Effects of age and streptozotocin-induced diabetes on contents and effects of substance P and vasoactive intestinal polypeptide in the lower urinary tract of the rat

Andersson , P. O., Fahrenkrug , J., Malmgren , A. & Uvelius , B. 1992. Effects of age and streptozotocin-induced diabetes on contents and effects of substance P and vasoactive intestinal polypeptide in the lower urinary tract of the rat. Acta Physiol Scand 144 , 361–368. Received 29 January 1 991 , accepted 11 October 1991. ISSN 0001–6772. Departments of Physiology, Clinical Pharmacology and Urology, University of Lund, Lund, Sweden and Department of Clinical Chemistry, Bispebjerg Hospital, Copenhagen, Denmark. The urinary bladder and urethral content of substance P and vasoactive intestinal polypeptide and the in vitro effects of the peptides on the bladder were studied at 6 weeks and 6 months of streptozotocin-induced diabetes in the rat. The results were compared with those obtained in age-matched control animals. Both short-term and long-term streptozotocin treatment induced a clearcut increase in bladder weight. Bladder substance P content was increased in both groups of diabetic animals but substance P concentration was similar in control and diabetic animals. Vasoactive intestinal polypeptide content was slightly higher in diabetic animals than in controls but vasoactive intestinal polypeptide concentration was significantly lower in the bladders from both short-term and long-term diabetic animals. The bladder contractile response to substance P was similar in all groups of animals and vasoactive intestinal polypeptide was found to be devoid of contractile or relaxatory effects in the rat bladder. No change in urethral weight was seen with diabetes. There were no clear-cut changes in the urethral contents or concentrations of substance P and vasoactive intestinal polypeptide. The study also enabled comparisons between younger (3 months) and older (9 months) rats. This comparison showed a decrease in the concentrations and contents of substance P and vasoactive intestinal polypeptide between young and older rats. The changes were seen in both the bladder and the urethra and were similar in diabetic and normal animals.     

Detection of Alterations in the Levels of Neuropeptides and Salivary Gland Responses in the Non-Obese Diabetic Mouse Model for Autoimmune Sialoadenitis

The salivary glands of non-obese diabetic (NOD) mice and BALB/c controls were evaluated for the stimulatory effects of the following neuropeptides; substance P (SP), vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY). Injection of either of the three neuropeptides in combination with the muscarinic–cholinergic agonist pilocarpine increased saliva flow rates in BALB/c mice while there was no observable augmentation to flow rates in pre-diabetic or diabetic NOD mice. Small increases in protein content of the stimulated saliva were observed in the BALB/c group of animals with the injection of any of the above neuropeptides in combination with pilocarpine. In pre-diabetic NOD animals, only VIP and NPY increased the protein content-ratio above pilocarpine alone. Radioimmunoassay determination of neuropeptide concentrations in the submandibular and parotid glands revealed reduced levels of SP with diabetes onset as compared with pre-diabetic NOD or BALB/c mice. The levels of NPY were similar between BALB/c and NOD animals except in the pre-diabetic parotid gland where NPY concentrations were 1.3-fold greater. On the other hand, VIP concentrations were substantially reduced in the submandibular gland of NOD mice, while in the parotid gland neuropeptide levels were evaluated 3.8-fold relative to BALB/c controls. Immunohistochemical staining of the parotid and submandibular glands for SP revealed primarily ductal cell staining which was reduced with diabetes onset in NOD animals. These findings further define the sialoadenitis observed in NOD mice to be due, in part, to a general loss of neurotransmitter responsiveness on the part of salivary gland cells.     

