Natriuretic peptides in cetaceans: identification, molecular characterization and changes in plasma concentration after landing

Dolphins are aquatic animals free from gravity, and this may have imposed significant changes in their cardiovascular status and its hormonal regulation compared with terrestrial animals. This study molecularly characterized two major cardiovascular hormones, atrial and B-type natriuretic peptides (ANP and BNP) and measured their changes in dolphin plasma concentrations in relation to the cardiovascular status of the animal. We initially identified ANP and BNP in three species of dolphins (Lagenorhynchus obliquidens, Phocoenoides dalli and Tursiops truncatus). ANP precursors are highly conserved in most mammals, but dolphin BNP precursors were more variable. In molecular phylogenetic analyses, dolphin ANP and BNP precursors grouped with those of artiodactyls, particularly to the camel peptides. The chromatographic characterization of tissue and plasma molecular forms using specific radioimmunoassays showed that the predominant ANP and BNP in the atrium are prohormone and mature peptide, respectively, whereas mature ANP and BNP are circulating in the dolphin blood. A mass spectrometric analysis showed that atrial BNP consists of 26 amino acids, rather than the 32-amino-acid form detected in other mammals. Finally, changes in plasma ANP and BNP concentrations were examined in captive bottlenose dolphins (Tursiops truncatus) after their pool was drained. Plasma ANP and BNP concentrations did not change after landing, unlike terrestrial mammals. Plasma angiotensin II and cortisol concentrations did not change either, showing minor stress after landing. Since landed dolphins show a different cardiovascular status on land than terrestrial mammals, plasma ANP and BNP concentrations seem to reflect the cardiovascular status characteristic of dolphins.     

Neurotrophin receptors and enteric neuronal development during metamorphosis in the amphibian<Emphasis Type="Italic"> Xenopus laevis</Emphasis>

During metamorphosis, the frog intestine goes through a dramatic shortening with extensive apoptosis and regeneration in the epithelial layer and connective tissue. Our aim was to study changes in the enteric nervous system represented by one inhibitory (vasoactive intestinal polypeptide; VIP) and one excitatory (substance P, neurokinin A; SP/NKA) nerve population and concomitant changes in neurotrophin receptor occurrence during this development in the gut of Xenopus laevis adults and tadpoles at different stages of metamorphosis (NF stages 57–66). Sections were incubated with antibodies against the neurotrophin Trk receptors and p75^NTR, and the neurotransmitters VIP and SP/NKA. Trk-immunoreactive nerves increased dramatically but transiently in number during early metamorphic climax. Nerves immunoreactive for p75^NTR were present throughout the gut, decreased in number in the middle intestine during climax, and increased in the large intestine during late metamorphosis. The percentage of VIP-immunoreactive nerves did not change during metamorphosis. SP/NKA-immunoreactive nerves were first apparent at NF stages 61–62 in the middle intestine and increased in the stomach and large intestine during metamorphosis. Endocrine cells expressing SP/NKA increased in number in stomach, proximal, and middle intestine during metamorphic climax. Thus, neurotrophin receptors are expressed transiently in neurons of the enteric nervous system during metamorphosis in Xenopus laevis and SP/NKA innervation is more abundant in the intestine of the postmetamorphic frog than in the tadpole.This study was supported by grants from the Swedish Research Council to S. Holmgren     

Immunocytochemical detection of orexin A in endocrine cells of the developing mouse gut.

Orexins are novel neuropeptides that were originally localized in neurons of the hypothalamus and neuronal fibers of the brain. Recently orexin A and its receptor have also been reported in neurons and endocrine cells of the gastrointestinal tract. Because no studies have been done at the embryonic period, we studied the appearance and distribution of orexin A during the development of mouse gastrointestinal tract using immunocytochemical methods. Immunoreactivity to orexin A was detected in neuroendocrine cells of the pyloric region of the stomach at gestational Day 14 and 1 day after in the small intestine. The numbers of immunoreactive cells progressively increased through development until the adult pattern was reached. Staining of reverse-face sections demonstrated that orexin A and serotonin co-localized in some endocrine cells of the mouse stomach and small intestine. These findings suggest that orexin A may be relevant in the growth and maturation of the gastrointestinal tract during intrauterine life.     

