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.     

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.     

Regulation of epithelial transport in the jejunal mucosa of the guinea pig by neurokinins

This study characterizes the actions of the neurokinins and calcitonin-gene related peptide (CGRP) on electrolyte transport across the mucosa of the guinea pig jejunum in vitro in a modified Ussing chamber. By following changes in short circuit current (Isc) induced by substance P (SP) and neurokinins A & B (NKA & NKB) in the presence and absence of tetrodotoxin (TTX) and atropine, it was established that two distinct neurokinin receptors are involved in the regulation of electrolyte transport. NKA preferentially activates a neuronal receptor since the actions of this neurokinin were inhibited by both TTX and atropine. SP, whose actions were reduced to a lesser extent by TTX and atropine, is considered to activate preferentially a receptor on the epithelial cells. The third neurokinin, NKB, appears to act non-selectively on both the neuronal and epithelial receptors. CGRP, which per se did not affect Isc, markedly potentiated the increases in Isc induced by SP and NKB, and thus acts synergistically with the epithelial neurokinin receptor. These results suggest that two distinct neurokinin receptors (the NK-1 and the NK-2) regulate epithelial transport in the jejunal mucosa of the guinea pig, and furthermore indicate that at least one of the peptides found in enteric nerves (i.e. CGRP) modulates the actions of neurokinins on epithelial cells.     

Distribution of neurokinin A-like and neurokinin B-like immunoreactivity in human peripheral tissues.

Using specific radioimmunoassay and immunocytochemistry for neurokinin A (NKA) and neurokinin B (NKB), distribution and localization of the two peptides in human peripheral tissues were studied. Both NKA-like immunoreactivity (NKA-LI) and NKB-like immunoreactivity (NKB-LI) were present in the walls of the gut and gall bladder and in the pancreas. In the gut, the values for NKA-LI were 0.56-35.73 pmol/g wet weight, while those in pancreas and gall bladder were 0.64-0.68 and 0.36 pmol/g wet weight, respectively. The values of NKB-LI were 0.45-2.66 pmol/g wet weight in the gut, 0.93-1.65 pmol/g wet weight in the pancreas, and 0.30 pmol/g wet weight in the gall bladder. The immunocytochemical reactivity to both peptides was localized to ganglia of the submucosal and myenteric nerve plexuses in the gut wall, and to neurons in the muscle layer and mucosa of the gut wall. Weak but positive NKA-LI appeared in nerve cells of the pancreas, while NKB-LI was not detectable in the pancreas. Conversely, in the gall bladder wall, NKA-LI was undetectable while a very faint NKB-LI was found in the muscle layer. The localization of NKA corresponded closely to that of NKB in the tissues although the relative concentrations of the peptides varied from organ to organ.     

Biologic activity of the tachykinins on lamb and sheep gallbladder

In this study, we examined the activity of the tachykinins (TKs) on lamb and sheep isolated gallbladder and whether the TKs are involved in the capsaicin-induced activity in these tissues. Substance P (SP) and physalaemin (PHYS) contracted lamb gallbladder, PHYS-induced striking tachyphylaxis. This tissue was nearly insensitive to neurokinin A (NKA), neurokinin B (NKB), septide, and capsaicin. As in lamb tissues, SP and PHYS both contracted sheep gallbladder although PHYS induced no tachyphylaxis. At doses that had no effect on lamb tissue, NKA, NKB, septide, and capsaicin contracted sheep gallbladder. Our findings indicate that TK receptors differ in adult and young ovine gallbladder. The activity of PHYS on lamb gallbladder could depend on the existence of an unusual binding site, carrying one or more residues critical for the N-terminal sequence present in PHYS but not in SP.     

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.     

Priming effects of mammalian tachykinins on human neutrophils.

The undecapeptide substance P (SP) is known to activate different cell types involved in inflammatory and immune processes. By evaluating primed stimulation of human neutrophils, we now demonstrate that SP (10 nM-0.1 mM) dose-dependently enhances superoxide anion production from cells stimulated by the phospholipid mediator Platelet Activating Factor (PAF). We also provide evidence that neurokinin A (NKA), which is released, as well as SP, from C fibers of sensory nerves, potentiates PAF-evoked superoxide anion generation, while neurokinin B (NKB) is ineffective.     

