Proteolytic Inactivation of Substance P and Neurokinin A in the Longitudinal Muscle Layer of Guinea Pig Small Intestine

Membrane vesicles, showing a 21 +/- 2-fold enrichment in the activity of 5'-nucleotidase and a 11 +/- 4-fold enrichment in the activity of angiotensin-converting enzyme relative to homogenate, were prepared from the myenteric plexus-containing longitudinal muscle layer of guinea pig ileum. Incubation of the vesicles with substance P and neurokinin A led to degradation of the peptides, and metabolites were isolated by reverse-phase HPLC and identified by amino acid composition. Cleavages of substance P between Glu6-Phe7, Phe7-Phe8, and Gly9-Leu10 and of neurokinin A between Gly8-Leu9 were observed and could be inhibited in a dose-dependent manner by phosphoramidon, an inhibitor of neutral endopeptidase 24.11. Formation of these metabolites was not completely inhibited by this agent, indicating that a phosphoramidon-insensitive form of endopeptidase 24.11 was present in the gut. Substance P was resistant to degradation by aminopeptidases, but neurokinin A was a substrate for bestatin-sensitive aminopeptidase(s), so that the neurokinin A (3-10) fragment represented the predominant metabolite in the chromatograms. The rate of formation of all the metabolites was not inhibited by enalapril and not enhanced by an increased Cl- concentration, indicating that angiotensin-converting enzyme was unimportant in the degradation process. Degradation of neurokinin A by the vesicles (Km 30 microM; Vmax 7.2 +/- 0.8 nmol min-1 mg of protein-1) was more rapid than degradation of substance P (Km 25 microM; Vmax 4.4 +/- 0.4 nmol min-1 mg of protein-1).     

Isolation of Neuropeptide-Containing Vesicles from the Guinea Pig Ileum

Three distinct vesicle fractions enriched 40-60 times in the neuropeptides substance P, somatostatin, and vasoactive intestinal peptide (VIP) were prepared from the myenteric plexus of guinea pig ileum by density gradient centrifugation in a small zonal rotor. Mean densities (in g X ml-1) and diameters (in nm) of the three classes of vesicles were: substance P, 1.123, 65; somatostatin, 1.138, 37; VIP, 1.148, 110; standard deviations were about 5%. These peaks were distinct from the peak of acetylcholine-containing vesicles of density 1.066 g X ml-1 and diameter 61 nm. When a relatively mild method of homogenization was used a second peak of acetylcholine appeared in the same region of the gradient as VIP and the VIP was larger. This may represent a class of vesicles containing both acetylcholine and VIP, though cosedimentation of two classes of vesicles of almost the same density and similar fragility, one containing VIP and the other acetylcholine, cannot be entirely excluded on present evidence.     

Neuropeptide-γ: A Peptide Isolated from Rabbit Intestine that Is Derived from γ-Preprotachykinin

The neurokinin A-like immunoreactivity in an extract of rabbit small intestine was resolved into two molecular forms by gel permeation chromatography. These components were purified to apparent homogeneity by reverse-phase HPLC. The primary structure of the larger component was established as the following: Asp-Ala-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val- Gly-Leu - Met.NH2. This amino acid sequence represents residues (72-92) of gamma-preprotachykinin, as predicted from the nucleotide sequence of a cloned cDNA from the rat. The peptide, termed neuropeptide-gamma, lacks residues (3-17) of neuropeptide K, and this segment is specified exactly by exon 4 in the preprotachykinin gene. The smaller form of neurokinin A-like immunoreactivity was identical to neurokinin A. Neuropeptide K was not present in the extract, demonstrating that the pathways of post-translational processing of beta- and gamma-preprotachykinins in the rabbit gut are different.     

