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The present study focused on the interactive pain regulation of endokinin A/B (EKA/B, the common C-terminal decapeptide in EKA and EKB) or endokinin C/D (EKC/D, the common C-terminal duodecapeptide in EKC and EKD) on chimeric peptide MCRT (YPFPFRTic-N[H.sub.2], based on YPFP-N[H.sub.2] and PFRTic-N[H.sub.2]) at the supraspinal level in mice. Results demonstrated that the co-injection of nanomolar EKA/B and MCRT showed moderate regulation, whereas 30 µmol EKA/B had no effect on MCRT. The combination of EKC/D and MCRT produced enhanced antinociception, which was nearly equal to the sum of the mathematical values of single EKC/D and MCRT. Mechanism studies revealed that pre-injected naloxone attenuated the combination significantly compared with the equivalent analgesic effects of EKC/D alone, suggesting that EKC/D and MCRT might act on two totally independent pathways. Moreover, based on the above results and previous reports, we made two reasonable hypotheses to explain the cocktail-induced analgesia, which may potentially pave the way to explore the respective regulatory mechanisms of EKA/B, EKC/D, and MCRT and to better understand the complicated pain regulation of [NK.sub.1] and μ opioid receptors, as follows: (1) MCRT and endomorphin-1 possibly activated different μ subtypes; and (2) picomolar EKA/B might motivate the endogenous NPFF system after [NK.sub.1] activation. Key words: endokinin A/B, endokinin C/D, MCRT, [NK.sub.1] receptor, opioid receptors, pain regulation, co-injection. L'etude presente s'est concentree sur la regulation de la douleur par l'interaction de l'endokinine A/B (EKA/B, le decapeptide C-terminal commun a EKA et EKB) ou de l'endokinine C/D (EKC/D, le duodecapeptide commun a EKC et EKD) et du peptide chimere MCRT (YPFPFRTic-N[H.sub.2], base sur YPFP-N[H.sub.2] et PFRTic-N[H.sub.2]) au niveau supra-spinal chez la souris. Les resultats ont demontre que la co-injection de quantites nanomolaires d'EKA/B et de MCRT generait une regulation moderee, alors que 30 µmol d'EKA/B n'avaient pas d'effet sur MCRT. La combinaison d'EKC/D et de MCRT produisait un effet anti-nocicepteur accru, qui etait presqu'egal a la somme des valeurs mathematiques d'EKC/D et MCRT pris individuellement. Des etudes des mecanismes sous-jacents ont revele qu'une pre-injection de naloxone attenuait l'effet de la combinaison de facon significative, avec des effets analgesiques equivalents a ceux d'EKC/D seul, suggerant que EKC/D et MCRT pourraient agir sur deux sentiers totalement independants. De plus, selon les resultats presentes ci-dessus et des rapports precedents, les auteurs ont propose deux hypotheses raisonnables afin d'expliquer l'analgesie induite par le cocktail, qui pourraient potentiellement paver la voie a l'exploration des mecanismes regulateurs respectifs d'EKA/B, EKC/D et MCRT, et mieux comprendre la regulation complexe de la douleur par les recepteurs [NK.sub.1] et opioides μ: (1) MCRT et l'endomorphine-1 ont possiblement active differents sous-types de recepteurs μ; et (2) des quantites picomolaires d'EKA/B pourraient mobiliser le systeme NPFF endogene apres l'activation de [NK.sub.1]. [Traduit par la Redaction] Mots-cles: endokinine A/B, endokinine C/D, MCRT, recepteur [NK.sub.1], recepteurs d'opioides, regulation de la douleur, co-injection., Introduction The tachykinin peptide family, which includes more than 40 isolated tachykinins, was considered as one of the largest peptide families in the animal organism (Severini et al. 2002). Their [...]

