Molecular characterization of a functional cDNA encoding the rat substance P receptor.

Substance P is a member of the tachykinin peptide family and participates in the regulation of diverse biological processes. The polymerase chain reaction and conventional library screening were used to isolate a complementary DNA (cDNA) encoding the rat substance P receptor from brain and submandibular gland. By homology analysis, this receptor belongs to the G protein-coupled receptor superfamily. The receptor cDNA was expressed in a mammalian cell line and the ligand binding properties of the encoded receptor were pharmacologically defined by Scatchard analysis and tachykinin peptide displacement as those of a substance P receptor. The distribution of the messenger RNA for this receptor is highest in urinary bladder, submandibular gland, striatum, and spinal cord, which is consistent with the known distribution of substance P receptor binding sites. Thus, this receptor appears to mediate the primary actions of substance P in various brain regions and peripheral tissues.

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.

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.

Structure, functions, and mechanisms of substance P receptor action.

Substance P is a member of a family of structurally related peptides, called tachykinins, that are involved in the regulation of many biologic processes. Diversity in the generation of multiple tachykinin peptides arises due to multiple genes encoding these peptides as well as by mechanisms of alternative RNA processing and differential posttranslational processing. The multiple peptides are neurotransmitters and/or neuromodulator substances, and they bring about their actions mainly by activating three primary types of receptors, NK-1, NK-2, and NK-3. The pharmacology and tissue locations of these receptor sites are discussed, as is their involvement in certain biologic responses. These three receptor sites have been molecularly characterized by cDNA cloning and functional expression, and all are members of the superfamily of receptors coupled to G-regulatory proteins. Second messenger systems established to be activated by tachykinin receptor stimulation include the hydrolysis of inositol containing phospholipids by a phospholipase C mechanism. The role of substance P in neurogenic inflammation and plasma extravasation is briefly discussed. The generation of new research tools recently in the tachykinin field should allow for a detailed examination of the mechanisms of peptide action, including a focus on receptor structure-function relations and regulation of receptor sensitivity.

Evidence that endopeptidase-catalyzed luteinizing hormone releasing hormone cleavage contributes to the regulation of median eminence LHRH levels during positive steroid feedback.

In previous studies we provided evidence that the degradation of LHRH is regulated so as to contribute to the establishment of appropriate levels of the decapeptide during the events leading to gonadotropin secretion in the first estrous cycle at puberty in the rat. In the present report, we present the first evidence that this apparent regulation of LHRH degradation can be studied in an experimental positive feedback model. We show that LHRH degradation in the median eminence was decreased 3 h after progesterone administration, at a time when LHRH content in this region is increasing, and when serum levels of LH remained at basal levels. Six h after progesterone administration, at the time of the LH surge, median eminence LHRH degradation was still low and LHRH content had fallen to basal levels. Additionally, we exploited this model to examine the mechanism of peptidase activity change by showing that blockade of noradrenergic neurotransmission by diethyldithiocarbamate abolishes the inhibition of LHRH degradation observed prior to the secretion of LH. We conclude that the degradation of LHRH by an endopeptidase may contribute to the regulation of LHRH levels appropriate for gonadotropin release, and that this can be studied in the ovariectomized, estrogen-progesterone-treated rat.

In vivo biosynthesis of hypothalamic luteinizing hormone releasing hormone in individual free-running female rats.

The present studies examined the feasibility of performing in vivo biosynthetic studies of hypothalamic LHRH in preoptic area cannulated individual ovariectomized, estradiol benzoate (EB)-progesterone (P) treated rats. One day after EB treatment, rats were bilaterally cannulated in the anterior ventromedial preoptic area (POA). The following day, rats were treated with P, and a mix of 3H-proline and 3H-leucine (500 muCi) was continuously infused (for 8 hr) into the POA using an Alzet osmotic minipump delivery system at a rate of 1 microliter/h/cannula. 3H-LHRH was acid-extracted from discrete hypothalamic regions (POA, medial basal hypothalamus [MBH]and median eminence [ME]) and was purified to constant specific activity by sequential High Performance Liquid Chromatography. 3H-LHRH accounted for 0.010%, 0.018% and 1.6% of the total tissue 3H recovered in the POA, MBH and ME, respectively. These results demonstrate the feasibility of performing physiological studies on the biosynthesis and transport of hypothalamic LHRH using POA cannulated free-running female rats.

