Co-existence and origin of peptidergic and adrenergic nerves in the guinea pig uterus

Summary The occurrence and distribution of peptidergic nerves in the guinea pig uterus was studied by means of immunocytochemistry using numerous neuropeptide antisera. Neuropeptide Y (NPY)-immunoreactive (IR) nerves were the most abundant, whereas substance P (SP)-, calcitonine gene-related peptide (CGRP)-, and neurokinin A (NKA)-IR nerves were less frequent, and peptide histidine isoleucine (PHI)-IR nerves were the most sparse. Chemical sympathectomy by means of 6-hydroxydopamine, and capsaicin treatment revealed the division of the peptidergic nerves into three separate populations: (1) NPY-IR nerves, which co-existed with adrenergic nerves, (2) SP-, CGRP-and NKA-IR nerves, which mutually co-existed, and (3) PHI-IR nerves. Parallel-running adrenergic/NPY-IR and SP-IR nerves could be found with very similar although not completely identical morphological appearance. Paracervical ganglia contained neurotensin-and dynorphin A-IR cell bodies in addition to cell bodies with immunoreactivities similar to those in prevertebral ganglia. Combined retrograde tracing with True blue and immunocytochemistry showed that the adrenergic and NPY-IR uterine nerves originate in paracervical and prevertebral ganglia. In the prevertebral ganglia the cellular origin was the same for adrenergic and NPY-IR nerves. In contrast, SP-, CGRP-,and NKA-IR nerves originated in dorsal root ganglia. At full-term pregnancy all the neuropeptide immunoreactivities had vanished, probably reflecting a fetus-induced general nerve degeneration.     

Development of substance P and neurokinin A immunoreactivity in ganglia supplying nerves to the submandibular glands of the rat

Developing submandibular, trigeminal and superior cervical ganglia, which provide innervation to the submandibular glands, were studied for substance P (SP)-and neurokinin A (NKA)-immunoreactive (IR) ganglion cells and nerve fibres in rat. These ganglia were examined by using an indirect immunofluorescence technique at daily intervals from the 16th day in utero (i.u.) until birth, and subsequently on the 2nd, 5th, 7th, 12th, 16th, 30th, 42nd postnatal day and in the adult (3 months). In the submandibular ganglion SP- and NKA-IR cells and fibres first appeared in considerable numbers on the 19th day i.u. (in one sample out of five on the 18th day i.u.), when more than 90% of the ganglion cells were immunoreactive to SP and NKA. The number stayed at more than 90% to the 7th postnatal day and then slowly decreased to the levels of adult animals (18% SP, 17% NKA). The first SP- and NKA-IR ganglion cells and fibres appeared in the trigeminal ganglion on the 18th day i.u. when they represented 7% (SP) and 4% (NKA) of the ganglion cells. The number of SP- and NKA-IR cells increased steadily, reaching a maximum at the time of birth when 68% (SP) and 74% (NKA) of the ganglion cells were immunoreactive. Thereafter they began to decrease toward the level of an adult rat (10% SP, 11% NKA). In the superior cervical ganglion only a few SP-and NKA-IR ganglion cells were detected from the 19th day i.u. to the fifth postnatal day. Positive ganglion cells were also occasionally found in the nerve trunks outside the superior cervical ganglion. From the seventh day onwards no SP- or NKA-IR ganglion cells were found. SP-and NKA-IR SIF (small intensively fluorescent) cells were detected from the 16th postnatal day onwards.     

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.     

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.     

Substance P and neurokinin A immunoreactive nerve fibres in the developing salivary glands of the rat

