Ultrastructural immunocytochemical localization of substance P in the cat small intestine

The immunocytochemical peroxidase-antiperoxidase technique was used to identify substance P (SP) in the cat small intestine at the ultrastructural level in the nerve plexuses. In the myenteric plexus the SP containing fibres form synapses with the other nerve terminals and run parallel to the circular muscle. The SP in these fibres is present in the vesicles of 80 to 120 nm or 150 to 250 nm in diameter. The results support the contention that SP containing nerve terminals are present in the enteric nervous system.     

Developmental studies on vasoactive intestinal peptide,substance P and calcitonin gene-related peptide in salivary glands of postnatal rats

The development of vasoactive intestinal peptide, substance P and calcitonin gene-related peptide in parotid, submandibular and sublingual glands of the male rat was followed by immunochemistry and immunocytochemistry. The total amounts of these peptides increased in surges during the first 8 weeks of the animal's life; one within 2–4 weeks and the other beginning 1–2 weeks later. Nerve fibres containing these peptides were present at birth showing a pattern of distribution similar to that in adults. During the first 4 weeks the nerve fibres increased in number.     

Coexistence of substance P and calcitonin gene-related peptide in the frog spinal cord

The localization of substance P (SP) and calcitonin gene-related peptide (CGRP) was studied in the untreated spinal cord of the frog using single or double immunohistochemical stainings. SP and CGRP appear to coexist in the primary afferent fibers and in the marginal and submarginal dorsal horn zones, as well as in the dorsolateral zone. In other parts of the spinal cord CGRP immunoreactivity was scanty while diffuse SP systems were seen, suggesting that the coexistence of the two peptides is restricted to primary afferent fibers.     

Ultrastructural evidence for the coexistence of calcitonin gene-related peptide and substance P in secretory vesicles of peripheral nerves in the guinea pig

Summary Using double immunogold staining procedures, calcitonin gene-related peptide (CGRP)-like and substance P (SP)-like immunoreactivities were localized at the ultrastructural level to guinea pig trigeminal ganglia, dorsal root ganglia and peripheral nerve fibres associated with the vascular system. CGRP-like and SP-like immunoreactivities were found consistently in large granular secretory vesicles (70–100 nm in diameter), and both peptide immunoreactivities were co-localized to the same vesicle in both sensory ganglion cells and within axons and their terminals in the adventitia and adventitial-medial border of the superior mesenteric artery. These results suggest that CGRP and SP are co-stored and may be released together from peripheral axons in the guinea pig.     

Dilatory responses to acetylcholine, calcitonin gene-related peptide and substance P in the congestive heart failure rat

It was examined to what extent congestive heart failure (CHF) in rats, induced by ligation of the left coronary artery, affects the vascular responses to the vasodilatory substances acetylcholine (ACh), calcitonin gene-related peptide (CGRP), and substance P (SP). After induction of CHF status, the basilar, mesenteric and renal arteries and the iliac vein were studied in vitro. Dilatory responses were determined in relation to pre-contraction by the thromboxane mimetic U46619. Sham-operated animals (Sham) served as controls. U46619 induced stronger contraction in CHF basilar and renal arteries compared with the corresponding segments in Sham. ACh induced concentration-dependent dilations in all vessels examined with no difference of maximum relaxation or potency between CHF and Sham. SP induced weak dilations in all arteries examined while the response was markedly attenuated in CHF iliac veins compared with Sham (Emax% 12.2 +/- 3.4 vs. 32.3 +/- 4.8, P = 0.01). The CGRP induced dilation in the CHF basilar artery was weaker (Emax% 18.6 +/- 6.5 vs. 66.9 +/- 5.0, P < 0.001) and less potent (pEC50: 8.2 +/- 0.2 vs. 9.0 +/- 0.2, P = 0.01) compared with Sham. Further, CGRP was less potent in the renal artery of CHF rats compared with Sham (pEC50: 8.1 +/- 0.2 vs. 9.5 +/- 0.3, P < 0.01). In the CHF iliac vein, CGRP was more potent compared with Sham (pEC50: 9.7 +/- 0.4 vs. 8.3 +/- 0.4, P < 0.05). It can be concluded CHF is accompanied by alterations in the vascular response to the dilatory substances studied. The changes differ between vascular beds and between the different substances.     

