Noncholecystokinin peptides in human serum which cause gallbladder contraction

In fasting human serum, cholecystokinin (CCK) is not the principal substance which causes $\text{in}$$\text{vitro}$ rabbit gallbldder contraction. Removal of CCK by affinity chromatography from fasting sera from 8 healthy adults reduced bioactivity only by 18 ± 4% (SEM). Unlike CCK, the bioactivity of serum was enhanced by 30 to 57% rather than destroyed by pronase and chymotrypsin respectively and was not inhibited by dibutyryl cGMP. Reduction of serum bioactivity by carboxypeptidase Y indicated that the bioactive substances in serum are peptides. On Sephadex G-50, bioactive substances eluted in positions different from any known form of CCK. Thus, the principal substances in fasting human serum causing $\text{in}$$\text{vitro}$ gallbladder contraction are not CCK but are most likely small peptides which act at receptors different from the receptors for CCK.     

Immunohistochemical demonstration of peptides, serotonin and dopamine-beta-hydroxylase-like material in the nervous system of the leech Hirudo medicinalis

The central ganglia of the leech, Hirudo medicinalis, were processed for the immunohistochemical localisation of bombesin-, substance P-, cholecystokinin-, vasoactive intestinal polypeptide-, enkephalin-, serotonin- and dopamine-beta-hydroxylase-related substances. To varying extents all of the substances were localised in neuropile processes, and all, with the exception of substance P, were associated with specific perikarya. The most prominent neuropeptides, in terms of the number of immunoreactive neurones, were cholecystokinin and vasoactive intestinal peptide. The dopamine-beta-hydroxylase positive neurones are thought to be octopaminergic, and the serotonin monoclonal antibody revealed positive staining in the Retzius cells. We were unable to demonstrate the coexistence of pairs of substances in any neurones in the leech ganglia.     

Distribution of serotonin-immunoreactive gut endocrine cells in chicks at hatching

Serotonin-immunoreactive, i.e. enterochromaffin (EC) cells were found to be widely distributed in the intestine of the newly hatched chick but sparse in the stomach, and being particularly abundant in the duodenum, upper ileum and rectum. Although in birds, as in mammals, EC cells are most abundant in the intestine, in the stomach they are far sparser than in mammals. Comparison of adjacent sections immunostained for serotonin and a peptide provided no evidence that EC cells in the hatching chick contain motilin or substance P, and that at least the great majority of bombesin-immunoreactive cells contain no serotonin: it is apparent that the mammalian pattern of distribution of peptides in EC cells does not occur in the chick, at least at hatching. Cross reaction of an antiserum to substance P with serotonin was discovered, suggesting the need for a review of existing evidence for co-localisation of this peptide with serotonin.     

Morphological features of the myenteric plexus of the stomach of the axolotl, Ambystoma mexicanum, revealed by immunocytochemistry.

Summary The general morphology of the intramural innervation of the myenteric plexus of the axolotl stomach has been investigated using antisera raised against neuron-specific enolase and a microtubule-associated protein. Additionally, the occurrence of serotonin and several peptidergic neurotransmitter/neuromodulator substances was studied.Immunoreactivity for galanin, vasoactive intestinal polypeptide, substance P and neuromedin U was found in both fibres and intrinsic perikarya, whereas the serotonin and calcitonin gene-related peptide-like-substance-containing nerve fibres seemed to be of extrinsic origin. The axolotl stomach myenteric plexus appeared to be devoid of enkephalin-, neuropeptide Y-, somatostatin-and bombesin-like immunoreactive nerve fibres and nerve cell bodies.Double labelling experiments revealed the presence of a subpopulation of substance P/calcitonin gene-related peptide-like immunoreactive nerve fibres. Contrary to mammals, no coexistence of neuromedin U and substance P was found. Our findings illustrate that besides a number of similarities, considerable species differences exist between urodeles and anurans with regard to the organization of the enteric nervous system.     

Changes in monoamine transmitter concentration in freshwater mussel tissues.

Freshwater mussels were analyzed for biogenic amine transmitter substances in gill tissue, suprabranchial nerve and blood. Gill tissue from normal pondwater-acclimated mussels contained significant amounts of monoamine neurotransmitter substances. In comparison with the suprabranchial nerve the gill tissue contained 42% of the dopamine, 7% of the serotonin and 490% of norepinephrine. Exposing the animals to deionized water (salt-depleted) resulted in a loss of transmitter substances from gill tissue, but serotonin reduction was modest. The mussel gill tissue content of serotonin and the precursor tryptophan was regulated at nearly constant levels. Serotonin is an important transmitter substance in mussels and the many functions it controls, including sodium transport regulation, would depend on its continued presence.     