Morphofunctional changes underlying intestinal dysmotility in diabetic RIP-I/hIFNβ transgenic mice

The pathogenetic mechanisms underlying gastrointestinal dysmotility in diabetic patients remain poorly understood, although enteric neuropathy, damage to interstitial cells of Cajal (ICC) and smooth muscle cell injury are believed to play a role. The aim of this study was to investigate the morphological and functional changes underlying intestinal dysmotility in RIP‐I/hIFNβ transgenic mice treated with multiple very low doses of streptozotocin (20 mg/kg, i.p., 5 days). Compared with vehicle‐treated mice, streptozotocin‐treated animals developed type 1 diabetes mellitus, with sustained hyperglycaemia for 3.5 months, polyphagia, polydipsia and increased faecal output without changes in faecal water content (metabolic cages). Diabetic mice had a longer intestine, longer ileal villi and wider colonic crypts (conventional microscopy) and displayed faster gastric emptying and intestinal transit. Contractility studies showed selective impaired neurotransmission in the ileum and mid‐colon of diabetic mice. Compared with controls, the ileal and colonic myenteric plexus of diabetic mice revealed ultrastructural features of neuronal degeneration and HuD immunohistochemistry on whole‐mount preparations showed 15% reduction in neuronal numbers. However, no immunohistochemical changes in apoptosis‐related markers were noted. Lower absolute numbers of neuronal nitric oxide synthase‐ and choline acetyltransferase‐immunopositive neurons and enhanced vasoactive intestinal polypeptide and substance P immunopositivity were observed. Ultrastructural and immunohistochemical analyses did not reveal changes in the enteric glial or ICC networks. In conclusion, this model of diabetic enteropathy shows enhanced intestinal transit associated with intestinal remodelling, including neuroplastic changes, and overt myenteric neuropathy. Such abnormalities are likely to reflect neuroadaptive and neuropathological changes occurring in this diabetic model.     

Distribution and origin of peptide-containing nerve fibers in the celiac superior mesenteric ganglion of the guinea-pig

The origin of the peptidergic nerve fibers and terminals in the celiac superior mesenteric ganglion of the guinea-pig was studied. The distribution of immunoreactivity to enkephalin, substance P, calcitonin gene-related peptide, cholecystokinin, vasoactive intestinal polypeptide/peptide histidine isoleucine, bombesin and dynorphin was analysed in intact animals and in animals subjected to various denervation and ligation procedures. The present results show that each of the connected nerve trunks carries peptidergic pathways and contributes to the peptidergic networks in the celiac superior mesenteric ganglion. Thus, the thoracic splanchnic nerves contain enkephalin-, substance P- and calcitonin gene-related peptide-immunoreactivity of which substance P and calcitonin gene-related peptide coexist in the same nerve fibers. In addition, cholecystokinin-, vasoactive intestinal polypeptide/peptide histidine isoleucine- and dynorphin-immunoreactivity is present in some fibers. All of these immunoreactivities are present in sensory neurons except enkephalin which probably originates in the spinal cord. The mesenteric nerves carry enkephalin-, calcitonin gene-related peptide-, cholecystokinin-, vasoactive intestinal polypeptide/peptide histidine isoleucine-, bombesin- and dynorphin-immunoreactive fibers from the intestine and are the main source for cholecystokinin, vasoactive intestinal polypeptide/peptide histidine isoleucine, bombesin and dynorphin fibers. Double-staining experiments indicate that many of these peptides are synthesized in the same enteric neurons. Also the intermesenteric nerve contains peptide-immunoreactive fibers to the celiac superior mesenteric ganglion from different sources, probably including the distal colon as well as dorsal root ganglia and spinal cord at lower thoracic and lumbar levels. The results are discussed in relation to earlier morphological and physiological studies supporting the view of a role of the celiac superior mesenteric ganglion in local reflex mechanisms involved in regulation of gastrointestinal functions.     

