Tachykinin multiplicity in rat central nervous system as studied using antisera raised against substance P and neurokinin A

Antisera were raised in rabbits against the tachykinins neurokinin A (NKA) and substance P (SP). All NKA-antisera tested cross-reacted markedly with NKB, kassinin and eledoisin in radioimmunoassay (RIA), but virtually not with SP and physalaemin. Also when used for immunohistochemistry, one of the NKA-antisera was found to be virtually without cross-reactivity with SP. The most specific SP-antiserum did not cross-react with NKA but to some extent with NKB at the immunohistochemical level. Using these two antisera, the same distribution pattern of immunoreactivity was seen in both the rat substantia nigra and dorsal spinal cord. In neutral extracts of the substantia nigra, all NKA-antisera used for RIA detected a major component which eluted at the position of NKA in reverse phase high performance liquid chromatography, while no or only little immunoreactivity was detected at the position of NKB. A major component of substance P-like immunoreactivity (SPLI) co-eluting with SP and one or two minor SPLI-components were also detected in these extracts. An SP-antiserum, which cross-reacted markedly with physalaemin, detected an additional rather prominent component. In neutral water extracts of dorsal spinal cord the component detected with the NKA-antisera at the position of NKB, as well as one of the SPLI-components not eluting in the position of SP, were much more prominent than in the corresponding extracts of substantia nigra. In acetic acid extracts of both tissues, only one major SPLI-component co-eluting with SP could be detected, while only very small amounts of immunoreactivity eluting at the position of NKA and NKB (dorsal spinal cord only) could be detected using the NKA-antisera. The present results illustrate the importance of the extraction method used in immunochemical studies and demonstrate that the relative proportions of various tachykinins are markedly different in the rat substantia nigra and dorsal spinal cord.     

Antisera raised against eledoisin and kassinin detect immunoreactive material in rat tissue extracts: tissue distribution and chromatographic characterization

Radioimmunoassays were developed for the tachykinins eledoisin (ELE) and kassinin (KAS) using antisera raised in rabbits. The antisera exhibited low (less than 0.1%) cross-reactivities to substance P (SP) and physalaemin (PHY), but crossreacted (with one exception, antiserum K7) to varying extents with neurokinin A (NKA) and neurokinin B (NKB). In the rat, the tissue distribution of the immunoreactive material detected by antiserum (E7) raised against ELE and by another antiserum (K1) raised against KAS both resembled that previously described for SP. Using the highly KAS-specific antiserum K7, no or only very low levels of immunoreactivity could be detected in extracts of various rat tissues. Gel permeation chromatography and ion-exchange chromatography of tissue extracts indicated that all antisera (except K7) detected the same population of immunoreactive molecules. One of the components was chromatographically indistinguishable from NKA. The tissue distribution of this component also resembled that of SP. Another immunoreactive component co-chromatographed with NKB at cation exchange chromatography. Acid tissue extracts, but not neutral tissue extracts, were found to contain immunoreactive components which appeared more basic than NKA and NKB. The total levels of immunoreactivity were higher in neutral than in acid tissue extracts. However, the ratio between the amounts of immunoreactivities in the two types of extracts varied considerably between tissues, indicating that tachykinin immunoreactive components may be present in different relative proportions in various tissues.     

Multiple tachykinins (neurokinin A, neuropeptide K and substance P) in capsaicin-sensitive sensory neurons in the guinea-pig

