Rapid elucidation of agonist-driven regulation of the neurokinin 1 receptor using a GPCR phosphorylation immunoassay.

Agonist-induced phosphorylation is a crucial step in the activation/deactivation cycle of G protein-coupled receptors (GPCRs), but direct determination of individual phosphorylation events has remained a major challenge. We have recently developed a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in 96-well plates, thus eliminating the need for western blot analysis. In the present study, we adapted this assay to three novel phosphosite-specific antibodies directed against the neurokinin 1 (NK1) receptor, namely pS338/pT339-NK1, pT344/pS347-NK1, and pT356/pT357-NK1. We found that substance P (SP) stimulated concentration-dependent phosphorylation of all three sites, which could be completely blocked in the presence of the NK1 receptor antagonist aprepitant. The other two endogenous ligands of the tachykinin family, neurokinin A (NKA) and neurokinin B (NKB), were also able to induce NK1 receptor phosphorylation, but to a much lesser extent than substance P. Interestingly, substance P promoted phosphorylation of the two distal sites more efficiently than that of the proximal site. The proximal site was identified as a substrate for phosphorylation by protein kinase C. Analysis of GPCR kinase (GRK)-knockout cells revealed that phosphorylation was mediated by all four GRK isoforms to similar extents at the T344/S347 and the T356/T357 cluster. Knockout of all GRKs resulted in abolition of all phosphorylation signals highlighting the importance of these kinases in agonist-mediated receptor phosphorylation. Thus, the 7TM phosphorylation assay technology allows for rapid and detailed analyses of GPCR phosphorylation.

Deciphering specificity and cross-reactivity in tachykinin NK1 and NK2 receptors.

The tachykinin receptors neurokinin 1 (NK1R) and neurokinin 2 (NK2R) are G protein-coupled receptors that bind preferentially to the natural peptide ligands substance P and neurokinin A, respectively, and have been targets for drug development. Despite sharing a common C-terminal sequence of Phe-X-Gly-Leu-Met-NH2 that helps direct biological function, the peptide ligands exhibit some degree of cross-reactivity toward each other's non-natural receptor. Here, we investigate the detailed structure-activity relationships of the ligand-bound receptor complexes that underlie both potent activation by the natural ligand and cross-reactivity. We find that the specificity and cross-reactivity of the peptide ligands can be explained by the interactions between the amino acids preceding the FxGLM consensus motif of the bound peptide ligand and two regions of the receptor: the ß-hairpin of the extracellular loop 2 (ECL2) and a N-terminal segment leading into transmembrane helix 1. Positively charged sidechains of the ECL2 (R177 of NK1R and K180 of NK2R) are seen to play a vital role in the interaction. The N-terminal positions 1 to 3 of the peptide ligand are entirely dispensable. Mutated and chimeric receptor and ligand constructs neatly swap around ligand specificity as expected, validating the structure-activity hypotheses presented. These findings will help in developing improved agonists or antagonists for NK1R and NK2R.

Ionic signalling mechanisms involved in neurokinin-3 receptor-mediated augmentation of fear-potentiated startle response in the basolateral amygdala.