In vitro evidence that vasoactive intestinal peptide is a transmitter of neuro-vasodilation in the head of the cat

Experiments have been undertaken to determine the nature of the atropine-resistant neurogenic dilation that can be demonstrated in vitro in cephalic arteries of the cat. Levels of vasoactive intestinal peptide (VIP) and substance P were measured in a number of arteries and related to the extent of the neurogenic dilation that can be elicited in vitro. There is no correlation between the tissue contents of the two peptides. A positive correlation was found between vasoactive intestinal peptide but not substance P content and neurogenic dilation. Vasoactive intestinal peptide but not substance P consistently caused a concentration-dependent dilation of cephalic arteries not subject to significant tachyphylaxis. Vasoactive intestinal peptide antiserum in concentrations that block the dilation to vasoactive intestinal peptide (10(-6) M) but not that due to papaverine, significantly reduced neurodilation of both atropinized and non-atropinized lingual arteries--the cephalic artery with the highest VIP content. These results suggest that vasoactive intestinal peptide and not substance P significantly contributes to the non-cholinergic neurogenic dilation observed in vitro in arterial segments from the head of the cat.     

Distribution of neuropeptides in the limbic system of the rat: the hippocampus

The distribution of several neuropeptides (vasoactive intestinal polypeptide, cholecystokinin octapeptide, substance P, neurotensin, methionine-enkephalin and somatostatin) in the hippocampal formation has been studied with immunocytochemical techniques. Numerous vasoactive intestinal polypeptide, cholecystokinin-octapeptide and somatostatin-positive cell bodies were found within the hippocampus and subiculum. Neurotensin-positive cell bodies were found within the subiculum, but no substance P or methionine-enkephalin-containing cell bodies were seen in either hippocampus proper or subiculum. Vasoactive intestinal polypeptide and cholecystokinin-octapeptide-positive cell bodies were predominantly located in the stratum moleculare and stratum radiatum of CA 1-2 regions and dentate gyrus, whilst somatostatin-containing cell bodies were found mainly in the stratum oriens. Nerve fibres containing each of the six peptides were found within the hippocampus. Large numbers of vasoactive intestinal polypeptide, cholecystokinin-octapeptide and somatostatin fibres innervated the pyramidal and granule cell layers, with smaller numbers in the stratum radiatum and fewer still in the stratum moleculare and stratum oriens. Other than a moderately dense neurotensin-positive fibre plexus in the dorsal subiculum, fewer neurotensin, substance P and methionine-enkephalin fibres were present. However, when present, these three peptides had a distribution restricted to a region close to the pyramidal layer in the CA 2/3 region and to the stratum moleculare of the CA 1 region. Peptide-containing fibres were identified entering or leaving the hippocampus in three ways, via (i) the fornix (all six peptides), (ii) the dorsal subiculum (neurotensin-positive fibres projecting to the cingulate cortex: somatostatin, vasoactive intestinal polypeptide, and cholecystokinin-octapeptide present in fibres running between the dorsal subiculum and occipito-parietal cortex) and (iii) the ventral subiculum (vasoactive intestinal polypeptide, cholecystokinin-octapeptide and somatostatin in fibres running between entorhinal cortex and hippocampus, and all six peptides in fibres crossing the amygdalo-hippocampal border). These findings indicate a major distinction between those peptides (vasoactive intestinal polypeptide, cholecystokinin-octapeptide, somatostatin, neurotensin) which are found in cell bodies intrinsic to the hippocampal formation and those peptides (substance P, methionine-enkephalin) which are found only in hippocampal afferents.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of neuroactive substances in the rat parabigeminal nucleus: an immunohistochemical study

The distribution of neurons and fibres that contain substance P, cholecystokinin-8, vasoactive intestinal polypeptide, corticotropin-releasing factor, calcitonin-gene-related peptide, choline acetyltransferase, tyrosine hydroxylase, somatostatin, leucine-enkephalin, and neuropeptide Y was examined in the parabigeminal nucleus of the rat by immunohistochemistry. Many choline acetyltransferase-like immunoreactive or calcitonin-gene-related peptide-like immunoreactive neurons were observed in the dorsal, middle and ventral subdivisions of the parabigeminal nucleus. A few corticotropin-releasing factor-like immunoreactive neurons were also seen in these three subdivisions. The double-immunostaining demonstrated that some choline acetyltransferase-like immunoreactive neurons in the dorsal and ventral subdivisions contained calcitonin-gene-related peptide. Fibres containing cholecystokinin-8, substance P or vasoactive intestinal polypeptide were abundant in the parabigeminal nucleus. Fibres containing cholecystokinin-8 were concentrated in the dorsal and ventral subdivisions, and the lateral margin of the middle subdivision, whereas many fibres containing substance P or vasoactive intestinal polypeptide existed in the lateral half of each subdivision. Fibres containing calcitonin-gene-related peptide or corticotropin-releasing factor were mostly observed around the immunoreactive neurons. Tyrosine hydroxylase-like immunoreactive fibres were scattered in the parabigeminal nucleus.     