The occurrence of tachykinins in sensory neurons of the guinea-pig was studied by means of radioimmunoassay combined with ion-exchange and high-performance liquid chromatography as well as by immunohistochemistry. Antisera raised against kassinin (antiserum K12), neurokinin A (NKA) (antiserum NKA2) and substance P (SP) (antisera SP25 and SP2) were used. Antiserum K12 detected NKA, neuropeptide K (NPK) and a component eluting in the position of eledoisin (ELE) in extracts of the lung and ureter. Neurokinin B (NKB) was, however, not found. Neutral water extraction favored recovery of NKA and of the ELE-like component, while NPK was found only in acid extracts. The SP antisera detected two immunoreactive components of which the major form coeluted with synthetic SP. Capsaicin pretreatment depleted all these various forms of immunoreactivity in several peripheral organs including the ureter and lung. The immunoreactivity detected by antisera K12 or SP25 in radioimmunoassay had a similar regional distribution pattern in peripheral tissues. Immunohistochemical examination revealed that antiserum NKA2 stained the same spinal ganglion cells as the SP2 antiserum. The distribution of capsaicin-sensitive nerve fibers stained by these two antisera was also identical in peripheral organs such as the ureter, inferior mesenteric ganglion, heart and lung. It is concluded that multiple tachykinins, including SP, NKA, NPK and an ELE-like peptide, are present in capsaicin-sensitive sensory nerves in the guinea-pig. This finding can most likely be related to the origin of SP, NKA and NPK from the same precursor molecule, subsequent posttranslational tissue processing and axonal transport to terminal regions.     

Antisera raised against eledoisin and kassinin detect elevated levels of immunoreactive material in plasma and tumor tissues from patients with carcinoid tumors

Radioimmunoassays based on antisera raised against the tachykinins eledoisin (antiserum E7) and kassinin (antiserum K12) were used to measure the concentration of tachykinin-like immunoreactivity (TKLI) in plasma from 52 healthy subjects. 65 patients with carcinoid tumors (of which 46 had symptoms of both flushing and diarrhoea), and 6 patients with endocrine pancreatic tumors. The antisera did not crossreact with substance P (SP). Elevated concentrations of TKLI, as compared with healthy subjects, were found in 75% of the carcinoid patients, but in none of the patients with pancreatic tumors. Tumor metastases from 8 of the carcinoid patients all contained TKLI. Ion-exchange chromatography of plasma samples and tumor tissue extracts indicated the presence of several immunoreactive molecular forms. The elution patterns of the immunoreactivity detected by antisera E7 and K12 were similar, indicating that the same molecular species are measured by these antisera. None of the components coeluted with synthetic SP. One of the immunoreactive components in carcinoid tumor extracts coeluted with synthetic NKA. The major immunoreactive components in plasma from the patients eluted in a position different from that of all currently known mammalian tachykinins. Tachykinin immunoreactive material detected in tumor tissue and plasma of patients with carcinoid tumor may play a role in the symptomatology of the carcinoid syndrome.     

Neurokinin B- and substance P-like immunoreactivity are co-localized in enteric nerves of rat ileum

The tachykinins (TKs) substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) have conserved C-terminal sequences and mediate similar physiological responses by activating neurokinin receptors found on neural and smooth muscle cells. Many enteric nerves express preprotachykinin A (PPT A) mRNA and synthesize SP and NKA. However, it is unclear if NKB is synthesized in enteric neurons as many antibodies developed against NKB also recognize other TKs. Therefore, the cellular distribution of NKB-like-immunoreactivity (NKB-ir) in rat ileum was examined using selective antisera raised against either synthetic Cys10-NKB or peptide 2 (P2), a non-tachykinergic peptide sequence in NKB precursor protein. NKB-ir and P2-ir had a similar distribution in varicose nerve fibers in submucosal and myenteric ganglia and almost all ganglia contained immunoreactive nerves. Few submucosal or myenteric neuronal somata contained strong immunoreactivity. Preabsorption of NKB or P2 antisera with their respective cognate peptides, but not with other TK peptides, abolished specific immunostaining. Finally, co-localization of NKB-/P2-ir with SP-ir suggested that most NKB-/P2-ir nerve fibers contain SP-ir, but some SP-ir nerves do not contain detectable NKB-/P2-ir. These results indicate that PPT B products P2 and NKB are localized in a subpopulation of enteric nerves containing TKs encoded by PPT A. Stimulation of these nerves may release NKB to activate local neurokinin receptors.     

