CD40, B7.1 or B7.2 molecules on RA FLS incubated with interferon-gamma (IFN-gamma) or IL-1beta or tumour necrosis factor-alpha (TNF-alpha) with or without SP. Our results indicate that SP potentiates the effect of proinflammatory cytokines on the expression of VCAM-1 on RA FLS. We verified the presence of specific SP (NK1) receptor mRNA. Using reverse transcription-polymerase chain reaction, we showed that RA FLS of patients express NK1 receptor mRNA. These results suggest that SP increase of cytokine-induced VCAM-1 expression acts via this specific SP receptor. Thus, during chronic inflammation RA FLS are at the interface between the immune and the nervous systems.

Cultured rat sensory neurones express functional tachykinin receptor subtypes 1, 2 and 3.

The neuropeptide substance P (SP) is known to play a key role in peripheral nociceptive processes. We investigated the in vitro pharmacological characteristics of functional tachykinin receptors expressed in dorsal root ganglia (DRG) sensory neurones by analysing intracellular free calcium concentration changes induced after stimulation by SP or specific tachykinin agonists. We observed that about 37% of the tested neurones were responsive to either SP or an NK1-, NK2- or NK3-specific agonist. Tachykinin-responsive neurones had a small soma diameter (<20 microm) and were sensitive to capsaicin. These results suggest the presence of NK1, NK2 and NK3 receptors in noxious sensory neurones.

Characterization of antisera specific to NK1, NK2, and NK3 neurokinin receptors and their utilization to localize receptors in the rat gastrointestinal tract.

Understanding the physiological role of tachykinins requires precise cellular and subcellular localization of their receptors. We raised antisera by immunizing rabbits with peptides corresponding to portions of the intracellular tails of the rat neurokinin 1, 2, and 3 receptors (NK1-R, NK2-R, NK3-R). Receptors were localized by immunofluorescence and confocal microscopy. NK1-R, NK2-R, and NK3-R were detected at the plasma membrane of transfected cells with minimal intracellular stores. Staining was abolished by preabsorption of the antisera with the peptides used for immunization. Nontransfected cells were unstained. Each antiserum only stained cells transfected with the appropriate receptor and did not stain cells transfected with the other receptors. Therefore, the antisera are specific and do not cross-react with other neurokinin receptors. We examined the distribution of the neurokinin receptors in the gastrointestinal tract of the rat. NK1-R was detected in myenteric and submucosal neurons and in interstitial cells of Cajal. NK2-R was localized to circular and longitudinal muscle cells and to nerve endings in the plexuses. NK3-R was detected in numerous myenteric and submucosal neurons. Some neurons expressed both NK1-R and NK3-R. Receptors were detected at the plasma membrane and in endosomes. Cells expressing the receptors were closely associated with tachykinin-containing nerve fibers. Thus, NK1-R and NK3-R mediate neurotransmission by tachykinins within enteric nerve plexuses, and NK1-R and NK2-R mediate the effects of tachykinins on interstitial and smooth muscle cells, respectively.

Detection of naturally expressed receptors for gastrin-releasing peptide and tachykinins using cyanine 3-labelled neuropeptides.

Peptides labelled with the fluorophore cyanine 3 were used to study naturally expressed neuropeptide receptors by confocal microscopy in continuous cell lines, primary cultures, and unfixed tissue. Swiss 3T3 fibroblasts bound cyanine 3-gastrin-releasing peptide at 4 degrees C, and internalized the peptide after 10 min at 37 degrees C. Internalization was specific, since it was blocked by incubation with unlabelled peptide. Primary cultures of myenteric neurons of the guinea pig incubated with cyanine 3-substance P at 4 degrees C had specific surface labelling. After 30 s at 37 degrees C, the peptide was internalized into vesicles in both the soma and neurites. Direct observation of live neurons showed movement of fluorescent vesicles to a perinuclear region after 30 min. Endocytosis was associated with a loss of surface binding sites. Unfixed whole mounts of guinea pig and rat ileum were incubated with cyanine 3-neurokinin A at 4 degrees C. After 5 min at 37 degrees C, Cy3-neurokinin A was specifically internalized in neurons and smooth muscle cells. After 30 min, a perinuclear labelling occurred in some cells. Labelling in rat neurons was diminished by the NK3-R antagonist SR142801. Thus, cyanine 3-neuropeptides are valuable tools to study expression and endocytosis of naturally expressed receptors.

Pharmacological evidence for neurokinin receptors in murine neuroblastoma C1300 cells.