Origin of peptide-containing fibers in the inferior mesenteric ganglion of the guinea-pig: Immunohistochemical studies with antisera to substance P,enkephalin, vasoactive intestinal polypeptide,cholecystokinin and bombesin

After different denervation procedures the guinea-pig inferior mesenteric ganglion was analysed by immunohistochemistry using antisera to substance P, enkephalin, vasoactive intestinal polypeptide, cholecystokinin and bombesin. The results demonstrate that each of the nerve trunks connected to the ganglion carries specific peptidergic pathways. Thus, the lumbar splachnic nerves contain substance P-immunoreactive primary afferent neurons, which to a large extent traverse the ganglion, and enkephalin-immunoreactive preganglionic neurons; the colonie nerves carry vasoactive intestinal polypeptide-, cholecystokinin- and bombesin-immunoreactive fibers from the distal colon to the ganglion; the hypogastric nerves contain vasoactive intestinal polypeptide-positive fibers from the pelvic plexus; and the intermesenteric nerve contains vasoactive intestinal polypeptide, cholecystokinin, substance P and enkephalin from divergent sources. By studying accumulations of peptides in ligated lumbar splanchnic, intermesenteric, hypogastric and colonic nerves the existence of these major peptidergic pathways was confirmed and evidence was obtained for additional, not so prominent, peptidergic projections. The results are discussed in view of earlier morphological and physiological studies.     

Calcium homeostasis in chronic streptozotocin-induced diabetes mellitus in the rat

Calcium homeostasis was studied in freely fed control, streptozotocin diabetic, long-term and short-term insulin-treated diabetic rats 7 wk after the induction of diabetes. In contrast to the short-term (5-12 day) diabetic rat model, intestinal absorption of calcium was markedly enhanced in chronically insulin-deficient animals. Moreover, conventional balance studies showed that these animals were in positive calcium balance despite severe hypercalciuria. Intestinal hyperabsorption of calcium in long-standing diabetic rats occurred despite low levels of circulating 1,25-dihydroxyvitamin D and hypercorticosteronism and was attended by hypercalcemia and suppression of both plasma parathyroid hormone (PTH) and urinary cyclic 3',5'-AMP (cAMP). Long-term insulin replacement completely normalized the intestinal hyperabsorption of calcium, corrected the plasma calcium, and significantly increased circulating PTH and urinary cAMP excretion. Insulin therapy also corrected the decreased plasma 1,25-dihydroxyvitamin D observed in untreated diabetic animals. Intestinal hyperabsorption of calcium appeared to be only partially corrected by short-term insulin therapy. The accumulated results reveal decided differences in calcium homeostasis and hormonal response between the rats with long-standing diabetes and those with diabetes of short duration.     