Pharmacological receptors for substance P and neurokinins

The three neurokinins identified in mammals, substance P, neurokinin A and neurokinin B, as well as their C-terminal biologically active fragments, have been used to characterize the responses of a variety of isolated organs. Three preparations selective either for substance P (the dog carotid artery), or for neurokinin A (the rabbit pulmonary artery) or for neurokinin B (the rat portal vein) are described. A neurokinin receptor classification is attempted using the neurokinins and their fragments to determine the order of potency of agonists. Three receptor subtypes have been identified: the NK-P, on which substance P (SP) is more active than neurokinin A (NKA) and neurokinin B (NKB), and the neurokinins are more active than their respective fragments; the NK-A on which NKA greater than NKB greater than SP, and some NKA fragments are more discriminative than their precursor; the NK-B on which NKB greater than NKA greater than SP, and fragments of NKB are less active than their precursor. Among the peptides studied, some potent compounds have been identified that could provide selective receptor ligands.     

Tachykinin immunoreactivity in the European common frog, Rana temporaria: localization, quantification and chromatographic characterization.

1. Tachykinin immunoreactivity has been localized, quantified and chromatographically-characterized in the brain, stomach, intestine and skin of Rana temporaria. 2. Antisera to mammalian substance P (SP) and neurokinin A (NKA) immunostained nerve fibres in all tissues except skin, and a population of mucosal endocrine cells in the intestinal epithelium. 3. Radioimmunoassay of tissue extracts identified SP immunoreactivity in all tissues but NKA immunoreactivity was restricted to the brain. 4. Chromatographic analysis of both frog tachykinins revealed that they possessed different physico-chemical properties than their mammalian counterparts.     

Tachykinin immunoreactivity in the european common frog,Rana temporaria: Localization,quantification and chromatographic characterization

1. Tachykinin immunoreactivity has been localized, quantified and chromatographicallycharacterized in the brain, stomach, intestine and skin of Rana temporaria.2. Antisera to mammalian substance P (SP) and neurokinin A (NKA) immunostained nerve fibres in all tissues except skin, and a population of mucosal endocrine cells in the intestinal epithelium.3. Radioimmunoassay of tissue extracts identified SP immunoreactivity in all tissues but NKA immunoreactivity was restricted to the brain.4. Chromatographie analysis of both frog tachykinins revealed that they possessed different physicochemical properties than their mammalian counterparts.     

Spinal action of neurokinins in the rat: effects on mean arterial pressure, heart rate, and vascular permeability

In urethane-anaesthetized rats, the intrathecal administration of 6.5 nmol of substance P (SP), neurokinin A (NKA), or neurokinin B (NKB) at the T8-T10 level of the spinal cord enhances mean arterial pressure and heart rate. However, in the pentobarbital-anaesthetized rat, while NKB produces no effect on mean arterial pressure, NKA produces a biphasic change and SP, a depressor response. All three neurokinins elicit a tachycardia. The following rank order of potency SP greater than or equal to NKA greater than NKB is observed in relation to these cardiovascular responses when either one of the two anaesthetics is used. The low cardiovascular activity of NKB cannot be attributed to its hydrophobicity, as the water soluble analogue of NKB, [Arg0]NKB, elicits a response as weak as the native peptide. In pentobarbital-anaesthetized rats, the intrathecal administration of 6.5 nmol of SP, also enhances plasma protein extravasation in cutaneous tissues of the back, the hind paws, and the ears. In this response NKA and NKB are either inactive (skin of hind paws) or less potent than SP (ears and dorsal skin). These findings agree with the hypothesis that in the rat spinal cord, the neurokinin receptor producing changes in mean arterial pressure, heart rate, and vascular permeability is of the NK-1 subtype.     