The distribution and origin of nerve fibers immunoreactive for substance P and neurokinin A in the anterior buccal gland of the rat

The distribution and origin of substance P (SP) and neurokinin A (NKA) were studied in rat in the anterior buccal glands, which are minor mucous salivary glands. Indirect immunofluorescence staining showed moderate SP and NKA innervation of salivary acini and interlobular ducts, whereas blood vessels were more sparsely innervated, and there were few nerve fibers in the stroma and around the intralobular ducts. About 10%–20% of the trigeminal ganglion cells showed equally strong immunoreactivity to both SP and NKA. Unilateral denervation of the branches of the trigeminal nerve caused complete disappearance of the stromal fibers and greatly reduced the number of all other SP-immunoreactive and NKA-immunoreactive nerve fibers. In the superior cervical ganglia, SP and NKA immunoreactivity was restricted to small intensely fluorescent cells; SP and NKA immunoreactivity was absent from principal ganglionic cells, and thus sympathectomy had no any effect on the number or distribution of fibers immunoreactive for SP and NKA in the anterior buccal glands. The fibers remaining after sensory denervation could have been of parasympathetic origin, indicating a dual origin of nerves immunoreactive for SP and NKA in these glands. The present data demonstrate that the major part of the glandular SP and NKA innervation in the minor salivary glands derives from the trigeminal ganglia. The distribution of the peripheral nerve fibers indicates that they may play a role in the delivery of potent neuropeptides involved in the vascular, secretory, and motor (myoepithelial cells) functions of salivary glands.     

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.     

Distribution and localization of neurokinin A-like immunoreactivity and neurokinin B-like immunoreactivity in rat peripheral tissue

Using specific radioimmunoassays and immunocytochemistry for neurokinin A (NKA) and neurokinin B (NKB), distribution and localization of these peptides in rat peripheral tissues were studied. NKA-like immunoreactivity (NKA-LI) was present in highest levels of 15.7–23.9 pmol/g wet wt. and NKB-like immunoreactivity (NKB-LI) was in levels of 0.33–0.67 pmol/g wet wt., throughout the gastrointestinal tract involving stomach, duodenum, jejunum, ileum and colon. Immunocytochemical analysis of gastrointestinal tract revealed that NKA-LI and NKB-LI localized in ganglia of both the submucosal and myenteric plexuses as well as varicose neurons in the mucosa and the muscle layer of the small and large intestine. On the other hand, high levels of NKB-LI were observed in oesophagus (0.83 ± 0.08 pmol/g wet wt.), adrenal (1.02 ± 0.21), head of pancreas (0.73 ± 0.06) and kidney (0.98 ± 0.05).

The present study shows the difference of localization of NKA-LI and NKB-LI in peripheral tissues and suggests that NKB may have some physiological role differing from that of NKA in peripheral tissues.     

Effects of some neurokinin A analogues on tachykinin-induced contraction of guinea pig trachea.

A series of analogues of neurokinin A (NKA) has been synthesized and characterized by testing for their abilities, in vitro, to contract guinea pig tracheal smooth muscle or to antagonize NKA-, NKB- and SP-induced contraction of this tissue. Substitution of NKA residues Gly8 or Leu9 by conformationally restricting amino acids produced peptides that were antagonists of NKA action, but the type and specificity of the antagonism depended on the size of the peptide. Thus, while [Ala5, Aib8, Leu10]NKA(2-10) showed no agonism and was a specific, competitive antagonist of NKA, [Ala5, Aib8, Leu10]NKA(4-10) was a noncompetitive antagonist of NKA and substance P (SP) and was itself a weak agonist at concentrations above 10(-7) M.     

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.     

The projections of chemically identified nerve fibres in canine ileum

Summary The projections of nerve fibres with immunoreactivity for the peptides enkephalin (ENK), gastrin-releasing peptide (GRP), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and vasoactive intestinal peptide (VIP) were studied in canine small intestine by analysing the consequences of lesions of intrinsic and extrinsic nerves. Of peptides present in fibres supplying myenteric ganglia, GRP, SOM and VIP were in anally directed nerve pathways, whereas ENK and NPY were in orally directed pathways. Pathways ran for up to about 30 mm. SP fibres ran for short distances in both directions in the myenteric plexus. The circular muscle was supplied with ENK, NPY, SP and VIP fibres arising from the myenteric ganglia, whereas most mucosal SP and VIP fibres were deduced to arise from submucous ganglia. There were projections of fibres reactive for ENK, GRP, SOM, SP and VIP from myenteric ganglia to submucous ganglia. Antibodies to tyrosine hydroxylase were used to locate noradrenaline nerve fibres supplying the intestine; these fibres all disappeared when extrinsic nerves running through the mesentery to the small intestine were cut. It is deduced that there is an ordered pattern of projections of peptide-containing fibres in the canine intestine.     