Effects of neuropeptides on rat cortical neurons: laminar distribution and interaction with the effect of acetylcholine

The effects of the microiontophoretic application of five different peptides (cholecystokinin octapeptide sulfated form, cholecystokinin octapeptide non-sulfated form, vasoactive intestinal polypeptide, angiotensin-II and substance P) on cortical neurons were studied in rats anaesthetized with urethane. Vertical electrode penetrations were made in the first somatic sensory cortex and the laminar position of the neurons determined by the reconstruction of the tracks based on extracellular dye deposits. The first type of effect observed was an excitation of some cortical neurons. These neurons were mostly found in infragranular layers, specially in layer Vb. Pyramidal tract neurons were more often excited by peptides than the cortical population taken as a whole. Substance P excited the largest percentage of neurons, followed by vasoactive intestinal polypeptide and cholecystokinin octapeptide sulfated form, whereas angiotensin II and cholecystokinin octapeptide non-sulfated form were the least potent in terms of frequency of neurons excited as well as of amplitude of the responses. The vast majority of the neurons excited by a peptide could also be excited by acetylcholine. A second and independent effect of peptides was observed: the neuronal excitation induced by acetylcholine could be depressed by the simultaneous application of peptide. This depressing effect was also the most frequently observed with substance P, followed by cholecystokinin and vasoactive intestinal polypeptide.     

The co-localization of neuropeptides in the submucosa of the small intestine of normal Wistar and non-diabetic BB rats.

Immunocytochemical double and triple staining techniques were employed on whole mounts of the submucosal plexus from normal Wistar and non-diabetic BB rat jejunum and ileum, to determine the patterns of co-localization of vasoactive intestinal polypeptide-, peptide histidine-isoleucine-, somatostatin-, neuropeptide Y-, calcitonin gene-related peptide-, substance P-, and galanin-immunoreactive nerves. Neuropeptide Y immunoreactivity was found in 38% of submucosal plexus neurons, within the same neuronal elements as vasoactive intestinal polypeptide immunoreactivity (39% of submucosal plexus neurons) and peptide histidine-isoleucine immunoreactivity. A small population (1% of submucosal plexus neurons) containing vasoactive intestinal polypeptide- and peptide histide isoleucine-like immunoreactivity without NPY-like immunoreactivity was also observed. A significant population of fibers containing vasoactive intestinal polypeptide and galanin immunoreactivity were observed in the mucosa and submucosa, although no cell bodies were detected which contained both neuropeptides. Galanin-like immunoreactivity was seen in a small (2% of submucosal plexus neurons) population, not co-localized with any of the other neuropeptides examined. All somatostatin-immunoreactive neuronal elements (18% of submucosal plexus neurons) contained calcitonin gene-related peptide immunoreactivity, just over half of which also contained substance P immunoreactivity. An additional 25% of submucosal plexus neurons contained calcitonin gene-related peptide- without somatostatin-like immunoreactivity and 28% of submucosal plexus neurons contained substance P without somatostatin-like immunoreactivity. Some degree of co-localization was seen between calcitonin gene-related peptide- and substance P-like immunoreactivity, however, this could not be directly quantified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of neuropeptides in the limbic system of the rat: the bed nucleus of the stria terminalis, septum and preoptic area

The distribution of the neuropeptides vasoactive intestinal polypeptide, cholecystokinin octapeptide, substance P, neurotensin, methionine-enkephalin and somatostatin has been mapped immunocytochemically in the bed nucleus of the stria terminalis, one of the major sites of termination for efferent projections from the amygdala. Immunoreactive fibres and terminals were distributed more or less topographically and largely in accordance with the previously described localization of peptide-containing cell bodies in the amygdala and the amygdaloid projection fields in the bed nucleus as described by neuroanatomical techniques. Thus, vasoactive intestinal polypeptide, which was found in some of the lateral amygdaloid nuclei, had a substantial projection to the lateral bed nucleus. The lateral bed nucleus also contained cholecystokinin-octapeptide, substance P, neurotensin and methionine-enkephalin immunoreactivity which probably derived from the central amygdaloid nucleus, whilst cholecystokinin-octapeptide, and especially substance P-containing fibres, were found in the medial bed nucleus and probably arise from cells in the medial amygdala. Reciprocal amygdalopetal projections were suggested by the presence of substance P- and somatostatin-containing cell bodies in the mediodorsal bed nucleus and vasoactive intestinal polypeptide cells in the lateral bed nucleus, but somatostatin otherwise had a widespread distribution. Numerous local peptidergic connections were also noted both within the bed nucleus and between it and adjacent structures, especially the preoptic area, hypothalamus and the basal ganglia. These data provide further evidence that neuropeptides play a major role in the connectivity of the limbic system and show that the bed nucleus of the stria terminalis is an important relay station, particularly between amygdaloid efferents and other forebrain areas.     

