Calcitonin gene-related peptide (CGRP) in capsaicin-sensitive substance P-immunoreactive sensory neurons in animals and man: distribution and release by capsaicin

The presence of calcitonin-gene related peptide (CGRP)-like immunoreactivity (-LI) in sensory neurons was established by immunohistochemistry and radioimmunoassay (RIA) in combination with high performance liquid chromatography (HPLC). CGRP-immunoreactive (-IR) nerve fibres were present in many peripheral organs including heart, ureter, uterus and gall bladder of guinea-pig and man. The distribution of CGRP-IR nerves in the dorsal horn of the spinal cord, of positive cell bodies in thoracic spinal and nodose ganglia and nerves in peripheral organs was closely related to that of substance P-LI. Double staining experiments revealed that in most cases peripheral CGRP-IR nerve terminals also contained SP-LI. However, different localization of SP- and CGRP-IR neurons was observed in the nucleus of the solitary tract as well as in the ventral horn of the spinal cord. In the heart, CGRP-IR nerves were associated with myocardial cells (mainly atria), coronary vessels, local parasympathetic ganglia as well as with the epi- and endocardia. Three to 4-fold higher levels of native CGRP-LI were observed in the atria than in the ventricles of the heart. HPLC analysis revealed that the major peak of CGRP-LI in the heart of rat and man had the same retention times as the synthetic equivalents. Systemic capsaicin pretreatment and adult guinea-pigs caused a loss of CGRP-IR terminals in the dorsal horn of the spinal cord as well as in peripheral organs including the heart. After capsaicin treatment, the content of CGRP-IR was reduced by 70% in the heart and by 60% in the dorsal part of the spinal cord. In superfusion experiments with slices from the rat spinal cord, a release of CGRP-LI was induced by 60 mM K+ and 3 microM capsaicin in a calcium-dependent manner.     

Capsaicin-induced bronchoconstriction and neuropeptide release in guinea pig perfused lungs.

In the guinea pig isolated perfused lung, we have examined the relationship between the effects of capsaicin and neuropeptide release and the possible existence of an axon reflex arrangement. Bolus injections into the pulmonary artery of capsaicin (1-100 pmol), substance P (10-1,000 pmol), and neurokinin (NK) A (10-100 pmol) produced a concentration-dependent bronchoconstriction, whereas calcitonin gene-related peptide (CGRP, 20-40 nmol) was without effect. Repeated administration of capsaicin at 40- to 60-min intervals was not associated with tachyphylaxis. These data support the presence of a NK2- (or NKA) type of tachykinin receptor in the guinea pig airways. Tetrodotoxin (0.3-3 microM) inhibited the effect of capsaicin, indicating that an axon reflex was operant. Capsaicin increased overflow of CGRP-like immunoreactivity (-LI) and NKA-LI, the latter only during concurrent infusion of the enkephalinase inhibitor phosphoramidon (3 microM). Phosphoramidon also increased overflow of CGRP-LI, suggesting that both NKA and CGRP were catabolized by a similar enzyme. The purine nucleoside adenosine did not cause any detectable overflow of CGRP-LI, indicating that neuropeptides may not be involved in adenosine-evoked bronchoconstriction and that bronchoconstriction per se does not induce neuropeptide overflow. Capsaicin and NKA had only minor effects on buffer flow, whereas substance P produced pulmonary vasoconstriction. These data clearly demonstrate that capsaicin acts via an axon reflex in the guinea pig airways. Supramaximal concentrations of capsaicin are needed to detect neuropeptide overflow, but the possibility exists that released neuropeptides mediate its effects.     

Innervation of lower airways and neuropeptide effects on bronchial and vascular tone in the pig

