A selective and extremely potent antagonist of the neurokinin-1 receptor

Sendide [Tyr6,D-Phe7,D-His9]-substance P(6-11) has been examined by measurements of ligand binding to crude membrane fractions and by functional tests on the spinally mediated behavioral response. Sendide potently displaced [3H]-labeled substance P (SP) binding to mouse spinal cord membranes in a competitive manner. In vivo, sendide, intrathecally co-injected with SP, competitively antagonized SP-induced scratching, biting and licking. The behaviors elicited by physalaemin, septide and [Sar9, Met(O2)11]-SP were also reduced by co-administration of sendide. Large doses of sendide were needed to reduce the action of neurokinin A, D-septide, neurokinin B and eledoisin. The in vitro and in vivo pharmacological profile of sendide demonstrated that it is a selective and extremely potent antagonist of the neurokinin-1 receptor.     

Multiple tachykinin receptors in guinea pig brain. High densities of substance K (neurokinin A) binding sites in the substantia nigra

Receptor binding characteristics of labelled substance P (SP) and substance K (SK) were investigated in guinea pig membrane preparations and by in vitro receptor autoradiography. The two ligands bind to single class of sites with Kd of 2.1 nM and Bmax of 37.0 fmol/mg protein for SP and 10.6 nM and 50.3 fmol/mg protein for SK. Ligand selectivity patterns markedly differ for the two ligands. On SP binding sites, SP greater than physalaemin greater than eledoisin greater than SK greater than kassinin greater than Neuromedin K. On the other hand, SK greater than kassinin greater than Neuromedin K greater than eledoisin greater than SP greater than physalaemin on SK binding sites. The autoradiographic distribution of SP and SK binding sites is also different. While the cerebral cortex, substantia nigra and cerebellum contain very low to low densities of SP sites, they are all rich in SK binding sites. These data clearly demonstrate the existence of at least two classes of tachykinin binding sites in guinea pig brain. Moreover, the high density of SK binding sites present in the substantia nigra suggests possible physiological roles for SK in this region.     

Biochemical characterization and autoradiographic localization of central substance P receptors using [I]physalaemin

The binding of [¹²⁵I]physalaemin to rat brain slices was investigated. Radiolabeled physalaemin bound with high affinity (K_d = 0.3 nM) to a single class of sites (B_max = 22 fmol/mg protein). Kinetic studies indicated that binding was time-dependent and all specific binding was reversible. Pharmacology studies indicated that specific [¹²⁵I]physalaemin binding was inhibited by structurally related peptides such as substance P and eledoisin. Biochemical studies indicated that specific binding of radiolabeled physalaemin was greatly reduced if the brain slices were pretreated with heat, trypsin or N-ethyl maleimide. Autoradiographic studies indicated that the [¹²⁵I]physalaemin binding sites were discretely distributed throughout the brain. Highest grain densities were present in the olfactory bulb, dentate gyrus, amygdala, superficial layers of the superior colliculus, subiculum, dorsal parabrachial nucleus, locus coeruleus, nucleus tractus solitarii and dorsal horn of the spinal cord. Moderate grain densities were present in the nucleus accumbens, olfactory tubercle, pyriform cortex, striatum, hippocampus, inferior colliculus and central gray of the midbrain. Low grain densities were present in most thalamic nuclei, the substantia nigra and cerebellum. The corpus callosum and controls treated with 1 μM unlabeled physalaemin had negligible levels of binding. The unique pharmacological and regional distribution data obtained suggest that [¹²⁵I]physalaemin may serve as a valuable probe to study central substance P receptors.     

Regional distribution of substance P, neurokinin alpha and neurokinin beta in rat spinal cord, nerve roots and dorsal root ganglia, and the effects of dorsal root section or spinal transection

The regional distribution of 3 mammalian tachykinins (substance P, neurokinin alpha and neurokinin beta) in the rat spinal cord and related structures was investigated using a method of radioimmunoassay combined with high performance liquid chromatography. Substance P and neurokinin alpha were found to be distributed in a very similar manner with fairly constant molar ratios i.e. ratios of substance P to neurokinin alpha were 3.69 in the dorsal root ganglia, 3.49 in the dorsal root and 3.09 in the dorsal horn of the cervical spinal cord. On the other hand, the distribution of neurokinin beta was different from other tachykinins; although concentrated in the dorsal horn, neurokinin beta in the dorsal root ganglia or in the dorsal roots was negligibly small in amount. When the cervical dorsal roots were sectioned unilaterally, substance P and neurokinin alpha were decreased in a parallel fashion in the dorsal horn, whereas neurokinin beta was not. In addition neurokinin alpha was selectively and significantly decreased in the dorsal horn of the intact side when compared to that in the unoperated control rat. Since the magnitude of a decrease of neurokinin alpha in molar basis was approximately the same as a decrease of substance P, these findings suggest that the neurokinin alpha and substance P-containing primary afferent fibres could project partly to the contralateral dorsal horn as well. When the thoracic spinal cord was transected, substance P (and neurokinin alpha) was decreased in the ventral part of the lumbar spinal cord, suggesting the presence of tachykinin(s)-containing descending fibres.(ABSTRACT TRUNCATED AT 250 WORDS)     