Compartment analysis of vascular effects of neuropeptides and capsaicin in the pig nasal mucosa.

The vascular effects of local infusion of capsaicin, substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) were monitored in an experimental model on the pig nasal mucosa. Arterial, venous and superficial mucosal blood flow (laser-Doppler flowmetry) as well as mucosal volume, reflecting changes in capacitance vessels were studied in parallel. All substances induced concentration dependent increases in the parameters studied with the exception of the decrease in the superficial mucosal flow induced by vasoactive intestinal polypeptide. This latter finding was interpreted as a stealing phenomenon and suggests that vasoactive intestinal polypeptide mainly exerts its vasodilatory effect in the deeper glandular layers of the nasal mucosa. The vasodilatory effect of capsaicin, except the laser-Doppler signal, was markedly reduced by pretreatment with a combination of the ganglionic blocking agent chlorisondamine and atropine implying that capsaicin evokes a central reflex with a final parasympathetic pathway and release of agents like vasoactive intestinal polypeptide. The remaining capsaicin response may depend on a local effect with axon reflexes and the release of sensory neuropeptides with actions on superficial mucosal blood flow.     

Distribution of vasoactive intestinal polypeptide-, substance P-, enkephalin-and neurotensin-like immunoreactive nerves in the chicken gut during development

The ontogeny and distribution of nerve cell bodies and fibres which contain vasoactive intestinal polypeptide-, substance P-, enkephalin- and neurotensin-like immunoreactivity have been studied in the chicken gastrointestinal tract, using immunocytochemistry. All four peptides were found in nerve fibres, with characteristic distribution patterns, which, in the cases of vasoactive intestinal polypeptide, substance P and methionine enkephalin were similar to those described for the mammalian gut. In addition, many of these fibres were shown to arise from intrinsic neurons, since immunoreactive nerve cell bodies for each of the peptides studied were observed. Neurotensin-immunoreactive nerves were confined to the upper part of the tract and neurotensin immunoreactive cell bodies were only observed in embryonic and newly hatched chicken gut. All four peptides were first observed at 11 days of incubation, or Hamburger-Hamilton stage 37,²⁰ in the upper part of the tract, particularly in the gizzard. Substance P and methionine enkephalin were subsequently seen in more caudal regions, while vasoactive intestinal polypeptide developed from each end of the tract. Adult patterns of immunoreactivity in nerve fibres were achieved during the first week after hatching. A striking observation was that immunoreactive neuronal cell bodies were much more abundant in the gut of young chickens and chicken embryos than in that of adult birds.     

Immunocytochemical identification of long ascending peptidergic neurons contributing to the spinoreticular tract in the rat

In the present study, we examined the peptidergic content of lumbar spinoreticular tract neurons in the colchicine-treated rat. This was accomplished by combining the retrograde transport of the fluorescent dye True Blue with the immunocytochemical labeling of neurons containing cholecystokinin-8, dynorphin A1-8, somatostatin, substance P or vasoactive intestinal polypeptide. After True Blue injections into the caudal bulbar reticular formation, separate populations of retrogradely labeled cells were identified as containing cholecystokinin-like, dynorphin-like, substance P-like or vasoactive intestinal polypeptide-like immunoreactivity. Retrogradely labeled somatostatin-like neurons were not identified in any of the animals examined. Each population of double-labeled cells showed a different distribution in the lumbar spinal cord. The highest yield of double-labeling occurred for cholecystokinin, with 16% of all intrinsic cholecystokinin-like neurons containing True Blue. These double labeled neurons were found predominantly at the border between lamina VII and the central canal region. About 11% of intrinsic vasoactive intestinal polypeptide-like neurons in the lumbar spinal cord were retrogradely labeled from the bulbar reticular formation. These neurons were found mostly in the lateral spinal nucleus, with only a few double-labeled cells located deep in the gray matter. Dynorphin-like double-labeled neurons were localized predominantly near the central canal; a smaller population was also seen in the lateral spinal nucleus. It was found that double-labeled dynorphin-like neurons made up 8% of all intrinsic dynorphin-like neurons. Retrogradely-labeled substance P-like neurons were rare; the few double-labeled neurons were found in the lateral spinal nucleus and lateral lamina V. These findings suggest a significant role for spinal cord peptides in long ascending systems beyond their involvement in local circuit physiology.     