The tachykinin peptides include substance P (SP), neurokinin A and neurokinin B, which interact with three G-protein-coupled neurokinin receptors, NK1Rs, NK2Rs and NK3Rs, respectively. Whereas high densities of NK3Rs have been detected in the basolateral amygdala (BLA), the functions of NK3Rs in this brain region have not been determined. We found that activation of NK3Rs by application of the selective agonist, senktide, persistently excited BLA principal neurons. NK3R-elicited excitation of BLA neurons was mediated by activation of a non-selective cation channel and depression of the inwardly rectifying K+ (Kir) channels. With selective channel blockers and knockout mice, we further showed that NK3R activation excited BLA neurons by depressing the G protein-activated inwardly rectifying K+ (GIRK) channels and activating TRPC4 and TRPC5 channels. The effects of NK3Rs required the functions of phospholipase Cß (PLCß), but were independent of intracellular Ca2+ release and protein kinase C. PLCß-mediated depletion of phosphatidylinositol 4,5-bisphosphate was involved in NK3R-induced excitation of BLA neurons. Microinjection of senktide into the BLA of rats augmented fear-potentiated startle (FPS) and this effect was blocked by prior injection of the selective NK3R antagonist SB 218795, suggesting that activation of NK3Rs in the BLA increased FPS. We further showed that TRPC4/5 and GIRK channels were involved in NK3R-elicited facilitation of FPS. Our results provide a cellular and molecular mechanism whereby NK3R activation excites BLA neurons and enhances FPS. KEY POINTS: Activation of NK3 receptors (NK3Rs) facilitates the excitability of principal neurons in rat basolateral amygdala (BLA). NK3R-induced excitation is mediated by inhibition of GIRK channels and activation of TRPC4/5 channels. Phospholipase Cß and depletion of phosphatidylinositol 4,5-bisphosphate are necessary for NK3R-mediated excitation of BLA principal neurons. Activation of NK3Rs in the BLA facilitates fear-potentiated startle response. GIRK channels and TRPC4/5 channels are involved in NK3R-mediated augmentation of fear-potentiated startle.

Neuropeptide Y, calcitonin gene-related peptide, and neurokinin A in brain regions of HAB rats correlate with anxiety-like behaviours.

Anxiety disorders are pervasive psychiatric disorders causing great suffering. The high (HAB) and low (LAB) anxiety-related behaviour rats were selectively bred to investigate neurobiological correlates of anxiety. We compared the level of neuropeptides relevant for anxiety- and depression-related behaviours in selected brain regions of HAB and LAB rats. Increased anxiety and depression-like behaviours of male and female HAB rats in the elevated plus-maze and forced swim tests were accompanied by elevated levels of neuropeptide Y (NPY) in the prefrontal (PFC), frontal (FC) and cingulate cortex (CCx), the striatum, and periaqueductal grey (PAG). Moreover, HAB rats displayed sex-dependent, elevated levels of calcitonin gene-related peptide (CGRP) in PFC, FC, CCx, hippocampus, and PAG. Higher neurokinin A (NKA) levels were detected in CCx, striatum, and PAG in HAB males and in CCx and hypothalamus in HAB females. Increased neurotensin was detected in CCx and PAG in HAB males and in hypothalamus in HAB females. Elevated corticotropin-releasing hormone (CRH) levels appeared in female HAB hypothalamus. Significant correlations were found between anxiety-like behaviour and NPY, CGRP, NKA, and neurotensin, particularly with NPY in CCx and striatum, CGRP in FC and hippocampus, and NKA in entorhinal cortex. This is the first report of NPY, CGRP, NKA, Neurotensin, and CRH measurements in brain regions of HAB and LAB rats, which showed widespread NPY and CGRP alterations in cortical regions, with NKA and neurotensin changes localised in sub-cortical areas. The results may contribute to elucidate pathophysiological mechanisms underlying anxiety and depression and should facilitate identifying novel therapeutic targets.

TRPM3 channel activation inhibits contraction of the isolated human ureter via CGRP released from sensory nerves.

