Biochemical mapping of cholecystokinin-, substance P-, [Met]enkephalin-, [Leu]enkephalin- and dynorphin A (1-8)-like immunoreactivities in the human cerebral cortex

The distribution of immunoreactive cholecystokinin, substance P, [Met]enkephalin, [Leu]-enkephalin and dynorphin was determined in the cerebral cortex of the human brain post mortem. Peptide radioimmunoassays in three selected zones of the cortical gray mantle (frontal, temporal, occipital) revealed significant regional differences, prompting to the development of a new dissection procedure for the complete mapping of peptide-like materials throughout the entire cerebral cortex. For this purpose, frozen cerebral hemispheres were cut rostrocaudally in 21 verticofrontal serial sections, from which the cortical gray matter was divided into 4-5 distinct zones. The peptides could be measured in each of the 93 dissected pieces of tissue, but their distribution was uneven. The most abundant was cholecystokinin, particularly in the anterior part of the frontal lobe and in the temporal cortex, where its levels reached 0.5 ng/mg of tissue. The regional distribution of cholecystokinin resembled that of substance P with a decreasing gradient from the frontal to the occipital pole, but absolute levels of substance P were hardly one tenth of cholecystokinin levels. The mean concentrations of the three opioid peptides were even less than those of substance P, and their regional distributions were markedly different. [Met]Enkephalin was concentrated in the occipital cortex, and [Leu]enkephalin in the temporal cortex. Dynorphin was the least abundant, even in the temporal cortex where the highest levels were found. The widespread and heterogeneous distribution of these peptides strongly suggests that each of them exerts specific functions in the human cerebral cortex.     

[Met5]enkephalin-Arg6-Phe7- and [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibres and neurons in the superior cervical ganglion of the rat

[Met⁵]enkephalin-Arg⁶-Phe⁷- (MEAP-) and [Met⁵]enkephalin-Arg⁶-Gly⁷-Leu⁸- (MEAGL-) immunoreactivity was studied by indirect immunohistochemistry in the superior cervical ganglion of the rat with specific antisera produced in rabbits against the corresponding synthetic opioid peptides. Several MEAP- and a few MEAGL-immunoreactive principal nerve cells were observed in the ganglion, while the small intensely fluorescent cells appeared as non-reactive. The superior cervical ganglion also contained dense networks of MEAP- and MEAGL-immunoreactive nerve fibres, which often formed basket-like structures around the principal nerve cells and small intensely fluorescent cells. After ligation of the preganglionic nerve trunk with simultaneous transection of the main postganglionic trunks, a distinct accumulation of both MEAP- and MEAGL- immunoreactivity was observed on both sides of the ligature. Ligation of the preganglionic nerve trunk caused a marked decrease in the number of both MEAP- and MEAGL-immunoreactive nerve fibres in the ganglion. Ligation of the main postganglionic nerve trunks with simultaneous preganglionic nerve division resulted in accumulation of MEAP- and MEAGL-immunoreactive material on the ganglionic side of the ligature in both the external and internal carotid nerve. After division of both the pre- and postganglionic nerve trunks, some immunoreactive nerve fibres and principal nerve cells were still observed in the ganglion. A few immunoreactive neurons and nerve fibres were also observed in the ganglion stellatum. A large number of MEAP- and MEAGL-immunoreactive nerve fibres was detected in the spinal cord at the levels C6-Th6. A few neurons in the intermediolateral cell column of the spinal cord at levels C8-Thl showed MEAP- but not MEAGL-immunoreactivity. The cultured superior cervical ganglion contained a few MEAP-immunoreactive neurons, and the fibre outgrowth showed immunoreactivity both to MEAP and MEAGL. In electron microscopy, MEAGL-immunoreactivity in the superior cervical ganglion was localized in nerve fibres containing neurotubules and in principal nerve cells.The present results demonstrate that the rat superior cervical ganglion contains both extrinsic and intrinsic MEAP- and MEAGL-immunoreactive nerve fibres. Most of these fibres are of preganglionic origin. Both the principal nerve and small intensely fluorescent cells are often surrounded by MEAP- or MEAGL-immunoreactive nerve fibres and may receive innervation by these fibres. Several ganglionic neurons projecting to the sympathetic target tissues show MEAP- and/or MEAGL-immunoreactivity. Since MEAP- and MEAGL sequences are included in the proenkephalin A molecule, but not in the other opioid precursor molecules, it is evident that a neuronal system containing proenkephalin-A-derived enkephalins, is present in the rat superior cervical ganglion.     