Ovarian regulation of hypothalamic and pituitary progestin-metabolizing enzyme activities.

Progesterone 5 alpha-reductase activity and 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) activities (NADH-linked) were measured in hypothalamic and pituitary tissues from rats during various stages of the estrous cycle and in ovariectomized rats treated with 17 beta-estradiol or vehicle. Enzyme assays were performed using 3H-labeled steroid substrates and reverse isotopic dilution analyses. Pituitary 5 alpha-reductase activity fluctuated over the estrous cycle, with peak levels observed on proestrus, estrus, and metestrus and lowest levels on diestrous 1 and 2 of 5-day cycling rats. Pituitary NADH-linked 3 alpha-HSOR activity also fluctuated over the estrous cycle, paralleling 5 alpha-reductase activity. The pituitary NADPH-linked 3 alpha-HSOR activity and three hypothalamic enzyme activities, the 5 alpha-reductase, NADH-linked, and NADPH-linked 3 alpha-HSOR activities, did not change over the estrous cycle. After ovariectomy (10 days), pituitary 5 alpha-reductase activity was elevated 10- to 12-fold relative to mean levels in intact rats, while the pituitary NADH-linked 3 alpha-HSOR activity was elevated 4- to 5-fold. Treatment for 3 days with estradiol benzoate (10 micrograms/day) significantly reduced both pituitary 5 alpha-reductase and NADH-linked 3 alpha-HSOR activities to about half the levels observed in ovariectomized rats. The pituitary NADPH-linked 3 alpha-HSOR activity was unaffected by ovariectomy. After ovariectomy, a small decrease was observed in the hypothalamic 5 alpha-reductase; no differences were observed in the NADH-linked or NADPH-linked 3 alpha-HSOR activities of the hypothalamus compared to mean levels in cyclic rats. The results suggest a role of the ovary in the regulation of the pituitary and hypothalamic progesterone 5 alpha-reductase and pituitary NADH-linked 5 alpha-dihydroprogesterone 3 alpha-HSOR activities.

Gonadal steroid regulation of substance P (SP) and SP-encoding messenger ribonucleic acids in the rat anterior pituitary and hypothalamus.

Substance P-like immunoreactivity (SP-LI) is present in the rat anterior pituitary (AP) and in hypothalamic neurons that may be involved in the control of AP secretion and/or reproductive function. The presence of multiple SP-encoding mRNAs and tachykinin peptides and their regulation by steroid hormones were examined in APs and hypothalami from normal, gonadectomized, and steroid-treated male and female rats. SP-encoding mRNAs were identified by nuclease protection assays of RNA, and tachykinin peptides were identified by combined HPLC-RIA of tissue extracts, beta- and gamma-preprotachykinin (PPT) mRNAs and SP, neurokinin A, and neuropeptide gamma peptides were identified in the AP. The alpha-, beta-, and gamma-PPT mRNAs and SP, neurokinin A, neuropeptide gamma, neuropeptide K, and neurokinin B peptides were present in hypothalamic tissue. Previous studies have established that in the AP, SP is differentially regulated by gonadal steroids; estrogen decreases and androgen increases AP SP. Steroid effects were further analyzed in experiments using RIAs to measure SP levels in the AP and median eminence (ME) of steroid- and oil-treated gonadectomized rats. To assess whether steroids alter steady state PPT mRNA levels and presumably SP synthesis in these tissues, potential effects on AP and hypothalamic SP-encoding mRNAs were determined. Ovariectomized rats treated for 10 days with estradiol benzoate showed a 50% decrease in AP SP and a 90% decrease in AP beta- and gamma-PPT mRNAs compared to ovariectomized oil-treated controls. Estradiol benzoate replacement had no effect on SP levels in the isolated ME, but did cause a 50% increase in alpha-, beta-, and gamma PPT mRNAs in the hypothalamus. Although there was no significant effect of testosterone propionate on AP SP levels in castrated males, 10 days of testosterone propionate replacement did cause a significant increase in beta- and gamma PPT mRNAs in the AP. No androgen effects were seen on either ME SP or hypothalamic SP-encoding mRNAs. These data demonstrate that estrogen up-regulates SP-encoding mRNAs in the hypothalamus, whereas it down-regulates SP-encoding mRNAs in the pituitary. These results implicate SP and other tachykinins derived from the SP gene as steroid-regulated modulators of AP secretion and possibly reproductive function.