AIMS: Sensory nerve activation modulates ureteral contractility by releasing neuropeptides including CGRP and neurokinin A (NKA). TRPM3 is a recently discovered thermosensitive channel expressed in nociceptive sensory neurons, and plays a key role in heat nociception and chronic pain. The aim of this study is to examine the role of TRPM3 activation in human ureter motility. MAIN METHOD: Human proximal ureters were obtained from fourteen patients undergoing nephrectomy. Spontaneous or NKA-evoked contractions of longitudinal ureter strips were recorded in an organ bath. Ureteral TRPM3 expression was examined by immunofluorescence. KEY FINDINGS: Spontaneous contractions were observed in 60% of examined strips. TRPM3 activation using pregnenolone sulphate (PS) or CIM0216 (specific TRPM3 agonists) dose-dependently reduced the frequency of spontaneous and NKA-evoked contractions, with IC50s of 241.7 µM and 4.4 µM, respectively. The inhibitory actions of TRPM3 agonists were mimicked by CGRP (10 to 100 nM) or a cAMP analogue (8-Br-cAMP; 1 mM). The inhibitory actions of TRPM3 agonists (300 µM PS or 30 µM CIM0216) were blocked by pretreatment with primidone (TRPM3 antagonist; 30 µM), tetrodotoxin (sodium channel blocker; 1 µM), olcegepant (CGRP receptor antagonist; 10 µM), or H89 (non-specific PKA inhibitor; 30 µM). TRPM3 was co-expressed with CGRP in nerves in the sub-urothelial and intermuscular regions of the ureter. SIGNIFICANCE: TRPM3 channels expressed on sensory terminals of the human ureter involve in inhibitory sensory neurotransmission and modulate ureter motility via the CGRP-cAMP-PKA signal pathway. Targeting TRPM3 may be a pharmacological strategy for promoting the ureter stone passage.

Effects of inhalation of sevoflurane at different concentrations on TRPV1 in airways of rats at different developmental stages.

Aim Determine changes in the expressions of the ion channel-TRPV1-and neuropeptides-NKA, NKB, calcitonin gene-related peptide (CGRP), and SP-in 14-, 21-, and 42-day-old rats after inhaling 1.5% and 2.6% sevoflurane. MAIN METHODS: A small in-house inhalation anesthesia chamber was designed to allow 14-, 21-, and 42-day-old rats inhale 1.5% and 2.6% sevoflurane, and rats in the control group inhaled carrier gas(1 L/min air +1 L/min O2). In addition, 14- and 21-day-old rats were pretreated with capsazepine, followed by inhalation of 1.5% and 2.6% sevoflurane or the carrier gas. The expression of TRPV1 in lung tissues was detected by Western blotting, whereas the expressions of NKA, NKB, CGRP, and SP in the trachea were detected by immunohistochemistry. KEY FINDINGS: After inhalation of 1.5% sevoflurane, the expression of TRPV1 in the lung tissues of 14- and 21-day-old rats was significantly increased compared with that in the control group, which was antagonized by capsazepine pretreatment. Moreover, inhalation of 1.5% sevoflurane markedly increased the expressions of NKA, NKB, CGRP, and SP in the trachea of 21-day-old rats and of NKB, CGRP, and SP in the trachea of 14-day-old rats. The expressions of these molecules were antagonized by capsazepine pretreatment. Conversely, inhalation of 2.6% sevoflurane decreased the expressions of NKA and NKB in the trachea of 42-day-old rats. SIGNIFICANCE: Sevoflurane did not upregulate the expression of TRPV1 in the airways of late-developing rats. This anesthetic may have a two-way effect on airways, resulting in considerable effects in pediatric clinical anesthesia management.

Neuroimmune/Hematopoietic Axis with Distinct Regulation by the High-Mobility Group Box 1 in Association with Tachykinin Peptides.

Hematopoiesis is tightly regulated by the bone marrow (BM) niche. The niche is robust, allowing for the return of hematopoietic homeostasis after insults such as infection. Hematopoiesis is partly regulated by soluble factors, such as neuropeptides, substance P (SP), and neurokinin A (NK-A), which mediate hematopoietic stimulation and inhibition, respectively. SP and NK-A are derived from the Tac1 gene that is alternately spliced into four variants. The hematopoietic effects of SP and NK-A are mostly mediated via BM stroma. Array analyses with 2400 genes indicated distinct changes in SP-stimulated BM stroma. Computational analyses indicated networks of genes with hematopoietic regulation. Included among these networks is the high-mobility group box 1 gene (HMGB1), a nonhistone chromatin-associated protein. Validation studies indicated that NK-A could reverse SP-mediated HMGB1 decrease. Long-term culture-initiating cell assay, with or without NK-A receptor antagonist (NK2), showed a suppressive effect of HMGB1 on hematopoietic progenitors and increase in long-term culture-initiating cell assay cells (primitive hematopoietic cells). These effects occurred partly through NK-A. NSG mice with human hematopoietic system injected with the HMGB1 antagonist glycyrrhizin verified the in vitro effects of HMGB1. Although the effects on myeloid lineage were suppressed, the results suggested a more complex effect on the lymphoid lineage. Clonogenic assay for CFU- granulocyte-monocyte suggested that HMGB1 may be required to prevent hematopoietic stem cell exhaustion to ensure immune homeostasis. In summary, this study showed how HMGB1 is linked to SP and NK-A to protect the most primitive hematopoietic cell and also to maintain immune/hematopoietic homeostasis.