The immunohistochemical localization of nine peptides in the sacral parasympathetic nucleus and the dorsal gray commissure in rat spinal cord

The sixth lumbar and first sacral spinal cord segments in the rat contain parasympathetic preganglionic neurons which innervate the pelvic viscera. There have been few studies, however, which have specifically considered the distribution of putative peptide neurotransmitters in these cord segments. The present paper describes and compares the immunohistochemical distribution of dynorphin (1-8)-, enkephalin-, somatostatin-, cholecystokinin octapeptide-, avian pancreatic polypeptide-, FMRF-NH2-, neurotensin-, and vasoactive intestinal polypeptide-like immunoreactivities in the dorsal gray commissure and sacral parasympathetic nucleus of the sixth lumbar and first sacral spinal cord segments in colchicine-treated rats. Antisera against all of the peptides, except avian pancreatic polypeptide, stained cells in the sacral parasympathetic nucleus. Dynorphin (1-8-), enkephalin-, and substance P-like immunoreactive cells were present in significantly greater numbers than somatostatin-, neurotensin-, cholecystokinin-, FMRF-NH2-, and vasoactive intestinal polypeptide-like immunoreactive cells. All of the antisera also stained fibers in the sacral parasympathetic nucleus in varying densities, and a fiber bundle which extended between the dorsal gray commissure and the sacral parasympathetic nucleus. Antisera against substance P and cholecystokinin stained a bundle of fibers that extended between the dorsal horn and the sacral parasympathetic nucleus. Antisera against somatostatin, cholecystokinin octapeptide, substance P and FMRF-NH2 stained an additional fiber bundle which extended between the lateral edge of the dorsal horn and the dorsal gray commissure. All the remaining antisera, except neurotensin, also stained fibers that extended between the sacral parasympathetic nucleus and the dorsal gray commissure, but in a sparser distribution. Immunoreactive cells were localized to the dorsal gray commissure in sections stained with each of the antisera. Dynorphin (1-8) and enkephalin antisera stained the greatest number of cells, followed by FMRF-NH2, neurotensin, somatostatin and avian pancreatic polypeptide. The smallest number of immunoreactive cells was present in substance P, cholecystokinin and vasoactive intestinal polypeptide immunostained sections. A significant difference was noted between the number of dynorphin, enkephalin, somatostatin, cholecystokinin, avian pancreatic polypeptide, FMRF-NH2, neurotensin and vasoactive intestinal polypeptide immunoreactive cells in the sacral parasympathetic nucleus and dorsal gray commissure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Compartment analysis of vascular effects of neuropeptides an capsaicin in the pig nasal mucosa

The vascular effects of local infusion of capsaicin, substance P (SP), calcitonin generelated peptide (CGRP) and vasoactive intestinal polypeptide (VIP) were monitored in an experimental model on the pig nasal mucosa. Arterial, venous and superficial mucosal blood flow (laser-Doppler flowmetry) as well as mucosal volume, reflecting changes in capacitance vessels were studied in parallel. All substances induced concentration dependent increases in the parameters studied with the exception of the decrease in the superficial mucosal flow induced by vasoactive intestinal polypeptide. This latter finding was interpreted as a stealing phenomenon and suggests that vasoactive intestinal polypeptide mainly exerts its vasodilatory effect in the deeper glandular layers of the nasal mucosa. The vasodilatory effect of capsaicin, except the laser-Doppler signal, was markedly reduced by pretreatment with a combination of the ganglionic blocking agent chlorisondamine and atropine implying that capsaicin evokes a central reflex with a final parasympathetic pathway and release of agents like vasoactive intestinal polypeptide. The remaining capsaicin response may depend on a local effect with axon reflexes and the release of sensory neuropeptides with actions on superficial mucosal blood flow.     