Immunohistochemical localization of [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8 in sympathetic and parasympathetic neurons and nerve fibers projecting to the rat submandibular gland.

The localization of [Met5]enkephalin, [Met5]enkephalin-Arg6-Gly7-Leu8, vasoactive intestinal polypeptide and tyrosine hydroxylase immunoreactivities was studied in the submandibular gland of adult Sprague-Dawley and Wistar rats using the indirect immunofluorescence technique. Immunoreactivities for [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8, a proenkephalin A-derived octapeptide, showed identical distributions. A large number of enkephalin-immunoreactive nerve fibers were detected around secretory acini, along intercalated ducts, convoluted granular tubules, intra- and interlobular ducts, as well as in close contact with blood vessels. The submandibular ganglia contained several enkephalin-immunoreactive neurons and nerve fibers. In the superior cervical ganglion numerous enkephalin-immunoreactive neurons and nerve fibers were also detected. Immunohistochemical co-localization studies indicated that [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactivities co-exist with vasoactive intestinal polypeptide in a subpopulation of neurons of the rat submandibular ganglia, in nerve trunks along the salivary ducts of the gland, and in nerve fibers around the acini. Uni- or bilateral superior cervical ganglionectomies for 1-4 weeks resulted in a complete disappearance of tyrosine hydroxylase immunoreactivity in the glandular parenchyma, while moderate tyrosine hydroxylase immunoreactivity was seen in some neurons of the submandibular ganglia. Abundant [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers were still seen around the acini and blood vessels, as well as close to salivary ducts. These operations did not affect the [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive neurons in the submandibular ganglia. Many principal neurons in the superior cervical ganglion contained both [Met5]enkephalin-Arg6-Gly7-Leu8 and tyrosine hydroxylase immunoreactivity. Nerve ligation experiments indicated that [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive sympathetic fibers project along the external carotid nerve. Accordingly, nerve fibers were found around the acini and blood vessels as well as in nerve trunks along the salivary ducts of the submandibular gland, showing co-localization of [Met5]enkephalin-Arg6-Gly7-Leu8 and tyrosine hydroxylase. Taken together, these observations suggest that the nerve fibers of the rat submandibular gland containing proenkephalin A-derived peptides are of both sympathetic and parasympathetic origin.     

Distribution of [Met5]- and [Leu5]-enkephalin-, vasoactive intestinal polypeptide- and substance P-like immunoreactivities in human adrenal glands

There is recent evidence that the amine storing cells of mammalian adrenal medulla also contain bioactive peptides. In the present study we examined human adrenal glands with the immunoperoxidase-bridge method using specific antisera raised against [Met⁵]- and [Leu⁵]-enkephalin, vasoactive intestinal polypeptide hormone (VIP), and substance P. Approximately one-third of the cells in the adrenal medulla demonstrated enkephalin-like immunoreactivity. The intensity of the immunostain varied among individual cells but did not appear to correlate with amine content, as determined by the formaldehyde-induced fluorescence of catecholamines. An abundant network of varicose fibre-like structures and dots, representing preterminal and terminal nerves, demonstrated vasoactive intestinal polypeptide-like immunoreactivity and were found in close proximity to medullary gland cells. Substance P-like immunoreactivity was observed in a few fibres in the medulla and cortex. However, we could not detect cells containing vasoactive intestinal polypeptide- or substance P-like immunoreactivity in adrenal glands. p ]The present findings suggest that human adrenal medullary cells contain both [Met⁵]- and [Leu⁵]-enkephalin and are richly innervated by peptidergic nerves containing vasoactive intestinal polypeptide. These peptides may modulate the release and effects of catecholamines in the adrenal medulla. The nerves with substance P-like immunoreactivity may represent a separate peptidergic neuronal system.     

Colocalization of GABA and [Met]enkephalin-Arg6-Gly7-Leu8 in the rat cerebellum

The distribution of [Met]enkephalin-Arg6-Gly7-Leu8-like immunoreactivity (MEAGL-LI) in the rat cerebellum was investigated by peroxidase anti-peroxidase immunocytochemistry using specific antiserum against MEAGL. MEAGL-LI positive neuronal perikarya were distributed in the granular layer, and they seemed to correspond to Golgi cells from their size and location. In addition, diffusely and weakly stained neuronal perikarya were also observed in the molecular layer. Immunoreactive fibers and terminals were found in the granular layer. Furthermore, examination of serial frozen sections (4-6 micron in thickness) from rats pretreated with colchicine clarified the colocalization of gamma-aminobutyric acid (GABA) and MEAGL in Golgi cells but not in the stellate cells.     