Adrenal expression of orexin receptor subtypes is differentially regulated in experimental streptozotocin induced type-1 diabetes

Orexins (hypocretins) are involved in the regulation of energy homeostasis and sleeping behavior. Orexins were also implicated in the regulation of neuroendocrine and autonomic functions. Recent data show the expression of orexin receptors within the hypothalamic-pituitary-adrenal (HPA) axis and suggest specific actions of orexins at the pituitary and adrenal glands. To further evaluate the role of orexin in the HPA axis, we investigated the mRNA expression of prepro-orexin (PPO) and orexin receptors within the HPA axis of streptozotocin-injected (STZ) rats showing type-1 like diabetes. PPO, as well as OX(1) and OX(2) receptor levels were analyzed by quantitative real-time PCR (qPCR). STZ rats were characterized by decreased body weight, plasma insulin, and leptin levels and by increased plasma glucose. Hypothalamic PPO mRNA levels were significantly reduced in STZ compared to non-diabetic control rats. No differences were found in the mRNA levels of hypothalamic or pituitary OX(1) and OX(2) receptors between control and STZ rats. In adrenals, OX(1) receptor mRNA levels were significantly elevated in STZ rats while OX(2) receptors were significantly reduced. Our results imply distinct functions of adrenal orexin receptor subtypes during type-1 like diabetes.     

An immunohistochemical study of the innervation of the large intestine of the toad (Bufo marinus)

The distribution of intrinsic enteric neurons and extrinsic autonomic and sensory neurons in the large intestine of the toad, Bufo marinus, was examined using immunohistochemistry and glyoxylic acid-induced fluoresecence. Three populations of extrinsic nerves were found: unipolar neurons with morphology and location typical of parasympathetic postganglionic neurons containing immunoreactivity to galanin, somatostatin and 5-hydroxytryptamine were present in longitudinally running nerve trunks in the posterior large intestine and projected to the muscle layers and myenteric plexus throughout the large intestine. Sympathetic adrenergic fibres supplied a dense innervation to the circular muscle layer, myenteric plexus and blood vessels. Axons containing colocalized calcitonin gene-related peptide immunoractivity and substance P immunoreactivity distributed to all layers of the large intestine and are thought to be axons of primary afferent neurons. Five populations of enteric neurons were found. These contained immunoreactivity to vasoactive intestinal peptide, which distributed to all layers of the large intestine; galanin/vasoactive intestinal peptide, which projected to the submucosa and mucosa; calcitonin gene-related peptide/vasoactive intestinal peptide, which supplied the circular muscle, submucosa and mucosa; galanin, which projected to the submucosa and mucosa; and enkephalin, which supplied the circular muscle layer.     

Social evolution in toothed whales

Two contrasting results emerge from comparisons of the social systems of several odontocetes with terrestrial mammals. Researchers have identified remarkable convergence in prominent features of the social systems of odontocetes such as the sperm whale and bottlenose dolphin with a few well-known terrestrial mammals such as the elephant and chimpanzee. In contrast, studies on killer whales and Baird's beaked whale reveal novel social solutions to aquatic living. The combination of convergent and novel features in odontocete social systems promise a more general understanding of the ecological determinants of social systems in both terrestrial and aquatic habitats, as well as the relationship between relative brain size and social evolution.     

Leptin regulation of prepro-orexin and orexin receptor mRNA levels in the hypothalamus

The aim of this study was to determine the effects of leptin treatment on prepro-orexin and orexin receptor expression in the rat hypothalamus. Adult male rats, food-deprived for 48 and 72 h, were treated one time with vehicle or leptin (10 microg, icv). Prepro-orexin mRNA content was measured by semiquantitative RT-PCR, Northern blot, and in situ hybridization; orexin receptor 1 and 2 mRNA content was quantified by Northern blot and/or semiquantitative RT-PCR. Our results indicate that leptin inhibits a fasting-induced increase in prepro-orexin mRNA and orexin receptor 1 mRNA levels in the rat hypothalamus, while orexin receptor 2 mRNA levels were unchanged in all situations evaluated. These data provide direct evidence for an additional mechanism of adaptation of the hypothalamus to food deprivation and for a new effect of leptin in the regulation of food intake.     

