De novo expression of the neurokinin 1 receptor in spinal lamina I pyramidal neurons in polyarthritis.

Spinal lamina I (LI) neurons play a major role in the transmission and integration of pain-related information that is relayed to higher centers. Alterations in the excitability of these neurons influence chronic pain development, and expression of the neurokinin 1 receptor (NK-1r) is thought to play a major role in such changes. Novel expression of NK-1r may underlie hyperexcitability in new populations of LI neurons. LI projection neurons can be classified morphologically into fusiform, pyramidal, and multipolar cells, differing in their functional properties, with the pyramidal type being nonnociceptive. In agreement with this, we have shown that spinoparabrachial pyramidal neurons seldom express NK-1r, in contrast with the other two cell types. In this study we investigated in the rat the long-term changes in NK-1r expression by spinoparabrachial LI neurons following the unilateral injection in the hindpaw plantar surface of complete Freund's adjuvant (CFA). Cholera toxin subunit B (CTb) was injected unilaterally into the parabrachial nucleus. Our results revealed that, ipsilaterally, pyramidal neurons were seldom immunoreactive for NK-1r both in saline-injected and in CFA-injected rats, up to 10 days post-CFA. However, a considerable number of pyramidal cells were immunoreactive for NK-1r at 15, 21, and 30 days post-CFA. Our data raise the possibility -- which needs to be confirmed by electrophysiology -- that most LI projection neurons of the pyramidal type are likely nonnociceptive in naive animals but might become nociceptive following the development of arthritis.

Morphological characterization of spinal cord dorsal horn lamina I neurons projecting to the parabrachial nucleus in the rat.

Many Rexed's lamina I neurons are nociceptive and project to the brain. Lamina I projection neurons can be classified as multipolar, fusiform, or pyramidal, based on cell body shape and characteristics of their proximal dendrites in the horizontal plane. There is also evidence that both multipolar and fusiform cells are nociceptive and pyramidal neurons nonnociceptive. In this investigation we identified which types of lamina I neurons belong to the spinoparabrachial tract in the rat and characterized them regarding the presence or absence of neurokinin-1 receptor (NK-1r) immunoreactivity. For this, cholera toxin subunit B (CTb), conjugated to a fluorescent marker was injected unilaterally into the parabrachial nucleus. Sections were additionally stained for the detection of NK-1r immunoreactivity and were examined using fluorescence and confocal microscopy. Serial confocal optical sections and 3D reconstructions were obtained for a considerable number of neurons per animal. Using immunofluorescence, we assessed the proportion of lamina I neurons belonging to the spinoparabrachial (SPB) tract and/or expressing NK-1r. The relative distribution of neurons belonging to the SPB tract was: 38.7% multipolar, 36.8% fusiform, 22.7% pyramidal, and 1.9% unclassified. Most of the SPB neurons expressing NK-1r were either multipolar or fusiform. Pyramidal SPB neurons were seldom immunoreactive for NK-1r, an observation that provides further support to the concept that most lamina I projection neurons of the pyramidal type are nonnociceptive. In addition, our study provides further evidence that these distinct morphological types of neurons differ in their phenotypic properties, but not in their projection patterns.

The distribution and regulation of vanilloid receptor VR1 and VR1 5' splice variant RNA expression in rat.

The vanilloid (capsaicin) receptor, VR1, is expressed in dorsal root ganglion and mediates the sensory response to vanilloids and other noxious stimuli. There is evidence for VR1 expression in CNS regions as well, but its function in these tissues is unknown. The identification of a rat VR1 5' splice variant and the rat stretch inhibitable channel, which are also expressed in dorsal root ganglia and CNS, raises the possibility that these and/or other VR1 variants may regulate VR1 activity. We have used a quantitative ribonuclease protection assay to characterize the central and peripheral expression of VR1 and VR1 variant RNA in the rat. The data confirm that VR1 is widely expressed in CNS, with highest RNA levels found in cerebral cortex, hippocampus, and cerebellum. VR1 RNA expression in dorsal root ganglia is approximately 28 times greater than in any other tissue sample studied. VR1 5' splice variant RNA is expressed at levels 12 times lower than VR1 in dorsal root ganglia, but at similar levels to VR1 in all other tissues examined. A VR1-related RNA expressed at high levels in kidney was detected, and was distinct from VR1 or stretch inhibitable channel. Our results also show that peripheral inflammation does not change VR1 RNA levels in rat dorsal root ganglia. Systemic resiniferatoxin administration, however, decreases VR1 expression in dorsal root ganglia by 65-80%, an effect that persists for at least 2 months. This study demonstrates that VR1 is expressed at high levels in dorsal root ganglia relative to other tissues and that VR1 5' splice variant is expressed at low levels in dorsal root ganglia compared to VR1. VR1 gene expression in dorsal root ganglia is regulated in response to systemic resiniferatoxin but not peripheral inflammation.