Immunohistochemical demonstration of peptides,serotonin and dopamine-β-hydroxylase-like material in the nervous system of the leechHirudo medicinalis

Summary The central ganglia of the leech,Hirudo medicinalis, were processed for the immunohistochemical localisation of bombesin-, substance P-, cholecystokinin-, vasoactive intestinal polypeptide-, enkephalin-, serotonin- and dopamine--hydroxylase-related substances. To varying extents all of the substances were localised in neuropile processes, and all, with the exception of substance P, were associated with specific perikarya. The most prominent neuropeptides, in terms of the number of immunoreactive neurones, were cholecystokinin and vasoactive intestinal peptide. The dopamine--hydroxylase positive neurones are thought to be octopaminergic, and the serotonin monoclonal antibody revealed positive staining in the Retzius cells. We were unable to demonstrate the coexistence of pairs of substances in any neurones in the leech ganglia.     

Localization of serotonin, cholecystokinin and somatostatin immunoreactivity in the lower respiratory tract of embryonic, foetal and postnatal sheep.

The lower respiratory tract of the sheep was studied by light-microscopical immunocytochemistry for serotonin, cholecystokinin, somatostatin, bombesin and calcitonin during different periods of lung development; embryonic, foetal and postnatal. At embryonic period only intraepithelial serotonin-containing cells as solitary neuroendocrine cells (NEC) and neuroepithelial bodies (NEB) were found. At foetal stages, immunoreactive cells to serotonin, cholecystokinin and somatostatin were observed in airway epithelium, as solitary NEC and NEBs, and in autonomic intrapulmonary ganglia as single or clusters of small intensely-fluorescent (SIF) cells. In postnatal sheep, serotonin- and cholecystokinin-containing cells were found within airway mucosa as solitary NECs and NEBs. No immunoreactive cells were observed with antiserum to bombesin and calcitonin. Quantitative studies showed that serotonin was the predominant substance, and that solitary neuroendocrine cells were more numerous in distal conducting airways and at foetal stages.     

Modulation of colonic motility by substance P, cholecystokinin and neuropeptide Y.

The effects of substance P, cholecystokinin and neuropeptide Y were examined on rabbit distal colonic motility. All three agents produced increased contractile activity but the mechanisms responsible differed depending on the agent tested. In the intact animal, peptide effects were measured under basal conditions and following exposure to atropine, tetrodotoxin and the alpha-adrenergic antagonist phentolamine. Administration of all three peptides resulted in a stimulation of colonic motility. Phentolamine did not significantly effect substance P-, cholecystokinin- or neuropeptide Y-induced activity. By contrast, the in vivo activity induced by cholecystokinin and neuropeptide Y, but not substance P, was nearly eliminated by tetrodotoxin. Only the neuropeptide Y response was partially atropine sensitive. In isolated colonic strips, cholecystokinin-induced activity, but not that produced by neuropeptide Y or substance P, was blocked by tetrodotoxin. Atropine did not significantly inhibit any of the hormone-induced contractions.     

Immunocytochemical study of the spinal distribution of substance P, enkephalins, cholecystokinin and serotonin in amyotrophic lateral sclerosis

Using immunocytochemical methods, a severe loss of substance P, but not of enkephalin, cholecystokinin and serotonin containing fibers was observed in lamina IX of the spinal cords from 4 amyotrophic lateral sclerosis cases. Substance P-fibers were decreased before degeneration of motoneurons. They were normal in the remaining spinal gray matter.     

Release of contractile agents from rat vas deferens by clonidine.

Clonidine induces contractile effects on the isolated rat vas deferens, but not on rat uterus or guinea-pig ileum. However, we have observed that if clonidine is incubated for about 10 min with a nutrient solution containing an isolated rat vas deferens, the resulting solution can contract an isolated rat uterus, or guinea-pig ileum indicating the involvement of a substance released from the vas. This contractile effect was partially reduced by naloxone and by serotonin antagonists, and by using a denervated vas, indicating that opioids, serotonin and eventually other substances released from nerve tissue of the vas can be involved.     

Relationship between serotonergic measures in periphery and the brain of mouse.