Characterization of the Role of NKA in the Control of Puberty Onset and Gonadotropin Release in the Female Mouse.

The tachykinin neurokinin B (NKB, Tac2) is critical for proper GnRH release in mammals, however, the role of the other tachykinins, such as substance P (SP) and neurokinin A (NKA) in reproduction, is still not well understood. In this study, we demonstrate that NKA controls the timing of puberty onset (similar to NKB and SP) and stimulates LH release in adulthood through NKB-independent (but kisspeptin-dependent) mechanisms in the presence of sex steroids. Furthermore, this is achieved, at least in part, through the autosynaptic activation of Tac1 neurons, which express NK2R (Tacr2), the receptor for NKA. Conversely, in the absence of sex steroids, as observed in ovariectomy, NKA inhibits LH through a mechanism that requires the presence of functional receptors for NKB and dynorphin (NK3R and KOR, respectively). Moreover, the ability of NKA to modulate LH secretion is absent in Kiss1KO mice, suggesting that its action occurs upstream of Kiss1 neurons. Overall, we demonstrate that NKA signaling is a critical component in the central control of reproduction, by contributing to the indirect regulation of kisspeptin release.

Tachykinin upregulation in atopic dermatitis.

Context: Atopic dermatitis (AD) is a chronic, inflammatory, itching skin disorder, which may worsen due to stress, depression and anxiety. Tachykinins may be involved in inflammation signaling as well as they may have a role in stress, depression and anxiety. Objective: This study aimed to measure the expression of tachykinin markers, in the skin of patients with AD, and the correlation of these tachykinins with clinical and psychodemographic parameters. Materials and methods: Twenty-eight adult patients with AD were investigated regarding tachykinin expression in skin biopsies, using an immunohistochemical technique. The patients were characterized with clinical and psychodemographic parameters. Results: The number of substance P and neurokinin (NK)A positive nerve fibers, as well as NKA positive mononuclear dermal cells, was increased in lesional compared to non-lesional skin. Interestingly, the depression score and the number of dermal NK-1 receptor (R) positive cells in lesional as well as in non-lesional skin showed a correlation. Conclusion: These findings indicate an upregulation of the tachykinergic system in the inflamed skin of AD.

NKA enhances bladder-afferent mechanosensitivity via urothelial and detrusor activation.

Tachykinins are expressed within bladder-innervating sensory afferents and have been shown to generate detrusor contraction and trigger micturition. The release of tachykinins from these sensory afferents may also activate tachykinin receptors on the urothelium or sensory afferents directly. Here, we investigated the direct and indirect influence of tachykinins on mechanosensation by recording sensory signaling from the bladder during distension, urothelial transmitter release ex vivo, and direct responses to neurokinin A (NKA) on isolated mouse urothelial cells and bladder-innervating DRG neurons. Bath application of NKA induced concentration-dependent increases in bladder-afferent firing and intravesical pressure that were attenuated by nifedipine and by the NK2 receptor antagonist GR159897 (100 nM). Intravesical NKA significantly decreased bladder compliance but had no direct effect on mechanosensitivity to bladder distension (30 µl/min). GR159897 alone enhanced bladder compliance but had no effect on mechanosensation. Intravesical NKA enhanced both the amplitude and frequency of bladder micromotions during distension, which induced significant transient increases in afferent firing, and were abolished by GR159897. NKA increased intracellular calcium levels in primary urothelial cells but not bladder-innervating DRG neurons. Urothelial ATP release during bladder distention was unchanged in the presence of NKA, whereas acetylcholine levels were reduced. NKA-mediated activation of urothelial cells and enhancement of bladder micromotions are novel mechanisms for NK2 receptor-mediated modulation of bladder mechanosensation. These results suggest that NKA influences bladder afferent activity indirectly via changes in detrusor contraction and urothelial mediator release. Direct actions on sensory nerves are unlikely to contribute to the effects of NKA.