The time of appearance and distribution of substance P (SP) and neurokinin A (NKA) immunoreactive nerve fibres in developing salivary glands of the rat were studied by the use of indirect immunohistochemical methods. The glands were examined at daily intervals from the 15th day in utero (i.u.) until birth, and subsequently on the 2nd, 5th, 7th, 12th, 16th and 30th postnatal day. The findings were compared to samples from adult. The first SP- and NKA-immunoreactive (IR) nerve fibres appeared on the 19th day i.u. in the parotid and submandibular glands and were abundantly distributed around developing ductal branches. In the mesenchyme around the developing ductal branches of the parotid gland the fibres appeared on the 20th day i.u. In the submandibular gland NKA-IR fibres appeared in the mesenchyme surrounding the developing ductal branches on the 19th day i.u. and SP-IR fibres on the 21st day i.u. Around blood vessels of both glands, SP- and NKA-IR fibres made their appearance only much later, on the second postnatal day. The number of SP- and NKA-IR nerve fibres in the developing salivary glands was already high on the 19th day i.u. when they were first detected. From this point up to the 16th postnatal day the glands were richly innervated by the fibres, but later the numbers slowly decreased to adult levels. The abundance of SP- and NKA-IR nerve fibres especially around the ductal branches and secretory structures in the developing salivary glands suggests a role in the functional maturation of salivary glands.     

Substance P and neurokinin A immunoreactive nerve fibres in the developing salivary glands of the rat.

The time of appearance and distribution of substance P (SP) and neurokinin A (NKA) immunoreactive nerve fibres in developing salivary glands of the rat were studied by the use of indirect immunohistochemical methods. The glands were examined at daily intervals from the 15th day in utero (i.u.) until birth, and subsequently on the 2nd, 5th, 7th, 12th, 16th and 30th postnatal day. The findings were compared to samples from adult. The first SP- and NKA-immunoreactive (IR) nerve fibres appeared on the 19th day i.u. in the parotid and submandibular glands and were abundantly distributed around developing ductal branches. In the mesenchyme around the developing ductal branches of the parotid gland the fibres appeared on the 20th day i.u. In the submandibular gland NKA-IR fibres appeared in the mesenchyme surrounding the developing ductal branches on the 19th day i.u. and SP-IR fibres on the 21st day i.u. Around blood vessels of both glands, SP- and NKA-IR fibres made their appearance only much later, on the second postnatal day. The number of SP- and NKA-IR nerve fibres in the developing salivary glands was already high on the 19th day i.u. when they were first detected. From this point up to the 16th postnatal day the glands were richly innervated by the fibres, but later the numbers slowly decreased to adult levels. The abundance of SP- and NKA-IR nerve fibres especially around the ductal branches and secretory structures in the developing salivary glands suggests a role in the functional maturation of salivary glands.     

An immunohistochemical study on the innervation of SP-IR nerves in the cerebral arteries of the bent-winged bat

The overall distribution of substance P (SP) immunoreactive (IR) nerves surrounding the cerebral arteries of the bent-winged bat were investigated immunohistochemically. In this microchiropteran species, the walls from the vertebral artery to the caudal part of the basilar artery have considerably well-developed plexuses of SP-IR nerves, whereas no demonstrable SP-IR fibers were found in the rostral part of the basilar artery, and in more rostrally located arteries the nerve supply was very sparse or occasionally lacking. This innervation pattern has not yet been established for the cerebral arterial systems of other mammals that have been studied under normal conditions, but it is very similar to the pattern of SP-IR innervation observed in the guinea pig and cat of which the trigeminal ganglia have been destroyed. From the combination of this and other immunohistochemical findings, it is suggested that SP-IR nerves innervating the vertebral and basilar arteries of the bent-winged bat originate from the upper cervical dorsal root ganglia (DRG) and enter the cranial cavity along the vertebral artery and through the meninges.     

An immunohistochemical study on the innervation of SP-IR nerves in the cerebral arteries of the bent-winged bat

Summary The overall distribution of substance P (SP) immunoreactive (IR) nerves surrounding the cerebral arteries of the bent-winged bat were investigated immunohistochemically. In this microchiropteran species, the walls from the vertebral artery to the caudal part of the basilar artery have considerably well-developed plexuses of SP-IR nerves, whereas no demonstrable SP-IR fibers were found in the crostral part of the basilar artery, and in more rostrally located arteries the nerve supply was very sparse or occasionally lacking. This innervation pattern has not yet been established for the cerebral arterial systems of other mammals that have been studied under normal conditions, but it is very similar to the pattern of SP-IR innervation observed in the guinea pig and cat of which the trigeminal ganglia have been destroyed. From the combination of this and other immunohistochemical findings, it is suggested that SP-IR nerves innervating the vertebral and basilar arteries of the bent-winged bat originate from the upper cervical dorsal root ganglia (DRG) and enter the cranial cavity along the vertebral artery and through the meninges.Abbreviations BA basilar artery - CSN cervical spinal nerves - ICS internal carotid system - SCG superior cervical ganglion - SNB sympathetic nerve bundle - VA vertebral artery - VBS vertebro-basilar system     