Differential Release of Endogenous 5-Hydroxytryptamine, Substance P., and Neurokinin A from Rat Ventral Spinal Cord in Response to Electrical Stimulation

Abstract: The release of endogenous 5-hydroxytryptamine (5-HT), substance P (SP), and neurokinin A (NKA) from superfused tissue slices of rat ventral lumbar spinal cord, where SP and NKA coexist with 5-HT in terminals of descending bulbospinal neurons, was investigated. Electrical field stimulation was performed using square-wave pulses of 2-ms duration and 30 mA stimulus intensity. The following four different patterns of stimulation were used: 2 Hz continuous, 20 Hz continuous, 20 Hz intermittent, and 50 Hz intermittent. 5-HT was measured in the slice superfusates by HPLC with electrochemical detection. SP and NKA were measured by radioimmunoassay. The release of 5-HT was significantly enhanced using all stimulation paradigms and the evoked release of 5-HT per pulse was independent of the stimulation frequency. The release was found to be calcium dependent and there was no increase in the efflux of 5-hydroxyindoleacetic acid in response to stimulation. At 2 Hz (continuous), no significant increase in the release of SP was observed. Stimulation at higher frequencies yielded a significant increase in the release of SP per pulse. At 20 Hz, the release was increased by 73% (continuous) and 74% (intermittent), and at 50 Hz (intermittent) by 175% of basal efflux. The evoked release of NKA was also frequency dependent. At 2 Hz (continuous), no significant increase in the release of NKA was observed. At 20 Hz (intermittent), the evoked release per pulse was increased by 33% and at 50 Hz (intermittent) by 53% compared with the basal efflux of NKA. The results suggest that coexisting neurotransmitters/neuromodulators in the spinal cord may be released in different proportions depending on the stimulation frequency and that only 5-HT is released when the nerve terminal is activated by low-frequency stimulation.     

Distinct binding sites for substance P and neurokinin A (substance K): co-transmitters in rat brain

The distribution of binding sites in rat brain for iodinated neurokinin A and iodinated substance P were compared using autoradiography. Distinct patterns of binding for the two iodinated tachykinins were noted. Binding sites for iodinated neurokinin A were noted in the olfactory bulb, cortex, supraoptic n., paraventricular n., certain amygdaloid n., hippocampus, medial habenula, interpeduncular n., n. of the tractus solitarius, and dorsal horn of the spinal cord. This pattern was in contrast to low levels of binding of iodinated substance P to the cortex, supraoptic n., paraventricular n., and the interpeduncular n., but substantial density of binding sites in numerous other regions.     

Subcellular Distribution of Mammalian Tachykinins in Rat Basal Ganglia

A combined differential and density gradient centrifugation procedure was used to study the subcellular localisation of the mammalian tachykinins in rat caudateputamen and substantia nigra. Substance P, neurokinin A, neuropeptide K, and neurokinin B were found to be concentrated in the synaptosomal fractions and in fractions containing heavy synaptic vesicles in both regions studied. In contrast, the catecholamines dopamine and noradrenaline had a more widespread distribution throughout the gradient. HPLC analysis of the immunoreactivity recovered showed that the tachykinin immunoreactivity coeluted with the relevant synthetic tachykinins, except in the soluble gradient fraction where neurokinin A immunoreactivity eluted in position consistent with neurokinin A3-10. These results suggest that, in the basal ganglia, the mammalian tachykinins are localised in fractions containing large dense cored synaptic vesicles. This vesicular localisation would be consistent with the proposed role of the tachykinins as neurotransmitters and neuromodulators.     

Distribution and origin of substance P and neuropeptide Y-immunoreactive nerves in the guinea-pig heart