The neuropeptide substance P manifests its biological functions through ligation of a G protein-coupled receptor, the NK1R. Mice with targeted deletion of this receptor reveal a preponderance of proinflammatory properties resulting from ligand activation, demonstrating a neurogenic component to multiple forms of inflammation and injury. We hypothesized that NK1R deficiency would afford a similar protection from inflammation associated with hyperoxia. Counter to our expectations, however, NK1R-/- animals suffered significantly worse lung injury compared with wild-type mice following exposure to 90% oxygen. Median survival was shortened to 84 h for NK1R-/- mice from 120 h for wild-type animals. Infiltration of inflammatory cells into the lungs was significantly increased; NK1R-/- animals also exhibited increased pulmonary edema, hemorrhage, and bronchoalveolar lavage fluid protein levels. TdT-mediated dUTP nick end labeling (TUNEL) staining was significantly elevated in NK1R-/- animals following hyperoxia. Furthermore, induction of metallothionein and [Na.sup.+]-[K.sup.+]-ATPase was accelerated in NK1R-/- compared with wild-type mice, consistent with increased oxidative injury and edema. In cultured mouse lung epithelial cells in 95% [O.sub.2], however, addition of substance P promoted cell death, suggesting the neurogenic component of hyperoxic lung injury is mediated by additional mechanisms in vivo. Release of bioactive constituents including substance P from sensory neurons results from activation of the vanilloid receptor, TRPV1. In mice with targeted deletion of the TRPV1 gene, acute hyperoxic injury is attenuated relative to NK1R-/- animals. Our findings thus reveal a major neurogenic mechanism in acute hyperoxic lung injury and demonstrate concerted actions of sensory neurotransmitters revealing significant protection for NKIR-mediated functions. tachykinins; neurogenic inflammation; oxidative lung injury; transient receptor potential vanilloid type 1; TRPV1 doi: 10.1152/ajplung.90509.2008

The primary afferent neurotransmitter triggering the spinal micturition reflex after complete spinal cord injury (SCI) in the rat is unknown. Substance P detected immunohistochemically in the sacral parasympathetic nucleus was significantly higher in 12 SCI rats than in 12 spinally intact rats (P = 0.008), suggesting substance P as a plausible candidate for the primary afferent neurotransmitter. The effects of the tachykinin NK1 receptor antagonist L-733060 on the spinal micturition reflex were then determined by performing conscious cystometry in an additional 14 intact rats and 14 SCI rats with L-733060 (0.1-100 [micro]g) administered intrathecally at L6-S1. L-733060 was without effect in intact rats, but blocked the spinal micturition reflex in 10 of 14 SCI rats and increased the intermicturition interval in 2 of 4 others at doses ranging from 10 to 100 [micro]g. Both phasic and nonphasic voiding contractions, differentiated according to the presence of phasic external urethral sphincter (EUS) activity, were present in most SCI rats. Both types of contractions were blocked by high doses of L-733060. Interestingly, there was a relative decline in phasic voiding contractions at high doses as well as a decline in contraction amplitude in nonphasic voiding contractions. In other respects, cystometric variables were largely unaffected in either spinally intact or SCI rats. L-733060 did not affect tonic EUS activity at any dose except when the spinal micturition reflex was blocked and tonic activity was consequently lost. These experiments show that tachykinin action at spinal NK1 receptors plays a major role in the spinal micturition reflex in SCI rats. spinal cord injury; bladder; tachykinins; external urethral sphincter

