The role of substance P-containing fibers in sympathetic ganglia: Effect of capsaicin

Capsaicin was given subcutaneously to guinea pigs and the effect on substance P-immunoreactive (SP-I) fibers in the celiac/superior mesenteric and inferior mesenteric ganglia was observed at 2 day and 8–10 day intervals. Capsaicin (125 mg) treatment led to almost total disappearance of SP-I fibers from all areas examined in both short- and long-term animals. This effect applied equally to the dense network of varicose SP-I fibers and to basket-like SP-I contacts with principal ganglionic neurons. The effect of capsaicin on SP-I fibers in the mesenteric ganglia provides a strong indication that these fibers represent a homogenous population of visceral sensory afferents. This is supported by other lines of anatomical evidence in the literature. Taken together with studies that have shown axodendritic contact of SP-I terminals on principal ganglionic neurons and neuro-modulatory effects of SP on these neurons, it may be hypothesized that SP-I fibers in the mesenteric ganglia represent collaterals of visceral sensory afferents forming a subspinal feedback arc.     

Cardiovascular interactions between methionine-enkephalin and substance P in the conscious dog

Interactions between the undecapeptide Substance P (SP) and the pentapeptide methionine-enkephalin (Met5-ENK) have been described in isolated organ systems and in baroreceptor reflex mechanisms. Previous studies in our laboratory have demonstrated that systemically injected Met5-ENK simultaneously increases mean systemic arterial pressure (MAP) and heart rate (HR) in the conscious, chronically instrumented dog. We have now evaluated cardiovascular interactions between SP and Met5-ENK. In this model, SP injected intravenously produces a rapid and transient decrease in MAP and increase in HR over the dose range from 1.0 to 10.0 ng/kg. SP does not appear to appreciably alter subsequent responses to Met5-ENK. At SP doses of 1.0 ng/kg, the peak hemodynamic response to SP and Met5-ENK (35 micrograms/kg) given together appears to represent a simple summation effect of both drugs on HR and MAP. However, at higher SP doses (5.0 ng/kg), the SP response predominates and is little altered by the presence of Met5-ENK. Thus, Met5-ENK does appear capable of modulating the hemodynamic responses to SP over certain dose ranges.     

Intrathecal substance P and somatostatin in rats: behaviors indicative of sensation

Biochemical, histochemical and neurophysiological data suggest that substance P and somatostatin are neurotransmitters for primary afferent neurons. This study used intrathecal administration of these peptides and others (neurotensin and vasoactive intestinal polypeptide) in chronically catheterized, environmentally adapted, freely moving rats to evaluate their effects on unconditioned behavior. Substance P and somatostatin each elicited behaviors which were dose related. The behaviors included caudally directed biting and licking along with hindlimb scratching, writhing and retching. The behavioral responses were rapid in onset (1 min) and, in the case of substance P, short in duration (3 min). Vehicle, neurotensin and vasoactive intestinal polypeptide were without effect. These results demonstrate the ability of substance P and somatostatin to induce behavior in rats upon intrathecal administration and extend previous studies in mice.     

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.     

Tetracaine blocks the responses of isolated acinar cells from rat parotid to carbachol but not to substance P

Carbachol and substance P stimulated ⁴⁵Ca²⁺ flux changes, ⁸⁶Rb⁺ efflux, and amylase secretion from acinar cells isolated fromrat parotid. The local anesthetic tetracaine blocked all of these measured responsed to carbachol, but none of the responses to substance P. Tetracaine must act at either the cholinergic receptor or at a subsequent transducing step in the cholinergic stimulus-response sequence. If tetracaine acts at one of the transducing steps between cholinergic receptor occupation and the physiological responses then the action of tetracaine must be at a locus in the cholinergic reaction scheme not shared by substance P, because tetracaine did not block any response of the parotid to substance P.     

Calcitonin gene-related peptide coexists with substance P in capsaicin sensitive neurons and sensory ganglia of the rat

Immunohistochemical and radioimmunoassay studies revealed that both CGRP- and SP-like immunoreactivity in the caudal spinal trigeminal nucleus and tract, the substantia gelatinosa and the dorsal cervical spinal cord as well as in cell bodies of the trigeminal ganglion and the spinal dorsal root ganglion is markedly depleted by capsaicin which is known to cause degeneration of a certain number of primary sensory neurons. Higher brain areas and the ventral spinal cord were not affected by capsaicin treatment. Furthermore CGRP and substance P-like immunoreactivity were shown to be colocalized in the above areas and to coexist in cell bodies of the trigeminal ganglion and the spinal dorsal root ganglia. It is suggested that CGRP, like substance P, may have a neuromodulatory role on nociception and peripheral cardiovascular reflexes.     

