A monoclonal antibody to the ligand-binding domain of the neurokinin 1 receptor (NK1-R) for the neuropeptide substance P

For PCR-based genotyping using polymorphic microsatellite markers, DNA from decomposed postmortem human tissues was fractionated into six groups according to molecular size. The minimum required amounts of this degraded DNA, for detecting alleles at five microsatellite loci (ACTBP2, CMAG, HUMTH01, CYP19, and LPL) and one minisatellite locus (MCT118) were investigated respectively. The allele patterns were detected by electrophoresis of the PCR products on a 6%-denaturing polyacrylamide gel following silver staining. The detection of alleles for the loci with large allele size required more template DNA with higher molecular size than for that with small allele size. Amounts from 0.3 ng to 5 ng were needed for allele detection on genomic DNA from fresh blood. When the decomposed DNA mixture was used as the template, approximately ten times the amount of genomic DNA was required to detect alleles at the three loci of LPL, CYP19 and HUMTH01, while 24 to 67 times was required for the loci, CMAG, ACTBP2 and MCT118. It was demonstrated that a minimum molecular, size and amount of template DNA was needed for amplifying alleles of the six loci, and degraded DNA less than minimum size in the samples would prevent the detection of the loci which have large allele size.     

Sparing of striatal neurons coexpressing calretinin and substance P (NK1) receptor in Huntington's disease

Immunohistochemical studies of the striatum in normal human subjects with a double-antigen localization method have revealed the presence of large and medium-sized aspiny neurons displaying immunoreactivity for both the calcium-binding protein calretinin and substance P (neurokinin-1) receptor. These large and medium-sized cells from two distinct classes of striatal interneurons, which together represent less than 3% of the total neuronal population of the human striatum. Observations made in four cases of Huntington's disease revealed that such doubly labeled interneurons are still present in the striatum of these patients, despite the marked atrophy of the structure. This study provides the first evidence for the existence of interneurons containing calretinin and expressing tachykinin receptors in the human striatum. It also demonstrates the selective sparing of these chemospecific striatal neurons in Huntington's disease.     

Differential expression of two isoforms of the neurokinin-1 (substance P) receptor in vivo

Recent pharmacological and biochemical studies have suggested that there may be more than one molecular form of the neurokinin-1 receptor (NK-1), a long and short isoform differing in the length of their cytoplasmic carboxyl-terminal tails, but no definitive evidence of the existence of such NK-1 receptor isoforms in tissue has been presented. To examine whether these different isoforms are expressed in vivo we have compared the distribution of high affinity substance P (SP) binding sites (visualized by autoradiography with [125I]SP), with the distribution of the C-terminal epitope of the full length receptor (visualized with a specific antibody against the extreme C-terminal sequence). The former method labels both long and short forms of the NK-1 receptor, while the latter labels only the long form of the protein. In the rat there is a close correspondence of [125I]SP binding and NK-1 immunoreactivity in the striatum, suggesting that the long isoform predominates in this tissue. In the parotid and submaxillary gland, there are very high levels of [125I]SP binding but only low levels of NK-1 immunoreactivity, suggesting that expression of the short form predominates in these tissues. These results imply that different tissues express different ratios of the two isoforms of the NK-1 receptor. This differential expression provides the theoretical basis for tissue specific pharmacological targeting of NK-1 receptors.     

Expression of substance P (NK1) receptor mRNA in human nose

A capillary zone electrophoretic method (CZE) was developed using an uncoated fused silica capillary for the separation and determination of the main tropane alkaloids. The applicability of the developed method for analysis of plant samples was examined by analyzing samples of transgenic Egyptian henbane Hyoscyamus muticus (L.) plants. A simple 40 mM phosphate buffer at pH 7.8 using a voltage of 20 kV was found the best for this purpose. The main tropane alkaloids, atropine and scopolamine as well as nor-(-)-scopolamine, and tropic acid, the precursor of tropane alkaloids, could be separated in less than 13 min. The linear concentration range for atropine was 5.00-140 microg ml(-1), for scopolamine 7.50-210 microg ml(-1) and for tropic acid 2.50-70.0 microg ml(-1).     

4-Alkylpiperidines related to SR-48968: potent antagonists of the neurokinin-2 (NK2) receptor

A series of 4-alkylpiperidine derivatives related to the potent neurokinin-2 (NK2) receptor antagonist SR-48968 (1) is described. Simple aliphatic derivatives were found to be poorly active, but appropriate placement of an alcohol functional group afforded compounds that were of similar activity to 1. Several representatives in this series, such as the 4-(1-hydroxy-1-ethylpropyl)piperidine (14), were found to exhibit oral activity in a model of labored abdominal breathing in guinea pigs. These results expand the latitude of substituents available in this region of this series of NK2 receptor antagonists.     