The tachykinin peptide bufokinin, isolated from the cane toad intestine, is important in intestinal and cardiovascular regulation in the toad. In this study, three tachykinin N[K.sub.1]-like receptor isoforms, bN[K.sub.1]-A, bN[K.sub.1]-B, and bN[K.sub.1]-C, encoding proteins of 309, 390, and 371 amino acids, respectively, were cloned from the toad brain and intestine. These isoforms differ only at the intracellular COOH terminus. The bN[K.sub.1]-A and bN[K.sub.1]-B isoforms are similar to the truncated and full-length forms of the mammalian N[K.sub.1] receptor, whereas bN[K.sub.1]-C is unique and does not correspond to any previously described receptor. RT-PCR studies demonstrated that three isoform transcripts are widely distributed in the toad with high expression in gut, spinal cord, brain, lung, and skeletal muscle. When expressed in COS-7 cells, bufokinin showed similar high affinity (I[C.sub.50] 0.6-0.8 nM) in competing for [sup.125]I-labeled Bolton-Hunter bufokinin binding at all receptors, but the binding affinities of substance P (SP) and neurokinin A (NKA) were very different at each isoform. When expressed in Xenopus oocytes, the truncated isoform, bN[K.sub.1]-A, was inactive, whereas bN[K.sup.1]-B and bN[K.sub.1]-C produced changes in chloride current when stimulated by tachykinins (minimum concentrations: bufokinin, 0.1 nM; SP, 1 nM; and NKA, 10 nM). A marked desensitization of the response was seen to subsequent applications of tachykinins, as experienced by the mammalian N[K.sub.1] receptor. In summary, our study describing three isoforms of N[K.sub.1]-like receptor from the toad suggests that the alternative splicing of N[K.sub.1] receptor is a physiologically conserved mechanism and raises a fundamental question as to the physiological role of each isoform. bufokinin; tachykinin receptors; sequencing and cloning; Xenopus oocyte expression; amphibian

The health burden of stress-related diseases, including depression and anxiety disorders, is rapidly increasing, whereas the range of available pharmacotherapies to treat these disorders is limited and suboptimal with regard to efficacy and tolerability. Recent findings support a major role for neuropeptides in mediating the response to stress and thereby identify neuropeptide systems as potential novel therapeutic targets for the treatment of depression and anxiety disorders. In preclinical models, pharmacological and/or genetic manipulation of substance P, corticotropin-releasing factor (CRF), vasopressin, neuropeptide Y and galanin function alters anxiety- and depression-related responses. Recently, specific and highly potent small-molecule neuropeptide receptor agonists and antagonists have been developed that can readily cross the blood--brain barrier. Clinical assessment of several compounds is currently underway, with antidepressant efficacy confirmed in double-blind, placebo-controlled trials of tachykinin N[K.sub.1] (substance P) receptor antagonists, and preliminary evidence of antidepressant activity in an open-label trial of a CR[F.sub.1] receptor antagonist.

Tachykinins interact with three neurokinin receptors (NKRs) that are often coexpressed by the same cell. Cellular responses to tachykinins depend on the NKR subtype that is activated. We compared the colocalization of NK1R and NK3R with [beta]-arrestins 1 and 2, which play major roles in receptor desensitization, endocytosis, and signaling. In cells expressing NK1R, the selective agonist SarMet-substance P induced rapid translocation of [beta]-arrestins 1 and 2 from the cytosol to the plasma membrane and then endosomes, indicative of interaction with both isoforms. In contrast, the NK3R interacted transiently with only [beta]-arrestin 2 at the plasma membrane. Despite these differences, both NK1R and NK3R similarly desensitized, internalized, and activated MAP kinases. Because interactions with [beta]-arrestins can explain differences in the rate of receptor resensitization, we compared resensitization of agonist-induced [Ca.sup.2+] mobilization. The NK1R resensitized greater than two-fold more slowly than the NK3R. Replacement of intracellular loop 3 and the COOH tail of the NK1R with comparable domains of the NK3R diminished colocalization of the NK1R with [beta]-arrestin 1 and accelerated resensitization to that of the NK3R. Thus loop 3 and the COOH tail specify colocalization of the NK1R with [beta]-arrestin 1 and determine the rate of resensitization. desensitization; endocytosis; tachykinins

The amyloid β protein is deposited in the brains of patients with Alzheimer's disease but its pathogenic role is unknown. In culture, the amyloid β protein was neurotropic to undifferentiated [...]