N-Acetylcysteine protects human bronchi by modulating the release of neurokinin A in an ex vivo model of COPD exacerbation.

AIMS: N-Acetylcysteine (NAC) reduces the risk of exacerbation of chronic obstructive pulmonary disease (COPD). Although NAC also has anti-inflammatory activity, the detailed mechanism leading to its protective role remains to be elucidated. We tested the impact of NAC against the effects of lipopolysaccharide (LPS) in an ex vivo model of COPD exacerbation, and investigated the role of neurokinin A (NKA) in this context. MAIN METHODS: Isolated airways from COPD patients were incubated overnight with LPS (100 ng/ml). NAC was tested at concentrations resembling the plasma levels elicited by oral administration of NAC at 200 mg/day (very low dose), 600 mg/day (low dose) and 1.200 mg/day (high dose). KEY FINDINGS: NAC at high concentrations normalized the peroxidase activity, H2O2, malondialdehyde (MDA), nitric oxide, glutathione (GSH), total antioxidant capacity (TAC), and interleukin 6 (IL-6) (overall change 34.32% ±â€¯4.22%, P < 0.05 vs. LPS-treated). NAC at low concentrations modulated peroxidase activity, H2O2, MDA, GSH, TAC, and IL-6 (overall change 34.88% ±â€¯7.39%, P < 0.05 vs. LPS-treated). NAC at very-low concentrations was effective on peroxidase activity, H2O2, GSH, and IL-6 (overall change 35.05 ±â€¯7.71%, P < 0.05 vs. LPS-treated). Binary logistic regression analysis indicated that the modulatory effect of NAC on NKA levels was associated with a reduction of pro-oxidant factors and IL-6, and selectively blocking the NK2 receptor abolished such an association. SIGNIFICANCE: This study demonstrates that, along with its well-known antioxidant activity, the protective effect of NAC against the detrimental effect of LPS is due to the modulation of NKA and IL-6 levels.

The Roles of Neurokinins and Endogenous Opioid Peptides in Control of Pulsatile LH Secretion.

Work over the last 15 years on the control of pulsatile LH secretion has focused largely on a set of neurons in the arcuate nucleus (ARC) that contains two stimulatory neuropeptides, critical for fertility in humans (kisspeptin and neurokinin B (NKB)) and the inhibitory endogenous opioid peptide (EOP), dynorphin, and are now known as KNDy (kisspeptin-NKB-dynorphin) neurons. In this review, we consider the role of each of the KNDy peptides in the generation of GnRH pulses and the negative feedback actions of ovarian steroids, with an emphasis on NKB and dynorphin. With regard to negative feedback, there appear to be important species differences. In sheep, progesterone inhibits GnRH pulse frequency by stimulating dynorphin release, and estradiol inhibits pulse amplitude by suppressing kisspeptin. In rodents, the role of KNDy neurons in estrogen negative feedback remains controversial, progesterone may inhibit GnRH via dynorphin, but the physiological significance of this action is unclear. In primates, an EOP, probably dynorphin, mediates progesterone negative feedback, and estrogen inhibits kisspeptin expression. In contrast, there is now compelling evidence from several species that kisspeptin is the output signal from KNDy neurons that drives GnRH release during a pulse and may also act within the KNDy network to affect pulse frequency. NKB is thought to act within this network to initiate each pulse, although there is some redundancy in tachykinin signaling in rodents. In ruminants, dynorphin terminates GnRH secretion at the end of pulse, most likely acting on both KNDy and GnRH neurons, but the data on the role of this EOP in rodents are conflicting.