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.     

Immunoreactivity for high-affinity choline transporter colocalises with VAChT in human enteric nervous system

Cholinergic nerves are identified by labelling molecules in the ACh synthesis, release and destruction pathway. Recently, antibodies against another molecule in this pathway have been developed. Choline reuptake at the synapse occurs via the high-affinity choline transporter (CHT1). CHT1 immunoreactivity is present in cholinergic nerve fibres containing vesicular acetylcholine transporter (VAChT) in the human and rat central nervous system and rat enteric nervous system. We have examined whether CHT1 immunoreactivity is present in nerve fibres in human intestine and whether it is colocalised with markers of cholinergic, tachykinergic or nitrergic circuitry. Human ileum and colon were fixed, sectioned and processed for fluorescence immunohistochemistry with antibodies against CHT1, class III beta-tubulin (TUJ1), synaptophysin, common choline acetyl-transferase (cChAT), VAChT, nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). CHT1 immunoreactivity was present in many nerve fibres in the circular and longitudinal muscle, myenteric and submucosal ganglia, submucosa and mucosa in human colon and ileum and colocalised with immunoreactivity for TUJ1 and synaptophysin confirming its presence in nerve fibres. In nerve fibres in myenteric ganglia and muscle, CHT1 immunoreactivity colocalised with immunoreactivity for VAChT and cChAT. Some colocalisation occurred with SP immunoreactivity, but little with immunoreactivity for VIP or NOS. In the mucosa, CHT1 immunoreactivity colocalised with that for VIP and SP in nerve fibres and was also present in vascular nerve fibres in the submucosa and on epithelial cells on the luminal border of crypts. The colocalisation of CHT1 immunoreactivity with VAChT immunoreactivity in cholinergic enteric nerves in the human bowel thus suggests that CHT1 represents another marker of cholinergic nerves.     

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.     

Glial cells,but not interstitial cells,express P2X7, an ionotropic purinergic receptor,in rat gastrointestinal musculature

Purinergic (ATP) neurotransmission is a component of the inhibitory response of the musculature in various regions of the gastrointestinal tract. So far, seven ionotropic purinergic receptors (P2X1-7) have been cloned. As specific antibodies become available, their respective distribution in the gastrointestinal tract can be elucidated. Here, we used high-resolution tricolor confocal microscopy, to study the distribution of P2X7-immunoreactive (-ir) cells in the muscularis propria of the rat stomach, small intestine, and colon. Smooth muscle cells, KIT-ir interstitial cells of Cajal, and CD34/SK3-ir fibroblastlike cells were P2X7-negative, whereas P2X7 immunoreactivity was observed in nerves and S100-ir glial cells. In all regions studied, P2X7 immunoreactivity was also observed in myenteric and submucosal ganglia, where perineuronal nerve endings appeared brightly labeled. Our observations suggest that purinergic signaling could influence the enteric glia through P2X7 receptors.     

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.     

Sensory innervation of the ear drum and middle-ear mucosa: Retrograde tracing and immunocytochemistry

Summary The distribution and origin of nerve fibers of presumed sensory nature in the ear drum and middle-ear mucosa of the rat were studied by a retrograde tracing technique in combination with immunocytochemistry.Application of True Blue (TB) on the ear drum or on the middle-ear mucosa labeled nerve cell bodies in the jugular, trigeminal, geniculate and cervical dorsal root ganglia (C₂–C₄). Judging from the number of TB-labeled nerve cell bodies the jugular and trigeminal ganglia contributed the major component to the sensory innervation of the ear drum and the middle-ear mucosa, while the contribution from the geniculate and cervical dorsal root ganglia was relatively minor.The majority of the TB-labeled nerve cell bodies contained calcitonin gene-related peptide (CGRP), whereas minor populations stored substance P (SP) and neurokinin A (NKA). Nerve fibers containing SP, NKA and CGRP were moderate in number in the middle-ear mucosa and few in the ear drum. Double immunostaining revealed that SP invariably coexisted with NKA in nerve cell bodies in the ganglia examined. The SP/NKA-containing nerve cell bodies constituted a subpopulation of those storing CGRP.The findings indicate that several ganglia project to the ear drum and middle-ear mucosa and that many neuropeptides are involved in the mediation of middle-ear sensitivity.