Photoaffinity labeling of mutant neurokinin-1 receptors reveals additional structural features of the substance P/NK-1 receptor complex.

Photoaffinity labeling, receptor site-directed mutagenesis, and high-resolution NMR spectroscopy have been combined to further define the molecular details of the binding of substance P (SP) to the rat neurokinin-1 (NK-1) receptor. Mutant NK-1 receptors were constructed by substituting Ala for Met174 and/or Met181: residues previously identified as the sites of covalent attachment of radioiodinated, photoreactive derivatives of SP containing p-benzoyl-L-phenylalanine (Bpa) in positions 4 and 8, respectively. Photoaffinity labeling of the M181A mutant using radioiodinated Bpa8-SP resulted in a marked reduction in photoincorporation efficiency compared to the wild-type receptor. In contrast, photoaffinity labeling of the M174A mutant using radioiodinated Bpa4-SP gave the unexpected result of an increase in the efficiency of photoincorporation compared to the wild-type receptor. Enzymatic and chemical fragmentation analysis of the photolabeled receptor mutants established that the sites of covalent attachment were not the substituted alanine, but rather the other methionine on the second extracellular (E2) loop sequence, that is not the primary site of attachment in the wild-type receptor. The results thus suggest a close spatial relationship between Met174 and Met181 on the NK-1 receptor. To evaluate this structural disposition, NMR analyses were performed on a synthetic peptide with a sequence corresponding to the entire E2 loop and segments of the adjoining transmembrane helices to anchor the peptide in the lipids used to mimic a membrane. The structural features of the E2 loop include a centrally located alpha-helix, extending from Pro175 to Glu183, as well as smaller alpha-helices at the termini, corresponding to the transmembrane regions. The two methionine residues are located on the same face of the central alpha-helix, approximately 11 A apart from each other, and are therefore consistent with the conclusions of the photoaffinity labeling results.

Developmental expression of neurokinin A and functional neurokinin-2 receptors in lung.

Peribronchial smooth muscle constriction causes airway stretch, an important mechanical force in developing lung. Little is known about factors influencing these spontaneously active muscle elements. We measured contractile activity of neurokinin (NK) receptors on fetal intrapulmonary smooth muscle by tracheal perfusion assay (n = 11). Injecting either capsaicin or the NK(2) receptor agonist [NLE(10)]NKA resulted in significant (P < 0.05) bronchoconstriction. A specific NK(2) receptor antagonist inhibited constriction caused by endogenous tachykinins released by capsaicin. We then examined NK(2) receptor (n = 44) and NKA (n = 23) ontogeny in human lung. NKA immunostaining was identified in peribronchial nerves in samples with gestational age >12 wk. NK(2) receptor protein was identified in peribronchial and perivascular smooth muscle. These results indicate that endogenous tachykinins released by the developing lung act via NK(2) receptors to cause smooth muscle constriction. We speculate that tachykinins could modulate lung development.

Arginine-vasopressin neurons in the rat hypothalamus produce neurokinin B and co-express the tachykinin NK-3 receptor and angiotensin II type 1 receptor.

Secretion of arginine-vasopressin (AVP) from the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei is induced by neurokinin B (NKB) and angiotensin. To characterize the mechanisms by which this occurs, we used immunohistochemical techniques to assess the ability of AVP-producing neurons to express NKB, NKB receptor (NK-3 receptor) and angiotensin II type 1 receptor (AT-1 receptor). Double fluorescence immunohistochemistry indicated that AVP-immunoreactive cell bodies in the PVN and SON, as well as their axon varicosities in the posterior pituitary, co-express NKB. Almost all AVP-neuron perikarya also expressed both the NK-3 receptor and AT-1 receptor. Thus, AVP-producing neurons in the PVN and SON, which are regulated by NKB, are themselves a source of NKB. Furthermore, the regulation of AVP release by these neurons by NKB and angiotensin II is mediated by the NK-3 receptor and the AT-1 receptor, respectively.