Origins and pathways of cerebrovascular nerves storing substance P and calcitonin gene-related peptide in rat

Origins and pathways of cerebrovascular substance P- and calcitonin gene-related peptide-positive nerves in rat were studied by immunohistochemistry combined with denervation experiments and retrograde axonal tracer technique. The two peptides have been found to coexist in one and the same neuron. After sectioning of the nasociliary nerve bilaterally the substance P/calcitonin gene-related peptide fibers in the rostral half of the circle of Willis and its branches were eliminated, whereas the number decreased in the caudal half of the circle of Willis and rostral two thirds of the basilar artery. Substance P/calcitonin gene-related peptide fibers in the internal carotid arteries, the caudal third of the basilar artery and the vertebral arteries were not affected by the nerve section. After application of the retrograde axonal tracer True Blue onto the proximal segment of the middle cerebral artery the dye accumulated in several Substance P/calcitonin gene-related peptide-containing cells in the ophthalmic division of the ipsilateral trigeminal ganglion and in a few cells in the maxillary trigeminal division and in the internal carotid miniganglion. No other cranial ganglia accumulating the dye contained any substance P/calcitonin gene-related peptide-positive cells. It is concluded that the rostral portion and part of the caudal portion of the cerebral vessels are innervated by substance P/calcitonin gene-related peptide-containing fibers from the trigeminal ganglion and the internal carotid miniganglion. The great majority of trigeminal fibers reach the vessels via the nasociliary nerve of the ophthalmic division, which enters the cranial cavity through the ethmoidal foramen, whereas fibers from the miniganglion project directly to the bypassing internal carotid artery. A probable pathway for the fibers from the maxillary division is suggested. The caudal portion receives, in addition, a supply from other sensory ganglia (lower cranial and/or upper cervical dorsal root ganglia).     

Nociceptive behavior after intrathecal injections of substance P, neurokinin A and calcitonin gene-related peptide in mice

Intrathecal injections of substance P (SP) or neurokinin A (NKA) in the mouse caused dose-dependent reciprocal hindlimb scratching, licking and biting responses directed to the caudal part of the body. NKA decreased the latency in the tail flick assay but like SP, did not alter the reactions in the hot plate test or the hypertonic saline assay. Although immunoreactivity of calcitonin gene-related peptide (CGRP) was detected in mouse spinal cord, CGRP caused no behavioral reactions, nor did it significantly affect thermo- or chemonociception or the scratching induced by SP. Since NKA-like immunoreactivity was found to be present in sensory neurons, NKA as well as SP are likely transmitters of nociceptive primary afferent neurons.     

Brain natriuretic peptide in the porcine spinal cord: an immunohistochemical investigation of its localization and the comparison with atrial natriuretic peptide, substance P, calcitonin gene-related peptide, and enkephalin

Immunohistochemistry was used to localize brain natriuretic peptide in the porcine spinal cord and to compare it with that of atrial natriuretic peptide, substance P, calcitonin gene-related peptide and [Met]enkephalin. Brain natriuretic peptide-immunoreactive varicose fibers were observed in lamina I and the inner portion of lamina II of the dorsal horn. Semiquantitative analysis showed that the highest density of brain natriuretic peptide-immunoreactive varicosities was in the lumbosacral and coccygeal segments. The distributional pattern of brain natriuretic peptide-immunoreactive nerve fibers in the spinal cord was unique and quite distinct from that of the other neuropeptides studied. These neuroanatomical findings suggest that brain natriuretic peptide may play a role in the regulation of nociceptive processing in the spinal cord, either alone or with bioactive substances.     

Intraspinal release of substance P and calcitonin gene-related peptide during opiate dependence and withdrawal.