Noradrenergic and peptidergic innervation of the mammary gland in the immature pig

The presence and pattern of coexistence of some biologically active substances in nerve fibres supplying the mammary gland in the immature pig were studied using immunohistochemical methods. The substances studied included: protein gene product 9.5 (PGP), tyrosine hydroxylase (TH), somatostatin (SOM), neuropeptide Y (NPY), galanin (GAL), calcitonin gene-related peptide (CGRP) and substance P (SP). The mammary gland was found to be richly supplied by PGP-immunoreactive (PGP-IR) nerve fibres that surrounded blood vessels, bundles of smooth muscle cells and lactiferous ducts. The vast majority of these nerves also displayed immunoreactivity to TH. Immunoreactivity to SOM was observed in a moderate number of nerve fibres which were associated with smooth muscles of the nipple and blood vessels. Immunoreactivity to NPY occurred in many nerve fibres associated with blood vessels and in single nerves supplying smooth muscle cells. Solitary GAL-IR axons supplied mostly blood vessels. Many CGRP-IR nerve fibres were associated with both blood vessels and smooth muscles. SP-IR nerve fibres richly supplied blood vessels only. The colocalization study revealed that SOM, NPY and GAL partly colocalized with TH in nerve fibres supplying the porcine mammary gland.     

Effects of trigeminal neurotransmitters on piglet pial arterioles.

We investigated effects of calcitonin gene-related peptide (CGRP), substance P (SP), and neurokinin A (NKA) on pial arterioles in newborn pigs. Pial arteriolar diameter was determined using a closed cranial window and intravital microscopy. Initial diameters were approximately 100 microns. Calcitonin-gene related peptide dilated pial arterioles by 22 +/- 8% at 10(-9)M and by 34 +/- 6% at 10(-8)M (n = 8), and this response was not significantly altered by prior administration of indomethacin (5mg/kg, iv) (n = 6) or administration of NG-methyl-L-arginine (5mg/kg, iv, and 10(-3)M in CSF) (n = 10). Substance P dilated arterioles at 10(-10)M through 10(-5)M (maximal response = 23 +/- 3%) (n = 6), and this response was unaffected by indomethacin administration (n = 6). In contrast, NG-methyl-L-arginine blocked much of the pial arteriolar dilation to SP. Unlike the other two peptides, NKA did not change pial arteriolar diameter. Radioimmunoassay determinations indicated that cerebrospinal fluid levels of 6-keto-prostaglandin F1 and prostaglandin E2 did not change appreciably during application of CGRP or SP. We conclude that CGRP and SP but not NKA are dilator stimuli in the piglet pial circulation. Dilation by CGRP probably involves direct activation of receptors on vascular smooth muscle, while SP probably partially dilates pial arterioles via release of an endothelium-dependent relaxing factor.     

Postnatal development of autonomic and sensory innervation of thoracic hairy skin in the rat