Possible induction of [Met]enkephalin-Arg6-Gly7-Leu8 immunoreactivity in neurons of the rat superior colliculus following eye enucleation

The distribution of [Met]enkephalin-Arg6-Gly7-Leu8 (MEAGL)-immunoreactive (-IR) neurons and its modification after enucleation have been investigated in the rat superior colliculus. In normal rats and on the ipsilateral side of monocular-enucleated rats, small sized vertically elongated fusiform-shaped weakly immunostained neurons were dispersed throughout the sublamina of the stratum griseum superficiale (SGS). In bilaterally enucleated rats and on the contralateral side of monocular-enucleated rats, many small strongly immunoreactive MEAGL-containing neurons, projecting processes horizontally or obliquely toward the surface, appeared in the deepest part of the SGS and the superficial part of the stratum opticum (SO), in contrast to the disappearance of the fusiform-shaped weakly stained neurons in the SGS. MEAGL-IR fibers increased in density throughout the sublamina of the SGS, being densest in the deep SGS, accompanying their increase in the neighboring SO. Sporadically found MEAGL-IR neurons in the deep SO and the stratum griseum intermediale did not show the detectable change of immunoreactivity. These results indicate that enkephalin biosynthesis is undergone by different type of neurons in the normal and the ocular-derived superior colliculus, and suggest that some neurons in the adult superior colliculus have a potentiality to express the peptidergic phenotype.     

Localization of [Met5]- and [Leu5]-enkephalin-like immunoreactivity in nerve terminals in human paravertebral sympathetic ganglia

Both [Leu⁵]- and [Met⁵]-enkephalin have been localized immunohistochemically in nerve fibres and in small, intensely fluorescent cells of adult human sympathetic ganglia. The nerve fibres showing enkephalin-like immunoreactivity formed a network varying in density around the sympathetic neurons, some being closely related to the perikarya. No labelled neuronal cell bodies were found. No structures within the ganglion were labelled after reaction with antibodies to vasoactive intestinal polypeptide, adrenocorticotrophin or substance P. No differences between the distributions of [Leu⁵]-and [Met⁵]-enkephalin-like immunoreactivities were found.The physiological roles of enkephalins are still unknown, but it is possible that they might act as neurotransmitters or neuromodulators in the human sympathetic nervous system.     

Met]enkephalin-Arg6-Gly7-Leu8-immunoreactive nerves in guinea-pig and rat lungs: distribution, origin, and co-existence with vasoactive intestinal polypeptide immunoreactivity.

[Met]Enkephalin-Arg6-Gly7-Leu8 is an endogenous opioid peptide, first isolated from the bovine adrenal medulla. Because this octapeptide is specifically contained in the amino acid sequence of preproenkephalin A but not in other opioid precursors like preproopiomelanocortin or preproenkephalin B, [Met]enkephalin-Arg6-Gly7-Leu8 has been regarded as a specific marker for preproenkephalin A and its derivatives. In this study, we examined the occurrence and origin of [Met]enkephalin-Arg6-Gly7-Leu8-immunoreactive nerves in the guinea-pig and rat lung by immunohistochemical techniques, using a specific antiserum against this peptide. In addition, we investigated the possible co-existence of [Met]enkephalin-Arg6-Gly7-Leu8 and vasoactive intestinal peptide immunoreactivity in neuronal elements of the respiratory tract. In both species, [Met]enkephalin-Arg6-Gly7-Leu8 immunoreactivity was localized in nerve fibers chiefly distributed to the trachea and major bronchi, where they were prevalent in smooth muscle bundles, in the lamina propria, around airway glands, and in the walls of pulmonary vessels, but were absent in airway epithelium. Slight differences in the distribution pattern of immunoreactive nerve fibers were noted between the two species: immunoreactive nerve fibers in the smooth muscle bundles were much more abundant in guinea-pigs than in rats, while those in the mucous glands were richer in rats than in guinea-pigs. Neither chemical sympathectomy by 6-hydroxydopamine, nor chemical sensory denervation by capsaicin, changed the density or distribution of [Met]enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers in the airway, suggesting an intrinsic source for these nerve fibers. Colchicine injection into the tracheal wall, to promote the accumulation of neuropeptides in nerve cell bodies, led to the visualization of [Met]enkephalin-Arg6-Gly7-Leu8 immunoreactivity in some neuronal cell bodies within airway ganglia. Immunostaining for [Met]enkephalin-Arg6-Gly7-Leu8 and for vasoactive intestinal polypeptide on serial adjacent sections of airway ganglia obtained from colchicine-treated tracheae, demonstrated the co-existence of these immunoreactivities in a population of nerve cell bodies in these ganglia. The immunohistochemical localization of immunoreactive [Met]enkephalin-Arg6-Gly7-Leu8 in nerve elements in guinea-pig and rat lungs provides a morphological basis for the possibility that preproenkephalin A-related opioid peptides may have a neuromodulatory role in mammalian airways and pulmonary vessels.     