Summary The localization and origin of substance P (SP)-, neuropeptide Y (NPY)-, and noradrenaline/tyrosine hydroxylase (NA/TH)-immunoreactive (IR) nerves in the guinea-pig heart were investigated by means of immunohistochemistry; quantitative analysis was performed by radioimmunoassay (NPY) and high performance liquid chromatography (NA). Both untreated animals and animals subjected to stellatectomy, combined stellatectomy and local capsaicin pretreatment of the vagal nerves or systemic application of capsaicin were studied. A dense network of SP-IR nerves was observed in the right atrium in different locations: (1) around local cardiac ganglion cells, (2) close to blood vessels, (3) within the myocardium, and (4) close to and within peri and endocardium.A moderately dense SP-innervation, mainly related to blood vessels, was found in the ventricles. Very dense networks of NPY and TH-IR nerve fibers with an overlapping distributional pattern around blood vessels and in the myocardium were seen in both the atria and the ventricles. In addition, some cell bodies in local cardiac ganglia were NPY-IR. Bilateral stellatectomy resulted in a reduction of SP-IR in the right atrium (55% of control), which was more pronounced after additional capsaicin pretreatment of the vagal nerves (44% of control).In the left ventricle no significant depletion of SP-IR was seen by either stellatectomy or combined stellatectomy and capsaicin treatment of the vagal nerves. It was not possible to establish any defined target areas within the heart for vagal or spinal SP-IR afferents by use of immunohistochemical methods. Systemic capsaicin treatment caused a total loss of SP-IR nerves in the heart. After bilateral stellatectomy the levels of NPY-IR and NA were reduced to about 10% of control in both the right atrium and left ventricle. In accordance, NPY and TH-IR nerves were also almost totally absent in the heart after bilateral stellatectomy.     

Substance P and substance K receptor binding sites in the human gastrointestinal tract: Localization by autoradiography

Quantitative receptor autoradiography was used to localize and quantify the distribution of binding sites for ¹²⁵I-radiolabeled substance P (SP), substance K (SK) and neuromedin K (NK) in the human GI tract using histologically normal tissue obtained from uninvolved margins of resections for carcinoma. The distribution of SP and SK binding sites is different for each gastrointestinal (GI) segment examined. Specific SP binding sites are expressed by arterioles and venules, myenteric plexus, external circular muscle, external longitudinal muscle, muscularis mucosa, epithelial cells of the mucosa, and the germinal centers of lymph nodules. SK binding sites are distributed in a pattern distinct from SP binding sites and are localized to the external circular muscle, external longitudinal muscle, and the muscularis mucosa. Binding sites for NK were not detected in any part of the human GI tract. These results demonstrate that: 1) surgical specimens from the human GI tract can be effectively processed for quantitative receptor autoradiography; 2) of the three mammalian tachykinins tested, SP and SK, but not NK binding sites are expressed in detectable levels in the human GI tract; 3) whereas SK receptor binding sites are expressed almost exclusively by smooth muscle, SP binding sites are expressed by smooth muscle cells, arterioles, venules, epithelial cells of the mucosa and cells associated with lymph nodules; and 4) both SP and SK binding sites expressed by smooth muscle are more stable than SP binding sites expressed by blood vessels, lymph nodules, and mucosal cells.     

Demonstration and Distribution of Kassinin-Like Material (Substance K) in the Rat Central Nervous System

Antiserum was raised against kassinin in rabbits. Cross-reactivity with other tachykinins was determined; these included substance K (100%) and substance P (0.1%). Peptides extracted from rat brain and synthetic tachykinins were chromatographed by reverse-phase HPLC. The major peak of kassinin-like material eluted at a time different from that of synthetic kassinin, eledoisin, physalaemin, neurokinin beta, and substance P but coeluted with substance K. Measurement of kassinin-like material in macrodissected and microdissected brain regions indicated that the distribution of kassinin-like material was similar to that of substance P.     