The antibody microprobe technique was used to study the release of immunoreactive substance P and immunoreactive calcitonin gene-related peptide within the lower lumbar spinal cord of anaesthetized spinalized cats pretreated twice daily for 3.5 days with increasing doses of morphine hydrochloride (2-20 mg/kg, i.p.). Both peptides were released in the region of the substantia gelatinosa during noxious cutaneous thermal stimulation or high-intensity electrical stimulation of a hind limb nerve. Intravenous administration of naloxone increased the nociceptive excitation of lumbar dorsal horn neurons, but did not alter the evoked release of immunoreactive substance P or immunoreactive calcitonin gene-related peptide in the superficial gray matter dorsal to these neurons. In addition, the release of both peptides was not significantly different to that detected under similar experimental conditions in opioid-naive cats. The results suggest that alterations in neuropeptide release from the central terminals of nociceptive primary afferent neurons do not occur during states of opiate dependence and withdrawal, and thus do not contribute to the characteristic signs of these phenomena in dependent animals.     

The role of substance P and calcitonin gene-related peptide in neurogenic plasma extravasation and vasodilatation in the rat

Immunohistochemistry combined with retrograde tracing has been used to show that of the afferent neurons supplying the dorsomedial surface of the hind paw, approximately 30% contain substance P and 50% calcitonin gene-related peptide immunoreactivity. Stimulation of the saphenous nerve causes plasma extravasation and antidromic vasodilatation in this area of skin. The roles of calcitonin gene-related peptide and substance P released from peripheral afferent endings in mediating these effects were examined using immunoneutralization. In pilot experiments, the binding of radiolabelled peptide to the immunoglobulin fraction of calcitonin gene-related peptide and substance P antisera was characterized in quasi-physiological conditions. Systemic administration of either substance P or calcitonin gene-related peptide antibodies caused a significant decrease (P less than 0.05) in plasma extravasation measured by the Evans Blue method in response to topical application of mustard oil (0.5%) to the skin, or of capsaicin (5 microM) to the saphenous nerve. Topical application of mustard oil also produced a 52.9 +/- 5.1% increase in skin red cell flux. This increase was significantly decreased by both substance P and calcitonin gene-related peptide antibodies. The results suggest that both peptides are involved in mediating neurogenic inflammatory responses.     

Morphine treatment induced calcitonin gene-related peptide and substance P increases in cultured dorsal root ganglion neurons

The mechanism of spinal tolerance to the analgesic effects of opiates is unclear at present. We have reported previously that calcitonin gene-related peptide-like immunoreactivity was significantly increased in primary afferents of the spinal dorsal horn during the development of morphine tolerance, suggesting that changes in the level of pain-related neuropeptides in dorsal root ganglion neurons may be involved [Menard D. P. et al. (1996) J. Neurosci. 16, 2342-2351]. In this study, we investigated if in vitro treatment with morphine can mimic the in vivo findings and induce increases in calcitonin gene-related peptide-like immunostaining in cultured dorsal root ganglion neurons from young (three-month-old) and middle-aged (10-month-old) adult rats. Following a repetitive exposure to morphine sulfate (1, 5, 10 microM) for six days, the number of calcitonin gene-related peptide- and substance P-immunoreactive neurons in cultured dorsal root ganglia from three- and 10-month-old rats was significantly increased. A lower concentration (0.5 microM) of morphine induced these increases only in dorsal root ganglion neurons from middle-aged rats. Morphine treatment was also found to increase the number of calcitonin gene-related peptide-immunoreactive neurons possessing multiple, long branches (i.e. with at least one branch >0.5mm). This apparent increase in the number of calcitonin gene-related peptide- and substance P-immunoreactive neurons observed following morphine treatment was blocked by naloxone, an opiate antagonist, indicating the involvement of genuine opioid receptors. No significant change in the number of neuropeptide Y- or galanin-immunoreactive neurons in cultured dorsal root ganglia was detected following any of these treatments.These data suggest that repeated exposure to morphine rather selectively increases calcitonin gene-related peptide- and substance P-like immunoreactivity in cultured dorsal root ganglion neurons. Moreover, the sensitivity to morphine-induced changes is greater in cultured dorsal root ganglion neurons from 10- compared to three-month-old rats. Hence, cultured dorsal root ganglion neurons can provide a model to investigate the cellular and molecular mechanisms underlying alterations in neuropeptide levels following repeated exposure to opiates and their relevance to the development of opioid tolerance.     