Expression of neuropeptides and anoctamin 1 in the embryonic and adult zebrafish intestine, revealing neuronal subpopulations and ICC-like cells

This immunohistochemical study in zebrafish aims to extend the neurochemical characterization of enteric neuronal subpopulations and to validate a marker for identification of interstitial cells of Cajal (ICC). The expression of neuropeptides and anoctamin 1 (Ano1), a selective ICC marker in mammals, was analyzed in both embryonic and adult intestine. Neuropeptides were present from 3 days postfertilization (dpf). At 3 dpf, galanin-positive nerve fibers were found in the proximal intestine, while calcitonin gene-related peptide (CGRP)- and substance P-expressing fibers appeared in the distal intestine. At 5 dpf, immunoreactive fibers were present along the entire intestinal length, indicating a well-developed peptidergic innervation at the onset of feeding. In the adult intestine, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), galanin, CGRP and substance P were detected in nerve fibers. Colchicine pretreatment enhanced only VIP and PACAP immunoreactivity. VIP and PACAP were coexpressed in enteric neurons. Colocalization stainings revealed three neuronal subpopulations expressing VIP and PACAP: a nitrergic noncholinergic subpopulation, a serotonergic subpopulation and a subpopulation expressing no other markers. Ano1-immunostaining revealed a 3-dimensional network in the adult intestine containing multipolar cells at the myenteric plexus and bipolar cells interspersed between circular smooth muscle cells. Ano1 immunoreactivity first appeared at 3 dpf, indicative of the onset of proliferation of ICC-like cells. It is shown that the Ano1 antiserum is a selective marker of ICC-like cells in the zebrafish intestine. Finally, it is hypothesized that ICC-like cells mediate the spontaneous regular activity of the embryonic intestine.     

Pituitary adenylate cyclase activating polypeptide (PACAP) in the gastrointestinal tract of the rat: distribution and effects of capsaicin or denervation

The expression of pituitary adenylate cyclase activating polypeptide (PACAP) was studied in the gastrointestinal tract (GI-tract) of normal rats using radioimmunoassay, chromatography, immunocytochemistry, and in situ hybridization. PACAP-38, PACAP-27, and PACAP-related peptide were demonstrated in all parts of the GI-tract, PACAP-38 being the predominant form confirmed by chromatography. PACAP-immunoreactive nerve fibers and nerve cell bodies were found in the myenteric ganglia throughout the GI-tract. PACAP-containing nerve cell bodies were also demonstrated in the submucous ganglia of the small and large intestine. The synthesis of PACAP in intrinsic neurons was confirmed by in situ hybridization. Double immunostaining showed that PACAP is present in calcitonin gene-related peptide-containing sensory nerve fibers as well as in vasoactive intestinal polypeptide (VIP)- or VIP/gastrin-releasing peptide (GRP)-containing (intramural) nerve fibers in the upper GI-tract and in anally projecting, intrinsic VIP-and VIP/nitric oxide syntase-containing nerve cell bodies and nerve fibers in the small and large intestine. Neonatal treatment with capsaicin significantly reduced the concentration of PACAP-38 in the esophagus, stomach, and colon. Extrinsic denervation decreased the PACAP-38 concentration in the stomach, while no change was observed in the small intestine. These results indicate that PACAP- immunoreactive nerve fibers in the GI-tract originate from both intrinsic (enteric) and extrinsic (presumably sensory) sources suggesting that PACAP may have diverse gastrointestinal functions.     