The tissue distribution and functional characterization of human VR1.

The irritant action of capsaicin is mediated by the vanilloid receptor, VR1, which is expressed in sensory neurons termed nociceptors. Capsaicin also desensitizes nociceptors and, thus, is useful clinically as an analgesic. Given the potential importance of VR1 in pain, we have cloned the human capsaicin receptor, hVR1, from a human dorsal root ganglia (DRG) cDNA library. Human VR1 protein is 85% identical to the rat VR1 and many of the amino acid differences are concentrated at the amino and carboxyl termini. VR1 is expressed in DRG as an approximately 4.2 kilobase RNA, and is also expressed in the central nervous system and in the kidney. Capsaicin (EC(50) = 853 nM), low pH (<5.5), and noxious heat (44 degrees C) activate hVR1 expressed in Xenopus oocytes. Subthreshold pH (6.4) sensitizes VR1 to capsaicin (EC(50) = 221 nM). This study demonstrates the similarity of human and rat VR1 in integrating multiple noxious stimuli.

Immunohistochemical localization of the neuropeptide Y Y1 receptor in rat central nervous system.

The diverse effects of neuropeptide Y (NPY) are mediated through interaction with G-protein coupled receptors. Pharmacological analysis suggests the Y1 receptor mediates several of NPY's central and peripheral actions. We sought to determine the distribution of Y1 protein throughout the rat central nervous system by means of indirect immunofluorescence using the tyramide signal amplification method and a novel, amino terminally-directed Y1 antisera. This antisera was verified as specific for Y1 by solution-phase competition ELISA, Western blot and in situ blocking experiments. High concentrations of Y1 immunoreactivity were found in the claustrum, piriform cortex (superficial layer), arcuate hypothalamic nucleus, interpeduncular nucleus, paratrigeminal nucleus, and lamina II of the spinal trigeminal nucleus and entire spinal cord. Moderate levels of Y1 immunoreactivity were found the in the main olfactory bulb, dorsomedial part of suprachiasmatic nucleus, paraventricular hypothalamic nucleus, ventral nucleus of lateral lemniscus, pontine nuclei, mesencephalic trigeminal nucleus, external cuneate nucleus, area postrema, and nucleus tractus solitarius. Low levels of Y1 immunostaining were distributed widely throughout layers II-III of the cerebral cortex (i.e., orbital, cingulate, frontal, parietal, insular, and temporal regions), nucleus accumbens core, amygdalohippocampal and amygdalopiriform areas, dentate gyrus, CA1 and CA2 fields of hippocampus, principal and oral divisions of the spinal trigeminal nucleus, islands of Calleja and presubiculum. These findings are discussed with reference to previously reported receptor autoradiography, immunohistochemistry and mRNA analyses to further support the role of Y1 in NPY-mediated biology.

Substance P and its receptor neurokinin 1 expression in asthmatic airways.

BACKGROUND: Neural mechanisms have been suggested to contribute to the pathogenesis of chronic asthma. The expression of neuropeptides such as substance P may be regulated by infectious pathogens, including Mycoplasma species. In contrast to substance P, the substance P receptor neurokinin 1 has not been examined at the protein level in asthmatic airways. OBJECTIVE: This study evaluated substance P and neurokinin 1 protein expression and mucus content in endobronchial biopsy specimens from normal control subjects and asthmatic subjects. Detection of Mycoplasma pneumoniae was performed in both biopsy and bronchoalveolar lavage specimens. METHODS: Biopsy specimens were collected from 10 normal control subjects and 18 asthmatic subjects before and after a 6-week treatment with a macrolide antibiotic (n = 11) or placebo (n = 7) and were stained for substance P, neurokinin 1, and mucus. M pneumoniae was evaluated by PCR. RESULTS: At baseline, compared with normal control subjects, asthmatic subjects demonstrated increased expression of substance P and neurokinin 1 and mucus content in the airway epithelium. Epithelial mucus content correlated with epithelial substance P expression (r (s) = 0.45, P =.04) and FEV(1) percent predicted (r (s) = -0.51, P =.019). After antibiotic treatment, both epithelial substance P and neurokinin 1 expression were significantly reduced in asthmatic subjects. M pneumoniae was found in 8 of 18 asthmatic subjects. Asthmatic subjects with M pneumoniae, compared with those without M pneumoniae, showed higher baseline epithelial neurokinin 1 expression, which was significantly reduced after antibiotic treatment (P =.02). CONCLUSION: Our data suggest that abnormalities in neural mechanisms may exist in the epithelium of asthmatic airways, and M pneumoniae is possibly involved in this process. Antibiotic intervention may be effective in the treatment of asthma partly through the downregulation of the neurogenic process.