便秘型肠易激综合征患者血清神经肽Y、P物质和5-羟色胺水平与焦虑抑郁的相关性

为探讨便秘型肠易激综合征(constipation-predominant irritable bowel syndrome, C-IBS)患者血清神经肽Y (neuropeptide Y, NPY)、P物质(substance P, SP)以及5-羟色胺(5-hydroxytryptamine, 5-HT)水平与焦虑抑郁的相关性,本研究选取确诊为C-IBS的患者35例,健康对照组35例,采用焦虑自评表(self-rating anxiety scale, SAS)和抑郁自评表(self-rating depression scale, SDS)评定C-IBS患者和健康对照组的焦虑和抑郁状况。酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)检测C-IBS患者和健康对照组血清NPY、SP以及5-羟色胺含量变化。结果表明,IBS-C中出现抑郁、焦虑或者无抑郁、焦虑的患者在各方面(性别,年龄以及病程)无统计学意义。与健康对照组相比,C-IBS组NPY和SP水平显著降低(t=7.96, p<0.01; t=5.63, p<0.01),5-HT则表现出显著性升高(t=8.06, p<0.001)。C-IBS组SAS和SDS评分结果与健康对照组相比有着显著性的升高(t=12.13, p<0.001; t=11.34, p<0.001)。C-IBS患者血清NPY与SAS和SDS评分均呈负相关(r=-0.62, p<0.001; r=-0.69, p<0.001)。C-IBS患者血清SP水平与SAS和SDS评分均呈负相关(r=-0.63, p<0.001; r=-0.57, p<0.001)。C-IBS患者血清5-HT与SAS和SDS评分均呈正相关(r=0.74, p<0.001; r=0.68, p<0.001)。本研究初步表明,C-IBS患者血清中NPY和SP水平降低,并与焦虑、抑郁评分呈负相关性,5-HT在C-IBS患者血清中升高,与焦虑、抑郁评分呈正相关性,NPY、SP以及5-HT可能在便秘型肠易激综合征出现焦虑、抑郁的过程中发挥重要作用。     

Comparison of responses by striatonigral substance P and neurokinin A systems to methamphetamine treatment

Multiple administrations of high doses of methamphetamine (METH) previously have been shown to significantly elevate the concentrations of substance P-like immunoreactivity in CNS regions associated with the basal ganglia. Recently, another tachykinin, neurokinin A (NKA), has been found to be closely associated with substance P (SP). While both neuropeptides exert comparable effects when locally injected, there are significant differences in their potencies apparently based on the relative concentrations of their unique receptors. Due to the controversy which has arisen as to their respective roles within the basal ganglia, we have evaluated and compared the responses of the striatal and nigral SP and NKA systems to METH treatment. We observed that multiple high doses of this stimulant increased the nigral and striatal concentrations of both neuropeptides in an identical fashion. Our observation that METH treatment did not alter the relative concentrations of SP and NKA suggests that responses of both transmitter systems, associated with the basal ganglia, parallel each other and are sensitive to the same regulatory mechanisms.     

Ligand Binding Kinetics of Substance P and Neurokinin A Receptors Stably Expressed in Chinese Hamster Ovary Cells and Evidence for Differential Stimulation of Inositol 1,4,5-Trisphosphate and Cyclic AMP Second Messenger Responses

Stably transfected Chinese hamster ovary cells expressing either the substance P receptor or neurokinin A receptor were constructed, isolated, and characterized. Equilibrium ligand binding studies performed on whole cells demonstrated that cell lines expressing either of these receptors contained a single class of high-affinity binding sites with an apparent KD of 0.16 nM for the substance P receptor and an apparent KD of 2.1 nM for the neurokinin A receptor. The higher affinity of substance P for its receptor was accounted for by both a greater association rate constant and a lesser dissociation rate constant. The time course and extent of ligand-stimulated inositol 1,4,5-trisphosphate mass increases in both cell lines were similar and displayed rapid and transient kinetics. Ligand-stimulated cyclic AMP accumulation was also apparent in the cell lines, although the time course and magnitude of the responses were substantially different, with the neurokinin A receptor mediating a greater and more prolonged response. These studies establish the presence of functional substance P receptors and neurokinin A receptors in the stably transfected cell lines and provide evidence for agonist-dependent differential stimulation of second messenger responses.     

Substance P Synthesis and Transport in Explants of Nodose Ganglion/Vagus Nerve: Effects of Double Ligation, 2-Deoxyglucose, Veratridine, and Ouabain