Orexin receptors: Multi-functional therapeutic targets for sleeping disorders,eating disorders,drug addiction,cancers and other physiological disorders

The orexin peptides (orexin A, orexin B) and their receptors (orexin receptor type 1, orexin receptor type 2) are involved in multiple physiological processes such as the regulation of sleep/wakefulness state, energy homeostasis and reward seeking. A result of this has been the development of small-molecule orexin receptor antagonists as novel therapies for the treatment of insomnia and drug addiction. Increased levels of signaling via the orexin peptide/receptor system may protect against obesity, while somewhat unexpectedly, orexins acting at orexin receptors induce dramatic apoptosis resulting in the significant reduction of cell growth in various cancer cell lines. Meanwhile, the orexin peptide/receptor system is also involved in cardiovascular modulation, neuroendocrine and reproduction regulation. This review summarizes the latest developments in deciphering the biology of orexin signaling as well as efforts to manipulate orexin signaling pharmacologically.     

Trk-neurotrophin receptor-like immunoreactivity in the gut of teleost species

Neurotrophins, acting through their high-affinity signal-transducing Trk receptors, are involved in the development, differentiation and maintenance of discrete neuron populations in the higher vertebrates. Furthermore, the presence of Trk receptors in some non-neuronal tissues, including the endocrine cells of the gut, could indicate an involvement of neurotrophins also in these tissues. Recently, neurotrophins and neurotrophin receptor proteins have been identified in the lower vertebrates and invertebrates, whose amino acid sequences are highly homologous with those found in mammals. The present study investigates the occurrence and distribution of Trk-like proteins in the neurons and gut endocrine cells in five species of teleost. Single and double immunolabeling was carried out on fresh and paraffin-embedded tissue using commercially available antibodies against sequences of the intracytoplasmic domain of the mammalian Trk. Western-blot analysis, carried out on samples of stomach and intestine of bass, identified proteins whose estimated molecular masses (140 kDa, 145 kDa and 143–145 kDa) were similar to those reported for full-length TrkA, TrkB and TrkC in the higher vertebrates. TrkA-like immunoreactivity was found in the enteric nervous system plexuses of three fish species. Trk-like immunoreactivity was observed in the endocrine cells as follows: sparse TrkA-like immunoreactive endocrine cells were detected only in the intestine; TrkB-like immunoreactive cells were detected only in the stomach; and TrkC-like immunoreactive cells were found both in the intestine of the carp and in the stomach of the bass, where they also showed TrkB-like immunoreactivity. These findings confirm the occurrence and distribution of Trk-like proteins in teleosts. These proteins are closely related to the Trk neurotrophin receptors of mammals. The functional significance of Trk-like proteins in both neuronal and non-neuronal cells of teleosts is still not clear. Received: 3 August 1998 / Accepted: 2 October 1998     

Localization and effects of orexin on fasting motility in the rat duodenum

The orexins [orexin A (OXA) and orexin B (OXB)] are novel neuropeptides that increase food intake in rodents. The aim of this study was to determine the distribution of orexin and orexin receptors (OX1R and OX2R) in the rat duodenum and examine the effects of intravenous orexin on fasting gut motility. OXA-like immunoreactivity was found in varicose nerve fibers in myenteric and submucosal ganglia, the circular muscle, the mucosa, submucosal and myenteric neurons, and numerous endocrine cells of the mucosa. OXA neurons displayed choline acetyltransferase immunoreactivity, and a subset contained vasoactive intestinal peptide. OXA-containing endocrine cells were identified as enterochromaffin (EC) cells based on the presence of 5-hydroxytryptamine immunoreactivity. OX1R was expressed by neural elements of the gut, and EC cells expressed OX2R. OXA at 100 and 500 pmol x kg(-1) x min(-1) significantly increased the myoelectric motor complex (MMC) cycle length compared with saline. Similarly, OXB increased the MMC cycle length at 100 pmol x kg(-1) x min(-1), but there was no further effect at 500 pmol x kg(-1) x min(-1). We postulate that orexins may affect the MMC through actions on enteric neurotransmission after being released from EC cells and/or enteric neurons.     

Orexins and feeding: special occasions or everyday occurrence?

Neurons expressing prepro-orexin, the precursor of orexin-A and -B, are found in the lateral hypothalamic area, a region classically implicated in driving feeding. Orexin-A induces feeding transiently when injected centrally, and food intake can be decreased when orexin action is disrupted by immunoneutralization of orexin-A, or by pharmacological blockade of orexin receptors, or by transgenic knockout of orexin. Here, we argue that orexin neurons may act to stimulate feeding in the short term, and that important regulatory signals may be a fall in plasma glucose (stimulatory), countered by satiety signals generated by eating, such as gastric distention (inhibitory).     