Circadian rhythm and the relationship between the concentration of serotonin (5HT) and related substances (5-hydroxyindoleacetic acid; 5HIAA and tryptophan; Trp) in mouse brain, stomach and blood have been studied. All factors underwent circadian changes in the brain and blood. 5HT and 5HIAA levels in the stomach showed no circadian fluctuation. The concentrations of 5HT in the brain and blood did not correlate. Significant correlations were found between other serotonergic parameters analyzed in brain, stomach and blood. A significant negative correlation was observed between brain 5HIAA and blood 5HIAA. The concentration of tryptophan in the brain was correlated with the plasma total tryptophan level. There was fairly significant correlation (p less than 0.06) between brain serotonin and plasma tryptophan levels. The brain serotonin and tryptophan levels were strongly correlated (R = 0.410, p less than 0.03). Significant negative correlation was found between serotonin in the blood and serotonin in the stomach as well as between its level in the brain and in the stomach. The significance of these findings and their relationship to the use of peripheral serotonergic system as a model of neurons are discussed.     

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.     

The characteristics of the effect of mediator substances on the dynamic oxygen tension in the gastric mucosa]

The studies on rats were carried out to determine dynamics of pO2 in the mucous membrane of the stomach under the effect of acetylcholine, norepinephrine, serotonin, histamine, prostaglandin E2 and ATP. As to the changes in pO2 the mediator substances were arranged as follows (from more intensive effect to less pronounced one): serotonin, acetylcholine, prostaglandin E2, norepinephrine, histamine and ATP. As to the duration of the action--PGE2 acetylcholine, serotonin, norepinephrine, ATP and histamine. Under the joint action of the mediator substances (serotonin, norepinephrine, histamine) with acetylcholine the effects of domination and modulation of acetylcholine effect are found.     

Colocalization of endothelin, vasopressin and serotonin in cultured endothelial cells of rabbit aorta.

The localization and colocalization of endothelin-1, arginine-vasopressin and serotonin in the endothelial cells of rabbit aorta in primary culture were investigated by preembedding and postembedding immunocytochemistry. These three substances were localized in the same population of cells, where they appeared in high proportions (greater than 60%). These findings indicate: 1) that the cell population is heterogeneous, and 2) that these substances are colocalized in some of the cultured endothelial cells. Double labeling in single cells has demonstrated the simultaneous presence of 1) endothelin and vasopressin, 2) vasopressin and serotonin, and 3) endothelin and serotonin. The immunolabeling dominated in the cytoplasmic matrix.     

The toxic effect of dermotrophic substances in tests in vitro

In the model system of tissue culture, the effect of dermatotrophic substances, such as the tenzides and conservatory substances, was tested. The cytotoxic effect of the substances was evaluated according to the vitality of the cells and the lactate dehydrogenase (LDH) activity in the suspension culture of human lymphocytes and further according to the migration inhibition of cells from the animal spleen fragments (SF). The cells obtained from rabbits, guinea pigs, mice and man were used. The highest correlation between the concentration of the substance in the cultivation medium and its toxic effect in the culture has been proved in the tests of LDH activity from the supernatant of the human peripheral lymphocytes culture and further in the migration inhibition test from rabbit SF. Both the tests are in mutual correlation. The determination of LDH activity represents a sensitive marker of the cell damage, the least sensitive is the estimation of the cell vitality. It has been proved that the system of suspension culture of human peripheral lymphocytes and the migration inhibition test from rabbit SF is suitable for the screening determination of the effect of dermatotrophic substances. The system of tissue culture compared to classical tests of skin irritation performed in vivo secures overall objectivity in evaluating the results and the possibility of making a number of parallels without any ethical limits.     

Occurrence of platelet-activating factor (PAF) in normal rat stomach and alteration of PAF level by water immersion stress

We detected platelet-activating substance in gastrointestinal areas, which was confirmed to be platelet-activating factor (PAF) on the basis of the following findings: 1) it comigrated with authentic PAF on thin-layer chromatography; 2) it did not aggregate PAF-desensitized platelets; and 3) its activity was completely antagonized by the receptor antagonists CV3988 and L-652,731. The level of PAF was determined with a bioassay method based on the release of [3H]serotonin from washed rabbit platelets. In the normal rat stomach, the level of PAF was high in the antrum (940 +/- 200 nmol PAF/mol phosphorus of original phospholipids), especially in the antral mucosa (1801 +/- 426 nmol/mol phosphorus of original phospholipids). The stomach PAF level was significantly altered by water immersion stress. Stress for a period of 1 h was associated with a decrease in the antral PAF level to 39 +/- 7% of that of untreated controls. This low PAF level persisted during stress. On the other hand, in the corpus, stress for periods of 1 and 3 h was associated with decreases in the PAF content, and further stress (7 h) resulted in restoration of the PAF level to normal. Furthermore, 7 h of stress was associated with distinct hemorrhagic lesions, which were prevented by CV3988 infused i.v. before the stress. This is the first report of an association between a decrease of the endogenous PAF level in animal tissues and tissue damage.     