The substance P content of peripheral tissues in several mammals

Levels of substance P immunoreactivity (SPI) were determined in several skin and mucosal areas, in parts of the sympathetic nervous system, the urinary, biliary and respiratory systems of cats, rabbits and guinea-pigs, and in various skin and mucosal areas of humans by radioimmunoassay. Salient findings are (1) The general distribution pattern of SPI in rabbits was similar to that in rodents. (2) The highest SPI tissue levels were found in the sympathetic nervous system, notably in guinea-pigs. (3) The guinea-pig also had the highest SPI levels in ureter, urinary bladder and bile duct. (4) The aorta, pulmonary artery and portal vein of the rabbit contained very low amounts of SPI, the concentration in the carotid sinus being several fold higher. (5) Skin SPI content was generally highest in the cat, especially in the hindpaw-pad, and lowest in abdominal and back skin. (6) SPI levels found in postmortem human skin and mucosal samples are comparable to those found in other mammals. The observations are discussed in view of the sensory innervation of the various tissues.     

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.     

Plasticity in expression of calcitonin gene-related peptide and substance P immunoreactivity in ganglia and fibres following guanethidine and/or capsaicin denervation

Summary This study was designed to investigate the effects of multiple denervation procedures on calcitonin gene-related peptide- and substance P-immunoreactive neurons in sympathetic and sensory cranial ganglia and in selected targets. Sympathectomy by long-term guanethidine treatment induced a pronounced increase in calcitonin gene-related peptide-immunoreactive and substance P-immunoreactive nerve fibres in all the tissues investigated, in contrast to a significant reduction of immunoreactive cell bodies. Neonatal capasaicin treatment abolished substance P immunoreactivity in many targets and caused a dramatic reduction of substance P-immunoreactive sensory nerve cell bodies; calcitonin gene-related peptide-immunoreactive nerve density was decreased, but the number of immunoreactive nerve cell bodies was unchanged. Guanethidine treatment of capsaicin-injected rats reversed the loss of calcitonin gene-related peptide-immunoreactive nerves, but not that of substance P-immunoreactive neurons. In the iris, capsaicin treatment had little effect on calcitonin gene-related peptide- and substance P-immunoreactive nerves, suggesting that in rats the majority of these fibres originate from capsaicin-insensitive neurons. The results suggest that the denervation procedures used in this study alter the synthesis and transport of neuropeptides in sensory neurons in conjunction with changes in the number of nerve fibres.     

Algesia and local responses induced by neurokinin A and substance P in human skin and temporal muscle

Neurokinin A (NKA), substance P (SP) and the two peptides combined (SP + NKA) were injected intracutaneously on the forearm and into the temporal muscle of healthy volunteers. Pain intensity, cutaneous wheal and flare responses and tenderness of the temporal muscle were quantitated. SP but not NKA induced cutaneous pain. This relates the algesic effect of SP to the specific N-terminal amino acid sequence of the peptide, not shared by NKA. NKA, however, potentiated the algesic effect of SP as SP + NKA induced a significantly prolonged cutaneous pain sensation. Both peptides induced wheals, but only SP induced flare. These results confirm previous studies relating wheal formation to the identical C-terminal amino acid sequence of the two peptides and flare reaction to the N-terminal part of SP. Injections into the temporal muscle did not cause pain or tenderness.     

Distribution and origin of bombesin,substance P and somatostatin in cat spinal cord

Bombesin (BN), substance P-(SP) and somatostatin (SRIF) were measured in individual laminae of the cervical, thoracic and lumbar (L) spinal cord of control cats, and in the L6 segment of cats receiving a spinal hemisection (L2) or deafferentation via dorsal rhizotomy at L6, 7, S1. The interlaminar distribution of BN, SP, and SRIF was remarkably similar. Highest concentrations were found in the superficial dorsal horn, and progressively less was found proceeding ventrally. Some intersegmental variations in peptide concentration within a single lamina were found. Dorsal rhizotomy caused a significant decline in BN, SP and SRIF in lamina I-III, therefore all three peptides appear to be contained in dorsal root ganglion cells. Evidence is presented for the existence of ascending BN and SP projections originating in lamina I-III and VII, for a descending SRIF pathway terminating in lamina VIII, and for an ascending BN path in lamina VIII. Dorsal root afferents to lamina VIII influence levels of BN, SP and SRIF.     