Eledoisin, substance P and related peptides: intracranial dipsogens in the pigeon and antidipsogens in the rat

The undecapeptide eledoisin caused vigorous and copious drinking within a minute or two of injection into the pigeon forebrain. Systemic injections of the same doses were ineffective. The relative efficacy of eledoisin and angiotensin II as dipsogens in the pigeon was similar to that of carbachol and angiotensin II in the rat. The related peptides eledoisin hexapeptide, physalaemin and substance P also caused some drinking, but they were less effective than eledoisin. In the rat none of these substances caused drinking. On the contrary eledoisin and substance P were found to depress angiotensin-induced drinking, but carbachol-induced drinking was not depressed to the same extent by these peptides. The preferential depression of angiotensin II-induced drinking resembles the effects of other vasoplegic drugs on this response in the rat, and may be related to the potent vasodilator properties of these peptides.     

Neuropeptide receptors in developing and adult rat spinal cord: An in vitro quantitative autoradiography study of calcitonin gene-related peptide,neurokinins, μ-opioid,galanin, somatostatin,neurotensin and vasoactive intestinal polypeptide receptors

A number of neuroactive peptides including calcitonin gene-related peptide (CGRP), substance P, neurokinin B, opioids, somatostatin (SRIF), galanin, neurotensin and vasoactive intestinal polypeptide (VIP) have been localized in adult rat spinal cord and are considered to participate either directly and/or indirectly in the processing of sensory, motor and autonomic functions. Most of these peptides appear early during development, leading to the suggestion that peptides, in addition to their neurotransmitter/neuromodulator roles, may possibly be involved in the normal growth and maturation of the spinal cord. To provide an anatomical substrate for a better understanding of the possible roles of peptides in the ontogenic development of the cord, we investigated the topographical profile as well as variation in densities of [¹²⁵I]hCGRPα, [¹²⁵I]substance P/neurokinin-1 (NK-1), [¹²⁵I]eledoisin/neurokinin-3 (NK-3), [¹²⁵I]FK 33–824 ([D-Ala², Me-Phe⁴, Met(O)ol⁵]enkephalin)/μ-opioid, [¹²⁵I]galanin, [¹²⁵I]T₀D₈-SRIF₁₄ (an analog of sornatostatin), [¹²⁵I]neurotensin and [¹²⁵I]VIP binding sites in postnatal and adult rat spinal cord using in vitro quantitative receptor autoradiography. Receptor binding sites recognized by each radioligand are found to be distributed widely during early stages of postnatal development and then to undergo selective modification to attain their adult profile of distribution during the third week of postnatal development. The apparent density of various receptor sites, however, are differently regulated depending on the lamina and the stage of development studied. For example, the density of μ-opioid binding sites, following a peak at postnatal day 4 (N), declines gradually in almost all regions of the spinal cord with the increasing age of the animal [¹²⁵I]substance P/NK-1 binding sites, on the other hand, show very little variation until P14 and then subsequently decrease as the development proceeds. In the adult rat, most of these peptide receptor binding sites are localized in relatively high amounts in the superficial laminae of the dorsal horn. To varying extents, moderate to low density of various peptide receptor binding sites are also found to be present in the ventral horn, intermediolateral cell column and around the central canal. Taken together, these results suggest that each receptor-ligand system is regulated differently during development and may each uniquely be involved in cellular growth, differentiation and in maturation of the normal neural circuits of the spinal cord, Furthermore, the selective localization of various receptor binding sites in adult rat spinal cord over a wide variety of functionally distinct regions reinforces the neurotransmitter/ modulator roles of these peptides in sensory, motor and autonomic functions associated with the spinal cord. © 1995 Wiley-Liss, Inc.     