Solution structures in SDS micelles and functional activity at the bullfrog substance P receptor of ranatachykinin peptides.

A set of novel tachykinin-like peptides has been isolated from bullfrog brain and gut. These compounds, ranatachykinin A (RTKA), ranatachykinin B (RTKB), and ranatachykinin C (RTKC), were named for their source, Rana catesbeiana, and their homology to the tachykinin peptide family. We present the first report of the micelle-bound structures and pharmacological actions of the RTKs. Generation of three-dimensional structures of the RTKs in a membrane-model environment using (1)H NMR chemical shift assignments, two-dimensional NMR techniques, and molecular dynamics and simulated annealing procedures allowed for the determination of possible prebinding ligand conformations. RTKA, RTKB, and RTKC were determined to be helical from the midregion to the C-terminus (residues 4-10), with a large degree of flexibility in the N-terminus and minor dynamic fraying at the end of the C-terminus. The pharmacological effects of the RTKs were studied by measuring the elevation of intracellular Ca(2+) in Chinese hamster ovarian cells stably transfected with the bullfrog substance P receptor (bfSPR). All of the RTKs tested elicited Ca(2+) elevations with a rank order of maximal effect of RTKA >/= SP > RTKC >/= RTKB. A high concentration (1 microM) of the neuropeptides produced varying degrees of desensitization to a subsequent challenge with the same or different peptide, while a low concentration (1 pM) produced sensitization at the bfSPR. Our data suggest differences in amino acid side chains and their charged states at the C-terminal sequence or differences in secondary structure at the N-terminus, which do not overlap according to the findings in this paper, may explain the differing degree and type of receptor activation seen at the bfSPR.

Distribution of mRNA for vanilloid receptor subtype 1 (VR1), and VR1-like immunoreactivity, in the central nervous system of the rat and human.

The cloned vanilloid receptor VR1 has attracted recent attention as a molecular integrator of painful stimuli on primary sensory neurons. The existence of vanilloid-sensitive neurons in the brain is, however, controversial. In this study, we have used an antibody and a complementary RNA probe to explore the distribution of neurons that express VR1 in rat and in certain areas of human brain. In the rat, we observed VR1-expressing neurons throughout the whole neuroaxis, including all cortical areas (in layers 3 and 5), several members of the limbic system (e.g., hippocampus, central amygdala, and both medial and lateral habenula), striatum, hypothalamus, centromedian and paraventricular thalamic nuclei, substantia nigra, reticular formation, locus coeruleus, cerebellum, and inferior olive. VR1-immunopositive cells also were found in the third and fifth layers of human parietal cortex. Reverse transcription-PCR performed with rat VR1-specific primers verified the expression of VR1 mRNA in cortex, hippocampus, and hypothalamus. In the central nervous system, neonatal capsaicin treatment depleted VR1 mRNA from the spinal nucleus of the trigeminal nerve, but not from other areas such as the inferior olive. The finding that VR1 is expressed not only in primary sensory neurons but also in several brain nuclei is of great importance in that it places VRs in a much broader perspective than pain perception. VRs in the brain (and putative endogenous vanilloids) may be involved in the control of emotions, learning, and satiety, just to name a few exciting possibilities.

Substance P and neurokinin-1 receptor expression by intrinsic airway neurons in the rat.

Tachykinins and their receptors are involved in the amplification of inflammation in the airways. We analyzed the expression of preprotachykinin-A (PPT-A) and neurokinin-1 (NK-1) receptor genes by intrinsic airway neurons in the rat. We also tested the hypothesis that PPT-A-encoded peptides released by these neurons fulfill the requisite role of substance P in immune complex injury of the lungs. We found that ganglion neurons in intact and denervated airways or in primary culture coexpress PPT-A and NK-1 receptor mRNAs and their protein products. Denervated ganglia from tracheal xenografts (nu/nu mice) or syngeneic lung grafts had increased PPT-A mRNA contents, suggesting preganglionic regulation. Formation of immune complexes in the airways induced comparable inflammatory injuries in syngeneic lung grafts, which lack peptidergic sensory fibers, and control lungs. The injury was attenuated in both cases by pretreatment with the NK-1 receptor antagonist LY-306740. We conclude that tachykinins released by ganglia act as a paracrine or autocrine signal in the airways and may contribute to NK-1 receptor-mediated amplification of immune injury in the lungs.