Substance-P is present in a subset of thyrotrophs in the human pituitary.

Substance-P immunoreactivity and tachykinin-like peptides are present in the pituitary gland of several mammalian species. In humans, however, the biochemical nature and cellular localization of pituitary substance-P has not been defined. We report here that substance-P-immunoreactive material is present in low concentrations in both the anterior and posterior lobes of the human pituitary gland. Gel chromatography and reverse phase high performance liquid chromatography indicate that the majority of the substance-P immunoreactivity in human pituitaries elutes as authentic substance-P and its oxidized derivative. Immunohistochemical studies showed substance-P-immunoreactive fibers and terminals in the posterior pituitary gland and occasional substance-P-immunoreactive cell bodies in the anterior lobe. The substance-P-immunoreactive cells were found to colocalize with a small subpopulation of TSH beta-immunoreactive cells (thyrotrophs). Substance-P immunoreactivity was also found in a pituitary microadenoma that contained numerous TSH beta-immunoreactive cells. These studies indicate that substance-P is present in the human pituitary gland, and they suggest a relationship between substance-P and thyroid function.

Distribution of neurons expressing neurokinin B in the rat brain: immunohistochemistry and in situ hybridization.

Neurokinin B (NKB) belongs to the family of neuropeptides named tachykinins. Members of this family such as substance P or neurokinin A have been proposed to function as neurotransmitters or neuromodulators. Searching for possible sites of action of NKB in the central nervous system, we have now investigated its distribution within the rat brain by immunohistochemical techniques and in situ hybridization. For immunohistology two different antisera directed against amino acid sequences within preprotachykinin B were used. One antiserum had been raised against a synthetic derivative of NKB; the other one was directed towards the amino acids 50-79 of preprotachykinin B, which are referred to as peptide 2. Essentially the same distribution of immunoreactive perikarya was obtained with both antisera and it closely corresponded to the cellular localization of preprotachykinin B mRNA. Neurons containing NKB immunoreactivity and mRNA were present in many areas including cerebral cortex, hippocampal formation, amygdaloid complex, bed nucleus of the stria terminalis, ventral pallidum, habenula, medial preoptic area, arcuate nucleus, and lateral mammillary bodies. Dense immunoreactive fibers were observed in various parts of the brain and were most prominent in the olfactory bulb and tubercle, the lateral olfactory tract, medial hypothalamus, around blood vessels of the median eminence and interpeduncular nucleus, amygdaloid nuclei, stria terminalis, subbrachial nucleus, and medial geniculate nucleus. Fibers of less intense staining were seen among other brain areas in the substantia nigra, the reticular formation, and the area of the nucleus of the solitary tract. Surgical lesion of the fasciculus retroflexus revealed that the dense fiber network observed in the interpeduncular nucleus originates from the ventral and dorsal parts of the medial habenula. Our data suggest a widespread and distinct distribution of neurons expressing NKB within the central nervous system, suggesting possible neuromodulatory roles of this neuropeptide for various brain functions.

Cellular localization of substance P- and neurokinin A-encoding preprotachykinin mRNA in the female rat brain.

To determine the locations of neurons in the rat brain expressing substance P and neurokinin A mRNA, we performed in situ hybridization with a radiolabeled cRNA probe that was complementary to alpha-, beta-, and gamma-preprotachykinin mRNA. Several types of controls indicated specificity of the labeling. Brain regions containing many labeled neurons include the anterior olfactory nucleus, layer II of the olfactory tubercle, the islands of Calleja, the nucleus accumbens, the caudate-putamen, portions of the amygdala and hypothalamus, the medial habenular nucleus, nuclei of the pontine tegmentum, several raphe nuclei, several portions of the reticular formation, and the nucleus of the solitary tract. Less frequent labeled neurons were also found in many other regions of the brain. These results extend many previous immunocytochemical studies of the locations of neurons containing immunoreactive substance P, neurokinin A, and neuropeptide K.

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.

Peptide-like immunoreactivity in anuran optic nerve fibers.