Influence of substance P on the behavioral changes induced by haloperidol in rats

Locomotor activity and grooming behavior of rats were recorded for a period of 30 min following intraventricular injections of substance P(SP) in doses of 0.60 and 2.50 microgram/rat. The lower dose of the peptide significantly increased locomotion for 10 min and time spent grooming for 25 min. The effects of the same two doses of SP on the hypokinesia induced by various pharmacological treatments modifying catecholaminergic systems were then examined. SP did not affect the behavioral depression produced by alpha-methyl-para-tyrosine (250 mg/kg), FLA-63 (25 mg/kg) and phenoxybenzamine (20 mg/kg). However, SP, in dose of 0.60 microgram/rat, systematically reversed the decrease in locomotor activity induced by a relatively small dose of haloperidol, 0.1 mg/kg. The dame dose of the peptide significantly counteracted the rigidity but not the hypokinesia and catalepsy resulting from the previous administration of a higher dose of haloperidol, 3 mg/kg. The results support the hypothesis that SP may exert direct or indirect function in motor behavior, possible via a modulatory action on brain dopaminergic systems.     

Age and strain differences in some behavioral effects of intracranial substance P

Intracerebroventricular (ICV) injections of substance P (SP) induce a vigorous reciprocal hindlimb scratching (RHS) syndrome, accompanied by extensive grooming behavior. There is a significant (approximately 1000X) difference in responsiveness to SP, as measured by RHS and grooming, in mice as a function of genetic strain (Swiss/Webster, C57 or DBA) and age. There was considerable specificity in the ability of drugs to increase responsiveness in the least responsive type of mouse (aged DBA/2J). Responding in old DBAs was enhanced by high doses of naloxone, suggesting the involvement of opioid peptides. Significant enhancement of responding by alpha-methyl tyrosine and propranolol, but not by phenoxybenzamine or haloperidol, indicated that beta-adrenergic systems are also involved. Similar manipulations of serotonergic systems were without effect.     

Assay of ezlopitant, a substance P receptor antagonist, and metabolites in biological matrices by gas chromatography with mass spectrometric detection: simultaneous analysis of a benzyl alcohol and alkene

A method for the analysis of the substance P antagonist ezlopitant and two active metabolites in serum using solid-phase extraction followed by GC–MS analysis is described. The linear dynamic range was 1.0 to 100 ng/ml and precision and accuracy over this range were within 15%. Upon injection of reconstituted sample extracts into the hot injector port of the gas chromatograph, the benzyl alcohol metabolite undergoes a small amount of spontaneous dehydration to the alkene metabolite. We have incorporated an additional hexadeuterated internal standard of the benzyl alcohol into the assay to permit measurement of the extent of dehydration in each sample. This novel approach should be generally applicable to the simultaneous determination of benzyl alcohols and corresponding alkenes by GC–MS when the possibility exists that the alcohol can undergo spontaneous dehydration to the alkene in the injector port of GC instrumentation.     

Ultrastructure and localization of substance P and met-enkephalin immunoreactivity in the human fetal gastric antrum

Summary The ultrastructural localization and relations of substance P- and met-enkephalin-labeled neuronal structures were examined in the wall of the human gastric antrum during early fetal life. By 14–16 weeks of gestation, clearly discernable neural plexuses and a well developed external muscle coat were present. In the submucous coat, neural plexuses varied from immature forms consisting of 1–4 neurites partially enveloped by Schwann cell processes to more mature plexuses where neurons were completely enclosed by Schwann cell processes. Neuronal profiles with substance P- and met-enkephalin-like immunoreactivities were observed in the submucous plexus. In the myenteric plexus met-enkephalin-like immunoreactivity was seen within cell bodies and neurites. By contrast, although substance P-like immunoreactivity was observed in neurites in the myenteric plexus, no substance P-labeled somata could be identified. Unlabeled terminals were seen in contact with both unlabeled dendrites and met-enkephalinergic neurons. An increase in electron density was observed at the sites of contact. These structures probably represent early stages in the development of synaptic specializations. In addition, met-enkephalin-labeled varicosities were seen in apposition to smooth muscle cells of the circular muscle coat. This suggests that antral smooth muscle cells are directly innervated by met-enkephalin neurons.     