Summary The occurrence and distribution of peptide-containing nerve fibres [substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), neuropeptide Y (NPY)] and noradrenergic nerve fibres [tyrosine hydroxylase (TH)- and dopamine beta hydroxylase (DBH)-positive] in the airways of the pig were studied by means of immunohistochemistry. SP- and CGRP-immunoreactive (-IR) nerve fibres were present close to and within the lining respiratory epithelium, around blood vessels, within the tracheobronchial smooth muscle layer and around local tracheobronchial ganglion cells. The content of CGRP- and neurokinin A (NKA)-like immunoreactivity (-LI) measured by radioimmunoassay (RIA) was twice as high in the trachea compared to that in the peripheral bronchi. SP was a more potent constrictor agent than NKA on pig bronchi in vitro. CGRP had a relaxant effect on precontracted pig bronchi. On blood vessels CGRP exerted a relaxant effect that was more pronounced on pulmonary arteries than on bronchial arteries. VIP/PHI-IR fibres were seen in association with exocrine glands and in the tracheobronchial smooth muscle layer. VIP-positive nerve fibres were abundant around blood vessels in the trachea but sparse or absent around blood vessels in the peripheral bronchi. This histological finding was supported by RIA; it was shown that the content of peptides displaying VIP-like immunoreactivity (-LI) was 18 times higher in the trachea compared to peripheral bronchi. VIP was equally potent as CGRP in relaxing precontracted pig bronchi in vitro. Both bronchial and pulmonary arteries were relaxed by VIP. NPY was colocalized with VIP in tracheal periglandular nerve fibres and in nerve fibres within the tracheobronchial smooth muscle layer. NPY was also present in noradrenergic (DBH-positive) vascular nerve fibres. The content of NPY was much higher (15-fold) in the trachea compared to small bronchi. NPY caused a contraction of both pulmonary and bronchial arteries. The bronchial smooth muscle contraction to field stimulation in vitro was purely cholinergic. A non-cholinergic relaxatory effect following field stimulation was observed after bronchial precontraction. Capsaicin had no effect on pig bronchi in vitro.     

Tachykinins mediate hypocapnia-induced bronchoconstriction in guinea pigs

The aim of this study was to determine whether hypocapnia causes bronchoconstriction by releasing tachykinins (TKs) from C-afferent nerves in airways. Hypocapnia-induced bronchoconstriction (HIBC) was induced in anesthetized vagotomized guina pigs by ventilating lungs with a heated humidified hypocapnic gas mixture for 15 min after sudden circulatory arrest. The intensity of bronchoconstriction was assessed by calculating changes in dynamic compliance and by measuring the relaxation lung volume at the completion of experiments. Visualization of the airways by tantalum bronchography showed constriction of segmental bronchi with relative sparing of more proximal airways. Hypocapnia-induced bronchoconstriction was prevented by prior administration of salbutamol aerosol. Three experimental interventions were used to investigate the role of TKs in HIBC: 1) repeated capsaicin injections to deplete airway sensory nerves of TKs, 2) treatment with phosphoramidon, an inhibitor of enkephalinase, the main enzyme responsible for TK inactivation, and 3) topical airway anesthesia. Capsaicin pretreatment markedly attenuated the hypocapnia-induced changes in dynamic compliance (P less than 0.0005) and relaxation lung volume (P less than 0.0002), whereas phosphoramidon augmented these changes (P less than 0.02, P less than 0.03, respectively). Topical anesthesia of airways with lignocaine postponed the onset of bronchoconstriction, whereas the longer-acting, more lipid-soluble local anesthetic, bupivacaine, almost completely prevented HIBC. We conclude that, in the guinea pig lung, HIBC is mediated by TKs that are released after the activation of bronchial axonal reflexes.     

感觉神经肽与支气管对肺动脉缺氧反应的影响

豚鼠离体肺动脉用辣椒素（Ｃａｐｓａｉｃｉｎ）耗竭感觉神经肽（ＳＮＰ）后,缺氧性收缩反应（ＨＰＶ）显著增高（Ｐ＜０．０１）。套有带完整上皮支气管的肺动脉的ＨＰＶ显著弱于不套或套有去上皮支气管肺动脉的ＨＰＶ（Ｐ＜０．０５）;支气管与肺动脉同时用Ｃａｐ处理后,此差异消失;只有外套的支气管先用Ｃａｐ预处理时,肺动脉ＨＰＶ仍显著强于溶剂预处理的对照组（Ｐ＜０．０５）;套有去上皮支气管的肺动脉缺氧反应显著强于不套支气管的肺动脉。结果提示：肺动脉Ｃ－感觉神经所释放的ＳＮＰ在肺动脉缺氧反应中具调节作用;支气管上皮层可释放舒血管物质调节ＨＰＶ,此物质与ＳＮＰ密切相关;支气管还可能释放缩血管物质,介导ＨＰＶ。     

Occurrence and effects of multiple tachykinins; substance P, neurokinin A and neuropeptide K in human lower airways