Unilateral section, crush, or ligation of the optic nerve was performed in Rana pipiens. Following optic nerve disruption, Substance P (SP)-, leucine-enkephalin (LENK)-, cholecystokinin octapeptide (CCK8)-, and bombesin (BOM)-like immunoreactivities were analyzed in the retinae and optic nerves. Peptide-like immunoreactivity developed in the retinal stump of disrupted optic nerves within 1 hour after surgery and was retained until at least 30 days. Peptide-positive staining in the retinal stump of the optic nerves was abolished by preabsorption of each of the antibodies/antisera with the corresponding synthetic substances. No massive peptide-like immunoreactivity was observed in the cerebral stump of the ligated side, nor in the contralateral, nonoperated, optic nerve. No change in the pattern of peptide-like immunoreactivity was apparent in the retina ipsilateral or contralateral to the experimental procedure. The optic tectum contralateral to the surgical procedure displayed those changes in peptide-like immunoreactivity described previously following retinal deafferentation (Kuljis and Karten, '82a, '83a). Peptide-like immunoreactivity in the stump retinad to the surgical procedure occurred in the form of beaded and fibrillar elements often ending in an irregular expansion near the lesion site. Fluorescent double-label antibody methods demonstrated that SP-like immunoreactivity is present in different processes than those containing LENK, CCK8, or BOM. Electron microscopical immunocytochemistry revealed that peptide-like immunoreactivity is contained within unmyelinated and possibly also within myelinated axons in the stump retinad to the traumatic procedure. Radioimmunoassay studies of SP demonstrated a four- to sixfold increase in SP-like content in the retinal stump of ligated nerves, compared with both the cerebral stump and with the contralateral nonoperated optic nerves. These findings demonstrate the presence of peptide-like immunoreactivity in retinal ganglion cell processes, which is compatible with either a posttraumatic expression of previously repressed peptide-like phenotypes, or, most likely, with the existence of various classes of peptide-containing retinal ganglion cells. The latter prospect strongly suggests that peptide-specific subsets of retinal ganglion cells terminate in highly specific laminae in the optic tectum (Kuljis and Karten, '81, '82a-83a) and presumably differ in their physiological role in vision.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

The distribution of substance P in turtle nervous system: a radioimmunoassay and immunohistochemical study.

The distribution of a substance P-like material in turtle brain, spinal cord, dorsal root ganglion, and retina was determined using radioimmunoassay (RIA) and immunohistochemistry. High levels of a substance P-like material were found in turtle neural tissue, particularly in basal telencephalon, hypothalamus, and tegmentum. In many regions, the concentration of a substance P-like material in turtle nervous tissue was found to be similar, in a region-to-region comparison, to that previously reported for birds and mammals, particularly for the more "phylogenetically conservative" parts of the nervous system (such as the basal ganglia, tegmentum, and hypothalamus). The slopes of substance P RIA dose-response curves for tissue extracts from nearly all regions of the turtle nervous system examined were parallel to a standard dose-response curve for synthetic substance P (SP). The immunohistochemical results, with anti-substance P antisera from guinea pig or rabbit, or with a monoclonal antibody, were consistent with the RIA data. Regions showing high concentration of an SP-like material by RIA were observed to contain numerous neurons and/or fibers containing an SP-like material. The immunohistochemical results provide evidence for the presence in turtle of numerous SP-containing pathways, several of which (e.g., an SP-containing strionigral pathway, an SP-containing striopallidal pathway and an SP-containing dorsal root ganglia-spinal dorsal horn pathway), have been described in birds and mammals. The present results thus suggest that the neuropeptide SP has had a largely stable evolutionary history as a transmitter or modulatory agent during amniote brain evolution.

Distinct and interactive effects of d-amphetamine and haloperidol on levels of neurotensin and its mRNA in subterritories in the dorsal and ventral striatum of the rat.

Striatal tissue concentrations of neurotensin, expression of neurotensin/neuromedin N (NT/N) mRNA, and numbers of neurotensin-immunoreactive neurons are increased by d-amphetamine (amph), which stimulates dopamine release in the striatum, and haloperidol (hal), a dopamine receptor antagonist with high affinity for D2-like receptors. The possibility that the effects of these drugs involve distinct subpopulations of striatal neurons was addressed in this study, in which the relative numbers and distributions of striatal neuron profiles containing neurotensin immunoreactivity and/or NT/N mRNA were compared following administrations of hal, amph, hal and amph co-administered, and vehicle. Fourteen striatal subterritories in caudate-putamen, nucleus accumbens, and olfactory tubercle were evaluated. Amph produced increases in the expression of neurotensin preferentially in the ventromedial and caudodorsal subterritories of the caudate-putamen, the rostrobasal cell cluster and lateral shell of the nucleus accumbens, and the olfactory tubercle. Haloperidol produced increased neurotensin expression in much of dorsal and ventral striatum, most prominently in the rostral, dorsomedial and ventrolateral quadrants of the caudate-putamen, and in the rostrobasal cell cluster, rostral pole, medial and lateral shell of the nucleus accumbens and the olfactory tubercle. The numbers of neurons responding to amph and hal in all subterritories following co-administration of the two drugs were significantly less than the summed numbers responding individually to amph and hal. Furthermore, in the subterritories where immunohistochemically detectable responses elicited by amph exceeded those produced by hal, co-administration of the two drugs resulted in responses comparable to those elicited by hal given alone. It is suggested that some of the reported anti-dopaminergic behavioral effects of basal ganglia neurotensin may be attenuated in conditions of reduced dopamine neurotransmission.