Summary Histochemical, immunocytochemical, and radioenzymatic techniques were used to examine the neurotransmitter-related properties of the innervation of thoracic hairy skin in rats during adulthood and postnatal development. In the adult, catecholamine-containing fibers were associated with blood vessels and piloerector muscles, and ran in nerve bundles throughout the dermis. The distribution of tyrosine hydroxylase (TH)-immunoreactive (IR) fibers was identical. Neuronal fibers displaying neuropeptide Y (NPY) immunoreactivity were seen in association with blood vessels. Double-labeling studies suggested that most, if not all, NPY-IR fibers were also TH-IR and likewise most, if not all, vessel-associated TH-IR fibers were also NPY-IR. Calcitonin gene-related peptide (CGRP)-IR fibers were observed near and penetrating into the epidermis, in close association with hair follicles and blood vessels, and in nerve bundles. A similar distribution of substance P (SP)-IR fibers was evident. In adult animals treated as neonates with the sympathetic neurotoxin 6-hydroxydopamine, a virtual absence of TH-IR and NPY-IR fibers was observed, whereas the distribution of CGRP-IR and SP-IR fibers appeared unaltered. During postnatal development, a generalized increase in the number, fluorescence intensity, and varicose morphology of neuronal fibers displaying catecholamine fluorescence, NPY-IR, CGRP-IR, and SP-IR was observed. By postnatal day 21, the distribution of the above fibers had reached essentially adult levels, although the density of epidermal-associated CGRP-IR and SP-IR fibers was significantly greater than in the adult. The following were not evident in thoracic hairy skin at any timepoint examined: choline acetyltransferase activity, acetylcholinesterase histochemical staining or immunoreactivity, fibers displaying immunoreactivity to vasoactive intestinal peptide, cholecystokinin, or leucine-enkephalin. The present study demonstrates that the thoracic hairy skin in developing and adult rats receives an abundant sympathetic catecholaminergic and sensory innervation, but not a cholinergic innervation.     

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

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

Neurohormonal peptides, serotonin, and nitric oxide synthase in the enteric nervous system and endocrine cells of the gastrointestinal tract of neotenic and thyroid hormone-treated axolotls (Ambystoma mexicanum)

Immunoreactivity against vasoactive intestinal polypeptide (VIP), neurotensin (NT), substance P (SP), calcitonin gene-related peptide (CGRP), gastrin/cholecystokinin (GAS/CCK), somatostatin (SOM), serotonin (SER), and nitric oxide synthase (NOS) was investigated in the gastrointestinal tract of the urodele Ambystoma mexicanum, the axolotl, by the use of immunohistochemical techniques. The study also compares the distribution patterns and frequencies of the neurohormones, and NOS in neotenic and thyroxine-treated (metamorphosed) individuals. GAS/CCK, SP, NT, SOM, and SER immunoreactivities occurred in endocrine mucosal cells and VIP, SP, CGRP, NTSER, SER, and NOS immunoreactivities in the enteric nervous system. The GAS/CCK-immunoreactive (-IR) cells were restricted to the upper small intestine. NT-IR and SP-IR endocrine cells were found in the entire gastrointestinal tract and were most prominent in the distal large intestine. The density of the SOM-IR cells decreased from the stomach toward the large intestine. SER-IR endocrine cells were found throughout the gastrointestinal tract, with particularly high densities in the stomach and distal large intestine. The VIP-IR enteric nerve fibers were the most prominent ones, present in all layers of the entire gastrointestinal tract, and supplied the smooth muscle and the vasculature. The SER-IR fibers exhibited similar distribution patterns but were less numerous. Very few NT-IR but many SP-IR fibers were found in the muscle and submucosal layers. The NT-IR fibers mainly supplied blood vessels, while the SP-IR fibers were also in contact with the smooth muscle. In the muscle and submucosal layers, CGRP-IR fibers were associated to the vasculature; CGRP immunoreactivity occurred also in a minority of SP-IR fibers. NOS-IR nerve fibers were in contact with submucosal arteries but were the least frequent . After metamorphosis provoked by exogenous thyroxine, the number of SOM-IR endocrine cells in the stomach mucosa was increased as well as the density of VIP-IR, SER-IR, and SP-IR nerve fibers in the gastrointestinal tract. It is proposed that the observed increases may reflect refinements of the neurohormonal system after metamorphosis.     