Substance P (SP), the widely distributed undecapeptide, is synthesized in cell bodies of vagal sensory ganglia and transported bidirectionally toward the CNS and thoracic and abdominal viscera. In explants of the guinea pig inferior (nodose) vagal sensory ganglion and attached 2 cm of distal vagus nerve, SP is synthesized within the ganglion and transported predominantly distally. The quantity of distal transport is similar to that observed in vivo and provides an index of ongoing synthesis within the ganglion. In this report, the model is further characterized. Double ligation of the explant distal to the ganglion demonstrates that all the transported peptide is derived from the ganglion; there is no evidence of intraaxonal processing of peptide precursor. Approximately 50% of the peptide is in a rapid transport vs. an apparent stationary compartment. Not only transport, but also synthesis, of SP was blocked by 20 mM colchicine. Ongoing SP biosynthesis is dependent on a nutrient medium [medium 199 (M-199)] and is partially inhibited with added fetal bovine serum (FBS; 10%): total explant content in M-199/FBS vs. M-199, 1,785 +/- 101 (n = 8) vs. 2,254 +/- 123 pg (n = 9); p less than 0.02. Addition of 2-deoxyglucose (2-DG) decreased both total SP synthesis and transport (total explant content for 2-DG vs. control, 986 +/- 94 vs. 1,391 +/- 111; p less than 0.05). Medium supplemented with glucose to a final concentration of 600 mg/100 ml or with glucose (300 mg/100 ml) with or without insulin (50 ng/ml) did not alter explant SP content or transport. Veratridine (5 X 10(-6) M) inhibited both SP synthesis and transport; ouabain (10(-4) M) also inhibited synthesis, but less so transport. Tetrodotoxin reversed the effects of veratridine. These studies demonstrate the usefulness of this model, which can examine factors regulating both synthesis and transport of sensory neuropeptides in vitro. The results suggest that SP synthesis/transport may be under tonic inhibition, perhaps by both neural and humoral mechanisms.     

Separation of Recycling and Reserve Synaptic Vesicles from Cholinergic Nerve Terminals of the Myenteric Plexus of Guinea Pig Ileum

Acetylcholine-rich synaptic vesicles were isolated from myenteric plexus-longitudinal muscle strips derived from the guinea pig ileum by the method of Dowe, Kilbinger, and Whittaker [J. Neurochem. 35, 993-1003 (1980)] using either unstimulated preparations or preparations field-stimulated at 1 Hz for 10 min using pulses of 1 ms duration and 10 V . cm-1 intensity. The organ bath contained either tetradeuterated (d4) choline (50 microM) or [3H]acetate (2 muCi . ml-1); d4 acetylcholine was measured by gas chromatography-mass spectrometry. As with Torpedo electromotor cholinergic vesicle preparations made under similar conditions the distribution of newly synthesized (d4 or [3H]) acetylcholine in the zonal gradient from stimulated preparations was not identical with that of endogenous (d0, [1H]) acetylcholine, but corresponded to a subpopulation of denser vesicles (equivalent to the VP2 fraction from Torpedo) that had preferentially taken up newly synthesized transmitter. The density difference between the reserve (VP1) and recycling (VP2) vesicles was less than that observed in Torpedo but this smaller difference can be accounted for theoretically by the difference in size between the vesicles of the two tissues. At rest, a lesser incorporation of labelled acetylcholine into the vesicle fraction was observed, and the peaks of endogenous and newly synthesized acetylcholine coincided. Stimulation in the absence of label followed by addition of label did not lead to incorporation of labelled acetylcholine, suggesting that the synthesis and storage of acetylcholine in this preparation and its recovery from stimulation is much more rapid than in Torpedo.     

Characterization of Neuronal and Endothelial Forms of Angiotensin Converting Enzyme in Pig Brain

The molecular forms of angiotensin converting enzyme (ACE; EC 3.4.15.1) in preparations of pig brain cortical microvessels and striatal synaptosomal membranes have been identified by immunoelectrophoretic blot analysis. The cortical microvessels contained only the endothelial form of the enzyme, Mr 180,000, which comigrated with pig kidney ACE on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast, the synaptosomal membranes contained only a smaller form of ACE, Mr 170,000, which represents the neuronal form of the enzyme. No significant differences in inhibitor sensitivity or substrate specificity were detected between the two forms of ACE. In particular, neurokinin A was resistant to hydrolysis by either microvessel or synaptosomal membrane ACE, and the pattern of hydrolysis of substance P by the two preparations was identical.     

