Neurokinin-1 Binding Research Articles

Immunolocalization of Full-length NK1 Tachykinin Receptors in Human Tumors

Biological effects of substance P (SP) are mediated by the neurokinin-1 (NK1) receptor that exists as a full-length and as a carboxy-terminally truncated isoform in humans. Although NK1 receptor mRNA and binding sites have been detected in certain malignancies, little is known about the cellular and subcellular localization of NK1 receptor protein in human neoplastic tissues. We developed and characterized a novel anti-peptide antibody to the carboxy-terminal region of the human full-length NK1 receptor. Specificity of the antiserum was demonstrated by (1) detection of a broad band migrating at molecular mass 70,000-90,000 Da in Western blots of membranes from NK1-expressing tissues; (2) cell-surface staining of NK1-transfected cells; (3) translocation of NK1 receptor immunostaining after SP exposure; and (4) abolition of tissue immunostaining by preadsorption of the antibody with its immunizing peptide. Distribution of NK1 receptors was investigated in 72 formalin-fixed, paraffin-embedded human tumors showing that NK1 receptors were frequently expressed in glioblastomas and breast and pancreatic carcinomas. Immunoreactive NK1 receptors were clearly confined to the plasma membrane and uniformly present on nearly all tumor cells. Development of this novel NK1 receptor antibody allows the efficient localization of NK1 receptor protein in human formalin-fixed, paraffin-embedded tissues. NK1 receptor visualization with this simple and rapid immunohistochemical method will facilitate identification of tumors with a sufficient receptor overexpression for diagnostic or therapeutic intervention using SP analogs.

Down‐regulation of amygdala preprotachykinin A mRNA but not 3H‐SP receptor binding sites in subjects affected by mood disorders and schizophrenia

Increasing evidence suggests that substance P (SP) neurokinin-1 (NK1) receptors are involved in stress and emotional responses, representing a potential target for the treatment of anxiety and depression in humans. Given the important role of the amygdaloid complex in the regulation of emotional behavior, we examined the mRNA levels of preprotachykinin A [PPT-A, a precursor of both SP and neurokinin A (NKA)] and 3H-SP binding sites in the amygdala of patients affected by bipolar disorder, major depression or schizophrenia as compared with matched control individuals. By means of in situ hybridization, a significant reduction of PPT-A mRNA expression levels was detected in the three diagnostic groups, mainly in the basal, lateral and accessory basal amygdaloid nuclei, but not in the temporal cortical area proximal to the amygdala. Receptor autoradiography performed on adjacent sections showed no change in 3H-SP binding sites as compared with controls. No significant correlation was found between levels of PPT-A mRNA expression or binding sites and subject age, gender, hemisphere side, cause of death or history of substance misuse (marijuana, alcohol, cocaine/amphetamine). An inverse relationship between PPT-A mRNA expression levels and lifetime antipsychotic treatment (Fluphenazine) in the schizophrenic and bipolar disorder groups was found. Post-mortem delay was also negatively correlated with NK1 binding sites. The results confirm an involvement of the tachykinins in psychiatric disorders, suggesting there is a generalized impairment of the SP system in the amygdala in mood disorders and schizophrenia rather than this being a disease-related phenomenon.

Regional decreases in NK-3, but not NK-1 tachykinin receptor binding in dystonic hamster ( dtsz) brains

Although the pathophysiology of primary dystonias is currently unknown, it is thought to involve changes in the basal ganglia–thalamus–cortex circuit, particularly activity imbalances between direct and indirect striatal pathways. Substance P, a member of the tachykinin family of neuropeptides, is a major component in the direct pathway from striatum to basal ganglia output nuclei. In the present study quantitative autoradiography was used to examine changes in neurokinin-1 (NK-1) and neurokinin-3 (NK-3) receptors in mutant dystonic hamsters ( dt sz), a well characterized model of paroxysmal dystonia. NK-1 receptors were labeled in 10 dystonic brains and 10 age-matched controls with 3 nM [ 3H]-[Sar 9, Met(O 2) 11]-SP. NK-3 binding sites were labeled in adjacent sections with 2.5 nM [ 3H]senktide. NK-1 binding was found to be unaltered in 27 brain areas examined. In contrast, NK-3 binding was significantly reduced in layers 4 and 5 of the prefrontal (−46%), anterior cingulate (−42%) and parietal (−45%) cortices, ventromedial thalamus (−42%) and substantia nigra pars compacta (−36%) in dystonic brains compared to controls. The latter effects may be particularly relevant in view of evidence that activation of NK-3 receptors on dopaminergic neurons in the substantia nigra pars compacta can increase nigrostriatal dopaminergic activity. Since previous studies indicated that a reduced basal ganglia output in mutant hamsters is based on an overactivity of the direct pathway which also innervates substantia nigra pars compacta neurons, the decreased NK-3 binding could be related to a receptor down-regulation. The present finding of decreased NK-3 receptor density in the substantia nigra pars compacta, thalamic and cortical areas substantiates the hypothesis that disturbances of the basal ganglia–thalamus–cortex circuit play a critical role in the pathogenesis of paroxysmal dystonia.