Establishment of highly specific radioimmunoassays for neurokinin A and neurokinin B and determination of tissue distribution of these peptides in rat central nervous system

Highly specific radioimmunoassays (RIAs) for neurokinin A (NKA) and neurokinin B (NKB) were developed. Antisera were produced by the procedure which involved immunization with NKA or NKB, both conjugated with keyhole limpet hemocyanin, and treatments with a tolerogenic conjugate of kassinin and a copolymer of D-glutamic acid and D-lysine (D-GL) to inhibit the production of cross-reactive antibodies against common C-terminal region of tachykinins. Cross-reactivities of anti-NKA antiserum (R704), thus produced, with NKB, kassinin, eledoisin were 12.6%, 10.6% and 11.5%, respectively. This was in sharp contrast with those of antiserum obtained from the rabbit not treated with kassinin-D-GL, these values corresponding to 129.0%, 42.5% and 94.4%, respectively. The cross-reactivities of R704 with substance P and physalaemin were 0.3% and 1.5%, respectively. This antiserum also bound 35.6% of neuropeptide K which contains NKA at its C-terminal. More importantly, anti-NKB antiserum (R707) obtained by the above tolerizing regimen was highly specific for NKB and the cross-reactivities with NKA, neuropeptide K, kassinin and other tachykinins were all less than 0.001%. RIAs using these specific antisera allowed us to measure directly NKA and NKB in tissue extracts without their fractionation by chromatography prior to RIAs. Measurements of immunoreactive NKA and NKB in different rat brain regions and spinal cord revealed that they are present with various ratios (NKA/NKB: 1.1-9.9) depending on the region.     

Tachykinin receptor subtype that mediates the increase in vascular permeability in guinea pig skin

To determine the tachykinin receptor subtype that mediates the increase in vascular permeability, we examined the rank order of potency of tachykinins for inducing plasma extravasation in guinea pig skin and the specificity of tachykinin-induced tachyphylaxis of the responses. Plasma extravasation of the skin induced by tachykinins was NK-1 (SP-P)-type response from the rank order of potency of mammalian and nonmammalian tachykinins. Tachyphylaxis of the vascular response was induced by intradermal preinjection of mammalian tachykinins and was tachykinin-specific. In substance P (SP) tachyphylaxis (where SP was preinjected), the response to SP, not to neurokinin A (NKA) or neurokinin B (NKB), was decreased. In NKA tachyphylaxis and NKB tachyphylaxis, the response to NKA, not to SP or NKB, and the response to NKB, not to SP or NKA, were decreased, respectively. Thus, we conclude that the apparent NK-1-type response is mediated through three mammalian tachykinin receptors, NK-1, NK-2, and NK-3, which are specifically stimulated by their preferred agonist, SP, NKA, and NKB, respectively.     

Degradative enzymes modulate airway responses to intravenous neurokinins A and B

We studied the effects of the neutral endopeptidase (NEP) inhibitor thiorphan (1.7 mg/kg iv) and the angiotensin-converting enzyme (ACE) inhibitor captopril (5.7 mg/kg iv) on airway responses to rapid intravenous infusions of neurokinin A (NKA) and neurokinin B (NKB) in anesthetized, mechanically ventilated guinea pigs. The dose of NKA required to decrease pulmonary conductance to 50% of its base-line value (ED50GL) was fivefold less (P less than 0.0001) in animals treated with thiorphan compared with controls. NKA1-8, a product resulting from cleavage of NKA by NEP, had no bronchoconstrictor activity. Similar results were obtained by using NKB as the bronchoconstricting agent. Captopril had no significant effect on airway responses to NKA or NKB. In contrast, both thiorphan and captopril decrease the ED50GL for substance P (SP). We also compared the relative bronchoconstrictor potency of NKA, NKB, and SP. In control animals, the rank order of ED50GL values was NKA much less than NKB = SP. NKA also caused a more prolonged bronchoconstriction than SP or NKB. Thiorphan had no effect on the rank order of bronchoconstrictor potency, but in animals treated with captopril, the rank order of ED50GL values was altered to NKA less than SP less than NKB. These results suggest that degradation of NKA and NKB by NEP but not by ACE is an important determinant of the bronchoconstriction induced by these peptides. The degradation by ACE of SP but not NKA or NKB influences the observed relative potency of the three tachykinins as bronchoactive agents.     