Pathway-specific patterns of the co-existence of substance P,calcitonin gene-related peptide,cholecystokinin and dynorphin in neurons of the dorsal root ganglia of the guinea-pig

Summary The co-existence of immunoreactivities to substance P (SP), calcitonin gene-related peptide (CGRP), cholecystokinin (CCK) and dynorphin (DYN) in neurons of the dorsal root ganglion (DRG) of guinea-pigs has been investigated with a double-labelling immunofluorescence procedure. Four main populations of neurons could be identified that contained different combinations of these peptides and had distinctive peripheral projections: (1) Neurons that contained immunoreactivity to SP, CGRP, CCK and DYN were distributed mainly to the skin. (2) Neurons with immunoreactivity to SP, CGPR and CCK, but not DYN, were distributed mainly to the small blood vessels of skeletal muscles. (3) Neurons with immunoreactivity to SP, CGRP and DYN, but not CCK, were distributed mainly to pelvic viscera and airways. (4) Neurons containing immunoreactivity to SP and CGRP, but not CCK and DYN, were distributed mainly to the heart, systemic blood vessels, blood vessels of the abdominal viscera, airways and sympathetic ganglia. Other small populations of DRG neurons containing SP, CGRP or CCK alone also were detected. Perikarya containing these combinations of neuropeptides were not found in autonomic ganglia. The peripheral axons of neurons containing immunoreactivity to at least SP and CGRP were damaged by chronic treatment with capsaicin. However, some sensory neurons containing CCK alone were not affected morphologically by capsaicin.These results clearly show that individual DRG neurons can contain many different neuropeptides. Furthermore, the combination of neuropeptides found in any particular neuron is related to its peripheral projection.     

Muscarinic Receptors in the Cochlear Nucleus and Auditory Nerve of the Guinea Pig

The specific-binding properties of l-[3H]quinuclidinyl benzilate, a muscarinic acetylcholine-receptor antagonist, were investigated in synaptic and other membrane preparations of the guinea pig cochlear nucleus and auditory nerve. Binding parameters for all experiments were consistent with a single binding site with a Hill coefficient of 1.0. The binding of the ligand was specific and of high affinity, with values of KD in the range of 30-80 pM. Bmax was 0.352 +/- 0.023 pmol/mg protein for the dorsal cochlear nucleus and 0.215 +/- 0.011 pmol/mg protein for the ventral cochlear nucleus. The dorsal cochlear nucleus/ventral cochlear nucleus ratio for density of muscarinic receptors (1.6/1.0) was maintained across two different buffer systems, which varied with respect to the inclusion of proteolysis inhibitors. The results for auditory nerve indicated a level of binding much below that of the cochlear nucleus, with Bmax = 0.052 +/- 0.011 pmol/mg protein. The results of specific-binding experiments for l-[3H]quinuclidinyl benzilate support a role for acetylcholine as a neurotransmitter in the cochlear nucleus. The greater density of muscarinic receptors in the dorsal cochlear nucleus may indicate greater cholinergic activity in the dorsal relative to the ventral cochlear nucleus.     

Focal Stimulation of the Mossy Fibers Releases Endogenous Dynorphins That Bind K1-Opioid Receptors in Guinea Pig Hippocampus

Physiological release of endogenous opioids in guinea pig hippocampal slices was detected in an in vitro competition binding assay using [3H]U69,593, a kappa 1-selective radioligand. Veratridine-induced opioid release caused a decrease in [3H]U69,593 binding that was blocked by either tetrodotoxin addition or the removal of calcium from the incubation buffer. Focal electrical stimulation of opioid peptide-containing afferent pathways resulted in a decrease in [3H]U69,593 binding, whereas stimulation of a major afferent lacking endogenous opioid immunoreactivity had no effect. The addition of 6-cyano-7-nitroquinoxaline-2,3-dione blocked the reduction in [3H]U69,593 binding caused by perforant path stimulation, but not the reduction caused by mossy fiber stimulation, suggesting that the primary source of endogenous kappa ligands was likely to be the dentate granule cells. Antisera against dynorphin A(1-8) or dynorphin B peptides inhibited the effects of mossy fiber stimulation in the [3H]U69,593 displacement assay. Antisera against other prodynorphin- and proenkephalin-derived opioid peptides had no effect. As shown by receptor autoradiography, the distribution of kappa 1 binding sites was limited to the molecular layer of the dentate gyrus and the presubiculum region of temporal hippocampal slices. These results indicate that prodynorphin-derived opioids released under physiological conditions from the mossy fibers act at kappa 1 receptors in the guinea pig dentate gyrus.     