Demonstration of substance P in aortic nerve afferent fibers by combined use of fluorescent retrograde neuronal labeling and immunocytochemistry

Combined use of the intraaxonal retrograde transport of the fluorescent marker ‘true blue’ with substance P (SP) immunocytochemistry has been used to trace the nodose ganglion projections of SP-containing neurons of the aortic depressor nerve. It has been found that (1) SP immunoreactive (SP-I) cell bodies are clearly demonstrable in clusters in the rostral part of the nodose ganglion without the aid of colchicine pretreatment; (2) ‘true blue’ is retrogradely transported to the nodose ganglion following its application to the central cut end of the aortic nerve; (3) ‘true blue’ fluorescence and SP fluorescent immunoreactivity can be visualized in the same tissue section and certain cell bodies in the nodose ganglia contain both SP-I and retrogradely transported ‘true blue’. These results indicate that the aortic nerve which projects from the aortic arch baro- and/or chemoreceptors to brainstem vasomotor centers contains SP-I afferent fibers which emanate from the nodose ganglion.     

Immunocytochemical localization of substance P in the neurohypophysis and hypothalamus of the mouse compared with the distribution of other neuropeptides

Summary Substance-P immunoreactivity has been located in semithin sections of mouse hypothalami and pituitaries and compared with the distribution of other hypothalamic peptides. In the mouse, nerve fibres and terminals reacting with antibodies against substance P (SP) were detected both in the external zone of the median eminence (ME) and in the neural lobe of the pituitary. Immunoreactive SP (ISP) axons of the ME did not react with antibodies against other peptides, i.e. arginine-vasopressin (AVP), oxytocin (OT), somatostatin and enkephalin, and were also negative with an antibody to serotonin. In the neural lobe, SP immunostaining occurred in AVP but not in OT axons. In the hypothalamus, ISP axons were widespread but conspicuously lacking in areas containing AVP neurones, i. e. in the suprachiasmatic nucleus and the clusters of AVP cells in the SO and PV nuclei. In contrast, multiple ISP endings were seen in contact with OT neurones. Immunoreactive cell bodies, only detected after colchicine treatment, belonged to two distinct classes of neurones: 1) single AVP neurones of the SO and PV nuclei; 2) specific (staining only for SP) neurones, scattered or grouped in different areas of the hypothalamus, not showing relationship with any circumscribed nucleus. These results reinforce the opinion that SP can be released as a neurohormone into the vascular portal system and can directly affect the pars distalis. The presence of immunoreactive SP in the neural lobe, which has not been reported in species other than the mouse, may have a different physiological significance.     

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.     

Immunoreactive substance P in the chicken gut: Distribution,development and possible functional significance

Summary The distribution and cellular localization of substance P in the chicken gut was studied by immunocytochemistry and immunochemistry. Substance P-containing nerve fibers are numerous in the gut wall. They occur in the smooth muscle layer as well as in the mucosa, where they are associated with blood vessels or surround the intestinal crypts. The fibers are particularly numerous in the myenteric and submucosal plexuses, where substance P-containing nerve-cell perikarya are also encountered. Substance P was found also in scattered endocrine cells of the small intestine, caeca and colon. Previously, bombesin-containing cells, which are numerous in the proventriculus, have been mistakenly identified as substance P cells due to crossreactivity of certain antisera against substance P. Immunochemistry revealed the highest concentration of substance P in the duodenum. The gel chromatographic behavior of chicken substance P differs slightly from that of synthetic bovine substance P, suggesting that chicken substance P differs structurally from mammalian substance P. Substance P-containing nerve fibers in the chicken gut develop slowly after hatching, apparently beginning in the duodenum; at approximately 20 weeks after hatching the distribution pattern is fully developed.A functional investigation was performed on the isolated chicken caecum to clarify the role of substance P in the contractile behavior of smooth muscle. Substance P contracted the caecum over a wide dose range; the contractile response was greater in 20 week-old chickens than in 4 and 10 week-old animals. Electrical field stimulation caused a relaxation of the caecum and a contraction upon cessation of stimulation. Neither of these responses, both of which are neurally mediated, were inhibited by adrenergic and cholinergic blockade. It is conceivable that the contractile response following electrical stimulation is caused by substance P released from nerve fibers in the smooth muscle.