Characterization of neuropeptide K processing in rat 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 levels, including the differential expression of protachykinin mRNA and the controlled secretion of tachykinin peptides from neurons. We suspect that proteolysis regulates extracellular neuropeptide K (NPK) and neurokinin A (NKA) concentrations and NPK is a precursor of NKA. Here, we provide evidence that proteolysis controls NPK and NKA levels in the spinal cord, leading to the formation of active C-terminal peptide fragments. Using high-resolution mass spectrometry, specific tachykinin fragments were identified and characterized. The metabolic stability in rat spinal cord fractions of NPK and NKA was very short, resulting in half-lives of 1.9 and 2.2 min respectively. Following the degradation of NPK, several C-terminal fragments were identified, including NPK1-26 , NKA, NKA2-10 , NKA3-10 , NKA5-10 and NKA6-10 , which conserve affinity for the neurokinin 2 receptor but also for the neurokinin 1 receptor. Interestingly, the same fragments were identified following the degradation of NKA. A specific proprotein convertases inhibitor was used and showed a significant reduction in the rate of formation of NKA, providing strong evidence that proprotein convertase is involved in C-terminal processing of NPK in the spinal cord, leading to the formation of NKA.

Effect of lipopolysaccharide on the responsiveness of equine bronchial tissue.

Recurrent airway obstruction (RAO) is a main characteristic of horses with severe equine asthma syndrome. The presence of bacterial lipopolysaccharide (LPS) in the airways of horses is thought to play a crucial role in the clinical expression of this disorder. This study pharmacologically characterized the effect of LPS on the responsiveness of equine bronchial tissue. Equine isolated bronchi were incubated overnight with LPS (0.1-100 ng/ml) and then stimulated by electrical field stimulation (EFS). The role of capsaicin sensitive-sensory nerves (capsaicin desensitization treatment), neurokinin-2 (NK2) receptors (blocked by GR159897), transient receptor potential vanilloid type 1 receptors (TRPV1; blocked by SB366791), and neurokinin A (NKA) were investigated. Untreated bronchi were used as control tissues. LPS (1 ng/ml) significantly increased the EFS-evoked contractility of equine bronchi compared with control tissues (+742 ± 123 mg; P < 0.001). At higher concentrations LPS induced desensitization to airways hyperresponsiveness (AHR; EC50: 5.9 ±â€¯2.6 ng/ml). Capsaicin desensitization and GR159897 significantly prevented AHR induced by LPS at EFS1-50Hz (-197 ±â€¯25%; P < 0.01). SB366791 inhibited AHR at very low EFS frequency (EFS1Hz -193 ±â€¯29%; P < 0.01 vs. LPS-treated bronchi). LPS (1 ng/ml) significantly (P < 0.01) increased 3.7 ±â€¯0.7 fold the release of NKA compared with control bronchi. LPS induces biphasic dysfunctional bronchial contractility due to the stimulation of capsaicin sensitive-sensory nerves, increased release of NKA, and activation of NK2 receptors, whereas TRPV1 receptors appear to play a marginal role in this response. The overnight challenge with low concentrations of LPS represents a suitable model to investigate pharmacological options that may be of value in the treatment of equine RAO.

From receptor binding kinetics to signal transduction; a missing link in predicting in vivo drug-action.

An important question in drug discovery is how to overcome the significant challenge of high drug attrition rates due to lack of efficacy and safety. A missing link in the understanding of determinants for drug efficacy is the relation between drug-target binding kinetics and signal transduction, particularly in the physiological context of (multiple) endogenous ligands. We hypothesized that the kinetic binding parameters of both drug and endogenous ligand play a crucial role in determining cellular responses, using the NK1 receptor as a model system. We demonstrated that the binding kinetics of both antagonists (DFA and aprepitant) and endogenous agonists (NKA and SP) have significantly different effects on signal transduction profiles, i.e. potency values, in vitro efficacy values and onset rate of signal transduction. The antagonistic effects were most efficacious with slowly dissociating aprepitant and slowly associating NKA while the combination of rapidly dissociating DFA and rapidly associating SP had less significant effects on the signal transduction profiles. These results were consistent throughout different kinetic assays and cellular backgrounds. We conclude that knowledge of the relationship between in vitro drug-target binding kinetics and cellular responses is important to ultimately improve the understanding of drug efficacy in vivo.