A substance P (neurokinin-1) receptor mutant carboxyl-terminally truncated to resemble a naturally occurring receptor isoform displays enhanced responsiveness and resistance to desensitization

Two isoforms of the substance P (SP) receptor, differing in the length of the cytoplasmic carboxyl-terminus by approximately 8 kDa, have been detected previously in rat salivary glands and other tissues. The binding and functional properties of these two isoforms have been investigated using full-length (407 amino acids) and carboxyl-terminally truncated (324 amino acids) rat SP receptors transfected stably into Chinese hamster ovary cells. Both the full-length and the truncated receptor bound radiolabeled SP with a similar Kd ( approximately 0.1 nM). The average number of high affinity SP binding sites per cell was 1.0 x 10(5) and 0.3 x 10(5) for the full-length and the truncated SP receptor, respectively. In both cell lines, SP induced a rapid but transient increase in cytosolic calcium concentration ([Ca2+]i), which consisted of the release of Ca2+ from intracellular stores and the influx of extracellular Ca2+. Both components are dependent on phospholipase C activation. Although the full-length and the truncated receptor utilize the same calcium pathways, they differ in their EC50 values (0.28 nM for the full-length; 0.07 nM for the truncated). These differences in responsiveness may be related to the observed differences in receptor desensitization. The truncated receptor, in contrast to the full-length receptor, does not undergo rapid and long-lasting desensitization. Cells possessing the short isoform of the SP receptor would thus be expected to exhibit a prolonged responsiveness.     

Coexpression of ligand-gated P2X and G protein-coupled P2Y receptors in smooth muscle. Preferential activation of P2Y receptors coupled to phospholipase C (PLC)-beta1 via Galphaq/11 and to PLC-beta3 via Gbetagammai3

P2 receptor subtypes and their signaling mechanisms were characterized in dispersed smooth muscle cells. UTP and ATP stimulated inositol 1,4,5-triphosphate formation, Ca2+ release, and contraction that were abolished by U-73122 and guanosine 5'-O-(3-thio)diphosphate, and partly inhibited (50-60%) by pertussis toxin (PTX). ATP analogs (adenosine 5'-(alpha, beta-methylene)triphosphate, adenosine 5'-(beta, gamma-methylene)triphosphate, and 2-methylthio-ATP) stimulated Ca2+ influx and contraction that were abolished by nifedipine and in Ca2+-free medium. Micromolar concentrations of ATP stimulated both Ca2+ influx and Ca2+ release. ATP and UTP activated Gq/11 and Gi3 in gastric and aortic smooth muscle and heart membranes, Gq/11 and Gi1 and/or Gi2 in liver membranes, and Go and Gi1-3 in brain membranes. Phosphoinositide hydrolysis stimulated by ATP and UTP was mediated concurrently by Galphaq/11-dependent activation of phospholipase (PL) C-beta1 and Gbetagammai3-dependent activation of PLC-beta3. Phosphoinositide hydrolysis was partially inhibited by PTX or by antibodies to Galphaq/11, Gbeta, PLC-beta1, or PLC-beta3, and completely inhibited by the following combinations (PLC-beta1 and PLC-beta3 antibodies; Galphaq/11 and Gbeta antibodies; PLC-beta1 and Gbeta antibodies; PTX with either PLC-beta1 or Galphaq/11 antibody). The pattern of responses implied that P2Y2 receptors in visceral, and probably vascular, smooth muscle are coupled to PLC-beta1 via Galphaq/11 and to PLC-beta3 via Gbetagammai3. These receptors co-exist with ligand-gated P2X1 receptors activated by ATP analogs and high levels of ATP.     

An immunocytochemical investigation of the relationship between substance P and the neurokinin-1 receptor in the lateral horn of the rat thoracic spinal cord

The relationship between substance P-containing axons and sympathetic preganglionic neurons possessing the neurokinin-1 receptor was investigated in the lateral horn of the rat thoracic spinal cord. Sympathetic preganglionic neurons were labelled retrogradely with Fluorogold. Sections containing labelled cells were reacted with antibodies against choline acetyltransferase, substance P and the neurokinin-1 receptor and examined with three-colour confocal laser scanning microscopy. In all, 95 sympathetic preganglionic neurons were examined and 79% of these were immunoreactive for the neurokinin-1 receptor. Substance P-immunoreactive axons not only made contacts with preganglionic neurons which were immunoreactive for the receptor but also made contacts with cells which did not express the receptor. Dendrites, labelled with immunoreactivity for choline actyltransferase, also received contacts from substance P-immunoreactive varicosities but this was not related to the presence or the absence of receptor. An electron microscopic analysis was performed to investigate the relationship between substance P-containing boutons and dendrites possessing the neurokinin-1 receptor. Immunoreactivity for substance P was detected with peroxidase immunocytochemistry and immunoreactivity for the receptor was detected with the silver-intensified gold method. Substance P-containing boutons made synapses with dendrites which were positively and negatively labelled for the receptor. Receptor immunoreactivity was not usually present at synapses formed by substance P boutons with neurokinin-1-immunoreactive dendrites. It is concluded that substance P may modulate much of the activity of sympathetic preganglionic neurons through an indirect non-synaptic mechanism.     