Ovarian Steroid Modulation of Neurokinin Contents in Hypothalamus, Pituitary, Trigeminal Nucleus, and Cervical Spinal Cord of the Ovariectomized Female Rat

Ovariectomized rats were treated with estradiol benzoate (EB) and progesterone in conditions known to negatively and positively regulate gonadotropin secretion. Injection with EB decreased the plasma concentration of substance P at the time of the positive feed-back exerted by EB on gonadotropin secretion, while having no effect on the plasma concentration of neurokinin A. In the hypothalamus, EB injection enhanced the substance P and neurokinin A content, while progesterone reduced the substance P content. In the anterior pituitary, the substance P content was increased after progesterone, and this increase was blocked by EB. Conversely, in the posterior pituitary, the substance P content was reduced after progesterone, and this effect was enhanced by EB. In the trigeminal nucleus, the substance P content was increased after progesterone and EB, while only progesterone affected neurokinin A content. Finally, in the cervical spinal cord, the substance P and neurokinin A contents were reduced after EB. We conclude that neurokinin contents are controlled by ovarian steroids not only in the hypothalamo-pituitary complex but also in the trigeminal nucleus and the cervical spinal cord.     

Spinal NK2 receptors contribute to the increased excitability of the nociceptive flexor reflex during persistent peripheral inflammation

The role of endogenous neurokinin A in changes in the excitability of spinal neurons during adjuvant-induced, peripheral inflammation was examined by determining the effect of a selective NK₂ receptor antagonist, GR103537, on the nociceptive flexor reflex in rats. Intrathecal administration of GR103537 (1.4–14 nmol) dose-dependently attenuated the increased activity of the flexor reflex ipsilateral to the inflamed paw. The activity of GR103537 at NK₂ receptors was confirmed by blockade of the facilitation of the reflex by neurokinin A but not substance P in normal rats. These results indicate that endogenous neurokinin A increases the excitability of spinal neurons during persistent peripheral inflammation.     

Neurokinin 1 receptor expression by neurons in laminae I, III and IV of the rat spinal dorsal horn that project to the brainstem

Large neurons in laminae III and IV of the spinal cord which express the neurokinin 1 receptor and have dendrites that enter the superficial laminae are a major target for substance P (SP)-containing (nociceptive) primary afferents. Although some of these neurons project to the thalamus, we know little about other possible projection targets. The main aim of this study was to determine whether all cells of this type are projection neurons and to provide information about brainstem sites to which they project. Injections of cholera toxin B subunit were made into four brainstem areas that receive input from the spinal cord, and the proportion of cells of this type in the L4 spinal segment that were retrogradely labelled was determined in each case. The results suggest that most of these cells (>90%) project to the contralateral lateral reticular nucleus (or to a nearby region), while many (>60%) send axons to the lateral parabrachial area and some to the dorsal part of the caudal medulla. However, few of these cells project to the periaqueductal grey matter. As lamina I neurons with the neurokinin 1 receptor appear to be important in the generation of hyperalgesia, we also examined projection neurons in this lamina and found that for each injection site the great majority possessed the receptor. These results demonstrate that dorsal horn neurons which express the neurokinin 1 receptor contribute to several ascending pathways that are thought to be important in pain mechanisms.     

Characterization and segmental distribution of 125I-Bolton-Hunter-labeled substance P binding sites in rat spinal cord

Substance P (SP) is widely distributed in the spinal cord and has been implicated as a neurotransmitter in several spinal cord neuronal systems. To investigate SP receptors in the spinal cord, 125I-Bolton-Hunter-SP (125I-BH-SP) was used to identify and characterize spinal cord binding sites for the peptide. The binding of 125I-BH-SP had the following characteristics: high affinity; time, temperature, and membrane concentration dependent; reversible; and saturable. The IC50 of SP in whole spinal cord was 0.46 nM as compared with 0.95, 60, and 150 nM for physalaemin, eledoisin, and kassinin. Four putative antagonists of SP were less than 0.0001 times as potent as SP in inhibiting 125I-BH-SP binding. IC50s were 5, 7.5, 7.0, and 45 microM for D-Pro2, D-Trp7,9-SP; D-Pro2, D-Phe7, D-Trp9-SP; D-Arg1, D-Pro2, D-Trp7,9, Leu11-SP; and D-Pro4, D-Trp7,9,10-SP(4-11), respectively. The lumbosacral section bound 3 times more SP than the cervical and thoracic sections, although IC50 for the cervical section was 0.06 of that for the lumbosacral and thoracic sections. The data suggest more than one class of binding site for SP in the spinal cord and indicate a direct role for SP in spinal cord functions.     