Distribution of galanin-1, -2 and -3 receptor messenger RNAs in central and peripheral rat tissues.

Galanin is a neuropeptide widely expressed in the central nervous system and periphery. In rat, three galanin-binding receptors have been cloned and characterized. We report the qualitative and quantitative distribution of galanin-1, galanin-2, and galanin-3 messenger RNAs in central and peripheral rat tissues by reverse transcription-polymerase chain reaction and solution hybridization/RNase protection assays, respectively. Galanin-1 messenger RNA was detected exclusively in the central and peripheral nervous system with highest expression in hypothalamus, amygdala, spinal cord and dorsal root ganglia. Galanin-2 messenger RNA was highly expressed in hypothalamus, dorsal root ganglia, and kidney with moderate expression in several other tissues. Galanin-3 messenger RNA was widely distributed at low to moderate levels in many central and peripheral tissues. The observed expression of multiple galanin receptors in several tissues including hypothalamus, anterior pituitary and spinal cord supports earlier pharmacological studies suggesting the presence of more than one receptor subtype in these regions. The presence of multiple galanin receptors in these tissues in conjunction with the detection of a single subtype, galanin-2, in tissues such as heart and intestine, illustrates the potential complexity of galanin-associated actions in rat central nervous system and periphery.

The distribution of neurokinin-1 and neurokinin-2 receptors in human central airways.

The precise locations of neurokinin (NK)-1 and NK-2 receptors in human airways, and their role in airway inflammatory diseases, have not been carefully examined. To determine the distribution of NK-1 and NK-2 receptors in human central airways, and to determine whether their distribution was different in smokers, we examined surgical specimens from patients undergoing lung resection for limited lung lesions. We mapped NK-1 and NK-2 receptors in four groups of subjects: four asymptomatic nonsmokers, seven asymptomatic smokers, seven symptomatic smokers with normal lung function, and eight symptomatic smokers with chronic airflow limitation. Tissues were immunostained with anti-NK-1- and anti-NK-2-receptor antibodies. Expression of NK-1 and NK-2 receptors was quantified through light microscopy and image analysis. Both NK-1 and NK-2 receptors were found in bronchial glands, bronchial vessels, and bronchial smooth muscle. Although no receptors were observed in the epithelium, receptors were occasionally found in nerves (NK-1) and in inflammatory cells (NK-2) such as T lymphocytes, macrophages, and mast cells. The distribution of both NK-1 and NK-2 receptors was similar in all the tissues examined in the four groups of subjects. These data show that NK-1 and NK-2 receptors are present in human central airways and that their expression is not modified by cigarette smoking.

Immunocytochemical localization of neurokinin B in the rat spinal dorsal horn and its association with substance P and GABA: an electron microscopic study.

Substance P and neurokinin B are tachykinins that derive from different precursors. Both tachykinins are known to be involved in the processing of pain-related information. Initial studies suggested an antinociceptive effect for neurokinin B, but more recent data indicate that neurokinin B facilitates nociception. Unfortunately, morphologic correlates are lacking, as little is known about the distribution of neurokinin B, especially at the ultrastructural level. Because of its potentially important role in the processing of pain-related information, we decided to investigate the synaptic interactions of neurokinin B-immunoreactive profiles in laminae I-III of the rat cervical spinal dorsal horn and their relation to substance P-immunoreactive structures. An antibody raised against a portion of the neurokinin B precursor peptide was used for the detection of neurokinin B. Neurokinin B-like immunoreactivity occurred in all superficial laminae, with the highest density in inner lamina II and the lowest in lamina III. Neurokinin B-like immunoreactive axonal boutons were mainly dome-shaped and established symmetric synaptic contacts with dendrites or cell bodies. Neurokinin B-like immunoreactivity was also detected in dendritic profiles in all superficial laminae. Some of these dendritic profiles were part of synaptic glomeruli in inner lamina II and lamina III. Double-labeling for neurokinin B and substance P showed a lack of appositions and synapses between neurokinin B and substance P-positive profiles. Furthermore, very few profiles double-labeled for the two peptides were observed. Double-labeling for gamma-aminobutyric acid (GABA) and neurokinin B showed a complete absence of neurokinin B/GABA co-localization. Furthermore, neurokinin B-positive profiles were never presynaptic to GABA-immunoreactive profiles, but frequently neurokinin B-positive dendrites were postsynaptic to GABA-immunoreactive boutons. These results suggest that neurokinin B participates in circuits separate from those involving substance P, as virtually no anatomic correlation was found between the two neuropeptides.