The neostriatal mosaic: basis for the changing distribution of neurokinin-1 receptor immunoreactivity during development

The pattern of neurokinin-1 receptor-like immunoreactivity (NK-1Rir) was mapped in perinatal and adult mouse striatum by using a new polyclonal antiserum. NK-1Rir was detected in the differentiating regions of the ganglionic eminences on embryonic day 12.5 (E12.5). NK-1Rir structures were enriched in the striatal patch compartment between E16.5 and approximately postnatal day 3 (P3); distributed more uniformly, within portions of both the patch and matrix compartments on P7; and enriched in the matrix compartment in the adult. Analysis of the phenotype of NK-1Rir cells on P2, P7, and in the adult suggested that cholinergic cells accounted for the majority of NK-1Rir cells early postnatally, with increasing contributions from somatostatinergic cells later postnatally. In the adult, approximately half of NK-1Rir cells were cholinergic and half were somatostatinergic. The transient enrichment of NK-1R-bearing cells and processes in the patch compartment which contains cells that express substance P (SP), a putative ligand for the NK-1R, may be a consequence of compartment formation or may be functionally important for compartment development.     

Conformational probes for elucidating the nature of substance P binding to the NK1 receptor: initial efforts to map the Phe7-Phe8 region

Three substance P analogs with conformation constraints in the Phe7-Phe8 region have been prepared in connection with an effort to differentiate two families of potential conformations for the binding of substance P to its NK1 receptor. While the analogs did not bind the NK1 receptor with high affinity, the synthesis of the analogs demonstrated the utility of a general method for constructing piperazinone based peptidomimetics.     

Human substance P receptor expressed in Chinese hamster ovary cells directly activates G(alpha q/11), G(alpha s), G(alpha o)

BACKGROUND: Abdominal pain is a common finding in patients with systemic lupus erythematosus (SLE), occurring in as many as half of all SLE patients in the course of their disease. The rheumatology and gastroenterology literature emphasizes etiologies of abdominal pain in patients with SLE such as peritonitis from polyserositis, dyspepsia from reflux, nausea and vomiting from bowel edema, ascites, mesenteric ischemia, pancreatitis, pneumatosis intestinalis from necrotizing enterocolitis, and hepatobiliary abnormalities. But in clinical practice, caring for SLE patients in a community teaching hospital, these seem to be rare entities. PATIENTS AND METHODS: A chart review study was performed of all patients with SLE with the diagnosis of abdominal pain admitted to a community teaching hospital between 1980 and 1995. RESULTS: Of 13 patients who presented with abdominal pain, 9 required surgical intervention for cholecystitis, perforated ulcer, colonic perforation, diverticulitis, and adhesions. There were no negative laparotomies for polyserositis or bowel edema, or cases of mesenteric infarction or ascites. CONCLUSION: Despite some unusual diagnostic possibilities in abdominal pain in SLE such as polyserositis and mesenteric infarction, and despite the superimposed problems of steroid therapy in most of the patients in this study, the majority of lupus patients with abdominal pain presenting at community hospitals have relatively conventional illnesses.     

Magnetic properties and magnetocaloric effects of manganite (La0.8Ho0.2)2/3Ca1/3MnO3 and (La0.5Ho0.5)2/3Ca1/3MnO3

We reported the magnetic properties and magnetocaloric effects (MCE) of (La_0.8Ho_0.2)_2/3Ca_1/3MnO₃ and (La_0.5Ho_0.5)_2/3Ca_1/3MnO₃ nanoparticles by sol-gel technique. With this method, we were able to obtain the samples with particle diameters ranging from 50 to 200 nm. In the (La_1-xHo_x)_2/3Ca_1/3MnO₃ compound, an external magnetic field induced a magnetic transition from an paramagnetic phase to a ferromagnetic phase above T_s=105-135 K, leading to magnetocaloric effects. The maximum value of ΔS_M was 1.19 J/(kg·K) at 100 K and 2.03 J/(kg·K) at 152 K for a magnetic field change of 5 T. Because both samples had large relative cooling power (RCP) and wide δT_FWHM, the study on systems with the (La_1-xHo_x)_2/3Ca_1/3MnO₃-related magnetic transitions may open an important field in searching good magnetic materials.