成人腹腔神经节的形态观察

目的观察成人腹腔神经节的位置及形态结构,为揭示其功能提供形态学资料。方法取死后24～48h的18具成人尸体的18个腹腔神经节,经石蜡包埋切片,苏木精-伊红、Nissl及Masson染色,观察该神经节的细胞构筑。结果腹腔神经节多位于从腹主动脉发出的腹腔干的两侧,其形态不规则,但轮廓清楚,质地坚实,易与其它结构区别;镜下观察到,该神经节被单层扁平细胞构成的被膜包裹,节内的神经元为多极形,尼氏体丰富,核大圆亮;卫星细胞的形态结构也完整;在节内的细胞间质中含有丰富的胶原纤维。神经节的所有细胞均无溶解现象。结论若能在成年病人死亡后24～48h及时固定,既可确保其腹腔神经节的形态结构完整,又可为神经生物学的教学和科研制作成良好的组织切片标本。     

Light inhibits the release of both [Met5]enkephalin and [Met5]enkephalin-containing peptides in chicken retina, but not their syntheses

The levels of native and cryptic [Met5]enkephalin in the chicken retina were found to vary during a 12:12 h light-dark cycle, both rising in the light and falling during the dark. Such variations could conceivably arise from (a) changes in the rate of release and subsequent degradation of native and/or cryptic [Met5]enkephalin, (b) changes in the rate of proenkephalin A synthesis, or (c) changes in the rate of proenkephalin A processing. Measurement of the rate of release of native and cryptic [Met5]enkephalin in vitro indicated that the increased rate of release of both of these forms of [Met5]enkephalin during the dark quantitatively accounted for the fall in their retinal levels during the dark. This indicated that the biosynthesis of proenkephalin A was not activated during the light-dark cycle. Molecular weight fractionation of retinal extracts also supported this idea, since the pool of high molecular weight precursors did not vary in size, suggesting that processing was not modulated during the light-dark cycle. Instead, the fall in both cryptic and native [Met5]enkephalin during the dark was due to their increased rate of release together with a rate-limiting conversion of high molecular weight [Met5]enkephalin-containing peptides to low molecular weight [Met5]enkephalin-containing peptides. The enkephalinergic cells of the retina seem to cope with physiological variations in demand by accumulating a large pool of peptide during periods of low stimulation (light), so that when stimulation and release is high (dark), the decrease in pool levels does not compromise the function of the cells and their postsynaptic targets.     

Distribution of p75 and trk-neurotrophin receptor proteins in adult human sympathetic ganglia

We investigated the expression of immunoreactivity (IR) for low- (p75) and high-affinity (trk proteins) neurotrophin-receptor proteins in adult human paravertebral-sympathetic ganglion neurons. Mouse monoclonal antibodies against the pan-neurotrophin-receptor p75, and rabbit polyclonal antibodies against specific epitopes of the intracytoplasmic domain on trk neurotrophin-receptor proteins were used in fresh unfixed and formaldehyde-fixed paraffin-embedded sympathetic ganglia. All adult human paravertebral-sympathetic neurons displayed trkA neurotrophin-receptor-like protein IR, 10% express trkC neurotrophin-receptor-like protein IR, 37–44% show p75 IR, and no IR was obtained for trkB neurotrophin-receptor-like protein. The intensity of immunostaining was independent of the neuron size. Labelling of non-neuronal tissues, especially blood-vessel walls, was observed for p75, trkA and trkC neurotrophin-receptor proteins. These results indicate that overlapping exists in the expression of p75 and trk neurotrophin-receptor proteins in adult human paravertebral-sympathetic neurons, and suggest that neurotrophins might act on these neurons.     