Food deprivation differentially modulates orexin receptor expression and signaling in rat hypothalamus and adrenal cortex

Although starvation-induced biochemical and metabolic changes are perceived by the hypothalamus, the adrenal gland plays a key role in the integration of metabolic activity and energy balance, implicating feeding as a major synchronizer of rhythms in the hypothalamic-pituitary-adrenal (HPA) axis. Given that orexins are involved in regulating food intake and activating the HPA axis, we hypothesized that food deprivation, an acute challenge to the systems that regulate energy balance, should elicit changes in orexin receptor signaling at the hypothalamic and adrenal levels. Food deprivation induced orexin type 1 (OX1R) and 2 (OX2R) receptors at mRNA and protein levels in the hypothalamus, in addition to a fivefold increase in prepro-orexin mRNA. Cleaved peptides OR-A and OR-B are also elevated at the protein level. Interestingly, adrenal OX1R and OX2R levels were significantly reduced in food-deprived animals, whereas there was no expression of prepro-orexin in the adrenal gland in either state. Food deprivation exerted a differential effect on OXR-G protein coupling. In the hypothalamus of food deprived rats compared with controls, a significant increase in coupling of orexin receptors to Gq, Gs, and Go was demonstrated, whereas coupling to Gi was relatively less. However, in the adrenal cortex of the food-deprived animal, there was decreased coupling of orexin receptors to Gs, Go, and Gq and increased coupling to Gi. Subsequent second-messenger studies (cAMP/IP3) have supported these findings. Our data indicate that food deprivation has differential effects on orexin receptor expression and their signaling characteristics at the hypothalamic and adrenocortical levels. These findings suggest orexins as potential metabolic regulators within the HPA axis both centrally and peripherally.     

Origin of secretin receptor precedes the advent of tetrapoda: evidence on the separated origins of secretin and orexin

At present, secretin and its receptor have only been identified in mammals, and the origin of this ligand-receptor pair in early vertebrates is unclear. In addition, the elusive similarities of secretin and orexin in terms of both structures and functions suggest a common ancestral origin early in the vertebrate lineage. In this article, with the cloning and functional characterization of secretin receptors from lungfish and X. laevis as well as frog (X. laevis and Rana rugulosa) secretins, we provide evidence that the secretin ligand-receptor pair has already diverged and become highly specific by the emergence of tetrapods. The secretin receptor-like sequence cloned from lungfish indicates that the secretin receptor was descended from a VPAC-like receptor prior the advent of sarcopterygians. To clarify the controversial relationship of secretin and orexin, orexin type-2 receptor was cloned from X. laevis. We demonstrated that, in frog, secretin and orexin could activate their mutual receptors, indicating their coordinated complementary role in mediating physiological processes in non-mammalian vertebrates. However, among the peptides in the secretin/glucagon superfamily, secretin was found to be the only peptide that could activate the orexin receptor. We therefore hypothesize that secretin and orexin are of different ancestral origins early in the vertebrate lineage.     

The enteric nervous system: region and target specific projections and neurochemical codes.

The goal of this report is to summarise the current knowledge on the projection pathways of enteric neurones innervating the muscle and mucosa in different regions of the gut. Combination of neuronal tracing, immunohistochemical and electrophysiological methods has allowed researchers to gain insight into the enteric hardwiring of specific target tissue in the gut. A polarised innervation pattern of the circular muscle was demonstrated for the stomach fundus/corpus and the ileum with descending pathways being primarily nitrergic while ascending pathways were primarily cholinergic. This characteristic hardwiring is thought to set in part the functional basis for peristalsis. A similar polarised innervation pathway was found for the enteric innervation of the mucosa in the stomach and large intestine but not in the small intestine. In both the stomach (myenteric neurones) and in the proximal and distal colon (submucosal neurones), ascending pathways to the mucosa are primarily cholinergic while descending pathways are primarily non-cholinergic. In the colon, results suggest that activation of both pathways induces a cross potentiation of cholinergic and vasoactive intestinal polypeptidergic mediated secretion. Furthermore, a large population of myenteric neurone s projecting to the mucosa in the small and large intestine are probably intrinsic primary afferent neurones sensitive to mechanical as well as chemical stimuli.     