Neurokinin A, neurokinin B and neuropeptide K in the rabbit iris: a study comparing different extraction methods

Neurogenic inflammation in the rabbit eye is thought to be partly mediated by tachykinins released from trigeminal sensory nerve fibres. In the present study we have investigated the occurrence of neurokinin A (NKA), neurokinin B (NKB), neuropeptide K (NPK) and related immunoreactive components in the rabbit iris-ciliary complex using neutral and different types of acidic media for extraction, reverse phase high performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA). The immunoreactive material detected with an antiserum reacting almost equally well with NKA, NKB and NPK consisted mainly of NKA, and small amounts of NPK but almost no NKB. Acidic media seemed to be more effective than neutral media for extraction of NKA and NPK. Acid extraction yielded also an NKA-immunoreactive component which eluted immediately before NKA while neutral extracts, on the other hand, contained a component which appeared behind NKA, i.e. in the position of NKA-(3-10) and NKA-(4-10). The present results indicate that NKA but not NKB may play a role in neurogenic inflammation in the rabbit eye.     

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

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

Calcitonin gene-related peptide and the lung: neuronal coexistence with substance P, release by capsaicin and vasodilatory effect

The occurrence and distribution of calcitonin gene-related peptide (CGRP) in the lower airways was studied by means of immunohistochemistry and radioimmunoassay (RIA) in combination with high performance liquid chromatography (HPLC). CGRP-like immunoreactivity (-LI) was observed in nerves from the epiglottis down to peripheral bronchi in rat, cat and guinea pig and also in human bronchi. Double staining revealed colocalization of CGRP-LI and substance P (SP)-LI in cell bodies of nodose and jugular ganglia as well as in axons and nerve terminals of the airways. Systemic capsaicin pretreatment induced a marked loss of the CGRP- and SP-immunoreactive (-IR) nerves in the lower airways. CGRP-IR was also present in epithelial endocrine cells and neuroepithelial bodies. The content of CGRP-LI as measured with RIA in guinea pig bronchi was significantly lower after capsaicin pretreatment. Analysis of human bronchial extracts revealed that CGRP-LI coeluted with synthetic human CGRP on HPLC. In the isolated perfused guinea pig lung capsaicin exposure caused overflow of CGRP-LI suggesting release from peripheral branches of sensory nerves. Both in vivo experiments in the guinea pig measuring insufflation pressure as well as in vitro studies on isolated guinea pig and human bronchi showed that whereas tachykinins contracted bronchial smooth muscle no contractile or relaxing effect was elicited by human or rat CGRP. However, CGRP caused relaxation of serotonin precontracted guinea pig and human pulmonary arteries. In conclusion, the presence and release of CGRP-LI from capsaicin sensitive nerves in the lower airways adds another possible mediator, in addition to tachykinins, of vascular reactions upon sensory nerve irritation.     

Calcitonin gene-related peptide (CGRP) in the nipple of the rat mammary gland

The distribution of nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP) was investigated by immunohistochemistry in nipples and mammary glands from lactating and non-lactating rats and compared to the immunoreactivity of other neuropeptides including substance P (SP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and somatostatin (SOM). The study revealed an extensive innervation of the mammary nipples, in which CGRP-immunoreactive (IR) nerve fibres were abundantly present in the epidermis, dermal connective tissue and intralobular connective tissue of the mammary gland parenchyma. Several of the dermal CGRP-IR fibres seemed to follow blood vessels, or formed ringlet-like structures. The latter were mostly observed in the dermal connective tissue of the nipple from the lactating rat and may have a mechanoreceptive function, e.g. for the suckling stimuli. The location of SP-IR appeared to be comparable to CGRP-IR, but in fewer fibres. Dense NPY-IR networks of nerve fibres were closely associated with the fascicles of smooth musculature in the core of the nipple base. In contrast, VIP-IR fibres were only sparsely present, and SOM-IR was not detected in the mammary nipples. The immunoreactive content of CGRP and SP was determined by radioimmunoassays. The total amount of immunoreactive CGRP was significantly higher in the nipples from the pregnant and the lactating rats when compared to SP. The maximum concentration of CGRP (65.9±4.0 pmol/g) measured in the nipples of the pregnant (day 10) rats exceeded almost ninefold the maximum concentration of SP (7.7±2.0 pmol/g). Thus, the immunoreactive content of CGRP in the nipples confirmed the immunohistochemical observations, and the present results strongly suggest that CGRP is a major neuropeptide in the rat nipple.     

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

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