Characterization of substance P- and [Met]enkephalin-immunoreactive neurons in the caudate nucleus of cat and ferret by a single section Golgi procedure

Modifications of the single-section Golgi-impregnation procedure of Gabbott and Somogyi are described. The modifications allow easier and more rapid preparation of the sections for Golgi-impregnation and easier handling of large numbers of serial sections. The technique consists of placing a section that has been treated with osmium tetroxide and potassium dichromate on a microscope slide and "sandwiching" it with a second microscope slide. The two slides are held together at one end by tape and the assembly is dipped into a solution of silver nitrate. Golgi-impregnation of neurons occurs within a few hours and is generally complete within 12 h. The technique has been applied to sections through the caudate nucleus of the cat and ferret in order to define the morphological characteristics of striatal substance P- and methionine enkephalin-immunoreactive neurons. Sections were first incubated to reveal the immunoreactive structures and then subjected to the Golgi method. Golgi-impregnated neurons that were immunoreactive for either substance P or methionine enkephalin had medium-size perikarya from which several dendrites emerged. The dendrites branched close to the perikaryon; secondary and higher order dendrites were densely laden with spines, as many as 15 spines per 10 microns of dendrite. It is concluded that both striatal substance P-containing and methionine enkephalin-containing neurons are of the medium-size densely spiny type. Medium-size densely spiny neurons may be homogeneous with respect to their somatodendritic morphology but heterogeneous with respect to their chemical characteristics and axonal morphology.     

Serotonin-, substance P- and glutamate/aspartate-like immunoreactivities in medullo-spinal pathways of rat and primate.

Serotonergic neurons of the medulla oblongata have been proposed to play a role in the control of sensory, motor and autonomic cells in the spinal cord. Many of these raphe neurons have been shown to contain the undecapeptide substance P as well as the tripeptide thyrotropin-releasing hormone, but evidence for the presence of an excitatory amino acid in these pathways has not yet been documented. In colchicine-treated rats, we have used a combination of retrograde tracing and tri-color immunohistofluorescence techniques to study co-localization of serotonin- and substance P- with glutamate- or aspartate-like immunoreactivities in medullary neurons and the possible spinal projections of these cells. In addition, the distributions of serotonin-, substance P- and glutamate-immunoreactive terminal fields in the dorsal, ventral and lateral horns of the spinal cord were examined with tri-color immunofluorescence in the rat and the primate Macaca fasciculata. In colchicine-treated rats, glutamate- and aspartate-like immunoreactivity was found in practically all serotonin- and substance P-immunoreactive neurons of the B1, B2 and B3 cell groups. Some of these neurons also contained wheat-germ agglutinin conjugated to inactivated horseradish peroxidase and colloidal gold particles retrogradely transported from the spinal cord. In the spinal cords of non-colchicine-treated monkeys and rats, striking co-localization of serotonin, substance P- and glutamate-like immunoreactivities was seen in large boutons, surrounding the dendrites and cell bodies of large alpha motor neurons in the ventral horn. These observations suggest the existence of spinally projecting serotonin/substance P neurons containing excitatory amino acids such as glutamate or aspartate.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Distribution of p75 and trk-neurotrophin receptor proteins in adult human sympathetic ganglia

We investigated the expression of immunoreactivity (IR) for low- (p75) and high-affinity (trk proteins) neurotrophin-receptor proteins in adult human paravertebral-sympathetic ganglion neurons. Mouse monoclonal antibodies against the pan-neurotrophin-receptor p75, and rabbit polyclonal antibodies against specific epitopes of the intracytoplasmic domain on trk neurotrophin-receptor proteins were used in fresh unfixed and formaldehyde-fixed paraffin-embedded sympathetic ganglia. All adult human paravertebral-sympathetic neurons displayed trkA neurotrophin-receptor-like protein IR, 10% express trkC neurotrophin-receptor-like protein IR, 37–44% show p75 IR, and no IR was obtained for trkB neurotrophin-receptor-like protein. The intensity of immunostaining was independent of the neuron size. Labelling of non-neuronal tissues, especially blood-vessel walls, was observed for p75, trkA and trkC neurotrophin-receptor proteins. These results indicate that overlapping exists in the expression of p75 and trk neurotrophin-receptor proteins in adult human paravertebral-sympathetic neurons, and suggest that neurotrophins might act on these neurons.     