In the present work we have studied the occurrence of different tachykinins (substance P (SP), neurokinin A (NKA) and neuropeptide K (NPK)) in human distal bronchi and pulmonary arteries by means of radioimmunoassay (RIA) and high performance liquid chromatography (HPLC). We have also compared the biological effects of different tachykinins on isolated human bronchi and pulmonary arteries in vitro. The concentration of immunoreactive SP using antiserum SP2 in the pulmonary arteries was higher (1.34 +/- 0.15 pmol/g) than in the bronchi (0.56 +/- 0.05 pmol/g). The contents of other tachykinins than SP measured using antiserum K12 was on the other hand considerably higher in the bronchi (0.33 +/- 0.14 pmol/g) than in pulmonary arteries (0.13 +/- 0.02 pmol/g). Immunoreactive materials corresponding to SP, NKA and NPK were identified in bronchial extracts by RIA combined with HPLC, which also indicated the presence of an eledoisin (ELE)-like component. In vitro studies showed that NKA was the most potent of the tachykinins as a bronchoconstrictor agent, being several hundred-fold more active than SP, acetylcholine and histamine. NPK had an intermediate potency. The bronchoconstrictor effect of NKA was unaffected by atropine, mepyramine and cimetidine. The tachykinins SP and NKA had on the other hand, a rather equal potency in inducing relaxation of serotonin precontracted pulmonary arteries. In conclusion, multiple tachykinins are present in lower airways of man. These peptides exert different biological activities whereby NKA is a very active bronchoconstrictor agent compared to SP while both NKA and SP have rather similar relaxatory activities of vascular smooth muscle.     

Capsaicin releases calcitonin gene-related peptide from the human iris and ciliary body in vitro.

Slices of human iris or ciliary body, obtained post-mortem (8-12 h after death, n = 5), were superfused in vitro with capsaicin (10 microM) and the immunoreactivity for substance P (SP-LI) or calcitonin gene-related peptide (CGRP-LI) was measured in the effluent. In the iris and in the ciliary body CGRP-LI was 3.71 +/- 0.74 pmol/g and 3.01 +/- 0.55 pmol/g and SP-LI was 6.68 +/- 0.75 pmol/g and 6.55 +/- 0.84 pmol/g, respectively. A first exposure to capsaicin increased the CGRP-LI outflow from the ciliary body (427 +/- 46 fmol/g/30 min), whereas a second challenge with the drug 30 min later, failed to significantly enhance the CGRP-LI outflow (21.8 +/- 15.6 fmol/g/30 min). Likewise, the capsaicin-evoked increase in CGRP-LI outflow from the iris slices (472 +/- 62 fmol/g/30 min) was no longer observed at the second drug administration (38.4 +/- 12.8 fmol/g/30 min). Capsaicin failed to increase the SP-LI outflow from either the iris or the ciliary body. Reverse phase HPLC analysis of CGRP-LI indicated that authentic CGRP was contained in the tissue and in the superfusate collected during exposure to capsaicin. The present results show that in the human iris and ciliary body, capsaicin releases CGRP possibly contained in terminals of sensory nerves.     

An immunocytochemical study of cutaneous innervation and the distribution of neuropeptides and protein gene product 9.5 in man and commonly employed laboratory animals.

The cutaneous nerves of rat, cat, guinea pig, pig, and man were studied by immunocytochemistry to compare the staining potency of general neural markers and to investigate the density of nerves containing peptides. Antiserum to protein gene product 9.5 (PGP 9.5) stained more nerves than antisera to neurofilaments, neuron-specific enolase (NSE), and synaptophysin or histochemistry for acetylcholinesterase (AChE). Peptidergic axons showed species variation in density of distribution and were most abundant in pig and fewest in man. However, the specific peptides in nerves innervating the various structures were consistent between species. Nerve fibers immunoreactive for calcitonin gene-related peptide (CGRP) and/or vasoactive intestinal polypeptide (VIP) predominated in all the species; those immunoreactive to tachykinins (substance P and neurokinin A [NKA]) and neuropeptide tyrosine (NPY) were less abundant. Neonatal capsaicin, at the doses employed in this study, destroyed approximately 70% of CGRP- and tachykinin-immunoreactive sensory axons; whereas 6-hydroxydopamine (6-OHDA) at the doses employed resulted in a complete loss of NPY and tyrosine hydroxylase (TH) immunoreactivity without affecting VIP, CGRP, and tachykinins. Thus, this study confirms that antiserum to PGP 9.5 is the most suitable and practical marker for the demonstration of cutaneous nerves. Species differences exist in the density of peptidergic innervation, but apparently not for specific peptides. Not all sensory axons immunoreactive for CGRP and substance P/NKA are capsaicin-sensitive. However, all sympathetic TH- and NPY-immunoreactive axons are totally responsive to 6-OHDA; but no change was seen in VIP-immunoreactive axons, suggesting some demarcation of cutaneous adrenergic and cholinergic sympathetic fibers.     