Effects of iontophoretically applied substance P, calcitonin gene-related peptide on excitability of dorsal horn neurones in rats

Spontaneous pain, allodynia and hyperalgesia are well known phenomena following peripheral nerve or tissue injury, and it is speculated that secondary hyperalgesia and allodynia, are generally thought to depend on a hyperexcitability (sensitization) of neurons in the dorsal horn. It is supposed that the sensitization may be due to various actions of neurotransmitters (SP, CGRP, excitatory amino acids) released from the primary afferent fibers. In this study, we examined effects of the iontophoretically applied SP and CGRP on the response to EAA receptor agonists (NMDA and non-NMDA) in the WDR dorsal horn neurones and see if the effects of SP or CGRP mimic the characteristic response pattern known in various pain models. The main results are summarized as follows: 1) SP specifically potentiated NMDA response. 2) CGRP non-specifically potentiated both NMDA and AMPA responses. Potentiation of NMDA response, however, was significantly greater than that of AMPA response. 3) 50% of SP applied cells and 15.8% of CGRP applied cells showed reciprocal changes(potentiation of NMDA response and suppression of AMPA response). These results are generally consistent with the sensitization characteristics in diverse pain models and suggests that the modulatory effects of SP and CGRP on NMDA and non-NMDA (AMPA) response are, at least in part, contribute to the development of sensitization in various pain models.     

Effects of intravenous calcitonin gene-related peptide (CGRP) and substance P on the blood-aqueous barrier in the rabbit

The irritation response of the rabbit eye to trigeminal nerve stimulation, which includes a breakdown of the blood-aqueous barrier (BAB), seems to be due to the release of substance P (SP) and calcitonin gene-related peptide (CGRP). In order to assess the relative importance of these two peptides for the barrier effect, and the role of arachidonic acid metabolites (AAM) in the response, we have studied the effects of intravenous injections of the peptides on the permeability of the blood vessels of the anterior uvea and the BAB using labelled albumins. At a dose of 120 pmol kg-1 there was marked leakage of labelled albumin into the aqueous humour in animals under pentobarbital anaesthesia. The leakage was enhanced by sympathotomy. In conscious animals 5 pmol kg-1 CGRP caused enhanced leakage from the blood vessels of the ciliary processes in those pre-treated with biperiden in order to abolish the cholinergic vasoconstrictor tone in the anterior uvea. 24 and 120 pmol kg-1 CGRP caused marked leakage of albumin and a breakdown of the epithelial part of the BAB. These effects were not modified by biperiden pre-treatment, but markedly reduced by pre-treatment with indomethacin. The protecting effect of indomethacin was lost when biperiden was given as well. SP did not cause a leakage with 5 nmol kg-1 and only moderate leakage with 25 nmol kg-1. This effect was abolished by pre-treatment with indomethacin but not if indomethacin was combined with biperiden.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cutaneous responses to substance P and calcitonin gene-related peptide in chronic urticaria: the effect of cetirizine and dimethindene