Resiniferatoxin-type phorboid vanilloids display capsaicin-like selectivity at native vanilloid receptors on rat DRG neurons and at the cloned vanilloid receptor VR1.

1 Although the cloned rat vanilloid receptor VR1 appears to account for both receptor binding and calcium uptake, the identification of vanilloids selective for one or the other response is of importance because these ligands may induce distinct patterns of biological activities. 2 Phorbol 12,13-didecanoate 20-homovanillate (PDDHV) evoked 45Ca(2+)-uptake by rat dorsal root ganglion neurons (expressing native vanilloid receptors) in culture with an EC50 of 70 nM but inhibited [3H]-resiniferatoxin (RTX) binding to rat dorsal root ganglion membranes with a much lower potency (Ki>10,000 nM). This difference in potencies represents a more than 100 fold selectivity for capsaicin-type pharmacology. 3 45Ca2+ influx by PDDHV was fully inhibited by the competitive vanilloid receptor antagonist capsazepine, consistent with the calcium uptake occurring via vanilloid receptors. 4 PDDHV induced calcium mobilization in CHO cells transfected with the cloned rat vanilloid receptor VR1 with an EC50 of 125 nM and inhibited [3H]-RTX binding to these cells with an estimated Ki of 10,000 nM. By contrast, PDDHV failed to evoke a measurable calcium response in non-transfected CHO cells, confirming its action through VR1. 5 We conclude that PDDHV is two orders of magnitude more potent for inducing calcium uptake than for inhibiting RTX binding at vanilloid receptors, making this novel vanilloid a ligand selective for capsaicin-type pharmacology. These results emphasize the importance of monitoring multiple endpoints for evaluation of vanilloid receptor structure-activity relations. Furthermore, PDDHV now provides a tool to explore the biological correlates of capsaicin-type vanilloid pharmacology.

Substrate turnover by transporters curtails synaptic glutamate transients.

Although inhibitors of glutamate transport prolong synaptic currents at many glutamate synapses, the cause of the current prolongation is unclear. Transport inhibitors may prolong synaptic currents by simply interfering with synaptic glutamate binding to transporters, by inhibiting substrate translocation, or by promoting accumulation of ambient glutamate, which may act cooperatively at receptors with synaptic glutamate. We show that reversal of the membrane potential of astrocytes surrounding the synapse prolongs synaptic currents but does not decrease the apparent affinity of transporters or significantly alter glutamate-dependent kinetics of macroscopic transporter currents in excised membrane patches. Positive membrane potentials do not affect binding of a nontransported glutamate analog, nor do positive membrane potentials alter the number of transporters available to bind analog. We also test the hypothesis that glutamate accumulation during uptake inhibition by transporter substrates is the direct cause of synaptic current prolongations. Transporter substrates elevate ambient glutamate near synapses by fostering reverse transport of endogenous glutamate. However, increases in ambient glutamate cannot account for the prolongations of synaptic currents, because a nonsubstrate transport inhibitor does not foster reverse uptake yet it prolongs synaptic currents. Moreover, exogenous glutamate does not mimic synaptic current prolongations induced by substrate inhibitors. These results provide strong support for a major role of substrate translocation in determining the time course of the glutamate concentration transient at excitatory synapses.

The cloned rat vanilloid receptor VR1 mediates both R-type binding and C-type calcium response in dorsal root ganglion neurons.