Evidence that calcitonin gene-related peptide contributes to the capsaicin-induced relaxation of guinea pig cerebral arteries.

Pretreatment with capsaicin caused a depletion of substance P (SP)-, neurokinin A (NKA)- and calcitonin gene-related peptide (CGRP)-like immunoreactivity (-LI) from the trigeminal ganglion, dura mater and cerebral arteries. The effect of capsaicin on isolated basilar arteries of guinea pig resulted in a biphasic relaxant response of histamine precontracted vessels. The first phase of the capsaicin-induced relaxation was absent in capsaicin-treated guinea pigs. Furthermore, repeated administration of capsaicin decreased the first but not the second phase of relaxation, supporting the view that a stored agent was released, while the second phase probably was due to a direct effect of capsaicin per se. The biphasic effect was not modified by the SP antagonist Spantide in a concentration that blocks tachykinin response (3.10(-6) M), nor by removal of the endothelium. There was no significant difference in pD2 values (-log concentration eliciting half maximum relaxation) between relaxations induced by SP, NKA, neurokinin B, neuropeptide K or CGRP in capsaicin pretreated as compared to vehicle-treated animals. These results are in support of the assumption that CGRP is involved in the capsaicin-induced relaxation caused by release of vasoactive agents from sensory afferent nerves.     

Sensory peptide neurotransmitters mediating mucosal and distension evoked neural vasodilator reflexes in guinea pig ileum

The aim was to determine the role CGRP and/or tachykinins released from sensory neural mechanisms in enteric neural vasodilator pathways. These pathways project through the myenteric plexus to submucosal vasodilator neurons. Submucosal arterioles were exposed in the distal portion of an in vitro combined submucosal-myenteric guinea pig ileal preparation, and dilation was monitored with videomicroscopy. Vasodilator neural reflexes were activated by gently stroking the mucosa with a fine brush or by distending a balloon placed beneath the flat-sheet preparation in the proximal portion. Dilations evoked by mucosal stroking were inhibited 64% by the CGRP 8-37 and 37% by NK3 (SR 142801) antagonists. When the two antagonists were combined with hexamethonium, only a small vasodilation persisted. Balloon distension-evoked vasodilations were inhibited by NK3 antagonists (66%) but were not altered by CGRP 8-37. In preparations in which myenteric descending interneurons were directly activated by electrical stimulation, combined application of CGRP 8-37 and the NK antagonists had no effect. Stimulation of capsaicin sensitive nerves in the myenteric plexus did not activate these vasodilator reflexes. These findings suggest that mucosal-activated reflexes result from the release of CGRP and tachykinins from enteric sensory neurons. Distension-evoked responses were significantly blocked by NK3 antagonists, suggesting that stretch activation of myenteric sensory neurons release tachykinins that activate NK3 receptors on myenteric vasodilator pathways.     

Endothelin as a putative sensory neuropeptide in the guinea-pig: different properties in comparison with calcitonin gene-related peptide