Renal sodium handling and blood pressure changes in gestational protein-restricted offspring: Role of renal nerves and ganglia neurokinin expression.

BACKGROUND: Considering long-term changes in renal sodium handling and blood pressure in maternal protein-restricted (LP) offspring, we assumed that the development of LP hypertension results from abnormal dorsal root ganglia (DRG) neurokinin expression associated with impaired responsiveness of renal sensory receptors, promoting a reduced urinary excretion of sodium. The present study investigates whether increased blood pressure in protein-restricted offspring would be associated with changes in the DRG cells and in renal pelvic wall expression of NK1R, SP and CGRP when compared to NP offspring. In addition, we assessed the tubular sodium handling, estimated by creatinine and lithium clearances before and after bilateral renal denervation in conscious LP offspring relative to age-matched NP counterparts. METHODS: Dams received a normal (NP) or low-protein diet (LP) during their entire pregnancy period. Male NP or LP offspring underwent bilateral surgical renal denervation before the 8-week renal functional test and blood pressure measurements. Immunofluorescence staining in DRG cells was assessed in optical sections by confocal laser scanning microscope. RESULTS: The current data demonstrated a sustained rise in blood pressure associated with a decrease in fractional excretion of sodium (FENa) by reducing post-proximal tubule sodium rejection in 16-wk old LP rats relative to age-matched NP counterparts. According to this study, bilateral renal denervation attenuated blood pressure and increased FENa in LP offspring. Furthermore, an immunohistochemical analysis showed a reduced expression of SP and CGRP in DRGs of LP when compared with NP rats. Renal pelvis of LP rats did not show a strong CGRP expression related to NP rats, whereas there was no change in SP immunostaining. CONCLUSIONS: These observations raise the possibility that impaired DRG and pelvic neurokinin expression associated with responsiveness of renal sensory receptors in 16-wk old LP offspring are conducive to excess renal reabsorption of sodium and development of hypertension in this programmed model.

Targeted high-resolution quadrupole-Orbitrap mass spectrometry analyses reveal a significant reduction of tachykinin and opioid neuropeptides level in PC1 and PC2 mutant mouse spinal cords.

Tachykinin and opioid neuropeptides play a fundamental role in pain transmission, modulation and inhibition. The proteolysis control of endogenous tachykinin and opioid neuropeptides has a significant impact on pain perception. The role of proprotein convertases (PCs) in the proteolysis of proneuropeptides was previously established but very few studies have shown the direct impact of PCs on the regulation of specific tachykinin and opioid peptides in the central nervous system. There is an increasing interest in the therapeutic targeting of PCs for the treatment of pain but it is imperative to assess the impact of PCs on the pronociceptive and the endogenous opioid systems. The objective of this study was to determine the relative concentration of targeted neuropeptides in the spinal cord of WT, PC1-/+ and PC2-/+ animals to establish the impact of a restricted PCs activity on the regulation of specific neuropeptides. The analysis of tachykinin and opioid neuropeptides were performed on a HPLC-MS/MS (High-Resolution Quadrupole-Orbitrap Mass Spectrometer). The results revealed a significant decrease of Dyn A (p<0.01), Leu-Enk (p<0.001), Met-Enk (p<0.001), Tach58-71 (p<0.05), SP (p<0.01) and NKA (p<0.001) concentrations in both, PC1-/+ and PC2-/+ animals. Therefore, the modulation of PCs activity has an important impact on specific pronociceptive peptides (SP and NKA), but the results also shown that endogenous opioid system is hindered and consequently it will affect significantly the pain modulatory pathways. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies.