BACKGROUND: Neuropeptides appear to participate in the pathogenesis of chronic urticaria. The purpose of this study was to investigate the cutaneous responses to substance P (SP) and calcitonin gene-related peptide (CGRP) in delayed pressure urticaria patients (DPUpt) and chronic idiopathic urticaria patients (CIUpt) compared to healthy adults (HA), and also to evaluate the effect of H1-antagonists on these responses. METHODS: Wheal (W) and flare (F) reactions to intradermally (ID) injected SP, CGRP, histamine (H), and diluent controls were evaluated in nine CIUpt, nine DPUpt, and nine HA at 3, 7, 15, 30, 60, and 120 min after injection. Maximal W and F, area under curve (AUC), and time of W and F disappearance were calculated at baseline and 4 h after a single dose of 20 mg cetirizine (Ce), 4 mg dimethindene (Di), or placebo (P), in a double-blind, placebo-controlled (DBPC), crossover, randomized study. RESULTS: CIUpt exhibited enhanced and longer lasting W reactions to SP and CGRP (AUC: P<0.05) than HA; SP- and CGRP-induced F (at maximal concentration) were larger and longer lasting in CIUpt than in HA (P<0.003 and P = 0.004, respectively). In the DPU group, SP-induced W and F responses were intermediate in magnitude compared to CIUpt and HA. In HA, SP-induced flares were significantly suppressed only by Ce (P<0.020), while both Ce and Di affected SP-induced W and F in the two patient groups (P<0.05). CGRP-induced flare inhibition by the two H1-antagonists was also greater in the urticaria patient groups than in HA. CONCLUSIONS: CIUpt and, to a lesser extent, DPUpt showed enhanced SP- and CGRP-induced W and F reactions. CGRP elicited an immediate W and F response, followed by prolonged erythema. H1-antagonists partially affected W and F reactions to SP and only the F response induced by CGRP; this effect was more pronounced in the urticaria patient groups than in HA. Overall, W and F cutaneous responses to SP were suppressed to a greater extent by Ce than Di.     

Calcitonin, katacalcin, and calcitonin gene-related peptide in the human prostate. An immunocytochemical and immunoelectron microscopic study

We recently described for the first time the presence of calcitonin immunoreactivity (CTIR) in a subpopulation of prostatic and urethral endocrine-paracrine (EP) cells. We now further evaluate the distribution of the CTIR cell, characterize the coexistence of serotonin and calcitonin, and for the first time describe the coexpression of calcitonin and other calcitonin gene family peptides (calcitonin gene-related peptide and katacalcin) in the CTIR cell. Finally, the morphological ultrastructure of the secretory granule of the CTIR cell is analyzed. The finding of multiple calcitonin gene family peptides in prostatic and urethral EP cells and the specific localization of calcitonin to secretory granules strongly suggest that the calcitonin gene is expressed in this region and the products stored in the EP cells. The relatively high levels of calcitonin reported in the semen and the dendritic and nondendritic morphological features of the CTIR cell, respectively, suggest a lumencrine (exocrine), paracrine, and possibly endocrine role for calcitonin. The production of calcitonin and related peptides by the prostate may have implications in various pathologic processes of the prostate.     

Distribution and origin of extrinsic nerve fibers containing calcitonin gene-related peptide, substance P and galanin in the rat upper rectum.

The distributions of nerve fibers containing calcitonin gene-related peptide (CGRP), substance P (SP) and galanin (GAL) were examined in the rat rectum of mutants rats, aganglionic rats (AGRs), which completely lack the intramural nerve cells in the large intestine, and of their normal littermates. The origin of extrinsic peptide-containing nerve fibers was examined using retrograde tracing combined with immunohistochemistry in normal rats. In the rectum of normal rats, CGRP-, SP- and GAL-immunoreactive varicose fibers were observed throughout all layers of the rectal wall, and immunoreactive nerve cells were present in the enteric ganglia of colchicine-treated rats. In the aganglionic rectum of AGR, a rich supply of CGRP-immunoreactive fibers was observed in the mucosa, around the blood vessels, and in the submucous and intermuscular spaces. SP- and GAL-immunoreactive fibers in the aganglionic rectum showed a similar distribution to CGRP-immunoreactive fibers but were less dense. These results suggest that most of CGRP-positive fibers in the rectum are extrinsic whereas a large part of SP- or GAL-positive fibers are intrinsic. Fluoro-gold injected into the upper rectum of normal rat labelled nerve cells (less than 10% of total ganglion cells) in the lumbar (L1 and L2) and lumbosacral (L6 and S1) dorsal root ganglia. More than half of nerve cells in the dorsal root ganglia (L6 and S1) projecting to the rectum were immunoreactive for CGRP, and less than 10% were immunoreactive for SP or GAL. Comparison of serial sections of the dorsal root ganglion revealed that about half of the CGRP-immunoreactive cells were also positive for SP or GAL. These results indicate that SP- or GAL-positive neurons projecting to the rectum are scarce in the dorsal root ganglia. The present investigation suggests that CGRP-containing nerves are visceral afferents forming a major component of the sensory innervation of the rat rectum, and SP- and GAL-containing nerves which share their extrinsic origins appear to form a lesser proportion of the sensory innervation.     