Both endothelin-(ET) and calcitonin gene-related peptide- (CGRP) like immunoreactivity (-LI) were present in a variety of organs and neuronal tissue of the guinea-pig as determined by radioimmunoassay (RIA). Neuronal tissues like dorsal root ganglia (DRG) contained by far the highest levels of both ET- (65 +/- 11 pmol/g) and CGRP-LI (34 +/- 5 pmol/g). The tissue levels of ET-LI remained unchanged after 6-hydroxydopamine and capsaicin-pretreatment, while CGRP-LI was markedly reduced after capsaicin. Chromatographic characterization revealed that the main portion of ET-LI in the DRG, right atrium and lung corresponded to synthetic ET-1. Immunohistochemical studies showed the presence of ET-LI in a few neurons of intact DRG and many neurons in DRG cell-cultures, partly co-existing with CGRP-LI. In the neuronal cells of DRG cultures the ratio between the ET- and CGRP-LI was 1:27 compared to 2:1 in intact DRG. 24 h after ligation of the sciatic or vagal nerves no accumulation of ET-LI was observed above the ligation, while CGRP-LI was increased 4-5-fold. Transection (10 days) of the sciatic nerve caused a 85-95% depletion of CGRP-LI in the distal skin, gastrocnemius muscle and trunk below the transection site, while in the proximal portion of the nerve CGRP-LI increased. No effects on ET-LI in these tissues were observed after sciatic nerve transfection. In release experiments on DRG cell cultures. Langendorff heart preparations or perfused guinea-pig lungs, potassium (60 mM), capsaicin or antidromic nerve stimulation evoked a clear-cut increase in the supernatant levels of CGRP-LI, suggesting release, while no effect on the ET-LI concentration was observed in the effluent. Furthermore, anoxia failed to influence the outflow of ET-LI from the heart and lung. It is concluded that ET-1-LI is present in high levels in spinal ganglia and ET-LI occurs in afferent cell-bodies, but in comparison with CGRP, ET shows remarkable inertness upon various experimental conditions including no evidence for axonal transport, loss after denervation or release. The neuronal ET-LI seems to increase under culture conditions, however. The possible function for the high content of ET-LI in the intact guinea-pig peripheral nervous system remains to be elucidated and may mainly be related to a non-neuronal pool considering the relatively low content of ET-LI compared to CGRP in cultured DRG cells.     

Localisation and neural control of the release of calcitonin gene-related peptide (CGRP) from the isolated perfused porcine ileum

By immunohistochemistry, CGRP-like immunoreactive (CGRP-LI) nerve fibres were found in the lamina propria along small vessels and in the lamina muscularis mucosae in the porcine ileum. Immunoreactive nerve cell bodies were found in the submucous and myenteric plexus. Upon HPLC-analysis of ileal extracts, CGRP-LI corresponded entirely to porcine CGRP plus smaller amounts of oxidised CGRP. Using isolated vascularly perfused segments of the ileum, we studied the release of CGRP-LI in response to electrical stimulation of the mixed extrinsic periarterial nerves and to infusion of different neuroblockers. In addition, the effect of infusion of capsaicin was studied. The basal output of CGRP-LI was 2.9+/-0.7 pmol/5 min (mean+/-S.D.). Electrical nerve stimulation (8 Hz) significantly increased the release of CGRP-LI to 167+/-16% (mean+/-S.E.M.) of the basal output (n=13). This response was unaffected by the addition of atropine (10(-6) M). Nerve stimulation during infusion of phentolamine (10(-5) M) with and without additional infusion of atropine resulted in a significant further increase in the release of CGRP-LI to 261+/-134% (n=5) and 240+/-80% (n=9), respectively. This response was abolished by infusion of hexamethonium (3x10(-5) M). Infusion of capsaicin (10(-5) M) caused a significant increase in the release of CGRP-LI to 485+/-82% of basal output (n=5). Our results suggest a dual origin of CGRP innervation of the porcine ileum (intrinsic and extrinsic). The intrinsic CGRP neurons receive excitatory input by parasympathetic, possibly vagal, preganglionic fibres, via release of acetylcholine acting on nicotinic receptors. The stimulatory effect of capsaicin suggests that CGRP is also released from extrinsic sensory neurons.     

Sensory neuropeptides and airway function.

Sensory nerves synthesize tachykinins and calcitonin-gene related peptide and package these neuropeptides together in synaptic vesicles. Stimulation of these C-fibers by a range of chemical and physical factors results in afferent neuronal conduction that elicits central parasympathetic reflexes and in antidromic conduction that results in local release of neuropeptides through the axon reflex. In the airways, sensory neuropeptides act on bronchial smooth muscle, the mucosal vasculature, and submucosal glands to promote airflow obstruction, hyperemia, microvascular hyperpermeability, and mucus hypersecretion. In addition, tachykinins potentiate cholinergic neurotransmission. Proinflammatory effects of these peptides also promote the recruitment, adherence, and activation of granulocytes that may further exacerbate neurogenic inflammation (i.e., neuropeptide-induced plasma extravasation and vasodilation). Enzymatic degradation limits the physiological effects of tachykinins but may be impaired by respiratory infection or other factors. Given their sensitivity to noxious compounds and physical stimuli and their potent effects on airway function, it is possible that neuropeptide-containing sensory nerves play an important role in mediating airway responses in human disease. Supporting this view are the striking phenomenological similarities between hyperpnea-induced bronchoconstriction (HIB) in guinea pigs and HIB in patients with exercise-induced asthma. Endogenous tachykinins released from airway sensory nerves mediate HIB in guinea pigs and also cause hyperpnea-induced bronchovascular hyperpermeability in these animals. On the basis of these observations, it is reasonable to speculate that sensory neuropeptides participate in the pathogenesis of hyperpnea-induced airflow obstruction in human asthmatic subjects as well.     