We found that neurokinin A (NKA) and neurokinin B (NKB) induce an increase in the concentration of intracellular free Ca2+ ([Ca2+]i) in murine neuroblastoma C1300 cells (EC50: NKA 87 +/- 13 nM, NKB 97 +/- 15 nM). Substance P (SP) also caused a transient Ca2+ increase, although the potency of SP was much less than that of NKA and NKB. The increase in [Ca2+]i induced by NKA and NKB was inhibited by SR 48,968, a selective antagonist for NK2, and [beta Ala8]NKA(4-10), a selective agonist for NK2, did not stimulate the increase in [Ca2+]i. NKA- and NKB-induced Ca2+ mobilization was not inhibited by CP-96,345 and [Trp7, beta Ala8]NKA(4-10), selective antagonists for NK1 and NK3, respectively. These results suggested that C1300 cells express endogenous NK2 neurokinin receptors that have different features from known NK2 receptors.

Involvement of neurokinin 1 and 2 receptors in viscerosensitive response to rectal distension in rats.

BACKGROUND/AIMS: Tachykinins participate in somatic pain and intestinal motility control. The role of tachykinin receptors in both colonic motor disturbances and visceral pain (abdominal contractions as an index of visceral pain) induced by rectal distension were investigated. METHODS: Rats were surgically prepared with electrodes implanted on the proximal colon and the abdominal striated muscles. Catheters were implanted in lateral ventricles of the brain. Rectal distension was performed by inflation of a balloon (0.1-1.6 mL) rectally inserted. CP-96,345 and RP-67,580 (neurokinin [NK] 1 antagonists) and SR-48,968 (NK2 antagonist) were injected intraperitoneally (IP) or intracerebroventricularly (ICV) 20 minutes before distension. GR-73,632 and GR-64,639 (NK1, NK2 agonists) were infused intravenously at 0.15 micrograms.kg-1.min-1. RESULTS: Rectal distension evoked a significant inhibition of colonic motility and an increase in abdominal contractions. CP-96,345 injected ICV (0.2-0.8 mg/kg) or IP (5-10 mg/kg) and RP-67,580 (0.2 mg/kg IP) eliminated distension-induced colonic inhibition but did not affect abdominal response. SR-48,968 did not affect colonic response but significantly reduced visceral pain (0.4, 0.8 mg/kg ICV: 5-10 mg/kg IP). GR-73,632 enhanced the rectal distension-induced colonic inhibition, whereas GR-64,349 induced a greater abdominal response. CONCLUSIONS: NK1 receptors mediate the rectocolonic inhibitory reflex, whereas NK2 receptors participate in visceral pain; both responses involve central structures.

Tachykinins contract the circular muscle of the human esophageal body in vitro via NK2 receptors.

BACKGROUND: The action of tachykinins in the circular muscle of the human esophageal body is not known. The present study aimed to determine the response to tachykinins and the receptor type mediating this response. METHODS: Specimen were obtained from organ donors or patients undergoing esophagectomy for cancer, and isometric tension in response to tachykinins was measured. RESULTS: Substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) evoked a concentration-dependent contraction with the following order of potency: NKA > NKB > SP. The neutral endopeptidase inhibitor, phosphoramidon, increased only the response to SP. [beta Ala8]NKA(4-10), a selective agonist of the NK2 receptor, produced a concentration-dependent contraction, whereas [Sar9,Met(O2)11]SP and [MePhe7]NKB, selective agonists of NK1 and NK3 receptors, respectively, had no effect. Contraction evoked by NKA was inhibited by the nonpeptide NK2 antagonist SR 48968 but not by the nonpeptide NK1 receptor antagonist CP-96,345, tetrodotoxin, or atropine. SR 48968 did not affect the response to carbachol. CONCLUSIONS: Tachykinins contract the circular muscle of human esophageal body by activation of NK2 receptors without involvement of neural mechanisms. Response to SP is modulated by a phosphoramidon-sensitive enzymatic activity.

[A new tachykinin receptor revealed by substance P analogues in the guinea pig ileum]. / Un nouveau récepteur des tachykinines révélé par des analogues de la substance P dans l'iléon de cobaye.

[Pro9]SP and septide have been described as selective agonists for the SP receptor (NK-1 type). These two peptides contract with a great efficacy the guinea-pig ileum, but unexpectedly septide was practically devoid of affinity for the NK-1 site labelled by 3H-[Pro9]SP. Like septide, SP analogues like SP-O-CH3, [Apa9-10]SP and [Pro9,10]SP share the same peculiar properties. In addition, the contracting activity of these peptides is not explained by an interaction with NK-2 or NK-3 sites. GR 71,251, a compound which has been described as NK-1 antagonist, was more potent in inhibiting the septide- and the [Apa9-10]SP- than the [Pro9]SP-evoked contracting responses. Altogether, these results suggest that septide, SP-O-CH3, [Apa9-10]SP and [Pro9,10]SP exert their high contracting activity in the guinea-pig ileum by acting on a new type of tachykinin receptor.