Alterations in neurokinin A-, substance P- and calcitonin gene-related peptide immunoreactivities in the caudal trigeminal nucleus of the rat following electrical stimulation of the trigeminal ganglion

We have carried out an immunohistochemical study on the presence of neurokinin A (NKA) and substance P (SP) in the rat caudal trigeminal nucleus (CTN) after electrical stimulation of the trigeminal ganglion (TG), used as an experimental model to induce alterations, some of which may occur during migraine attacks (release of vasoactive peptides from perivascular trigeminal axons and neurogenic inflammation). Both unilateral, 30 min electrical stimulation (5 Hz, 5 ms, 0.1-1 mA) of the TG and 5 min stimulation with a slight increase in the stimulating parameters (7.5 Hz, 5 ms, 1.4 mA) caused a significant depletion of the NKA and SP immunoreactivities (-IR) of the TG nerve central terminals in the ipsilateral CTN. Calcitonin gene-related peptide (CGRP)-IR of the ipsilateral CTN was also studied in the CTN using the increased stimulating parameters and a marked depletion of CGRP-IR was also observed following TG stimulation. Such depletion may be due to the release of neuropeptides from the trigeminal central terminals. These findings suggest that NKA, SP and CGRP could act as neurotransmitters at the first central synapses of the trigeminal nociceptive pathway to transmit the sensory stimuli to the higher brain centers.     

Co-localisation of substance P-, bombesin- and peptide histidine isoleucine (PHI)-like peptides in gut endocrine cells of the dogfish Scyliorhinus stellaris

Summary The gastrointestinal tract of cartilaginous fishes, like that of higher vertebrates, is known to contain endocrine cells and nerves immunoreactive for a wide variety of peptides, some of which have been structurally characterised. Since we have found that substance P-, bombesin-and peptide histidine isoleucine-like immunoreactivities are similarly distributed in the endocrine cells of the dogfish pyloric stomach, we have tried to establish whether any of these peptides are co-localised. The cells were compared in thin serial sections with both ligh-and electron microscopical immunocytochemistry. Double immunolabelling was also used to show two immunoreactive peptides in the same tissue section. Further characterisation of the immunoreactivity was attempted by preabsorbing the antibodies with various peptides or synthetic fragments of peptide molecules. Immunoreactivity for all three peptides was frequently present in the same cells, whereas antibodies to other peptides such as gastrin and somatostatin marked different cells. Electron microscopy indicated that all the secretory granules in three morphologically different cell types reacted with antibodies to all three peptides. Dual localisation of unrelated peptides in endocrine cells or nerves is established in many cases, but triple localisation is as yet unusual. The immunoreaction for bombesin-like peptides is different in endocrine cells and nerves, indicating that dogfish bombesin may be present in two forms, in agreement with biochemical evidence.     

Met5-enkephalin- and Met5-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers in the pig female reproductive system

The localization of Met5-enkephalin (ME) immunoreactivity in the female genital organs of the rat, guinea pig and pig was studied by indirect immunofluorescence method. In the rat and guinea pig, no ME immunoreactivity was observed in the uterus, fallopian tube or ovary. In the pig uterus and fallopian tube ME-immunoreactive nerve fibers were observed in muscular and submucose layers as well as around the blood vessels. In the pig ovary, ME immunoreactivity was localized in nerve fibers in medullary and cortical parts of the organ. Met5-enkephalin-Arg6-Gly7-Leu8 (MEAGL) immunoreactivity was also studied in the pig uterus, where its distribution was similar to that of ME. The present results suggest that the pig genital organs receive innervation by nerve fibers containing proenkephalin A-derived peptides, which may have a role in modulation of neurotransmission in these organs.     

Intramural distribution of Met5-enkephalin-Arg6-Gly7-Leu8 in sphincter regions of the human gut

The intramural distribution of Met5-enkephalin-Arg6-Gly7-Leu8 (MERGL) was studied in the oesophago-cardiac, pyloric, ileo-caecal and sigmoid-recto-anal regions of the human digestive tract. Serial samples encompassing each area were separated into mucosa, submucosa and muscularis externa and extracted for radioimmunoassay. Comparatively low levels of MERGL immunoreactivity were measured throughout the cardiac junction. Conversely, a remarkable peak of MERGL concentration was detected at the pyloric junction, in both submucosa and muscularis. A progressive decrease in tissue levels of the same peptide, most evident in the submucosa, was detected on the proximal side of the ileo-caecal region. In the distal sigmoid colon and rectum MERGL concentrations showed a rapid decline, down to very low levels in the anal canal. The results may suggest the involvement of an enkephalinergic mechanism in the control of the human pylorus.