Role of tachykinins in hyperpnea-induced bronchovascular hyperpermeability in guinea pigs

Isocapnic dry gas hyperpnea causes bronchoconstriction in guinea pigs that is mediated by release of tachykinins from airway sensory nerves. Exogenous neuropeptides can induce microvascular leak. Therefore we tested whether dry gas hyperpnea also elicits bronchovascular hyperpermeability by measuring Evans blue-labeled albumin extravasation along the airways of mechanically ventilated guinea pigs. We found that 1) room temperature dry gas hyperpnea increased Evans blue extravasation in extrapulmonary and intrapulmonary airways as a specific consequence of local airway heat/water losses, 2) capsaicin pretreatment ablated the bronchoconstrictor response to dry gas hyperpnea and reduced bronchovascular leak only in intrapulmonary airways, 3) phosphoramidon given to capsaicin-pretreated animals partially restored dry gas hyperpnea-induced bronchoconstriction and increased the vascular hyperpermeability response to hyperpnea in intrapulmonary airways, and 4) propranolol administration had no important effects on any of these airway responses. We conclude that dry gas hyperpnea causes bronchovascular hyperpermeability in guinea pigs. Tachykinins have a dominant role in this response in the intrapulmonary airways, although another mechanism may also contribute to the microvascular leak in the extrapulmonary airways.     

Effect of neuropeptides released from sensory nerves on blood flow in the rat airway microcirculation.

Stimulation of sensory nerves in the airway mucosa causes local release of the neuropeptides substance P and calcitonin gene-related peptide (CGRP). In this study we used a modification of the reference-sample microsphere technique to measure changes in regional blood flow and cardiac output distribution produced in the rat by substance P, CGRP, and capsaicin (a drug that releases endogenous neuropeptides from sensory nerves). Three sets of microspheres labeled with different radionuclides were injected into the left ventricle of anesthetized F344 rats before, immediately after, and 5 min after left ventricular injections of capsaicin, substance P, or CGRP. The reference blood sample was withdrawn from the abdominal aorta and was simultaneously replaced with 0.9% NaCl at 37 degrees C. We found that stimulation of sensory nerves with a low dose of capsaicin causes a large and selective increase in microvascular blood flow in the extrapulmonary airways. The effect of capsaicin is mimicked by systemic injection of substance P but not by CGRP, suggesting that substance P is the main agent of neurogenic vasodilation in rat airways.     

Galanin-, neuropeptide Y- and enkephalin-like immunoreactivities in catecholamine-storing paraganglia of the fetal guinea pig and newborn pig

Summary The occurrence and distribution of several neuropeptides and transmitter enzymes have been investigated by means of indirect immunofluorescence histochemistry in preaortal and carotid body-like paraganglia of the fetal guinea pig and the newborn pig. Preaortal paraganglia from the celiac and inferior mesenteric ganglion regions in fetal guinea pigs showed cell bodies immunoreactive (IR) for tyrosine hydroxylase (TH), dopamine -hydroxylase (DBH), neuropeptide Y (NPY), galanin (GAL) and metenkephalin (ENK). Almost all cells were IR for TH and DBH, whereas NPY-like immunoreactivity (-LI), GAL-LI and ENK-LI occurred less frequently. Direct double-labeling revealed the coexistence of NPY/GAL, NPY/ENK and GAL/ENK in paraganglion cells from the celiac and inferior mesenteric region. Nerve fibers and terminals were IR for ENK; fibers IR for calcitonin-gene-related peptide (CGRP) were present in the inferior mesenteric ganglion region. Preaortal paraganglia cells from the newborn pig showed TH-LI, DBH-LI, GAL-LI and ENK-LI, the distribution pattern being similar to that seen in the guinea pig; however, NPY-LI was absent. Carotid-body-like paraganglia from the newborn pig showed cell bodies IR to TH, GAL and ENK. Few cells were seen with DBH-LI. A rich supply of nerve fibers with CGRP-LI was present; some fibers exhibited ENK-LI and CCK-LI. In the adjacent superior cervical ganglion, ganglion cell bodies showed immunoreactivity to TH, DBH and NPY. A small number of cells were positive for GAL, CGRP and vasoactive intestinal polypeptide (VIP). Physiological activation of the paraganglia, leading to release or increase in catecholamines, may also change the content of the neuropeptides present in the paraganglia.     