Nerve growth factor induces mechanical allodynia associated with novel A fibre-evoked spinal reflex activity and enhanced neurokinin-1 receptor activation in the rat.

A single dose of nerve growth factor (NGF, 1 microgram/g, i.p.) administered to rats aged between postnatal days (PND) 12 and 14 resulted in a behavioural hypersensitivity of the hindlimb flexion withdrawal reflex to mechanical stimuli which developed 2 h after NGF and remained significant for 24 h. Heat hyperalgesia occurred some 4 h following NGF injection and lasted for 24 h. Isolated spinal cords were prepared from animals treated with NGF and were maintained in vitro for physiological and pharmacological analysis of lumbar spinal reflex activity. Repetitive, low-frequency group I/II A beta-fibre stimulation evoked a novel wind-up response after NGF injection similar to that produced by C-fiber group III/IV stimulation in normal animals. The neurokinin-1 (NK1) receptor antagonist RP67580 reduced the C fiber-evoked responses following NGF treatment but not in naive preparations. The novel A beta fiber-evoked wind-up response was also reduced by RP67580. The NGF-induced changes in NK1 receptor responses occurred in the absence of any detectable changes in either spinal cord NK1 receptor dose-response relationships or NK1 receptor mRNA levels. These findings are likely to be related to the behavioural allodynia observed in the present study and to central excitability changes observed after chronic inflammation where NGF levels are increased.

Solution hybridization-nuclease protection assays for sensitive detection of differentially spliced substance P- and neurokinin A-encoding messenger ribonucleic acids.

In this chapter we discussed methods that can be used for the sensitive detection and quantitation of differentially or alternatively spliced mRNAs as well as mRNAs of low abundance. Although mechanisms responsible for splicing (and differential splicing in particular) have not been fully determined, many RNAs derived from a variety of genes have been observed to undergo the process. The impact of splicing with regard to the expanded potential of gene expression emphasizes the usefulness of the solution hybridization-nuclease digestion technique described here, compared to Northern blot analysis. The use of radiolabeled cRNA(s) provides for an assay of both high specificity and high sensitivity. While end-labeled cDNA probes can be used, they do not have the sensitivity inherent in the assay performed with uniformly radiolabeled cRNAs. If multiple mRNAs are derived from a single gene as a result of differential or alternative precursor RNA splicing, however, the results with a cRNA probe may initially appear to be quite complicated, and end-labeled cDNAs may yield more easily interpretable results. Nonetheless, both types of probes are useful in the context of gene expression analysis, and it is clear that for routine purposes of quantitation cRNA probes in solution hybridization-nuclease protection assays are clearly more desirable than RNA blot analyses due to their truly quantitative nature as well as ease of assay.

Tetrapeptide tachykinin antagonists: synthesis and modulation of the physicochemical and pharmacological properties of a new series of partially cyclic analogs.

We report on the synthesis and the pharmacological properties of a new series of tachykinin antagonists based on the pseudopeptide pharmacophore cyclo[-Abo-Asp(D-Trp-Phe-N(Me)Bzl)-] which contains the 2-azabicyclo[2.2.2]octane-3(S)-carboxylic acid (Abo) residue. Variation of the substituents on the tryptophan indole nitrogen was shown to modulate water solubility and transport properties of the analogs as well as potency in classical in vitro response and binding assays. One water-soluble compound, 16, in which the substituent was 3-carbonylpropionate, strongly prolonged the reaction time in the mouse hot-plate test both after iv or oral administration and was devoid of degranulating activity in rat peritoneal mast cells.

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.