Tac1 Signaling Is Required for Sexual Maturation and Responsiveness of GnRH Neurons to Kisspeptin in the Male Mouse.

The tachykinins substance P (SP) and neurokinin A (Tac1) have emerged as novel regulators of kisspeptin/GnRH release. Recently, we documented that SP modulates reproductive function in the female mouse. Here, we extended this characterization to the male mouse. Tac1-/- male mice showed delayed puberty onset. They also presented significantly decreased expression levels of Pdyn (dynorphin) and Nos1 (nitric oxide synthase) in the mediobasal hypothalamus and elevated Gnrh1 levels. Unexpectedly, the response of Tac1-/- mice to central kisspeptin or senktide (neurokinin B receptor-agonist) administration was significantly decreased compared with controls, despite the preserved ability of GnRH neurons to stimulate luteinizing hormone release as demonstrated by central N-methyl-D-aspartate receptor administration, suggesting a deficit at the GnRH neuron level. Importantly, we demonstrated that kisspeptin receptor and SP receptor (NK1R) heterodimerize, indicating that changes in the SP tone could alter the responsiveness of GnRH neurons to kisspeptin. Finally, electrophysiological recordings from arcuate Kiss1 neurons showed that, although virtually all Kiss1 neurons responded to NKB and senktide, only half responded to an NK1R agonist and none to the neurokinin A receptor agonist at a 1-µM dose. In summary, we provide compelling evidence for a role of Tac1 in the control of reproductive function in the male mouse, suggesting a predominant central action that may involve a change in the balance of neural factors that control GnRH expression.

Neuromedin: An insight into its types, receptors and therapeutic opportunities.

Neuropeptides are small protein used by neurons in signal communications. Neuromedin U was the first neuropeptide discovered from the porcine spinal and showed its potent constricting activities on uterus hence was entitled with neuromedin U. Following neuromedin U another of its isoform was discovered neuromedin S which was observed in suprachiasmatic nucleus hence was entitled neuromedin S. Neuromedin K and neuromedin L are of kanassin class which belong to tachykinin family. Bombesin family consists of neuromedin B and neuromedin C. All these different neuromedins have various physiological roles like constrictive effects on the smooth muscles, control of blood pressure, pain sensations, hunger, bone metastasis and release and regulation of hormones. Over the years various newer physiological roles have been observed thus opening ways for various novel therapeutic treatments. This review aims to provide an overview of important different types of neuromedin, their receptors, signal transduction mechanism and implications for various diseases.

Kinetic binding and activation profiles of endogenous tachykinins targeting the NK1 receptor.

Ligand-receptor binding kinetics (i.e. association and dissociation rates) are emerging as important parameters for drug efficacy in vivo. Awareness of the kinetic behavior of endogenous ligands is pivotal, as drugs often have to compete with those. The binding kinetics of neurokinin 1 (NK1) receptor antagonists have been widely investigated while binding kinetics of endogenous tachykinins have hardly been reported, if at all. Therefore, the aim of this research was to investigate the binding kinetics of endogenous tachykinins and derivatives thereof and their role in the activation of the NK1 receptor. We determined the binding kinetics of seven tachykinins targeting the NK1 receptor. Dissociation rate constants (koff) ranged from 0.026±0.0029min-1 (Sar9,Met(O2)11-SP) to 0.21±0.015min-1 (septide). Association rate constants (kon) were more diverse: substance P (SP) associated the fastest with a kon value of 0.24±0.046nM-1min-1 while neurokinin A (NKA) had the slowest association rate constant of 0.001±0.0002nM-1min-1. Kinetic binding parameters were highly correlated with potency and maximal response values determined in label-free impedance-based experiments on U-251 MG cells. Our research demonstrates large variations in binding kinetics of tachykinins which correlate to receptor activation. These findings provide new insights into the ligand-receptor interactions of tachykinins and underline the importance of measuring binding kinetics of both drug candidates and competing endogenous ligands.