Release of calcitonin gene-related peptide in the pig nasal mucosa by antidromic nerve stimulation and capsaicin

The overflow of calcitonin gene-related peptide like-immunoreactivity (CGRP-LI) in the nasal venous effluent upon antidromic stimulation of the maxillary division of the trigeminal nerve with 6.9 Hz for 3 min or upon capsaicin (0.3 mumol bolus injection) were analysed in the nasal mucosa of sympathectomized pentobarbital anaesthetized pigs. The overflow of CGRP-LI upon antidromic stimulation displayed a slower appearance in the venous effluent than the overflow upon bolus injection of capsaicin. The vascular effects as revealed by the arterial blood flow, the venous blood flow, the blood volume of the nasal mucosa, i.e., the filling of the capacitance vessels and the superficial mucosal blood flow as revealed by the laser-Doppler signal were also studied. Antidromic stimulation of the trigeminal nerve as well as capsaicin bolus injection induced a marked vasodilation which was parallel to the overflow of CGRP. However, capsaicin bolus injection also resulted in a marked increase in the mean arterial blood pressure which may be due to reflex activation of sympathetic fibers. In conclusion, we have demonstrated that chemical stimulation with capsaicin as well as antidromic stimulation of nasal sensory nerves in sympathectomized animals induces both vasodilation and overflow of CGRP-LI in vivo. This indicates that CGRP may contribute to the sensory regulation of the microcirculation in the nasal mucosa.     

Association between histamine-containing mast cells and sensory nerves in the skin and airways of control and capsaicin-treated pigs

Summary The association between mast cells (visualized by routine staining and immunohistochemistry for histamine) and capsaicin-sensitive nerves (containing calcitonin gene-related peptide (CGRP) and substance P (SP)) was studied in the pig. In the 1-ethyl-3(3-diethylaminopropyl)carbodiimide (EDCDI)-fixed skin tissue, histamine-containing mast cells and CGRP/SP-positive nerves were found in close association around blood vessels. In the EDCDI-fixed airway mucosa, only single histamine-containing mast cells were detected. However, many alcian blue-positive mast cells were found, sometimes close to the airway epithelium where CGRP/SP-containing nerve fibres were absent 2 days after systemic capsaicin pretreatment, but no changes in the number and distribution of tissue mast cells, granulocytes or lymphocytes, or the number of blood leukocytes were detected. Local injection of allergen, histamine and capsaicin into the skin of pigs actively sensitized with ascaris antigen caused a rapid light red-flare (vasodilation) reaction. Allergen and histamine, but not capsaicin, also produced plasma protein extravasation. In contrast to the absent flare, the protein extravasation response still occurred in capsaicin-treated pigs. The sensitivity to ascaris antigen was mediated by an IgE-like antibody. We conclude that a functional and morphological relationship exists between histamine-containing mast cells and capsaicin-sensitive sensory nerves in the pig skin. Mast cells and sensory nerves are also found in the airway mucosa and appear to be closely associated with the epithelium.     

Distribution of adrenomedullin-containing perivascular nerves in the rat mesenteric artery

Distribution of adrenomedullin (AM)-containing perivascular nerve fibers was studied in rat mesenteric arteries. Many fibers containing AM-like immunoreactivity (LI) were observed in the adventitia. AM-LI fibers were abolished by cold storage denervation or capsaicin but not 6-hydroxydopamine. Double immunostainings showed colocalization of AM-LI with calcitonin gene-related peptide (CGRP)-LI. The dorsal root ganglia had many AM-positive cells and AM mRNA detected by RT-PCR. Electron microscopy study revealed high proportions of immunogold labeling for AM and colocalization of both AM-LI and CGRP-LI in unmyelinated nerve axons. These results suggest that AM-containing perivascular nerves are distributed in the rat mesenteric artery.