Effect of sensory neuropeptides on canine bronchial and pulmonary vessels in vitro

Sensory neuropeptides may be important in the noncholinergic component of parasympathetic vasodilation in the tracheobronchial circulation. We studied the effects of substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and calcitonin gene-related peptide (CGRP) on the isolated canine bronchial artery and used pulmonary artery and vein of similar size for comparison. CGRP (10pM-300nM) was a potent relaxant of the bronchial and pulmonary arteries, and the pulmonary vein with equal potency in all vessels. SP in low concentrations (10pM-100nM) caused vasodilation of the precontracted bronchial artery and in high concentration (10-100 microM) contracted the vessel from resting tone. SP also relaxed the pulmonary artery and vein. NKA and NKB caused relaxation in all three vessels. All of the vascular effects of the sensory neuropeptides were concentration-dependent. The order of potency of the neuropeptides in the bronchial and pulmonary artery was SP greater than NKA greater than CGRP greater than NKB. In the pulmonary vein NKB caused a much larger relaxation than SP and NKA but it was less potent than either NKA or CGRP. Capsaicin (1 microM) caused a large contraction of the bronchial artery, similar in magnitude to the contraction caused by high dose of SP. Neuropeptide Y was also studied and found to cause no consistent constriction of any of the vessels studied. In conclusion, CGRP is a universal dilator of the bronchial and pulmonary blood vessels. SP and NKA exert their main effect on arterial vasomotor tone, whereas NKB is the only tachykinin producing marked dilation of the pulmonary vein.     

Neurokinin A and neurokinin B in the human retina

Very recently, the authors found levels of neurokinin (NK) A-like immunoreactivities in the human retina which were more than five times higher than those of substance P (SP). The present study aimed to find out how many of these immunoreactivities can be attributed to NKA and NKB and then the exact distribution pattern of both NKA and NKB was evaluated in the human retina and compared with that of SP. For this purpose, NKA-like immunoreactivities were characterized in the human retina by reversed phase HPLC followed by radioimmunoassay using the K12 antibody which recognizes both NKA and NKB. Furthermore, the retinae from both a 22- and 70-year-old donor were processed for double-immunofluorescence NKA/SP and NKB/SP. The results showed that NKA contributes to approximately two thirds and NKB to approximately one third of the immunoreactivities measured with the K12 antibody. NKA was found to be localized in sparse amacrine cells in the proximal inner nuclear layer, in displaced amacrine cells in the ganglion cell layer with processes ramifying in stratum 3 of the inner plexiform layer and also in sparse ganglion cells. By contrast, staining for NKB was only observed in ganglion cells and in the nerve fiber layer. Double-immunofluorescence revealed cellular colocalization of NKA with SP and also of NKB with SP. Thus, the levels of NKA and NKB are more than three and two times higher than those of SP, respectively. Whereas the distribution pattern of NKA is typical for neuropeptides, the localization of NKB exclusively in ganglion cells is atypical and unique.     

Immunohistochemical and chromatographic studies of peptides with tachykinin-like immunoreactivity in the central nervous system of the lamprey

The distribution and chemical properties of compounds with tachykinin-like immunoreactivity (TK-LI) in the spinal cord and brain of lampreys (Lampetra fluviatilis and Ichthyomyzon unicuspis) were investigated by means of immunohistochemistry and various chromatographic methods combined with radioimmunoassay. The distribution of TK immunoreactive fibers in the lamprey spinal cord was investigated with 13 different TK antisera which gave positive staining in pilot experiments. The antisera were raised against substance P (SP) (n = 6), physalaemin (PHY) (n = 1), neurokinin A (NKA) (n = 2), kassinin (KAS) (n = 2) or eledoisin (ELE) (n = 2). Pre-incubation of these antisera with their corresponding TKs abolished or reduced the immunostaining. Four different patterns of distribution were found with the 13 antisera, and they did not seem to be related to the TKs against which the antisera were raised. The different patterns could be explained by assuming the presence of the three different TKs. Six different antisera, raised against SP (n = 2), KAS (n = 2) or ELE (n = 2), were used for radioimmunoassay. The TK-LI material eluted as several separate components in various chromatographic systems. The central nervous system (CNS) of the lamprey did not contain measurable amounts of SP, NKA, neurokinin B (NKB), KAS or ELE. The present data imply that the lamprey CNS contains at least three different TKs probably different from SP, PHY, NKA, NKB, KAS or ELE; these are possibly new, not earlier described TKs. The three hypothetical TKs differ in their distribution.     