Behavioural effects of tachykinins and related peptides

Substance P (SP) and related tachykinins administered either intracerebroventricularly or directly into the ventral tegmental area of the mesencephalon of rat brain caused increased locomotor activity, grooming behaviour and wet dog shakes. Kassinin, eledoisin, neurokinin A and DiMe-C7, agonists with some selectivity for the SP-E-receptor elicited the greatest locomotor activity and wet dog shake responses, whereas SP and physalaemin which are more selective for the SP-P-receptor were most effective in eliciting the grooming response.     

Cannabinoid CB1 receptor facilitation of substance P release in the rat spinal cord,measured as neurokinin 1 receptor internalization

The contribution of CB1 receptors in the spinal cord to cannabinoid analgesia is still unclear. The objective of this study was to investigate the effect of CB1 receptors on substance P release from primary afferent terminals in the spinal cord. Substance P release was measured as neurokinin 1 (NK1) receptor internalization in lamina I neurons. It was induced in spinal cord slices by dorsal root stimulation and in live rats by a noxious stimulus. In spinal cord slices, the CB1 receptor antagonists AM251, AM281 and rimonabant partially but potently inhibited NK1 receptor internalization induced by electrical stimulation of the dorsal root. This was due to an inhibition of substance P release and not of NK1 receptor internalization itself, because AM251 and AM281 did not inhibit NK1 receptor internalization induced by exogenous substance P. The CB1 receptor agonist ACEA increased NK1 receptor internalization evoked by dorsal root stimulation. The effects of AM251 and ACEA cancelled each other. In vivo, AM251 injected intrathecally decreased NK1 receptor internalization in spinal segments L5 and L6 induced by noxious hind paw clamp. Intrathecal AM251 also produced analgesia to radiant heat stimulation of the paw. The inhibition by AM251 of NK1 receptor internalization was reversed by antagonists of μ‐opioid and GABA_B receptors. This indicates that CB1 receptors facilitate substance P release by inhibiting the release of GABA and opioids next to primary afferent terminals, producing disinhibition. This results in a pronociceptive effect of CB1 receptors in the spinal cord.     

Disruption of the substance P receptor (neurokinin-1) gene does not prevent upregulation of preprotachykinin-A mRNA in the spinal cord of mice following peripheral inflammation

The neuropeptide substance P is thought to play an important role in nociception, although the function of the peptide remains controversial. Following peripheral inflammation there is a pronounced upregulation of substance P expression both in sensory neurons and in postsynaptic neurons within the spinal cord. We have examined the levels of expression of mRNA encoding substance P and dynorphin following the development of inflammatory hyperalgesia in mice in which the substance P receptor gene, also known as the neurokinin-1 receptor gene, has been disrupted by homologous recombination. We show that inflammatory hyperalgesia following injection of complete Freund's adjuvant develops normally in animals that lack the neurokinin-1 receptor and that expression of mRNAs encoding substance P and the neuropeptide dynorphin are upregulated regardless of the genotype of the mouse. This suggests that substance P activity is not required for the development and maintenance of inflammatory hyperalgesia and that the upregulation of substance P expression is mediated by neurotransmitters other than substance P.     

Substance p analogue blocks sp-induced facilitation of a spinal nociceptive reflex

Intrathecal administration of 10 micrograms (6.5 nmoles) of substance P to the lumbar spinal cord of the awake, restrained rat had a biphasic effect on reaction time in the tail-flick test. This effect consisted of an initial decrease at one min after administration, followed five min later by a smaller increase in reaction time. When substance P was given after prior intrathecal administration of 1.55, 3.1 or 6.2 nmoles of [D-Pro2, D-Phe7, D-Trp9]-substance P, the reduction in latency could be blocked in a dose-related manner. The analogue alone failed to alter reaction time but did produce a flaccid paralysis in some cases. Our results support the possibility that substance P is involved in transmission of nociceptive information at the spinal level, but indicate that it does not participate directly in the fast tail withdrawal response. The mechanism of the flaccid paralysis is not understood, but it appears to be a cumulative effect of more than one dose and is likely associated with motor rather than sensory neurones.     