Substance P and neurokinin A metabolism by cultured human skeletal muscle myocytes and fibroblasts

A recent study determined that cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts degraded angiotensins and kinins via neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11) and aminopeptidase N (APN; EC 3.4.11.2). Due to the possible importance of other peptides to skeletal muscle blood flow and function, the present study looked specifically at the metabolism of the neurokinins substance P (SP) and neurokinin A (NKA) by skeletal muscle peptidases. The results show that SP is degraded not only by NEP-24.11, but also sequentially by dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5)/APN. NKA is unaffected by DAP IV but is metabolized by NEP-24.11 and APN. NEP-24.11 was inhibited by phosphoramidon (IC₅₀ = 80 nM), thiorphan and ZINCOV, DAP IV by diprotin A (IC₅₀ = 8 μM), and APN by amastatin (IC₅₀ = 50 nM) and bestatin (IC₅₀ = 100 μM). Skeletal muscle myocyte and fibroblast metabolism of SP and NKA may regulate local skeletal muscle vascular and extravascular functions including SP- and NKA-mediated nerve-induced vasodilation. Inhibition of both NEP-24.11 and DAP IV/APN may increase skeletal muscle blood flow and decrease peripheral vascular resistance via potentiation of local neurokinin levels.     

Immunohistochemical evidence for different pathways immunoreactive to substance P and calcitonin gene-related peptide (CGRP) in the guinea-pig stellate ganglion

The colocalization of immunoreactivities to substance P and calcitonin gene-related peptide (CGRP) in nervous structures and their correlation with other peptidergic structures were studied in the stellate ganglion of the guinea pig by the application of double-labelling immunofluorescence. Three types of fibre were distinguished. (1) Substance P⁺/CGRP⁺ fibres, which sometimes displayed additional immunoreactivity for enkephalin, constituted a small fibre population of sensory origin, as deduced from retrograde labelling of substance P⁺/CGRP⁺ dorsal root ganglion cells. (2) Substance P⁺/CGRP^– fibres were more frequent; some formed baskets around non-catecholaminergic perikarya that were immunoreactive to vasoactive intestinal polypeptide (VIP). (3) CGRP⁺/substance P^– fibres were most frequent and were mainly distributed among tyrosine hydroxylase (TH)-immunoreactive cell bodies. The peptide content of fibre populations (2) and (3) did not correspond to that of sensory ganglion cells retrogradely labelled by tracer injection into the stellate ganglion. Therefore, these fibres are throught to arise from retrogradely labelled preganglionic sympathetic neurons of the spinal cord, in which transmitter levels may have been too low for immunohistochemical detection of substance P or CGRP. CGRP-immunoreactivity but no substance P-immunolabelling was observed in VIP-immunoreactive postganglionic neurons. Such cell bodies were TH-negative and were spared by substance P-immunolabelled fibre baskets. Retrograde tracing with Fast Blue indicated that the sweat glands in the glabrous skin of the forepaw were the targets of these neurons. The streptavidin-biotin-peroxidase method at the electron-microscope level demonstrated that immunoreactivity to substance P and CGRP was present in dense-cored vesicles of 50–130 nm diameter in varicosities of non-myelinated nerve fibres in the stellate ganglion. No statistically significant difference in size was observed between vesicles immunolabelled for substance P and CGRP. Immunoreactive varicosities formed axodendritic and axosomatic synaptic contacts, and unspecialized appositions to non-reactive neuronal dendrites, somata, and axon terminals. Many varicosities were partly exposed to the interstitial space. The findings provide evidence for different pathways utilizing substance P and/or CGRP in the guinea-pig stellate ganglion.