Distribution of neurokinin-2 receptors in the guinea-pig gastrointestinal tract

The distribution of neurokinin-2 (NK2) tachykinin receptors was investigated by immunohistochemistry in the guinea-pig oesophagus, stomach, small and large intestine. Receptor immunoreactivity occurred at the surfaces of smooth muscle cells throughout the digestive tract. Nerve fibre varicosities in enteric ganglia were also immunoreactive. In myenteric ganglia, these varicosities were most numerous in the ileum, frequent, but less dense, in the proximal colon and caecum, rare in the distal colon, extremely infrequent in the rectum and duodenum, and absent from the stomach and oesophagus. Reactive varicosities were rare in the submucous ganglia. Reactive nerve fibres in the mucosa were only found in the caecum and proximal colon. Strong NK2 receptor immunoreactivity was also found on the surfaces of enterocytes at the bases of mucosal glands in the proximal colon. Receptors were not detectable on the surfaces of nerve cells or on non-terminal axons. Reactivity did not occur on nerve fibres innervating the muscle. Denervation studies showed that the immunoreactive varicosities in the myenteric plexus of the ileum were the terminals of descending interneurons. Immunoreactivity for nitric oxide synthase was colocalised with NK2 receptor (NK-R) immunoreactivity in about 70% of the myenteric varicosities in the small intestine. Bombesin immunoreactivity occurred in about 30% of NK2-R immunoreactive varicosities in the small intestine. Received: 10 April 1996 / Accepted: 13 May 1996     

Adaptations to pressure in the RBC metabolism of diving mammals

Marine mammals are known to dive up to 2000 m and, therefore, tolerate as much as 200 atm. of hydrostatic pressure. To examine possible metabolic adaptations to these elevated pressures, fresh blood samples from marine and terrestrial mammals were incubated for 2 h at 37 degrees C under 136 atm (2000 psi) of hydrostatic pressure. The consumption of plasma glucose and the production of lactate over the 2-h period were used to assess glycolytic flux in the red cells. The results indicate that glycolytic flux as measured by lactate production under pressure can be significantly depressed in most terrestrial mammals and either not altered or accelerated in marine mammals. The data also suggest that there is a significant shift in the ratio of lactate produced to glucose consumed under pressure. Interestingly, human and dolphin blood do not react to pressure. These combined data imply a metabolic adaptation to pressure in marine mammal RBC that may not be necessary in human or dolphin cells due to their unique patterns of glucose metabolism.     

Islet amyloid polypeptide (IAPP) in the gastrointestinal tract and pancreas of man and rat

Summary An immunohistochemical study for islet amyloid polypeptide (IAPP) was made on the gastrointestinal (GI) tract and pancreas of man and rat, using antisera raised against a synthetic peptide of C-terminal human IAPP (24–37) and a synthetic peptide of rat IAPP (18–37). A large number of IAPP-immunoreactive cells were found in the pyloric antrum, and a small number in the body of the stomach in both man and rat. Cytoplasmic processes extended out from the bipolar peripheral region of the immunoreactive cells, rather like neuronal processes, and some appeared to make contact with other immunoreactive cells. In addition, small numbers of immunoreactive cells were also seen in the duodenum and rectum, whereas they were absent from the jejunum, ileum and large intestine. An examination was made for evidence of colocalization of IAPP-immunoreactive material with material immunoreactive for gastrin, somatostatin, vasoactive intestinal polypeptide, pancreatic polypeptide, insulin, and glucagon, but none was found. IAPP-immunoreactive cells were also found in the pancreas of non-diabetic and non-insulin-dependent diabetic patients, but they were completely absent from a patient with insulin-dependent diabetes mellitus despite the presence of IAPP in the plasma. The results of these studies suggest that the peptide may have a biological role in situ in the GI tract and, in addition to the pancreas, may be a possible source of plasma IAPP.