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.     

Blockade of nitric oxide decreases the renal vasodilatory effect of neuropeptide Y in the insulin-treated diabetic rat

 The effect of 50 days of streptozotocine-induced diabetes mellitus (blood glucose 20 mmol/l) on contraction and relaxation of isolated renal and intrarenal arteries in rats were examined. Strong and similar contractions were induced by potassium (60 mM), 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1) in renal and intrarenal arteries in diabetic and control rats. The vasodilatory reactivity, after precontraction with 5-HT, of neuropeptide Y (NPY) was similar to that of acetylcholine (ACh), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) and was similar in diabetic and control rats. The relaxing effect of NPY was decreased (40%) only in the diabetic group by blockade of nitric oxide synthase with N ^G-nitro-L-arginine methyl ester (10^–4 M) and by blockade (50%) of NPY with α-trinositol (10^–6 M). In conclusion, the present study showed that diabetes mellitus in the rat is associated with normal vasoconstrictive and vasodilatory capacities. However, the vasodilatory response to NPY was largely eliminated by blockade of nitric oxide synthesis only in the diabetic animals. This indicates that the vasodilatory effect of NPY in diabetes mellitus may be dependent on nitric oxide synthesis. Received: 12 November 1996 / Received after revision: 10 March 1997 / Accepted: 7 April 1997     

Uptake of 5-hydroxydopamine into non-sympathetic nerves of guinea-pig uterine artery in late pregnancy

Summary Perivascular nerve fibres of the uterine artery of virgin and late pregnant guinea-pigs were examined under the electron microscope following loading with 5-hydroxydopamine, a marker for catecholamine uptake, and immunohistochemistry for dopamine hydroxylase, neuropeptide Y, vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide. Varicosities, loaded with 5-hydroxydopamine labelled vesicles, and immunoreactive axons were counted in whole transverse sections of uterine arteries. Localization of the immunoreactivities in 5-hydroxydopamine-labelled vesicles was also studied. Colocalization of substance P and dopamine beta hydroxylase immunoreactivities was investigated at the light microscopic level. Both total and relative number of varicosities with 5-hydroxydopamine-labelled vesicles in a whole section of the artery increased in late pregnancy (61.2 ± 10.2 versus 24.5 ± 3.2 in virgin, representing 35% and 27% respectively, of all varicosities). Also the number of neuropeptide Y, vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide-immunoreactive axons increased, but their relative proportion remained unchanged. In virgin guinea-pigs only calcitonin gene-related peptide and neuropeptide Y immunoreactivities were associated with varicosities loaded with small dense-cored vesicles, while in late pregnancy 5-hydroxydopamine-labelled vesicles were also seen in a number of vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide-immunoreactive axons. Double immunolabelling for dopamine hydroxylase and substance P immunoreactivity showed that substance P immunoreactivity was not present in dopamine hydroxylase-immunoreactive axons of the uterine artery, of neither virgin nor late pregnant guinea-pigs. It is concluded that vascular hypertrophy of the uterine artery in late pregnancy is associated with an increase in the number of perivascular nerve fibres, that involves many, if not all of the subpopulations of neurons supplying the uterine artery. Also 5-hydroxydopamine-labelled varicosities were increased, but the results of the present study indicate that some of the nerve fibres that are able to take up 5-hydroxydopamine in late pregnancy are not sympathetic (i.e are sensory and/or parasympathetic in origin). The relevance of these findings in pregnancy is discussed.