Lowering of interstitial fluid pressure in rat trachea after substance P alone and in combination with calcitonin gene-related peptide

Neurogenic inflammation is mediated following a release of sensory neuropeptides including calcitonin gene-related peptide (CGRP) and substance P (SP). The release of peptides can be mediated chemically with capsaicin, or electrically by stimulation of the vagal nerve, both inducing vasodilation, plasma protein extravasation and lowering of interstitial fluid pressure (Pif) which will contribute to the enhancement of oedema formation. AIM: Lowering of Pif has previously been demonstrated following intravenous (i.v.) treatment with CGRP, but it was not possible to demonstrate that SP had this effect under the same condition. METHODS: Micropuncture measurements of Pif in the submucosa, without opening of the trachea, was conducted on rats anaesthetized with pentobarbital sodium (50 mg kg-1) and cardiac arrest was induced with i.v. KCl. RESULTS: Pif in vehicle-treated animal averaged -1.7 +/- 0.4 (SD) mmHg (n = 9). Intravenous injection of SP induced significant lowering of Pif compared with control, both at low dose (0.47 nmol kg-1 body weight) with 1 min distribution time (P < 0.007, -4.2 +/- 2.3 mmHg) and at high dose with seconds of distribution time (P < 0.03, -4.2 +/- 1.6 mmHg). The same response was observed after treatment with SP co-injected with CGRP. CONCLUSIONS: Substance P alone or in combination with CGRP is able to induce a rapid lowering of Pif showing that this peptide is a potent agent in increasing the hydrostatic driving pressure initially transporting fluid into the tissue during an acute inflammatory reaction.     

Distribution of calcitonin gene-related peptide in the rat peripheral nervous system with reference to its coexistence with substance P

This immunocytochemical study, using a double-staining method, showed that calcitonin gene-related peptide-like immunoreactive structures are widely distributed in the peripheral nervous system and that many of them coexist with substance P-like immunoreactive structures in single sensory ganglion cells. Neurons positive for calcitonin gene-related peptide but negative for substance P were detected in sensory ganglia. These cells were large (about 30-45 micron in diameter); these primary sensory neurons containing calcitonin gene-related peptide can probably act independently of substance P. There were neurons containing calcitonin gene-related peptide without substance P in the pterygopalatine ganglion, although these cells were less numerous than in the sensory ganglia. In consecutive sections, calcitonin gene-related peptide-like structures occurred in thyroid parafollicular cells, which also contain calcitonin. This suggested that messenger RNA for producing calcitonin gene-related peptide is also present in the thyroid, and like calcitonin, calcitonin gene-related peptide may have a peripheral physiological role.     

Different patterns of immunolocalization of calcitonin gene-related peptide and substance P in sympathetic ganglia of normotensive and genetically hypertensive rats

Calcitonin gene-related peptide (CGRP) and substance P (SP) immunoreactivity were investigated in the superior cervical ganglion of normotensive and genetically hypertensive Otago Wistar rats by an immunoperoxidase method. CGRP- and SP-positive varicose axons invested separate subpopulations of ganglion cells, neither of which contained neuropeptide Y. The densities of CGRP axons were similar in normotensive and hypertensive rats while the numbers of SP axons were several times higher in the hypertensive strain. Decentralization of the ganglion or chronic capsaicin treatment removed all immunoreactive terminals, indicating that both axon populations are likely to be collaterals from thoracic sensory afferents.