Pharmacology of neurokinin receptors.

The neurokinins are a group of naturally occurring peptides with the common C-terminal sequence Phe-X-Gly-Leu-Met.NH2. They include substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). SP and NKA are coded on the same gene, the PPT-A, while NKB is coded on a separate gene, the PPT-B. Neurokinins are present in the central nervous system and in peripheral organs where they exert various actions. They act on three receptors--NK-1, NK-2, and NK-3--characterized through pharmacological, biochemical, and histochemical studies. Selective agonists for each neurokinin receptor were developed and evaluated on isolated smooth muscle preparations containing only one neurokinin receptor type. All three neurokinin receptors were cloned and expressed in Xenopus oocytes. Relative affinities of those receptors to neurokinins are the same as in their respective smooth muscle preparation. Finally, the mechanism of action of SP on histamine release from rat peritoneal mast cell has been studied and a direct activation of G proteins by peptides with basic amino acids is proposed as a working hypothesis.     

Characterization of Substance P-Like Immunoreactivity and Tachykinin-Encoding mRNAs in Rat Medullary Thyroid Carcinoma Cell Lines

Rat thyroid tissue and three rat medullary thyroid carcinoma cell lines, 6-23, WE4/2, and CA77, have been examined for substance P (SP) and SP-like peptide expression. Analysis by combined HPLC and radioimmunoassay revealed the presence of SP in thyroid and 6-23 cell extracts. The presence of SP-encoding mRNAs was also detected in 6-23 cells by solution hybridization-nuclease protection analysis. SP-encoding mRNA expression was increased (fourfold) by maintaining the 6-23 cells in low serum (2%) for 4 or 10 days. The 6-23 cells also expressed other SP-like immunoreactive species, which were chromatographically and immunologically distinct from established tachykinin peptides. WE4/2 cells did not contain SP but did display SP-like immunoreactivity (SPLI), which migrated like the unidentified SPLI in 6-23 cells. CA77 cells did not contain SP or SP-encoding mRNA but did contain SPLI that migrated identically to the unidentified SPLI in the other cell lines. This novel SPLI was detected with an antiserum directed against the SP carboxyl terminus and to a lesser extent with an antiserum directed against the neurokinin A carboxyl terminus, but it showed minimal cross-reactivity using an antiserum directed against the midportion of SP. Treatment with 50 mM KCl resulted in secretion of this SPLI from CA77 cells. Gel filtration analysis demonstrated that this novel SPLI had an apparent molecular weight of approximately 1,000. These results are discussed in terms of cell lines that express tachykinin peptides and in terms of the molecular nature of the new SPLI detected in CA77 cells.     

Immunochemical characterisation of tachykinin immunoreactivity in the nervous system of the garden snail,Helix aspersa

1. Circumoesophageal ganglia and foot muscle of the garden snail. Helix aspersa, were subjected to immunocytochemistry using antisera to the tachykinins, substance P (SP), neurokinin A (NKA), kassinin (KAS) and eledoisin (ELE).2. Immunoreactivity in neuronal somata and fibres was detected only with the SP antiserum.3. SP and NKA radioimmunoassays were performed on extracts of Circumoesophageal ganglia. In common with immunocytochemistry, immunoreactivity was only detected with the SP antiserum.4. Gel permeation chromatography of extracts resolved a single peak of immunoreactivity eluting slightly later than synthetic mammalian SP. Reverse-phase HPLC of immunoreactive fractions resolved two immunoreactive peptides representing oxidised and reduced forms of a single peptide.5. These data suggest that the nervous system of H. aspersa contains a single tachykinin with C-tenninal structural characteristics similar to mammalian SP.     