Substance 48/80 and experimental inflammation

Substance 48/80 (condensation product of p-methoxy-N-methylphenethylamine with formaldehyde) is known to cause release of histamine and serotonin from the granules in mastocytes. The effect of this substance on the course of experimental inflammation was studied in rats. It was found that substance 48/80 produced local reactions (paw oedema, pleural exudate), while after systemic administration it exerted an antiexudative and antioedematous effect. It is suggested that local signs after administration of the substance are caused, probably, by histamine and serotonin release from the mastocytes and participation of these substances in the inflammatory reaction, since this reaction was partly blocked by antihistamine and antiserotonin agents. The mechanism of inhibition of the inflammatory reactions after systemic administration of the substance is discussed.     

The role of biogenic amines in the neurohumoral mechanisms of postradiation gastrostasis in rats

In experiments with rats it was shown that pharmacological substances that deplete the biogenic amine pool or selectively arrest dopamine and serotonin receptors prevent or considerably reduce the postirradiation gastrostasis. It is suggested that activation of dopamine- and serotonergic inhibiting mechanisms of regulation of motor-evacuative function of the stomach is responsible for the postirradiation delay in gastric evacuation.     

Presence of ranatensin-like and bombesin-like peptides in amphibian brains

Antibodies specific for the carboxyl-terminal regions of bombesin and of ranatensin were used to study the tissue distribution of substances immunochemically similar to these two peptides in three amphibian species. Brain extracts of Rana catesbeiana, Rana pipiens, and Xenopus laevis all contained considerable quantities of both types of immunoreactivity, with measured concentrations as high as several hundred pmol per g tissue. The two antibodies used in this study had very low crossreactivity (less than 1% by RIA) with the other peptide. In addition, gel filtration revealed different elution profiles for the two immunoreactive substances extracted from amphibian brains. Immunocytochemistry revealed differences in localization within nerve fibers and cell bodies and specific absorption by the appropriate peptide. Ranatensin-like peptides were also present in high concentrations in skin of the two Rana species but not in that of Xenopus. Bombesin-like peptides were more abundant in the stomach of all three species. Significant amounts of substance P/phylasaemin-like immunoreactivity also were detected in the brains of all three species. It is concluded that ranatensin-like peptides are not confined to the skin and can be included as central nervous system neuropeptides in amphibians. These two groups of peptides are not species-specific since both are found in brain and stomach of amphibians whose skin contains only ranatensin-like peptide or neither.     

Luminal amino acid sensing in the rat gastric mucosa

Recent advancements in molecular biology in the field of taste perception in the oral cavity have raised the possibility for ingested nutrients to be "tasted" in the upper gastrointestinal tract. The purpose of this study was to identify the existence of a nutrient-sensing system by the vagus in the rat stomach. Afferent fibers of the gastric branch increased their firing rate solely with the intragastric application of the amino acid glutamate. Other amino acids failed to have the same effect. This response to glutamate was blocked by the depletion of serotonin (5-HT) and inhibition of serotonin receptor(3) (5-HT(3)) or nitric oxide (NO) synthase enzyme. Luminal perfusion with the local anesthesia lidocaine abolished the glutamate-evoked afferent activation. The afferent response was also mimicked by luminal perfusion with a NO donor, sodium nitroprusside. In addition, the NO donor-induced afferent activation was abolished by 5-HT(3) blockade as well. Altogether, these results strongly suggest the existence of a sensing system for glutamate in the rat gastric mucosa. Thus luminal glutamate would enhance the electrophysiological firing rate of afferent fibers from the vagus nerve of the stomach through the production of mucosal bioactive substances such as NO and 5-HT. Assuming there is a universal coexistence of free glutamate with dietary protein, a glutamate-sensing system in the stomach could contribute to the gastric phase of protein digestion.