[(3)H]Resiniferatoxin (RTX) binding and calcium uptake by rat dorsal root ganglion (DRG) neurons show distinct structure-activity relations, suggestive of independent vanilloid receptor (VR) subtypes. We have now characterized ligand binding to rat VR1 expressed in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells and compared the structure-activity relations with those for calcium mobilization. Human embryonic kidney cells (HEK293/VR1 cells) and Chinese hamster ovary cells transfected with VR1 (CHO/VR1 cells) bound [(3)H]RTX with affinities of 84 and 103 pM, respectively, and positive cooperativity (Hill numbers were 2.1 and 1.8). These parameters are similar to those determined with rat DRG membranes expressing native VRs (a K(d) of 70 pM and a Hill number of 1.7). The typical vanilloid agonists olvanil and capsaicin inhibited [(3)H]RTX binding to HEK293/VR1 cells with K(i) values of 0.4 and 4.0 microM, respectively. The corresponding values in DRG membranes were 0.3 and 2.5 microM. HEK293/VR1 cells and DRG membranes also recognized the novel vanilloids isovelleral and scutigeral with similar K(i) values (18 and 20 microM in HEK293/VR1 cells; 24 and 21 microM in DRGs). The competitive vanilloid receptor antagonist capsazepine inhibited [(3)H]RTX binding to HEK293/VR1 cells with a K(i) value of 6.2 microM and binding to DRG membranes with a K(i) value of 8.6 microM. RTX and capsaicin induced calcium mobilization in HEK293/VR1 cells with EC(50) values of 4.1 and 82 nM, respectively. Thus, the relative potencies of RTX (more potent for binding) and capsaicin (more potent for calcium mobilization) are similar in DRG neurons and cells transfected with VR1. We conclude that VR1 can account for both the ligand binding and calcium uptake observed in rat DRG neurons.

Ectopic substance P-immunoreactive boutons are preferentially presynaptic to neurokinin-1 receptor immunoreactive dendrites in the spinal white matter of transgenic mice.

A recent immunocytochemical study has shown that substance P (SP) preferentially innervates targets expressing the neurokinin-1 receptor (NK-1r) in the superficial spinal dorsal horn of the rat. Based on these findings, we decided to further investigate the relationship between SP and the NK-1r in a transgenic mouse model in which SP fibres are ectopically located. Double-labelling immunocytochemistry at both the light and electron microscopic levels was performed to study the association between SP and the NK-1r in the spinal white matter of both control and transgenic mice. Light microscopy revealed NK-1r-immunoreactive (IR) dendrites in the white matter of the dorsolateral funiculus in both control and transgenic mice. In transgenic mice, but not in controls, SP-IR fibres were observed in close proximity to the NK-1r-IR dendrites in the white matter. At the ultrastructural level, SP-IR boutons were apposed to NK-1r-IR dendrites in the dorsolateral funiculus of transgenic mice, and a synapse was frequently observed as well. These results indicate that, even in conditions in which SP fibres are ectopically located, they still preferentially innervate targets expressing the NK-1r.

Neurokinin-3 receptor distribution in rat and human brain: an immunohistochemical study.

Autoradiographic and immunohistochemical studies have shown that the neurokinin-3 receptor is widely distributed in the rodent CNS. Expression of the neurokinin-3 receptor in human brain, however, has been debated. These conflicting findings, as well as the poor resolution of autoradiographic images, prompted us to develop a polyclonal antibody against an oligopeptide derived from the carboxy-terminus consensus sequence of both the rat and human neurokinin-3 receptor ([C]ASTTSSFISSPYTSVDEYS, amino acids 434-452 of the rat neurokinin-3 receptor). Western blot analysis of both human and rat brain tissue revealed a major band in the molecular weight range 65,000-67,000, the proposed molecular weight of the neurokinin-3 receptor based on its amino acid sequence and presumed glycosylation state. The distribution of selective high affinity neurokinin-3 receptor agonist [3H]senktide binding and neurokinin-3 receptor immunoreactivity were virtually identical in the brains of male Fischer 344 rats. The highest concentrations of neurokinin-3 receptors were observed in cortical layers IV-V; the basolateral amygdaloid nucleus; the hypothalamic paraventricular, perifornical and supraoptic nuclei; the zona incerta; and the entopeduncular and interpeduncular nuclei. [3H]senktide binding and neurokinin-3 receptor immunoreactivity were compared in homologous cortical areas of the human and rat brain. In contrast to the rat, autoradiographic analysis of normal control human brains (35-75 years) revealed a distinct and predominant superficial cortical labeling in the glia limitans and the cortical layer I. However, neurokinin-3 receptor immunoreactivity could be found not only in the superficial cortical layers, but also on pyramidal neurons and astrocytes in the neuropil and white matter. These findings suggest species differences in both the cellular and anatomical distribution of the neurokinin-3 receptor.