Action of intrathecal capsaicin and its structural analogues on the content and release of spinal substance P: Selectivity of action and relationship to analgesia

Intrathecal injections of capsaicin (CAP) and 4 other homovanillic acid (HMV) derivatives related to the structure of CAP were carried out. Capsaicin, 1-nonenoylvanillylamide (NVA), HMV-dodecylamide (DCA) (but not HMV-cyclohexylamide (CHA) or HMV-hexadecylamide (HDC] reduced the spinal content of substance P (SP), as measured by radioimmunoassay (RIA), and increased the tail-flick latency. Similar injection of kainic acid and piperine reduced levels of SP but failed to affect the tail-flick latency. None of the agents used affected spinal levels of cholecystokinin (CCK) or vasoactive intestinal peptide (VIP) as measured by RIA. In experiments using in vivo superfusion of the rat spinal cord, CAP, DCA and NVA were found to stimulate release of SP. Capsaicin had no effect on the levels of CCK or VIP immunoreactivity in the spinal superfusate. A tachyphylaxis to the effect of CAP and DCA on spinal SP release was demonstrated. Pretreatment with either agent blocked the releasing effect of the second. Pretreatment with an inactive analogue (HDC) had no effect on the subsequent activity of CAP. Kainic acid and piperine did not induce release of SP from the spinal cord. The relative selectivity of spinally administered capsaicinoids with regard to their effects on the content and release of peptides known to be contained in primary afferents and the presence of a similar structure-activity relationship for depletion and release of SP, desensitization and antinociception suggest the presence of a specific receptor site associated with a specific population of primary afferents through which pain information may pass. Whether SP is an 'afferent pain transmitter' is not clear, but at the least, it appears to serve as a marker for a population of afferents acted upon by spinally administered capsaicinoids.     

The effects of substance P and other peptides on spinal neurons of the frog

The effects of substance P and other peptides were studied on the neurons of isolated spinal cord of the frog. Substance P and certain related peptides, which have a common C-terminal sequence, -Phe-X-Gly-Leu-Met-NH₂ (where X=Ile, Tyr or Phe), exerted a remarkably strong excitant action on spinal motoneurons. On a molar basis, substance P was about 200 times, physalaemin 1500 times, and eledoisin 2000 times more active thanl-glutamate in depolarizing the spinal motoneuron in 0.4 mM Ca-Ringer's solution. Since the depolarizing action of substance P and related peptides persisted after the synaptic transmission was blocked by Ca-deficient (0–0.2 mM) Ringer's solution or by tetrodotoxin (10^?8?10^?7 g/ml), it was concluded that these peptides have a direct action on the motoneurons to induce the depolarization.Another group of peptides, bradykinin, angiotensins I and II, caused the depolarization of the spinal motoneurons by a trans-synaptic mechanism. These peptides probably activated the interneurons and thus caused the release of the excitatory transmitter from the nerve terminals synapsing with motoneurons.The possibility that substance P may be an excitatory transmitter of primary sensory neurons was discussed.     

Functional substance P receptors on a human astrocytoma cell line (U-373 MG)

[125I]Bolton Hunter conjugate of substance P ([125I]BHSP) can bind to human astrocytoma membranes in a monophasic and saturable manner with a Kd of 0.57 +/- 0.17 nM and a Bmax of 67.8 +/- 5.5 fmol/mg protein. The rank order of potency of tachykinins and related analogues as inhibitors of [125I]BHSP binding to astrocytoma membranes and intact cells correlated with their relative abilities to stimulate uridine incorporation into nucleic acid. The observed specificity pattern conformed to that reported for the NK1 tachykinin receptor with SP much greater than eledoisin greater than neurokinin A greater than neurokinin B and [Glp6, L-Pro9]SP(6-11) much greater than [Glp6, D-Pro9]SP(6-11).     

Substance p anatogue blocks sp-induced facilitation of a spinal nociceptive reflex

Intrathecal administration of 10 μg (6.5 nmoles) of substance P to the lumbar spinal cord of the awake, restrained rat had a biphasic effect on reaction time in the tail-flick test. This effect consisted of an initial decrease at one min after administration, followed five min later by a smaller increase in reaction time. When substance P was given after prior intrathecal administration of 1.55, 3.1 or 6.2 nmoles of [D-Pro²,D-Phe⁷,D-Trp⁹]-substance P, the reduction in latency could be blocked in a dose-related manner. The analogue alone failed to alter reaction time but did produce a flaccid paralysis in some cases. Our results support the possibility that substance P is involved in transmission of nociceptive information at the spinal level, but indicate that it does not participate directly in the fast tail withdrawal response. The mechanism of the flaccid paralysis is not understood, but it appears to be a cumulative effect of more than one dose and is likely associated with motor rather than sensory neurones.