Gene therapy for the management of pain: part II: molecular targets.

TREATMENT of chronic pain, particularly of neuropathic etiology, is extremely difficult and resistant to many available pharmacologic therapies. Current analgesic agents may be limited with regard to analgesic efficacy or side effects. Newer and experimental pharmacologic agents may also have significant limitations. By targeting a specific receptor or other specific protein targets, a gene therapy approach to the treatment of pain may provide greater analgesic efficacy without the limitations associated with current pharmacotherapy. Advances in the field of gene therapy, along with significant increases in our understanding of the neurobiology of nociception and knowledge of the fundamental genetic structure of many nociceptive targets, have made gene therapy for the management of pain a conceivable reality. In part I of this review, we introduced the basic concepts of gene therapy with an emphasis on the available tools (e.g., viral vectors and antisense oligonucleotides) and strategies for upregulating antinociceptive or downregulating pronociceptive targets. In part II, we summarize current knowledge regarding the nociceptive role, molecular biology, and antisense and knockout data of several novel nociceptive targets for gene therapy. We base our selection of the targets included in this review on the three aforementioned criteria. The targets selected are the best characterized and, in our opinion, most likely amenable to the gene therapeutic approach. A simple but feasible strategy and potential gene therapy targets for the management of pain are summarized in figure 1. However, the list is admittedly incomplete, and the readers are referred to other recent reviews cited in part I of this review for a broader perspective on potential targets for the management of pain.

An N-terminal fragment of substance P, substance P(1–7), down-regulates neurokinin-1 binding in the mouse spinal cord

Injected intrathecally, substance P (SP) down-regulates neurokinin-1 (NK-1) binding in the spinal cord and desensitizes rats to the behavioral effect of SP. N-terminal fragments of SP, such as SP(1–7), induce antinociception and play a role in desensitization to SP in mice. The goal of this study was to assess the abilities of N- and C-terminal fragments of SP to down-regulate NK-1 binding. Binding of [ 3H]SP to mouse spinal cord membranes was inhibited by SP, CP-96,345, and to a lesser extent by SP(5–11), but not SP(1–7), consistent with these binding sites being NK-1 receptors. Injection of SP(5–11) intrathecally did not affect the affinity ( K d) or concentration ( B max) of [ 3H]SP binding. However, injection of 1 nmol of SP(1–7) decreased the B max of [ 3H]SP binding in the spinal cord at 6 h after its injection just as this dose of SP decreased the B max at 24 h. These data suggest that the N-terminus of SP is responsible for down-regulation of NK-1 binding. As SP(5–11) did not down-regulate NK-1 binding, activation of NK-1 sites does not appear necessary or sufficient for down-regulation of SP binding. In contrast, SP(1–7), in spite of its inability to interact with NK-1 sites, did down-regulate SP binding, suggesting an indirect mechanism dissociated from NK-1 receptors.

Binding sites for [ 3H][Sar 9, Met(O 2) 11]substance P in rat bain and guinea pig ileum

[ 3H][Sar 9, Met(O 2) 11]substance P (SP) with high specific activity (32 Ci/mmmol) was used to study neurokinin-1 (NK-1) binding sites on rat brain and smooth muscle membranes of the guinea pig ileum. The specific binding of [ 3H][Sar 9, Met(O 2) 11]SP was shown to be saturable, reversible and increased in parallel with the protein concentration. Scatchard analyses of equilibrium binding experiments revealed that [ 3H][Sar 9, Met(O 2) 11]SP binds to a class of non-interacting binding sites in rat brain membranes ( K d = 2nM, B max = 56fmol/mg of protein ) and ileum muscle membranes ( K d = 2nM, B max = 194fmol/mg of protein) . Competition of [ 3H][Sar 9,Met(O 2) 11]SP, 125-BH[Sar 9, Met(O 2) 11]SP and 125I-BH·SP with different tachykinin-related peptides gave the following rank order of potencies: SP>physalaemin> [Sar 9,Met(O 2) 11]SP> N-Ac[Arg 6,Sar 9,Met(O 2) 11]SP(6–11) >neurokinin A (NKA)≥eledoisin≥neurokinin B (NKB)> [MePhe 7]NKB(4–10) > [β-Ala 8]NKA(4–10) . A very similar pattern was observed on ileum muscle membranes. [ 3H][Sar 9, Met(O 2) 11]SP was found to be highly selective for NK-1 binding sites in rat brain and in the intestinal tissue. Binding showed good correlation with the biological activity of tachykinins and related peptides. From these data it can be suggested that (a) the NK-1 receptor characterized in the central nervous system is identical to the one in the periphery, (b) the NK-1 binding site of the muscle membranes appears to be similar to the contractile receptor of the guinea pig ileum and (c) the functional site mediating relaxation of the dog carotid artery is similar to the contractile receptor of the guinea pig ileum.