[3H]Neurokinin B and 125I-Bolton Hunter Eledoisin Label Identical Tachykinin Binding Sites in the Rat Brain

[3H]Neurokinin B ([3H]NKB) of high specific activity (75 Ci/mmol) was synthesized for study of its binding to crude synaptosomes from the rat cerebral cortex. The specific binding of [3H]NKB (75% of total binding) was temperature dependent, saturable, and reversible. Scatchard analyses and Hill plots showed the existence of a single population of noninteracting binding sites (KD = 4.3 nM; Bmax = 123 fmol/mg of protein). Competition studies indicated the following rank order of potencies among tachykinins: NKB greater than eledoisin (E) greater than kassinin greater than physalaemin greater than neurokinin A (NKA) greater than substance P (SP), a result suggesting that NKB might be the endogenous ligand for [3H]NKB binding sites. It is of interest that 127I-Bolton Hunter (BH) NKA (127I-BHNKA) was much more potent than NKA in inhibiting the specific binding of [3H]NKB, which raises certain questions concerning the use of 125I-BHNKA as a ligand for NKA binding sites in the brain. These results, as well as those obtained with different SP analogues, show a close similarity to those obtained previously with 125I-BHE binding to cortical synaptosomes. This suggested that the two ligands labeled identical binding sites. In addition, using either [3H]NKB or 125I-BHE as ligands, similar displacement curves were obtained with increasing concentrations of NKB and 127I-BHE. The similarity of the [3H]NKB and 125I-BHE binding sites was further confirmed by comparison of their localization on rat brain sections by autoradiography. The distribution of binding sites for [3H]NKB and 125I-BHE was identical throughout the brain, and the highest density of binding sites for the two ligands was found in layers IV and V of the cerebral cortex, the paraventricular nucleus of the hypothalamus (magnocellular part), and the ventral tegmental area.     

Characterization of the effects produced by neurokinins and three agonists selective for neurokinin receptor subtypes in a spinal nociceptive reflex of the rat

In the awake restrained rat the intrathecal (i.th.) administration of 6.5 pmol-40 nmol of substance P (SP), neurokinin A (NKA) or one of two selective NK-1 receptor agonists [Pro9, Met(O2)11]SP, denoted ana1 and [beta-Ala4, Sar9, Met(O2)11]SP , denoted ana2 decreased reaction time (RT) to a noxious radiant heat stimulus in a dose-related manner. The following rank order of potency was observed in relation to this response: ana1 = ana2 greater than SP much greater than NKA. The decrement of tail-flick latency was greatest at 1 min and RT returned to the basal level within 6-11 min post-administration. However, in some rats SP produced a small increase in RT (anti-nociception) at 6-11 min post-administration. The i.th. administration of neurokinin B (NKB) or a selective NK-3 receptor agonist [beta-Asp4, MePhe7]NKB), denoted ana3 induced an antinociceptive effect which was greatest at 1 min and lasted less than 11 min after NKB or more than 30 min after ana3 administration. The magnitude of the increase in RT produced by 65 pmol-40 nmol doses of these peptides is ana3 much greater than NKB much greater than SP. The effect of NKB (8.0 nmol) was significantly blocked (P less than 0.005) by prior i.th. administration of naloxone (opioid antagonist) but not by idazoxan (alpha 2-adrenoceptor antagonist), [Thi5,8, D-Phe7]BK (kinin antagonist), or following bilateral adrenalectomy. From these results, we conclude that NKB-induced antinociception is mediated by the spinal release of an opioid and not through a BK or NA mechanism. The results also suggest that the nociceptive and antinociceptive effects of neuro-kinins are mediated by the activation of NK-1 and NK-3 receptor subtypes respectively, in the rat spinal cord.